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PROGRESS IN

Nucleic Acid Research and Molecular Biology Volume

44

This Page Intentionally Left Blank

PROGRESS IN

Nucleic Acid Research and Molecular Biology edited by

WALDO E. COHN

KlVlE MOLDAVE

Biology Division Oak Ridge National Laboratory Oak Ridge, Tennessee

Department of Molecular Biology and Biochemistry University of California, lrvine Irvine, Cal$ornia

Volume 44

ACADEMIC PRESS, INC. Harcourt Brace Jooanooich, Publishers San Diego New York Boston london Sydney Tokyo Toronto

This bock is printed on acid-free paper. @

Copyright 0 1993 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system. without permission in writing from the publisher.

Academic Press, Inc. 1250 Sixth Avenue, San Diego, California 92101-4311 United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NW 1 7DX

Library of Congress Catalog Number: 63- 15847 International Standard Book Number: 0-12-540044-6 PRINTED IN THE UNITED STATES OF AMERICA 9 3 9 4 9 5 9 6 9 7 9 8

BE

9 8 1 6 5 4 3 2

1

Contents

ABBREVIATIONSAND SYMBOLS ........................................

ix

.......................

xi

SOME

ARTICLES PLANNED FOR FUTUREVOLUMES

Structure and Action of Mammalian Ribonuclease (Angiogenin) Inhibitor Frank S . Lee and Bert L . Vallee I. I1. 111. IV. V.

Purification. Physicochemical Properties. and Occurrence . . . . . . . . . . Primary Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibitory Properties .......................................... Biologic Role ................................................ Concluding Remarks .......................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 6 10 20 24 26

Bacterial Adenylyl Cyclases Alan Peterkofsky. Aiala Reizer. Jonathan Reizer. Natan Gollop. Peng-Peng Zhu and Niranjana Amin I . The Action of cAMP as a Transcription Regulator in Escherichiu coli ............................................ I1. Regulation of cAMP Levels in E . coli .... I11. Structure and Expression of the E . coli Ade (cyu) Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. The Phosphoenolpyruvate:sugarPhosphotransferase System . . . . . . . . V. Regulation of E . coli Adenylyl Cyclase Activity by the Phosphoenolpyruvate:sugar Phosphotransferase System . . . . . . . . . . . . VI. Regulation of E . coli Adenylyl Cyclase Activity by Other Factors . . . . VII . AdenylyI Cyclases in Bacteria Other Than E . coli ................. VIII. Sequence Comparisons . . . . . . . . . . .................. IX. ATP-Binding Sites ...................... ................... X . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ..................................................

32 34 35 36

38 41 44 53 56

62 62

Initiation of Transcription by RNA Polymerase 11: A Multi-step Process Leigh Zawel and Danny Reinberg I . The Structure of Class I1 Promoters ............................. I1. RNA Polymerase I1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V

68 69

vi

CONTENTS

Transcription Factors and Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preinitiation and Initiation Complexes and Motifs . . . . . . . . . . . . . . . . . Activation and the General Transcription Factors . . . . . . . . . . . . . . . . . Repression of Class I1 Gene Transcription . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . , . . . . . . . . . , . . . . . . . . . . , . . . . . . . . . .

111.

IV. V. Vl.

75 94 100 102 105

Regulation of Repair of Alkylation Damage in Mammal ion Genomes Sankar Mitra and Bernd Kaina Historical Perspective . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . Unusual Repair of Ofi-Alkylguanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Multistep Repair of N-Alkylpurines . . . . . . . . . . . . . . . . . . . . . . IV. Properties of Mammalian Ofi-Methylguanine-DNA Methyltransferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Cloning of Mammalian Alkylation Repair Genes by Phenotypic Rescue of E . coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... .. VI. Regulation of Mammalian M G M T and MPG VII. Role of DNA Methylation in Methyltransferas VIII. Inclucihility of Alkylation Repair Genes . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Alkylating Drug Resistance and Regulation of DlVA Repair . . x. Amplification of th e and Drug Resist ......... ................ XI. Outlook . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

11. 111.

109 112 114 116 118 120 128 129 132 135 136 137

Cell Delivery and Mechanisms of Action of Antisense Oligonucleotides Jean Paul Leonetti, Genevihve Degols, Jean Pierre Clarenc, Nadir Mechti and Bernard Lebleu I. 11. 111.

IV. V. \'I.

VII.

Historical Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . From the Antisense Approach to the SNAIGE Concept Limitations of thc SNAIGE Approach . . . . . . . . . . . . . . . . . , . . . . . . . . . Internalization and Targeting of Oligonucleotides In tracelliilar Distribution of OIigonucIeotides . . . . . . . . . . . . . . . . . . . . . Mechanisms of Action of Antisense Oligonucleotides in the VSV Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and Perspectives ...... Hefcrences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

143 145 148 153 160 161 163 164

Enzyme Organization in DNA Precursor Biosynthesis Christopher K . Mathews 1. 11.

Enzyme Organization and Contemporary Enzymology . . . . . . . . . . . . . E d y Evidence for dNTP Coinpartmentation . . . . . . . . . . . . . . . . . . . . ,

167 171

CONTENTS

I11. T4 dNTP Synthetase: A Multienzyme Complex for Deoxyribonucleotide Synthesis ................................. IV. Is the T4 dNTP Synthetase Complex Linked to DNA Replication Machinery? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Enzyme Organization in Bacterial Cells ......................... VI . Organization of dNTP Synthesis in Eukaryotic Cells . . . . . . . . . . . . . . . VII . General Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii 177 181 186 187 200 201

Identification and Characterization of Novel Substrates for Protein Tyrosine Kinases Michael D . Schaller. Amy H . Bouton. Daniel C . Flynn and J. Thomas Parsons I . Detection of Phosphotyrosine-containing Proteins . . . . . . . . . . . . . . . . . I1. Receptor Protein Tyrosine Kinases .............................. I11. Oncogenic Protein Tyrosine Kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Novel Strategies for the Identification of Substrates . . . . . . . . . . . . . . . V. Tyrosine Phosphorylation: Molecular Consequences . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

207 208 211 215 222 224 229


Abbreviations and Symbols All contributors to this Series are asked to use the terminology (abbreviations and symbols) recommended by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN) and approved by IUPAC and IUB. and the Editors endeavor to assure conformity. These Recommendations have been published in many journals (I.2) and compendia (3) and are available in reprint form from the Office of Biochemical Nomenclature (OBN); t h 9 are therefore considered to be generally known. Those used in nucleic acid work, originally set out in section 5 of the first Recommendations ( I )and subsequently revised and expanded (2.3). are given in condensed form in the frontmatter of Volumes 9-33 of this series. A recent expansion of the one-letter system ( 5 ) follows. (5) SINGLE-LETTER CODERECOMMENDATIONS. Symbol

Meaning

Origin of symbol Guanosine Adenosine (ribo)Thymidine (Uridine) Cytidine

R Y M

K S Wb

G or A T(U) or C A or C G or T(U) G or C A or T(U)

puRine pyrimidine aMino Keto Strong interaction (3 H-bonds) Weak interaction (2 H-bonds)

C or T(U) T(U) or C C or A A or T(U)

D’

A or G or G or G or

N

G or A or T(U) or C

aNy nucleoside (i.e., unspecified)

Q

Q

Queuosine (nucleoside of queuine)

H B V

not not not not

G; H follows G in the alphabet A; B follows A T (not U); V follows U C; D follows C

‘Modified from h c . Natl. Acad. Sci. LISA. 83, 4 (1986). bW has been used for wyosine, the nucleoside of “base Y” (wye). ‘D has been used for dihydrouridine (hU or H, Urd).

Enzymes

In naming enzymes, the 1984 recommendations of the IUB Commission on Biochemical Nomenclature ( 4 ) are followed as far as possible. At first mention, each enzyme is described eirher by its systematic name or by the equation for the reaction catalyzed or by the recommended trivial name, followed by its EC number in parentheses. Thereafter, a trivial name may be used. Enzyme names are not to be abbreviated except when the substrate has an approved abbreviation (e.g., ATPase, but not LDH. is acceptable).

ix

ABBREVIATIONS AND SYMBOLS

X

REFERENCES 1. JBCtQ1.527 (1%); &hem 5, 1445 (1966); BIlO1. 1 (1966); ABB 115, I (1966), 129, I (1%9); and e1sewhere.t General. 2. EJB 15, 203 (1970); JBC 245, 5171 (1970); JMB 55. 299 (1971); and e1smhere.t 1. “Handbook of Biochemistry” (G. Fasman. ed.), 3rd ed. Chemical Rubber Co., Cleveland, Ohio, 1970. 1975, Nucleic Acids. Vols. I and 11, pp. 3-59. Nucleic acids. 4. “Enzyme Nomenclature“ [Recommendations (1984) of the Nomenclature Committee of the IUB]. Academic Pms. New York, 1984. 5. EIB 150, I (1985). Nucleic Acids (One-letter system).t Abbreviotions of Journal Titles

Journah

Abbreviotions used

Annu. Rev. Biochem. Annu. Rev. Genet. Arch. Biochem. Biophys. Biochem. Biophys. Res. Commun. Biochemistry Biochem. J. Biochim. Biophys. Acta Cold Spring Harbor Cold Spring Harbor Lab Cold Spring Harbor Symp. Quant. Biol. Eur. J. Biochem. Fed. Proc. Hoppe-Seyler’s Z. Physiol. Chem. J. h e r . Chem. Soc. J. Bacteriol. J. Biol. Chem. J. Chem. Soc J. Mol. Biol. J. Nat. Cancer Inst. Mol. Cell. Biol. Mol. Cell. Biochem. Mol. Gen. Genet. Naturs New Biology Nuclek Acid Research Proc Natl. h d . Sci. U.S.A. Proc Soc Exp. Riot. Med. progr. NucI. Acid. Res. Mol. Biol.

ARB ARGen ABB BBRC Bchem BJ BBA CSH CSHLab CSHSQB EJB FP ZpChem JACS J. Bact. JBC JCS

JMR JNCI MCBiol MCBchem MGG Nature NB NARCS PNAS PSEBM This Series

?Reprints available from the Office of Biochemical Nomenclature (W.E. Cohn, Director).

Some Articles Planned for Future Volumes The DNA Binding Domain of the Zn(ll)-containing Transcription Factors JOSEPH E. COLMAN AND T. PAN mRNA Binding Proteins in Eukaryotic Cells TOMDONAHUE AND K. GULYAS Specific Hormonal and Neoplastic Transcriptional Control of the Alpha 2u Globulin Gene Family

PHILIPFEIGELSON Molecular Biology in the Eicosanoid Field

COLIND. FUNK Cellular Transcriptional Factors Involved in the Regulation of HIV Gene Expression

RICHARDGAYNOR AND C. MUCHARDT tRNA Structure and Aminoacylation Efficiency

R~CHARD G I E G ~JOSEPH ~, D. PUGLISIAND CATHERINE FLORENTZ Control of Mitochondria1 Biogenesis in Yeast

LES GRIVELLAND H.

DE

WINDE

snRNA Genes: Transcription by RNA Polymerase II and RNA Polymerase 111 NOURIA HERNANDEZ AND LOBO

s.

Enzymology of Homologous Recombination in the Yeast Saccharornyces cerevisiae w. -D. HEYERAND RICHARDD. KOLODNER Regulation of mRNA Stability in Yeast

ALLANJACOBSON AND

s. PELTZ

Signal-transducing G Proteins: Basic and Clinical Implications

MICHAEL A. LEVINE Synthesis of Ribosomes LASSE LINDAHL AND

J. M. ZENGEL

Nitrogen Regulation in Bacteria and Yeast

BORIS MAGASANIK Immunoglobulin Gene Diversification by Gene Conversion WAYNE T. MCCORMACK, LARRY TJOELKER AND

w.

CRAIGB. THOMPSON xi

xii

SOME ARTICLES PLANNED FOR FUTURE VOLUMES

ADP-ribosylation Factors

JOEL MOSS AND MARTHA VAUGHAN Regulation of Eukaryotic mRNA Entry into Polysomes by Initiation Factor Phosphorylation

ROBERT E. RHOADES Mammalian 6-Phosphofructo-2-kinase/fructose-2,6-biphasphatase:A Bifunctional Enzyme

GUY G . ROUSSEAU AND LOUISHUE Analysis of Rice Genes in Natural and Transgenic Plants

R A Y WU, XIAOLAN

DUANAND

DEPING X U

Structure and Action of Mammalian Ribonuclease (Angiogenin) Inhibitor 1 FRANKs. LEE AND , BERT L. VAL LEE^

1 1

Center for Biochemical and Biophysical Sciences and Medin'ne Harvard Medical School Boston, Massachusetts 02115

I. Purification, Physicochemical Properties, and Occurrence . . . . . . . . . . . .

A. Inhibition Constants

2

....

.......................

20

References . . . . .

A exceptionally potent protein RNase2 inhibitor occurs in the tissues of mammalian species (1, 2). While this 50-kDa protein inhibitor is commonly employed experimentally to inhibit RNA degradation by adventitious RNases (3-18), it undoubtedly possesses a physiologic significance of its own. Among the functionally diverse mammalian RNases that it inhibits are some able to induce biologic activities that include neovascularization, ataxia, and paralysis, and others that possess potent helminthotoxic or antispermatogenic properties (reviewed in 19-22). Our original interest in this inhibitor stemmed from our efforts to identify factors involved in the regulation of organogenesis in general, and blood vessel development in particular. The culmination of this work was the isolation of angiogenin, a 14-kDa protein initially purified from the conditioned medium of the human adenocarcinoma cell line HT-29 and subsequently from normal human plasma (23,24).Angiogenin is a potent inducer 'To whom correspondence may be addressed. ZAbbreviations: RNase, ribonuclease; CAM, chorioallantoic membrane; RNase A, bovine pancreatic ribonuclease A; PRI, human placental RNase inhibitor; HRAS, Harvey ras oncogene homologue; IGF2, insulin-like growth factor 2; HBB, p-globin; INS, insulin.

1 Progress in Nucleic Acid Research and Molecular Biology, Vol. 44

Copyright 0 1993 by Academic Press, Inc. All nghts of reproduction in any form reserved.

2

FRANK S. LEE AND BERT L. V A L I X E

of blood vessel growth in the chick chorioallantoic membrane (CAM) and the avascular rabbit cornea (23, 25, 26), with neovascularization in the CAM assay occurring at angiogenin doses as low as 0.5 ng. Angiogenin exhibits specific, saturable binding to calf pulmonary endothelial cells (27). It stimulates phospholipases C and A, in endothelial cells at concentrations as low as 0.1 ng/ml, but is not an endothelial cell mitogen (28, 29). The 35% identity of the angiogenin primary structure to that of bovine pancreatic ribonuclease A (RNase A) is a most unexpected feature (30, 31). Three residues catalytically essential in RNase A (Lys-41, His-12, and His-119) are fully conserved in angiogenin. Importantly, the catalytic activity of angiogen is distinct from that of RNase A or other RNases, and this, in turn, distinguishes it from all other angiogenic factors (32). These findings provided a unique opportunity to examine naturally occurring ribonuclease inhibitors for their antiangiogenic properties, and this resulted in the finding that the human protein RNase inhibitor abolishes both the angiogenic and ribonucleolytic and phospholipase C-stimulating activities of angiogenin (28, 33). Beyond pointing toward angiogentn regulation as a possible physiologic function for this inhibitor, these experiments indicated the importance of obtaining further information about this inhibitor as a basis for novel approaches toward antiangiogenesis. Antiangiogenic agents could potentially play important roles in the clinical treatment of a wide variety of diseases, including cancer, diabetic retinopathy, and rheumatoid arthritis (34). This review focuses on recent studies of the mammalian ribonuclease inhibitor and, in particular, human placental ribonuclease inhibitor (PRI). Far less is known about this protein inhibitor of ribonucleases than about protein inhibitors of proteinases, for which a vast literature exists (reviewed in 35-37). Nevertheless, recent studies have revealed distinctive properties of this family of proteins, properties of interest from the point of view of understanding their relation to the inhibition of the activities of the mammalian RNase superfamily of enzymes, including angiogenin in particular.

1. Purification, Physicochemical Properties, and Occurrence Early work on the protein RNase inhibitor from various mammalian tissues has been reviewed (I, 2). Hence the present discussion is confined to certain important aspects of that period and focuses on more recent studies. Historically, the existence of an RNase inhibitor was first inferred from the presence of latent RNase activity in the high-speed supernatant fraction from guinea pig liver homogenates (38).Acidification of the extract resulted in an increase in measurable RNase activity, presumably due to the dissociation of an RNase-inhibitor complex. Many critical studies (39-44) on the corre-

MAMMALIAN RIBONUCLEASE (ANGIOGENIN) INHIBITOR

3

sponding inhibitor from rat liver demonstrated that it specifically inhibits bovine pancreatic ribonuclease A. These studies suggested that it was a protein, based on the susceptibility of the activity to proteases, acid conditions (0.05 M HCl), heat (65OC, 5 min), and sulfhydryl reagents [p-(hydroxymercuri)benzoate, N-ethylmaleimide, iodoacetate] (39, 41, 43). Initial attempts to isolate the inhibitor (40-42) were hampered by its sensitivity to air-oxidation, dilution to low protein concentrations, freezethaw cycles, and exposure to metals that often contaminated the RNA preparations employed to assay inhibitor activity (40, 42, 45-48). The use of reducing and metal-chelating agents in buffers employed during isolation facilitated maintenance of activity (42, 45, 48, 49). There have been many reports of partial purification of activity (11, 50-58). A critical advance in the purification of this inhibitor was the use of RNase A-Sepharose &nity chromatography (59).This allowed for the isolation of PRI and subsequently other inhibitors from mammalian sources in quantities sufficient for physicochemical analysis (59-62). The salient features of these inhibitors can be summarized as follows. They are acidic (pZ -4.7), heat- and acid-labile, sulfhydryl-dependent proteins of M, -50,000. They form 1:1 complexes with the highly basic (PI >9), acid-stable, mammalian pancreatic RNase (M, -14,000). They inhibit the enzymatic activity of this mammalian RNase but not that of Escherichia coli RNase I or the fungal RNases N,, T,, or U, (52, 63-65). Their amino-acid compositions are marked by high contents of Leu (-20% on a molar basis) and Cys (-7%) (Table I) (60-62, 66-68). Differences between these features and those of certain other inhibitors have been reported. For example, the inhibitor isolated from bovine lens is reported to have an M , of -30,000 (69, 70) while that from porcine brain is reported to have relatively low Leu and Cys contents (65).However, it is difficult to evaluate these observations because the stated specific activities of the protein preparations examined suggest that they were not pure. The inhibitor is a cytoplasmic protein, and the high-speed (60,000105,000 g) supernatant fraction of tissue homogenates is the source of virtually all inhibitory activity. Consistent with this, there is no evidence for signal peptides in the cDNA sequences encoding the human placental, HeLa cell, and porcine kidney inhibitors (66-67, 71).Free inhibitory activity can be detected directly in tissue homogenates. At the same time, latent RNase activity can be detected by treatment of the homogenates with sulfhydry1 reagents, presumably by inactivation of the sulfhydryl-dependent, bound inhibitor. Therefore, the inhibitor is apparently in stoichiometric excess over the enzyme(s) inhibited; the degree of molar excess has been estimated at three- to eight-fold (2, 72).The inhibitor constitutes -0.01% of the totaI protein in cytoplasmic extracts (2). Nuclear, mitochondrial, and

TABLE 1 AMINO-ACID(hMPOSITIONS

OF

MAMMALIAN HIHONLICLEASE

~NHIBI'IOHS"

~

Amino acid Asx

GlX Ser Cly His

Thr Ala Pro Tvr Val Met Ile Leu Phe

LYS CYS TrP Total Ref.

H 11 man placenta"

Huinan

HeLa"

Porcine liver","

Bovine brain"

44 59 45 32 5 24 13 32 14 3 25 2 12 92 4 16 32 6

44 59 46 32 5 23 13 32 14 3 25 2 12 92 4 16 32 6

40 60 38 37 7 22 23 32 16 4 19 2 9 98 0 14 30 5

43 65 40 53 5 20 20 38 18 5 22 2 9 88 3 15 30 5

460 66

460 67

456 68

481 60

aArnino-acid compositions reported in residues/mole protein. bFrom amino-acid sequence. CIdentical to porcine kidney inhibitor. dFrom amino-acid analysis of protein. "Not determined.

-.

nat

Ovinr liver"

Mti riiir liver"

Rat lived

47 6 19 21 37 1.5 5 24 2 9 91 4 14 30 5

48 62 42 51 5 20 19 35 17 4 23 1-2 10 92 4 15 27 6

55 63 13 33 3 19 22 30 I6 5 20 3 13 94 3 21 26-27 5

59 60 43 33 6 20 23 29 17 5 22 2 10 89 5 20 31 5

8 20 2 13 66 7 18 31 NUE

483

482

475

479

-

61

61

61

61

62

Bovine liver"

47 62 45

testis"

55 61 42 40 7 23 25 30 15


5

microsomal cell fractions, in contrast, contain little or none of the inhibitor (73, 74). Small amounts of inhibitor appear to be associated with mRNAribosomal particles (64,75). The RNase inhibitor has been detected in all mammals and in almost all tissues examined, which include lung, heart, parotid, esophagus, stomach, intestine, liver, pancreas, kidney, immature uterus, placenta, ovary, testis, thyroid, adrenal, thymus, spleen, reticulocyte, brain, muscle, and fat tissue (39, 58-63, 76-82). While most studies have assayed for the presence of inhibitor by inhibition of RNase activity, others have also documented its presence using anti-inhibitor antibodies (83) or the inhibitor cDNA (84). Among the tissues or cells examined, only mature uterus (82, 85, 86) and lymphocytes (87, 88) lack detectable levels of the inhibitor. It is not clear whether there is in fact a complete absence of it in these instances or whether it is present in quantities below the limit of experimental detection. In any case, it can be said that these inhibitors constitute a family of related, cytoplasmic proteins ubiquitous or nearly so in mammalian tissues. Extracellular fluids, which contain a variety of RNases, have also been examined for the presence of the inhibitor. Monoclonal antibodies directed against PRI detect immunoreactive material in human serum that would correspond to a concentration of 2 to 3 pg/ml if this material were PRI (89). However, when mammalian serum is either examined for latent RNase activity (90) or assayed directly for RNase inhibition (39, 58), no evidence for inhibitory activity is found. The RNase inhibitor may be present in serum in an inactive form; alternatively, it may be active but at undetectable levels. There is no evidence for the presence of inhibitor in either urine or saliva (55, 77). RNase inhibitory activity has been detected in nonmammalian organisms, including starfish nucleoli (91)and cytoplasmic extracts of insects (92), frogs (93,94), and chickens (78, 95, 96). As with the mammalian protein, the presence of the nonmammalian inhibitor has been demonstrated either by latent RNase activity or by direct inhibitory activity toward the endogenous RNase of the organism examined. These nonmammalian inhibitors do not appear to inhibit mammalian pancreatic RNase (49, 78). However, in the converse situation, the mammalian RNase inhibitor does inhibit at least one nonmammalian RNase, that from the pancreas of the snapping turtle (R. Shapiro, personal communication). This RNase is -30% identical in primary structure to mammalian pancreatic RNase; it is conceivable that some nonmammalian species may possess as yet undetermined inhibitors of mammalian pancreatic RNase. None of the nonmammalian inhibitors described have been purified to homogeneity. Gel filtration of crude cytoplasmic extracts suggests that the mass of the complex between frog inhibitor and its endogenous ribonuclease

6

FRANK S. LEE AND BERT L. VALLEE

is -130 kDa (93, 94). It is not known whether the difference in M, observed between this complex and the mammalian RNaseaRNase inhibitor complex reflects an intrinsic difference in the inhibitor, such as in molecular weight or inhibition stoichiometry.

II. Primary Structure The primary structures of RNase inhibitors from human placenta (66, 71), HeLa cells (69, and porcine kidney (84)have been derived from the cDNAs. In addition, that for the porcine liver inhibitor, identical to that from porcine kidney, comes from the amino-acid sequence (68).The primary structures of inhibitors from human placenta and HeLa cells are 99% identical and differ only at two residues, 422 and 423 (Arg and Gln, respectively, in the placental inhibitor; Ser and Glu, respectively, in the HeLa cell inhibitor). Those from human and porcine sources are 77% identical, and it may be noted that the human and porcine inhibitors bind to RNase A with comparable af€inity (see Section 111, Inhibitory Properties). The porcine inhibitor, 456 residues in length compared to 460 for the human placental inhibitor, is modified by Nacetylation at its N-terminus (68). The N-terminus of the human placental inhibitor is also refractory to Edman degradation and probably is modified by N-acetylation as well (66). The internal repeat structure is a salient feature of the inhibitor aminoacid sequence. It has been described variously as consisting of either (1) seven to eight 57-residue repeats, with each 57-residue repeat consisting of an internal duplication of a half-repeat (66, 67) or (2) 15 alternating repeats, with the alternating repeats consisting of either 28 or 29 residues each (68). The descriptions are essentially identical and for the sake of convenience the inhibitor is here described as consisting of seven direct, uninterrupted, internal repeat units, each exactly 57 amino acids in length (Fig. 1).These repeat units comprise nearly 90% of the molecule and are flanked by shorter N- and C-terminal segments that display a weak homology to the strong internal repeat motif. The average identity between any two repeat units is -40%. In the primary structure of PRI, 76-78% of either the leucines or the cysteines is conserved (present in at least four of the seven repeats) within the repeat units, compared to only 29% for the acidic residues (Asp Glu) (Fig. 1).Repetitive structural features reflected by the high degree of conservation of leucine and cysteine residues may constitute a common scaffold on which other residues, e.g., polar ones, determine specificity of interaction (67, 68). In this regard, it should be noted that the high conservation of cysteine residues does not imply the presence of conserved disulfide bonds, since chemical modification of these residues suggests that all are present in

+

LDIQCEELSDARWAE

1

-~

30 87

144 201

258 315 372 429

VESVR

SWRV I S

FIG. 1. Primary structure of PRI, highlighting the seven direct internal repeats (adapted from 66). The first residue in each line is numbered at the left. Shaded background is present for identical residues that occur in at least four of the seven repeats.


8

the reduced form (66, 84). The absence of disulfide bonds in PRI is consistent with its cytoplasmic localization, since the cysteines of cytoplasmic proteins are generally in the reduced form (97). Leucine-rich repeat units are not unique to RNase inhibitor. Related repeats, which are close to or exactly 21 residues long, have been identified in an increasing number of both mammalian and nonmammalian proteins (Fig. 2) (66-68). These proteins include the a and @ subunits of platelet glycoprotein Ib (the platelet receptor for von Willebrand factor) (98, 107, 108),the lutropin-choriogonadotropin receptor (103), the leucine-rich cxzglywprotein (99), the proteoglycan core protein (100,101,109, 110),the 83kDa subunit of human carboxypeptidase N (102), yeast adenylate cyclase ( I @ ) , and the Drosophila chaoptin and Toll-encoded proteins-the last two being factors involved in photoreceptor cell and embryonic development, respectively (105, 106). These proteins represent a widely divergent group of molecules, some that are membrane receptors and others that have catalytic or structural roles; collectively, they comprise extracellular, integral membrane, and intracellular proteins. In many of these proteins-such as the RNase inhibitor, the @-subunit of platelet glycoprotein Ib, the extracellular domain of the lutropin-choriogonadotropin receptor, the leucine-rich a,-glycoprotein, and chaoptin-the leucine-rich repeat units constitute most of the protein. Hence, these units may be intimately involved in the function of the protein. In others, such as the 83-kDa subunit of human carboxypeptidase N and yeast adenylate cyclase, the leucine-rich repeat units are distinct from

PRI (human) GPIba (human) LRG (human)

L-s -G

--v

-B

-a -&

- I

-@

PG40 (human) CPN (human) LHCGR (rat) AdCyc (yeast) Chaoptin (Drosophila) Toll (Drosophila)

FIG.2. Comparison between a portion of the repeat wnsensus sequence of PRI and those of the a subunit of platelet glycoprotein Ib (GPlba) (98),leucine-rich a2-glywprotein (LRG) (99),fibroblast proteoglycan core protein ( P O ) (100,101). the 83-kDa subunit of carboxypeptidase N (CPN) (102). the lutropin-choriogonadotropin receptor (LHCGR) (103), adenylate cyclase (AdCyc) (104). chaoptin (109, and the ToUencoded protein (106). Shaded background indicates residues identical among consensus sequences. Aliphatic residues (V, L, and I) are indicated by a.


9

the catalytic portions of the molecule. Thus, they may not be involved in the primary activity of the protein, and may have other functions. Though the three-dimensional structures of any of these proteins, including RNase inhibitor, have yet to be determined, the high degree of conservation of certain residues, particularly leucine, in the repeats of all of these proteins suggests the possibility of common structural motifs. It has been hypothesized that the repeats may be important for protein-membrane or proteineprotein interactions (99, 105,106). The implication, then, is that this leucine-rich repeat unit may provide a versatile motif for the attainment of diverse, specific molecular interactions. It remains to be seen whether any of these proteins share common functional properties attributable to the leucine-rich repeat. In this regard, it should be noted that this leucine-rich repeat is distinct from the leucine-zipper motif associated with the dimerization of various transcription factors, in which there is a regular periodicity of leucines every seventh residue (111). Northern blot analysis of human placental, monocyte, or various porcine tissue mRNAs employing the inhibitor cDNA as a probe reveals in all cases a single message of -1.8 kb in length (66, 67,71,84).While a single message size is detected in these studies, there is molecular heterogeneity in the inhibitor mRNA, since the inhibitor cDNA sequences from human placenta and HeLa cells differ in both sequence and length at their 5’ ends, and there are at least three additional human 5’ variants (71).The difference in size between the longest and shortest of these variants in 383 nucleotides, and the sequences suggest that up to four exons are potentially spliced into a site in the 5’ untranslated region, raising the possibility of translational regulation (71).Curiously, one of the 5‘ variants encodes for a protein lacking the first five residues, which comprise a pentapeptide sequence duplicated at the N-terminus of PRI. Whether this alternative cDNA produces a functional protein in uiuo is not known, but it should be noted that the porcine inhibitor, which lacks this N-terminal pentapeptide repeat, binds to RNase with an affinity comparable to that of PRI (see Section 111, Inhibitory Properties). Southern blot analysis of human DNA shows that the inhibitor is encoded by a single locus (71).Further localization studies employing in situ hybridization techniques and human-rodent hybrid cell lines indicate that the inhibitor gene is on chromosome subband llp15.5 (112,113). Other genes mapped to this subband include those for the Harvey ras oncogene homologue (HRAS), insulin-like growth factor 2 (IGF2), P-globin (HBB), and insulin (INS) (114). Chromosomal abnormalities involving band l l p 1 5 have been identified in a variety of neoplasms, including Wilm’s tumor, rhabdomyosarcoma, breast tumors, acute lymphoblastic leukemia, and acute myeloid leukemia (113).It will be of interest to examine whether RNase inhib-

10


itor expression, which could conceivably affect intracellular RNA turnover (see Section IV, Biologic Role), is altered in any of these disorders. The inhibitors from both human placenta and porcine kidney have been expressed as recombinant proteins. That from human placenta has been expressed from E. coli in a yield of 50 Fg/g of wet cells (1 mg/4 liters of culture) (115), while that from porcine kidney has been expressed from Saccharomyces cerecisiae in a yield of 200 Fg/g of wet cells (84).As with the native inhibitor protein, affinity chromatography employing RNase ASepharose is a critical step in the isolation of the recombinant protein. The protein expressed from E. coli lacks a modified N-terminus, in contrast to that expressed from S. cerecisiae and the native protein, both of which are modified at the N-terminus and refractory to Edman degradation. Otherwise, the physicochemical, immunological, and inhibitory characteristics of the recombinant inhibitors are virtually identical to those of the native proteins. This equivalence in the case of the inhibitor expressed in E. coli confirms that eukaryotic posttranslational modifications are not essential for inhibitory activity. In this regard, there is no evidence of glycosylation of the native protein (59).

111. Inhibitory Properties A. Inhibition Constants A striking feature of the mammalian ribonuclease inhibitor is its exceptionally potent inhibition of RNase, with extremely low K,values that reflect both rapid association and very slow dissociation rates. Kinetic studies concerning the angiogenin. PRI interaction illuminate these features. Association rate constants, obtained by stopped-flow kinetic measurements of the 50% fluorescence enhancement that accompanies the binding of PRI to angiogenin, indicate a two-step binding mechanism (116). The first step involves rapid formation of an enzymeeinhibitor complex, EI, followed by a slower isomerization of EI to a tight enzyme-inhibitor complex, EI*: E

+I

Ki +

EX

ki? S

El*

k-2

The values of K , and k, are 0.53 F M and 97 s - l , respectively, while the apparent second-order rate constant of association at protein concentrations . \V. Chung, K. Fnjikawa, F. S. Hagen, T. Papayannopoulou and 6. J. Roth, PNAS 84, 5615 11987). 99. N. Takahashi. Y. Takahashi and F. W. Putnarn, PNAS 82, 1906 (1985). 1 0 0 . T Krusius and E. Ruoslahti, PNAS 83, 7683 (1986). 101. L. Patthy, J h f B 198, 567 (1987). 102. F. Tan. D. K. Weerasinghe, R. A. Skidgel, H. Tamei, R. K. Kaul, I. B. Roninson, J. W. Schilling and E. G. Erdos, JBC 265, 13 (1990). 103. K. C. McFarland, R. Sprengel, H. S. Phillips, M. Kohler, N. Rosemblit, K. Nikolics, D. L. Segaloff and P. H. Seeburg, Science 245, 494 (1989). 1 0 4 . T. Kataoka, D. Broek and M. Wigler, Cell 43, 493 (1985). 105. H. Keinke, I>. E. Krantz, D. Yen and S. L. Zipursky, Cell 52, 291 (1988). 106. .:C Ha~hiniotrt,K. L. Hudson and K. V. Anderson, Cell 52, 269 (1988). 107. K. Titani. K. Takio, M. Handa and 2. M . Ruggeri, PNAS 84, 5610 (1987). 108. J. A. Lopez, 1). W.Chung, K. Fujikawa, F. S. Hagen, E. W. Davie and G. J. Roth, PNAS 85, 2135 (1988). 109. I,. W Fischer, J. D. Terniine and M. F. Young, ]BC 264, 4571 (1989). 1 1 0 . P. J , Neanir, H. U . Choi and L. C. Rosenberg, ]BC 264, 8653 (1989). I l l . W.H. Landschulz, P. F. Johnson and S. L. McKnight, Science 240, 1759 (1988). 112. S. M . Zneiiner, I>. Crawford, N. R. Schneider and B. Beutler, Genornics 8, 175 (1990). 113. S. Weremowicz. E. A. Fox, C. C. Morton and 8. L. Vallee, Genomnics 8, 717 (1990). 114. C . Junien and 0.W.McBride, Cytogenet. Cell Genet. 51, 226 (1989). 115. P. S . Lee and 9. L. Vallee, BBRC 160, 115 (1989). 116. F. S. Lee, 11. S. .4uld and B. L. Vallee. Bchern 28, 219 (1989). 117. F. S . Lee. R . Shapiro and B. L. Vallee, Bchem 28, 225 (1989). 118. N. M . Green, A& Protein Chetn. 29, 85 (1975). 119. R. Shapiro and B. L. Vallee, Bchern 30, 2246 (1991). 120. P. M . Turner, K. 51. Lerea and F. J. Kull, BBRC 114, 1154 (1983). 121. J. XZ. Fominava, J. M. Garcia-Segura, M . Ferraras and J. G. Gavilanrs, RJ253,517 (1988). 122. J. F. Morrison, B B A 185, 269 (1969). 123. J. F. Morrison and C. T. Walsh, Adc;. E n z y n d . Relat. Areas Mol. Biol. 61, 201 (1988). 224. J. A. Luthy, M. Praissman, W.R. Finkenstadt and M . Laskowski, Jr., JBC 248, 17EU (1973. 125. U . Quast, J. Engel. H. Heumann, C. Krause and E. Steffen, Bchem 13, 2512 (1974). 126. J. R’.Williams, J. F. Morrison and R . G . Dnggleby, Bchern 18, 2567 (1979). 127. R. Shapiro and J. F. Riorclan, Bchern 23, 5234 (1984). 128. H. C . B~ill,N.A . Thornberry, M.H. J. Cordes, A. A. Wtchett and E. H. Cordes, ]RC 260, 2952 (1985). 129. R. Huber and W. Bode, in ”Proceedings of the 11th FEBS Meeting on Regulatory Proteolytic Enzymes and Their Inhibitors” (S. Magnusson, M. Ottesen, B. Foltmann, K. l h n o and H. Neurath, eds.), p. 15. Pergamon, Oxford, 1978.


