Molecular Biology of the Gene, Fifth Edition

J

..

Molecular Biology of the Gene ~~~~· FIFTH EDITION

J . 1

I

•

l

l\

I

}

BAKER BELL GANN LEViNE

LOSICK

'

\

\

iS

.,

za.

~ ~

~ ...

-!

:I

~

e

0

;;; 0

c.

f'o:;n

f t;:

NtA

NS

Lys

• Chalo-tenninaling or •nonsense· codons

t Also used"' tlacteria to ~ 1tte ntiator tonnyJ-Met-tRNAINel

~ ..

11 t

'n'lE' &QSir.io t l:ase pili's.

~nd 1~

o(

~tu,

A

hydrogen bonds t;.et._, t he

NOT FOR SALE

'C:.!

• !

Molecular Biology of the Gene F

I

F T

H

E D I TI

ON

James D. Watson Cold Spring Hiitbot I..aboratory

Tania A. Bake r Massachusetts lnstitutP of Teclmology

Stephen P. Bell Massachusetts Institute of Technology

Alexander Gann Cold Spring Harbor Laboratory Pross

Michael Levine University of California. Berkeley

Richard Losick Harvard Universit y

--PE:ARSON

Ht"f\iamin

Cummim.!.S

NOT FOR SALE

Bet1jamin C..ummin,gs Publisher: Jim Smith As.~iflte

Project Editors: Alexand ffl F11Howes,

Jeanne ~les"ky Senior Production Editor: Corinne Benson Manufacturing Manager; Pmn Augspwger Senior Marketing Manager: Josh Frost Production Management and Text Design~ Elm Stl'eet Publishing Services, lnc. Mt!dia Development and Production: Scienr..e Tochnologies and David Marcey, CLU Questions for Website; Peter Follette Art Studio: Dragonfly Media Group Olmpositor: Progressive lofonnation 1echnologies Cover Image; Tomo Narashima

Cold Spring Harbor Laoo.-atory Press Publisher and Sponsoring &litor; John Inglis Editorial Director; Alexander Gann Editorial Development Manag*'r: ]11n Argentinft Project Manager and flcwdopmentall-::ditor; Kaaren Janssen Project Coordinntor: Maryliz Dir.ke£Son Editorial Oevelopment Assistant: Nora Rke Crystal structure images: leemor joshua-Tor Cover concept sketch: Erica Baade, MDC Grc1phics Cover Designers: Denise Weiss, Ed Alkeson

JSBN 0-321-22368-3

t..opyright © 2004 Pearson Educal iort, rnc.. publishing as Benjamin Cummings. t30t SansorneStroot. San Francisco, CA 94111 . AH rights reserved. Man ufactured in the United StAtes of America. This publication is protected by Copyr·tght anu permission should be obtained from the publisher prior to any prohibited rep1·oduction. storage in a retrieval system, or transmission in any form or by any means, electronic, mechanical, photocopying, recotding, or like wise. To obtain permission(s) to uSP. m aterial from this w ork. please submit a written request to Pearson Edur.ation, Inc., Permissions Departlllent. 1900 E. Lake Ave nu e . Glenview, IL 60025. For infon natton regarding permissions, call647/486/2635. Many of the designations used by manufacturers and sellers to distinguish their products are claimed Vaf,~e of 0 -0 Rd~se in Nudt:te Ac;:ul Synfhesis 64

Q -0 Spliu, Char.:tctetize Most Biosynthelic Reactions 65 Summary 67 Bibliography 6 7

Pos iriv~

llG 6 1 ACTIVATION OF PREClJRSORS IN GROlW TRANSFER REACTIONS 61 ATP Versatiliry in G roup Transfer 62 O

Coupling of Neg.ltive wirh

47

N T FOH SALE

~.

xiii

Detailed Contents

C HA P T ER

5

Weal< and Strong Bonds Determine Macromol~cular Structure 69 HIGHER..ORDER STRUCTURES ARE DETERMINED BY INTRA# AND

Different Protein Functions Arise frnm

INTERMOLECULAR INTERACTIONS 69 DNA Can Fom1 ;;J Regular Helix 69 RNA Form:s a Wide Varic£y of S tructutts

71

Chemical features ofPrOtt"tn Bvilding Bloch

71

The Pepti~ Bond 71 There A re Four Levels of Protein Structure 71 ex llclices an\.t J3 Sheers Are the Cumnton ronn~ of Seccln\.lh)'

180

16S

8

The Replication of DNA

181

THE CHEMISTRY OF DNA SYNTHESIS DNA Synthesis Requires Deoxynudcoside

182

T riphosphates and a Primer:Template Junction

182

DNA ls SynrhesizeJ by Extend ing the 3' End of rhe Primer I 83

Hydrolysis of Pyrophosphates ls the Dnving f orce for DNA SymhesL~

183

THE MECHANlSM OF DNA POLYMERASE 184 DNA Polymerases U:sc a S ingle Ac tive Site ro Catalyze DNA Synthesis 184

DNA Polymernses Resemble a Hand {bat Grips rhe Primer.Temrlare Junction 186 DNA Polymerases Are Prncesstve Enzymes 188 Exonudeascs Pr<x:trcad Newly Synthcsa.red DNA 191 THE REPLICATION FORK 192 Rorh Stronds of DNA Arc Synthesized Together at the Replication Fork

RNA Primers Must Be Remov.:pansiDn of Tritlle Repwt5

Excision Repair Enzymes Retr10ve Oanu:tgeJ

Bases by a Base·Ftipping Mechanism 248

Nudet)tide Excision Repair Enzymes deavt Damaged

Clluses Diseo.se 237

ON A on Either S i"d e of the lesion

250

M~match

Recombtnarion Repa irs DNA Breaks by Retriev1~ St-qucnce Information from Undamaged DNA 253

DNA DAMAGE 242

Translesion ONA Symhe;as Enables Rcplication

DNA Undergoes llamagc Spont Double~trand Break Repair Model More Accurately Describes Many

Recombination Evencs 264 Bux l 0-l Huw w Resolt.e a RocumhmatiJJn httermediate ~lith T~·o I fulliday ]unctiuns 266 Douhlc,Suaml~i DNA Breaks A rise b}· Numcruus Means and lnitiare Homologous Rccombinarion 267

HOMOLOGOUS RECOMBJNATlON PROTEIN MACHINES 268 The Red3CD l lelkase/Nudease Processes Broken DNA Molecules fo.- Rccombinatlon 269 RccA Protein Assembles on Single·Strancled DNA and Prom('l{esSmmd 1nvasion 272

Newly Base,Paired P~mners Are Established within rhe RecA Filament

Ret:A

Homol~

274

Are Prcscm

in All Ormmisms

275

RuvAB Complex Spedfically Recogn izts 1 JollidC'ly Junctions Rnd Promotes Branch Migration 276 RuvC C leaves Specific DNA Strands at the Holliday Juncrion ro hnish RL-comhination 276

C H AP 'I !-R

HOMOLOGOUS RECOMBINATION IN EUKARYOTES 278 Homologous Recombinat ion Has Additional Functions in Eukacyotes 278 Homo logous Recombination Is Required for Chrotnosorne Segregation Juring Meiosis 279 Programmed Generation of Double-Stranded DNA Rrcaks Occurs duri n~ Mcietailed Contents

EF-G Dr;vf'S Tnm:;location by Displacing the tRNA Bound to the A Site 445 EF-Tu ~C DP

GDP/GTP Exchange and GTP Hydrolysis Contro l the Function of the Cl~s 1l Rele-~se f actor 450 Tile Ribosome Recycling Factor Mimics a IRNA 450

aru.l Ef-G,QDP Must Exchang~ GDr

for GTP Prio£ to Partici{'atl~ in a New Rt1Und

of Elongauon 446 A Cycle o( Peptigenic Choice 52 1

MetR Acrivate:s Tr.anscripti.Dn by Twisting Promoter DNA 501

Growth Omdition:; of£_ coli C onrrol r.he Stabilety of C.U Protein and thus rhe Lytic/Lysogenic Chnic.e 5ZZ

Some Repressors Hold RNA Polymerase at the Promoter Rather rhan Excluding It

T ran~criptional Amat~nnination in }.. Development 523

Sit.es Far fiom the Gene

500

S02

AraC aru.l Contro l iJf the araBAO C)peton hl· Antiactivation 503 EXAMPLES OF GENE REGULATION AT STEPS AFTER TRANSCRIPTION INITIATION 504 Amino Acid Biosynrhetk Opemns Are O.Jnttolloo by Premature Transcriptio n Termirutti.on )().4 LHAP II: t
phila

Ate Organized in Specwl Chromosome Clusrets 627 MORPHOLOOICAL CHANGES IN CRUSTACEANS AND INSECTS 630 Arthropods Are Remarkably Diverse 630 Olanges in Vbx Exp~essiotl Explain Modifications in limbs aanong the Crustnceans 6l0 Why Insects Lade Ah:Jominallimhs 63 t Modification d Fltght Limb~ Might Arise from the Evt.llution of Regul
666

Genome-Wiul' AnAlyses 667 OJmparative Genome Analysis 669

PROTEINS 672 Specific Proteins C..an Be Purilied

from C ell Extmcts 671 Purilic~ tion of~ Protvercd in C . d t."gllns

Bacteria Exchange DNA by Sexual C--OnJugation, f'hage,Mediated Transductit'ln, a nd DNA-Mediated Transformatt~..m 688

Mut dotninant allele is represented by t1 capital let t~r ancl the r(.-ccssivf" nJle1e by the lowercase lett~r. It is important to notice that ~gi ven gamete contains only one of the nvo copies (one a llele} of the genes p resent in the organism it comes from (foe example, either R Of' r, but never both) and that lht:l two t}'pes of gametes ace produced in equal numbers. Thus . there is a 5()-.50 chant.:e that a given gamete from an F 1 p£'.a will contain a particular gene (R or r}. This choice is purely .random. We do not expect to fillC:l exoct 3:1 rati o~ when wa examine a limited n umber ofF2 progeny. TbP. ratio will sometimes be sl ightly higher and <Jther t imea slightly lower. But as we look at increasingly larg~r samples. we expect that the ratio of peas with the dominant trait to peas with the recessive trait will approximate the 3:1 ratio more and more closely. The reappearance of the recessive characteristic in the F2 generation inrlicates that recessive alleles are neither morlifled nor lost in the F 1 (Rri gen eration , but that the dominant and reces.-dvf:l genes are

parenlal genf31'ation RR

rr

t

t r

R

Rr 1

r

(

,.

Rr /

,.

Rr /

rr

/

FIGURE 1- 1 How Menders first Jaw

(ll1depeftclent MS~l ~$ lhe l!l fat"IO of dominanliD recessive phenotypes CII'I\Oftllhe F:t progeny. R reptesa1CS ~ domnant gene: a"'f3 r the recesSIVe gene The ftU'1d seed repre>eflts the domi~ phenotype.. the wnnlded seed ~ recESSive phenotype

8

7'/ie Mendelian View of the World

independently transmit1ed and so are able to segregate independently during the formation of sex cells.. This principle of independent segregation is frequeptly referred to as Mende t•s first law.

parental gooeralion

X

Some Allel~ Are Nt!ither Dominant Nor Recessive

A

gametes

ln the crosses reported by Mendet on e member of each gene pair was deady dominan t to the other. Such behavior, however, is not universaJ. Sometim~ the heterozygous phenotype is intermediate between the two homozygous p h enotypes. For example, the cross between a pure-breeding red sn apdragon ( Anti1rhinum) and a purebreeding wh ite variety gives F1 progeny of the intBJ"mediate pink color. Tf these F1 progeny are crossed among themselves, the resulting F2 progeny contain red. phik, and white flowers in the pro portion of 1:2:1 (Figure 1-2). Thus, it is possible here to distinguish heterozygolcs from homozygd cs by their phenotype. We aJso see lhat Mendel's laws do not depmd on wh~ther one allele of a gene pair is dominant over the other.

a

(

F1 genernliorl

f A

l

Aa 1 gametes

t a )

A

Principle of Independent Assortment

>::male

f I CURE 1-2 1be inheritance of flower color in tfle snapdragon. One parent is homozygous b red flowas (AA) and the

other- homozygous for white floll\"efS (oa). No domoance IS present and the heterozygous F\ flowers ae pink. The l :2 : 1 ratio of red, p1nk, and white l"lowef5111 the r, j:Yogeny lS shown by ~ropnate colonng..

