Human Molecular Genetics 3

,

HUMAN MOLECULAR GENETICS

3

This book is dedicated Frank Strachan

to the memory (1921-2004).

of

HUMAN MOLECULAR GENETICS

3

THIRD

EDITION

Tom Strachan BSc PhD FMedSci Pn!{essor C!{HI/lllan

iViolecl/lar Gel/eries al/d Sciel/r!fie Director, II/sriwre of HI/lllall Ullil/ersir)' of ,\"elllcosrle, ,"\:elllcosrle-llpoll-1j1Ile, UK and

Andrew P. Read MA PhD FRCPath FMedSci Professor (!{ HI/lllall Gelleries, UllilJcrsir)' cif i\1al/ehesre/; Mallehesre/;

UK

G3 ~y?o~~~~n~s ~,~~;nce LONDON AND NEW YORK

Gel/eries,

Vice President: Denise Schanck Producrlon

Editor: Fran Kingston

IIlustrotion: Touchmedia, Abingdon, UK New Media Editor: Michael Morales Copyeditor: Moira Vekony Typesetter: Saxon Graphics. Derby, UK © 2004 Garland Publishing All rights reserved. No part of this publication any means-electronic,

mechanical,

may be reproduced,

photocopying,

recording,

stored in a retrieval system or transmitted

or othclvvise-wirhou[

prior written

permission

in any form or by of the copyright

holder.

ISBN

0-8153-4184-9

Library of Congress Cataloging-in-Publication Strachan, T. Human

molecular

genetics

/ Tom Strachan, Andrew

Data I~ Read. __3rd ed.

p.em.

Includes bibliographical references and index. ISB 0-8153-4 I84-9 (pbk.) -- ISBN 0-8 I53-4 I822 (hardback) -- ISBN 0-8153-4183-0 (diskette) 1. Human molecular genetics. I. Read, Andrew P, 1939- II.Titie. QH431.S787 2003 61 I '.01816--dc22 2003017961 Published by Garland Science, a member of the Taylor & Francis Group, 29 West 35th Street, New York, NY 10001-2299, 4 Park Square, Milton Park,Abingdon, axon OX14 4RN.

and

Distributed in the USA by Fulfilment Center Taylor & Francis 10650 Toebben Drive Independence. KY 4 I051, USA ToU Free Tel.: + I 800 634 7064: E-mail: [email protected] Distributed in Canada by Taylor & Francis 74 Rolark Drive Scarborough, Ontario M 1R 4G2, Canada Toll Free Tel.: + I 877 226 2237; E-mail: [email protected] Distributed Thomson Cheriton

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of Human

Molecular

has been printed on difTerent paper srock. Printed

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States of America.

15 14 13 12 11 10 9 8 7 6 5 4 3 2

Genetics,

Third

Edition.

The

CQIl[l;Il(

is identical

to the first printing,

however,

it

Contents

Abbreviations

XXIII

Preface

XXVII

Supplelnentary

learning

aids

XX\'lI1

Before we start - Intelligent use of the Internet PART ONE: Chapter

t

XXIX

1

The Basics DNA structure

and gene expressIOn

3

1.1.1

Building blocks and chemical bonds in DNA, RNA and polypeptides DNA, RNA and polypl'ptidt's arc: large polymers defined by a linear sequence of simple repeating

1.1.2

Covaknr

1.1

bunds conrcr

stability: weaker

noncovalenr

bonds facilitate

imermolecular

o

1.2.1

The struc[Ure

A structure

Box t.l 1.2.2

DNA

6

1.2.3.

The

and replication

of DNA

Examples

n:plicltion DNA

8

is an 3ntiparalld

double

of the il11portance

is SL'1111-con=oO

12' H

3'

5'

° I

o~p-o-

3' end

3'end

°

5CH2 I ° I

Major groove

O-f>=oO

H ° 12, 13' HC-CH ,.1/1 '\1 4' C H C 1........ /.1 0 ITJ"""flji 5CH, ........1,. I HC III ° f>=oO

~H, 51/° CH 4'1\1 HC-CH 31 12' OH H

of DNA is a double-stranded,

-TA

......fCG,

I

-TA:~ AT-

?

5'

-I-0

I-

5'end

anti parallel

I nm

,I

helix.

(A) Antiparallel nature of the two DNA strands. The two strands are anti parallel because they have opposite directions for linking of 3' carbon atom to 5' carbon atom. The structure shown is a double-stranded trinucleotide whose sequence can be represented as: 5' pCpGpT-OH 3' IONA strand on left) / 5' pApCpG-OH 3' IONA strand on right) (where p ; phosphodiester bond and -OH = terminal OH group at 3' end). This is normally abbreviated by deleting the 'p' and'OH' symbols and giving the sequence on one strand only (e.g. the sequence could equally well be represented as 5' CGT 3' or 5' ACG 3'). (8) The double helical structure of DNA. The two strands are wound round each other to form a plectonemic coil. The pitch of each helix represents the distance occupied by a single turn and in the BDNA structure Isee text) accommodates 10 nucleotides.