29

P. Blackburn and B. L. Jailkhani, JBC 254, 12488 (1979). P. Blackburn and J. G. Gavilanes, JBC 255, 10959 (1980). J. S. Roth and D. Hurley, BJ 101, 112 (1966). P. Blackburn and J. G. Gavilanes, JBC 257, 316 (1982). K. A. Palmer, H. A. Scheraga, J. F. Riordan and B. L. Vallee, PNAS 83, 1965 (1986). F. S. Lee and B. L. Vallee, B c h m 28, 3556 (1989). R. Shapiro, E. A. Fox and J. F. Birordan, Bchem 28, 1726 (1989). R. Shapiro and B. L. Vallee, Bchem 28, 7401 (1989). M. D. Bond and B. L. Vallee, Bchem 29, 3341 (1990). A. R. Fersht, JMB 64, 497 (1972). A. R. Fersht, Bchem 26, 8031 (1987). A. R. Fersht, J.-P. Shi, J. Knill-Jones, D. M. Lowe, A. J. Wilkinson, D. M. Blow, P. Brick, P. Carter, M.M.Y. Waye and G. Winter, Nature 314, 235 (1985). 142. D. M. Lowe, G. Winter and A. R. Fersht, Bchem 26, 6038 (1987). 143. J. A. Wells, D. B. Powers, R. R. Bott, T. P. Graycar and D. A. Estell, PNAS 84, 1219 (1987). 144. J. W. Harper and B. L. Vallee, Bchem 28, 1875 (1989). 145. J. Hofsteenge, A. Vicentini and S. R. Stone, BJ 275, 541 (1991). 146. F. S. Lee and B. L. Vallee, Bchem 29, 6633 (1990). 147. F. S. Lee and B. L. Vallee, PNAS 87, 1879 (1990). 148. J. Hofsteenge, C . Servis and S. R. Stone, JBC 266, 24198 (1991). 149. K. Shortman, BBA 61, 50 (1962). 150. R. C. Imrie and W. C. Hutchison, BBA 108, 106 (1965). 151. R. Raghow, Trends Bioch. Sci. 12, 358 (1987). 152. G . Brawerman, Cell 57, 9 (1989). 153. E. N. Brewer, L. B. Foster and B. H. Sells, JBC 244, 1389 (1969). 154. C. Quirin-Stricker, M. Gross and P. Mandel, BBA 159, 75 (1968). 155. P. Ross0 and M. Winick, J. Nutri. 105, 1104 (1975). 156. R. L. Grief and E. F. Eich, FP 30, 360 (1971). 157. P.V.N. Murthy and J. M. McKenzie, Endocrinol. 94, 74 (1974). 158. R. J. Wojnar and J. S. Roth, Cancer Res. 25, 1913 (1965). 159. Y. Suzuki and Y. Takahashi, J. Neurochem. 17, 1521 (1970). 160. D. K. Liu, E. E. McKee and P. J. Fritz, Growth 39, 167 (1975). 161. 2. Kiss and F. Guba, FEBS Lett. 108, 185 (1979). 162. P. Fuhge and K. Otto, ZpChem 358, 1203 (1977). 163. E. M. Sajdel-Sulkowska and C. A. Marotta, Science 225, 947 (1984). 164. M. R. Morrison, S. Pardue, K. Maschoff, W.S.T. Griffin, C. L. White, J. Gilbert and A. Roses, Biochem. SOC. Trans. 15, 133 (1987). 165. K. Maschoff, C. L. White, L. W. Jennings and M. R. Morrison-Bogorad, J. Neurochem. 52, 1071 (1989). 166. L. M. Jones and J. T. Knowler, J. Neurochem. 53, 1341 (1989). 167. B. W. Little and W. L. Meyer, Science 170, 747 (1970). 168. R. L. Grief and E. F. Eich, Metabolism 26, 851 (1977). 169. B. J. Ortwerth and R. J. Byrnes, Exp. Eye Res. 12, 120 (1971). 170. J. S. Roth, ABB 60, 7 (1956). 171. J. Tabachnick, Radiat. Res. 15, 785 (1961). 172. N. Kraft, K. Shortman and D. Jamieson, Radiot. Res. 39, 655 (1969). 173. H. Hilz, M.-M. Oldekop and B. Bertram, ZpChem 349, 1475 (1968). 174. E. Ambellan and V. P. Hollander, PSEBM 127, 482 (1968). 175. R. G. von Tigerstrom, Can J. Biochem. 50, 244 (1972). 130. 131. 132. 133. 134. 135. 136. 137. 138. 139. 140. 141.

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176. S. C. Park, S. S. Choi and K. Y. Lee, SeouEJ. Med. 18, 31 (1977). 177. N . K. Sarkar, FEBS Leff. 4, 37 (1969). 178. J. S. Roth, S . Hilton and H. P. Morns, Cancer Res. 24, 294 (1964). 179. J. Hofsteenge, R. hlatthies and S. R. Stone, Bchem 28, 9806 (1989). 180. R. Shapiro, J. W. Fett, D. J. Strydom and B. L. Vallee, Bchem 25, 7255 (1986). 181. G . J. Gleich, D. A. Loegering, M. P. Bell, J. L. Checkel, S. J. Ackerman and D. J. McKean, PNAS 83,3146 (1986). 182. D. K. St. Clair, S. M . Rybak. J. F. Riordan and B. L. Vallee, PNAS 84, 8330 (1987). 183. K. J. Hamann, R . L. Barker, D. A. Loegering, L. R. Pease and G. J. Gleich, Gene 83,161 (1989). 184. H. F. Rosenberg, D. G . Tenen and S. J. Ackerman, PNAS 86, 4460 (1989). 185. D. J. McLaren, C.G.B. Peterson and P. Venge, Parasitology 88, 491 (1984). 186. G. J. Gleich and C. R. Adolphson, Adu. Itnmunol. 39, 177 (1986). 187. H. .4.Molina, F. Kierszenbaum, K. J. Hamann and 6 . J. Gleich, Am. 1.Trop. Med. Hyg. 38, 327 (1988). 188. E. Leone, L. Greco, R. K. Rastogi and L. Iela, J. Reprod. Fed. 34, 197 (1973). 189. J. Matousek, Experientia 29, 858 (1973).

Bacterial Adenylyl Cyclases ALAN PETERKOFSKY*,~ AIALA REIZER,~JONATHAN REIZER,1NATANGOLLOP,* PENG-PENG ZHU* AND NIRANJANAAMIN* *Laboratory of Biochemical Genetics National Heart, Lung and Blood Institute Bethesda, Maryland 20892 ?Department of Biology University of Cal!&niu, San Diego La Jolla, Calqornia 92093 I. The Action of cAMP as a Transcription Regulator in Escherichia coli 11. Regulation of CAMP Levels in E. coli . . . . .

IV. The Phosphoeno1pyruvate:sugarPhosphotransferase System . . V. Regulation of E. coli Adenylyl Cyclase Activity by the Phosphoeno1pyruvate:sugarPhosphotransferase System . . . . . . . . VI. Regulation of E. coli Adenylyl Cyclase Activity by Other Factors A. Activators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

...

35 36 38

......

.............................................

.............

..........................

X. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

...........

32

34

..........................

41 41 43 44 44 44 49 53 56 61 62 62

Adenosine 3',5'-cyclic monophosphate M CAMP)^ is a signaling molecule found in many, but not all, bacterial species. It has also been identified as an 'To whom correspondence may be addressed. 2Abbreviations: CAMP, adenosine 3',5'-cyclic monophosphate; CRP, cAMP receptor protein; R, regulatory subunit; C, catalytic subunit; CRE, cAMP response element; CREB, cAMP response element binding protein; P2, major promoter of cyu gene; ITS, phosphoenolpyruvate:sugar phosphotransferase system; Enzymes 11, membrane-bound sugar-specific transporters (permeases) of the PTS; protein 111, soluble sugar-specific phosphocarrier protein of the PTS; IIIdc, glucose-specific soluble phosphocarrier of the PTS; Enzyme I, PTS phosphocarrier protein, phosphorylated by PEP; PEP, phosphoenolpyruvate; HPr, PTS phosphocarrier protein phosphorylated by the phosphorylated form of Enzyme I (Enzyme I-P); crr, the gene for IIIdc (mutation of this gene confers on E. coli the catabolite repression-resistance phenotype); PDE, CAMP phosphodiesterase.


Copyright 0 1993 by Academic Press, Inc. All rights of reproductlon in any form reserved.

32

ALAN PETERKOFSKY ET AL.

important regtilator in eukaryotic cells. The mode of action of the nucleotide in bacteria is to convert the transcription factor known as cAMP receptor protein (CKP) from an inactive to an active form. The active form of CRP promotes the expression of unique sets of genes, generally referred to as inducible genes. The focus of this review is on the enzyme (adenylyl cyclase) that effects the synthesis of CAMP in various bacterial species. It is not intended to be an exhaustive review of the literature in this area, consonant with the style for this series. Kather, the content will reflect our current major interests. The adenylyl cyclase from Escherichiu coli has been a major subject of research interest ever since it was identified as the probable point for physiological regulation of CAMP levels in that organism and therefore a prime candidate for a protein mediator of the catabolite repression response mechanisin. Much of the emphasis in our laboratory in Bethesda has been on the elucidation of the factors controlling the activity of adenylyl cyclase in E . coli. The emphasis on that area is reflected in the review. The elucidation of the sequences of numerous adenylyl cyclases has made it possible to pose questions concerning the relatedness of the enzymes from various sources. The review contains a compendium, not previously assembled, of sequence homology comparisons for all the adenylyl cyclases whose sequences are currently known. On the basis of these analyses, we have organized some of these enzymes into four groups that suggest relatedness in either an evolutionary or functional sense. We have utilized a combination of “eyeball” and computer-assisted analyses to search for an ATP-binding site characteristic of these cyclases. The liberty is taken of suggesting an adenylyl cyclase “consensus,” which we are currently evaluating. Our aim is to provide an overview of bacterial adenylyl cyclases to the generalist as well as to provide food for thought for the adenylyl cyclase aficionados.

I. The Action of cAMP as a Transcription Regulator in Escherichiu coli CAMPfunctions as a cytoplasmic element mediating some reactions crucial for efficient cellular function. An activity that has been found only in eukaryotic cells is the CAMP-dependent protein kinase ( 1 ) . This enzyme is a well-known target of the action of cAMP as a second messenger, in which action this ligand transmits a signal generated by an extracellular hormone. The manner in which cAMP acts on the CAMP-dependent protein kinase invohees a release of the catalytic moiety of the enzyme from a complex in which its activity is inhibited as a result of binding to a regulatory subunit. The structure of the inactive holoenzyme is generally described as R,C,

BACTERIAL ADENYLYL CYCLASES

33

(where R indicates a regulatory subunit and C indicates a catalytic subunit); the interaction with cAMP leads to the formation of a complex of CAMP with R, and free active C. An important downstream function for cAMP in many eukaryotic cells involves the regulation of a family of CAMP-inducible genes (2).The cAMP response element (CRE) on such genes is believed to function as an enhancer, that is to say, it is located at a substantial distance from the regulated gene and can exhibit activity in either orientation. The mechanism by which these elements respond to cAMP involves the action of protein kinase A (3). The current model for the activation of these genes via cAMP requires a phosphorylation by protein kinase A of a transcription factor known as CREB (CAMPresponse element binding protein). The phosphorylation changes the conformation of CREB, allowing it to interact with a transcription complex and to thereby stimulate gene expression. The biochemistry of the action of cAMP in E . coli has been thoroughly defined and shown to be different than that in eukaryotic cells. In E . coli, cAMP serves as a coactivator of gene transcription for a substantial number of inducible genes (see Fig. 1).The key feature of this mechanism requires the interaction of CAMP with the pleiotropic transcription activator CRP (4). Genes coding for the enzymes required for the catabolism of a variety of sugars, such as lactose, are important but not exclusive targets for CRP. One essential regulatory feature of the structure of CRP is that the protein exists as a homodimer of the 210-aminoacid polypeptide. This dimeric structure is characterized by the presence of two domains: one for DNA binding (in the carboxyl-terminal region), and one for interaction with

ATP

-

-D

cAMP @LAMP

FIG. 1. The action of CAMP as a transcription regulator. cAMP interacts with cAMP receptor protein (CRP) to convert it to a form that binds specifically to certain promoters (crosshatched region). This binding stimulates RNA-polymerase-dependent transcription of unique genes (solid bar). The result is the accumulation of specific protein products.

34


cAMP (in the amino-terminal region). In the absence of CAMP, CRP takes on a conformation that eliminates its capability to interact effectively with specific promoters; this conformation (the inactive conformation) also makes CRP relatively resistant to degradation by proteolytic enzymes. Upon binding ofcAMP, CRP changes its conformation to the active one, which shows an enhanced affinity for double-stranded DNA. Thus, the function of cAMP in transcription in E . coli is to change the structure of CRP from the inactive state to the active one. The demonstration that it is possible to isolate mutated forms of CRP that can function as transcriptional regulators in the absence of cAMP (5a) proves that it is the activated structure of CRP, rather than CAMP, that is essential for transcription activation. In general, the promoters that are activated by CRP are characterized by the sequence 5'-TGTGAN4KACA-3' (5b),suggesting that this sequence is essential for CRP binding and thereby activation of transcription of CAMPinducible genes. CRP contains a helix-turn-helix DNA-binding motif. Recognition helices (one from each monomer) insert into the major groove of DNA at the TGTGA sequence. This interaction (as well as other contacts) promotes a bending of the DNA. The CRP-DNA complex facilitates the binding of RNA polymerase, which further increases the bending of the promoter DNA. It is currently assumed that this highly bent form of DNA (the open complex) is essential for the initiation of transcription of inducible genes.

II. Regulation of cAMP levels in E. coli As pointed out previously (6), CAMP levels can be regulated in three obvious ways. These patterns are depicted in Fig. 2 . The rate of the conversion of ATP to cAMP by the enzyme adenylyl cyclase might be regulated up or down by a variety of effectors. This type of regulation has been a major focus of interest for us, and was reviewed in 1989 (7). It is discussed in detail in Sections V and VI. The synthesized cAMP can, under the appropriate conditions, be eliminated from the cells. A variety of studies (8) have determined that there is an energy-dependent discharge of the nucleotide, and that the rate of this discharge is not proportional to its rate of synthesis (9). The relationship derived is that the intracellular cAMP pool size is proportional to the combined rate of cAMP degradation and excretion. Studies (10)using E . coli membrane vesicles suggest that the excretion of cAMP is carrier mediated and that the rate of nucleotide efflux is subject to regulation by both the magnitude of membrane energization as well as dose of carrier. The third factor involved in the regulation of cAMP levels is the degradation of cellular CAMP by cAMP phosphodiesterase (EC 3.1.4.19), the product of the cpd gene. Mutants

35


energy

q

ATP --@-CAMP

FIG.2. Regulation of cAMP levels in E . coli. cAMP is produced from ATP by the enzyme adenylyl cyclase (AC). As described in Sections V and VI, the activity of this enzyme is controlled by numerous factors (also, see Fig. 4). cAMP is a substrate for the enzyme cAMP phophodiesterase (PDE), the product being 5' AMP. The effect of this enzyme on cAMP levels is discussed in Section 11. Another factor that plays a role in regulating intracellular cAMP levels is the energy-dependent excretion of the nucleotide (wavy arrow). This aspect of CAMP metabolism is alluded to in Section 11.

deficient in this enzyme have higher steady-state levels of CAMP, indicating the importance of this activity as a regulator of nucleotide levels. An additional modulatory factor in maintaining cellular levels of CAMP is the cAMP binding protein, CRP. Strains carrying mutations in the gene (crp)for this protein accumulate larger than normal levels of cAMP both intracellularly and extracellularly (11). A typical crp mutant synthesizes cAMP approximately 25 times faster than does a wild-type strain. A strain deficient in both CRP and cAMP phosphodiesterase had a 100-fold higher intracellular cAMP level and an excretion rate 150-fold higher than the wildtype straing (12). The double-mutant strain was suggested to be a possible source for the industrial production of CAMP,although even higher levels of CAMP in culture media could probably be obtained using strains in which the gene for adenylyl cyclase is incorporated into a plasmid wherein the expression of the gene is under the control of a powerful promoter (13).

111. Structure and Expression of the E. coli Adenylyl Cyclase (cya) Gene Both the structural gene and promoter elements of the E. coli cya gene have been cloned (14, 15). The strategy used for the cloning involved the recognition that a host strain of E. coli deficient in the cya gene forms white colonies on a lactose MacConkey plate, while a strain expressing the cya gene forms red colonies. In this way, hybrid plasmids expressing the cya gene were easily selected. Isolated cya clones programmed the synthesis of a protein of M, -85,000. The analysis of the promoter region of such clones indicated the presence

36


of two sequences capable of binding CRP. This observation was of interest in the context of providing a possible explanation for the finding mentioned in Secticin I1 that E . coli strains deficient in CRP overproduce CAMP.The idea was then pursued that transcription of the cya gene might be negatively regulated by the complex of CRP with CAMP, a logical type of feedback inhibition. The transcription from the major promoter (called P2) of the cya gene was inhibited in Gitro by the CAMP-CRP complex (16). Further evidence for an interaction of CAMP-CRP with the cya promoter was derived from DNAse-I footprinting studies that showed an overlapping interaction of CAMP-CRP and RNA polymerase with the promoter region. The magnitude of the repression of cya transcription by CAMP-CRP in Salmonella typhimurium, a close relative of E . coli, has recently been quantitated (1 7). The fraction of cya expression through the P2 promoter (80%of total transcription) was repressed approximately eightfold by CAMP-CRP. A clone of the cya gene from E . coli was used to determine the complete nucleotide sequence of the structural gene (18).The data showed that the enzyme contains 848 amino acids, and indicated that the translation initiation codon is TTG, rather than the typical ATG. This observation, together with the finding that the ribosome binding site is weak, provided a partial explanation for the low level of adenylyl cyclase protein found in cells (19). The notion that the TTG initiation codon imposes a negative translational regulation was validated (20) by showing that mutating the TTG codon to ATG increased the expression of the cya gene three- to sixfold. The first extensive purification of E . coli adenylyl cyclase used as starting material a strain that overproduces the enzyme fivefold due to an episome carrying the cya gene in combination with a crp deletion background. The preparation of essentially homogeneous protein required a seven-step procedure that achieved an approximately 17,000-fold purification. More recently, a construct was made in which the cya gene was placed under the control of powerful promoter and ribosome binding sites derived from the A bacteriophage (13). After promoter activation in a strain containing such a construct, the amount of adenylyl cyelase corresponded to approximately 16% of the total protein. A three-step purification procedure was developed for the preparation of substantial amounts of essentially homogeneous enzyme.

IV. The Phosphoenolpyruvate: Sugar Phosphotransferase System Cellular levels of CAMP in E . coli are relatively high in cells depleted of a carbon source and are decreased substantially when such cells are exposed to a variety of transportable sugars (21). This regulation derives from a sugar-

37


SUGAR

FIG. 3. The phosphoenoZpyruvate:sugarphosphotransferase system. This transport system utilizes phophoenolpyruvate (PEP) to concomitantly translocate and phosphorylate numerous sugars into E. coli as well as some other organisms (see Section IV). The proteins Enzyme I (EI) and HPr are cytoplasmic proteins (not sugar-specific)used for the transport of all PTS sugars, while enzymes designated 11 and/or I11 are sugar-specific and, in general, membrane-associated. As indicated in the figure and discussed in Section V, a complex of the PTS proteins, designated by the bracket, is believed to regulate the activity of E . coli adenylyl cyclase (AC).

dependent inhibition of adenylyl cyclase activity. Numerous studies have implicated the sugar transport system known as the phosphoenolpyruvatemgar phosphotransferase system (PTS) as an important regulatory element for the control of adenylyl cyclase activity in E. coli. A general description of the PTS is outlined in Fig. 3. The multiprotein system catalyzes the following overall reaction: phosphoenolpyruvate

+ sugar

(out) -+ pyruvate

+ sugar-P

(in)

It is important to note that the total reaction involves both a sugar phosphorylation and a transmembrane movement of the sugar. The membrane-

38


associated recognition molecules that are specific for the family of sugars transported by this system are referred to as Enzymes 11; the genes for these enzymes are scattered on the E . coli genetic map (22). There appears to be at least one active cysteine residue in the typical Enzyme 11, and it has been proposed that this may be a site of phosphorylation. This suggests that the phosphoprotein is an intermediate in the sugar transport pathway. Consistent with the notion that there is an active phosphoenzyme intermediate, there is some evidence that Enzymes I1 can catalyze an exchange transphosphorylation reaction from a sugar-]? to free sugar. Transport of some sugar substrates of the PTS (for example, glucose) requires a soluble sugar-specific protein referred to as protein 111 (in the case of glucose, the designation is IIIaic). We point out in Section V that IIIgIc functions both as a phophocarrier in the transport reaction as well as a regulator of some physiological processes. The activity of E . coli adenylyl cyclase is one of the processes regulated by IIIglC. There are two phosphocarrier PTS proteins that are soluble and function with all the Enzymes 11; that is, they are not sugar-specific. The protein directly phosphorylated by PEP is designated Enzyme I and the phosphorylated form of Enzyme I can phosphorylate the protein known as HPr. In E . coli, the genes for Enzyme I, HPr, and protein IIIglCare linked in an operon in the order listed above. These genes have been cloned (23).Transcription from the promoter region driving the expression of the genes for Enzyme I and HPr is influenced positively by CRP (24). Extracellular glucose increases transcription of the pts operon; the mechanism of this effect appears to involve the conversion of the Enzyme I1 for glucose to the dephosphorylated form, which serves as a transcription activator. The operon for the general PTS proteins has been sequenced (25, 26). Recently, expression vectors have been constructed for overexpression of the genes for Enzyme I, HPr, and IIIgIc (13),and methods for the purification of the proteins on a large scale from extracts have been reported (27). The availability of large quantities of these proteins is expected to assist in the clarification of their roles in the regulation of adenylyl cyclase activity (7).

V. Re ulation of E. coli Aden lyl C clase Activity by the

9,

J r :

Phosp oeno1pyruvate:sugar hosp otransferase System As mentioned in Section IV, addition of glucose to a suspension of washed E . coli cells provokes a rapid decrease in the level of CAMP in the cells, an effect that is due to an inhibition of adenylyl cyclase activity. A variety of other sugars can substitute for glucose to cause this inhibition (21). The important property determining the specificity for inhibition is that the sugar can be transported. Therefore, induction of the transport system for a


39

number of sugars, such as mannitol or fructose, confers on cells the ability of those sugars to inhibit adenylyl cyclase activity. These studies provided a basis for the notion that the process of sugar transport across the cell membrane is responsible for the inhibition of adenylyl cyclase activity, consistent with the original idea (28) that passage of catabolites into cells is an essential aspect of the process of catabolite repression. A direct demonstration that glucose inhibits adenylyl cyelase activity was made by using E. coli cells made permeable by treatment with toluene (29). In this system, the adenylyl cyclase catalytic unit is presumed to be in a complex with appropriate regulatory factors that permit the signal transduction from glucose to the requisite components of the system. Consistent with the notion of a regulatory system being responsible for the glucose-dependent inhibition of adenylyl cyclase activity is the observation that glucose does not inhibit the enzymatic activity in broken cell extracts. The requirement for unique Enzyme-I1 components of the PTS for sugar-dependent inhibition of adenylyl cyclase activity was established by using mutants in one or the other of the Enzymes I1 specific for glucose. The product of the ptsG gene is a carrier that accommodates both glucose and amethylglucoside; a strain carrying mutated ptsG loses both the ability to phosphorylate a-methylglucoside via phosphoenolpyruvate (PEP) and to show a-methylglucoside-dependent inhibition of adenylyl cyclase activity. Similarly, the product of the ptsM gene is a carrier that promotes the transport of both glucose and 2-deoxyglucose; a strain of E. coli mutated in ptsM is deficient in both phosphorylation of 2-deoxyglucose and 2-deoxyglucosedependent inhibition of adenylyl cyclase activity (30).An important ramification of the observation that either one of these mutants allowed complete inhibition of adenylyl cyclase activity by glucose or the appropriate glucose analog was that there is only one population of adenylyl cyclase molecules and that the signal for sugar inhibition of the enzyme through different sugar-specific carriers was probably due to a common downstream component in the transport system. The realization that a common factor of the PTS might be responsible for either an activation or an inhibition of adenylyl cyclase activity, depending on the presence or absence of a sugar substrate for the PTS, led to the hypothesis that the condition of phosphorylation of one or more of the PTS phosphocarrier proteins could modulate adenylyl cyclase activity (31).The idea proposed was that the phosphocarrier(s) had to be in the phosphorylated condition to support a high level of enzyme activity; therefore, the explanation for sugar-dependent inhibition of adenylyl cyclase activity was that the process of PTS-dependent sugar phosphorylation resulted in a concomitant dephosphorylation of the PTS carrier. In the framework of the phosphorylation-dephosphorylation regulation

40


model for adenylyl cyclase (32),there was consideration of the role in physiologic regulation of enzyme activity of the other small components of the PTS. It was pointed out that PEP is the phosphate donor in the PTS and, conversely, pyruvate and PTS sugars are phosphate acceptors in the PTS. There are data in support of the idea of a push-pull or opposing mechanism for regulation of adenylyl cyclase activity in which phosphate donors (PEP) activate and ph6sphate acceptors (glucose or pyruvate) deactivate the enzyme. Further, a system for trapping PEP (ADP plus pyruvate kinase) can inhibit adenylyl cyclase activity in permeabilized cells; this suggests that there is normally a dynamic mechanism for maintaining a flux of phosphate groups into the PTS proteins that results in a modulated level of adenylyl cyclase activity. The precise nature of the interaction of adenylyl cyclase with the PTS has not heen cemented. Based on the repression behavior of E . coli mutants deficient in protein IIIglC( 3 4 , it was proposed that IIIRlc forms a regulatory complex with adenylyl cyclase, promoting an increase in enzyme activity when it is in the phosphorylated condition (34, 35). The position that other investigators, including our group, have taken is that a complex of the general PTS proteins (Enzyme I, HPr, and IIIglc)with adenylyl cyclase is the physiologically relevant species of the enzyme (36). This idea is represented in Fig. 4. Experimental support for that model has come from studies (36)in which homogeneous preparations of the PTS proteins Enzyme I, HPr, and IIIg'. were added to crude extracts of E . coli, resulting in a reconstitution of regulatory properties of the enzyme. It is

Inhibitors

Adenylyl Cyclase Activators FIG. 4. Inhibitors and activators of E . coli adenylyl cyclase activity. As described in Section V, the proteins of the PTS designated Enzyme I, HPr, and Enzyme IIIglc are proposed to form a complex at the cell membrane. Adenylyl cyclase is believed to interact with the complex, resulting in a diminution of the enzyme activity. The evidence for an inhibitory effect of the CAMP receptor protein (CRP) is discussed in Section VI. The roles of a variety of activators of adenylyl cyclase activity are dso enumerated in Section \'I. These activators are EF-Tu, nucleotides, PEP, and Pi.

41


noteworthy that the addition of Enzyme I to the extracts led to a dosedependent inhibition of adenylyl cyclase activity and that this inhibition became more severe when all three PTS proteins were added to the extracts; the interpretation of this finding was that adenylyl cyclase has interaction sites for not only IIIg'" but also for Enzyme I and HPr. Since the effects observed were with proteins in the unphosphorylated condition, the possibility must be considered that the mechanism of regulation of adenylyl cyclase activity involves an inhibition by unphosphorylated PTS proteins that is relieved by phosphorylation of the proteins. The properties of a family of precisely constructed mutants in which specific portions of the pts operon were deleted provided further evidence for the importance of PTS proteins other than II1glc for the regulation of adenylyl cyclase activity (37). A strain in which the crr gene (for IIIgl') was deleted was characterized by a level of cAMP 14-fold lower than a wild-type strain. Importantly, a further deletion of the gene for Enzyme I led to a further (2- to %fold) decrease in the level of cAMP production. Another aspect of cellular organization relevant to the regulation of adenylyl cyclase activity involves the possible assembly of a complex of PTS proteins at the cytoplasmic membrane. A follow-up to previous studies showing that membrane vesicles, which are essentially completely depleted of cytoplasmic proteins, actually contain some Enzyme I and HPr (38, 39) was carried out (40). Immunoelectron microscopy of frozen thin sections of E . coli was performed using antibody directed against Enzyme I. This type of study showed that a substantial fraction of the cellular Enzyme I was localized at or close to the cytoplasmic membrane. It seems reasonable to assume, therefore, that under physiological conditions the general proteins of the PTS may gather together at the membrane in a functional complex and that this complex may serve as a matrix for interaction with adenylyl cyclase (see Fig. 4).

VI. Regulation of E. coli Aden lyl Cyclase Activity

'f:

by Other actors

A. Activators As mentioned in Section V, there is a role for PEP as an activator of adenylyl cyclase activity. The mechanism of this activation relates to the role of this metabolite as a phosphate donor to PTS phosphocarriers. The notion is that PTS proteins in the phosphorylated condition promote a higher activity state of adenylyl cyclase than do PTS proteins in the unphosphorylated condition (32). The importance of inorganic orthophosphate as an activator of adenylyl

42


cyclase was realized as an offshoot of the development of the permeabilized cell system for the study of regulatory aspects of adenylyl cyclase activity (29). Treatment of cells with toluene allows the cellular pool of small factors to become depleted by dilution into the surrounding medium while maintaining physiologically significant protein-protein interactions. In this system, the assay for adenylyl cyclase in the absence of added phosphate reveals an activity that is characteristically low and insensitive to inhibition by PTS sugars. Since the addition of phosphate to French-press extracts of E. coli does not stimulate adenylyl cyclase activity, the locus of action of Pi appears not to be directly on the catalytic unit of the enzyme. The observation that potassium phosphate stimulates both adenylyl cyclase and PTS activities in permeabilized cell preparations (41) led to the proposal that the effects of these ions on adenylyl cyclase activity are mediated via some effect on the PTS. In this regard, it is noteworthy that phosphate stimulation of the cyclase is not observed in permeable cell preparations from a mutant strain of E . coli deficient in PTS proteins. The characteristic features of adenylyl cyclase in toluene-treated cells in the presence of Pi are that both the V,, and the K , for ATP are increased. Since the stimulatory effect of phosphate on adenylyl cyclase activity is abolished by transportable PTS sugars, it was proposed (29) that the PTS proteins exercise a dual regulation of adenylyl cyclase activity. First, the phosphorylation state of the PTS proteins, determined by the availability of a transportable sugar, dictates the activity level of the complex. Second, when sugars are transported via the PTS, the pool size of Pi is decreased due to the accumulation of sugar phosphates. It should be emphasized that the maximal stimulation of adenylyl cyclase activity requires Pi concentrations of 20-40 mM, concentrations that are within the normal physiological range (42). Exposure of intact cells to glucose or other PTS sugars results in a rapid decrease of the internal phosphate pool to approximately 20%of the original level (43).These effects have been convincingly demonstrated by the use of nlP nuclear magnetic resonance (NMR) spectroscopy (44). The kinetic properties of adenylyl cyclase vary substantially depending on whether the activity is measured in permeabilized cells, where the enzyme is assumed to be interacting with appropriate physiological regulators, or in broken cell extracts, where the enzyme is assumed to be dissociated from such regulators (41). In the permeable cell system, adenylyl cyclase produces sigmoidal substrate-vs. -velocity plots, suggesting an allosteric interaction. These studies provide a basis for thinking that, under physiological conditions, the adenylyl cyclase complex has two ATP-binding sites, one catalytic and the other regulatory. Since the allosteric kinetics requires the presence of PTS proteins, it has not yet been clarified whether the allosteric regulatory site is actually located on the adenylyl cyclase protein or on one of the PTS proteins.


43

Since eukaryotic adenylyl cyclases are generally regulated by proteins that specifically bind GTP, an investigation was made of the possibility that E. coli adenylyl cyclase exhibits a G-protein interaction as well (45). The discovery was made that EF-Tu, which is the most abundant protein in E. coli and serves as an elongation factor in the process of protein synthesis, specifically activates adenylyl cyclase. At a weight ratio of approximately 250:l (EF-Tu:cyclase), which is close to the normal cellular ratio, the stimulation of adenylyl cyclase was approximately 70%. The likely association of EFTu with adenylyl cyclase may provide a partial explanation for the previously observed association of a portion of the cellular EF-Tu pool with the cell membrane (46).The suggestion has been made, but not yet proved, that EFTu forms an essential part of the adenylyl cyclase regulatory complex, and that the allosteric kinetics reported with ATP may normally be due to the interaction of GTP with EF-Tu. Further studies in reconstituted systems may resolve this issue.

B. inhibiton Some factors that promote inhibitory effects on adenylyl cyclase have been described. It was mentioned in Section V that exposure of intact or permeable cells to a transportable PTS sugar results in an inhibition of adenylyl cyclase. The mechanism of this effect (a combination of dephosphorylation of PTS proteins and decrease of the cellular Pi pool) has been discussed in Section V. Pyruvate is also an inhibitor of adenylyl cyclase when the enzyme is in its coupled form. The mechanism of this regulation (36) is believed to be analogous to that involving the transportable PTS sugars; it drains phosphate from PTS proteins through a reversal of the PTS, and it concomitantly decreases the pool size of Pi. CRP is the mediator of the physiological effects of CAMP (see Section I); the complex cAMPCRP is a transcription activator. Interestingly, mutants deficient in CRP produce abnormally large amounts of CAMP (11).This observation has led to the proposal that CRP functions ordinarily as a downregulator of the adenylyl cyclase complex, although this protein has no effect on the activity of partially purified preparations of the enzyme (45). In keeping with the notion that CRP is a component of a multiprotein adenylyl cyclase complex, the elevation of CAMP levels characteristic of the absence of CRP requires the presence of Enzyme I, HPr, and II1glc of the PTS (47). The mechanism of the apparent interaction of CRP with the adenylyl cyclase complex has not yet been clarified, although a likely scenario is that CRP inhibits adenylyl cyclase by interacting with the complex when the PTS proteins are phosphorylated, resulting in only a suboptimal degree of enzyme activation. In the absence of CRP, the phosphorylated PTS proteins would be expected to lead to a higher level of enzyme activity. It is tempting to speculate that the physiological significance of the interaction of CRP with

44


the adenylyl cvclase complex is to mediate a down-regulation of the enzyme by CAMP;this would effectively be a type of product inhibition. The implication of this model would be that only CAMP-CRP, but not free CRP, would serve as an inhibitor of the adenylyl cyclase complex. This is clearly an area that deserves further analysis.

VII. Adenylyl Cyclases in Bacteria Other Than E. coli A. Mycoplasma Members of the genus Mycophina belong to the class Mollicutes (organisms with no cell walls) (48). These bacteria, with a genome size of 1155 kb, corresponding to one-quarter to one-fifth of the genome size of E . coli or Bacillus subtilis (@), are regarded as the smallest and simplest self-replicating organisms (50).It has been suggested (51) that they arose by evolution with loss of some of the genome from a branch of the eubacterial tree that contains gram-positive eubacteria containing DNA with a low G + C content. The assumption has been made (52) that the minimum number of genes in Mycoplasma (approximately 350) represents the conservation of only essential functions. Mycoplas~nucapricolum contains CAMP (53).In wild-type strains of this organism, the intracellular level of cAMP is reduced after exposure of the cells to sugars transported by the PTS. As is the case in E . coli, the level of cAMP in the cells is inversely proportional to the amount of PTS sugar substrate in the growth medium; depletion of the sugar in the medium concomitant with cessation of growth leads to an increase in the cellular cAMP level, and addition of sugars back to the cells results in a drop in the concentration of CAMP. The conclusion from these studies (53) is that the activity of the Mycoplasma adenylyl cyclase is regulated by the PTS as it is in E . coli and that this type of metabolic regulation must be very important since it is evolutionarily preserved in an organism with only “essential” genes. Since the level of cAMP was found to be consistently higher in glucose-grown compared to fructose-grown cells, it is likely that the expression of adenylyl cyclase is metabolite-controlled. The analysis of PTS components in M . capricolum indicated that this system may be even more complex than that in E . coli, since the Enzyme-I protein appears to be a large product (220 kDa) containing three different subunits. In contrast, the E . coli Enzyme-I protein in its physiologically active form is a homodimer consisting of subunits of M, approximately 70,000.

B. Sordetella pertussis and Bacillus anthracis Adenylyl cyclases are secreted by the pathogenic microorganisms Bacillus anthracis, Bordetella pertussis, and Brevibacteriurn liquefaciens.