Mendel extended his breeding experiments to peas differing by more than on e characteristic. As before. he started with two strains of peas, each of whic..h bred pure when mated with itself. One of the strains had round yeHow seeds; the other. wrinkled green seeds. Since round and yellow are dominant over wrinkled and green. lbe entire F1 gene.rJ ation produced rormd yellow seeds. The f 1 generation was then crossed within itself to produce a number of F 2 progeny. which were examlned for seed appearance (phenotype). In addition to the two original phenotypes (.round yellow: wrinkled gree n). two new types (recombinants} emerged: wrinkled yellow and rouTJd green. Again Mendel found he could interpret the results by the postulate of genes. if h e assumed that each gene pair was indepcndtmtly trans· mitted to the gamete during se.x-cell for-mation. This interpretation is shown i n Figure 1-3. Any one gamete contains only one type of allele from e.Bch gene pair. Thus , the gametes p roduced by an F1 (RrYy} will have the composition Rl~ Ry, rY. or ty, but never Rr, Yy. l'Y. or RR. Furthermore, in this example. all four possible gametes are produced with equal frequency. There is no tendency of genes arising from one parent to stay together. As a result, the Fz progeny ph enotypes appear in the ratio nine round yellow, three round green, thn~e w rinlded yellow, and one wrinkled green as depicted in the Punnett square, named after the Rcitish mathematician who introduced it, in the lower part of Figme 1-3. This principle of independent assortment is frequently called Mendet-s second law.

CHROMOSOMAL THEORY OF HEREDlTY A principal reason foe the original failure to appreciate Mendel's di~­ covery was the absence of firm facts about the behavior of chromosomes duri.ng meiosis and mitosis. This knowledge was available, however. when Mendel's lows were confirmed in 1900 and was seized upon in 1903 by American biologist Walter S. Sutton. Jn his dassic paper ..ThP Chromosomes .in Heredity," Sutton emphasized the importance of the fact tbat the diploid chromosome group consists of two

GenB Linlroge cmd Crossing Over

parental gereralloo

f IGURE 1-3 How Mendel's second

>
. The Rand Y aleles are dam· nart <Wef r arld y. "fue genotypeS of the vanous parents and proge!ly are m.cated by letter ~.and four diHerent phenotypes are disllnguJShed by wnprtate shadirlJ.

~

.PY

"'

ry

gametes

l

)

J

F1 genemlion

Rr'ly

' '

RV

l

F2 generation gametes

•

Ry

'

rY

gametes

l

9

'

~ ry

J

morphoJogica11y similar sets anrl th at, during meiosis, every gamete .reeeives only one ch romosome of each homologo us pair. He then used this fact to explain Mendel's results by assuming that genes are parts of the chromosome. He postulated that th e yellow- an d green-seed genes are carried on a certain pair of chrom osomes a n d tha t the round- and wrinkled-seed genes are carried on a different pair. This h ypothesis immediately explains the expeTimentally ohservP.d 9:3:3:1 segregation :ratios. Although Sutton's paper did not prove the chromosomal theory of heredity. it wa s immensely important. for it brought together for the first time the independent disciplines of genetics (the study of breeding experiments) and cytology (the stt.tdy of cell structure).

GENE LINKAGE AND CROSSING OVER Men der s principle of independent assortment is based on the fact that genes located on different chromosomes behave in dep endently during meiosis. Often. however, two gp.nes do not assort independently because they are located on the same chromosome {linked genes: see Box 1-2, Genes Ar~ Linked to Chromosomes). Many

Box 1-J ~Ale linked to Chromosomes

lnitialy, ~ breeding experiments used genetic dfferences a..eady ergan and his ~ng colabaatO"S, geneticists Calvin B. Bridges. 1-lerrnam 1 Muler, and Alfred H. Stutevant. They wotked ~ the tW-ty fly Drosophi1o me/anogaster. The r«st 1Tlt.Jt3nt found WdS a male with Vvtlite eyes instead of lhe normal red eyes.. The V'hte-eyed varian£ appeared spontaneously in a Cl.Jinxe bottle of

a

red-eyed flies. Because essertlaly dJ Drosophila foood ., nature have red eyes, the gene leadng to red eyes was referred to as the wi1d-type gene; the gene leadilg to white eyes 11\'tJS caled a mlMlt gene (aUele). The Vutute-eye mul'i.lnt gene was immedidtely used in breeding expesiments (8oK 1-2 f.g~e 1), wifh the stribng result that the behavior of the aneJe completely paralleled the cistribution of an X chromosome (that 1s,. was sex..Jinked). This finding immediately suggested that this gene might be located on the X chromosome. together with those genes controlling sex. This hypothesis was q.ndfy confirmed by addruoncA genetic crosses using newly ISOLated mutant genes. Many of these adrlitiONI mu~t genes also were sec-linked b

parental getlef< abc. six recombinant genotypes are found (Figura 1-7}. They fall into three groups of reciprocal pairs. The rarest of these groups arises from a double crossover. By looking for the least frequent class, it is often possible to insta ntly confirm (or deny) a postulated arrangeme nt. The results in Figure 1·7 inunediately confirm the ocder hinted at by the two-factor crosses. Only if the ord er is a-c-b d oes the fact that the rare recombinants are AcB and oCb make sense. The existence of m ultiple crossovers means that the amount of recombination between the outside markers a and b (ab) is usually less than the sum of the recombinAtion frequencies between a and c (cw) anrl c and b (c o). To obtain a more accurate approximation of the distance between t he outside markers. we caJculate the probability (oc X cb) that when a crossover occurs between c and b. a crossover also oc:x:utS between a and c, and vice versa (cb X ac). This probability subtracted from the sum of the frequ encifls expresses more occmatPJy the amount of recombination. The sU:nple formula ab:::::: or

~-

cb - 2{ac)(cb)

is applicable in all cases where the occurrence of one crossover does not affect the probability of anoth(),r crossover. Unfortunately,

1-6 Assignment of h tentative order of thtee g8\es on the basi.s of three two-factor crosses.

30%

f I c; U It l

25%

a

c

b

ChromOS(lme Mapping A

c

B X

tJ

A

c

a

c

A

c

a

c

l ~

67 5%

B

A

c

b

a

c

B

..

A

c

b

a

c

' l

b

c

1 B

A

c

b

a

c

b

A

c

B

a

e

~

!

B

A

c

tJ

a

c

b

A

c

B

a

c

7.5%

22.5%

lhi:> otass anses 'from a ~:~rea~< us Purple Speck

Dachs E.xtra vein

Wing Leg \lenat100

Frtnged

Wing

Strap Streah Trefoil Truncate Vest.glal

Curved

Wli'Q Abdorrlnal band Ctmmosome2

Omrr.at.Oa Body color VenatiOn Eye color Thorax n-ark

W1119 Pattem Panern Wing

Wtng

Group3

Band

Panern

flank

Eye colOr

Beaded Cream Ill Oef01med

Wing Eye cob

Floug)

Eye

Safrann

Eye

Sep.a

Dwarl El:lcrly

&ze of t:xxty Bodyeo40r

Giant

Sazeof body

Sooty Spirefess Spead Tndent

Eye color Eye color Body color Sptne Wrng

Pattern

K1dney

Eye

Lovv Ct0SS1n9 0\lef

Truncate

Maroon

Chl'omosome3 Eye color

Peach

Eye COlor

~te ocelli

WKlQ Pattern S.mpleeye

Wing

Eyeless

Eye

Wtitehe&:l

Groop4 Bent

*TI1e mAal ens laU 111to kxxmkage IJOIJPS Smcetourchrornosomeswere cy101ogicai!Yobsefved h s lnQcaled that !he geo"eS &e silueted on thtl m arrosomes Notice thai nua~ 11'1 vant1t1s yenes can acllo alter a srrve chw"acto. 5\Jch a:. 1>00y COlor tn difterenl ways.

The Origin of Gtmelic \/Driubility through Mutation~

~5

nonnal

Ted eyes

StraJgt\t

Sf~ht

wings

wings

long wings

tong

red e yes

1egs

104

99.2

~

~

B

long ~rae

'• ,

• •

_;;t:;;

long wings

75.5

-======= =t="" C

61

.t

vg

54.5

48.5

•

b

pt'

-t.-

3i 13 ~ ==== -l d dp

0

~

al

' 1.,

black

dad'ls

body

(shofl tegs)

mutant

THE ORIGIN OF GENETIC VARIABILITY THROUGH MUTATIONS It now became possible to understand the hereditary variation that is found throughout the biological world and that fonns the basis of the theory of evolution. Genes at'c normally copied ~xactly during chromosome duplication. Rarely. however. changPS (mutations) occur in genes to givfJ rjse to altered forms~ most- but not oil- of which ftmction less well than the wUiHype alldPS. This process is necessarily rare; otherwise, many genes would be changed during every cell cycle, and offspring would not ordinarily resemble their parents. ThHe is, instead, a strong advantage in the.re being a smaJI but finite mutation rate; it provides a constant source of new variability, nocessMy to allow plants and animals to ndapt to a constantly c ha11ginp. ph}' sical and biological cnvironmont. Surprisingly. h owever, the results of the Mendelian geneticis ts were not avidly seized upon by ihe classical biologists. then the authorities on tbe evolutionary relations between the various forms of life. Ooubts were rajsed ahout whether genetic chsng~ of thP. type studied by Morgan a nd his students wr.re sufficiP.nt to permit the evolution of radically new structures, like wings or eyes. ln~t£'ad, these biologists believed that there must also occur more powerful .. macromutations," and that it was these events that .allowed great evolutionary advances. Gradually, however, doubt!; vanished. largely as a result of the efforts of the mathematic:a) geneticists Sew all Wright, Ronald A. Fisher, and John Burden Sanclernon Haldane. ThP.y shownd that, consid~ring the great .age of Earth, the relatively low mutation rates found for Drosophila genes. together with only mild selective advantages, would be sufficient to allow the gradual act-'Umulation of new favorable attributes. By the 1930s, biologists began to reevaluate their knowledge on the origin of species and to understand tbe work of the mathematical gen~licil)ts. Among th~se new Darnrinians were biologist Julian 1luxley

dumpy wings

aristatess {shOO Sfistae)

(a grandson of Darwin's original publicist, Thomas Huxley). geneticist Theodosius Dobzbansky. paleontologist George Gaylord Simpson, nnd ornithologist Ernc;t Ma}'T. ln the 1940s all four wrotP. major work.c;. each showing from his speciAl viewpoint how Mendelianism and Oarwinism were indeed c.:ompatible.

EARLY SPECULATIONS ABOUT WHAT GENES ARE AND HOW THEY ACT Almost immediately after the rediscovery of Mendel's laws, geneticists began to speculate about both the chemical structure of the gene and the way it acts. No real progress could be made. however. because the chemicnl identity of the genetic material remained unknown. Even the realization that both nuclek acids and proteins are present in chromosomes did not really help, since lhe sttuclure of neither was at aU understood. The most fruitful speculations focused attention on the fact that genes must be, in some sense, self-duplicating. Their structure must be. exactly copied every time one chromosome becomos two. This f.\cl immediately raised the profound chentica l question of how a complicated molc:culr~ could oo precisely {X)pied to yield e act replicas. Some physicists also beaune intrigued with the gene. and when quantum mechanics i"l•trSt on the scene in the )ate 1920s, the possibility arose that in order to understand the gene. it wou ld first be necessary to roast'U" the suhtleties of the most advanced theoreliral phys i ~. Such tbottghts. however, never really took root. since it was obvious lhat even the best physicists or theoretit:al chemists would not concern themselves with a substance whose structure KtiJJ awaited elucidation. There was only one fact that they might ponder- Muller and L J. Stadler·s independent 192 7 c:liscoverlcs that X-rays induce mutations. Since there is e greater possibility that an X~ray will hit a Jaeger gene than a smaOer gene, the frequency of mutations induced in a given gene by a given X-ray dose yidds an estimate of the size of this gene. But even here, so many special assumptions were required that virtually no one. not even Mullf!f and Stadler themselves, took thfl estimates ve ry seriously.