1.2

DNA STRUCTURE AND REPLICATION

(B)

11

OH 3' end

I

A

G C

5'end ~

T T

C ~

G - C Acceptor arm

C-G A-U

Um-A

U-A G-C D-arm

GU

G C C CU U 'A I I I I I mG 5 m Cm5CGGG

G-CG

UGACUUG

Key:

- Hydrogen bond

GAG . \ C'

J' ,'~1 ,

r

YU//

m5C G

I I I I

G

A A~ U U AG C--r.~ ~

Anticodon arm C - G

AGACCA

T'I'C arm

'Co-"

I

Oajc. jJC('r

G-C CC -Gm5C U Anticodon

Figure 1.7:Intramolecular hydrogen bonding in DNA and RNA. (A) Formation of a double-stranded hairpin loop within a single DNA strand. The highlighted sequences in the DNA strand above represent inverted repeat sequences which can hydrogen bond to form a hairpin structure Ibelowl. (B) Transfer RNA (tRNA) has extensive secondary structure. The example shown is a human tRNAGlugene. Minor nucleosides are: D, 5, 6-dihydrouridine; '1', pseudouridine (5-ribosyl uracill; m5C, 5-methylcytidine; m'A, l-methyladenosine. The cloverleaf structure is stabilized by extensive intramolecular hydrogen bonding, mostly byWatson-Crick G-C and A-U base pairs, but also with the occasional G-U base pair. Four arms are recognized: the acceptor arm is the one to which an amino acid can be attached lat the 3' end); the T'I'e arm is defined by this trinucleotide; the D arm is named because it contains dihydrouridine residues; and the anticodon arm contains the anticodon trinucleotide in the center of the loop. The secondary structure of tRNAs is virtually'invariant: there are always seven base pairs in the stem of the acceptor arm, five in the T'I'C arm, five in the anticodon arm, and three or four in the D arm.

and

numbers

of constituent

proteins

and

protein

subunits.

Most DNA polymcrases in mammalian cells use an individual DNA strand as a template for synthesizing a complementary DNA strand and so are DNA-directed DNA pol)'merases. Unlike I,-NA polymerases, DNA polymerases absollfrely

5'

Original

+

3'

require the 3' -hydroxyl end of a base-paired primer strand as a substrate for chain extension. Therefore, a previously synthesized RNA primer (which is synthesized by a primase) is required to provide the free 3-0H group that DNA polyIlH;rascs need to start DNA synthesis. There arc more than 20 different types of DNA polymerase in mammalian cells (Friedberg fr al., 2000; 2002), which can be conveniently grouped into three major classes (see TaVIe 1.2):

Original

~ classical (high fidelitJ') DNA-directed DNA pol)'merases. Gfchese, two are involved in standard chromosomal DNA replication, being specific for synthesizing DNA from the leading strand or lagging strand (DNA polymerases 1\ and " respectively), two are dedicated to DNA repair ([3 and E)

Parental DNA

Figure 1.8: DNA replication is semi-conservative.

5'

3'

New

Original

Daughter strand

5'

3'

Original New Daughter strand

The parental DNA duplex consists of two complementary, antiparallel DNA strands which unwind and then individually act as templates for the synthesis of new complementary, antiparallel DNA strands. Each of the daughter DNA duplexes contains one original parental DNA strand and one new DNA strand, forming a DNA duplex which is structurally identical to the parental DNA duplex. Note;this figure shows the result of DNA duplication but not the way the process works (for which, see Figure 1.9).

~APTER

ONE

I

DNA STRUCTURE AND GENE EXPRESSIO

5

5

3'

5'

3'

3'

"'"

.9

t:

o

~

.~

c. (I'

'0 C E Q)

Q)

>

o

~

.Ia-tl- Heficase

3 5' Leading strand

3' 5'

3' 5'

3 5' Lagging

3'5'

strand

Figure 1.9: Asymmetry of strand synthesis during DNA replication. The helicase unwinds the DNA duplex to allow the individual strands to be replicated. As replication proceeds, the 5'~3' direction of synthesis of the leading strand is the same as that in which the replication fork is moving, and so synthesis can be continuous. The synthesis of the lagging strand, however, is in the direction opposite to that of movement of the replication fork. It needs to be synthesized in pieces 10kazaki fragments), first A, then 8, then C which are subsequently sealed by the enzyme DNA ligase to form a continuous DNA strand. Note: the synthesis of these fragments is initiated using an RNA primer. In eukaryotic cells, the leading and lagging strands are synthesized, respectively, by DNA polymerases Sand" (see Table 1.2).