45


The enzymes from B . pertussis and B . anthracis are considerably stimulated by calmodulin, a protein supplied by the target cell but absent from the bacteria. The uncontrolled levels of CAMP generated as a result of the bacterial invasion of the target eukaryotic cell reduce the capability of leukocytes and macrophages to kill the bacteria (54). The mechanism by which the B . pertussis toxin, containing adenylyl cyclase, penetrates cells is directly through the plasma membrane rather than by the expected receptor-mediated endocytosis (55)(see Fig. 5). Uptake of the toxin is inhibited by gangliosides, indicating a lipid-dependent mecha-

I-]

secretion

Bacteria

*

200 kDa

ofAC

protein C

n

binding (Ca-dependent) and penetration I

Activation by Calmodulin

Host cell

FIG.5 . The mechanism of action of Bordetellu pertussis adenylyl cyclase. As described in Section VII,B, adenylyl cyclase (AC) is secreted from bacteria as a protein of approximately 200 kDa. This enzyme can, in the presence of calcium (Ca), bind to and penetrate eukaryotic cells. As a result of the penetration, domains for the binding of calmodulin (CaM) and for catalytic activity become localized in the intracellular space. The result is that toxic levels of CAMPcan accumulate in the cells. Eukaryotic cells are equipped with an ATP-dependent mechanism for degrading the intracellular portion of the AC; this degradation mechanism allows eukaryotic cells to alleviate the toxicity produced by the elevation of CAMP levels. N and C represent the N-terminus and C-terminus of the protein, respectively.

46


nism of target cell invasion (56). In contrast to that of the B . pertussis enzyme, the mechanism of penetration of the B . unthrucis adenylyl cyclase is by way of receptor-mediated endocytosis (56);this endocytic uptake depends on the presence of an additional protein. The entry into cells of the B . pertussis adenylyl cyclase requires millimolar concentrations of calcium. The suggestion has been made (54)that the metal requirement is for the interaction of the enzyme with the membrane of the target cells. The current thinking is that the adenylyl cyclase penetrates the plasma membrane of the target cells but remains associated with the membrane in such a way that the catalytic and calmodulin binding domains become exposed to the cellular cytoplasm. It should be noted that exposure of Chinese hamster ovary cells to B . pertussis led to the expected elevation of CAMP in the cells (58).Unexpectedly, electron microscope analysis indicated that intact bacteria invade the cells. These studies suggested an alternate mechanism for cell intoxication involving a sequential adherence of the bacteria to the cells followed by entry of the bacteria, rather than a transmembrane transport of the bacterial toxin. I n B . pertussis, adenylyl cyclase is initially synthesized as a 200-kDa protein (61).The molecular mass of the invasive form of the enzyme determined by equilibrium sedimentation is 175-178 kDa (57).The 200-kDa form of the cyclase is necessary for invasive activity (57). Digestion by trypsin converts this large form to a smaller protein (45-50 kDa), a size similar to that generally found in culture supernatants as the secreted form of the enzyme. The fraction of cell-associated adenylyl cyclase ( M , 215,000) was 28%, with the remaining 72% of the catalytic activity found in culture supernatants ( M , 45,OOO). When the cell-associated activity was incubated with an extract of the bacteria, the 215-kDa species was degraded to the 45-kDa form (62). Interestingly, the bulk of the adenylyl cyclase found in cell culture media appears to be the 47-kDa species; however, this form of the protein is not toxic and is therefore probably not important in the etiology of whooping cough. The weight of evidence is that the smaller form is derived from the larger one by proteolytic digestion. Column chromatography on wheat germ lectin-agarose was used to separate, from the B . pertussis culture medium, an additional protein factor that conferred on the purified adenylyl cyclase the ability to invade neuroblastoina cells (59)with a concomitant increase in cellular CAMP concentration. Once inside the target cells, the adenylyl cyclase is rather unstable. The maintenance of continued high intracellular levels of CAMP depends on the continual presence of the cyclase in the extracellular space, in order to effect the constant replacement of enzyme inactivated by proteolysis. This proteolysis by a host-cell enzyme appears to occur by an ATP-dependent mecha-


47

nism (60). Nonhydrolyzable analogs of ATP can substitute for ATP, suggesting that the binding of the substrate induces some conformational change that makes the enzyme susceptible to degradation. Genes related to adenylyl cyclase synthesis and secretion in B . pertussis are located on an operon composed of four genes (63-65). cyaA codes for cyclolysin (200 kDa), which contains both adenylyl cyclase and hemolytic activities (66).The hemolytic determinant has been localized to the 3' region of the molecule by the examination of deletions. The other three genes (cyaB, cyaD, and cyaE) are involved with the transport of the cyclolysin protein. The gene for the adenylyl cyclase has been cloned in E . coli (67). The translation product is a precursor of the active enzyme, which is 1706 amino acids long. The amino-terminal end of this precursor (450amino acids) contains the calmodulin-activated enzyme activity. In a similar vein, the adenylyl cyclase gene from B . anthracis was cloned in E . coli. In this case (68), the clever selection method used depended on the restoration of adenylyl cyclase activity of a cya- strain that expressed the gene for calmodulin, which substantially activates the B . anthracis enzyme. The 43-kDa form of adenylyl cyclase from B . pertussis has two domains. The N-terminal region (residues 1-235) harbors the catalytic activity; the Cterminal region (residues 236-399) contains the calmodulin binding domain. Both domains are essential for the enzyme to display a high activity (69). These two domains of the enzyme may be separated after cleavage of the protein by trypsin into two fragments (70). The domain that interacts with calmodulin shows a tryptophan 242 fluorescence shift as a result of binding the activator. The B . pertussis adenylyl cyclase is similar to the enzyme from rat brain in that both are activated by calmodulin. Antibodies produced by the two enzymes cross-react, indicating a structural similarity (71). It has therefore been suggested that the two enzymes may be related evolutionarily. (However, see Section VIII for a discussion of the protein-sequence comparisons.) The site-directed mutagenesis approach was taken to identify amino-acid residues essential for binding calmodulin by the B . pertussis cyclase (72). Tryptophan 242 was identified as an amino acid important for tight binding of the activator. A change of the Trp to Asp reduced the &nity for calmodulin by a factor of 1000. These data are consistent with the previous suggestions that the C-terminal tryptic fragment (residues 236-399) of the adenylyl cyclase harbors the calmodulin binding domain. The DNA sequence of the B . anthracis edema factor (which has adenylyl cyclase activity) has an open reading frame of 2400 bp encoding an 800aminoacid protein. This precursor protein is processed to form a mature protein of 767 amino acids by a secretory mechanism (73). A 24-aminoacid sequence in the central region is homologous to a comparable sequence in

48

ALAN

PETERKOFSKY ET AI,.

the N-terminal portion of the cyclase from B . pertussis (74)(see Section VIII for a discussion of sequence homologies of these two proteins). The calmodulin binding domain of the B . anthracis adenylyl cyclase was labeled with a photofinity crosslinker that was coupled to calmodulin. Analysis of CNBr and N-chlorosuccinimide cleavage products allowed the localization of the binding region to the 150 amino acids at the C-terminus of that protcin. The N-terminal region of the B . anthracis cyclase encodes the binding site for the protective antigen, one of the components of the exotoxin that is a receptor binding protein, mediating the entry of the cyclase into target cells. There is no sequence in the B . anthracis adenylyl cyclase showing a similarity to the calmodulin binding site of the B . pertussis adenylyl cyclase, suggesting different mechanisms for calmodulin activation in the two enzymes (75) (see Section VIII and Fig. 7 therein). The cyu genes from B . anthrucis and B . pertussis differ in the percentage of G + C ( B . anthrucis = 65% G + C, B . pertussis = 29% G + C). However, the two adeiiylyl cyclases contain three highly conserved amino-acid domains. Both contain consensus sequences similar to that found in many ATPbinding proteins (76)(see Section IX, ATP-Binding Sites). 3’-Anthraniloyl-2’deoxyATP, a fluorescent analog of ATP, is a competitive inhibitor of the adeiiylyl cyclases from B . pertussis or B . unthracis (77)with a K , of about 10 pM. The fluorescent nucleotide was displaced by either ATP or 3’-dATP. The fluorescence of the nucleotide was enhanced when the adenylyl cyclase was complexed to Ca2+ and calmodulin. Mutagenesis studies (78) of the B . pertussis adenylyl cyclase show that lysine 58 is essential for catalytic activity. Replacement of this residue by inethionine resulted in loss of activity. Further studies (73) showed that mutagenesis of lysine 58 or lysine 65 to glutamine led to a decrease of catalytic activity by a factor of approximately 1000. These data are in keeping with the model that the N-terminal tryptic fragment of the adenylyl cyclase contains the catalytic activity. The enzyme also contains a conserved sequence typical of some ATP-binding proteins (DxD, corresponding to aspartic acid residues 188 and 190). Amino-acid replacement studies (79) led to the proposal that the carboxyl side chains of these aspartic acid residues coordinate with the Mg2+ of the Mg-ATP complex. The adenylyl cyclase of B . anthracis (edema factor) contains 800 amino acids, compared to a chain length of 1706 amino acids for the B . pertussis enzyme. Residues 342-358 of the B . anthracis enzyme and 54-70 of the B . perkussis enzyme contain sequences that represent ATP-binding consensus sequences (76). These two regions are almost identical (15 out of 17), indicating a high degree of conservation. i t has been suggested that lysines 346 arid 353 of the B . anthrucis cyclase may be essential since the corresponding


49

lysine residues of B . pertussis (58 and 65) have been shown by site-directed mutagenesis experiments to be required for activity. Mutation of Lys-346 to Gln led to a major decrease in the capability of a truncated form of the B . anthracis enzyme to bind a fluorescent analog of ATP (3’-anthraniloyl-2’-deoxyATP),resulting in loss of catalytic activity (80). These data suggest that a major function for Lys-346 is to enhance the binding of ATP to adenylyl cyclase. The protein has two probable ATPbinding A-type consensus sequences (see Section IX) with lysines at positions 346 and 353 as well as a B-type consensus sequence +X++[P] (where 4 is a hydrophobic residue) (100) containing a glutamate at position 436. The two proposed A-consensus sequences are overlapping and opposite (81). Mutagenesis of Lys-346 or -353 to Met resulted in loss of adenylyl cyclase activity. Mutagenesis of Glu-436 to Gln resulted in only a 25% loss of activity, suggesting that the proposed B-type sequence is not essential for activity. In summary, the adenylyl cyclases from B . pertussis and B . anthracis form a unique class of enzymes designed for expression of their activities in eukaryotic cells. Although they share with the adenylyl cyclase from bovine brain the property of being activated by calmodulin, there is no extensive sequence similarity (see Section VIII on sequence homology) of the bacterial and brain enzymes. These bacterial adenylyl cyclases constitute an interesting example of a parasitic relationship whereby the enzymes produced in the bacteria are activated by a protein (calmodulin) produced in the host cell.

C. Rhizobium Rhizobium species are of great practical interest due to their potential for a symbiotic relationship with specific plant tissues. The conversion of Rhizobium species from free-living cultures to the bacteroid form in plant tissues represents a type of differentiation. The involvement of CAMP in this process as well as in the regulation of gene expression in the different phases of this organism has been the subject of numerous investigations. It has been speculated that rhizobia, growing under symbiotic conditions, have the capability to simultaneously repress ammonia assimilatory enzymes (i.e., glutamine synthetase, glutamate synthase, and glutamate dehydrogenase) concomitant with the derepression of nitrogenase. Studies of the effect of CAMPon the growth of Rhizobiumjaponicum (82)showed that while 5 mM cAMP is toxic to the cells, 1 mM cAMP leads to a repression of the three NH4+ assimilatory enzymes. The effects were two- to threefold for glutamine synthetase and glutamate synthase and two- to ninefold for glutamate dehydrogenase. Since the inclusion of 1 mM AMP, ADP, or ATP in the growth medium did not show these effects, it was concluded that the repression is specific for CAMP. A model was suggested in which glutamate is transported from the plant to the Rhizobium bacteroid as a possible energy

50

ALAN PETERKOFSKY E T AL.

source that also represses ammonia assimilation, and that cAMP is an intracellular messenger that mediates the repression of the NH, assimilatory enzymes. A n important characteristic of Rhizobium meliloti is its capability to fix CO,. The fixation of CO, is repressed by a number of citrate cycle intermediates. There appears to be no connection of CAMP with this repression, since the cellular levels of the nucleotide are invariant under conditions of repression or derepression (83). On the other hand, hydrogen utilization by R . japonicum is influenced by the carbon source in the growth medium. Growth in the presence of malate has been associated with significantly lower hydrogenase levels than growth in the presence of glucose, suggesting some form of cataholite repression (84). Addition of CAMP (1 mM) to the growth medium overcame the malate-dependent inhibition of hydrogenase. Since the effect of CAMP was eliminated in the presence of protein or RNA synthesis inhibitors, it was concluded that new protein synthesis was required for the antagonism of the malate effect on hydrogenase activity. Transformation of the cells with a plasmid expressing the cya gene (the gene for adenylyl cyclase) of R . meliloti relieved malate-dependent repression of hydrogen uptake. The alleviation of the repression was accompanied by an approximately fourfold increase in the cellular CAMP level. While these observations suggested a role for CAMP in the regulation of H, metabolism in Rhizobium, the mechanism for modulation of CAMP levels in the organism remains undefined. It was speculated (84) that malate serves as a catabolite repressor in R. juponicum by leading to a reduction in cAMP levels, which then results in a decrease in hydrogenase activity by a mechanism that remains to be clarified. Using a gene bank from R. meliloti (comprising 1200 clones) in the broad host-range vector pRK290, the cyu gene was expressed in E . coZi, where it weakly complemented a cyu deletion (85, 86). Hybridization studies indicated that the cya gene is conserved in a variety of R . meliloti strains but not in other Rhizobiutn strains or in E . coli. While the cya gene from E . coli is repressed by growth in media supplemented with glucose, no such effect was observed for the Rhizobium cyu gene. The CAMP levels in the complemented cyu deletion strains were approximately 14% of that found in wildtype strains. The Rhizobium adenylyl cyclase expressed in E . coli was subject to catabolite repression. P-Galactosidase activity decreased after the addition of glucose in both wild-type strains as well as in the cyu deletion strain complcniented with the Rhizobium enzyme, although the repression was not as severe in the complemented strain. It is not clear from these studies what the mechanism is of catabolite repression mediated by the Rhizobium adenylyl cyclase. First, it has been suggested that rhizobia are devoid of the PTS +


51

(for glucose and fructose), a necessary component for catabolite repression in

E. coli. Second, the adenylyl cyclase of Rhizobium appears to be smaller than the E . coli enzyme ( M , -20,000), a size that may not include a domain for regulation by the PTS. It should be pointed out that no one has reported the presence of a cAMP receptor protein in any species of Rhizobium. A function for cAMP in transcription regulation should require the presence of such a cAMP binding protein. The sequence of the cya gene from R. meliloti revealed that the gene product has an M, of about 20,000 (87). The low M, of the protein suggests that it is composed of a catalytic domain with no regulatory domain, although the possible existence of a larger form carrying a regulatory domain is not excluded. The enzyme was purified and characterized as a lac fusion protein. It is unusual in its catalytic behavior, in that the K , is 4 mM compared to the K , of 0.6 mM for the E. coli cyclase. Another unusual property of the enzyme is its sensitivity to inhibition by GTP (a concentration of GTP equal to the substrate concentration gave approximately 35% inhibition). While the enzyme could interact with GTP, it did not synthesize cCMP. A comparison of the amino-acid sequence of the protein with that of other adenylyl or guanylyl cyclases reveals no obvious similarity with other bacterial adenylyl cyclases. However, there is a significant level of similarity with the adenylyl cyclases from yeast and bovine brain as well as the guanylyl cyclases of rat and bovine origin. These comparisons suggest a common origin for the adenylyl cyclase from Rhizobium and an enzyme from eukaryotes that might originally have had the capacity to synthesize both cAMP or cCMp (see Section VIII for a discussion of sequence similarities). Some experiments suggest that R. meliloti may have two cya genes (88). A cloned cya gene from this organism was used to construct cya-lac fusions in order to study the expression of the gene. The finding that there was little expression of the fusions prompted the construction of mutations in the cya gene. The authors discovered that a gene disruption of the cya gene decreased the levels of cAMP in the cells from 30 to 70%, but did not totally abolish the accumulation of the nucleotide. Therefore, they proposed that there may be an additional cya gene in these cells. It was noteworthy that the cya gene disruptions had no significant effects on growth, nodulation, or nitrogen fixation. A restriction fragment of DNA from Bradyrhizobium japonicum (a slowgrowing species) containing the cya gene was cloned into pBR322 (89).The clone was detected by complementation of Acya strains of E. coli. The transformants produced cAMP and reversed the Acya-encoded phenotype by allowing growth on sugars that require cAMP (arabinose, mannitol, ribose, and xylose) and regaining motility, which also requires the presence of CAMP. The cloned cya gene showed no sequence similarity to the E. coli

52


cya gene, as determined by DNA hybridization. The expression of the B . juponicuin cyu gene in E . coli led to lower cAMP levels (approximately 30%) than in wild-type E . coli. Slow-growing strains of Rhizohium have been examined for their caCell cultures took up the sugar by a mechapability to transport glucose (90). nism that depended on membrane energization, while bacteroids showed essentially no glucose uptake. The mechanism of glucose uptake was further explored in cell-free preparations. The cytoplasmic fraction of cultured cells phosphorylated glucose by an ATP-dependent reaction; there was no PEPdependent sugar phosphorylation. The bacteroids showed an ATP-dependent phosphorylation reaction in both the membrane and soluble fractions. A major conclusion from these studies was that there is no demonstrable PTS activity for glucose in these slow-growing rhizobiu. Rhizobium legzmtinosarurntakes up fructose by a process that is inhibited by azide, 2,4dinitrophenol, or carbonyl cyanide m-chlorophenylhydrazine, suggesting that the mechanism of the transport involves membrane energization (92).In a mutant that does not metabolize the transported fructose, the sugar accumulates in the nonphosphorylated form. These studies indicate that this organism does not transport fructose by the PTS. The properties of adenylyl cyclase and cAMP phosphodiesterase (PDE) in bacteroids of B . japonicum have been investigated (91). Adenylyl cyclase activity was found in the membrane fraction of the bacteroids, but not in any plant fraction. CAMP phosphodiesterase was found both in the soluble and membrane fractions. The adenylyl cyclase activity was stimulated four- to fivefold by sodium dodecyl sulfate (0.01%). Adenylyl cyclase activity increased approximately threefold during aging of the nodules (low at day 17 and higher at day 21), while the membrane-bound phosphodiesterase decreased about 40%. The authors suggest that CAMP may play a role in symbiosis. Adenylyl cyclase activity increased at least 10-fold after the bacteroids were broken in a French-pressure cell, indicating that the enzyme is located within the bacteroids. None of the following compounds affected the bacteroid adenylyl cyclase activity: Gpp(NH)p, forskolin, fluoride, glutamate, glutamine, hydroxybutyrate, pyruvate, pyrophosphate, PEP, Ca2+, NH4+, NL41)+,and NADH. Pi, which is a potent stimulator of E . coli adenyiyi cyclase activity, was not tested. The patterns of adenylyl cyclase and CAMP phosphodiesterase activities suggest that the level of CAMP increases during the development of symbiotic N, fixation. In summary, it is likely that cAMP functions in Rhizobium physiology as a mediator of catabolite repression as well as in the bacteroid-plant interaction in nodules. Since it appears that rhizobia do not transport sugars by the PTS, and the unusually small size of the adenylyl cyclase in this organism may not allow for a PTS-dependent type of regulation of adenylyl cyclase activity, it is


53

reasonable to speculate that a new mechanism for regulation of the activity of adenylyl cyclase in Rhizobium may be uncovered by further studies of this organism.

VIII. Sequence Comparisons The complete coding sequences of 12 adenylyl cyclases from a variety of sources have been deduced (in the case of Salmonella typhimurium, 50% of the sequence, the amino-terminal half, has been reported). In the published reports of these sequences, the authors have frequently shown sequence comparisons with other proteins. We have constructed a complete matrix of all the binary sequence similarity analyses, using the FASTA program (93). The results are shown in Fig. 6, together with pertinent information about the sizes of the proteins. It is noteworthy that there is considerable variation in the sizes of the different adenylyl cyclases; the chain lengths vary from 193 amino acids (A.meliloti) to 2026 amino acids (Saccharomyces cereuisiae). Presumably, this is a reflection of the differences in regulation mechanisms associated with the various enzymes. The results of the binary comparisons indicate that there is no common extensive sequence motif that is characteristic of all adenylyl cyclases, even though they catalyze the same enzymatic reaction. The data in Fig. 6 do indicate, however, that there are certain groups (four of them) that demonstrate significant relatedness. The members of these groups are denoted by shaded cells in Fig. 6 and the sequence similarities within each group are schematically depicted in Fig. 7. The first group (A, Fig. 7) consists of the adenylyl cyclases from S . typhimurium, E . coli, Erwinia chysanthemi, and Pasteurella multocida. All of these are gram-negative bacteria and may be closely related in an evolutionary sense. The regions of identity of these adenylyl cyclases encompass essentially the complete length of the coding sequences. The second group (B, Fig. 7) includes adenylyl cyclases from B . anthracis and B . pertussis. These two organisms produce toxins, of which adenylyl cyclase is one component. Further, these two enzymes are activated by calmodulin. The enzymes from B. anthracis and B . pertussis are dissimilar in that the former contains 800 amino acids, while the latter contains 1706 amino acids. Only the N-terminal 450-aminoacid region (isolated as a 45-kDa protein) of the B . pertussis adenylyl cyclase is necessary for calmodulin-activated catalytic activity. The alignment shown in Fig. 7 indicates that the region of the B . anthracis sequence from residue 303 to residue 688 is homologous to the region of the B . pertussis sequence from residue 15 to residue 407. It was pointed out in Section VII,B that the N-terminal portion of the B. pertussis adenylyl cyclase (residues 1-235) contains the catalytic domain while the

FIG.6. Sequence comparisons of prokaryotic and euhryotic adenylyl cyclases. The figures in each cell represent sequence comparisons of the adenylyl cyclases from the two organisms intersecting the matrix (defined as a binary comparison), The numbers in the table correspond to the percent identity in the segments compared, and to the number of amino acids in the aligned segment (in parentheses). Values in brackets denote the number of standard deviations higher than that obtained with 100 comparisons of randomized sequences of these protein segments. The shaded cells represent the 11 examples of homologies that are statistically significant. The FASTA (word size = 1) and the RDF2 programs (93)were used to assess similarity and to determine the comparison scores, respectively. The numbers in parentheses below the sources of the enzymes indicate the number of amino-acid residues in the enzyme, except in the case of S. typhimurium, where only the amino-terminal 419 residues out of a total of approximately 850 amino acids of the sequence have been published. Full genus names and references for the published sequences of the various enzymes are as follows: Rhizobium meliloti (89, Salmonella typhimuriuin (109, Escherichia coli (18), Erwinia chrysanthemi (108), Pasteurella multocida ( l o g ) , Bacillus anthracis (73), Bordetella pertussis (67), Breoibacterium liquefaciens (110), Saccharomyces cerevisiae (ill), Schizosaccharomyces ponibe (103), Bos taurus (104), and Rattus norcjegicus (112).

55


0

2

4

6

8

10

12

14

16

18

20

Chainlength

I

I

(xl 00)

Regions of sequence homology

A1

&16

E. coli E. chrysanthemi P.rnuitocida

833

B

C1

1706

997~~26 1$ ,7 , 1692

861

I

1

447

679

B anthracis 6.pemssis

1144

1134

S. cerevisiae

s

pomae

R. norvegicus B. taurus R. meblon

FIG.7. Schematic depiction of homologous regions in four distinct groups of eukaryotic and prokaryotic adenylyl cyclases. The four depicted classes are (A) gram-negative bacteria, (B) gram-positive pathogens, (C) yeast, and (D) mammalian Rhizobiurn. [A sequence alignment for the adenylyl cyclase from Salmonella typhirnuriurn is omitted from this figure because the complete sequence has not been published (see legend to Fig. 6 ) , ]The cross-hatched areas represent the regions of sequence homology. The numbers at the left and right termini of the sequences correspond to the amino-terminal and carboxy-terminal residues, respectively. The numbers at the left and right of the cross-hatched regions represent the amino-terminal and carboxy-terminal ends of the homologous sequences. The apparent discrepancies in the number of residues shown in the homologous regions in this figure and in the data of Fig. 6 are due to the placement of gaps in the sequence alignments by the FASTA program.

region from residues 236-399 harbors the calmodulin-binding domain. The 150 amino acids at the C-terminus of the B . anthracis enzyme contain the calmodulin-binding region. These data are consistent with the idea that there is a common ancestral origin for the two catalytic and calmodulinbinding domains, in contrast to the previous suggestion (82) that there is no homology in the calmodulin-binding sites of the two enzymes. The third group (C, Fig. 7) of adenylyl cyclases corresponds to the enzymes from s. cerevisiae and Schizosaccharomyces pombe. It has previously been reported that the catalytic domains of both of these adenylyl cyclases reside in the carboxyl-terminal regions. It is therefore not surprising that the region of sequence homology of these two proteins is in the C-terminal portion. The amino-terminal regions of these proteins are presumably involved with regulatory activities, which may differ in the two proteins. It has been pointed out that the adenylyl cyclase from S. cerevisiae is a target for

56


the action of the RAS protein while the adenylyl cyclase from S. pombe is not. The lack of sequence homology in the N-terminal regions of these two cyclases supports the notion that the regulation of adenylyl cyclase activity in these two organisms proceeds by different mechanisms. The last group of adenylyl cyclases (D, Fig. 7) includes the enzymes produced by rat and bovine species and by the bacterium R. meliloti. The N-terminal halves of the rat and bovine enzymes contain the regions of sequence homologies, suggesting that the catalytic centers or calmodulinbinding domains are located in those segments of the enzymes. It is noteworthy that there is a stretch of approximately 130 amino acids beginning at residue 313 of the bovine enzyme that is highly homologous to the Nterminal 130 amino acids (two-thirds of the complete sequence) of the adenylyl cyclase from R. meliloti. These observations suggest that there is an essential conserved region in these three proteins. It is noted in Section IX that the presumptive regions for ATP binding in these three proteins occur in other regions of these sequences. It is a surprising observation that the sequence of the R. meliloti enzyme is more closely related to eukaryotic enzymes than to other bacterial enzymes. The significance of this relationship remains to be established.

IX. ATP-Binding Sites Since the substrate for the adenylyl cyclase reaction is ATP, it might be surmised that a common feature of all adenylyl cyclases should be a sequence or domain that recognizes this nucleotide. X-Ray crystallographic (94) and NMR studies (95) as well as affinity-labeling experiments (96) on rabbit muscle adenylate kinase have helped to define the ATP-binding site for this enzyme. Additional studies with a variety of other enzymes that interact with ATP have led to the proposal that a typical ATP-binding domain is glycine-rich, contains a basic residue, and can undergo a conformational change upon binding ATP (95, 97-99). The typical so-called A-type ATP-binding domain can be expressed by the following consensus sequence (where slashes indicate “either/or”): (G/A)(X,)(G/A)(H/K/R)(X,--J(S/T/K/R/H) (100).This sequence is referred to as pattern 2 in Table I. Mutagenesis studies of a variety of enzymes that interact with ATP suggest that the basic residue (usually lysine) adjacent to the conserved G or A in the flexible loop sequence is important for activity. Replacement of this amino acid by a variety of others (Met, Ile, Glu, Gln, or Arg) results in changes in activity ranging from 2.5- to %fold decrease (I?. coli F,-ATPase) (101)to essentially complete loss of activity (B. pertussis or B. anthrucis adenylyl cyclase) (53, 54). We have carried out an alignment analysis of the all published adenylyl cyclase sequences (of which there are 12) as indicated in Table I and Fig. 8.

TABLE I OCCURRENCE OF CONSENSUS SEQUENCES IN

Bovine brain (type 1)

Rat olfactory tissue (type 111)

S. pombe S . cereoisiae 8 . pertussis

B . unthrucis

P. multocidu E . chrysanthemi

E . coli

S. typhimurium"

R. meliloti B . liquefuciens

VARIOUS

ADENYLYL CYCLASES

1042 GvsvKGkgemLT 1048 GKGemLT

26 AagpgGRR 202 AtlvpAKR 433 AaglpGKvH 780 AgaisGRS 989 AgvigARR

1104 GpifvKGkgeLlT

447 AggipGRvH

1110 GKGeLlT

1051 AgvigARK

1423 GsknevlyRGLS 1521 GefklKGLdT 1871 GehklKGLeT 54 GvatKGLgvhakS 825 GgidiasrKGerpaLT 826 GidiasrKGerpaLT 1165 GrggddilRGgLgldT 1167 GgddilRGgLgldT 1168 GddilRGgLgldT 1490 GrgldagaKGvfLS 1492 GldagaKGvfLS 1496 GaKGvfLS 1499 GvflslgKGfasLmdepeT 1505 GKGfasLmdepeT 174 GKGisLdiiS 342 GvatKGLnehgkS 375 GqqlaveKGnLenkkS None 51 GylegkvpHGicLfS 55 GkvpHGicLfS 82 GelsapdrKGeLpiT 51 GyldgnvpKGicLyT 55 GnvpKGicLyT 82 GmsvqdppKGeLpiT 708 GaishnklHGLS 82 GmtpqdppKGeLpiT None None

None None 26 GikavAKeK 59 GlgvhAKS 252 AvgteARR 349 AygvaGKS

347 GlnehGKS 417 GiilkGKK None 189 AvrmaGKR

190 AvrlaGKR

190 AvrlaGKR None None

~

"Pattern 1 is the proposed consensus sequence characteristic of adenylyl cyclases (see text), while pattern 2 is the consensus sequence proposed previously (100)to be a typical ATP-binding site. The numbers correspond to the first residue of the segment shown. Amino-acid residues shown in bold capital letters correspond to conserved residues of the consensus sequences, while amino-acid residues shown in lowercase letters correspond to nonconserved residues (indicated as X in the patterns). The data were generated by use of the FINDPATTERNS program (102). Wnly the first 419 amino acids of the sequence have been published.

58


ENIYXE SOURCE

SEOUENCE

RamB

------- )

bovine brain (type 1)

1048-1054

G(

rat olfactory (type 111)

1104-1116

G(---pifv) K G(-kge) L(----1) T

S. pombe

1521-1530

G(---efkl) X G(----) L(----d) T

S. cerevisiae B. pertussis

1871-1880

G(---ehkl) K a(----) L(----e) T

54-66

(3 (---- vat) EG(----)

B. anthracis

342-354

G (---- vat) g G(----) L(nvhgk) 8

P. multocida

249-265

G(aslwg1y) K a(----) I(dapyk) 8

E. chrvsanthemi

82-96

G(e1sapdr) K G(---e) L(---pi) T

E. coli

82-96

G(msvgdpp) K G(---e) L(---pi) T

S . tvDhimurium

82-96

G(mtpgdpp) X G(---e) L(---pi) T

R. meliloti B. lisufaciens

161-172

G(---taka) K G(rsta) L

126-134

G(-----mv) ReG(---a) L(-----) T

CONSENSUS

X G(--em) L(-----) T

L(gvhalc) S

G X o - 7 (H/X/R)GXo-,LX,-,(S/T)

Ftc. 8. Sequence of presumptive regions for ATP binding in various adenylyl cyclases. For a discnssion of this figure, see Section IX. The underlined lysine residues of the adenylyl cyclases from B. pertussis and B. anthrocis have been shown, by site-directed mutagenesis experiments, to be essential for activity.

{However, see Addendum at the end of this section.) The results of this type of analysis have allowed us to develop an alternative consensus sequence, which is found in essentially all these enzymes [G(qP7)(H/K/R)G (q-4) L(q)-5)(S/T)].This sequence is referred to as pattern 1 in Table I. A unique feature of this consensus sequence is that a basic residue precedes a glycine residue, whereas in the A-type sequence (pattern 2) a basic residue follows a glycine residue. It may be that this inversion of conserved amino acids is due to the nature of the adenylyl cyclase reaction (cleavage of ATP between the 01 and p phosphates) compared to that of the typical kinase or ATPase reaction that involves cleavage of ATP between the p and y phosphates. It should also be noted that this consensus contains a conserved leucine (or isoleucine) residue between the second conserved glycine and the conserved serine/threonine residue. On the basis of this proposal, it would be predicted that the indicated sequences for the various adenylyl cyclases would play an important role in the enzymes’ function. As noted above, mutagenesis stud-

BACTERLAL ADENYLYL CYCLASES

59

ies on the adenylyl cyclases from B . pertussis or B . anthracis have indicated the importance of the conserved lysine in this sequence [see the two underlined lysine (K) residues in Fig. 81. A search using the FINDPATTERNS program in the GCG software package (Version 7.0) (102)was made of the 12 adenylyl cyclase sequences to determine the presence or absence of the modified consensus described here (pattern 1)or the previously defined ATP-binding consensus (100)(pattern 2; see Table I). The type-I adenylyl cyclase of bovine brain contains one region (beginning at either residue 1042 or 1048) that matches pattern 1 (Table I and Fig. 8). There are five examples of adherence to the pattern 2 consensus. Two of these sequences (beginningat residues 202 and 780) occur in transmembrane regions and two of the sequences (beginning at residues 433 and 989) occur in cytoplasmic regions (104,113). It is possible that either or both of these sequences play a role in catalysis. The adenylyl cyclase from rat olfactory tissue (type 111) contains one region (beginning at either residue 1104 or 1110) that matches pattern 1. There are two sequences (beginning at residues 447 and 1051) that fit pattern 2. Since all of these sequences occur in regions proposed to be cytoplasmic (113),any one or more of them are possible candidates for essential ATPbinding sites. It is important to note that, in both S. pombe and S . cerevisiae, there are no sequences that match pattern 2. Since the sequence in S. pombe beginning at residue 1521 is homologous to the only match to pattern 1 of s. cerevisiae (103),that sequence has been selected as the best ATP-binding site candidate of S. pombe (Fig. 8). Since the active site of the S. cerevisiae adenylyl cyclase lies in the region of residues 1609-2026, the identification of the consensus sequence from residues 1871-1880 as an ATP-binding site is reasonable. Table I shows that there are 11 sequences in B. pertussis and three in B . anthracis that match pattern 1. Since the B . pertussis sequence beginning at residue 54 is homologous to the B . anthracis sequence beginning at residue 342, these two sequences have been selected as the best candidates for essential ATP-binding sites (Fig. 8). It is noteworthy, in this regard, that both lysine residues in each of these sequences have been shown by site-directed mutagenesis studies to be essential for catalytic activity (53,54). Another reason for choosing the indicated sequences is that they fall within the catalytic domains of the proteins. The P. rnultocidu adenylyl cyclase sequence has no perfect matches to either pattern 1 or 2. The sequence from residues 249-265 has been selected as a possible ATP-binding site since it only deviates from a pattern 1 consensus by a change of the conserved leucine to an isoleucine residue (Fig. 8). The sequences of the adenylyl cyclases from E . chrysanthemi, E . coli,

60


and S. typhimurium are nearly identical. The sequences beginning at residue S5 of the E . chysanthemi and E . coli adenylyl cyclases match pattern 1 and are possible ATP-binding sites, but the sequences beginning at positions 82 adhere to the consensus for all three proteins and therefore are more likely to be ATP-binding regions. In the case of pattern 2 sequences, there is one match to the consensus for all three proteins (beginning at residue 189 or 190); these sequences cannot be eliminated from consideration as ATP-binding regions sirice they all fall in the N-terminal domains of the cyclases, which are believed to harbor the catalytic site (115). The sequence of the R . rneliloti adenylyl cyclase (87) has no perfect matches to either the Chin et al. (100)consensus or the consensus proposed here [fable I). However, the sequences that begin at residues 132 (GMNKDYGTSVL) and 161 (GTAKAKGRSTAL)are variants of the consensus proposed it1 this study. In this variation, the Lys-Gly sequence (shown in bold type) may be interrupted by as many as two amino acids, and the conserved serine or threonine @old italics) precedes rather than follows the conserved leucine (bold type). In Fig. 8, the sequence beginning at residue 161 has been selected as the best candidate for an ATP-binding site. It is noteworthy that the R . meliloti enzyme is the smallest reported adenylyl cyclase (193 amino acids) and also has an unusually high K , (4mM), compared to the value of approximately 0.6 mM for the comparable E . coli lac2 fusion enzytne (87) or 0.21 mM for the wild-type enzyme (41). It is therefore possible that the unusually high Km is a reflection of the nonadherence in Rhizobirim to the ATP-binding consensus observed in other adenylyl cyclases. The B . liquefaciens adenylyl cyclase has no perfect matches to either pattern 1 or 2 (Table I). The sequence beginning at residue 126 has been selected as a good candidate for the ATP-binding site since it deviates from the pattern 1 consensus only by placement of a single amino acid (glutamic acid) between the conserved R and G (Fig. 8). Obviously, much experimental work will be necessary to evaluate these predictions of the location of ATP-binding sites in adenylyl cyclases. The question arises of whether the suggested consensus for adenylyl cyclases is restricted only to that class of enzymes. A search was made through a protein-sequence database (SwissProt) for the occurrence of the consensus sequence using the FINDPATTERNS program in the GCG package from the University of Wisconsin. Approximately 60 bacterial sequences contained the consensus sequence. Of the 60 sequences, 36 were for proteins that bind nucleotides. The search revealed that many of these proteins also contain the motif in which the basic amino acid follows a Gly or Ala residue (100). Interestingly, some of the sequences identified were for aminoacyl-tKNA synthetases (for alanine, phenylalanine, and lysine), enzymes


61

that are similar to adenylyl cyclases in that they produce PP, from ATP. The enzyme responsible for the synthesis of ppGpp, which involves a pyrophosphoric transfer, also contains the adenylyl cyclase consensus sequence. A number of E. coli enzymes that interact with NAD, which contains a pyrophosphoric unit (e.g., NADP-specific glutamate dehydrogenase and NADH dehydrogenase), have the consensus sequence. Another enzyme found to contain the consensus sequence is guanylyl cyclase (bovine and rat), an enzyme that is quite analagous in reaction mechanism to that of adenylyl cyclase. It therefore appears that the consensus sequence found in all adenylyl cyclases sequenced thus far is not a specific signature for those enzymes, but is found as a more general sequence in a variety of enzymes that bind nucleotides as well as in numerous enzymes that have not yet been shown to interact with nucleotides. Using this consensus paradigm, it will be useful to further explore the importance of Gly, Lys (or His or Arg), Leu, and Ser (or Thr) residues for the function of various adenylyl cyclases or guanylyl cyclases. We speculate that the function of glycines is to impart flexibility, that of the lysine is to interact with one of the phosphoric groups of ATP, that of the leucine is to provide some hydrophobic interaction with the adenosine moiety of ATP, and that of the serine (or threonine) residue is to facilitate the a-phosphoric transfer to the 3’-OH of the adenosine of ATP. A recent characterization of a calmodulin-activated adenylyl cyclase from bovine brain (105) demonstrated that two domains of the protein are required for full catalytic activity. The authors suggested that adenylyl cyclases and guanylyl cyclases may require an interaction of two domains for maximal catalysis. It is likely that E. coli adenylyl cyclase requires a domain in addition to the Gly-82-Thr-96 region for activity. In support of this idea is the demonstration that an in-frame deletion of a 25-aminoacid segment corresponding to residues 118-142 of E. coli adenylyl cyclase produces a protein that is catalytically inactive (106).The Trp-118-Asn-142 region has a helical structure (predicted by a Chou-Fasman analysis performed using the PEPTIDESTRUCTURE program in the University of Wisconsin GCG analysis package) with three repeated leucines spaced seven residues apart, reminiscent of a leucine-zipper motif, It might be speculated that the Gly-82-Thr-96 domain interacts with the Trp-118-Asn-142 domain to form a complete catalytic unit.