PRELIMINARY ATIEMPTS TO FIND A GEN&PROTEIN RELATIONSHIP The most fruitfu l early endeavors to find a relationship between genes and proteins examined the ways in which gene changes affect which proteins are present it1 the cell. At first these studies were difficult, since no one knew anything about the proteins thai were present in structures such as the eye or tl1e wing. ft soon becaTJ'I(! clear that ge nes wilh simple metabolic functions would be easier to study than genes affecting gross stntctures. One o f the first usefl.t] examples camts from a study of a hereditary disease affecting amino acid metabolism. Sponta~ neous mutations occur in humans affecting the ability to metaboJize the amino acid phenylaJanine. When individuals homozygous for the mutant trait eat food containing phenylalanine. their inability to convert the ~unino acid to tyrosine causes a toxir. levcJ of phenylpyruvic acid to build up in the bloodstream. Such diseases, examples of "iuborn errors of metabolism," suggested to English physician Archibald

SUITlJTlOry

17

E. Garrod, as early as 1.909, that the wHd-type gene is responsible for the presence of a parth::ul;:tr enzyme. nnd that in a homoeygous mutant . the enzyme is congenitall )' ~bsent. Gal-rod's general hypothesis of a gene-enzyme relationship was extendfld in thfl 1930s by work on flower pigments by Haldane and Rose Scolt-Moncrjeff in England. studies on the hair pigrnenl or Jhe

guinea pig by Wright in the United Stales, and research on the pigments of insect eyes by A. Kuhn in Germany and by Boris Ephrussi and GeoJge W. BeadJe, working first in France and then in California In all cases, the evidence 1-evealcd that a particular gene affec1ed a particular step in the formation or the rer-.pective pigment whose ~ence changed, say, the color of a fly's eyes frool red to ruby. However, the lack of fundamental knowledge about the structures of the relevant enzymes r uled out deeper examination of the gene-en z.yme relation· ship~ and no assurance could be given either that most genes control tbe synthesis of proteins (by then it was suspected thai all enzymes were proteins ) or that all proteins am undnr gene control. As early as 1936, it became apparent to the Mend elian gen eticists

that future experiments of the sort successful in elucidating the basic features of Mendelian genetics were unli.k ely to yield productive evidence about how genes act. lnstead. it would be necessary to find biological ob}eoo 2 ( shown 10 red) In OOdltior~ RNA has the pynmidine base tJdifted l'llJcieostdes exiSt 11"1 the sttl.JCll..n>· ~ ; pseudouridine. T - tix>thymidlf'lE:, DHU = 5,6-0lhydr~, l = ~. m'G = l-rnethylguanosme. m' - 1~10sine. and m7G -= N,Ndmettm~

34

fI G U R IE

Nucleic A.c ids Convey Genetic Information

2•15

Transcription and

translation. The nucJeotidt:>s of mRNA are a5Sef1lbled to fonn a canplement&y cq>y of ooe strand of DNA Each group or three 15 a codoo that is complernel1tary to a grot..p (lf 1htee nudeol:ldE:s in the anticodon region (lf a specific tRNA rno~ec~Ae. When bdse pairtng OCOJTS, an amino arid canied at tlle other end (lf tt1e tRNA molea.Ae IS added to the growing protein chain.

•., CGic~ cc iiOC~ cc Uu

13'

mRNA

l

8fT1inO acid

(rigure 2-17}. In bacteria, the same enzyme makes each of the maj{)r RNA classes {ribosomal. transfer, and messenger). u sing appropriate segments of chromosomal DNA as their templates. Direct evidence that DNA lines up the corrnct ribonucleotide precursons came from seeing how tbe RNA base composition varied with the addition of DNA molecules of different AT/GC ratios. ln every enzymatic synthesis. the RNA AU/GC ratio was roughly similar to the DNA AT/GC ratio (Table 2~2).

F I GU RE

2-16 Diagram oh

polyribosome. Eadl fibosome .maches a1 a start signar at the s· end of an mRNA chain end synthesizes a pclypeJl(icle as it proceeds along me molerule. Several nbosomes f"lla'Y be a~ to OCle rnRNA

molecute at one time;

1he emire assembly is caled a polyrbJsome

ril:!osoo"e subunits

released

The Celllf'al Dogmo

site of nt.lCiectide addiliof\ to growing RNA strand

Ft G U R E

35

2-17 Etuymatic synthesis of

RNA upon it DNA temp~. Qdalyred by

RNA polymerase-

During transcription, only one of the two strands of DNA is used as a template to make R""lA. This makes sense, because the messages carried by the two stranrls, being complementary blJt not identk.al, are expected to code for completely different polypeptides. The synthesis of RNA always proceeds in a fixed direction, beginning at the 5' end and concluding "'rith the 3'-end nucleotide (see Figure 2-17}. By this time, there was firm evidence for the postulAted movemenl of RNA from the DNA-containing nucleus to the ribosome-containing cytlasm. (Source: Coortf'sy of O.M. Prescott. U~rSity of Cdorado Medici!! School: reproduced from 1.964. Ptogr. Nucleic Acid Res. Ill: .35, With perrrjsroo.)

TABU

2-3 The Genetic~ second position

uuu we WA UUG

F'tle

leu

cuu

cue ~

0

= r.o

-

CUA

ucu ucc

Ser

UAC

UCG

1m I!1S

ccu

CAU

UCA

CCC Lev

UAU

CCA

CAC

Pro

CAA

ax;

CCG

CAG

AlJU

PO)

MU

Tyr

lJGU

Cys

UGC

$lcp

1!1!.1

stop

UGG

His

an

CGU CGC

CGA

Alg

()

~

E

I;:

AlJC

le

PC-A

NJA AUG

Met

GUU

G\JG

Tk

Val

APe AAA

/lCG

A./>13

GCU

GAU GAC

GCC

GUC

GtlA

N:.C

GCA

GCG

Ala

AGU Asll

lys

~

/1.00

Ser

Arg

GGU Asp

GGC

GAA

GAA

GAG

AGC

G1u

GGG

-a

::t'

eGO

Gty

Eswhlishing the Direction of Prorein Synthesi&

37

ESTABLISlUNG THE DIREC"'TION OF PROTEIN SYNTHESIS The nature of the genetic code. once determined. led to further questions about how a polynucleotide chain directs the synthesis of a polypeptide. As we hi!Ve seen here and shaH discuss in more detail in Chapter 6, polynucleotide chains (both DNA and RNA) are synthesized in a 5' - 3' direction. But what about the growing polypeptide chain? Is it assembled in an amino-tenninal to carboxyl-terminal direction. or the opposite? This question was ;mswered in a classic experiment 'in which a ceJl-free system was u sed for carrying out protein synthesis. The call-free system was cceated u sing an extract from immature ted blood cells (known as reticulocytes) from a rabbit. which are efficient factories for the synthesis of the n-and {:\-globin subunits of hemoglobin. The cell-free system was treated with a radioactive an1ino acid for a very few seconds {less than the time required to synthesize a comple te globin chain) after which (Jrotein synthesis was immediately stopped. A brief radioactive labeling regime of this kind is known as a pulse or puke-labeling. Nex1. globin chains that had completed their growth during the period of the pulse-labeling were separated from incomplete chains by gel electrophoresis (Chapler 20). The full-length polypeptides were then treated with an enzyme. the protease trypsin, that cleaves proteins on partic ular sites in the polypetide cha in ~ thereby generating a series of peptide fragements. ln the final step of the e:xpe.riment, the amount of radioactivity that had bP.en incorporntad into each peptide fragment was measured (Figwe 2 -1 9).

a

•

~

NH2

:=

(

FtGU RE

COOH

)

2·19

into • growing MPePtide main. lhe expern-nental detaits Me described lf"' the text {a) DrstfilxJtloo of racioac:bvity amoog ~ted chains after a short peood of labeling. (b) lncorptXation of label plotted as a func1ion of postioo of the peptide Wthin the c~ed chaifl.

b

• • ~

•

·;;;:

'5

"'

•

0

'6 ~

• • NH2

COOH position of peptide

lnmfpOritlion of label

Keep in mind that the globin chains were at various stages of completion during the period of t he pulse (Figure 2-19a). Thus, nascent chains that had only just started to be synthesized would be unlikely to have reac hed cmnplatiun during the period of the pulse because the time of the pulse-Labeling was less than the time required to synthesize a complete globin chain. On the other hand, globin chains that we.r e almost full length would be highly likely to have reached completion during the pulse. Also, keep in mind that only chains that had reached full length during the time of the pulse were isolated and subiected to trypsin treatment. lt. therefore, fol lows that the b'ypsin-generated peptides w.i th the leas t amou.nt uf radioacrive amino acid (normalized to the ~e of the peptide) should have derived from r~ions of the globin protein that w ere the first to be synthesized. Conversely. peptides with the g.reatest amount or raclioac• ivily BhouJd hat•e derived from regions of the protein that were the last to be synthesized. The results of the experiment are shown in .f'igure 2· 19b. As you can see. radioactive rnbeling was lowest foc peptides from the aminotennina l region of globin and greatest for peptides from the c.a.rboxylterminal region. We. therefore. conclude that the direction of protein synthesis is from the amino-terminus to the Girhoxyl-tenninus. rn other words. during protein synthesis the first amino add to Le incorporated into the nascent chain is the amino acid at the amino terminal end of the protein and tbe last to be incorporated is at the carboX)•l-terrninus.

Stan and Stop Signals Are Also Encoded within DNA Initially, it w as guessed thttt transll'! tion of an mRNA molecule w ould

commence a t one end and finish when the e ntire mRNI\. message had been read into amino acids. But, in fact. translatjon both starts and stops at internal positions. Thus. signals must be pTcsent wilhin DNA (and its mRNA products) to initiate and tenninate tra nslation. First to be workHd out w ere the stop signals. Three seperete coclons (UAA. UAG. and UGA). first .known as nonsense l:odons, do not d ired the addition of a varticular amjno acid. Instead, those codons serve as translational stop signals (sometimes called stop codons). More cornplicared is the way translational start signals arc encoded. The amino acid methionine starts all polypeptide cltains. but the trlpl(lt (AUCJ th~r c..od es for thP.se initialing mP.thionjnes a lso codfls for methionine residues that have internal locations. The AUG codons. at which polypeptide chains start, are p.rf1(Jeded by specific purine-rich blocks of nucleolides that serve to attach mRNA to ribosomes (see Chapter 14}.