Addendum Since these data were assembled, the sequences of two additional eukaryotic adenylyl cyclases (the rat-brain, type-11, calmodulin-insensitive enzyme and the rat-testis, type-IV, calmodulin-insensitive enzyme) have been published (113, 114). Examination of the two new sequences indicates that

62

ALAN PETERKOFSKY ET

AL.

they both contain regions in their C-terminal cytoplasmic domains that adhere to the characteristic adenylyl cyclase consensus proposed in this section. The relevant sequences in the type-I1 enzyme (114)include residues 1060- 1072 (GiinvKGkgdLkT) (conserved residues in bold capital letters). The comparable sequences in the type-IV adenylyl cyclase (113) includes residues 1036- 1048 (GvikvKGkgqLcT). These data provide compelling support for the importance of the consensus sequence [ G(&-7)(H/K/R)G(X,--4)L(q-,)(S/T)] or a slight modification of it for the catalytic activity of adenylyl cyclases. As more adenylyl cyclase sequences become available, it may be useful to modify the proposed sequence motif. A more general consensus that accommodates all the available sequence data would be G(&-7)(H/KW(&- ~ ) G ( ~ - ~ ) ( L / I ) ( & - ~ ) ( S / T ) .

X. Conclusions Adenylyl cyclases are responsible for the synthesis of an important small regulatory factor. Since variations of the cAMP level at the targets have profound effects on cell metabolism, it is of utmost importance to strictly control the activities of the adenylyl cyclases. In the case of E . coZi, the mechanism for regulating the enzyme activity involves a phosphorylationdephosphorylation of PTS proteins. The state of phosphorylation of these proteins is determined by the relative concentrations of the substrates phosphoenolpyruvate and sugar. There are, in addition, several other factors, of which P, is noteworthy, that exert an important influence on the activity of E. coli adenylyl cyclase. The adenylyl cyclases produced by the invasive B. anthracis or B . pertussis are relatively inactive outside of the eukaryotic host cells. After entry, the host contributes the activators calcium and calmodulin, resulting in a profound increase in adenylyl cyclase activity. The net result is that toxic levels of cAMP accumulate. Numerous genes for adenylyl cyclases have now been cloned and sequenced. Studies have been initiated in a number of laboratories to delineate structure-function relations of some of these proteins. The popular approach being utilized takes advantage of the techniques of site-directed mutagenesis of the structural genes for the enzyme under study. It is anticipated that, over the next few years, an enormous insight into the manner in which adenylyl cyclases interact with substrates and modulators will be achieved.

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hitiation of Transcription by RNA Polymerase II: A Multi-step Process LEIGHZAWEL AND DANNYREINBERG' Department of Biochemistry Robert WoodIohnson Medical School University of Medicine and Dentistry of New Iersey Piscataway, New Iersey 08854

I. The Structure of Class I1 Promoters ............................... 11. RNA Polymerase I1 . . . . . . . A. The C-terminal Domain o B. Carboxy-terminal Domain Kinases ............................. 111. Transcription Factors and Systems ................................. A. Transcription Factor IID ..................... B. Transcription Factor IIB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Transcription Factor IIF D. Transcription Factor IIE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Transcription Factor IIH ............................. F. Transcription Factor IIA ...................................... G. Other Transcription Systems . . . . . IV. Preinitiation and Initiation Complexes and Motifs . . . . . . . . . . . . . . . . . . . A. Complexes on TATA-containing Promoters ....................... B. Complexes on TATA-less Promoters ............................ C. Insights Regarding Initiator-mediated Initiation D. Cooperation between TATA and Initiator Motifs . . . . . . . . . . . . . . . . . . V. Activation and the General Transcription Factors .................... VI. Repression of Class I1 Gene Transcription .......................... References .....................................................

68 69 69

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7s 7s 81 83 86 89 89 93 94

94 97 98 100 100 102 105

The last decade has witnessed an explosion of information pertaining to gene regulation in higher eukaryotes. This has led to discoveries in gene splicing, the development of systems that can mimic specific initiation of transcription in uitro, and the discovery that some oncogenes can directly affect transcription of specific genes, among others. This review focuses primarily on studies, some completed but most still in progress, on the initiation of transcription of protein-coding genes. In the 1960s and 1970s, we learned a great deal about transcription in prokaryotes. Studies revealed the presence of an activity in bacterial cells 1To whom correspondence may be addressed. 67 Progress in Nucleic Acid Research and Molecular Biology, Vol. 44

Copyright Q 1993 by Academic Press. Iuc. All rights of reproduction in any form reserved.

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that could, in a DNA-dependent fashion, catalyze the synthesis of all RNA molecules. This is in contrast to eukaryotic cells, which contain three distinct RNA polymerases, each containing from 8 to 14 polypeptides and responsible for transcribing its own set of genes: RNA polymerase I (RNAPI), which transcribes ribosomal RNA; RNAPII, the RNA polymerase of protein-coding, or class 11, genes; and RNAPIII, which transcribes 5-S rRNA and tKNA genes. In keeping with the theme of this review, only RNAPII is discussed here, and only as it pertains to transcription initiation. [For a more comprehensive overview of RNAPII structure and function, see Young (1). For RNAPII-catalyzed elongation, see Spencer and Groudine (2) and Kerppola and Kane (2a).] Bacterial sigma (cr) factors, which are integral components of bacterial RNAP holoenzyme, are essential to the enzyme’s function. Sigma factors recognize promoter sequences and position the core component of RNA polymerase at transcriptional start-sites. Following start-site selection and initiation, sigma factors dissociate from the core enzyme, which then proceeds to catalyze RNA synthesis. In contrast to bacterial RNAP, mammalian RNAPII cannot specifically2 initiate transcription on its own. In order to fimction efficiently, mammalian RNAPII requires a set of auxiliary factors, otherwise known as “general transcription factors” (GTFs), present in crude cell extracts (3, 4). The last decade has seen substantial progress in the purification and characterization of the GTFs such that present day in vitro transcription systems are reconstituted with highly purified components. To date, seven GTFs have been identified and extensively purified, and four have been cloned (IID*, IIA, IIB*, IIF*, IIE*, IIH, and IIJ, where the asterisk denotes factors whose cDNAs have been cloned).

I. The Structure of Class II Promoters The transcription research field blossomed some 13 years ago when transcription processes were first duplicated in a cell-free system reconstituted with crude cellular extracts (3).In this work and in subsequently developed systems, it became clear that accurate transcription by RNA polymerase I1 is entirely dependent on the presence of promoter-containing eukaryotic DNA (3, 4). It has since become clear that sequences of the DNA template act as signals that direct transcription factors and RNA polymerase to the initiation ‘Abbreviations: RNAP, RNA polymerase; TF, transcription factor; CTF, General transcription factor; Inr, initiator; CTD, carboxy-terminal domain; Ad-MLP, adenovirus major late promoter; TBP, TATA binding protein; TAF, TBP-associated factor; RAP, RNAP-associated protein. %Specific, or accurate, initiation refers to transcription initiation that occurs at discrete start-sites in the promoter region, the same sites utilized in uiuo. If only purified RNAPII is incubated with promoter containing DNA under in oitro transcription conditions, transcripts are svnthesized nonspecifically at spurious locations along the template.

RNA POLYMERASE I1 TRANSCRIPTION INITIATION

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site. Four classes of cis elements have been identified in the promoters of class two genes. The TATA box and the initiator (Inr) region constitute the first two of these classes and are considered minimal promoter elements. One or both of them appear to be present in all protein-coding genes, and each is independently capable of directing the formation of a transcriptioncompetent complex. RNA polymerase I1 and the general transcription factors, collectively known as the basic transcription machinery, function through these minimal promoter elements. The other two classes, consensus sequence elements (which are recognized and bound by specific DNA binding proteins) and enhancers, are considered variable elements, because their presence or absence and the particular order of arrangement in which they occur are gene-specific. It is the combination of all these cis elements that gives a promoter its characteristic strength.

II. RNA Polymerase II The RNAPII of the yeast Saccharomyces cerevisiae has been a useful prototype in the study of eukaryotic RNAPII, as features such as hnction and subunit structure have been highly conserved. The yeast RNAPII is composed of 11 polypeptides with apparent masses ranging from 220 to 10 kDa. HeLa cell RNAPII contains 10 subunits ranging from 240 to 10 kDa (Fig. 1).The genes encoding all 11 yeast subunits have been cloned and shown to be essential for wild-type growth. Unlike the bacterial core enzyme, RNA polymerase I1 activity has not been reconstituted from purified subunits; it is thus unclear whether all of the polypeptides are genuine subunits as opposed to associated factors. An interesting finding that shed light on the possible function of the two largest RNAPII subunits is that significant amino-acid sequence homology exists among the two largest subunits of yeast and Drosophila RNAPII, yeast RNAPI and RNAPIII, and the p and p' subunits of Escherichia coli RNAP. These subunits are thought to be involved in DNA and nucleotide binding (5, 6). Biochemical and genetic (7, 8) experiments have implicated the yeast 32and 16.9kDa subunits as components important to promoter recognition. Mutant forms of RNAPII devoid of these two subunits behave like wild-type forms with respect to promoter-independent initiation, chain elongation, and recognition of pause sites. However, these mutants are inactive with respect to promoter-directed initiation in vitro.

A. The C-terminal Domain of RNA Polymerase II The largest subunit of eukaryotic RNAPII contains an unusual C-terminal domain (CTD) consisting of multiple repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Such a domain is not present in prokaryotic

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LEIGH ZAWEL AND DANNY REINBERG

FIG. 1. Polypeptide composition of phosphorylated (110) and nonphosphorylated (IIA) forms of human RNAPII. Silver staining ofa 5-17% SDS-polyacrylamide gel on which purified human RNAPII was electrophoresed, showing the polypeptide composition of (A) the 110 and (B) the IIA forms of RNAPII. Migration of molecular-weight protein standards are indicated to the left of A and to the right of B. Subunit composition of RNAPII is indicated to the right of A and to the left of B.

RNAP \ r in eukaryotic RNAPI and RNAPIII. The length of the repeat appears to correlate with the genomic complexity of the organism. For example, the heptapeptide sequence is repeated, with some degeneracy, 26-27 times in yeast, 42-44 times in Drosophih, and 52 times in mouse and man (Fig. 2). Owing to a high content of serine and threonine residues in the


71

muse Drosophila Yeast 1 YSPTSPA FCVSSPG YSPTSPN 2 YEPRSPGG YTASSPG FSPT SPT 3 YTPQSPS YSPTSPA GASPN 4 YSPTSPS YSPTSPS YSPSSPN 5 YSPTSPS YSPTSPS YSPTSPL 6 YSPTSPN YSPTSPS YA SPR 7 YSPTSPS YSPTSPS YASTTPN 8 YSPTSPS YSPTSPS FNPNSTG 9 YSPTSPS YSPTSPS YSPSSSG 10 YSPTSPS YSPTSPS YSPTSPV 11 YSPTSPS YSPTSPS YSPTVQ 12 YSPTSPS YSPTSPS FGSSPS 13 YSPTSPS YSPTSPS FAGSCSN I 14 YSPTSPS YSPTSPS YSPGN A 1 5 YSPTSPS YSPTSPS YSPSSSN 16 YSPTSPS YSPTSPS YSPNSPS 1 7 YSPTSPS YSPTSPS YSPTSPS 18 YSPTSPS YSPTSPA YSPSSPS 19 YSPTSPS YSPTSPS YSPTSPC 2 0 YSPTSPS YSPTSPS YSPTSPS 2 1 YSPTSPS YSPTSPS YSPTSPN 2 2 YSPTSPN YSPTSPS YTPVTPS 2 3 YSPTSPN YSPTSPN YSPTSPN 24 YTPTSPS YSPTSPS YS ASPQ 2 5 YSPTSPS YSPTSPG YSPASPA 2 6 YSPTSPN YSPGSPA YSQTGVK 2 7 YTPTSPN YSPKQOEQKHNENENSR YSPTSPT 2 8 YSPTSPS YSPPSPSDG 2 9 YSPTSPS YSPGSPQ 3 0 YSPTSPS YTPGSPQ 3 1 YSPSSPR YSPASPK 32 YTPQSPT YSPTSPL 3 3 YTPSSPS YSPSSPQ 34 YSPSSPS HSPS SQ 35 YSPTSPK YSPTGST 3 6 YTPTSPS YSPTSPR 3 1 YSPSSPE YSPNClsI 3 8 YTPASPK YSPSSTK 3 9 YSPTSPK YSPTSPT 4 0 YSPTSPK YTPTARN 4 1 YSPTSPT YSPTSPM 4 2 YSPRPK YSPTAPSH 4 3 YSPTSPT YSPTSPA 4 4 YSPTSPV YSPSSPTFEESED 4 5 YTPTSPK 4 6 YSPTSPT 47 YSPTSPU 18 YSPTSPT 4 9 Y SPTSPKGST 50 YSPTSPG 5 1 YSPTSPT 52 YSLTSPAISPDDSDEEN

FIG.2. The C-terminal domain of RNAPII contains a heptapeptide repeat conserved through evolution. Sequences of the heptapeptide repeats from the CTD of yeast, mouse, and Drosophih RNAPII are indicated. Note the limited degeneracy in primary amino-acid sequence and the divergence with respect to the total number of repeats observed in different species. [Adapted from Young (I).]

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heptapeptide, the CTD can be highly phosphorylated (phosphotyrosine has not been detected). As a result of this phosphorylation, the largest subunit of RNAPII, which contains the CTD, can be resolved into two major forms on SDS-polyacrylamide gels. The form called 110 has the lowest mobility at 240 kDa (Fig. 1A) and contains the CTD in its most highly phosphorylated state. The IIA form (Fig. 1B) is 215 kDa, unphosphorylated, and probably a primary translation product in oiuo. A third form, IIB, is 180 kDa and lacks most or all of the CTD. From IIB is observed only in oitro and is thought to be a proteolytic artifact of purification. WHATIs

THE

FUNCTION OF THE C-TERMINAL DOMAIN?

Deletion mutants that result in the loss of more than half of the repeats in mouse, Drosophila, and yeast are lethal, suggesting that this domain has an essential role in uioo (Ba, 9, 9a, 9b). Although the heptapeptide repeats are highly conserved among eukaryotes, significant deviations from the consensus sequence and in the overall length of the CTD occur in Drosophila, yeast, and hamster. To assess the significance of these species-specific differences, Allison et al. (9) made yeast RNAPII fusions by replacing the yeast CTD with that from Drosophila and hamster. The hamster fusion-containing yeasts were viable, whereas the Drosophila fusion mutations were lethal. Interesting, the Drosophila CTD, though it has 44 repeats, is much more degenerate in sequence than the hamster or the yeast. The hamster CTD bears 52 repeats, which are more homologous to the yeast CTD and may be functionally redundant. It was postulated that these repeats may provide sites for protein-protein contacts regulating transcription. One possible function of the CTD may be to mediate transcription activation by upstream regulators. Saccharomyces cerevisiue strains harboring a wild-type CTD (26 heptatpeptide repeats), a CTD with 13 repeats, or a CTD with 38 repeats are equally capable of mediating activation by the strong acidic activator Gal4 (10).However, when a battery of Gal4 deletion mutants containing activating region deletions were assayed for transcription activation, in all cases, the shorter CTD suppressed activation whereas the longer CTD enhanced activation relative to the wild-type CTD. Thus a longer CTD seems to complement Gal4 deletion mutations. It was postulated that the heptapeptide repeat is a functionally redundant motif that can interact with the activation domains of some regulatory proteins. In this manner, RNAPII could be recruited to promoters where further contacts between RNAPII and the GTFs position the enzyme, defining transcriptional start sites. Though from the data it is unclear whether such CTDIactivator contacts are direct or indirect, the model is plausible and warrants further analyses. In collaboration with Y.Aloni (The Wiezmann Institute), we have demonstrated


73

that the CTD specifically interacts with TFIID (IOU), a finding that strengthens this particular model. Alternatively, others have shown that the CTD is unnecessary for Spl-(II) and MLTF-mediated (12) activation in uitro. Perhaps the CTD mediates stimulation of only a subset of activators, or only in the context of a subset of specific promoters. Interestingly, the IIB form of RNAPII, which contains no CTD, can accurately transcribe the actin 5c and Ad-ML promgters in uitro (9b, 13). A requirement for the CTD in uitro is observed with the murine DHFR promoter, a TATA-less promoter (14). While these observations may suggest a requirement for the CTD with TATA-less promoters, preliminary data from our laboratory and from P. Farnham et at. (Unpublished) indicate that the requirement for the CTD in basal transcription may be determined by the promoter utilized, and more specifically, by the class of initiator motif present therein. Another viewpoint is that the CTD may act to remove, or to overcome the effects of, factors that negatively regulate transcription. This is suggested by the observation that wild-type viability is restored to yeast strains containing CTD truncations upon deletion of the repressor, SIN1 (144. There is physical evidence suggesting that the CTD can intercalate into DNA nonspecifically (15).Such an activity could strengthen RNAPII binding and/or initiation complex stability. Perhaps CTD phosphorylation releases the CTD/DNA interaction, thereby facilitating the transition from initiation to elongation (16). To investigate the functional significance of CTD phosphorylation, Dahmus and Laybourn (17) followed changes in the CTD phosphorylation state through the transcription cycle. They found that (1)the nonphosphorylated IIA form stably associates with the preinitiation complex, (2) the I10 form can be isolated from actively elongating complexes, and (3) the conversion of RNAPIIA to RNAPIIO occurs prior to the formation of the first phosphodiester bond. A model was presented in which the unphosphorylated polymerase preferentially associates with the promoter-bound initiation complex, and the subsequent phosphorylation of the CTD potentiates the transition from transcription initiation to elongation (Fig. 3). Several additional lines of evidence further support this model. (1) Crosslinking experiments demonstrated that the RNAPII, which was actively elongating, was in the I10 form (18, 19). (2) Antibodies with a preference for IIA preferentially inhibit initiation over elongation (20, 21). (3) Lu et al. (22) have recently developed a method to purify the RNAPIIA and RNAPIIO forms to apparent homogeneity from HeLa cells. Using pure forms of the enzyme, it was shown by gel shift assay that, in agreement with Dahmus’ model, the IIA form preferentially associates with the assembling initiation complex. Furthermore, the efficiency with which the I10 form of the polymerase associ-


74

Elonaation:

DNA GTFs

CTD Kinase(s)

/

Phosphoprotein

FIG.3. Phosphorylation of RNAPII modulates the transition from transcription initiation to elongation. The IIA form of RNAPII associates with the assembling preinitiation complex. One or more protein kinases trigger the elongation phase by phosphorylating the CTD. The action of a phosphatase would reset the cycle. [Adapted from Buratowski and Sharp (12).]

ates with the complex can be increased substantially by subjecting the I10 polymerase to phosphatase treatment.

B. Carboxy-terminal Domain Kinases A great deal of work has gone into identifying the cellular protein kinase(s) that modify the CTD. Dahmus and Laybourn proposed that the CTD kinase may be one of the general transcription factors present in the preinitiation complex (17). TFIIH was recently found to contain a specific CTD kinase activity drastically stimulated by factors that direct the polymerase and TFIIH to the promoter (224. Dynan and co-workers have isolated a kinase with specificity for the CTD that depends on DNA for phosphate transfer for catalysis (23). The TFIIH kinase and the kinase reported by Dynan can be distinguished by their DNA requirements. Whereas the TFIIH kinase is stimulated only by DNA elements that can direct the formation of a transcription complex, the other is nonspecific in this respect. Corden and Cisek (24) purified a murine kinase as a heterodimer of 58and 34-kDa moieties that had specificity for the CTD. Interestingly, the smaller subunit of the heterodimer has been identified as the cdc2 gene product, a protein involved in cell-cycle control and previously shown to be a component of the M-phase-promoting factor (MPF), an M-phase-specific histone H1 kinase. This protein is a homologue of the Saccharmyces pombe c&2-encoded protein and the S. cerevkiae cdc28-encoded protein, both of which are known to be cell-cycle regulators (25). It will be of interest to determine if CTD phosphorylation changes with the cell cycle. A yeast kinase with specificity for the CTD, purified to near homogeneity (26), contained three subunits of 58, 38, and 32 kDa. Since none of these


75

subunits cross-reacts with anti-cdc2 antibodies, this kinase is immunologically distinct from MPF kinase. The gene encoding the largest subunit, CTK1, was cloned and found to contain a protein kinase catalytic subunit motif(27). CTK1- mutants are viable but grow at reduced rates, indicating that this particular kinase is not essential. Interestingly, the RNAPII isolated from extracts of CTK1- cells was phosphorylated but not to the extent observed in wild-type cells. Thus, at least two CTD kinases are thought to exist in S. cereuisiae. One is the CTKl kinase, which phosphorylates RNAPII, converting RNAPIIA to RNAPIIO. The other kinase phosphorylates the yeast CTD, but not to the extent that the RNAPIIA form is converted to the RNAPIIO form. A similar situation has been observed in Aspergillus niduluns, where at least two CTD kinases have been described (27a). Kinase I, a serine kinase contained in a single polypeptide of 57 kDa, can phosphorylate RNAPII in solution, but cannot convert IIA to I10 unless it is contained within a preinitiation complex. Kinase 111, on the other hand, preferentially phosphorylates RNAPII in solution and cannot phosphorylate RNAPII when it is associated with the preinitiation complex. By converting free RNAPIIA to RNAPIIO, kinase I11 essentially limits the pool of RNAPII that can effectively enter into the preinitiation complex. Thus, in Aspergillus, the function of kinase 111 may be to regulate overall levels of class-I1 gene transcription. We have identified two CTD kinases from HeLa cell extracts (22). Both kinases behave similarly to Aspergillus kinase I with respect to their preference for complex-associated RNAPII. One of the kinases was identified as the MPF kinase. The other kinase activity is TFIIH.

I11. Transcription Factors and Systems As discussed in the introduction, eukaryotic RNAPII is unable to catalyze accurate transcription without the assistance of auxiliary factors. To date, seven required activities have been characterized. They are referred to as general transcription factors (GTFs) and recent years have witnessed much progress in the purification of these factors from HeLa cells (Fig. 4).Of these seven factors, only one, TFIID, contains a DNA binding activity with specificity for the TATA box. The other factors and RNAPII enter into the transcription cycle by protein-protein interactions.

A. Transcription Factor IID TFIID was first identified as an activity in Drosopkilu nuclear extracts that can specifically bind TATA-containing promoter regions of class I1 genes (28).Though this observation was made in 1984, further characterization of

Phosphocellulose

I 0.1 M (KCI)

I

I 0.3 M (KCI)

r

1

S-sepharose

I

I

Hepari agarose

1.0 M (KCI)

DEAE-Sspha-1

DEAE-52

,

DEAE-saphacd

Ompharose

I

I 0.5 M (KCI)

’

I

I

i

IIE.IIF.IIH.BTF3

I

IID

SP-sepharose

IIE

I I

Phenyl superose

Superder 200

Mom S Hydroxylapatile

I I

Phenyl superose

)I Phenyl

I

S-sepharose

+

superose

I

m

IIH

I

IIJ

Ts IIH

IIE

FIG.4. Scheme of the purification of the human class41 general transcription factors. [For details, see text and Flores et al. (51).]


77

TFIID has been slow, due to difficulties encountered in purifying the activity. However, using a partially purified factor, it was demonstrated that TFIID is the first factor to enter into the transcription cycle; it provides a foundation for the association of the other GTFs and RNAPII (28u-28c). A breakthrough was the discovery in 1988 that an activity in yeast can functionally replace human TFIID in a reconstituted system and support basal transcription levels (29, 30). The yeast protein has an apparent mass of 27 kDa, and, much like the human factor, exhibits DNA binding activity specific for the TATA box. Yeast TFIID protects a 20-nucleotide region centered around the TATA motif from DNase-I cleavage. The cDNA encoding yeast TFIID was cloned by several groups 1year later using reverse genetics (31-34). This represented the first GTF to be characterized at the sequence level. Significantly, the gene encoding yeast TFIID had been described previously as the SPTIS gene, a Tyl mutation suppressor, the absence of which results in cell death (35, 36). Not long after the yeast sequence was determined, cDNAs encoding Arubidopsis, Drosophila, and human TFIIDs were cloned by homology using degenerate primers and PCR (37-41). For a long time it was thought, due to the degeneracy of the TATA motif and the fact that specific responses were mediated through only certain TATA sequences (41u, 41b), that there are multiple TFIID genes encoding proteins whose binding specificities varied with different TATA sequences. With the exception of Arubidopsis, where two genes encoding TFIID were found (41), only one TFIID gene appears to exist in most eukaryotes. Thus, it seems that one molecule can recognize variant TATA motifs with different afhities and mediate different responses to defined stimuli. This versatility might be facilitated by the association of the TATA binding protein with different factors. The cloning of TFIID from a variety of species was facilitated by the highly conserved nature of TFIID, particularly in the carboxy-terminal half of the protein. The C-terminal180 amino acids of the human and Drosophila clones are 88% identical, while the yeast sequence shares 80% with either the human or Drosophila proteins (Fig. 5). In contrast, the N-terminal domain of TFIID is highly divergent across different species. The conserved C-terminal region of TFIID contains the following noteworthy structural motifs. (1)An imperfect 78-aminoacid direct repeat; these repeats may provide the molecule with an element of symmetry. Such a design may be important in recognizing a “directionless” TATA. The area between these repeats has been postulated to be involved in contacting TFIIA (41c).(2) A central basic core with an abundance of lysines (120-156); this region has the potential to form an a-helix with all basic residues oriented on one side, suggestive of a DNA binding or protein-protein interaction role. Individual basic residues within this region do not seem to be

78


Conserved Core Domain

Basic Region

(T

Homology

FIG,5 . TFIID structure is highly conserved in evolution. Schematics of the structure of TFIID from man, Ihosophila, Aruhidopsis, and yeast are shown. The shaded area extending from residues 155 to 355, with respect to human TFIID, denotes a region highly conserved among the different TATA binding proteins. Percentages indicate degree of sequence identity in the cnre domain relative to human TATA binding proteins. Horizontal arrows indicate direct repeats. The lightly shaded region indicates a highly basic region. The darker shaded area bears homology to prokaryotic u factors. In the N-terminal domain, black bars represent regions rich in serine, threonine, and proline. The shaded regions in the N-terminal domain of the human and Drosuphilu proteins signib Q-runs. [Adapted from Hoffmann et 02. (39).]

important for DNA binding or basal transcription (39). Perhaps this region is important to activation. (3) Sigma homology (197-240); this region bears sequence similarity to the portion of bacterial sigma factors known to interact directly with the - 10 element of bacterial promoters. Although the C-terminal domains from yeast and human TFIID are highly homologous and equally capable of supporting basal transcription in uitro, human TFIID is unable functionally to replace endogenous yeast TFIID in uico. This was not due to differences in the N-terminal domain because when this portion of the molecule was deleted, no detrimental effect on cell viability was observed (41d,41e). Analyses using humadyeast hybrid TFIIDs indicate that no single region within the conserved domain is responsible for the functional differences between the two proteins. Hather, a number of subtle polymorphisms throughout the entire conserved domain may cumulatively predispose a TFIID molecule to being functional in one species but not another. In striking contrast to the C-terminal domain, the TFIID N-terminal domain is highly divergent. In yeast, the TFIIDs N-terminal domain is only SO-60 residues (N-terminal to the N-terminal direct repeat), whereas in Drosophila and man it contains 173 and 155 residues, respectively, which


79

share only 24% identity. Overall, this portion of the molecule contains very few charged residues. Two motifs stand out, but in keeping with the overall divergence of the N-terminus, they are not strongly conserved across species: (1)The most notable feature is a stretch of 38 consecutive glutamines occurring in the central portion of this domain in humans. In Drosophilu there is a run of 6 and 8 glutamines (Q-runs) in the same respective position, while in the yeast protein there is no such motif. The function of these Qruns is unknown, but it is interesting to consider that the transcriptional activator S p l also contains them. (2) A Pro-Met-Thr tripeptide is repeated five times in human and four times in Drosophilu (it is absent from yeast) near the junction between the amino-terminal domain and the basic core. Consistent with the TFIID bipartite structure, biochemical studies indicate that the conserved domain can function somewhat independently of the amino-terminal domain. Mutational analyses demonstrate that in the yeast, Drosophila, and human clones, the highly conserved C-terminal domain is sufficient to provide DNA binding to the TATA motif and basal transcription activities (33u, 37, 40).Apparently, this portion of the protein is fully sufficient to participate in the formation of an active transcription complex. What then is the function of the divergent N-terminal domain? Recombinant TFIID is capable of supporting basal level transcription, but is unable to respond to activators (37, 42). Transcriptional activation as mediated by MLTF (39) or S p l (37) requires native human or Drosophilu TFIID. As discussed below in further detail, this apparently reflects the requirement for a novel class of “adaptor” molecules, termed coactivators, which are thought to bridge TFIID, or perhaps other GTFs, to transcriptional activators such as Spl. Mutant Drosophilu TFIID molecules lacking the Nterminal domain are incapable of mediating an S p l response (42),suggesting that the divergent N-terminal region may mediate interactions with coactivators. If this were the case, one would expect that interactions between TFIID and coactivators would be species-specific, since this region of the protein is highly divergent across different species. In fact, recombinant human or yeast TFIID is incapable of modulating activation mediated by Drosophila coactivators and S p l (42). Compelling in viuo evidence in support of this model has been presented by Zhou et ul. (43).By complementing an S . cereuisiae TFIID-deficient yeast strain with amino-terminal deletion mutants they demonstrated that (1)the conserved carboxy-terminal domain of yeast TFIID is sufficient for cell viability, but that (2) an acidic region (residues 48-57), just amino-terminal to the conserved domain, is required for a transcriptional response to upstream factor stimulation and normal cell growth. Biochemical studies using recombinant TFIID support earlier observations suggesting that, in viuo, human TFIID is part of a large protein com-

80

LEIGH ZAWEL AND DANNY REINBERC

plex (28~).When Drosophila, yeast, or human cDNAs encoding TFIID are used to overexpress the protein in E . coli, or in insect cells, the purified protein not only binds specifically to the TATA box but also protects, from DNase-I cleavage, a 20-nucleotide region centered around this motif (37, 38, 40). This protection is in stark contrast to the protected region observed with partially purified native mammalian TFTID, which protects a region encompassing 75 nucleotides centered around + 1 on the adenovirus major late promoter (Ad-MLP). A similar discrepancy is found when apparent mass is considered. Endogenous human TFIID activity elutes from a sizing column with an apparent mass of over 100kDA, yet the cloned protein’s 339 amino acids comprise a 38-kDa polypeptide. Glycerol gradient sedimentation and immunoprecipitation analyses indicate that TFIID exists in the cell in a multiprotein complex with at least six polypeptides ranging in mass from 32 to 150 kDa (44). Because the protein-protein interactions between TBP and the TBP-associated factors (TAFs) are extremely tight, it has been difficult to resolve and characterize the components of this complex. By performing conventional chromatography in the presence of the denaturant urea, however, TBP can be separated from the TAFs. Interestingly, while urea-purified TBP is unable to support activated transcription, addition of the urea-purified TAFs fully restores an activation response. This, together with the fact that the TAFs are dispensable for basal transcription, suggests that one or several of these TAFs function as coactivators. In initiation complex assembly, the binding of TFIID to the TATA motif appears to be the first step. The TFIID-DNA complex provides a recognition site for the association of the other GTFs and RNAPII (45, 46). As discussed above, the TATA binding protein (TBP) appears to have the ability to interact with many factors. Two factors that effect basal transcription, TFIIA and TFIIB, probably interact with the conserved domain of TFIID, since both yeast and human TFIID can interact with mammalian TFIIA and TFIIB proteins. Association of TFIIA with TBP can occur in solution, in the absence of DNA, as well as with a TFIID molecule bound to the TATA motif (Fig. 6) ( 4 6 ~ )TFIIB . can also interact with TFIID. It is unknown whether this interaction occurs in the absence of DNA. However, when TFIID is bound to the TATA motif, TFIIB can stably associate, producing the DB complex (Fig. 6). The association of TFIIA with the TFIID-DNA complex is not required for TFIIB binding nor does it preclude the association of TFIIB with the TFIID-DNA complex. Rather, the presence of TFIIA appears to increase the overall stability of the resultant DAB complex. The association of TFIIB with a bound TFIID is required for the association of the other GTFs and RNAPII with promoter sequences.

81


TATAH Inr

transciiption complexes FIG.6. Formation of the DA and DB complexes. TFIID recognizes and binds to the TATA motif. This is the first factor to associate with the promoter. TFIIA and TFIIB are equally capable of binding to the TFIID-DNA complex. If TFIIA binds before TFIIB, TFIIB can bind the DA-DNA complex, generatingthe DAB complex. The presence of TFIIB (in either a DB or a DAB complex) is necessary for the association of RNAPII and the other GTFs. Inr, Initiator.