THE ERA OF GENOMlCS With the elucidation of the central dogma, it became clt>.ar by the mid-Hl60s how the gerwtic blueprint contained in the nudeotlde

sequence could determine phenotype. This meant that profound insights into the nature of living things and their evo)uti.on would be rever~ led

from DNA SefJUencP.s. Jn recent yr.ars the Rdvent of .rapid. automated DNA sequencing methods has led to the dett'rmi nation of

Summary

39

complete genome sequences for a wide variety of organisms. Even the human genome, a s ingle copy of which is composed of more them 3 bi Ilion base pairs, has been elucidated and shown to contain more than 30,000 genes. During the upcoming years. many more complete genome assemblies will be available from a broad spectrum of organisms, including puplars. sponges, jellyfish. crustacflans, stta urchins, frogs, and dogs. ln the future it should be possible to extend the interpretation of genome sequences beyond the identification of genes and their encoded proteins. Other classes of ONA sequences mediate replication. chromosome pairing, recombination. and gene regulation. rt is possible to envision a day when comparative DNA sequem!e analysis will reveal basic 1nsights into the origins of complex behavior if1 humans, such as the acquisition of language, as well as the mechm1isms Wlderlying the evolutionary diversification of animal body plans. The purpose of the forthcoming Ghapters is to provicle a firm foundati o n for unde.rstandl.ng how DNA functions as the template for biological comple.xity. The remaining chapters in Part t , review the basic chemistry and biol<Jgy relevant to the main themes of this book. Parl 2, Maintenance of the Genome, describt'ls the structure of the genetic material and its faithful duplication. Part 3, Expression of the Genome, shows how the genetic instructions contained in DNA is converted into proteins. Part 4, Regulation. describes strategies for differential gene activity that are used to gone.rate complexity within organisms (for example, embryogenesis) a11d diversity among organisms {for example. evolution). Finally, Port 5, Methods, describes Vaiious laboratory techniquP.s, bioinformatics approaches, anrl mode) systems that are commonly u~ed to inv.-. stigate biological problem. uf badttria could be gt~t tetlaiUy tr'~nsformcrl 'With a f'ubstance derived from a hoot-killed pathogenic .stta.in. Avery. McC~rty. a11d Macleod subRequently demonstrated that the transforming substam·e was DNA. Furtl1er evidenc~ tha.t DNA is the genetic material Who~ Building on Chargaff's ruJ~ and franklin and Wilk.iru;' X-ray di.ffrac:tiot1 studies, Warson and Crick proposerl a douhle.h,elkal structure of DNA. In this mode), two polytJucle(Jtide chair~s are twisted around each other to form a regular cloubltl helix. The two ch~int~ within the double helix are bPlcl togp,ther by hydrogen bond~ between pairs of ~~~es. Adeni.ne is alw~ys joinc~d to chymine. and guanine is aJways bondoo to cylo8ine. The e~isteoce of the base pairs means that the sequence of nucleotide5 a long the two mains are not identit;aL but t:omp i~MT~entary. 'fhfl finding of lhi..c; relatiunshtp suggested a mechanism for the replication of DNA in whir:h each ruanrl sl:li'Ves as a tern plate foe its complement. Proal for Uti!' hvpothesls Cl:ln~ from (~) the ol>servation of

MeseJ~n

and Stahl that the two strands of each double he-

Ux separate during each round of DNA replication, rable negative charge, whereas the two hydrogen atoms together h(lve an equal amotmt of positive charge. The center of the positive charge is on one side of the center of the negative charge. A combination of separdted positive and negative charges is ctive binding force at physiologk..al temperatures only whell several atoms in a given molecule are bound to several atoms in another molecule. Then the en~rgy of interaction is mm;h greattH' than the diSS<X:iating tendf'.ncy resulting from random thermal movements. For several atoms to inl~r­ at:t effectively, the molecular fit must be precise, since the distance separating any two interacting atoms must not bo much greater than the sum of their van dcr Waals radii (Figure 3-6). The strength of interaction rapidly approaches zet'O when this distance is only slightly exc~ded. Thus, thP. strongest type of van der Waals contact arises when a molecule t:ontains a cavity exactly compfemcntary in shape to a protruding group of another molecule, as is the case with an antigen and its specific ;mtibody (Figure 3~7 ) . In this instance, the binding energies sometimes can be as large as 20 to 30 kcal/mol , so that antigen-antibody complexes seldom faiJ apart. The bonding pattern of polar molecules is rarely dominated by van dar Waal5 intf!ractions. sinue such molecult>s can acqv ire a lower energy state (lose more free energy) bv forming other types of bonds.

I ABLE

47

3-2 \WI dft waals Radii of the Atoms in Biologiall Molec:ules der Waals radius (A)

tl8fl

Atom H

12

tv

1.5

()

1.4

p

19

s

1 85

CH3 group

20

~If

17

tflid

..

b

L...J

1A

l-11 f)Wnple$ of \'an~ Waals (hydrophobic) bonds between the nonpolill" side lf0Cip5 of amino adds. The hydrogens are not thcfecated lf"ldMdt..raty_For the sk of clarity, the van d€f Waals 001 are reduced by l()Ofo. The strlJOural fO!i'l'lulas adJ(!Cef'lt to each space-filling dra\Ning indicate the Clllaflgemenlof lhe atoms (a) Phenylalanc~euane bond. (b) Pheflylafanl,-,ephen~tfle bond (Soun:e· Adapted frorn Sd1eraga HA. T1le proreins, 2nd e&t.on p. 527. Copyright © Harold Schffilga. U'X'd W1 permiSSIOn.) fIG U R l

third molecule that h as a surface complementary to alanine. A methyl group is present in alanine but not in glycine. When alanine is bound to the third molecule, the \an der Waals contacts around the methyl group yield 1 kcal/mol of energy. whkh is not released when gJycine is bound instead. From Equation 3-~. we know that this smaJJ energy difference alone would give only a factor of 6 between the binding of alanine and glycine. However. this their products. by mom powerful bcm(ls, they woulcl act much mom s1ow1y.

Weak Bonds Mediate Most Protein:DNA and Protein:Protein Interactions As we wiU see throughout the book. interaeticms bernreen proteins and DNA, and between proteins and other proteins, lie at the h.Se weak bonds can .~:esull in " stable aggll!gate. The (act that doubl&-helical DNA never falls apart spontaneously demonstrates the extrerne stability possible in such an aggregate.

BIBLIOGRAPHY General References Brandon C. and Tooze J. 1999. Introduction to proLflin strl!cture. \.,arlaud Publishing. New York. Creighton T.E. 1992. Proteins: Struc.ft.jfP. (,md mnlecJJlur properties. 2nd edition. W. H. FreP.rnan. New York. - - - 1983. Pmtoins. heeman" San Francisco. Donohue J. 1968. Selected topics in hydrogen bonding. In Structural chemistry ond molcculw b1ol~·· (ed_ A. Rlch and N. IJavirlson). pp. 443- 465. Freeman, San Francisoo. Fersht A. 1999. S tt·ucture cuuf mcchUI11sm in prolein science· II RUide to enzyme rotolysis und protein {oldir1g. W.H. freeman, New York. Cray lLB. 1964. Electrons afld chemical bondillg_ EenJ810in Cummings, Menlo Jlaik. California. Klotz f M . 1 oo:;. Enorgy dwn~s in biuchelmCul r eUcfl()llS. AcarlefTiic Pre$, New York. Kyle }. 1995. MecllUilism in protein chemif>try. Carland Publishing, New York.

- - - 1995. Struct1.1re in protein chemistry. Carland

Publishing. New York.

LehtJinger A.L. 197 1. Bioenergetics, 3rd edition. Benjamin Cummings, Menlu Park. California. Lesk A. 2000. Introduction Eo prol t::itl orchilec.ture: The slnJClural biology of proleins. Oxfurd Univorsity Press. New York.

Marsh R.E. 1966. Some comments on hydrogen bonding in purine and pyrimidine bases. In Structural chemistry ond molecular biology (ed. A. Rich and N. Davidson). pp. 485- 4a9. Freeman. San Francisco.

Morowitz H.J. 1 970. Entropy fot biologists. Academic Press, New York Pauling L. 1960. The flature of the ch,.~mical bond, 3rd edition. Cornell Univen;ity ~-.s.1thaca. New York Tinoco r. (ed.). Sau E'.r Iitive IJ.G, the other \1\tith a negative AG. Instead. a coupled reaction is achieved by two or

more successive reactions. These are aJways group-transfer reactions: reactions. not involving oxidations or reductions, in which molecules exchange functional groups. The enzymes that catalyze these reactions are called transferases. Consider the reaction (A- X) ·f- (B- Y) - - (A- B)

+ (X-

Y).

(Equatioo 4-8]

In this example. group X is exchanged with component B. Crouptransfe r reactions are arbitrarily defined to exclude water as a partacipant. When wa ter is involved , (A-B) + (J 1-GH ) -

( A-QJ-I)

+ (B-H).

(Equation 4-91

This reaction is called a hyd rolysis, and the enzymes involved are called hydrolases. The group-transfer .reactions that interest us here are those involvi ng groups attache«l by high-energy lxmds. WhP.n such a high~en ergy group h: lronsf~rrcd to a.n a ppropriate acceptor molec'.lle, it becomes attached to the occoptor by a high-energy bond. Croup transfer thus aJlows the trans fer of high-energy bonds from one molecule to dn othc r. For example, Eqttations 4·10 and 4·11 show how energy present in ATP is transferred to form GTP, one of the prccwsors ~d in RNA synthesis:

AdP.nosine-0-Q ... @ + Guanosine-&~ Adenosine-0-0 + Guanosine-0 - 0 Adenosine-0-&-~ + Guanosine - G - O Adenosine- 0-G + Cuanosine-0-G-0.

~uation

4-101

JEq. 4-ttl

The high-energy 0- group on GTP a llow s it to 11nite spontaneously with a nother molecule. t."TP if; thus an example of what is called an aclivated molecuJe; corr~ pond ingl y. the process of transferring a high· energy group i'l CR11ed group activation.

ATP Versatility in Group Transfer ATP synthesis has a k ey cole in the controlled trappins of the energy of molecules that serve as energy donors. ln both oxidative and photosynthetic phosphorylations, energy is used to synt hesize ATP from A OP am] phosphate: Adenosine--G-O +

0 + energy -

Adenosine-0-0-0 IEqualion 4-12)

Because ATP is the original biological recipient of h igh-energy groups, it m uf;t bo thr: 1\tarting point or a variety of w..actions in which highenergy groups are transferred to low~nergy molecu1cs to give them the potentia l to react spontoneou.c;;ly. ATP·s contra! role u tilizes the fact that it conta ins two high-<morgy bonds whose splitting releases specific groups. T1tis is seen in Fi~ 4-4, which shows three important groups arising from ATP: 0-G. a pyrophosp hate group: - AMP, a n adenosyl m onophosphate group: a nd -0. a phospha te group. ft is important to notice that theso high-energy groups reta in their highenergy quality only when transferred to an appropriate acceptor group to a molecule. For example, a lthough the transfer of a COO group yields a high -energy COCJ-9 acylphosphate group, the tra.nsfer of the same group to a sugar hyd•·o:xyl group (- C- OH), as in the fonnation of g lucose-6~ph os phatc. gives rise to a low-ene rgy bond (less than 5 kcal!mol decrease in ~G upon hydrolysis).

Ac;ti~·olion

fl/ Frecum1rs m Croup Tronsfrr Re(Jdions

ATP

R- C

0

4-4 inwofma ATP. F I (i U R f

A

A

• - o- Q-Cil + ~

' o-Q + e-~

AMP

PDP 0

o-e + •- c,o-~ - AMP

Activation of Amino Acids by Attachment of AMP The activation of an amino acid is achieved by transfer of a n AMP group from ATP to the COO group of the amino aci d . as sh own in EqURtion 4-13: H

R

H

H

I I ~o H - ~- c-c + Adenosine--G- O - O I I "-o-

H

R

O

1 J ~ H- N·- c-c +c -o I I 'o- Q-Adenosine H H IEquation 4-131

(In the equation, R represents the specific side group of the amino acid ) The enzymes that catalyze this type of reaction are caJled aminoal:yl syntbelases. Upon activation, an mnino acid (AA) is thermodynamically capable o f being efficiently used for prote in synthesis. Nonethelnss, the AA-AMP r.omplexes ara not the direct pmtion ~use the high-energy G ~G molecl.lle , creatal when the M - AMP moleculE' is

formed. is broken down by the enzyme pyrophosr.hatase to low-energy groups. Thus. the reverse reaction, G -G + AA-AMP- ATP + AA. cannot occur. Almost e~ II biosyflthP.lic reactions result in the relem>e of G-G. Almost as soon as il (s mAde. it is enzymatically broken down to two p hosphate molecules. thereby making a ~ve.-sal of the biosyf!thetic reactiQn \mpossible. 'l'hc great u tility of the 0 - 0 split provides an explanation fO£ why ATP, not ADP. is the primary energy donor. ADP cannot llat.isfer a h igh-energy group and at the same time produce 0 -G groups as a by-product.