B. Transcription Factor IIB TFIIB was first described as an activity present in phosphocellulose 0.5 M KC1 washes, which was absolutely required for transcription from class-I1 promoters and which copurified with a protein of 30 kDa (47). It was thought to participate in the formation of stable preinitiation complexes, as it was required to establish heparin-resistant transcription. Consistent with such a role, TFIIB recognizes the D-DNA or the DA-DNA complex and binds to it, generating the DB or the DAB-DNA complex in gel mobility shift assays (45, 46). At the time of its initial characterization, it was observed that free TFIIB could associate with crude TFIIE, a preparation that contained TFIIF and TFIIH, but could not directly associate with RNAPII in solution (47). Also around this time, BTF3, a 27-kDa transcription factor that behaved chromatographically like TFIIB, was reported (48). Even though BTFS was known to form a stable complex with RNAPII and TFIIB did not, there were many who believed that these two proteins were the same. It was not until both BTFS (49) and TFIIB (50) were cloned and shown to be distinct based on primary sequence that this controversy was resolved. The role of BTF3 in transcription, if any, is currently unknown (51). Reverse genetics has been used to obtain the cDNA clone encoding TFIIB (50). The polypeptide exhibits a molecular mass of 33 kDa. The nucleotide sequence of human TFIIB predicts an open reading frame encoding a polypeptide of 316 amino acids with a calculated molecular mass of

82


34.8 kDa. TFIIB overexpressed in E. coli (IIBr) is indistinguishable from purified native TFIIB with respect to (1)gel mobility shift assay, i.e., TFIIB can recognize and bind to the DA complex forming the DAB complex; the DABr-DNA complex is recognized by RNAPIIIIIF and subsequently assembling components; (2) basal transcription, i. e., reconstituted transcription reactions including IIBr in place of native TFIIB display similar or identical levels of transcription; and (3) activated transcription, i.e., in contrast to cloned TFIID, IIBr is capable of supporting stimulated transcription as mediated by S p l and MLTF (50). Recently emerging has been the model that TFIIB may be critical to interactions between the initiation complex and upstream activators. There is evidence suggesting that the synthetic acidic activator Gal4-AH (51a) stimulates transcription by recruiting TFIIB to the assembling complex (52) and that TFIIB association with the assembling preinitiation complex is the rate-limiting step in initiation. The acidic activator may stimulate transcription levels by recruiting and maintaining TFIIB in the promoter region. Consistent with this model, it was shown that a direct and specific interaction occurs between TFIIB, native or recombinant, and the VP16 activating region. This interaction appears to be important for activation because a mutant VP16, which is unable to support activation, also failed to interact with TFIIB (53) (see also Section V). Southern and Northern blot analyses indicate only one human gene encoding TFIIB. Computer searches fail to detect any genes with sequences substantially similar to TFIIB. An imperfectly repeated motif of 76 amino acids is present in the carboxy-terminal half of the protein (Fig. 7). That a structurally similar motif is also present in TFIID suggests these repeats may be functionally important. Deletion analyses indicate that these repeats are required for TFIIB to associate with the DA complex (53a).These mutations, as well as other deletions located anywhere in the protein, result in a TFIIB that is transcriptionally inactive, suggesting that the protein is overall very compact. In the 20 or so amino acids separating these repeats, there is a region bearing some similarity to prokaryotic sigma factors. Also nested between these repeats is an -15 aminoacid domain with the potential to form an arnphipathic a-helix containing hydrophobic residues along one side and charged basic residues along the other (Fig. 7). We suspect that this region may contain residues that contact VP16. Single point mutations in which a basic residue along the charged side of the putative amphipathic a helix is changed to a neutral residue behave like wild-type forms with respect to VP16 binding, but double point mutations in this area exhibit reduced or no binding to VP16 columns. If this entire motif is deleted, VP16 binding is abolished ( 5 3 ~ ) .


83

FIG.7. Structural features of human TFIIB. Top: schematic of TFIIB polypeptide. Arrows in shaded regions denote imperfect direct repeats. The region bearing homology to prokaryotic u factors is located between repeats and overlaps slightly with the N-terminal repeat, as indicated; + + + +, depicts putative amphipathic a-helix with cluster of charged basic residues oriented along one side. Bottom: helical wheel depiction of putative amphipathic a-helix (residues 185-202). Shaded region emphasizes cluster of charged basic residues, which may represent contact region with Ga14-VP16.

+

C. Transcription Factor IIF In early reconstituted transcription systems, TFIIF was present in crude TFIIE preparations (54). This fraction was interesting, not only because it was absolutely required for transcription, but also because it was shown, using glycerol gradient analyses, that it bound tightly to RNAPII in solution (47). The 30- and 74-kDa polypeptides that make up TFIIF were first isolated by exploiting this particular property. When Greenblatt and colleagues fractionated calf thymus or HeLa cell-derived extracts over columns containing immobilized RNAPII, they isolated three major RNAPII-associating proteins, RAP30, RAP74, and RAP38 (55). RAP38 is equivalent to TFIIS, also known as SII, an elongation factor that affects the efficiency by which RNAPII passes through pausing sites (56, 57, 57a). TFIIF was purified to homogeneity from nuclear extracts using a functional transcription assay, and

84


the 30- and 74-kDa polypeptides were immunologically identical to RAP30 and RAP74 (58,59). TFIIF activity elutes from a gel filtration column with an apparent molecular mass of 220 kDa, suggesting that it exists in solution as an a2Pzstructure. In addition to being essential for transcription initiation, TFIIF has the ability to stimulate transcription elongation (60). The mammalian cDNAs encoding RAP30 (61)and RAP74 (61a, 61b)have been cloned. The sequence of RAP30 is rich in basic residues and contains several interesting motifs. (1)The central portion of the RAP30 249 amino acids (residues 93- 165) is homologous to two noncontiguous regions of E. coli u70 (Fig. 8). These two regions are the only regions conserved among all bacterial and bacteriophage u factors. They are postulated to contain contact sites with the core component of bacterial RNAP. Consistent with this proposed function, TFIIF can bind to E . coli RNAP and subsequently be displaced by bacterial u70 (62). (2) The amino-terminal portion of the protein (residues 36-42) features a consensus nucleotide binding motif. (3) Just C terminal to the cr70 homology domain (residues 164-174), a region of homology exists with the proposed DNA binding region of CREB (Fig. 8). Curiously, the calculated molecular mass of the polypeptide encoded by

RAP 30 Similaity to 070 4

C /-

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/Nucleotide\, Bindina Motil-

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RAP 74 428 437 450

+-+-+-+-

517

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FIG. 8. Structural features ofthe srnall and large sullunits of human TFIIF. Top: schematic of KAPJO, the small subunit of TFIIF. Residues 36-42 contain a nticleotide-binding motif represented b y horizontal bars. The shaded region in the center of the protein bears sequence similarity to prokaryotic u factors. Blackened region (residues 165-174)bears homology to the D N A binding doniain of CREB. Bottom: scheniiltic of RAP7.1, the large stibunit of TFTIF. The centcr of the protein contains a region rich in charged amino acids. shown here as charge cliisters. Tlie C-terminal domain cwntairis a region with homology to a 1iiicleotide-lindirig motif idiagonal lines) and a region with homology to RNAPI (shaded area).


85

RAP74 cDNA is 58.2 kDa. Both subunits ofTFIIF are phosphorylatable and the largest subunit is extensively phosphorylated in vivo. RAP74 contains a globular N-terminal domain, a highly charged central domain containing clusters of acidic and basic residues, and a globular C-terminal domain, which includes two interesting motifs (Fig. 8). The first is a weak region of homology with a subdomain (region IV) present in yLast RNAPI and other eukaryotic RNAPs. The function of this region is unknown. The other motif is a region of homology to the phosphate binding loop of thymidine kinase. This is intriguing in light of the fact that the large subunit of TFIIF has been proposed to contain an ATP-dependent DNA helicase activity, the probable function of which is to melt the DNA during initiation of transcription (61). Recombinant RAP74 does not, however, contain intrinsic adenylation or helicase activity, be it in the presence or absence of RAP30. Also, highly purified, transcriptionally active native TFIIF is devoid of helicase, as well as any kinase or phosphatase activities (59). If TFIIF is involved in one or several of these enzymatic activities, it most likely requires additional polypeptides present within the preinitiation complex. Mechanistically, TFIIF appears to have the critical role of associating with RNAPII and delivering the enzyme to the assembling preinitiation complex on the DNA (Fig. 9). Buratowski et al. have proposed, based on gel shift experiments, that RNAPII can associate directly with the DAB complex (45). However, when highly purified or recombinant factors are used, neither TFIIF nor RNAPII can join the DAB complex alone (63). When TFIIF and

FIG.9. Model for the association of RNAPII with the promoter. RNAPII, in association with TFIIF, recognizes the DAB or the DB protein-DNA complex generating the DAB-PoIF or DB-PoIF complexes, respectively.

86


RNAPII are added together to the DAB complex, a slower migrating DABPolF complex is formed. This RNAPII-recruitment function is particularly in keeping with the u homology described in RAP30. Killeen and Greenblatt have observed (63a)that TFIIF or RAP30 can suppress nonspecific binding of RNAPII to DNA. Interestingly, the 30-kDa subunit can, independent of the large subunit, recruit RNAPII to the DAB complex. The DAB-PolRAP30 complex is, however, less stabile to electrophoresis than DAB-PolF, suggesting that RAP74 may stabilize the complex. Though RAP74 does not appear to be obligatory for the recruitment of RNAPII, studies in which binding complexes were disrupted and probed with antibodies indicated that both the 30- and the 74-kDa subunits are retained in the DAB-PolF complex (63). In reconstituted transcription systems, RAP30 is unable to replace TFIIF. The formation of the DB-PolF or DAB-PolF complex provides the foundation for the association of the remaining factors required for basal transcription, TFIIE, TFIIH, and TFIIJ.

D. Transcription Factor IIE TFIIE copurifies with TFIIF and TFIIH through phosphocellulose, DEAE-Sephacel, and gel-filtration chromatography; thus early TFIIE preparations contained a variety of activities, making its characterization a slow and difficult process. The eventual purification of TFIIE to apparent homogeneity resulted in the following observations: (1) TFIIE is essential for accurate in oitro transcription from class-I1 promoters; (2) activity copurifies as a heterodimer of 34 and 56 kDa, and the stoichiometry appears to be 1:1; (3)gel filtration analyses indicate that the factor appears to exist in solution as a tetramer, with a native apparent mass of 200 kDa (64,65). Assembly of TFIIE into the initiation complex occurs after the association of RNAPII/IIF with the DAB complex (65).In gel mobility shift assays, the association of TFIIE with the DAB.PolF complex produces a slower migrating DABSPolFE complex. The prerequisite that RNAPII must be stably bound prior to TFIIE association is in agreement with the observation that TFIIE and RNAPII cosediment in glycerol gradients (47, 6Sn) and suggests that the association of TFIIE with the complex may be mediated, in part, through an interaction with RANPII. Both subunits of TFIIE have been cloned from HeLa cDNA libraries using reverse genetics (66).The open reading frame of the p56 cDNA encodes a polypeptide of 493 amino acids with a predicted mass of 49.5 kDa. The 291 amino acids of p34 comprise a polypeptide of 33 kDa. Both subunits have been cxpressed in E . coli. In gel mobility shift assays, rTFIIE behaves identically to native TFIIE. Recombinant TFIIE can replace native TFIIE in a reconstituted system and support basal, as well as activated, transcription. These experiments revealed that, in contrast to initial observations


87

based on the renaturation of purified, native subunits, neither TFIIE subunit can functionally replace TFIIE independently. The factors used in the initial experiments were most likely contaminated with the small TFIIE subunit. The sequences of p34 and p56 are not closely related to any existing proteins in the data base. In both subunits, however, there are several interesting motifs that may provide some clues as to the functional role of TFIIE (Fig. 10). In stark contrast to one another, the 56-kDa subunit is highly acidic, with a PI of 4.5, whereas the 34-kDa subunit is highly basic, with a PI of 9.5. In particular, there is a stretch of acidic residues near the C-terminus of p56 and a stretch of basic residues in the corresponding region of p34 (Fig. 10A). The opposite charges probably contribute to the strong subunit interaction observed in gel filtration analyses. Consistent with observations suggesting an interaction of TFIIE with RNAPII and other GTFs is the presence in p56 of a sequence with the potential to form an amphipathic a-helix with a cluster of basic residues on one face and hydrophobic residues on the other. A cluster of cysteine residues arranged in the pattern CX,CX,,CX,C is contained in p56 (Fig. 10B). Similar zinc-finger patterns have been noted in a number of nucleic-acid binding proteins, including the steroid hormone family of receptors, Spl and others. This region shares considerable sequence homology with the zinc-finger motifs present in the UvrA and UvrB proteins of E . coZi. These proteins are components of the ABC excinuclease, a DNA-repair enzyme consisting of three subunits, UvrA, UvrB, and UvrC (67). Significantly, UvrA has DNA-binding activity, albeit with a preference for damaged DNA. As yet, TFIIE is not known to possess any independent DNA-binding activity and so the significance of this domain remains to be determined. As noted in Fig. lOC, p56 contains a region bearing homology to a consensus sequence present in the catalytic loop of several kinases, including RAFl kinase, protein kinase C, and Src kinase among others. This motif is especially similar to a domain common to the protein kinase C family. Though the 56-kDa subunit lacks an accompanying consensus sequence for nucleotide binding, there is such a motif in p34 (Fig. 10D). This region in p34 is similar to the nucleotide binding site present in the human multidrugresistance protein (68). Previous studies have suggested that TFIIE contains an ATPase activity (69).While potential kinase and nucleotide binding motifs are present in TFIIE, biochemical studies attempting to demonstrate kinase or ATP-binding activity using recombinant TFIIE have failed. Homogeneous preparations of native TFIIE that are transcriptionally active are devoid of ATPase, topoisomerase, and DNA helicase activities (65). The meaning of these motifs in TFIIE is unknown, but it is tempting to speculate that

88


A 34K

26-42

122-135

SK

Nucleotide BS-2

I I@

Basic 9

56K a-Helix

Zinc finger

Kinase consensus

Acidic

B F

C

56K PKCp PKCG Raf 1 cdc2 Src MIW

MDR-1 pfMDR WHITE

T+

~ E DQ Y N v vII T N[M@

[ K P

I I I I V F R F

I I L

AR AR A S

E E E NUCLEOTIDE BINDING SITE 2

FIG.10. Structural features of human TFIIE. (A) Schematic of p34 and p56. In p34, S and K represent runs of serine and lysine residues, respectively. The shaded area in the center of the 34-kDa protein represents a region bearing homology to a nucleotide-binding motif. Sequence similarity of this region with MDR-1 nucleotide-binding motifs is further illustrated in D. Blackened area represents a region rich in basic residues. In p56, diagonal bars depict region predicted to form amphipathic a-helix. C-C--C-C represents a sequence that could form a zinc finger, also depicted in B. The crosshatched box indicates kinase consensus sequence; the similarity to other kinases is shown in C. The shaded area depicts region rich in acidic residues. (B) Schematic of the potential zinc-finger structure within p56. Circles indicate residues that are

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TFIIE, in association with other GTFs, perhaps TFIIH (see Section II1,E) may constitute an ATPase or a kinase activity. The association of TFIIE with the preinitiation complex requires the DAB-PolF complex, and gives rise to the DAB-PolFE complex. This provides the foundation for the association of TFIIH.

E. Transcription Factor IIH When TFIIF preparations were purified by phenyl-Superose chromatography, a previously uncharacterized, essential basal transcription activity was discovered (51).The elution profile of this activity, named TFIIH, is distinct from TFIIF as determined by Western blot analyses using antibodies against RAP30 and RAP74. The use of TFIIH in this system does not overcome the requirement for any of the previously described GTFs. TFIIH is required for transcription from a variety of class-I1 promoters, including those for P-globin, Hsp70, Ad-MLP, and Ad-IVa2. TFIIH activity coelutes with five polypeptides that migrate on SDS-polyacrylamide gels with apparent masses of 90,60,43, 41 and 35 kDa (69a). The cDNA encoding the 60kDa subunit has been isolated (69b). TFIIH activity elutes from a' sizing column with an apparent mass of 230 kDa. TFIIH enters the preinitiation complex after TFIIE and before TFIIJ. We have recently detected a kinase activity associated with THIIH that can phosphorylate the CTD of RNAPII (22a).

F. Transcription Factor IIA Regarding its role, its requirement, and even its polypeptide composition, TFIIA is perhaps the most controversial of all the general transcription factors. Egly et al. purified a 43-kDa protein from HeLa cells to apparent homogeneity; this protein contained basal transcription stimulatory activity and appeared to act early in initiation complex formation (70). Surprisingly, it possessed a number of intriguing similarities to the filamentous structural protein, actin. In addition to having the same molecular weight as actin, this protein cross-reacted with anti-actin antibodies, could self-polymerize and -~ ~

~~

identical (unbroken) or similar (broken) to the first zinc finger of UvrA. Residues were found to be identical (asterisk) or similar (caret) to a region of the UvrB protein. (C) Comparison of the region of p56 containing a kinase consensus sequence with segments p and 6 of protein kinase C, the kinase-related transforming proteins Rafl and Src, and the yeast cell-division control proteins cdcz and MIW. Conserved residues are boxed and shaded. The region proposed to be the catalytic loop is underlined. (D) Comparison of the nucleotide-binding motif present in p34 with similar motifs in the human MDR-1 protein, the Plosmodiumfalciparum MDR protein (pfMDR), and the Drosophila white gene product (WHITE). Conserved residues are boxed and shaded. Residues thought to constitute the nucleotide-binding fold are underlined. [From Peterson et al. (66).]

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depolymerize, and could bind and inactivate DNase I. Actin’s known localization in the nuclear matrix further fueled speculation that it might have a role in transcription. It seems likely that these observations (70)were due to trace, nondetectable amounts of TFIIA that copurified with actin. Subsequently, it was demonstrated that fractionation of TFIIA on H P E hydroxylapatite and TSK-phenyl columns separates TFIIA activity from actin (28c). Numerous actin-free, transcriptionallly active TFIIA preparations have subsequently been described. Samuels and Sharp isolated from calf thymus an activity that could functionally replace HeLa cell fractions containing TFIIA (71).This activity is associated with three polypeptides of 19.6, 19.1, and 12.8 kDa. The native molecular mass was estimated to be around 30 kDa based on gel filtration and sedimentation analyses. Reconstitution of TFIIA activity from individual polypeptides was not attempted; thus, the polypeptide composition of TFIIA was not conclusively established. Reinberg and Roeder described HeLa TFIIA as a factor required for specific initiation of transcription; it was eluted from phosphocellulose by 0.1 M KC1 (28c). Curiously, there is a variable requirement for TFIIA in a reconstituted system that depends on the purity of the TFIID used. The most crude TFIID preparation, that derived from the 1.0 M KCI wash of phosphocellulose, abolished the requirement for TFIIA. Functional analyses suggested that a specific interaction occurred between TFIID and TFIIA in the absence of DNA (28c).The mass of TFIIA was estimated to be 84 kDa. Later, using TFIID &nity chromatography, TFIIA was isolated as a heterotrimer of 34, 19, and 14 kDa (46a). Usuda et al. (72)recently reported that TFIIA activity from HeLa cells is contained in a single polypeptide of 38 kDa with a native mass ranging from 90 to 160 kDa. Purified preparations of p38 stimulated a reconstituted transcription reaction; however, renaturation of p38 from protein gels was not attempted. It thus remains unclear whether TFIIA activity is contained in the single polypeptide. TFIIA activity from wheat germ extracts was also reported to be contained in a single polypeptide of 35 kDa (73).This protein could also replace human TFIIA in a reconstituted transcription system. Significantly, Burke et al. (73) renatured the 35-kDa protein from protein gels and recovered transcription activity. This finding is particularly surprising in view of the multisubunit composition reported for TFIIA from HeLa cells (46a, 72), calf thymus (71),and yeast (74).Perhaps in the experiments using renatured protein, one or more TFIIA subunits cross-contaminate other transcription factor preparations used in the system. TFIIA has recently been purified as a heterodimer of 32 and 13.5 kDa from S . cereuisiae (74).The genes encoding each subunit have been cloned. Yeast TFIIA expressed in E . coli is functional in a yeast transcription system


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and can functionally replace human TFIIA in a reconstituted HeLa cell system. Both subunits are necessary for yeast TFIIA activity, and more importantly, the genes encoding both subunits are essential (744. Further complicating the TFIIA picture is a recent report introducing TFIIG as an essential specific initiation factor that can partially be replaced by TFIIA (75). TFIIG, which elutes from P11 with TFIID, is required to reconstitute transcription in the absence of TFIIA; the converse holds true as well. The addition of TFIIG to transcription reactions saturated with TFIIA results in a two- to threefold stimulation, suggesting that the two factors are not functionally equivalent. No polypeptide composition was reported for TFIIG. Recent findings from our laboratory can perhaps reconcile several years of conflicting observations regarding the role and requirement of TFIIA. In the course of extensively purifying HeLa cell TFIIA, Cortes et al. (46a) found that the factor can be separated into two distinct activities, termed TFIIA and TFIIJ. TFIIA purified by yeast TFIID &nity chromatography is composed of three polypeptides of 34, 19, and 14 kDa. TFIIA stimulates basal transcription when native HeLa cell TFIID is used, but is without effect in a system reconstituted with bacterially produced TFIID. A model is proposed in which one of the polypeptides that tightly associates with native TFIID negatively affects TFIID activity. TFIIA is thought either to force the dissociation of this negative component, NC, or to effect an alteration in TFIID conformation such that the influence of NC is removed (Fig. 11). This model is consistent with recent observations (76) regarding a fraction called USA, isolated from the 0.8-1.0 M KCI wash. USA has the following properties: (1) it represses basal transcription (as a result of components named NC1 and NC2) and (2) it enhances MLTF- and Spl-mediated activation (due to another component, PC1). If TFIID and TFIIA fractions are incubated with DNA prior to the addition of USA, then the effects of USA are drastically reduced. What we have described as NC displays the same characteristics as the NC1 component of USA, namely, it is a repressor of basal transcription, the effect of which can be removed by TFIIA. NCl can specifically alter the mobility of the TFIID*DNA complex and is competed for this binding by TFIIA. In our laboratory an activity (called Dr-2) was isolated that may be equivalent to NC1 and to the negative component that copurifies with native TFIID (see also Section VII). TFIIJ, which is separated from TFIIA during hydroxylapatite chromatography, is required for transcription when bacterially produced TFIID is used, but has only a modest effect with native TFIID. The chromatographic behavior of TFIIJ helps to explain its variable requirement. TFIIJ was originally purified from crude TFIIA preparations derived from

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FIG. 11. TFIIA removes a negative component from native TFIID. Model of the possible function of TFIIA in transcription. TBP represents the TATA binding protein, TFIID. (A) Recombinant TBP is devoid of TBP associating factors (TAFs), including the negative cofactor (NC), a component that inhibits TFIID activity. Thus, the activity of recombinant TFIID is high and the association of TFIIA with TBP is without an effect. (B) Native TFIID is shown to be complexed with several TAFs, including NC. In the absence of TFIIA, native TFIID activity is lower than that observed with bacterially produced TFIID. The association ofTFIIA with native TFIID is thought either to induce a conformational change in the TFIID complex or to displace NC directly and, as a result, stimulate TFIID activity.

the 0.1 M KCl phosphocellulose fraction. We have determined that TFIIJ activity can also be recovered from the 0.8-1.0 M KCl phosphocellulose fraction, the same fraction that contains TFIID and TFIIG. Thus, native TFIID preparations are likely already to contain TFIIJ. Complementation analyses with TFIIH indicate that this activity is also present in the TFIIG fraction (51, 75). Thus, TFIIG seems to be a combination of TFIIH and TFIIJ (see Fig. 4). In complex formation, TFIIA can associate with the TFIID-DNA com-


93

plex. Furthermore, TFIIA can associate with all the preinitiation complex intermediates thus far characterized: DB, DB-PolF, DB-PolFE, etc. These observations, together with the finding that TFIIA is not required for transcription when recombinant TBP is used in place of native TFIID, suggest that TFIIA association with the promoter region is not necessary for the formation of a transcription-competent complex. TFIIJ, on the other hand, is required (when TBP is used) and is the last factor to enter into the preinitiation complex. TFIIJ recognizes and associates only with the DB-PolFEH complex, resulting in the formation of the DB-PolFEHJ complex.

G. Other Transcription Systems In addition to the set of HeLa cell transcription factors defined initially by Roeder et al., a number of other systems have been developed. Fractionation of yeast nuclear extracts has resulted in the identification of at least five fractions that are required, in addition to RNAPII, for efficient class two transcription ( 7 6 ~ )These . activities have been designated factors a, b, c, d, and e. Factor d is homologous to TFIID, and factor a may correspond to TFIIE. Factor b has been purified to homogeneity, is comprised of 87, 75and 50-kDa polypeptides (76b), and has recently been shown to contain a kinase specific for the CTD of RNAPII. We believe that factor b is homologous to TFIIH in our system. Factor e is equivalent to human TFIIB (unpublished observations). Further purification of factors a, b, d, and e reveals that factor c is not required for transcription (76b). Seven factors (designated factors 1-7) have been fractionated from Drosophila Kc cell extracts (76c). Of these seven, only one, factor 5, is clearly homologous to a component of our system. Like TFIIF, factor 5 is a heterodimer of comparable size (34 and 86 kDa); it stimulates the elongation rate of RNAPII, and associates with RNAPII in solution (764. A basal transcription system using factors purified from Drosophilu embryos has yielded three main fractions in addition to RNAPII that are required for basal transcription: TFIID, TFIIB, and TFIIE/F (76e). TFIID (40, 40a) and TFIIB (J. Kadonaga, personal communication) have been cloned from Drosophila. Drosophila RNAPII, TFIIB, and TFIIEIF are functional in the context of a HeLa cell reconstituted system. Native DrosophiZa TFIID is a highly complex fraction that, as suggested by the following observation, may contain other basal factors. A reconstituted transcription system in which recombinant TFIID is substituted for native TFIID cannot mediate transcription from the Kruppel gene promoter. This promoter, but not the alcohol dehydrogenase promoter, also requires TFIIZ, an activity that copurifies with TFIID. TFIIA cannot substitute for TFIIZ, and it does not appear to be required in this system (J. Kadonaga, personal communication). A set of five initiation factors, designated a,P-y, 6 , E, and T, have been

94


fractionated from rat liver extracts. Factors a,P-y, and E have been purified to homogeneity. The a activity is contained in a single 35-kDa polypeptide and most closely resembles TFIIB (76f).Factor P-y is a heterodimer of 30 and 70 kDa that shares immunological identity with TFIIF (76g). Factor E is composed of 34- and 58-kDa polypeptides (76h) and may be equivalent to TFIIE. Using a functional transcription assay, T was shown to be the rat liver homologue of TFIID (76h). The exact polypeptide composition of 6 has not been determined. Interestingly, 6 has been reported to contain a closely associated DNA-dependent ATPase activity (76i), the proposed function of which is to participate in activation of the preinitiation complex (see Section IV). Chambon and colleagues and Weissman and colleagues have independently developed HeLa cell-derived systems. The latter group identified a set of six factors, designated FA, FB, FC, FD, FE, and FF (76j).Factor FC seems to be equivalent to TFIIF, based on its polypeptide composition and its ability to associate in solution with RNAPII (76k).Factors FA and FE have been purified to near homogeneity; both are 33-kDa polypeptides. Factor FA may be equivalent to TFIIB (764, however, the identity of the remaining factors with respect to our system is unclear. Similarly, the Chambon group (76rn, 76n)has isolated several factors that are clearly homologous to components of our system, and others that are not as easily reconciled. Their BTFl and STF appear to be equivalent to TFIID and TFIIA, respectively. BTFS has no clear homologue in our system (see also Section 111).We believe that BTFS is equivalent to TFIIH based on polypeptide composition ( 6 9 ~ )BTF4 . seems to be equivalent to TFIIF (76n).

IV. Preinitiation and Initiation Complexes and Motifs A. Complexes on TATA-containing Promoters At least seven different protein factors, in addition to RNAPII, operate via the TATA element to modulate basal transcription: IID, IIA, IIB, IIF, IIE, IIH, and IIJ. Transcription is preceded by the assembly, on promotercontaining DNA, of all the factors in a highly ordered fashion (Fig. 12). Much of what is known about this assembly process has come from template competition studies (28u, 28b), kinetic analyses on the association of factors (28c), and DNA binding assays in which native gels are used to resolve, electrophoretically, complexes formed between labeled promoter-containing DNA and purified GTFs (45, 63). TFIID is the first GTF to associate at the promoter. As described earlier, it is the only factor possessing specific DNA binding activity. DNase-I footprinting studies using cloned human TFIID indicate that, when bound


95

FIG. 12. Preinitiation complex formation on TATA-containing promoters. Model depicts the order of assembly of the GTFs and the transition from initiation to elongation as mediated by a CTD kinase. The formation of the DAB and of DAB-PolF complexes is illustrated in Figs. 6 and 9, respectively. TFIIE, followed by TFIIH, and then TFIIJ, are next to assemble. As also depicted in Fig. 3, this model indicates that the nonphosphorylated IIA form of RNAPII associates with the preinitiation complex. The action of a CTD kinase that converts RNAPIIA to RNAPIIO is thought to be at least one event required to activate the complex. Transcription begins when nucleotides (NTPs) are supplied to an activated complex. The action of a phosphatase that converts RNAPIIO to RNAPIIA is required for RNAPII to reenter the cycle.

at the TATA, TFIID protects a 20-nucleotide region centered around the TATA motif from -36 to -17 on the Ad-MLP (37). Though TFIIA can associate with the TFIID-DNA complex, as well as with all of the subsequently formed intermediates, it is dispensable for transcription, provided that bacterially produced TFIID is used (see Section 111,F). Formation of the DA complex is characterized by an increase in the nucleotide region protected from DNase-I or chemical cleavage. These nucleotides map upstream of the TATA motif with respect to the start site. This footprint is not characterized by any sequence specificity since the binding of TFIIA is mediated entirely through its interaction with TFIID. TFIIB associates with the DAaDNA complex, resulting in the formation of the DAB complex (45, 46). Early observations from the Sharp laboratory (45) using a heterologous system that included native yeast TFIID and mam-

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malian factors indicated that the association of TFIIB with the DA complex resulted in protection of nucleotides around the transcription start site from DNase-I cleavage (Fig. 6). Similar results were obtained using all human factors (46). Interestingly, when recombinant TFIID, either human or yeast, was used to form the DAB complex, it was found that association of TFIIB required sequences downstream of the TATA motif, but resulted in protection only of the TATA motif and not of the transcription start site (46). While this observation has been overlooked by many in the field, it is possible that TFIIB-induced protection over the transcription start site is not due directly to TFIIB, but rather to a factor that exists in association with native human or yeast TFIID. RNAPII is next delivered to the DAB-promoter complex by TFIIF, thereby creating the DAB-PolF complex (63). The studies of Flores et al. (63)demonstrated that the association of RNAPII with the preinitiation complex is strictly dependent on TFIIF. This is in contrast to the findings of Conaway et al. (76g),who proposed that a and Py, the rat liver homologues of TFIIB and TFIIF, respectively, act in combination to promote binding of RNAPII at the TFIID-DNA complex. Consistent with models proposed by Dahrnus and others (19, 78), the IIA (nonphosphorylated) form of RNAPII is more efficiently incorporated into the assembling complex than is the phosphorylated, I10 form (22). The subsequent phosphorylation of the CTD is thought to be a key step in the transition from initiation to elongation. The association of TFIIF with RNAPIIA significantly extends the DNase-I-protected region of the Ad-MLP toward and beyond the transcription start site, from -42 to +17 (63). Gel mobility shift assay has shown that TFIIE, followed by TFIIH, recognizes and associates with DAB-PolF forming the DABSPolFE and DAB-PolFEH complexes, respectively (51, 65). Experiments in which the amounts of TFIIE and TFIIH are varied indicate that these two factors may bind cooperatively. Finally, TFIIJ binds, completing assembly of the transcription-competent preinitiation complex (Fig. 12). Preliminary DNase-I footprinting experiments from our laboratory indicate that the association of TFIIE, TFIIH, and TFIIJ does not significantly extend the protected region beyond what is observed with DAB-PolF (-42 to + 17). Currently, chemical footprinting methodologies are being employed to further characterize the DNA contact regions of these, as well as elongated, complexes. It has long been known that ATP hydrolysis between the P and y phosphates is required for accurate initiation of transcription to occur (79). This hydrolysis seems to occur subsequent to the complete assembly of the initiation complex but prior to the formation of the first phosphodiester bond. The role of ATP hydrolysis is unknown but some of the more popular speculations


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include (1) to phosphorylate one or several of the GTFs such that the complex becomes activated, (2) to facilitate the conversion of RNAPIIA to RNAPIIO, and (3) to provide energy for the formation of the first phosphodiester bond. The events that establish the open complex, a transcription-ready intermediate in which the template DNA has been at least partially unwound, are poorly understood. Once this is established, all that is needed for transcription to ensue is ribonucleoside triphosphates. If nucleotides are present, promoter clearance, the process by which the activated complex beings to move down the DNA in the elongation stage, will follow. It is believed that some of the GTFs remain at the promoter and are available for reinitiation, while others proceed along with the elongating complex and may eventually recycle back to the promoter. Factor u homologies have been found in TFIID, TFIIB, and TFIIF, and may exist in still other GTFs not yet cloned. In eukaryotes, it appears that the multiple roles of the bacterial u factors have been distributed among several of the mammalian GTFs. For example, TFIID has replaced the promoter binding function and IIF has replaced the polymerase delivery function. This dispersion of function allows for the greater regulatory complexity typical of eukaryotic gene expression.

B. Complexes of TATA-less Promoters Functional analyses (28a, 28b, 28c) indicate that the binding of TFIID to the TATA motif is the first step in the formation of a transcription-competent complex and provides a nucleation site for the association of the other GTFs and RNAPII. This has also been confirmed by DNA binding studies (45,46, 28a). It is now known that a large number of class-I1genes contain promoters that lack any recognizable TATA element. Most of these are “housekeeping” genes, genes active in all cells and transcribed at a reduced rate. Though these promoters are generally not as strong as TATA-containing promoters, they can, to a somewhat lesser extent, modulate accurate transcription initiation. In uitro reconstitution experiments using TATA-less promoters indicate that transcription requires all the GTFs, including TFIID, the TATA-binding protein (80-82). The question then becomes, in the absence of a TATA motif-an element known to play an important role in start site positioningwhat provides the entry for RNAPII and the other GTFs, and how is accurate initiation maintained? In an effort to understand what alternative control elements work to direct transcription in such promoters, Smale and Baltimore made deletion mutants in the TATA-less terminal deoxynucleotidyltransferase (TdT) gene promoter and assayed for specific transcription in vivo. They demonstrated that 17 nucleotides surrounding the transcription start site contained the

9a

LEIGH ZAWEL AND DANNY FZEINBERG

information necessary to direct transcription initiation independently (80). This element was termed the initiator (Inr). Recently, many TATA-less promoters have been carefully scrutinized and it appears that Inr elements can be grouped into families based on their nucleotide sequences. The Inr present in the TdT is composed of 11nucleotides, 5‘-GCCCTCATTCT-3’, with the A residue serving as the transcriptional start site. The Inr present in the TdT promoter is similar to that present in the adenovirus-endcoded major late promoter, a TATA sequence containing promoter, and the adenovirus IVa2 promoter, a promoter containing an unusual functional TATA motif located downstream from the transcription start site (the boldface sequences denote nucleotides conserved among these three Inrs) (81). Our studies demonstrate that the Inr present in the Ad-ML and Ad-IVa2 promoters is weakly recognized by RNA polymerase I1 (82). Another class of Inr is that present in the TATA-less promoter of the dihydrofolate reductase (dhfr)gene, which shows no sequence similarity to the TdT-Inr, but does contain a recognition site for a specific DNA binding protein (83,84). A third distinct class of Inr is located within the TATA-less porphobilinogen deaminase (PBGD) promoter. Like the TdT-Inr, the conserved 5’-GxxCTCAxxxT-3’ motif is present in the PBGD promoter, with the A residue representing the transcriptional start site (85). The PBGD-Inr also contains a binding site for a specific DNA binding protein immediately 3’ to the transcriptional start site (+3 to +12). Mutations in the protein binding site or at - 1 and + 1 abolish transcription (85). Interestingly, transcription from the PBGD promoter initiates at a precise site and appears to be independent of TFIID, the TATA binding protein (85). Another distinct class of Inr is that present in the promoter of the ribosomal protein S16 (rpS16) gene. The initiation of transcription in the rpS16 gene is defined by and within a polypyrimidine tract (86). An element distinct from the TATA motif is located in the -30 region, which does not fix + 1 initiation but affects the levels of transcription and can be substituted by a TATA motif (86). Thus, it appears that there are different classes of Inr elements, and that this DNA element is present in all promoters regardless of the presence (AdML) or absence (TdT, dhfi, PBGD, rpS16) of the TATA motif.

C. Insights Regarding Initiation-mediated Initiation The fact that most TATA-less promoters still require all the GTFs, including TFIID, suggests that, with the exception of the TATA-recognition event, the overall initiation mechanism may not be that different from what is thought to occur in TATA-containing promoters. Consistent with this idea, Carcamo et al. (82) demonstrated, using functional transcription assays and gel mobility shift assays, that factors TFIID, TFIIB, TFIIF, and RNAPII are all required for transcription from a TATA-less promoter and can form a


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specific complex on a DNA fragment containing an Inr motif. This complex was specifically competed by an oligonucleotide containing a wild-type TdT Inr motif but not by oligonucleotides containing mutations in the nucleotides conserved among the TdT, MLP, and IVa2 Inrs. The involvement of TFIID was substantiated by the observation that oligonucleotides containing a TATA motif effectively competed DNA-protein complexes founded on Inrcontaining DNA fragments. We have demonstrated that RNAPII weakly but specifically recognizes the Ad-MLP and IVa2 Inrs, and in so doing can provide a foundation for the association of the other GTFs. The conserved nucleotides in the Inr were required for recognition by RNA polymerase I1 (82). By this model, RNAPII provides the entry for the rest of the basal machinery in TATA-less promoters (Fig. 13). In TATA-less promoters, which contain Inrs distinct from that present in Ad-MLP, TdT, and IVa2, a specific factor may bind to the Inr,

FIG.13 Preinitiation complex formation on TATA-less promoters. Schematics compare pathways of preinitiation complex formation on a TATA-containing promoter (left) and a TATAless promoter (right). In the absence of a TATA motif, the initiator (Inr) is thought to direct the formation of the transcription complex. As shown on the right, RNAPII recognizes and binds weakly to the initiator. An Inr-bound RNAPII provides a nucleation site for the association of the other general transcription factors. Subsequent complex assembly stabilizes the association of RNAPII with the initiator. Note that data to support this model have been obtained only for the initiator present in the Ad-MLP, TdT, and IVa2 promoters, which contain the GxxCTCAxxxT motif. TBB, TATA binding protein; NC, negative cofactor.

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which can interact with RNAPII and or one or more of the GTFs. The Inrprotein complex could provide a nucleation site for the association of the other GTFs and RNAPII. Recent studies indicate that, with a construct containing no TATA and multiple S p l sites upstream from the Inr, an additional factor-the tethering factor-is required for optimal Spl-mediated activation. This factor appears to have no role on promoters containing the TATA motif (86~).

D. Cooperation Between TATA and lnitiatior Motifs Various lines of evidence suggest that, when present simultaneously, the TATA and Inr function cooperatively to interact with the transcription machinery to ensure specific initiation. (1) Significantly, double mutants containing base changes in the TATA and Inr are transcriptionally nonfunctional, a drastic effect not observed when either element is singularly mutated (87). (2) When the TATA motif of the Ad-MLP is replaced with random sequence, resultant null mutants exhibit greatly decreased transcription levels but accurate initiation is maintained both in vitro and in vivo (80, 82). We believe that transcription-competent complexes can form on either TATA or Inr motifs, but that, due to steric constraints, there is mutual exclusivity. A complex built solely on an Inr element has only one anchorage point. As a result, it slides, and multiple start sites, though all in the vicinity of the CAP site, are observed. The presence upstream or downstream of a second element recognized by a component of the preinitiation complex (such as the TATA, which is recognized by TFIID, or the Inr, which can be bound by RNAPII), or a site recognized by a specific transcription factor, such as an Inr-binding protein, imparts upon the complex a second anchorage point, greater stability, and the capacity to initiate transcription from a discrete nucleotide.

V. Activation and the General Transcription Factors Transcription of protein-coding genes can be stimulated by a large array of DNA-binding proteins otherwise known as sequence-specific transcription factors or transcriptional activators. The classical transcriptional activator protein is most simply characterized as having two main domains: (1) a sequence-specific DNA-binding domain that recognizes modular sequences occurring in a promoter one, two, three, etc. time(s), depending on the gene, and (2) an activation domain, the portion of the molecule genuinely responsible for stimulating transcription. Activator proteins have been loosely classified into groups based on the properties of their activation domains. For example, the yeast activator Gal4 and herpes simplex virion protein VP16 are thought of as acidic activators because their activation


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domains are rich in aspartate and glutamate residues. Alternatively, the activation domains of Spl and CTF are rich in glutamine and proline residues, respectively. The mechanism by which these proteins stimulate transcription is unclear. Two lines of evidence suggest that the mechanism exploits a theme common to all class-I1 promoters: (1)any one activator can stimulate transcription in a wide variety of different promoters; (2) any one activator functions cooperatively and synergistically in combination with other activators, be they of the same or of a different activator class. Transcription initiation is thought to be the target of these regulatory proteins. The multistep, highly ordered assembly process through which the GTFs and RNAPII associate into a transcription-competent complex provides a multitude of steps that can be regulated. Insight into which steps or, more specifically, which factors are the targets of interactions with upstream activators has come from studies using affinity columns containing immobilized activator molecules such as VP16 or just the activating domain coupled to a carrier such as protein A or glutathione S-transferase (GST). TFIID is bound tightly and selectively by VP16 in this manner (88). Importantly, when control columns containing mutant forms of VP16 were tested, there was a correlation between the strength of the activator (in terms of its potential to stimulate transcription) and its ability to bind TFIID (89). In addition to this work, there is some indirect evidence suggesting that TFIID is a target for activation. The binding of MLTF within the AD-MLP (90) and the binding of Gal4 (91) and the EZV promoter upstream factor (92) to their respective binding sites affects the binding of TFIID to the TATA motif. As discussed in Sections III,A and IV,A, TFIID binding to the TATA motif represents the first step in the formation of the preinitiation complex. This bi,nding, which is slow (28c, 93, 94), may be facilitated by upstream factors such as VP16 and MLTF. In addition to TFIID, TFIIB is selectively bound by the activating region of VP16 (52). A mutation in the activating region of VP16 that reduced the activation potential of the protein but not the overall net negative charge eliminated the interaction with TFIIB, suggesting that the interaction is specific. This specific interaction was also observed with recombinant TFIIB (53).Indirect evidence implicating TFIIB as a target for acidic activators was also obtained using a functional transcription assay in which the DNA template was immobilized on agarose beads (52). In this system, transcription complexes formed on the template are stable to washing, and complete RNA synthesis can occur. Complex formation stalled at the point where TFIIB enters, indicating that TFIIB binding was the rate-limiting step. The inclusion of the synthetic acidic activator Gal4-AH (514 in preincubations, prior to washing, resulted in more efficient binding of TFIIB. It was postulated that Gal4-AH stimulates transcription by accelerating TFIIB recruitment to

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the promoter and perhaps by maintaining the factor there through multiple rounds of initiation. Through independent collaborations with M. Green and J. Greenblatt, we have recently learned that TFIIH can also interact with VP16 (unpublished observations). Thus it seems that transcription activation may happen at many levels, as a result of interactions between an activator molecule and several of the initiation complex components. The fact that an activator may contact several GTFs helps to explain the phenomenon of synergy, whereby two activators stimulate transcription multiplicatively, as opposed to additively. It is important to stress that while specific interactions between an acidic activator and one or more of the GTFs can be demonstrated, this interaction is not necessarily sufficient for activation. For example, it was mentioned earlier that recombinant TFIID proteins cannot participate in activation, yet an interaction between the acidic activation domain of VP16 and recombinant TFIID proteins can be demonstrated. Furthermore, while an interaction between VP16 and three of the GTFs has been observed, addition of Gal4-AH or Ga14-VP16 to a highly purified reconstituted transcription system containing native TFIID resulted in no activation (A. Merino and D. Reinberg, unpublished observations). Ongoing studies have resulted in the isolation of a protein fraction that is necessary, in addition to the GTFs and native TFIID, for activation. The further fractionation of this material has resulted in separation of at least three components, two of which, Dr-1 and Dr-2, interact with TFIID. Surprisingly, addition of these two factors to reconstituted transcription systems results in repression of basal transcription. Thus it is possible that activation of transcription, as defined by in uitro experiments, involves at least two separate processes: (1)the removal of factors that negatively effect transcription, i.e., antirepression (also see Section VI) and (2) true stimulation of transcription. Note that all the studies described here have used only the acidic activators. The mechanisms by which S p l and other activators stimulate transcription may be quite distinct and perhaps provide even greater complexity to transcriptional regulation. The mechanisms driving transcription activation are just starting to be understood. The coming years promise great advances in our understanding of this very important phenomenon.

VI. Repression of Class II Gene Transcription It is now becoming clear that just as a number of diverse mechanisms have evolved to stimulate class I1 gene transcription, a variety of mechanisms also exist that repress it.


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Recently, histone-mediated repression has received a great deal of attention. Histones were long thought to be simple, structural, nonspecific DNAbinding proteins abundant in chromatin. Since most experimental systems utilize purified, “naked DNA, the effects of histones in transcription have traditionally been overlooked. The picture now emerging is one in which the formation of functional transcription complexes on DNA is in direct competition with the assembly of the DNA into nucleosomes (95). This realization alters the way in which we think about activators. Activators are now thought to work at two levels: by contacting one or several of the GTFs and stabilizing or accelerating complex formation, and by binding DNA in the promoter and essentially freeing this from histone-mediated interference. This has been defined as antirepression (96). A very different repression mechanism involves Dr-1, a 19-kDa protein isolated by J. Inostroza and D. Reinberg (unpublished). Dr-1 was isolated from HeLa cell extracts as an activity that can form complexes with TFIID and that, when added to transcription assays, inhibits transcription. Dr inhibits transcription in two ways: (1)by binding to TFIID molecules bound at the TATA motif-TFIID-Dr complexes are not recognized by IIA, IIB, etc. (Fig. 14), and by the ability of (2) Dr to disrupt preformed initiation complexes such as DABePolF. Interestingly, EIa and SV40 large-T antigen can disrupt TFIID-Dr complexes, indicating that Dr function may be tightly regulated, particularly within the context of cellular growth-signaling pathways (V. Kraus, J. Inostroza, J. Nevins, and D. Reinberg, unpublished observations). NC1 (negative cofactor l), which was introduced in Section III,F, represents yet another repressor of basal transcription (76). NC1 consists of one or more polypeptides in the size range of22-28 kDa that can associate with and mod+ the mobility of TFIID-DNA complexes in gel mobility shift assays. It is this interaction with TFIID that is thought to result in down-regulation of basal promoter activity. Increasing concentrations of TFIIA can displace NC1 and compete for TFIID binding. We have isolated a protein fraction, Dr-2, which may be equivalent to NC1. Like NC1, Dr-2 represses transcription by binding to TFIID and its effect can be overcome by TFIIA and or by an acidic activator (A. Merino and D. Reinberg, unpublished observations). As discussed in Section III,F, we suspect that Dr-2 is a negative regulator that associates with native TFIID and that is displaced by the association of TFIIA with TFIID. All the repressors described here inhibit basal transcription through protein-protein interactions. There have been numerous reports of specific gene transcription being repressed as a result of protein-protein interactions. The Id protein is a helix-loop-helix (HLH) protein lacking a basic, DNA-binding region (97). When it dimerizes with other HLH proteins, they

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FIG. 14. Dr is a repressor of basal transcription. Model illustrates the ability of Dr to associate with TFIID molecule bound to the TATA motif. This association prevents the further assembly of the preinitiation complex. Though not indicated here, Dr is also capable of disrupting preformed preinitiation complexes and of associating with TFIID in the absence of DNA.

cannot bind to their recognition sites, which are present in different enhancers, and myogenesis, or Ig gene expression, is inhibited. Another specific inhibitor of transcription is IKB, a factor interacting with NF-KB in the cytosol, which inhibits the translocation of NF-KB to the nucleus, thereby repressing transcription of NF-KB-responsive genes (98-100). This should not imply that protein-protein interaction is the only repression mechanism. Examples exist in which repression is the direct result of a protein binding to DNA. Drosophilu P-element transposase binds to the TATA region of the P-element promoter and blocks association of RNAPII and the


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GTFs (101). Similarly, the homeodomain protein encoded by engruiled inhibits preinitiation complex formation by binding to the TATA box of the Drosophilu Hsp70 as well as other eukaryotic promoters (102). As we move nearer to a transcription system fully reconstituted with cloned proteins, the identification of specific repressors and activators of basal transcription from cell extracts probably will become more routine. Our understanding of how gene expression is regulated will remain incomplete until this family of factors is better defined. ACKNOWLEDGMENTS We thank our colleagues in the transcription field for providing information prior to publication. We extend sincere apologies to those who, due to the enormous amount of material that had to be condensed, we failed to acknowledge. We thank Roberto Weinmann Masayori Inouye, Michael Dahmus, Richard Young, David Arnesti, Robert Tjian, Osvaldo Flores, and Nancy Stone for comments on the manuscript and all members of the Reinberg laboratory for helpful discussions. L.Z. is supported by NIH Training Grant in Molecular and Cellular Biology GM 08360. D.R. is supported by the NIH, the National Science Foundation, the American Cancer Society, the New Jersey Commission on Cancer Research, the Foundation of the University of Medicine and Dentistry of New Jersey, and Robert Wood Johnson Medical School; he is the recipient of an American Cancer Society Faculty Research Award. REFERENCES 1. R. A. Young, ARB 60,689 (1991). 2. C. A. Spencer and M. Groudine, Oncogene 5, 777 (1990). 2a. T. Kerppola and C. M. Kane, FASEB J . 5, 2833 (1991). 3. P. A. Weil, D. S. Luse, J. Segall and R. Roeder, Cell 18, 469 (1979). 4. J. L. Manley, A. Fire, A. Cano, P. Sharp and M. Gefter, PNAS 77, 3855 (1980). 5. L. A. Allison, M. Moyle, M. Shales and C. J. Ingles, Cell 42, 599 (1985). 6. J. Biggs, L. L. Searles and A. Greenleaf, Cell 42, 611 (1985). 7. A. Ruet, A. Sentenac, P. Fromageot, B. Winsor and F. Lacroute, JBC 255, 6450 (1980). 8 . A. M. Edwards, C. M. Kane, R. A. Young and R. D. Kornberg, JBC 266, 71 (1991). 8a. M. Nonet, D. Sweetser and R. Young, Cell 50, 909 (1987). 9 . L. A. Allison, J. K. Wong, D. Fitzpatrick, M. Moyle and J. C. Ingles, MCB 8,321 (1988). 9a. M. S. Bartolomei, N. F. Halden, C. R. Cullen and J. L. Corden, MCBiol8, 330 (1988). 9b. W. A. Zehring, J. M. Lee, J. R. Weeks, R. S. Jokerst and A. L. Greenleaf, PNAS 85,3698 (1988). 10. L. A. Allison and C. J. Ingles, PNAS 86, 2794 (1989). 10a. A. Usheva, E. Maldonado, A. Goldring, H. Lu, C. Honbavi, D. Reinberg and Y. Aloni, Cell 69, 871 (1992). 1 1 . W. A. Zehring and A. Greenleaf, JBC 265, 8351 (1990). 12. S. Buratowski and P. A. Sharp, MCBiol 10, 5562 (1990). 13. W. Y. Kim and M. E. Dahmus, JBC 264, 3169 (1989). 14. N. E. Thompson, T. H. Steinberg, D. A. Aronson and R. R. Burgess, JBC 264, 11511 (1989). 14a. C. L. Peterson, W. Kruger and I. Herskowitz, Cell 64, 1135 (1991). 15. M. Suzuki, Nature 344, 562 (1990). 16. J. L. Corden, Trends Biochem. Sci 15, 383 (1990).

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Regulation of Repair of Alkylation Damage in Mammalian Genomes SANKARMITRA*,~AND BEFWD K A I N A ~ *Biology Division Oak Ridge National Laboratory Oak Ridge, Tennessee 37831 fznstitut fur Genetik und Toxikologie Kemforschungszentnm, Karlsruhe 0-7500 Karlsruhe, Germany I. 11. 111. IV. V.

Historical Perspective ........................................... Unusual Repair of 06-Alkylguanine ............. Multistep Repair of N-Alkylpurines ................................ Properties of Mammalian 06-Methylguanine-DNA Methyltransferases . . Cloning of Mammalian Alkylation Repair Genes by Phenotypic Rescue

109 112 114 116

................................................... .............

118 120 128 129 132 135 136 137

Mammalian MGMT and MPG

1. Historical Perspective DNA repair is a universal and ubiquitous process that is essential for survival. Lethal and mutagenic damages in DNA result not only from exposure to external chemical and physical agents but also from spontaneous chemical reactions, in particular, deamination of cytosine (and S-methylcytosine) to uracil (and thymine) and spontaneous loss of purines (1).Such alterations, if left unrepaired, would result in C * T transition mutations and in apurinic/apyrimidinc (AP)2 sites, which usually block replication (2-4). 1 To whom correspondence may be addressed. Present address: Sealy Center for Molecular Science, Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch H-52, Galveston, Texas 77555. 2 Abbreviations: Ada, product of Escherichia coli adu gene regulating adaptive response; A M , product of E. coli alkA gene, a component of ada regulon; AP, apurinic/apyrimidinic CHO, Chinese hamster ovary; CNU, N sites; BCNU, 1,3-bis(2-chloroethyl)-l-nitrosourea; chloroethyl-N-nitrosourea; CREB, CAMP response element binding protein; DM, double minute chromosome; DMS, dimethyl sulfate; DHFR, dihydrofolate reductase; DTIC, dacarba-


Copyright 0 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.

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SANKAR MITRA AND BERND KAINA

A wide variety of damages can be induced in DNA in uiuo by physical agents, such as ultraviolet light and ionizing radiation, and by a plethora of chemical agents, some of which are as simple in structure as vinyl chloride or ethylene oxide, or as complex as the polynuclear aromatic hydrocarbons. With all chemical mutagens, the reactive species is an electrophile that attacks various nucleophilic targets in the bases and phosphates of DNA (5, 6). In most cases, the chemical agent is not itself reactive, but is activated via oxidative metabolism in detoxification pathways (7). Simple alkylating agents, such as N-alkylnitrosamines, include many known carcinogens, mutagens, and toxic agents. Some of them, e.g., methyl methanesulfonate (MMS), alkylate directly in an S,2 reaction. Others, particularly those requiring metabolic activation, e.g., N-alkylnitrosamines, generate a reactive alkylcarbonium ion as the intermediate (6). The hallmark of these simple aliphatic chain-containing agents is their reaction with a number of sites in DNA. The distribution of the adducts at various sites depends not only on the chemical structure of the alkylating agent but also on the alkyl group. For example, ethyl methanesulfonate (EMS) alkylates oxygen sites at a much higher proportion than its congener, methyl methanesulfonate (8). Most simple alkyl adducts of DNA bases, unlike the bulky adducts, do not block DNA replication absolutely. However, they are often toxic and/or mutagenic (9). Since the original proposal of Loveless more than two decades ago, that among these adducts 06-alkylguanine is the critical mutagenic and carcinogenic lesion (lo),a variety of in uiuo and in uitro experiments have shown conclusively that 06-alkylguanine is indeed the primary mutagenic lesion (11-14). Organotropism studies of carcinogenesis induced by N-alkylnitrosamides, e.g., N-methyl-N-nitrosourea (MNU), and N-alkylnitrosamines also suggest a causal link of 06-alkylguanine and tumor Recent experiments on mammary tumor induction induction in rats (15,16). by MNU in rats causing activation of the Ha-ras oncogene are consistent with the notion that tumorigenesis resulting from G + A transition mutations in the oncogene is due to the formation of 06-alkylguanine, which zine; ERCC, human gene correcting excision repair defect by cross complementation; HeCNU, N-hydroxyethyl-N-chloroethyl-nitrosourea;HPRT, hypoxanthine phosphoribosyl phosphotransferase; HSR, homogeneous staining region in a metaphase chromosome; MAG, 3-methyladenine-DNA glycosylase; MDR, multiple drug resistance; EMS, ethyl methanesulfonate; Mer- , Mex- , 06-methylguanine repair defective; MGMT, 06-methylguanine-DNA methyltransferase; MMS, methyl methanesulfonate; MNNG, N-methyl-N’-nitro-N-nitrosoguanidine; MNU, N-methyl-N-nitrosourea; MPG, N-methylpurine-DNA glycosylase (same as MAG for eukaryotes);Ogt, product of E . coli ogt gene for constitutive repair of 06-alkylguanine damage; PCR, polymerase chain reaction; RFLP, restriction fragment-length polymorphism; Tag, 3-methyladenine-DNA glycosylase (product of E . coli tug gene). UDS, unscheduled DNA synthesis, due to excision repair.

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behaves like adenine during DNA replication (14). While the N-alkylpurines have not been shown experimentally to be mutagenic, they can be indirectly mutagenic because their removal, either in spontaneous chemical reaction or during excision repair, results in the formation of AP sites. As already mentioned, AP sites normally prevent DNA replication. However, under special circumstances they can also lead to mutations (17). Even though the N-alkylpurines are not directly mutagenic, they are toxic as indicated by the fact that agents such as MMS and dimethylsulfate (DMS), which induce mostly N-methylpurines and methylphosphotriesters and only minute amounts of 06-methylguanine compared to N-nitrosamines and N-nitrosoamides, are quite cytotoxic in cultured cells, and it is believed that the methyl adducts, particularly N-methylpurines, contribute significantly to the overall toxicity (18-20). Hypersensitivity of N-methylpurine repair-deficient mutant strains of Escherichia coli to alkylating agents has directly confirmed the cytotoxic nature of 3-methyladenine in DNA (21, 22). N-Alkylpurines in DNA may also contribute to other biological effects, especially induction of chromosomal aberration. There is a correlation between N-alkylation level and clastogenic efficiency of various alkylating agents (23). Studies with mice exposed to MMS suggest that N-methylpurines in DNA cause both dominant lethal and somatic mutations as well as reciprocal heritable translocations in a stage-specific fashion during germ cell differentiation and embryonic development (24-26). Exposure to DMS and MMS caused induction of tumors of the central nervous system. Such data strongly argue for a carcinogenic role, direct or indirect, of the N alkylpurines in DNA (18, 27). N-Methylpurines in general, and 7-methylguanine in particular, have been implicated in the aging process on the basis of circumstantial evidence (28).Excision repair in response to exposure to alkylating agents is deficient in older animals (29, 30). At the same time, 7-methylguanine appears to accumulate in the DNA of aging mice (31). At the teleological level, the evolution of repair systems for alkyl adducts may be related to the fact that even in the absence of exposure to external alkylating agents, spontaneous methylation of various nucleophilic sites in DNA may occur in vivo by nonenzymatic reaction with S-adenosylmethionine (32, 33). This review is not intended to be a comprehensive compilation of the findings embodied in the vast literature on alkylation damage and DNA repair. Such information can be found in several recent reviews (21, 34-36). Our focus is a review of our current understanding of the molecular basis of the regulation of alkylation damage repair in mammals. Such molecular studies were not possible until the recent success in the cloning of the

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alkylation repair genes. By way of background, we first review the basic mechanisms of repair proteins.

II. Unusual Repair of 06-Alkylguanine Similar mechanisms are operative for the repair of alkyl adducts in the DNAs of all organisms investigated. “Excision repair” is the general mechanism of removal of DNA damage in which a segment of a DNA strand, including the damage, is enzymatically removed. The integrity of the DNA is subsequently restored by de novo synthesis in the gapped duplex (37). In the case of 06-alkylguanine, an unusual repair mechanism exists in all organisms. First discovered in E. coli in 1980, the repair process involves a direct stoichiometric transfer of the alkyl group from the guanine adduct to the repair protein, resulting in its inactivation (38, 39). The amino-acid residue accepting the alkyl group is a unique cysteine (40). The S-alkylcysteine produced is stable, and the repair protein does not regenerate. Thus this is the only repair protein that, while not a true enzyme, restores the integrity of DNA in a single-step reaction. While the protein has been variously named by different workers, its commonly accepted name is 06-methylguanine-DNA methyltransferase (EC 2.1.1.63),abbreviated by us as MGMT (21,41).MGMT has been accepted by the Human Gene Mapping Nomenclature Committee as the formal name of the human gene and the protein (41).The bacterial and yeast methyltransferase genes and polypeptides have been given distinct names (21).In this review, we use the term MGMT as a generic name for all 06-methylguanine-DNA methyltransferases. MGMT activity has been detected in all organisms tested so far (35, 4244). Even though the overall sequences of various MGMTs are rather different, the sequence surrounding the alkyl acceptor cysteine residue is highly conserved (21,41,45,46)(Fig. 1).The reason for the evolutionary conservation of this unique repair protein is not clear. It is possible that among all mutagenic lesions in DNA, 06-alkylguanine holds a unique position in that even though it does impede DNA polymerase (12), it does not act as an absolute replication block. Additionally, its invariable tendency to base-pair with thymine makes it imperative to repair this lesion in the fastest possible way prior to replication (47). The other well-characterized adduct induced by alkylating mutagen and known to be directly mutagenic is 04-alkylthymine (48, 49). However, it is induced to a much smaller extent than 06-alkylguanine (48). Furthermore, the 04-alkylthymines are also substrates, although poor ones, for the E. coli MGMTs (Ada and Ogt proteins) (21,45, 50-52). The situation is not as clear for the mammalian system. The human MGMT appears to act on 04methylthymine in DNA, but at an extremely slow rate (51, 53),and it does

Mouse MGMT

I S Y Q Q L A A L A G N P K A A R A V G G A M R S N P V P I L I P C H R V V R S D G A I G I H

n

R a t MGMT

S Y Q Q L A A L A G N P K A A R A V C G A M R S N P V P I L I P C H R V I R S D G A I G N

Human MGMT

S Y Q Q L A A L A G N P K A A R A V G G A M R G N P V P I

E. coli Ada

E. coli

Ogt

6. subtilis D a t - 1

n

V V P C H R’V I G R F V P C H R V I G K N S A L T G

-

FIG. 1. Sequence homology among MGMTs of mammalian and bacterial origin around the alkyl acceptor site. The boxes enclose regions with identical sequences. [With permission from Shiota et al. (175) and the ACS.]

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not appear to act on 04-ethylthymine at all (54).An alternative pathway for the repair of 04-alkylthymine in DNA in mammalian cells has been detected but not characterized (55). It is not surprising that MGMT accepts higher alkyl groups from 0 6 alkylguanine, but the finding that E. coli and mammalian MGMTs show different rates of reaction with different alkyl residues was unexpected. Bulky base adducts, irrespective of their chemical nature, distort the DNA helix and are removed by the excision repair pathway. The excision repair systems remove 06-alkylguanine, especially if large alkyl groups are involved (56). Such results are consistent with the observation that the major MGMT in E. coli (Ada protein) reacts poorly with 06-ethylguanine and is nearly inactive with propyl and butyl derivatives (57, 58), with rates of 10.1% that for 06-methylguanine. In contrast, mammalian MGMT reacts with ethyl to butyl derivatives of guanine at a significant rate (57, 59). For example, rat MGMT removes 06-ethylguanine from DNA at about onethird the rate of removal of 06-methylguanine (57). While the repair mechanism of large alkyl adducts of guanine has not been studied in mammalian cells, the fact that the loss of 06-methylguanine does not occur in vivo in MGMT-negative (Mex-) cells indicates that the excision repair pathway does not contribute significantly to the repair of 06-methylguanine in mammalian cells. In any case, it is tempting to speculate that evolution of repair pathways is coordinated so that the 06-alkylguanine adducts that act as replication blocks are repaired by a more complex mechanism, in contrast to 06-methylguanine, which warrants a more immediate repair.

111. Multistep Repair of N-Alkylpurines In contrast to 06-alkylguanine, N-alkylpurines, the other major adducts induced by simple alkylating agents, are repaired in multiple steps. These involve removal of the bases by DNA glycosylases followed by excision repair of the resulting AP sites. With the exception of 06-alkylguanine (and 04-alkylthymine), the repair of abnormal bases or bases modified with small adducts occurs via specific glycosyl bond cleavage of the base, and appears to follow a common pathway in all organisms (2). Many such DNA glycosylases have been identified and characterized. N-Alkylpurine-specific glycosylases are also ubiquitous. Escherichia coli AlkA protein, the major glycosylase for N-alkylpurines, also utilizes 02-methylpyrimidines as substrates (60). Repair of these pyrimidine adducts in mammalian systems has not been investigated. In contrast to the situation with 06-alkylguanine, the N-methylpurine-DNA glycosylases have not been extensively studied. We have adopted the abbreviation MPG for this class of enzymes, which has been accepted by the


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Human Gene Mapping Nomenclature Committee. The DNA glycosylases, unlike MGMT, are true enzymes; they act catalytically. The MPGs from a number of mammalian sources have been purified to various degrees, and so far, unlike E. coli, mammals appear to contain a single MPG. Interestingly, mammalian MPG behaves more like the inducible E. coli AlkA protein (3-methyladenine-DNA glycosylase 11) rather than the constitutive Tag protein (3-methyladenine-DNA glycosylase I) (61,62). The protein has a broader substrate range (including 7-alkylguanine and 3alkylguanine in addition to 3-alkyladenine) than the E. coli Tag protein, which is specific for 3-alkyladenine in DNA (21). The enzymological parameters of the MPGs have not been studied. Thus it is not proved that the same MPG is expressed in different tissues or cultured cell lines of an organism. There are also significant discrepancies in the molecular weight values reported in the literature (62). Recently, cDNAs and coding sequences of the MPG gene from human, rat, and yeast have been isolated (63-66). Based on the deduced amino-acid sequence, as expected, the mammalian proteins share a significant homology. However, these proteins are quite different from the two E. coli MPGs and the yeast enzyme (MAG protein) (Fig. 2). The yeast MAG polypeptide on the other hand, does share a significant proportion of conserved aminoThus the situation with MPG acid sequences of the E. coli AlkA protein (64). is in sharp contrast to the highly conserved active-site region of MGMT. Similarly conserved sequences in MPG could have suggested the locations of their active sites, which have not been identified as yet. However, as we discussed in a recent paper, the glutamine and arginine residues that may be involved in the recognition of adenine and guanine, respectively, are conserved in these proteins (Fig. 2). The arginine is not conserved in the E. coli Tag protein, which does not recognize the N-alkylguanines as substrates. The AP endonucleases play a central role in DNA repair because AP sites are produced not only by the action of DNA glycosylases, but also by spontaneous loss of purines as mentioned above. The excision repair of noninstructional AP sites involves cleavage of phosphodiester bonds on either

E. sol1 Tag Yeast XPC

......so> K K R E N Y R A c P x Q F D . P v K v A A n q E E D v < 77.. . __.... 82> Y F I R L A S T I L S Q Q I S C Q . . A A E S I K A m < l O 8 ...

FIG. 2. Sequence comparison among MPGs of mammalian, E . coli, and yeast cells. The numbers represent the positions of the amino-acid residues preceding or following the sequences shown. The boxes represent sequence identity. [With permission from Chakravarti et al. (@).I

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side of the AP site by AP endonucleases (AP lyases types I and II), excision of the damaged sites by exonucleases, and resynthesis of the gapped region, using the complementary DNA strand as the template. Commensurate with its importance in maintenance of DNA integrity, multiple AP endonucleases are present in all organisms (62,67-69). The E. coli AP endonucleases-one major (Ex0111protein) and several minor species-have been characterized; some contain associated and specific DNA glycosylase activities as well (6872). Only a few mammalian AP endonucleases have been identified, and fewer human enzymes have been purified to homogeneity and studied in detail (73, 74). The excision repair of AP sites in DNA involves a number of other proteins, including DNA replication complexes; these have not been characterized in mammalian systems. Nevertheless, it is reasonable to state that MGMT, MPG, and AP endonucleases are the key proteins involved in repair of alkylation damage in DNA. However, this review is confined to MGMT and MPG. AP endonucleases have recently been reviewed elsewhere (67-69).

IV. Properties of Mammalian 06-Methylguanine-DNA Methyltransferases Until the recent cloning of cDNAs (41, 65, 66), which allows purification of significant quantities of the plasmid-encoded mammalian proteins from E . coli cells, highly purified mammalian MGMT and MPG were not available for biochemical investigation. In addition to the low concentration of these proteins in mammalian tissues, purification procedures of the human MGMT in general led to extensive loss of material apparently due to “stickiness” of the protein (75). MPG had been extensively purified from several mammalian cell lines and tissues, but the final preparations were far from homogeneous (61, 62). Some biochemical studies have been carried out with partially purified human and rodent MGMTs. One of the unresolved questions about the MGMT reaction is the fate of the alkylated protein after alkyl group transfer. It is reasonable to suggest that the alkylated and active MGMT may have significantly different conformations and thus an altered affinity for DNA. Thus the early observation (76), that the methylated MGMT has a much larger Stokes radius than the unmethylated MGMT, was consistent with this idea of an alkylation-induced change in conformation of the polypeptide. However, we could not confirm this observation in our more recent series of experiments (75, 77). On the other hand, we did find evidence for a subtle change in polypeptide conformation as indicated by a change in isoelectric point following methylation (75). The issue of how surveillance by repair proteins for the presence of

ALKYLATION DAMAGE REPAIR I N MAMMALIAN GENOMES

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lesions in the large mammalian genome occurs is not clear. The repair proteins, like other DNA-specific proteins (e.g., repressors), may have two components in their mode of recognition for DNA, one for a nonspecific lowaffinity binding, and the other for a high-affinity binding for specific recognition (78). As expected, MGMT was found to be chromatin-bound (77, 79). Although a nuclear location of other repair proteins has not been uniformly demonstrated, all of the characterized repair proteins have varying degrees of affinity for DNA (2, 74, 75).We observed, by affinity chromatography on DNA-cellulose, that the non specific DNA binding of human MGMT was unaffected by the methylation status of the protein (75). However, the importance of the 06-alkylguanine-specificbinding was uncertain because the rate of the MGMT reaction was inversely proportional to the amount of DNA, either in the single-stranded or the duplex form (75). RNA is not as good a competitive inhibitor as is DNA (75). These results predict that 06-alkylguanine repair in the presence of a vast excess of unaltered DNA may be far less efficient than its repair in uitro when a limited amount of DNA is present. However, we would need to reinvestigate such inhibition of MGMT when DNA is present in the form of chromatin before drawing a definite conclusion. The other unusual property of the human (and presumably all mammalian) MGMT is inhibition by salt. While similar studies have not been carried out with the bacterial MGMTs, the human MGMT reacts in physiological ionic strength (0.2 M) at about one-fifth the rate of that in the absence of added salt (10 mM ionic strength of the buffer). The inhibition is not ionspecific and may be related to the reduced affinity for DNA in higher ionic strength (75, 80, 81). Nonetheless, it appears that 06-alkylguanine repair should be significantly slower in uivo than the observed rate in uitro. Even though the primary sequence of all MGMTs characterized so far surrounding the alkyl acceptor cysteine residue is remarkably conserved (Fig. I), their reaction rates can be widely different, in addition to the differences in their substrate preferences, as already discussed. We have determined that the second-order rate constant of the E. coli Ada protein is at least 103 times that of partially purified human MGMT (75, 82). While it should eventually be possible to explain such differences by the structural differences of these polypeptides, it makes sense that the bacterial protein should have a much higher reaction rate, because the shorter interval between DNA replication cycles warrants rapid repair of mutagenic lesions. It should also be noted in this context that the number of MGMT molecules in mammalian tissues is in the range of 3 X 104 to 20 X 104 per cell (35, 83, 84) compared to 10 molecules or less per cell in uninduced E. coli (85, 86). Because the mammalian genome is more than lo00 times the size of the E. coli genome, and because the mammalian cells have about 104 times the

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MGMT of E. cob, the slower reaction rate of the former may be partially offset by its larger amount.