BIBLIOGRAPHY Genet'al References Kornberg A 1962. On th e metabolic significan ce of phosphorolytic and pyr,ophosphotolytic reactions. In Hori· 2ons in biochemistry (ed. M. lJ.bods in enzymology: Enzyme .kinP.tit.•s and mechanism: Detfll'tion ond characteriza-

Voet D., Voet J.G., and Pratt C. 2002. Fundamentals of bio. chemistry. John Wiley & Sons, New York..

CHAPlER

Weak and Strong Bonds Determine 1Aacrounolecular Structure NA. RNA. and protein are aU polymers of simple building blocks. As we learned in Chapter 4, synthesis of these polymel'S depends on the controlled, cat aly~ed linkage oJ activated building blor.ks. For DNA and RNA, these building blocks are nuclcotides (sec Figure 2-11 ). For prolt~ins. the lruilding blocks arc the 20 ami no acids donated from thoir activa led intermediates. the clonor tRNAs. Assemhly or thesP. chains requires bTeakage of multiple high-energy bonds for the. addition of each building block. For all these molecules , the order of the constih.tenl bu ilding blocks d(ltermines their genetic and biochemical function. Weak bonds play a critical role in determining the structure and function of theso polymers. The primary information of RNA, DNA. and proteins is the order ot' their covalently-linked building blocks. NrverthPJess, it is only after they have formed extensive additional weak bonds between their different parts that these polymers adopt characteristic shapes that allow them to carry out theil' functions. The hydrogen bonds and ionic, hydrophobic:, and van dcr Waals interaf:tions described in Chapter 3 direct proteins to form critical binding sites and ONA to assume its dotsble helical stn•cture. lndood, the disruption of these interactions (by heat or detergent, for example) without disruption of coval ent bonds completely destroys the activity of all but a few biological polymers. In this chapter we briefly describe tho structua-..: of biological macromolecules and the forces that control thflir shape. DNA and RNA are discussed br.iefly here and morn thoroughly in Chapter 6. We then focus on the diverse structures of proteins. The final sections of the chapter focus on the interactions between proteins and nucleic acids. an activity central to many of the processes we will c.ncounteT in this bo ok, and the control of protflin fnnction by allostery.

D

OU T L INE

• Higher-order 5tructutes h e Oetermmed by Intra- and lntermolecoli:lr lnteredkJns (p. 69)

• The Specific Conformatim of a Protein ResUts from Its Pattem of Hydrogen Bonds (p. 18)

• Mosl Prot@ins Are MOOolar, Conlall"'lng Two ()(Three Dorru~ms (p 81)

• weak Bonds Cooectty POSitiOO Proteins .;!1011g DNA ard RNA Molecules {p. 84)

• Alostery: Regulation of a Protetn's F\mctim by Cl1ang111g Its Shape {p. 87)

HIGHER..QRD~R STRUCTURES ARE DETERMINED BY INTRA.. AND INTERMOLECULAR INTERACTIONS DNA Can Form a Regular Helix DNA molecules usually have regular helical configurations. This is because most DNA molecules contain two antipata1ld polynucleotide strands that have complementary structures (see Chapter 6 for more details). Both in1ernal and external noncovalent bonds stabilize the structure. The t\.-vo stranflS are .held togflthcr by hydrogen bonds beh.-voon pairs of complementary purines and pyrimidines (Figure 5-1). Advnine is Always hydrogen-bonded to thymine, whfilroas guanine is 69

70

Weuk a111l Stroug Bands flt>l£-.rmine Macromoler.vfar Structure

FtGU RIE 5-1 The hydrogen--bonded

base pails of DNA. The flguc shows the position and leng1h of the hydrogen bonds betvveen the base pairs_n,ec0\.31ent boods between 1he atorns within eadl base are shown, but double ilrld s~ bonds have broken at the t~. they can refoml {luwer left) or addfttonal borlds can breaJ;.

hydrogen-bonded to cytosine. In addition. virtually all the surface atoms in the sugar and phosphate groups form bonds to water molecules. Tbe purine-pyrimidine base pairs are found in the center of the DNA molecule. This arrangement allows their flat s.urfaces to stack on lop of each other. creating shared ('1T - 1T) electrons between the bases and limiting their contret with water. This arrangemt-mt, known as base stacking. would be much less satisfactory if only one polynucleotide chain were present. Because pyrimidines are smaller than the purinns. single-stranded DNA would result in the unfavorable exposure of hydrophobic surface between adjacent bases. Thr. presence of r.omplementary base pairs in double-helical DNA makes a regular strur.turo possible, since ear.h base pair is of the same si7:e. The double-helical DNA molecule is very stable for two reasons. First, disruption of the double helix would bring the hydrophobic purines ano pyrimidines into greater contact with water, which is very unfavorable. Second. rloublc-stranded DNA molecules contain a v·ery large number of wook bonds, arranged so that most of them cannot break without simultaneously breaking many others. Thus, for example, even though thermal motion is constantly breaking apart the purine-pyrimidine pairs at the ends of each molecule, the two chains do not usually full apart because other hydrogrm bonds 1n the molecule nre stilt intact (.Figure 5-2). Once a given bond is broken. th~ rnost likely next event is thfl rcfonning of the same hydrogen bonds to restore the original molecular configuration. rathur than the breaking of additional bond-;. Sometimes. of course, the first breakage is followed by a second. and so forth. Such multiple breaks, however. are quite rare. so that double helices held togethct' by ffi{)J'C than ten base pairs arc very stable at room temperature. When ONA stranns do comn apart without reforming. this typically stm-ts at on£! end of th~ mo)e}{:ule. and proceeds inward. This is hecause

the interactions between the bases aJ thE> end of the DNA are the least supported by adjacent interactions. That is, they have only one n eighboring OOse pa ir to bn]p sect.J.re the infP.rnction. As df.>SCrlbed in ffiOre det11il below, U1e sam e principle-the use of multiple Wflak bondsgoverns the stability of proteins. Ordered collections of secondary bonds become less anrl less s table as their temperature is raised above physio logical tcmporaturos. At elevated temperatures, the simultaneous breakage of several weak bonds is more frcqunnt. After a s ignjficant numbfir h :~~ve broken, a molecule usually loses its original fonn (the pTocess of denaturation) and nssumes an inaclive. or den atured. configuration. Thus, as the temperature rises, more interactions are required to maintain the double-stranded natura of' DNA.

RNA Forms a Wide Variety of Structures In oontl'ast to the highly regular stmcture of the DNA double helix. RNA is usually found as a single-stranded molecule. Some RNA molecules (such as messenger RNAs) function as transient carriers of genetic information and arc constantly associated w ith proteins and thus do not have an indetJc n ncnt, slable, tert iary l'old . Othe r RNA moleculES fold into uniql.!t1 te rtiary structures. For these RNAs, intramolecular interactions between distinct regions load to the formation of specific elements of sCQJndarv structum. These interactions are prindpally between the bases of the RNA and include traditional Watson-Crick base pairing, unusual base pairing found only in RNA, and hydrophobic base stacking. RNA differs from DNA in that the ribose sugar of the backbone carril:lS a 2'-hydroxyl group. ln the fo lded structure of RNA molecu les, these 2'-hydroxy] groups often partici· pate in interactions that stabilize the structure. The binding of divalent m etal ions {such as Mg2 1 • Mn2 + , and Ca2 ' ) to the RNA is often critir.ftl. to the formntion of a stable. folded conformation a1CCtein to adopt a uni~e conformaoon. Although progress l!i being made in the prediction d protcin stJtJcUe based on amil'\o acid sequence, the WI detemlination of the energetic constraints of a partirular seqt~ence is still beyond the most powerful computaticnll approoches. Nevet1heless, prediction of ceftain secondary structt.Sal elements (such as the common a. heix structLfe introduced below) &s beeoo1ing increasingly reliable. The ina~ large number of available ~entaly-detennined s1ructures has provided an important resource for making protein stJucttre predctions based on amino acid 5e9Jence These atcrnic stn..ldUres have helped to define fam~ies of ai'T'lif10 acid Seq.JeflCeS 1Mt share reklted thr~ shapes By companrlg the seqJences of protans d unkncP.rvn strumne with those lhat have been defer-mined, it is often pa55ible to mal<e stnJctLfcj predictions based on the identified sintLanty. Co~ this lnformat10n ~lh computer algorithms that predict secondary structlxes is prcMog to be a powerllJ method fer pre&ting how protens fold The ~erm ootW is 1M lh€se approaches wit allow at least an approxrn.ate struc· ttre to be predicted for any r:mtein fran tts primary sequ6iee alone

quarternary

W~k

011d Stro1Jg &ncft; Detru-mi lle Mac:romoiP.CtJlar Structure

F f Ci U R f 5-8 A pclypeptide main folded into a helical CCf'lfiguration calted the a helix. (Soorce: MoleoJar structure a~ed from PaUing L 1960_ The llOture of the chemical bond and rhe strudl.lfe of mofeaJies and crystals: An tntroductJOO to ~structural~ 3rd e6lit..n, p, SOO. Copyrigl1t © 1960 Cornell University. Used by permLSsion of d-.e publisher.)

0

5.4A (3.6 rcsidtles}

conformation such that hydrogen bonds can fonn betwe<m carbonyl groups of one (3 strand and NH groups on the adjacent strand. 'I)'pically. a region of I) sheet is composed of four to six separate stretches of poly(lflptide (each forming ao individual ~ strand). each eight to ten amino acids in length in the~ sheet, adjacent amino acids are related by a rotation of 180° and thus their respective side groups emerge from opposite sides of the

~

sheet (see Figure 5~9b).

13 sheets L'Ome i.n predominantly one of two forms. These diHer in the relative orientations of their chains (Figure 5-1 0). In one, the adja~ cent chains run in the same amino-to-carboxyl direction to produce a parallel 13 sheet. In the other. the adjacent chains run in opposite directions to yield an antiparallel 13 sheet Although less common, there are also f3 sheets that have both parallel and antiparallel compo~ nents. In both parallel and antiparallelp sheets, all the peptide groups lie approximately i11 the plane of the sheet. Strucrural studies have -revealed that in most cases the inrlividual sb:11nds o f p sheets tend to be twisted along their length in a right-handed manner (Figure 5-11). Thus, instead of flat sheets of protein. regions of~ sheet tend to curve to generate a compact protein module. For a protein to fold properly, both the backbone and the side chains must adopt conformations that maximize favorable interactions. The a helix and f3 sheet are both very stable c onfoTmatlons of the polypeptide backhone. But for each side chain to make the maxi· mum number of weak bonds, proteins have to adopt more varied

HiF/Ier-Ordet· S trudures A re Determmed by Intra- and l.ntermolecwar Interactions

a top-vtew

77

b stdeview

~sheets are held ~ by hydrogen bonds. (a) A J3 sheet~ shown from above Note that the oxyger5 and Mrogens of the backbone are fdly hydrogeclxlnded. (b) A ~ sheet stlown from a side \llt'W. ll'ris illustrates the locatiOI'I of the side 8JOt4)S. v.hct1 alternate between emergtr~g from atxNe « be4ow the plane of the (> shee-t (Source: MoltnJar strucllxe adilpled from Pauling l 1960. The nature of the chemiroJ bond and the SlnJCture of mcieaJies and uystols: An introducrion to modern structural chemistty. 3rd e.arls to their N·t~rminal end being positively charged. This positively..c;harged end frequently makes weak internctions with the phosphate backbone adjacent to the major groove. The heHx·turn ~helix motif was the first protein motif involved in sequence-specific DNA binding to be identified. This motif is composed of two adjacent o. helices that are separated by a short turn (Figure 5-21]. One a helix, C'.allod the recognition helix. is responsib)c for DNA recognition. The second a helix is located approximately perpendicular to I he first n helix. Although these two helices form the core of the DNA recognilion motif, other nearby regions of helix-tum-helix DNA-binding proteins freqtmntly stabilize the arrangement of these two Clt hfllices and contact the DNA. Other DNA-binding motifs also insert n helices into the major groove, such as the zinc finger and leucine zipper DNA· binding motifs (as we shall discuss in Chapter 17). Whereas the use of an o helix is the predominant fonn of specific DNA recognition. some proteins do use different strategies. An extTt:lme example of this is seen with the TATA-binding protein lTBP). which determines the site of transcriptional initiation at many e ukaryotic promoters (see Chapter 12). TBP uses an extensive Tegion of 13 sheet to recognize the minor groove of the so-caJJoo TATA-box (Figure 5-22). So, in this case. we see the use of 13 shed instead of o hel ix and interact ions with the minor groove rather than the major groove (for a detailoo discussion of this maller, see ChaJlter 12).

5-20 SchematX of intetitdion between the recognition ltetix of~ reptessor monomel' and maju groove of operatot DNA. (Soorce: Adapted from Jordan SR aod Pabo CO. l988. Stfucttre of lhe larrbda «Dffl>lex at 2..5 Aresolooon. ~ :242 693-899. Cop~fl~t cr' 1988 Amencan Assoc:laOC('I klr the~ or soeoce F I (i U R E

~ Vv'dl pen11isslon )

Proteins Scan along DNA to Locate a Specific DNA, Binding Site Many DNA-binding proteins make substantiaJ contacts with t he DNA backbone as well a.o; with the specific base pairs of their recognit ion sites. Mediating these backbone contacts are patches of positivelycharged amino acids located at sites very dose to those that bind to the base pairs. These associations rely primarily on electrostatic attraction between these positive patches and the negatively-cllarged phosphate backbone of the DNA. Because the backbone has a similar negatively-charged surface, regardless of the sequence, these proteinDNA backbone contacts contribute substant ially both the specific and nonspecific affinity of a protEJin for DNA. Thus. even a highly spocific DNA-binding protein wiiJ have a substantial affinity for nonspccilk DNA sites as well. For example, thfl affinity of some well-charac:tm-ized regulators of gene ex-pression (such as fhe lactose repressor) for t heir recognition sequences is about 1l1i-fold greater than their affinity for nonspecific DNA. As a consequence. in the cell these proteins are typkally bound at a number of nonspecific sites as weJl as at their specific target sequence. This is due to the m uch larger nu.mber of nonspecific sites r.ompared to the sp«:ific: sites. Indeed, ewtty nucleotide in thn genome

FaGtJRE

5-21 Ceomettyoh.

..~~ lhesdlematic shows t'NO rronomers of A repressor tlound to the q>erator. The helices 1n eadl monomer are la-

be4ed

t lO :;. II IS heliJI 3 vh::t11nset15 ..liD l ht!

groove as ~ itl r!fSUre 5-10. (Source Jldclp~ frcm 10rdiY'I S.R 1'JI'ld P.ixJ C.O. 1988 Strudure o1 the tambda ClJI11I)IeX at 2 5 Aresolu~

bcnSocnce242' 893-899, f 2b, page89S.

COpynght ~ 1988 American Assooanon for the Advosed by Watson and end:. f Of this \AA:Jik t.at ~ bacteriophage as lhe 1irst model system of molec:tJiar biology (Chaplet 2 1}.

CH A PTER

The Structures of DNA and RNA

T

he discovery that DNA is the prime genetic molecule. carrying all the he~ditary information within chroro.osomes, immediately focused attention on its structure. It was hoped th~t knowledgP. of the structure would reveal how DNA carries the genetic messages that are replicated when chromosomes divide to produce two identit~ copies of themselves. During the late 1940s and early 1950s. several re:>earch group5 in the United States and. in E\lrupe engaged •n serious efforts-both cooperative and rival-to understand bow the atoms of ONA are linked together by covalent bonds and how the resulting molecules ru~ arranged in three-dimtmsionaJ space. Not surprisingly, there irutially were (ears that DNA might have very complit:aterl and perhaps b\za.rre structures that differed radically fmm o ne gene to another. Great relief, if not general elation, was thus expressed when the fundamental ONA stnu,i ure was found to be the double helix. It told us that all genes have roughJy the same throo-dimensi onc:~l form and th4-1.f the differenL'eS between two genfJS res.ide in the un.ler and number of their four nucleotide building blocks along the complemental'y strands. Now. some 50 :years after the discovery of the double helix, this simple description of the genetic material remains true and has not bad to be apprnciab1y altered to accommodate new findings. Nevertheless, we have come to realize that the structure of DNA is not quite as unifocm as was fi.rsl thought. Fnr e:xample, the chromosonm of some small viruses have single-stranded. not doubl&stranded, molecules. Moreover, the precise orientation of the base pairs varies slightly from base pair to base pair in a manner that is influenced by the local DNA ~qucnco. Some DNA seql..tellces even permit the doub lo heli x tn twist in the )eft-handed sense, as opposed to the right-handed sense originally fonnuJated for DNKs general structure. And while some DNA moleScnpt, MdScrlpt. and Raster 3D)

As we have seen, the energetics of the double bel i_x favor the pairing of each base on one polynucleotide stra nd with tha complementary base on the othor str.and. Sometimes. however. individual bases ca.n protrude from the d ouble helix in a remarkable phenomenon known as base Hipping shown in Figure 6-8 . As we shall see in Chapter 9, certain enzymes that m ethylate bases or remove damaged bases do so with the base in om extra-heUcal cunfigwatiun in which it Is flivped out from the double helix, enabling the base to sit in the catalytic cavity o£ the enz}'ffie. Furthermore. enzymes involved in homologous recombination and DNA repair are believed to scan DNA for h omolog}' or Jesions by flipping out one base after another. This is not energetically expensive because only one base is Aipped out at a time. Clemly. DNA is more flexible than might be asswned at first glance.

DNA Is Usually a Right-Handed Double Helix Applying the handedness ml e from pllysics. we can see that each of the polynud ootide chains in lhe double helix is rigbl-handed . ln your mind 's eye. hold yo ur rigllt hand up to the DNA molecule in Figure 6-9 with your thumb pointing up and along the long axis nf thP. helix and your fingers following the grooves in the helix. Trace a long om~ strand of the heHx in the direction in which your thumb is pointing. Notice that yo u go arounn the helix in the same direction as your fingers are pointing. This does not wurl< if you u~ your le ft .hcmrl . Thy it! A ronsequttnco of tho helical na ture of DNA is its periodicity. Each base pair is displaced (twisted) from th e pre\'ious one by about ~6c. Thus. in lim X-ray crystal structure of DNA it takes a stack of about 10 base p airs to go complete ly aro und the helix (360' ) (see Figure 6-la). That is. tht:l helical periodicity is generally 10 base pairs per turn of U1e he lix. For further discussion, see Box 6-1, DNA Has 10.5 Oase Pairs per Turn of the Helix in Solution: The Mica Experiment.

The D()uble Helix Has Minor and Major Grooves As a result of lhe double-helical stn.cture of the two chains, the DN A molecule is a lung extended polymer with two grooves that are not equal in size to each o ther. Wh y are there a minor groove and a major groove? II is a simple con._iliiS with respect to the fldlx am. In CldditJon, the Aform has a central hcle (bottom). fus h€lical form is adopted by RNA- DNA Mel RNA- RNA ~'aces. (c) Z DNA lS a left-h.vlded helix dnd has a zigz.ag (hence 7) ~ (Soutce: Courtesy of C ~and P. B. Dervan.)

a BONA

b A ()IJA

c ZONA

nN" Structur f' a

b

average 10.5 base py heating

I

-

_

DNA molecule

·-·--;---·---·--·-.·----·--·- ·---·--· --·--·-·.-._- _-_-_(--< sI

cool slowly and start to refiature

mssiog region a

I

a

I

COI1tinue to renatl.l'e :

----- - - - -

-

--

FIGURE 6-14 Reanneafing 4lnd hybridlutioo. A mx\tJte of two othe!wtse IdentiCal double-slrande:l DNA rrdec.lles, ooe flOrmal vvdcHype Ctit\ and the other a mAant nissing a short SITetch of nucJeotides (matted as region • WI red), afe denatu-ed by healillg. The denaued DNA molecules are dowed to renature

t7,t ilobation just below the melting ~1.11e- This ueatment rest-Cls in two types of reflat~Xed molet.Ue5. ~Of CCJfll>l€tely ret1a1:1.Jre d molecules lfl W1ICh two ~tary wjd-type strands ftfum a l'e1lx and tvvo axnplementary m.Jl.xll strands reform a llefil< 1he Oil ter type are ~!d m~. compO&ed of .1 w4d-type and a mutn strand, ~a short u~ Coop of DNA (regKJO a)

Ole type 6

tO.. sevet·a l i ndispensab le techniques in molecular biology, such .t1S &ruthem blot hybridization {see Chapter 20) and DNA mic:roarray analysis (see Chapter 18. Bux 18-1}. Important insights into the properties of the double helix were obtained from classic experiments carried out it1 the 1950s in which the denaturation of UNA was studied under a variety of con ditions. In these experiments, DNA denalurntion was monitored by measuring the absorbance of ultraviolet light passed through a solution of DNA. DNA

- ~-

109

110

Th~ Stnmtu~ of nN,A

ami RNA

maximally absorbs ultraviolet light at a wavelength of about 260 run. It is the bases that are principally responsible fur this absorption. When the temperature of a solution of DNA is raised to near the boiling point of water. the optica1 density, called absorbance. at 2fi0 nrn markedly increases, a phenomenon kno·w n as hyperchromicity. The explanation for this increase is that duplex DNA absorbs less u ltraviolet light by about 40% than do individual DNA chains. This hypochromicity is due to base s tacking. which diminishes the c.apacity of the bases in duplex DNA to ~bsorb ultraviolet light. lf we p!ot tl1e uptic·dl de11sity of DNA as a function of temperature. Wfl observe that u.e im.:rease in absurptiuo ou;urs ClbJ'uptly over a relatively narrow temperature ra11ge. The midpoint of this transition is the melting point or Tm(Figure 6-15). Like ice.. DNA melt~; it undergoes a transition from a highly ordered double-helical structure to a much less ordered structure of individual strands. The !'harpness of the increase in absorbance al the m elting temperature tel ls us that the denaturation and renaturation of complementary DNA strands is a highly cooperative. zippering-lU..e process. Renaturation, fur example, probably occurs by means of a slow nucleation process in wJ1ich a relati"ely small s tretch of bases on one strand find and pair with their complement on the co mplementary strand (midrlle panel of Figure 6· 14}. The remainder nf the two strands th en rapidly z jpper-ttp from the nu cleation site to refo,.m an extended double he1ix (lower panel of Figure 6-14). The melting temperature o f ONA is a characteristic of each ONA that is largely d etennined by the G:C content of the ONA and the ionic strength of the solution. The higher the pe~nt of G:C base pairs in the ONA (and hence thP. lower the content of A:T base pairs). the higher the melting point (Figure 6-16). Like\.vise, the higher the saiL ooncentralion of the solution, the greater the temp erature at which the DNA denatures. How do we explain this behavior? G:C base pairs contribute more to the stability of DNA than do A:T b.T!se pairs because of the greater number of hydrogell bonds for tbe fonner (three in a G.:C base pair \1ersus two for A:T) but a lso importantly. because the stCJcking interactions of G:C base pairs with adiacent base pairs are mote favorable than the corresponding interactions of A:T base pairs with their neighboring base pairs. The effect of ion if.:: sfJWgth reflects another fundamental feature of the double helix. The backbones of the two DNA strands contain phosphoryl

l

FIGURE 6-15 DNA~c:urve.

single siTanded

~


..d by cations, thereby stabilizing the helix. Conversely. at low ionic strength the unshielded negative charges render the helix less stable.