P

V. Clonin of Mammalian Alkylation Re air Genes %y Phenotypic Rescue of E. co i Several strategies have been routinely used for isolation of cDNAs and genomic clones of mammalian genes. The simplest approach would be to screen mammalian cDNA libraries with the analogous cloned genes of E. coli as probes. However, it appears unlikely that a sequence homology at the nucleotide level exists in the bacterial and mammalian genes over a stretch of 15-20 nucleotides to make it unique even in the most conserved regions of polypeptides. This is due to a large difference in the bias of codon usage between mammalian and bacterial genes (87). In fact, it did turn out that the nucleotide sequences encoding the absolutely conserved pentapeptide ProCys-His-Arg-Val are different in different organisms (Fig. 1). Alternative procedures for cloning genes include screening cDNA or expression libraries with oligonucleotides or antibodies as probes (88). In both cases, nearly homogeneous protein (at least as pure as that present in a polyacrylamide gel band) is necessary before the oligonucleotide sequence (inferred from the peptide sequence) can be deduced or an antibody produced. The approach utilized successfully elsewhere in cloning several human DNA repair genes (ERCC genes) was to transfect repair-deficient CHO cells with human DNA, isolate repair-proficient transfectants, and then retrieve the human DNA containing the repair gene from the transfectants (89). Early attempts to clone the human MGMT gene were based on this approach because Mex- CHO cells could readily be made Mex+ with human DNA (90-92). However, such efforts uniformly failed because of two limitations of the approach; the possibility of activation of endogenous hamster gene in Mex+ cells could not be eliminated, and the method can be successful only if the mammalian repair gene is smaller than the DNA fragments used for transfection. It turned out, as elaborated below, that the human MGMT gene (>150 kb) is much larger than the DNA fragments used for transfection. Thus the MGMT activity in all Mex+ Chinese hamster ovary cells were encoded by the activated endogenous gene as shown directly by the lack of human MGMT gene sequences in these cells (93). In our continuing efforts to clone human MGMT cDNA, we tried other, more involved procedures, such as differential screening of cDNA libraries of Mex+ and Mex- human cells. In principle, this technique should succeed if the differential expression of only a few genes, including MGMT, occurs in the selected pair of cells. The identity of MGMT cDNA among the


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putative clones could be established subsequently by more extensive screening of a variety of Mex- cells and by isolating Mex+ clones from them after transfection with an expression vector derived from the cloned cDNA. In practice, construction and screening of subtraction libraries are difficult. We and at least one other group failed to isolate MGMT cDNA clones after expending a significant amount of effort (K. Tano, R. S. Foote, and M. Mitra, unpublished experiments; A. Fornace, personal communication). Another potential approach for cloning the mammalian MGMT genes (cDNA) is based on transient complementation of Mex- cells with microinjected mRNA transcribed from a cDNA expression library (94). However, this procedure is extremely labor-intensive. We finally entertained the simple idea, based on our past results about the comparative properties of human and E. coli MGMTs, that the human protein expressed in E. coli may be active and thus that a&- E. coli may become resistant to an alkylating agent as a result of this expression. This idea was supported by the earlier complementary experiment that the E. coli Ada protein confers resistance to alkylating agents on mammalian cells (95-96). The concept of phenotypic rescue is not new and had been used in the past for cloning eukaryotic genes in bacteria (97). However, the genes cloned earlier are involved in intermediary metabolism. We and L. Samson's group were the first to exploit this strategy for cloning eukaryotic DNA repair genes (41,63). The limitation of this strategy should also be obvious. It is unlikely that mammalian repair proteins that function in oligomeric protein complexes would be functional in E. coli. Furthermore, appropriate mutants of E. coli must be available for cloning. Finally, a simple way to challenge the cells in order to eliminate the repair-defective mutant cells should be feasible. The strategy of cloning of DNA repair genes (and their cDNAs) by positive selection is extremely powerful. However, it is important to note two parameters that affect such selection. First, the discrimination in killing of repair-positive and repair-negative E. coli may vary widely for different repair genes and genotoxic agents. Second, the mammalian repair proteins expressed in E. coli may not fully confer the wild-type phenotype. We postulated that the screening strategy should be effective even with a lower ratio of discrimination between the control mutant of E. coli and the repair protein-positive cells if' a suboptimal condition of selection is used. While this will undoubtedly lead to an increase in the level of background noise, a significant enrichment of the desired clones could be achieved by cyclic exposure to an appropriate genotoxic agent and enrichment of the plasmid by cyclic transformation. We exploited this approach in cloning human MGMT and human MPG cDNAs from a HeLa cell cDNA expression library (41, 66). Phenotypic

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screening was subsequently used elsewhere for cloning cDNAs of rat MGMT and MPG (65,98). Human MGMT cDNA has been cloned independently by others using other strategies (53,99).

VI. Regulation of Mammalian MGMT and MPG In view of the fact that persistence of alkyl adducts in DNA leads to severely adverse biological consequences, it is surprising that the repair activity, at least for alkylation damage, is highly regulated. The regulation of MGMT has an important practical implication in the therapeutic effectiveness of the alkylating drugs, such as procarbazine, DTIC, and derivatives of CNU (e.g., Carmustine), which exert their cytotoxic action via formation of 06-alkylguanine derivatives (20). There is an excellent correlation between the development of resistance to these drugs and increased levels of MGMT in tumors of brain and other organs (100-103). In a few cases where such correlation was not observed, it appears likely that the critical cytotoxic lesions may be N-alkylpurines, and the drug resistance may be due to elevated levels of MPG and AP endonucleases involved in repair of these lesions (104). Regulation of enzyme activity can occur at various levels. While the regulation at the gene level, i.e., at the level of transcription without any other concomitant change, may be the common mechanism of controlling enzyme activity, other more complex ways (e.g., change in the stability of mRNA, the half-life of the active protein, and translational control) have been shown to be operative for some well-regulated proteins (105, 106).The molecular basis of regulation of DNA repair proteins in general, and of MGMT and MPG in particular, have not been extensively studied. In the limited experiments carried out so far, MGMT appears to be regulated exclusively at the level of transcription (41, 107, 108, 168). In one extensive study with a number of human brain tumor lines, a good correlation was observed among the MGMT activity, the amount of MGMT polypeptide measured immunochemically, and the amount of MGMT message (103). Even fewer data are available for mammalian MPG. However, we observed recently that cells of rat origin have a much lower level of stable MPG message than do human and mouse cells, even though all of these cells have comparable MPG activity (66). Thus it appears that the regulation of rat MPG activity occurs at a level other than transcription of the gene.

A. Tissue-Specific Level of Expression Ever since the proposal that 06-alkylguanine, in spite of its being a minor alkyl adduct, is the critical mutagenic and carcinogenic lesion (lo),extensive studies of its removal from rodent tissues have been carried out. After the


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observation that the adduct persists to a much higher extent in brain than in liver, with an intermediate level in the kidney, of rats exposed to N-alkylnitrosamines, and with the subsequent discovej of MGMT, a correlation of MGMT activity and the rate of removal of 06-alkylguanine was documented (92, 109, 110). Because it is much easier to quantitate MGMT activity than the 06-alkylguanine adduct in DNA, the levels of MGMT in different tissues of various eukaryotes have been assayed (42, 44,111,112). The liver has uniformly been shown to contain the highest, and brain and lymphocytes the lowest, activity (0.2-0.01 that in the liver). We were surprised to find that the MGMT activity in mouse ovary is as high as that in mouse liver. The methyltransferaseactivity in other organs also vaned over a range of three- to fivefold (113). The significance of tissue-specific variation in MGMT level is not clear. One possible explanation is that liver DNA may be the target for the highest level of adduct formation, because the active species for alkylation are often produced by P-450-mediated metabolic activation of alkylating mutagens and carcinogens (114).Furthermore, the need for repair may be rather low in brain because the adduct formation in this organ is low, probably due to inefficient transport of the alkylating species across the blood/brain barrier, and because the alkyl adduct may be tolerated in nondividing cells. On the basis of this hypothesis, we expected that the highest level of other alkylation repair proteins would be present in liver as well. It was therefore extremely surprising to observe that, in mouse, the MPG level is the highest in stomach, higher than that in the liver, and that the brain has also a high level of activity (113, 115). It appears that a better understanding of the tissuespecific levels of DNA repair proteins can be achieved only after a comprehensive study of the variety of DNA repair pathways is completed.

B. Age-Dependent Modulation of the Methyltransferase (MGMT) and Glycosylase (MPG) Activities Many investigators have proposed a linkage between aging in mammals and accumulation of lesions in DNA or their misrepair (28,30). As discussed, while some of these lesions could be induced by exposure to environmental agents, others, including alkyl adducts, may arise from spontaneous endogenous chemical reaction. 7-Methylguanine accumulates in older mice that have not been deliberately exposed to alkylating agents (31). The observation that excision repair, measured as “unscheduled DNA synthesis” (UDS), needed for the elimination of N-methylpurines was significantly reduced in mice exposed to MNU is indicative of inefficient repair of N-methylpurines from the DNA of older animals (29). Thus these results support the possibility of accumulation of 7-methylguanine, although its half-life in DNA due to spontaneous release is approximately 150 h.

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We measured MPG activity in several organs of two inbred stocks of mice of three different age ranges. The enzyme activity of cell-free extracts was calculated as units per microgram of total DNA in the extracts and therefore represents relative activity per diploid cell. It was surprising to observe that the MPG activity was lower both in suckling animals and in mature adults than in young adults. The same trend was observed in all four organs tested, namely, liver, lung, brain, and ovary, and in both strains of mice (113). However, we cannot conclude from these results alone that N-alkylpurines will accumulate in the DNA of older animals without exposure to alkylating agents, because even the reduced glycosylase MPG activity may be more than enough to remove the small amount of N-methylpurines (e.g., 7-methylguanine) spontaneously induced. Nevertheless, it appears reasonable that a systematic study should be undertaken to determine the level of alkyl adducts in several organs of a test animal as a function of age following a chronic low level of exposure to alkylating agents. The methyltransferase activity is lower in human and rodent fetal tissues than in adults (116-118), but no systematic measurement of MGMT in human tissues as a function of age has been carried out. Again, our results show that suckling mice have lower MGMT activity than young adults, analogous to the situation with MPG. However, in contrast to MPG, MGMT activity is not lower in mature adults than in young adults. Finally, whether the age-dependent changes in MGMT and MPG activities are a reflection of the transcription rates of their genes, or result from altered stability of their mRNAs and their translation, is not known. While the significance of these results in regard to the change in level of alkylation repair in aging is not clear, it is obvious that the MGMT and MPG activities may not be coordinately controlled in viuo in mammals. The situation in E. coli is very different, in that the inducible repair of all alkylation damages is under a single control (21,119).

C. Cell-Cycle-Specific Regulation of the Methyltransferase Many genes, e.g., those encoding chromosomal DNA replication, are regulated in a cell-cycle-dependent fashion in mammalian cells. Thus, the expression of DNA polymerases ci and 6, dNTP synthesizing enzymes, and proliferating cell nuclear antigen (PCNA) is activated just prior to or early in the S phase (120, 121). Cell-cycle dependence of the repair of DNA adducts is also critical, because the lack of repair prior to DNA replication will lead either to replication block or to mutation due to misreplication. Both the activity and the mRNA level of uracil-DNA glycosylase (the enzyme that removes uracil generated in DNA due to deamination of cytosine) is higher in proliferating cells than in resting cells, presumably beaause of the need to remove the mutagenic lesion prior to DNA replication (122). By the same token, we expected the transferase activity to be the greatest


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in cells prior to the S phase. It was therefore surprising that, in a mouse cell line parasynchronized by serum starvation, MGMT activity appeared to be minimal 6-8 h before the onset of S phase (123). These results were later verified at the level of transcription of the MGMT gene in diploid human fibroblasts. In these cells a relatively high level of MGMT mRNA [compared to the amount of 3-phosphoglyceraldehyde dehydrogenase (GAPDH) mRNA measured as a control] was observed in the Go phase after serum starvation. Following supplementation of serum, the MGMT mRNA level declined (relative to GAPDH and p-actin mRNA) with a minimum just prior to the S phase (Fig. 3) (124). This is in contrast to some other enzymes of DNA

C

.-0

1.0

v)

v)

E

:a:

0.8

a z K

0.6

E

5 I

0.4 eMGMTIGAPDH O M G M T l a c t in

FIG.3. Cell-cycle-dependent modulation of expression of MGMT mRNA in a human fibroblast line. Time 0 represents the addition of serum to serum-starved GMlO cells. The upper graph shows the kinetics of thymidine incorporation in DNA. The lower graph shows the level of MGMT mRNA normalized to the level of glyceraldehyde-phosphatedehydrogenase (GAPDH) or p-actin.

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metabolism that are present at a very low level in resting (Go) cells (120, 121). The significance of the unexpected cell-cycle-dependent fluctuation of the transferase level is not clear. Because little is known about the cell cycle dependence of the glycosylase and other repair proteins in mammalian cells, it is not possible to draw any general conclusion about this phenomenon.

D. Epigenetic Regulation of the Methyltransferase in Cell Lines and Human/Rodent Cell Hybrids A remarkable feature of regulation of MGMT, so far unique among all repair proteins, is its complete extinction in some cultured cell lines. Many human cell lines of both human fibroblast and lymphoblast origin, after transformation with SV40 and Epstein Barr virus, lose their ability to counter the toxic effects of MNNG, and a concomitant loss of 06-methylguanine repair activity was observed (125, 126). The MGMT-negative cells were called Mex- or Mer-. Subsequently a large number of Mex- (Mer-) cells were identified among human and rodent cell lines (127-130). In addition to the virus-transformed lines, some 20-30% of the lines derived from human tumors are Mex- (131). Except for a few cell lines (e.g., GMll), all diploid human cell lines are Mex+. The G M l l line is unusual because, unlike a similar but unrelated line GM10, it is Mex+ at a low passage level, but loses MGMT activity as a function of age (132, 133). After 20-30 cell doublings, the MGMT activity was undetectable, although no other macroscopic and karyotypic changes in the cells could be detected. The situation in rodent cells is somewhat different. While many tumorderived human cell lines are Mex-, in the case of rodents, a number of “normal” cell lines derived from different tissues are also Mex-. In fact, none of the routinely used hamster cell lines, including Chinese hamster ovary (CHO), lung (V79) cells, and Syrian hamster kidney (BHK21), has detectable MGMT activity (128, 132). Syrian hamster fibroblasts become Mex- after two or three passages of the Mex+ primary culture (134).One of four embryonic mouse lines is Mex- (135). In an attempt to map the structural and regulatory locus for human MGMT, we investigated the MGMT activity of Mex+ human/Mex- mouse or hamster cell hybrids, which selectively lose the human chromosomes. In one such study, none of the hybrids, derived from human skin’fibroblasts W138 and mouse RAG lines, had detectable MGMT activity (132).These and similar results with humadhamster hybrids, in which some or all of the human chromosomes were present, preclude an unequivocal assignment of a human chromosome linked to MGMT gene. As a control, we did observe that the Mex+/Mex+ hybrids had the Mex+ phenotype. These results suggest that Mex- is the dominant phenotype.


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On the other hand, most hybrids of Mex+ human/Mex- human cells are Mex+ (84). This supports an earlier study (136). Thus it appears that the epigenetic control of the Mex phenotype is much more complex than if simply caused by a single trans-acting factor. Analysis of MGMT activity of a panel of Mex+ human/Mex- CHO cells suggests the possibility that the MGMT activity in some of the hybrids arose from activation of the endogenous hamster gene and were not encoded by the human gene. A recent surprising observation is that the extinction of MGMT in Mex- human lymphoblastoid cells is accompanied by the loss of expression of unrelated genes, namely, thymidine kinase and galactokinase (137) and overexpression of a ribosomal protein gene (138). Because thymidine kinase and galactokinase genes are located on chromosome 17 and the MGMT gene is on chromosome 10 (99, 139), it appears possible that a nonspecific trans-acting factor encoded by a gene on chromosome 17 contributes to the Mex+-toMex- transition. One obvious explanation of the Mex- phenotype is the loss of MGMT structural genes. This possibility could not be investigated until the recent cloning of MGMT cDNA and chromosomal mapping of the gene. Although such studies have not been carried out extensively, it is clear that loss of the structural gene is precluded by the observation of reactivation of the MGMT gene in the Mex- cells under certain circumstances. Based on the evidence discussed in Section IX, both V79 and CHO cells appear to have the complete MGMT gene. The appearance of Mex- lymphoblastoid lines following viral transformation of Mex+ cell lines suggests that the transition from Mex+ to Mexphenotype is a stochastic process (130, 134, 140). Furthermore, the Mexcells did revert to Mex+ phenotype, although at a very low (10-7-10-s per cell per generation) frequency (141).It is intriguing that these numbers are in the same range as that of the spontaneous mutation rate of mammalian genes, such as that governing HPRT (142).However, there is no evidence as yet that the Mex- phenotype is a result of mutation in the MGMT gene as discussed above. Nevertheless, questions have often been raised as to whether the Mexphenotype is an artifact of cell culture, or whether Mex- cells occur naturally. For example, were the original tumor tissues from which Mex- cell lines were established also Mex-? A recent study provides an answer to this question (143).It appears that a small number of excised liver tumors from humans has no detectable MGMT activity. What is the molecular basis of the nearly complete extinction of MGMT in many Mex- cells? Does it involve positive or negative regulatory elements, or both? Is there a unlfying mechanism that can explain the appearance of the Mex- phenotype in the diverse circumstances described

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above? The answers to these questions will be evident only after a comprehensive understanding of the promoter and enhancer elements of MGMT gene and the trans-acting factors that recognize them. We have recently cloned the 5' proximal region of the transcribed sequence of the human MGMT gene and observed promoter activity in a segment of the region by measuring transient expression of a reporter gene (chloramphenicol acetyltransferase) (144).As is common for many housekeeping genes, the promoter region has no TATA or CAAT boxes, but is extremely G-C-rich and contains a repeat of the 5'-CCGCCC motif. Gradual reduction in the promoter strength observed with sequential deletion of the promoter fragment suggests that multiple regulatory elements control MGMT expression. However, the nature of these elements and of the sequence-specific transcription factors and their interaction in regulating the MGMT level have yet to be elucidated. It was somewhat surprising that, in our preliminary experiments, no difference was observed in the MGMT promoter-driven transcription of the reporter gene in transfected Mex+ and Mex- cells (K. Tan0 and B. Kaina, unpublished experiment), (145).Thus, the lack of MGMT transcript in Mex- cells is unlikely to be caused by the loss of specific transcription factors or to the presence of a specific repressor in those cells that recognize the cloned promoter fragment. One way to investigate the role of trans-acting factors for MGMT regulation that may be encoded by genes located in distinct chromosomes is to reexamine the genetic origin of MGMT expressed in Mex+ human/Mexrodent somatic-cell hybrids (132).Because of the nearly identical size of the MGMT polypeptides and mRNAs in human and rodent cells, it is rather dimcult to establish such a genetic origin. However, a polyclonal antibody directed against a unique peptide sequence of human MGMT not conserved in the mouse coding sequence has recently been raised (146,147). It does not recognize the mouse and presumably other rodent MGMTs (147)and should therefore be useful in establishing unequivocally whether a specific human chromosome is responsible for activating the rodent MGMT gene. One trivial explanation for MGMT extinction is the presence of mutations in the regulatory sequence that would prevent synthesis of MGMT transcripts. However, this appears unlikely for most of the Mex- cells in view of the observations on Mex- -+ Mex+ transition stated earlier. Other arguments against this explanation are as follows. First, we and several others have detected no gross deletion or rearrangement of the MGMT gene It in Mex- human and rodent cells by Southern blot analysis (41,108,168). also appears unlikely that in many of the pseudodiploid Mex- cells (e.g., CHO cell line), both copies of the MGMT gene will be mutated to yield the Mex- phenotype. Only two Mex- lines that also lack the MGMT gene were


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identified, the human HeLa/S3 (MIT) clone (41) and rat line 208F (158).A restriction fragment length polymorphism (RFLP) was observed in the MGMT gene in Mex- HeLa MR line (158),but whether the polymorphism is related to the lack of MGMT transcription is not clear. Second, although no MGMT mRNA was detected in Mex- cell mRNA by Northern blot analysis (41, 99,107,168), a minute amount of the MGMT message was identified by the more sensitive PCR method in Mex- human tumor and CHO cells (108, 158).This suggests that, in most Mex- cells, the MGMT gene is not inactivated by mutation and that the lack of MGMT activity is caused by repression of the gene. Finally, Mex+ cell clones have been isolated from Mex- human lymphoblastoid cells without any selective treatment (130, 141)by simple selection with an alkylating agent in the case of V79 cells (148),and by transfection of CHO cells with human DNA (90-93).

E. Lack of Correlation between Methyltransferase Repression and Oncogene Expression Because of the Mex- phenotype of a significant number of tumor cells, and because infection with tumor viruses such as EBV and SV40 often leads to a Mex- phenotype in the resulting transformed cells, it was of interest to investigate whether the extinction of the MGMT gene is related to oncogene

FIG. 4. Northern blot analysis of induction of MGMT and MPG in mRNA H4IIE cells following treatment with X-rays, MMS, and MNNG. Lane 1, control; 2, 24 h after 200-rad Xrays; 3, 48 h after 200 rad; 4, 24 h after 300 rad; 5, 48 h after 300 rad; lanes 6 and 7, 24 and 48 h after exposure to 0.2 mM MMS; lanes 8 and 9,24 and 48 h after 10 pM MNNG treatment; lanes 10 and 11,24 and 48 h after 20 p M MNNG treatment; lanes 12 and 13,24 and 48 h after 30 p.M MNNG treatment. GAPDH mRNA levels were determined as an internal control.

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activation. No available information supports the possibility of a direct connection. In has been observed that SV40 transformation of human diploid cells does not immediately give rise to the Mex- phenotype (140).It appears that the Mex+-to-Mex- transition usually occurs at the time of crisis during the establishment of cell lines. Immortalizing cells with SV40 large T antigen led to both Mex+ and Mex- strains. These results suggest that MGMT extinction is not a direct consequence of expression of viral oncogenes. This conclusion is further supported by the observation that activity after transient overexpression of c-Ha-ras, c-fos, and v-mos oncogenes in Mex+ NIH 3T3 cells is not associated with a change in the level of MGMT mRNA (149; B. Kaina, unpublished).

VII. Role of DNA Meth lation in Methyltransferase Gene f;egulation A. Level of Methylation of MGMT Gene Sequences in Mex+ and Mex- Cells In view of the extensive substitution of 5-methylcytosine for cytosine in CpG sequences in mammalian genomes, and a large body of evidence in support of the dogma that transcriptional activity of a gene is inversely correlated with the level of its methylation (primarily at the 5’ ends of genes), it was attractive to investigate whether the Mex- phenotype is related to hypermethylation of the MGMT gene. It is now believed that methylation of cytosine causes a change in the local configuration of the gene that negatively affects its promoter activity, and that binding of some transcription factors (e.g., CREB) to specific recognition elements in the promoter is sensitive to methylation (150, 151).Even though the promoters are enriched in CpG sequences “CpG islands” (150), the dinucleotides are also present in the coding sequences of genes. Because the promoter elements and their sequences of the MGMT gene have not been fully identified, we made a preliminary investigation of the methylation sequence of the exon and surrounding sequences of the M GMT DNA isolated from Mex+ and Mex- cells. A nearly identical pattern of methylation was observed in three independent studies with DNA isolated from SV4O-transformed and tumor lines with Mex- phenotype (152). In all cases, the presence of m5C in CpG sequences was determined by comparative probing of genomic DNA digested with restriction endonuclease isoschizomers HpaII and MspI in Southern blots. Both enzymes recognize the 5’-CCGG sequence in DNA but HpaII cannot digest DNA when the internal C is methylated. The DNA of all Mex+ cells expressing MGMT at various levels was methylated to a much higher level than the DNA of Mex-


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cells (152). While this unexpected result is the opposite of the pattern previously observed in general, it is by no means unique. For example, a direct correlation between methylation and gene expression has been observed in the major histocompatibility complex gene H-2K of the mouse (153).Similarly, there was no correlation between DNA methylation and transcriptional inactivation in the chicken lysozyme gene (154). However, we should keep in mind that the genes in the two last examples are developmentally regulated, unlike MGMT and other DNA repair genes that should be typical “housekeeping” genes. Is it possible that there is no causal relationship between methylation of exon sequences and gene activity for MGMT? Despite attempts to explain the phenomenon, it is not clear how and why an increase in the methylation level of cytosine can lead to opposite effects for different genes. We hope that answers to these questions will be forthcoming, once we have dissected the promoter sequence and functions of the human and rodent MGMT genes.

B. MGMT Activation with 5-Azacytidine 5-Azacytidine, a potent agent for inhibiting methylation of cytosine, has been routinely used to demonstrate the role of 5-methylcytosine in gene activation (155), even though the drug has other activities and affects genes that do not contain 5-methylcytosine (156).Treatment of murine sarcoma virus-transformed Mex- NIH 3T3 cells with 5-azacytidine led to stable expression of MGMT in these at about the same level as that of the nontransforming parent cells (157). Attempts by us and others to reproduce these results with Mex- Chinese hamster lines (CHO and V79) and with human HeLa MR cells have been unsuccessful (158; W. C. Dunn and S. Mitra, unpublished; R. S. Day, personal communication). With V79 (Chinese hamster) cells, 5-azacytidine treatment gave rise to cell clones that acquired resistance to HeCNU. However, these cells still lacked MGMT activity and were not cross-resistant to other alkylating agents, e.g., MNNG (158). A strong correlation of MGMT activity and CNU resistance has been observed for the hamster and other mammalian cells. Thus it appears that the induction of MGMT by 5-azacytidine cannot be achieved in all Mexcells and that activation of other, as yet undefined, genes may also be responsible for alkylation resistance.

VIII. Inducibility of Alkylation Repair Genes The seminal discovery in the understanding of alkylation damage repair in E. coli was that of “adaptive response” during which E. coli cells treated with a subtoxic dose of a simple alkylating agent became resistant to the toxic and mutagenic effects of the same and other alkylating agents (159). Subse-

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quent studies showed that several genes in the ada regulon involved in alkylation repair are coordinately induced during the adaptation treatment (21,119). The 39-kDa Ada protein with its distinct amino- and carboxylterminal domains has methyltransferase activities for alkyl phosphotriesters and 06-alkylguanine in DNA, respectively. The alkyl acceptor residues in the protein are Cys-69 and Cys-321 (21).The Ada protein methylated at Cys-69 acts as the inducer of its own as well as one of the glycosylase (aZkA) genes in addition to other genes of unknown function (21,160). Adaptive response has been observed in some other bacteria as well (46, 86,161).Experiments to identify an adaptive response in mammalian cells gave intriguing results. Early in uiuo studies showed a two- to threefold increase in MGMT activity in livers of rats chronically fed with dimethy1-Nnitrosamine (162).It was later observed that a similar increase in MGMT level can also be induced by a variety of hepatotoxic agents (35).Partial hepatectomy and ionizing radiation, and even interferon inducers and hormones, are effective inducers of MGMT (35,163). Many of the inducing agents are not unrelated to DNA repair because these apparently do not cause DNA damage. Thus it is possible that the apparent induction of MGMT arises from the stimulation of cell proliferation. It is surprising that MGMT induction was not observed in livers of other rodents (e.g., mice and hamsters). In in uitro experiments with cultured cell lines, we and others did not observe an induction of MGMT activity following exposure to various alkylating and other DNA-damaging agents (128,164).However, such an induction was indeed seen only in several rat and a human hepatoma, and a rat rhabdomyosarcoma line treated with alkylating agents and UV light (148, 164a, 165).More recently, a 5- to 10-fold increase in MGMT activity concomitant with an increased protection against alkylating agents for neoplastic transformation was observed in mouse C3H 10T1/2 cells after exposure to Xrays (166,167). These results discount the possibility that the adaptive response is generally present in mammals. In a few exceptional cases in rodent tissues and cell lines where there is some evidence of MGMT induction, it is clear that this was due to a response to nonspecific DNA damage. Thus MGMT activity increased in H4 and C3H 10T1/2 cells after exposure to X-rays and bleomycin and restriction endonucleases, all of which cause DNA strand breaks (165,166,168).It is now evident that mammalian cells are capable of a DNA damage-inducible global response in which induction of specific genes involved in repair of DNA damage as well as other seemingly unrelated genes (e.g., oncogenes) occurs (169,170).It is interesting that MGMT and MPG genes have not been identified so far among the damage-inducible repair genes except in a few rodent cells. However, DNA polymerse @, the enzyme involved in “very-short-patch excision repair of DNA lesions, including N-

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alkyl adducts following the removal of MPG, is strongly inducible by alkylating agents (171). It was not possible, prior to the cloning of alkylation repair genes, to establish that the increase in MGMT activity in mammalian cells pretreated with DNA-damaging agents reflects true induction at the level of transcription. Alternate explanations, e. g., stabilization and increased half-life of MGMT by the damaging agent, could not be eliminated. Our recent results, as well as the experiments of others, have now established that the increase in MGMT activity in rat cells exposed to alkylating agents is indeed due to an increase in transcription of the gene (168, 172) (Fig. 4). In H41IE and FTO-2B, both of which are rat hepatoma cells that express liver specific enzymes (e.g., tyrosine aminotransferase), a two- to fivefold increase in MGMT mRNA was observed after exposure to MNNG, MNU, MMS, HeCNU, X-rays, and UV light. Furthermore, MGMT transcription could also be induced by PuuII, a restriction endonucleae producing blunt ends in DNA (168).That the accumulation of mRNA was prevented by actinomycin D, an RNA-synthesis inhibitor, indicates that this increase in RNA level was due to de nouo synthesis. The induction response appears to be late and long-lasting. Thus, MGMT mRNA accumulation continued up to 72 h after exposure of the rat cells to MNNG; addition of actinomycin D 6 h after MNNG prevented the increase (168).Abolition of this induction at both protein and mRNA levels by the protein-synthesis inhibitor cycloheximide suggests that de nouo protein synthesis is essential for this inducible response (168). Because MGMT activity appears to be correlated with the state of digerentiation in rat hepatoma cells (173),it would be worthwhile to test whether the inducibility of the MGMT gene is linked to a specific liver function. A 10-fold increase in MGMT mRNA level was also observed in uiuo in the liver of rats exposed to 2-acetylaminofluorene (173). However, no studies on the possible induction of MGMT transcription in other rat tissues and cultured cells have come to our attention. No MGMT induction was observed in human hepatoma and fibroblast lines under the conditions that induce the MGMT gene in rat hepatoma lines (168). In view of the coordinated regulation of inducible MGMT (Ada) and MPG ( A M ) proteins in E. coh, it was obvious to ask whether MPG is simultaneously induced with MGMT in the rat cells and liver exposed to genotoxic agents. Figure 4 shows our results. The MPG gene is indeed induced in H4 cells at the same time MGMT is induced (172).However, the level of induction of the gene is at best about twofold under the condition in which more than a fivefold increase was observed in the MGMT mRNA level (172, 172a, 172b, 172c). It should also be noted that, unlike for MGMT, MPG induction has not been shown as yet to be due to de nouo RNA synthesis. While the inducibility of MGMT (and MPG genes) in rat liver and

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hepatoma cells has been unambiguously established, why is it that such a phenomenon cannot be clearly demonstrated in human fibroblasts or even other rodent cells and tissues? Furthermore, why is the 2- to 10-fold induction of MGMT observed in rat cells much lower than the several hundredfold induction observed in E. coli? One possible answer to the latter question is that even though, on the basis of genome size, the number (2 x 104 to 10 X 104) of MGMT molecules per cell in mammalian cells is not much different from that of 20 MGMT molecules per cell in E. coli (85, 86), each mammalian cell has 103 more MGMT molecules than do the bacteria. A large increase in MGMT activity may not be necessary when a high basal level is present. The answer to the first question is more uncertain. Is it possible that the MGMT gene in other rodents and even humans is also damage inducible, just as in rats, but that the optimum conditions for such induction have not been established? Alternatively, because the induction phenomenon both at the level of activity and transcription is limited to rat liver cells with a few exceptions (165, 166, 168), is it possible that the regulatory region of the rat MGMT (and MPG) gene is uniquely different from those of other mammals? The cloning and a comprehensive characterization of the MGMT gene from different mammals will be needed for an answer to this question. We may make a point in this context that the primary sequences of the rat and mouse MGMTs are nearly identical and are significantly different from that of the human protein (174, 175).

IX. Alkylating Dru Resistance and Regulation of %NA Repair Alkylating drugs constitute a major class of antitumor agents that exert their cytotoxic action by damaging DNA in various ways. It thus follows that repair competence of cells for the potentially lethal lesions should have a direct impact on the ultimate therapeutic potency of these drugs. It has long been known that a large variation exists in the tissue-effectiveness of drugs in general. In particular, development of resistance to drugs is a common phenomenon among tumors. While the precise nature of critical DNA adducts that primarily contribute to cytotoxicity has not been elucidated for many alkylating drugs, extensive studies have been carried out for the class of CNU derivatives (20, 176). These drugs cross-link DNA strands by forming the intermediate 06-chloroethylguanine (20, 100, 176). Several other drugs (e.g., procarbazine and DTIC) may also exert their cytotoxic effect by inducing 06-alkylguanine (176). Furthermore, all of the alkylating agents, as expected, also induce in DNA N-alkyl adducts that are removable by MPG. MGMT activity has been measured in a variety of tumor cell lines and


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xenografts, and an excellent inverse correlation has been observed between the MGMT level and the cytotoxic response of cells to CNU and its derivatives. A clear correlation between MGMT level and CNU (HeCNU) resistance was also shown in CHO cells expressing human MGMT (177).While these results suggest that alkylation of guanine at the 0-6 position is the major toxic event induced by CNU, certain exceptions were observed where the BCNU (or HeCNU)-resistant cells did not have a high level of MGMT (158,178;S. C. Schold, personal communication). It has been proposed that, in certain BCNU-resistant human glioma lines, repair of N-alkyl adducts is enhanced (101). With the availability of a human MPG cDNA clone, it should now be possible to confirm whether alkylation resistance in some cells is due to an elevated expression of the MPG gene, and whether coordinate regulation of MGMT and MPG is a common phenomenon in resistance to alkylating drugs. These studies have also underscored another important issue, namely, the relative toxicity of different alkyl adducts in DNA. Our recent results reconcile a recent controversy about the toxic nature of 06-alkylguanine (179, 180). We found that MGMT expression protects cells from MNNG, MNU, and, to a certain extent, EMS and MMS-induced cell killing, but not at all from ENU toxicity (177). The differential resistance toward various alkylating agents has led us to propose the hypothesis that, in addition to 06alkylguanine, a second group of cytotoxic alkyl adducts are induced by alkylating drugs (92). These could be one or several N-alkylpurine adducts. The relative contribution to the overall toxicity of a drug will depend on the relative proportion of the class of adducts induced by it (92, 177).

A. Activation of the Endogenous Methyltransferase Gene and Drug Resistance As stated in Section VI, MGMT-positive clones were isolated from MexCHO cells after transfection with human DNA. In all cases, the selective agent was CNU or its derivatives and the MGMT activity was due to derepression of the endogenous gene. The obvious question is whether the human DNA activates transcription of the hamster MGMT by providing a trans-acting factor. This situation will be analogous to the MGMT regulation in humadrodent cell hybrids described in Section VI,D. Isolation of Mex+ clones from Mex- V79 cells by simple selection in the presence of CNU (148) suggests that Mex+ cell populations arose without involvement of foreign DNA. However, as we have discussed (92), we did not observe reversion of Mex- to Mex in the nontransfected CHO cells used as a control, in the presence of CNU. Second, the MGMT activity in the Mex+ V79 clone appears to be much lower than that present in the CHO transfectant lines isolated by others (92, 148). +

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In any event, regardless of the mechanism, development of CNU resistance is in most cases directly correlated with MGMT activity. A similar correlation was not observed for MPG. We showed that, at least with our MGMT-positive transfectants, in uiuo removal of O6-methy1guaninebut not of N-methylpurines was affected in the CNU-resistant cells (91).

6. Expression of the Glycosylase Gene and Alkylating Drug Resistance Unlike MGMT-negative cells, no MPG-negative cell line has yet been isolated. It is therefore not possible to establish a correlation of MPG repair activity and sensitivity to alkylating drugs. However, we investigated the possibility of increased drug resistance as a result of overexpression of MPG. MMS-resistant CHO cell lines have been isolated either as spontaneous variants or after transfection with human DNA (19,181). These MGMTnegative cell lines do not have a higher level of MPG activity, nor do they express a higher level of MPG mRNA than the control cells. Furthermore, neither the removal of 3-methyladenine in DNA nor the MPG mRNA level was altered in an MMS-hypersensitive mutant derived from CHO cells (182).We have tentatively concluded from these results that N-alkylpurines in DNA in Mex- cells may be toxic, but during the multistep repair of these adducts, MPG activity may not be rate limiting. It appears that this repair protein is present in excess in normal cells. Thus a specific increase in MPG activity will not affect the overall repair of N-alkyl addwcts in DNA. The increased alkylation resistance of various MGMT-negative cells would then be due to increased expression of an activity involved in a subsequent step, e.g., AP endonuclease. We have obtained additional support for this hypothesis by carrying out complementary studies in which CHO cells were transfected with a human MPG cDNA expression vector. Several clones expressing up to seven times the MPG mRNA and MPG activity of the control cells were isolated. None of these overexpressing cell lines is more resistant to MMS, DMS and ENU than the parent line (183).