Some DNA Molecules Are Circles It was initially believed that all DNA molecules are linear and have nvo free end s. lndeed, the chromosomes of eulcaryotic cells eacl1 contain o single (extremely long) DNA molecule. But now we know that some DNAs are circles. For example, the chromosome of tl1e small monkey DNA virus SV40 is a circular. double-helical DNA molecule of about 5,000 base pai.J's. Also. most (but not all) bacterial chromosomes are circular; E. coli has a circular chromosome of alxmt 5 million base pairs. Additionally. many bacteria have small autonomously replicating genetic elements known as plasmids. which are generally circular DNA m olecules. lntereslirlgly, some DNA molecules are sometimes linear and sometimes circular. The most well-known example is that o f the bacteriophage .A. a DNA virus of E. coli. The phagp, A genome is a linear double-stranded molecule in the v irion pCU'ticle. However. when the A genome js jnjectoo in to an E. coli cell dm j ng infecliun. the DNA circularizes. This occurs by base--pairi ng between single-stranded regions that protrude from the ends of the DNA and that have complementary seqmmces. also known as ''sticky encls. ••

DNA TOPOLOGY As DNA is a flexible structure. its exact molecular parameters are a function of both the sunuunding ionic environment anfl the nature of the DNA-binding prote.ins w ith which it is complexed. Because their ends are free, linear DNA molecules can freely rotate to acconunodate

tt1

fiGURE 6- 16 DependenceofDNA denabHation on G + C content and on salt

a!Clc:enhation. The greater the G + C content. lhe higher the lerl1Jlefature must be Ia denature the DNA strarld. DNA from differerw SOLXces was dissot.ted 111 solttiOnS of low (red lif)e) and ll1gh (green line) conc€1'\lJ;;ttions of sd1 at pH 7.0. The poots represent the l~ture at v.hdl the DNA denatuted, grct*ted agairt>t the G + c c.oment. (Sourc.e: Data from Marf11Uf J. and Ooty P. 1962. Journal of Molectr lor Brology 5. 120. Copyright ~ l%2, \...nh perm iSSion fTom Elsevier Soence.)

changes in the number of times the two chaiJ1s of the double helix twist ah(mt oach other. But if t'hc two ends are covalently linked to form a circular DNA molecule and if there are no interruptinns in the sugar-phosphate backbones f the two strands, t.h en the absolute n umber of times the chains can twist about each other cannot change. Such a covalently closed. cin:u1ar DNA is said to be topologica lly oonstrained. Even the linear DNA molecules of eukaryotic chromosomes are subject to topological constraints d ue to their extreme length, en lrainmonl in c hromatin, and interaction with other cellular components (see Chapter 7). Despite these constraints, DNA particip<stes in n umerous tly namic processes iJl the cell. For exampl11, the two strands of the double helix, which are twisted around each other, must rapidly separnto in order for DNA lo b e duplicated and tu be transcrjbed into RNA. Th us, understanding the topology of DNA and how the cell lll' is explained below.

Prokaryotes Have a Special Topoisornerase that Introduces

Supercoils into DNA Both prokaryotes and eukarytoes have type rand type n topoisomerases 1hat are c-a pable of removing supercoils from DNA. In addition. however, prokaryotes have a special type H topoisomcrase known as DNA gyrase that i.ntroduQJS. rather than removes. negative supercoils. DNA gyrase is responsible for the negative suporcoiling of chromosomes in prd.a.ryotcs. 'fhis n egative su perooiling facilitates the unwinding of thfl DNA duplex, which stimulates many reactions of DNA including initiation of bot h transcription and DNA repJication.

FIGURE 6-22 Schematic mechanism ot action fm topoisomerase f. The enzyme cus a ~gle Slrand ol.re PNA du~ passes lhe u11wt svand th~ the break. then reseals the break. The process 1'K1eases tht> tinklf'lg ntJI'l'ltEr by + 1. ~

e ~

Ll< =n

pass strand

niek

§

thl~break

and ligate

(

•

€ ~

U.s promote several otlw.r reactions important to maintaining the proper DNA st:ructul'e within cells. The enzymes use the same transient DNA break and strand pas:;age reaction lhat they use to relax DNA to carry out these reactions. Topoisomerases can both catenate and decatenate circular DNA molecules. CireulaT DNA m oloculf'.s are said to be catenated if they are linked together 1ike two rings of a r;ha.in (Figure 6-23a). Of these two activities. the ability of topoisomcrases to decatenate DNA is of clear biological importance. As we 'INiiJ see in Chapter 6, catenated DNA rnolecules are conunonly produced as a round of DNA replication is finished {see Figure 8-33), Topoisomerases play the essential role of nnlinking these DNA molecules to allow them to s~parate into the two daughter cells for coil division . Decatenation of two covalently closed circular DNA molecules requires passage of the two DNA strands of one nlolecuJe through a double-stranded l:lw.ak in the second DNA molecule. Tltis reaction thorefore depends on a type 11 topoisomerasc. The requirement for decatenation explains why type n topoisomerases are essential ce11ular proteins. However, if at least one of the two catenAtP.d DNA molecules carries a nick or a gap, then a type I enzyme may also unlink the two molecules (Figure 6-23b). Although we often focus on circuJar DNA molecules when oonside~:­ ing topological issues, the long Jinear chromosomes of cukaryotic organisms also P.xperience topologic.al problems. For exampiP, during a round a type 11 topoisomerase

0

F I GURE

calenalioo ,

"decalenatioo

b type I topoisomernse

catenatioo , 'decatenation

c type It topoisomerase

d t~ nropcisomerase

6-23

T~s

decatenate. disentilngte. and \W\knot OHA.. (a) Type" topO\SOfT1efase ca11 catenate and decalenate covalently dosed, ciro.Jal DNA moleetlt.-5 by ~troduong a dotble-stftmded break "' ooe ONA a11d pass.ng the otha DNA rnolt1:ult ltvoogh the beak (b) Type I tcpcliSOmerases can my cater)ate i'll1d decateoate molecules if one DNA sband ha$ a nd or a saP· 1hts tS because these enzyrres dedve amy 011€0NA stlafld~t a time. (c) &rtangledlong lnear rnA molecules, f,eneraled, !Of exanple. during the replic.Jtioo of etJcaryotic du'OITtOSOfl'll:S. can be disentangled by a topoc SOfr1eii!Se. (d) rnA 1-.ncts can als-o be ur*notted by topOISanerase action,

of DNA replication. the two double-stranded daughter DNA molecules will often become entangled (Figure &23c~ These sites of entanglement, jusl like the links between m tenated DNA molecules. block the ~para­ lion of the daughlm chromosomes during mitosis. Tbf'.refow., DNA disantanglelllont. generally cataly~P.d by a type n lopoisomcrase, is also required for a successful round of DNA replication and ceU d ivision in

eukaryotE?S. On occasioo, a DNA molecule becomes knotted (Figure &23d). Por example , some s1te-spocific recombination reactions. which we shall discuss in Uf>lail in Chapter 1 t . give rise to knotted DNA products. OncE' again, a type n topoisomerase can "untie" a knot in d uplex DNA. If the DNA m olecule is nicked or gapped, theh a lype J enzyme cun also do this job.

Topoisomerase1i Use a Covalent Protein-DNA Linkage to Cleave and Rejoin DNA Strands To perform thoir fundions, topoisomerases must cleave a DNA slmnd (or two s trands} and then rejoin the cleaved s trend {or strands). 1bpoisomerases aro able to promote both DNA cleavage and rejoining without the assistance of other p roteins or high -energy co-factors (for example. ATP; also see btllow) because thPy use a cnvalent-intermediate mochanism. DNA cleavage oa: w~ whtm a tyrosin~ residue in the active site of the topoisomerase attacks a ph osph.odiester bond in the backbone of the target DNA (Figure 6-24). This attack generates a break in the DNA. wh.(?t'eby the topoisomc~BSE: is covalently joined to one of the broken ends vi a a phospho-tyrosine linkage. The other end of the DNA termiMtes with a free UH group. This e nd is also held tightly by the enzyme, as we will see below. The phospho-tyrosine linkage con serves the en ergy of the pJtosphodiester bond that was cleaved. Therefore, the DNA ca11 be re-sealed simpJy by reversing the Ol'iginal reaction: the OH group from 011e broken DNA end attacks the phospho-tyrosine bond reforming tbe DNA p hosphodiASte r bond. This reaction rejoins the DNA strand and releases th e topni~ somerase. which can th en go on to catalyze another reaction cycle. Although as noted abo' 'e, type 0 topoisomerases requite ATP-hydrolysis for activity. the energy releo.c;cd by this hydrolysis is used to promote conformational changes in the topoisomerase--DNA complex mtber than to deave or rejo in CNA.

Topoisomerases Form an Enzyme Bridge and Pass DNA

Segments through Each Other Between the steps of DNA cleavage and DNA rejoining, tbe topoisomerase promotes passage of a second segment of DNA through the break. Topoisomecase function thus requires that DNA cleavage, strAnd passage. and DNA rejoin ing all occur in a highly coordinated manner. Structures o( Sfllleral different topoisomei'oses have provided .insight into h ow the reaction cyde oreurs. Here w e will explain a rnorle1 for huw a type f topoison'lora~ relaxos DNA. To initiate a relaxation cycle, the topoisomerase binds to a segment o f duplex DNA in which the two strands are melted (Figure 6-25a). Melting o f the DNA sll"and.s is favored in highly negatively supercoiled DNA (soe above}. ma.king this DNA an exaillent substrate for relaxation. One ()t the DNA strands binds in a cleft ln the enzyme that plaCt'!S it lll'.str tJw

DNA Topology

a

b

s------------------------------------~ ~

s·c.._____________,>J'OH

_ ________

s·~·

>:r

d

(?

5"-·_ __

__,)3'C!:!

lA:'--w~-----"---">:r :r--- 0~

FIGURE

6-24

TopoisofMfMes deaw DNA using a

awatent tyrosine-DNA inlermediirte.

(a) SdlematJc of the c1eavage af1d rejOI(llflg react lOr\. For Slmpltc.ty, only a ~ strand of ONA IS sf1wm. See Figtl"e 6 25 lor a IT\OI'l! reaflstlc p tcttxe. The same ~ es used by lype fl topoisomerases, a~ two enz.ymf' Stbu'uts are requre:Q ooe to cleave each of lhe two ONA strand$ Topotson' emses wrnetunes M 10 lhe 5 · Side: and somelmes to the 3 • Side. (b) Close-up view of the phospho-tyroone

ro.ralent irteiTI"leliate.

tyrosine intermediate (Figure 6-25b). The success ofthe reaction requires that the othor end of the newly cleaved DNA is also tightly bound by the enzyme. After cleevaga, the topoisortlf!rase undergoes a larga conlonnational change to open up a gap in the cleaved strand . with the enzyme bridging the gap. The second tuncleaved) DNA strand then pa.ssas though the gap, and binds to a DNA-binding site in an internal ·•donutshaped., hole in the protein (Figure 6-25c}. After strand passage occws, a second oonformationa1 change in the topoisomernse-DNA complex brings the cleavf!d. DNA ends back together (Figure 6-25 d)~ rejoi.ning of the DNA strand occurs by attack of the OH enrl on th e phosophotyrosine bond (see above). After rejoining. the enzyme must open up one final time to release the DNA (Figure 6-25e). This product DNA is identical to the starting ONA m olecule. except that the linking numbec h as

boon increased by one. This general n1eehanism, in which the enzyme providP.s a " protein bridge" during the 6tmnd passage reaction can also be applied to the type J( topoisomerases. The ty pe IJ enzymes, however, are dimeric (or in som e cas~..s tetrarneric). Two topoisomerase subunits, with their active s ite tyrosine residues. are requ ired to cleave the two DNA strands and mal-e the rloubJo-stranded ONA bmnk that is an essential feature of the type lf topoisom£>.rase n 1echanism.

"120

The Structures of DNA nnd RNI\

cleavage and

opening of gate

a

c

b

DNA releaw

e

d

f 1c; u R£ 6-25 Model for the reaction cyde catalyzed by a type I topoisomerase. The f1gtre shows a SEries of proposed steps for the relaxation of one turn of a negativEly ~coded paasm.d ONA. The 1\'110 strands of DNA are shown as datk gray (and not dra\'\.'0 to scale) The four domains of the prulem are labeled in patle1 (a). Domain I1S shown 1r1 red, I is blue. lit is grei!n, and N is orange. (SO\xce: Adapted

from (h.}mpoux J. 200L DNA lclp<JisomE;fases. Annual Review of Btochemstry 70: 369-413. Copyright f) 2001 by ~I RelnEMIS.. '1/WJW.annu.""reviews.org.)