C. The Tolerance Phenomenon and Alkylation Resistance An alternative hypothesis for a lack of correlation of drug resistance with increased DNA repair has recently been proposed by several groups who have cloned Mex- human and rodent cell lines that are resistant to MNNG and yet are unable to remove 06-methylguanine. These cell lines were isolated either by simple selection (180,184-186) or cloned following their transfection with genomic human DNA (181,188).Because these lines were uniformly MGMT-negative and showed no difference in the removal of Nalkylpurines (181,184,187),it appears that the cells developed tolerance to 06-alkylguanine and/or other toxic lesion(s). Interestingly, the cells were


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cross-resistant only to other methylating agents but not to UV light, CNU derivatives, and mitomycin C. It is surprising that they were also resistant to 6-thioguanine (181, 185, 188, 189; B. Kaina and G. Fritz, unpublished result). This resistance appears to be due to tolerance of the analog rather than inhibition of its incorporation into DNA (190). It was unexpected that resistance to cell killing was not associated with a reduction in mutation and sister-chromatid exchanges induced by alkylating agents (187, 191). The molecular mechanism of tolerance to alkylating agents is not clear, but it appears that cellular toxicity to alkylating drugs may involve a number of seemingly unrelated processes. For example, rodent cells that did not express the metallothionein gene but that were made metallothioneinpositive by transfection became resistant not only to heavy metals but also to simple alkylating agents, e.g., MNNG and MNU, as well as to other antitumor drugs such as cisplatin and chlorambucil(192-194). It should be noted that the metallothionein gene is inducible by various DNA-damaging agents (195, 196). It is thus possible that metallothionein may indirectly regulate DNA repair or tolerance functions, perhaps by control of the intracellular level of zinc (192).

X. Amplification of the Methyltransferase Gene and Drug Resistance The link between drug resistance and amplification of genes whose products provide the resistance to the specific antimetabolites is well documented (197,198). In the earlier studies, the drugs used in such experiments had specific targets. For example, methotrexate inhibits dihydrofolate reductase in a competitive fashion. An increased resistance to methotrexate may arise from the increased activity of DHFR expressed from multiple copies of its gene. In the case of “multiple drug resistance,” the MDR protein reduces the intracellular concentration of a variety of drugs (e.g., adriamycin and vincristine) by acting as an effluxing pump (199). Thus the increased resistance of cells to these drugs is due to an increased level of the MDR protein as a result of gene amplification. In contrast to the above examples, amplification of mammalian DNA repair genes has not previously been observed. We reasoned that a higher level of expression of MGMT that results in an increased cellular resistance to CNU may sometimes result fiom an amplification of the MGMT gene. Thus, in a recent study with NIH 3T3 cells, we showed that cells chronically exposed to increasing concentrations of CNU give rise to clones with 3-10 times higher levels of MGMT activity, which in turn resulted from three- to fivefold increase in the number of copies of the MGMT gene (200). A detailed analysis showed that the MGMT gene amplification was not accom-

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panied by a similar amplification of MPG, nor was there an amplification of DHFR and MDR genes. Because the mouse MGMT gene is located at the distal end of chromosome 7 (201),we looked for but did not observe amplification of any other gene on chromosome 7, including IGF-2, which is located nearest the MGMT gene. There was also no evidence of DM chromosomes or HSR in chromosome 7, the classical indicators of high-level gene amplification (198). This was the first demonstration of amplification of a DNA repair gene in mammals. There are some unusual features of this amplification. We never observed more than 10 copies of the gene in spite of attempts for stepwise selection for variants carried out in the usual way. These results are in sharp contrast to the hundreds of copies of DHFR genes observed in methotrexate-resistant cells. However, the low copy number of the MGMT gene is consistent with the lack of DM chromosomes and HSR sequences associated with its amplification (198). We should point out here that our inability to isolate cells with a high copy-number of the repair gene may be due to the fact that we could not use a selecting agent that specifically targets MGMT or acts in an indirect fashion, e.g., an alkylating agent that exclusively produces 06-alkylguanine. In contrast to target-specific drugs such as rnethotrexate, C N U produces a number of toxic lesions. Even though 06-alkylation of guanine may be the predominant event for toxicity, the cells could not handle a higher concentration of C N U because the other minor toxic adducts would reach significant levels and become major contributors to cell killing. This has indeed been shown with cells transfected with an MGMT expression vector (177). We would thus predict that resistance to high concentrations of alkylating drugs would require simultaneous amplification of mope than one DNA repair gene. This prediction can be tested experimentally. What is the biological significance of the amplification of DNA repair genes? In contrast to the common occurrence of gene amplification in cultured (particularly rodent) cells, amplification of genes (mostly those for multiple drug resistance and oncogenes) is rather rarely observed in uiuo and also exclusively in tumors (202). Increased activity of proteins responsible for drug-resistance results more commonly from an up-regulation of its gene than as a result of increased gene dosage (199).While the same situation may be true for DNA repair genes as well, it is important to investigate how often resistance to alkylating drugs does result from amplification of MGMT and other DNA repair genes, both in uiuo and in cultured cells in uitro.

XI. Outlook Research on the molecular biology of mammalian DNA repair in general and alkylation damage repair in particular has entered an exciting phase with


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the recent success in the cloning of a number of DNA repair genes and cDNAs from human and rodent sources. The availability of nucleic-acid and antibody probes for many of the alkylation repair genes and proteins, and elucidation of the structure and identification of the regulatory elements of these genes, provide an opportunity for a comprehensive understanding of regulation of alkylation damage repair. The prospect of large-scale production of the human alkylation repair proteins in E. coli for the subsequent determination of their structure by X-ray crystallography and NMR looks quite good. Rational design of drugs €or specific inhibition of repair proteins may be possible when the three-dimensional structures of these proteins are known. Inhibitors could be very important as adjuvant therapeutic agents for alkylating drugs. Antisense oligonucleotides could also be used for inhibiting transcription and/or translation of repair genes both in uiuo and in uitro. In contrast to inhibition of repair genes at the level of their expression, these genes could also be inactivated in cultured cells by homologous recombination. Starting with mutations in repair genes of pluripotent embryonic stem cells, repair-deficient or repair-negative mice could be generated. Such animals may make excellent models for mutagen, carcinogen, and aging studies. Finally, an understanding of the molecular basis of repair regulation may lead to targeted mutagenesis for up-regulation of the alkylation repair genes in the mouse, particularly, MGMT. Such a change may also profoundly affect the mutagenic, carcinogenic, and toxic responses of the animals to environmental agents. ACKNOWLEDGMENTS The work described in this article was supported at Oak Ridge National Laboratory by the Office of Health and Environmental Research, U.S. Department of Energy, under contract DEACO5-84OR21400 with the Martin Marietta Energy Systems, Inc. and by U.S.P.H.S. Grants CA 31721 and CA 53791, and at Karlsruhe Kernforschungszentrum by Deutsche Forschungemeinschaft KA 724-22. We would like to thank Dr. Rufus Day for his critical reading of the manuscript and for suggestions that significantly improved its quality. Finally, we gratefully acknowledge our colleagues’ contributions, which made this review possible. REFERENCES 1 . T. Lindahl, This Series 22, 135 (1979). 2. T. Lindahl, ARB 51, 61 (1982). 3. S. K. Randall, R. Eritja and B. E. Kaplan, JBC 262, 6864 (1987). 4. B. Straws, Adv. Cancer Res. 45, 45 (1985). 5. E. C. Miller and J. A. Miller, in “Chemical Carcinogens” (C. E. Searle, ed.), pp. 737-762. American Chemical Society, Washington, D.C., 1976. 6. P. D. Lawley, in “Chemical Carcinogens and DNA” (P. L. Glover, ed.), pp. 1-36. CRC Press, Boca Raton, Florida, 1979. 7. J. K. Selkirk and M. C. MacLeod, Biosci. 32, 601 (1982). 8. D. T. Beranek, Mutat. Res. 231, 11 (1990). 9. B. Singer and D. Grunberger, in “Molecular Biology of Mutagens and Carcinogens,” Plenum, New York, 1983.

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Cell Delivery and Mechanisms of Action of Antisense Oligonucleotides JEANPAULLEONETTI, GENEVIBVE DEGOLS,JEAN NADIR PIERRECLARENC, MECHTI, AND BERNARD LEBLEU~ UA CNRS 1191 G d d t q u e Mol&ulaire Unioersitd de MontpeUier I1 Sciences et Techniques du Languedoc 34095 Montpellier Cdder 5, France Historical Background . . ............................ AIGE Concept . . . . . . . From the Antisense Approac Limitations of the SNAIGE Approach ...... Internalization and Targeting of Oligonucleoti Intracellular Distribution of Oligonucleotides Mechanisms of Action of Antisense Oligonucl in the VSV Model ....................... VII. Conclusion and Perspectives .............. References .............................

I. 11. 111. IV. V. VI.

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1. Historical Background Down-regulating or specifically turning off the expression of individual genes represents a powerful tool for studies of their biological role in both in uitro cell cultures and in uivo in animal or plant models. This should also have great potential for therapeutic applications, as in the search for more selective antiviral or antineoplasic drugs. The antisense concept, in which mRNA translation is controlled by chemically synthesized complementary oligonucleotides (or oligomers), originated as early as 1967 (1).At about the same time, the DNA-mRNA hybridarrested cell-free translation assay was reported (2), while Zamecnik and Stephenson described the control of Rous sarcoma virus expression by synthetic oligodeoxynucleotides (3). It has since been realized that gene expression may be finely tuned in prokaryotes by complementary RNAs. This particular strategy has been used frequently for the control of bacterial plasmid expression. Although it is not our purpose to review this broadly documented field (see 4 for a recent 1

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review), two aspects appear important to mention. First, various strategies have been exploited; these include translation arrest by complementary RNA, RNase-III-mediated degradation of the target RNA, and transcription interference. Second, extensive genetic analysis has led to the unexpected “kissing” model, which postulates an interaction between complementary sequences initiated at the tip of a stable loop structure in the antisense moiety (5). To our knowledge, this has not been exploited for the design of synthetic RNA or DNA antisense oligomers. A broader approach to specific gene regulation has been demonstrated by Inouye (prokaryotes) and by Weintraub (eukaryotes) in which the control of various genes is achieved in cells transfected (or microinjected in the case of Xenopus laevis oocytes) by complete genes or gene fragments inserted in inverse orientation downstream from various promoters (reviewed in 6-8). Several transgenic species with an altered gene expression pattern have been obtained as well. As an example, altered phenocopies have been obtained by the injection of antisense RNAs in Drosophila embryos (9). Similar studies with plants appear to be one step closer to potential applications, i.e., the successful down-regulation of the polygalacturonase gene in transgenic tomato plants results in a better control of fruit ripening (10). Paralleling these advances in genetic manipulations, rapid progress in oligomer chemistry has rendered the automated synthesis of oligodeoxyribonucleotides accessible to nonspecialized laboratories, using either phosphoramidite (11)or, more recently, hydrogen-phosphonate chemistry (12). This has stimulated a growing number of studies, demonstrating the potential of the antisense oligomer approach (reviewed in 13-15). Reliable protocols for the synthesis of oligoribonucleotides have only recently emerged, due to the difficulty of devising appropriate protecting groups for the 2’-OH group of the ribose moieties (reviewed in 16). Initial successes have also allowed the synthesis of oligomer analogs with modifications within the internucleotidic linkages, the sugar moiety, or more recently, the base. Derivatization of oligomers with reporter groups, peptides, or lipids at various positions is also possible (reviewed in 17). Collaborative efforts of chemists and biologists both in the academic arena and in an increasing number of companies have been devoted to the potential diagnostic and therapeutic applications of nucleic acids; this will undoubtedly lead to more progress in the near future. Whether transfection of antisense genes (or of genes coding ribozymes) or administration of chemically assembled synthetic oligomers will have the brighter future cannot now be ascertained. The first approach bears the problems and the potential of gene therapy. The latter is closer to the problems classically encountered in drug use, e.g., target access and recognition, metabolism, and toxicity; these are reviewed in Section 111.

ANTISENSE OLIGONUCLEOTIDE ACTION

145

II. From the Antisense to the SNAIGE Concept Since these early pioneering efforts, the antisense concept has been vastly expanded. Apart from being antimessengers, synthetic oligomers provide several other strategies for the artificial regulation of gene expression. These include triple-helix formation, inhibition of protein binding, and artificial nucleases, such as ribozymes. We therefore propose SNAIGE (Synthetic or Small Nucleic Acids Znterfering with Gene Expression) as a generic term to describe more appropriately these various approaches (Fig. 1).

A. The Antisense Approach Most of the early studies were performed with oligodeoxyribonucleotides complementary to mRNAs; hence, the antimessenger or antisense concept. Although extensive studies have been performed in both cell-free extracts and intact cells (i.e., X. Zaeuis oocytes), the precise mechanisms involved have seldom been unraveled. A physical interaction between sense and antisense sequences was proposed initially, either at the mRNA level or at the pre-mRNA level in eukaryotic cells. This in turn would lead to an inhibition of mRNA processing, nucleocytoplasmic transport, or mRNA translation. Alternatively, RNA.DNA hybrids are substrates for cellular or viral RNase H, which hydrolyzes the RNA strand of these hybrids and effectively converts antisense oligomers into powerful sequence-specific nucleases. The precise role played either by physical interaction or by RNase-H-mediated cleavage in intact cells is still a matter of conjecture and might vary with the biological system under consideration; these aspects have been reviewed in detail recently (18) and are discussed in Section VI. Likewise, it has generally been difficult to assess whether the primary site of action of an antisense oligomer is within the cytoplasm (e.g., mRNA translation) or the nucleus (e.g., pre-mRNA maturation and transport) (see also Section V).

B. The Triple-Helix Concept The association of a homopyrimidine DNA sequence in the major groove of duplex DNA was originally demonstrated in 1957 (19). Hydrogen bonding can be achieved between thymidine or protonated cytosine in the third strand and, respectively, the conventional A-T and G-C base-pairs in the double helix, following the so-called Hoogsteen rules. This results in the sequence-specific annealing of a pyrimidine sequence to homopurine-homopyrimidine tracts in DNA. Interestingly, appropriate sequences appear to be relatively widespread around regulatory regions in natural genes, thus opening prospects for the control of gene expression. This could, in princi-

dsoligomer

m

deDNA

Oligomer

m

mRNA

Oligomer

m

mRNA


147

ple, take place in various ways. First, a stable triple helix could block the progression of RNA polymerase or interfere with the recognition of DNA binding proteins essential for transcription initiation or for the integration of a viral genome, to give just a few examples. Second, synthetic oligomers forming triple helices could be used to concentrate various DNA-binding drugs, thereby greatly increasing their specificity; the latter might be as diverse as intercalating, cross-linking, or DNA-hydrolyzing groups. The feasability of these various approaches has been documented in cellfree model systems using photocrosslinking (20) or cleavage (21) agents (see also 18 and 22 for reviews). The restrictions imposed by the initial Hoogsteen pairing rules might be overcome, since additional possibilities are being found; for example, a purine-rich oligomer can be associated in antiparallel orientation to the pyrimidine strand of double-stranded DNA through G.(T.A) hydrogen bonds (23). Another potential limitation to the formation of a triple helix in intact cells resides in their high sensitivity to pH and ionic conditions. Additional rules allowing stable annealing at physiological pH have now been demonstrated (24) and allow the sequence-specific control of c-myc gene transcription in intact cells (25).

C. Synthetic

Ribozymes

Studies on the structures and autocatalytic properties of plant and animal ribozymes have defined the consensus sequences required for their nuclease activity; the best defined is the “hammerhead structure (reviewed in 26). Other ribozymic activities have also been described as an essential step in delta hepatitis virus multiplication in human hepatocytes (27). Although the alignment of the catalytic moiety and of the cleavage site is provided by intramolecular hydrogen-bonding in the natural ribozymes, both entities may belong to separate molecules. Synthetic hammerhead ribozymes can thus be engineered for the sequence-specific cleavage of any complementary RNA sequence; the only target requirement is a G-U-H sequence (H = “not G”) on the 5’ side of the cleavage site (28). The feasability of the approach has been demonstrated in cell-free experiments on various targets. Ribozyme-mediated degradation of various RNAs has also been demonstrated in cell cultures, although problems dealing with cleavFIG. 1. The SNAIGE concept: different strategies can potentially be used to inhibit gene expression by synthetic oligomers. (1) Competitionof transcription factors with double-stranded oligomers. (2) Competition with transcription factors, or transcription blockage with triplehelix-forming oligomers. (3) Inhibition of the mRNA functions with antisense oligomers. (4) Cleavage of the mRNA by a ribozyme. (5) Competition of RNA binding proteins (i.e., transactivators) with synthetic ribonucleotides.

148

JEAN PAUL LEO N EV I ET AL.

age yield and stability of the catalytic RNA still must be solved (for example, 29, 30). Progress in oligomer chemistry now allows for the design of composite synthetic ribozymes combining ribo- and deoxyribonucleotides. A better knowledge of ribozyme fine structure and mode of action will obviously be helpful in defining simple and more efficient structures for possible therapeutic use.

D. Sense Oligonucleotides Gene expression is conditioned by sequence-specific interactions between nucleic acids and regulatory proteins at both genomic and mRNA levels. Synthetic double-stranded or single-stranded oligomers can thus be used as competitors for these proteins. This approach obviously requires appropriate knowledge about the sequence and the structure of these protein binding sites. As an example, double-stranded oligomers or analogs thereof have been introduced into cells by direct microinjection (31)or incubated with intact cells (32), and have been successful in regulation of gene expression, most probably through competition with transcription regulatory proteins. Trans-activating proteins represent another class of possible targets, e.g., Tat and Rev, two gene-regulating proteins expressed in HIV-infected cells. Increasing knowledge of the structural elements essential for the interaction between these proteins and their RNA binding sites, e.g., TAR (33)and RRE (34),will allow the design of competitor oligomers. A last example illustrates both the potential and some of the pitfalls of synthetic oligomers. Phosphorothioate oligomer derivatives designed as antisense oligomers aiming at interfering with HIV expression turned out to be efficient but non-sequence-specific inhibitors of the viral reverse transcriptase through competition with genomic RNA for the enzyme template binding site (35).

111. limitations of the SNAIGE Approach As summarized above, the SNAIGE concept is straightforward and, in principle, should give rise to specific gene-expression modifiers. However, difficulties have often been underevaluated, giving rise to a flurry of illcontrolled data and of failures, as well as undisputed successes. The main problems encountered in in uitro utilization of synthetic oligomers deal with metabolic stability, cell penetration, intracellular distribution, availability of the nucleic acid or protein target, and processing of target-oligomer complexes; these points are dealt with in this section. Further, in uiuo applications must cope with large-scale production and manufac-


149

turing costs, toxicology, mutagenicity, immunogenicity, and body distribution; this has been discussed in a few recent publications (36,37).Finally, we have contributed to the development of antibody liposomes (vide infru) as a first step toward site-specific delivery of the encapsidated antisense oligomers.

A. Target Choice The choice of a target sequence is not always easy, due to (1)a lack of knowledge about the three-dimensional structure of most RNA targets within their natural environment; (2) the near impossibility of predicting accessible nucleic acid sequences within ribonucleoprotein complexes or chromatin structures; and (3) our as yet poor understanding of the rules governing sense-antisense or nucleic acid-protein interactions. Splice sites on premRNAs (38),as well as 5’ untranslated regions on mRNAs, generally appear as the most efficient targets in the antisense approach, but exceptions have been documented (reviewed in 18) (Fig. 2).

B. Metabolic Stability The metabolic stability of oligonucleotides is low due to the action of nucleases (mainly 3’-exonucieases) in extracellular fluids, endocytic compartments, and the intracellular environment. Various analogs modified at either the internucleotidic phosphate backbone [e.g., methylphosphonates (39) or phosphorothioates (40)],the sugar configuration [e.g., a-oligomers (41)or e’-O-methyl oligomers (42)],or the 3’ end have been synthesized and are adequate solutions to this particular problem. The abundant literature along these lines has been extensively reviewed recently (13, 17). Reconciling these modifications with the structural features required for sequencespecific nucleic acid recognition at a useful T,, and eventual processing of the hybrids by RNase H, have, however, turned out to be more difficult than initially foreseen. Moreover, little is still known about the toxicity and the mutagenicity of the metabolites arising from oligomer analogs. Let us illustrate these points with a few examples. The a-anomeric oligomer analogs can be assembled with good coupling yields using standard automated methods (43).They hybridize, with good T , values, to their complementary targets, although in parallel orientation (45), and are not recognized by most nucleases (44). However, 15-mer oligomers specific for vesicular stomatitis virus (VSV) or interleukin-6 are devoid of biological activity either in a cell-free translation assay or in intact cells (45);absence of processing of these DNA-mRNA hybrids by RNase H might be an explanation. On the other hand, an oligomer complementary to the cap site of P-globin mRNA inhibits its translation in reticulocyte lysates (46).Likewise an a-oligomer complementary to the primer binding site in pglobin mRNA, taken here as a model system, inhibits its transcription by

Nuclear events

0

'2,

Nuclear or cytoplasmic events

Cytoplasmic events

Ribosome

mRNA

FIG.2. Possible mechanisms of action of antisense oligomers. The mechanism of action is p r l y understood, but is generally supposed to inhibit translation. However, this inhibition can be direct or quite indirect due to interferences in the nuclei or the cytqplasm of the cells. The hybridization of the oligomer can change the mRNA structure and inhibit splicing (1)or nucleocytoplasmic transport (2). The mRNA oligonucleotide duplex can be recognized by RNase H and subsequently the mRNA can be degraded (3). When located at the 5' end of mRNA, the oligomer can inhibit the binding of translation initiation factors (4). The oligomers can also directly inhibit the translation of the mRNA by ribosomes (5).

151


MoMuLV reverse transcriptase (47). Taken together, these experiments seem to restrict the use of a-oligomers as sequence-specific inhibitors to non-RNase-H-dependent effects. Methylphosphonate derivatives combine the two advantageous properties of being uncharged and resistant to nucleases. Yet their biological activity in various models is somewhat disappointing; they must be added to cell culture media in a 50-200 pM range (48). This might be due to lack of recognition by RNase H, cell penetration through diffusion rather than receptor-mediated endocytosis, or chirality of the modified phosphate backbone. Interestingly, psoralen methylphosphonate derivatives photoactivatable by UV become active in the same models around 5-10 pM (38). An alternative approach consists of the association of unprotected oligomers with drug delivery systems such as liposomes, lipoprotein particles, nanoparticles, or protein conjugates, as developed initially by our group (see Section IV).

C. Cell Uptake and lntracellular Compartmentalization Another problem encountered with the use of synthetic oligomers deals with cell uptake and intracellular compartmentalization (Fig. 3). Oligomers

I Antibody targeted liposome FIG. 3. Endocytosis of free oligomers as compared to their targeted counterparts. Oligomers are taken up by the cells by receptor-mediated or fluid-phase endocytosis. Poly(L1ysine)-conjugatedoligomers interact with the negative charges of the cellular membranes, and are taken up by nonspecific receptor-mediated endocytosis. On the contrary, oligomers encapsulated in antibody-targeted liposomes are taken up by specific receptor-mediated endocytosis. The oligomers accumulate in the endocytic compartments, and must escape from these compartments to reach their target in the cytoplasm or in the nuclei.

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JEAN PAUL 1.EONElTI ET AL.

are now believed to be taken up by pinocytosis and/or by receptor-mediated endocytosis after binding to cell surface proteins; several candidate receptors have recently been described and partially characterized (49, 50). Conjugation to synthetic polypeptides such as poly(L-lysine)(51)and to lipids such as cholesterol (52), or encapsidation in antibody-targeted liposomes (XI), increases cell uptake and biological efficacy, strongly suggesting cell uptake of unmodified oligomers as a limiting step; this point is detailed in Section IV. Whether oligomers are used in their free form or in association with the delivery systems mentioned briefly above, their internalization involves receptor-mediated (or fluid-phase) endocytosis. Escaping the endocytic compartments to reach intracellular targets in the cytoplasm or in the nucleus is another problem. Trapping of the oligomers in endocytic compartments and/or degradation by lysosomal nucleases might be a strong limitation to this approach. Tools allowing the cytoplasmic delivery of oligomers should overcome these potential problems. However, neutral methylphosphonate analogs that bypass endocytosis suffer other limitations, as outlined above. An unexpected feature of the intracellular behavior of oligomers has arisen from microinjection studies that indicate a rapid diffusion to the nucleus (%, 55). Whether this favors the interaction of synthetic oligomers with nuclear targets or segregates oligomers in the nuclei is not known and is discussed in Section V.

D. Fate of Oligonucleotide-Target Hybrids The fate of oligomer-target hybrids represents an additional ill-understood event with relevance to biological efficacy. As discussed above, physical association through hybridization could lead to the activation of endogenous RNase H with an expected increment in inhibitory activity (45, 56, 57). A new generation of oligomers engineered to destroy their target RNA (or DNA) or to covalently bind to them is now being studied in many laboratories. It includes oligomers linked to alkylating, free-radical generating, or photoactivable moieties (Fig. 4). Oligomers conjugated to intercalating drugs, with the aim of increasing the binding constant to their DNA or RNA target should also be mentioned (reviewed in 17 and 58). In addition, cells are equipped with a collection of RNA unwinding activities, particularly evident for the ribosome machinery. This would explain why translation elongation cannot be blocked by antisense oligomers unless RNase H is able to destroy mRNAs (Fig. 2). Unwinding and, more recently, unwinding-modifying activities have been documented in both X. Zueois embryos (59) and in mammalian cells (60). Such activities impair the biological efficacy of antisense RNAs in developing X. Zueuis embryos (59).It deserves additional study, since a better understanding of these unwinding


153

-cross-linkingof the oligomer and the mRNA (alkylatingagents, psoralen) -alteration of the mRNA by the functionalized oligomer (porphyrin) -stabilizationof the interactionsbetween the oligomer and the mRNA (acridine) FIG.4. Strategies to increase the interaction properties of antisense oligomers.

activities might ultimately allow the design of oligomer analogs or of protecting groups preventing their recognition.

E.

Side Effects

Finally, we have to cope with often-unforeseen side-effects of oligomers and their analogs. Striking examples have been documented in various attempts to control in uitro HIV infection by synthetic oligomers. In & nouoinfected T-lymphocyte cell-lines, little sequence specificity of antisense oligomer was found. A polycytidylate with phosphorothioate internucleotidic linkages (SdC),, turned out to be the most active compound, with an EC50 (concentration giving a 50% inhibition) -0.5 pM (61). Non-sequencespecific biological activity probably results from competitive inhibition for substrate binding on viral reverse transcriptase (35)or from interferences with virus adsorption to the CD4 membrane receptor (62). Likewise, oligomer-cholesterol derivatives probably act through sequence-independent mechanisms. We have initiated a comparative study of 12-mer oligomers complementary to the Tat splice acceptor site in collaboration with the group of J. L. Imbach (Lab. Chimie Bio-Organique, Universitk Montpellier 11);our results also exhibit little or no sequence specificity, and the following order of efficiency: a or PS % a or Met P > p.

IV. Internalization and Targeting of Oligonucleotides As previously mentioned, one of the main problems in using synthetic oligomers is to get a sufficient number of molecules into the cells and prevent hydrolysis before they reach their target. Despite the presence of putative receptors at the cell surface (49,50), it seems that oligomers are not internalized very efficiently. Much interest has been devoted in our group to develop tools allowing protection of antisense oligomers against serum nu-

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JEAN PAUL LEONE'ITI ET AL.

clease degradation, efficient transfer across the plasma membrane, and the possible targeting to cells expressing specific determinants at their surface (Fig. 5). Conjugation to poly(L-lysine) (51) or encapsulation in antibodytargeted liposomes (53) have provided efficient ways to deliver oligomers into cells. The efficiency of cellular uptake of antisense oligomers was significantly improved. However, both of these tools present some limitations, as we discuss below.

Liposome -encapsulated oligomers Small unilamellar liposome

0 I

0-P-0 I

Oligomer linked b poly(L-Lysine) wc

NH,-CH-COO

I

w ? ) 4

I Ntl,

I (y%lr (NH -CH-COO\nNH-CH-COO(NH

I I

-CH-COO),NH

I

-CH-COOH

I

(CH3,

( 7 1 4

(7YI4

NY

NY

NHZ

FIG.5. Specific and nonspecific targeting of antisense oligomers. To increase their uptake in the cells, oligomers have first been synthesized with an adenosine at their 3' end. After oxidation of the ribose, the dialdehyde formed reacts with the a-amino groups of poly(r,-lysine), leading after reduction to an N-morpholine ring. Oligomers have also been encapsulated in small unilamellar liposomes linked to protein A. These liposomes can be efficiently targeted to cell surface determinants by monoclonal antibodies.


155

A. Poly(L4ysine) Conjugation of Oligonucleotides Poly(L-lysine)is a well-known polypeptidic carrier; it has been used to potentiate the uptake of various drugs and macromolecules such as methotrexate and horseradish peroxidase (63, 64).We have conjugated oligomers to this carrier in order to potentiate their antisense properties. Oligomers were chemically linked to the €-amino groups of polylysine through an Nmorpholine ring following periodic acid oxidation and borocyanohydride reduction of their 3' end ribose (65). The best results were obtained by coupling oligomers ranging in size from 10 to 15 nucleotides to M , 14,000 polylysine. Statistically, the 14-mer sequence occurs once in the RNA of a higher eukaryote. 3'-Modified natural oligomers have been used throughout in order to minimize problems and possible adverse effects associated with alternative chemistries.

B. Biological Activity of Oligonucleotide-Poly(L-lysine) Conjugates The efficiency of polylysine conjugates was illustrated in several biological models (51, 66-69). Initially, conjugated oligomers of the appropriate sequence were demonstrated to inhibit VSV multiplication (51, 65). More recently, similar approaches were successful in developing an antiproliferative activity with anti-c-myc oligomers (67') or to decrease the cytopathic effects of HIV-1 in de nouo-infected MT4 T-lymphocytes (66). Polylysine-conjugatedoligomers complementary to the 5' end of the VSV N protein mRNA are 10 to 50 times more active than unconjugated oligomers on L929-infected cells. Sequence-specific antiviral activities of such conjugates are observed at concentrations lower than 1pM (as summarized in Table I; see also 51). As a point of comparison, methylphosphonate derivatives inhibit VSV expression in a 50 pM concentration range (39). In the c-myc oncogene model, it is important to mention that nonconjugated oligomers only exhibit biological efficacy when incubated in a culture medium devoid of serum nucleases (as obtained by heat decomplementation of the serum or by omitting the serum at the time of experimentation). In contrast, polylysine-conjugated oligomers are active without any manipulation of the culture medium (67). The mechanisms through which polylysine increases the antisense effect of oligomers are not clearly understood. We have demonstrated that the uptake of fluorescently tagged oligomers conjugated to polylysine is accelerated and increased as compared to unconjugated material (70). The conjugate is taken up by a nonspecific receptor-mediated endocytic pathway. It seems to accumulate in acidic compartments, where proteolysis of the carrier would release some oligonucleotide material. Data involving pOly(Dlysine) and inhibitors of the endocytic pathway are in line with this scheme. Other effects of poly(L-lysine) cannot be excluded in the potentiation of the

TABLE I EFFECTSOF ANTISENSE NUCLEOTIDESON VSV MULTIPLICATION^

Oligomer target

5’ end of N mRNA 5’ end mismatch of N mRNA Internal site of N mRNA Intergenic region (-) Control oligomer Viral polymerase binding site

VSV reduction

++ + ++ -

In uitro translation

In uitro transcription

Primary viral transcription

Viral transcription

+

-

-

++

-

nd nd

nd nd -

nd nd

++ nd

-

nd

-

nd

++ nd

015-Mer oligonucleotides complementary to various sites on VSV mRNAs or genomic RNA have been compared for inhibition of antiviral activity, translation in reticulocyte lysates complemented with RNase H, cell-free transcription from isolated virions, primary transcription in actinomycin D-treated cells, and total virion transcription in cells. + +, Drastic (10- to 100-fold reduction) inhibitory activity; +, moderate (iosynthesisand, 188-189. 19s- 196 DNA precursor biosyntbesis, enzymes in. see k:nzynies in D N A precursor biosynthesis DNA repair. alkylation daniage repair in genome5 and, 132-135 dNTP, enzyines i n DN.4 precursor hiosynthesis and compart mentat ion, 188- 196 multienzyme aggregates. 196;-198 organization. 171- 177 replication. 198-200 synthesis i n eukaryotic cells, 187 dNTP synthetase. T4, D N A precursor biosynthesis anti, 177-181 Drug resistance, alkylation damage repair i n genomrs and D N A repair, 132-135 gene amplificatictn, 135-136

enzymes in D N A precursor biosyntliesis and, 171-177 Eukaryotic cells. enzymes in D N A precursor hiosynthesis and, 187-200

F Focal adhesion, protein tyrosine kinase substrates and. 217-220

G Genomes, alkylation damage repair in, see Alkylation damage repair in genomes Clycosylase gene, alkylation damage repair in genomes and, 134 GTPase-activating protein, protein tyrosine kinase substrates and, 215

E

H

Enzymes, protein tyrosine kinase substriates and, 208, 210 Enzymes in f)N..\ precursor biosyntliesis,

Hybrids alkylation damage repair in genomes and, 1%-127 antisense oligonucleotides and, 152-153

200

231

INDEX

0

Hydroxyurea inhibitors, enzymes in DNA precursor biosynthesis and, 194-195

I Inhibitors bacterial adenylyl cyclases and, 43-44 enzymes in DNA precursor biosynthesis and, 194-195 mammalian ribonuclease, see Mammalian ribonuclease inhibitor Initiation of transcription in RNA polymerase 11, see RNA polymerase I1 transcription initiation

L Liposomes, antisense oligonucleotides and,

158-160

Oligonucleotides, antisense, see Antisense oligonucleotides Oncogenes alkylation damage repair in genomes and,

127-128 protein tyrosine kinase substrates and,

211-215

P Phosphatidylinositol 3-kinase, protein tyrosine kinase substrates and, 211, 214 Phosphorylation, protein tyrosine kinase substrates and, 208, 210-211, 216-224 Phosphotyrosine, protein tyrosine kinase substrates and, 207-208 Poly(L-lysine), antisense oligonucleotides and, 155, 157-158 Polyanions, antisense oligonucleotides and,

157-158

M Mammalian ribonuclease inhibitor, 1-2, 24-

25 biologic role, 20-24 inhibitory properties binding site, 12-20 constants, 10-12 experimental applications, 20 mode, 12 properties, 2-6 structure, 6-10 06-Methylguanine-DNA methyltransferases (MGMT), alkylation damage repair in genomes and activation, 128-129 properties, 116-118 regulation, 120-128 sequences, 128-129 N-Methylpurine-DNA glycosylase (MPG), alkylation damage repair in genomes and, 120-128 Monoclonal antibodies, protein tyrosine kinase substrates and, 216-222 Mycoplasm, bacterial adenylyl cyclases and, 44

Promoters, RNA polymerase I1 transcription initiation and, 94-98 Protein, enzymes in DNA precursor biosynthesis and, 183-186 Protein tyrosine kinase substrates, 205-207 detection of proteins, 207-208 identification, 215-222 oncogenes, 211-215 phosphorylation, 222-224 receptors, 208-211

R Replication, enzymes in DNA precursor biosynthesis and, 181-186, 189-191, 198-

200 Rhizobium, bacterial adenylyl cyclases and, 49-53 Ribonuclease inhibitor, mammalian, see Mammalian ribonuclease inhibitor Ribozymes, synthetic, antisense oligonucleotides and, 147-148 RNA polymerase I1 transcription initiation,

67-68 domains, 69-75

232

INDEX

motifs, 98-100 promoters, 94-98 repression, 102-105 structure, 68-69 transcription factors, 75-76, 93-94 activation, 100-102 TFIIA, 89-93 TFIIB, 81-83 TFIID, 75-81 TFIIE, 86-89 TFIIF, 83-86 TFIIH, 89

S Sequences alkylation damage repair in genomes and, 128-129 bacterial adenylyl cyclases and, 53-56 SNAIGE concept antisense oligonucleotides and, 145-146 limitations, 148-153 synthetic ribozymes, 147-148 triple helix, 145, 147 sense oligonucleotides and, 148 Substrates, protein tyrosine kinase, see Protein tyrosine kinase substrates

Sugar transport, bacterial adenylyl cyclases and, 36-41

T T4 dNTP synthetase, DNA precursor biosynthesis and, 177-181 Tolerance, alkylation damage repair in genomes and, 134-135 Transcription, bacterial adenylyl cyclases and, 32-34 Transcription initiation, of RNA polymerase 11, see RNA polymerase I1 transcription initiation Transferase repression, alkylation damage repair in genomes and, 127-128 Triple helix, antisense oligonucleotides and, 145, 147 Tyrosine kinase substrates, protein, see Protein tyrosine kinase substrates

V Vaccinia virus, enzymes in DNA precursor biosynthesis and, 199-200

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