A B C D

--FIG U R l

6-26 Sdlematic of

elecbophoretK separation of DNA topoisornels. l ane A represents relaxed a tlldt.'d orcular DNA; lane B, linea~ ONA; lane C, highly supe«:Qiled ca::DNA; and lane D, a ladder of tClpOISOr11ei'S.

DNA Topoisomers Can Be Separated by Electrophoresis Covalently closed. circular DNA molecules of the same length but of different linking numoors are called DNA topoisomers. Even though topoisoroers have the same molecular weight, they can be separated fron1 each other by electrophoh"$iS through a gel of agarose (see Chapter 20 for an explanation of gel electrophoresis). The basis for this separation is that the gre-rller the writhe, the more compact the shape of a cccDNA Once again. think of how supercoiling a telephone cord causes it to become more compact. The more compact the UNA. the more easily (up to a point} it is able to migrate through the gel matrix (Figure 6-26). Thus. a fully relaxed creDNA migrates more slowly than a highly supercoiled topoisomer of the same circular DNA. Figure &27 shows a ladder of DNA topoisomers resolved by gel electrophoresis. Molecules in acljacent rungs of the ladder differ from each other by a linking number differenre of just one. Obviously. electrophoretic mobility is highly sensitive to the topological state of DNA (see Box &2. Proving that DNA Has a Helical Periodicity of abo ut 1 0.5 Base Pairs pBr Turn from the Topologic-dl Properties of DNA Rings).

Ethidiurn Ions Cause DNA to Unwind Ethidium is a large, flat. multi-ringed cation. Its planar shape enables ethidium to slip. oc intercalate, between the ~1acked base pairs of DNA

DNA Topology

1Z1

Box 6-~ Proving that DNA Hou a Helical Periodicity of about to.s Base Pairs per Tum froln lhe Topological Properties of DNA Rings

The observation that DNA tq:>otsomers can be separated from each Olher etec~bcafty IS the basis for a SlfTlJie e>q:>emtent that ptlM!S lhat DNA has a helical pencdcity of about 10.5 base pairs per tum If"\ so\ADn. Consider three cccONAs of sezec; 3,990, 3,995, and 4,011 base pairs that were relaxed to CDJT1)1ell00 by treatment with type I tqxiscrnerase. ~ subtected to ~ through agarose, the 3,990- and 4,01 1-base-pair DNAs echibit essenbaUy idef"lticat

Due to thermal fluouaOon. tqxllsomerase treatment iKtUally generates a naJTON spec1rum of topotsomers, bu: for ~ let us consider lhe rnd:>iity of onfy ttle most abmdant topoisomer (tmt correspondf"€ to the ce£:00A ., its most re1axed state). The rnobnlties of the rmst abl.l1dartt t~ for the 3,990and 4,01 1-base-pair DNAs are in&stingutShable b&(Jllse lhe 2 1-base-pa.r difference between them tS negfigible ccrnpared to the stzes of the rings. The most abundant topcisomer for lhe 3,995-base-pair ring, hcAA'ever, tS found to mgrat.e slighlty more f"?t)idly than the other tv.to nngs even though it is only 5 base pails larger than the 3,990-base-paer ring. How are~ to explain this anomaly? The 3,990- and 4,0 11 base-par rings i1 their most retaxed states are expected to ha..-e linmg numbers equal to Lk?, that cs, 300 tn the case of the 3.990-base-pair ring (dtviang lhe sll£ by 10.5 base piW>) and 382 in the case of ti-e 4,ot 1-base-pair ring_BecdJse Ll< 15 e halves of ead1 st£m coo~ with a loop, b4Jt none of the three stems need be a IO shown by a red F l CURE

Secondary

C!fTOW; approxlfTli')te mllii'Tlal S\Jbstrate strands

are labeled in bk.le; (U) uracil: {A) adert4ne; {C) c.y10Slne; (G) BU8f\lfle (b) 1lle hammerhead ribol.yme deavage reactton 1rwolves an •ntermediary state &.lnng whrll Mg(OH) m complex with lhe ribozyrrE (shown in green) am as a general base catillyst to ren10Vt.' a protor, from tt.e 2•-11\lith cytosine. This strict base pai1ing reflects the hxed locations of hydrogen atoms in the purine and pyrimidine bases in the forms of those bases found in DNA. Adenine and cytosine :almost always exist in the amino as opposed to tht> imino tautomeric: forms, w h ereas guanine and thymine a lmost always exist in the keto as opposed to enol {()rms. The complementarity between the bases on the two s trands gives DNA it:s self-coding character. The two slr.mds of the d ouhle heliK fall apart (denaturel upon exposure to high temperature. extremes of pH. Of' any agent thaf causes the breakag.t of hydrogen bonds. Upon sJow return to normal cellular conditiom. the dena r:ured s ingle strands can s pecificalJy reassociate to bipJ~ · cally active d ouble helices (renature or anneal). ONA in solution has a helical periodicity of about 10.5 base pairs per tl1m of the fu!lix. The stacl-Jng of base pairs upon each ethel' creates a llelix with two grooves. Decause the sugars protrude from the bases at an angJe of about 120 comes of agt~. Cel/90: 833- 840 .

CHAPTER

Chromosomes, Chromatin, and the Nucleosome n Chapter fl, we considered the structure of DNA in isolation. Within lhe ceU, however, DNA is associated with proteins and each DNA and its associated protein is called a chromosome. This organization holds true for prokaryotic and eukaryotic cells anrl even for viruses. Packaging of the DNA into chromosomes serves several impmtant functions. First, 1hc chromosome is a compact form af the DNA that readily nts inside the ceU, Second, packaging the DNA into chromosomes serves to protect the DNA from damage. Completely naS...ed DNA mQ}ecules are relatively unstabla in cells. 1n contrast, chromosomal DNA is ext remely stable. allowing ihe in[ommtiQn encoded by the DNA to be .reliably passed on. Third. onJy DNA packaged into a clrromosome can be transmitted efficiently to bclth daughter ceJls each time a cell divides. Finally. the chromoS()rne confers an overall organization to each molecule of DNA. This organization facilitates gene expression as weU as the recombination between parental chromosomes that generates the diversity observed among different indlvirluals of any organism. Half of the molecular mass of a eukaryotic chromosome is protein. ln eukaryQtic cells, a given region of DNA with its associated proteins is oiHed chromatin and the maiority of the associated proteins are small, basic proteins callad bic;tones. Although not nearly as abwulant. other proteins, frequently referred to as the non-histone proteins, are assQci· ated witb the chromosome. These proteins indude the numerous DNAhinding proteins that regulnte the transcription, replication. repair, and recombination of cellular DNA. Each of these topir:s will be discussed in more detail in the next five c.hapters. The proteins in chromatin perform another essential function: they compact the DNA. The following calculation makes 1he importance of this function clear. A human cell c ontains 3 X 109 bp per haploid set of chromosomes. The thickness of ea.c h base pair (the .. rise") is 3.4 A. Therefore, i( the DNA moJ(.-cules in a haploid set Qf chromosomes were laid out end-to-end, the total length of DNA would be apptoximately 1010 A, or 1 meter! For a d ipl oid cell (as human cell5 typic,;aJiy are}, this length is doubled to 2 meters. Since the diameter of a typi!=al human cell nucleus is only 10-15 J.I.ITleters, it is obvious that the DNA must be compacted by several orders of magnitude to fit in such a small space. Aow is tbis achieved? Most UJmpaction in human cells (and all other eukaryotic cells) is the result of the regular association of DNA with histones to fonn structures called nucleosomes. The formation of nucleosomes is the first step in a process that allows the DNA to be folded into much more compac:t strur:tures that .reduc::e the linear length by as much as 10,000-fold. Compacting the DNA d oes not come without a cost. Asso-

I

O UTL.IN E

• Ctlr()fi)()SOOle Sequence and Diversfty

.at

'12.1

3

la- 2

L~near

12.5

6 5

2 2

4

2

linear linear Linear

Micl'of)udet.R5 5

Micronucleus 2

Linear

Macronucleus 225 22 19 +X andY 22+XafldY

Macronucleus 10- 10.000

(budding yeast) Schi.tosaccharomyces pombB (f'8Sion yeast)

C. elegans (rOIJldwOITT)) ArabtdopsiS ttnliana (weed) Drosophila me1anogaster (frllit fty} Tetiahymena thermophtlus (protozoa) Fugv rvbripes (fish) Mus musculus (l'l"'IU$e) Homo sspqlefJS

linear tu•ear

2

Ltnear

thousands {for example, in the mac-.ronucleus of the protozoa Tetrahymena. Table 7-1). Circular and linear chromosomes ead1 pose specific challenges that must be overcome for maintenance and repJication of the genome. Circular d rromosomes require topoisomerases to sepa.rate the daughter molecules after they are replicated. Without these enzymes, the two daughter molecules would remain interlocked, or catenated. with one another after replicaHon. In oontrast, the DNA ends of the linear eukaryotic chromosomes have to be protected from enzymes that normally degrade DNA end s and present a different set of difficulties during DNA replication. as we shall see in Chapter 8.

Every Cell Maintains a Characteristic Number of Chromosomes Prokaryo1ic cells typically have only one complete copy of their chromosome(s) that is packaged into a structurn called the nucleoid (Figure 7-lb). When prokaryotic c:::eUs are dividing rapidly. however. portions of the chromosome in the process of replicating are present in two and sometimes even four copies. Prokaryotes also frequently carry one or more smaller independent circular DNAs. called plasmids. Unlike the la.-ger chromosomal D NA. plasmids typically are n ot ~n­ tial fur bacterial growth. instead, they cnrry genes that confer desirable

traits to the bacteria, such as antibiotic resistance. Also distinct from chromosomal DNA. plasmids can be present in many complete copies per cell. The majority of cukaryotic cells are diploid; thnt is. they contain two copies of ear.h r.hrornosome {see F•gure 7·1c)~ The two copies of a given chromosome are called homologs; one is derived from each

180 220 (MicrootlCieus)

2

2

97 125

365 2.500 2.900

ta:t

ChromDSCJmes. Cl!romotin, and tht! Nuc:lP.OStJme

FIGURE 7-1 Comparison of typical prokaryotic and ~cceU. (a) lhe &meter of a typiCal eukaryotJc reH ts - 10 JJ.ffi. The typjc:al ~tic C£1 is - 1 JI.ITI tong. (b) Prol<aryotic chwmosomal ONA is

b haplOid bacteria

loomes. Chmmabn. end the NuckN>I'OirlB

bacteria, single-cell eukaryotes, and multicellular cukaryotes-sec Chapter 19). it is not surprising that genome size is roughly correlated with m1 organism's Bpparcnt complnxity. Thus. prokaryotic cells typicaUy havo gcnomes smaller than 10 megabases (Mb). The genomcs of singJ&ccH cukaryolcs are typically less than 50Mb, although the m()f() L-omplex protozoans c-.m have genomes greater than 200 Mb. Mu]ticcJluJar organisms have even larger genorncs that can reach sizes grouter rhan 100,000 Mb. Although thRI'P- is a Coh'tl)at ion between genome size and organism complexity, it is far from perfect Many organisms of apparently similar complexitie s have very different genome sizes: a fruit fly has a genome approximately 25 times smaJler than a locust and the rice genome ~about 40 times smaJJcr than wheat (S£le Table 7-2}. ln these ('.Xamples, the munber of gnnes rather than the expansion in genome size appears to be more closely related to organism complexity. This becomes df'.ar when we examine the rf'Jativc gene densities of dilfcrent genomes.

The E. coli Genome Is Composed almost Entirely of Genes The great majority of the single chromosome of the bacteria E. coli encodes proteins o:r structural RNAs (Figure 7-2). The majority of the nona...-oding sequences are d{ldicateNA) uut is only expressed by certain types of viruses that require this en zyme to reproduce. But, ns a side effect of infe