Jnd

011t.'

is elL-clicHed to replicating

chondri,l

~ error-prolle vJriery

and

repairing

I11lrO-

DNA-directed

o( difTerent

DNA

DNA pO]Ylllcrases

polymerases. have been

A wide identified

with very low fidelity of DNA replic,tion (e.g. the error rote for DNA polymerosc L (iota) is 20000 times that for DNA polymerose E) Some are known to be very highly expressed

Box t .2;

jn

illllllllllC

their low fidelity of DNA replication suggests involvement in 13 and T lymphocytes (see Section

in hYPlTlTllltation

DNA ('Y):

system

cells, and this obscrv:1tion

plus

10.0);

~ RNA-directed DNA polymerases. Some DNA polymerases synthesize DNA by using an R.NA tl·mpbte. and so have been described as reverse trallscri]Jtases (see Sections 1.2.4 and 1.3.1). They include an activity present ill the enzyme te/omerase which is responsible for n.:plicHing the ends of

proteins used in the DNA replication machinery, . '" linear chromosome using the reverse transcriptase of the Topoisomerases start the process of DNA unwinding by nicking enzyme telomerase. Ibreaking) a single DNA strand. As a result, the tension holding the helix in its coiled and supercoiled form is released. Primases. DNA polymerases find it difficult to start synthesizing Major

classes,of /,

'-~'"

Helicases accomplish unwinding of the original double strand, once supercoiling has been eliminated by a topoisomerase. DNA polymerases synthesize new DNA strands. DNA polymerases are complex aggregates of several different protein subunits, olten including DNA proof-reading and nuclease activities so any wrongly incorporated bases can be identified and a local stretch of DNA containing the error can be cut away and then repaired. In cells, DNA typically replicates by new DNA synthesis from an existing DNA strand template, using DNAdirected DNA polymerases, and a wide variety of such DNA polymerases exist in mammalian cells. In more specialized situations, DNA can be synthesized in cells from RNA templates using RNA-directed DNA polymerases lalso called reverse transcriptasesl as in the case of synthesizing the ends of a

de novo from a bare single-stranded template. Instead, a primer is required with a 3' OH group onto which the polymerase can attach a dNTP. Primases attach a small RNA primer to the singlestranded DNA to act as a substitute 3' OH for DNA polymerase to begin synthesizing from. This RNA primer is eventually removed by a ribonuclease and the gap is filled in using a DNA polymerase, then sealed using a DNA ligase. Ligases catalyze the formation of a phosphodiester bond given an unattached but adjacent 3' OH and 5' phosphate. Single-stranded binding proteins are important to maintain the stability of the replication fork. Single-stranded DNA is very labile, or unstable, so these proteins bind to it while it remains single stranded and keep it from being degraded.

_____________________

1._3

I

RNA

TRANSCRIPTION

AND

GENE

EXPRESSION

~

Table 1.2: Major classes of mammalian DNA polymerase (Al High fidelity (classical) DNA-directed DNA polymerases. DNA polymerase class

"

~

'Y

8

E

Location

Nuclear

Nuclear

Mitochondrial

Nuclear

Nuclear

General DNA replication

Synthesis and priming of lagging strand

mtDNA replication

Synthesis of leading strand

3'--1' 51 exonuclease8

No

No

Yes

Yes

Yes

By base excision

mtDNA repair

By nucleotide and base excision

By nucleotide and base excision

DNA repair function

'Serves as a proof-reading

activity.

(8) low fidelity (error-prone) DNA-directed DNA polymerases. DNA polymerase ~ (zetal

Expressed in mutating Band T cells; involved in hypermutation?

DNA polymerase TJ letal

Mutates A and T nucleotides during hypermutation?

DNA polymerase,

Very low fidelity of replication; thought to be involved in hypermutation

(iotal

DNA polymerase Il.lmu)

Highly expressed in Band T cells; thoughtto

be involved in hypermutation

(e) RNA-directed DNA polymerases (reverse transcriptases). Telomerase reverse transcriptase L1NE-l/endogenous

(Terti

Replicates DNA at the ends of linear chromosomes

retrovirus reverse transcriptase

.i,

Occasionally converts mRNA and other RNA into cDNA which can integrate elsewhere

'J'

into genome

\

f" I.',

" , r; linear

chromosollles

(Section

2.2.5)

plus reverse

Uses encoded by some highly el1dogellous retrovirus cbsses.

repetitive

transcripDNA

and

DNA viruses which generally ~~~~~~~~~~~ viruses

Llsually

1.2.4 Viral genomes are frequently maintained replication rather than DNA replication DNA

i~ the heredit:uy

for the

material

hereditary

Despite

the

materia\.

many

DNA

ubiquity

in all present-day

molecules of DNA

different

cells and we

cellular

classes of present-day

Jose

position

resIdues

alld so DNA

being a stable carrier is also error-prone being DNA

carry

about 10000 replication.

only

is much

of genetic with times

hy more

rogen

RNA

atoms than RNA

mutJtional snull

load.

viral

RNA

but have the advantage

genomes of rapid

some

replication complementary

integrates

into

like

error

RNA

rates. DNA

host

an RNA-directed

polymerases.

Following (cDNA),

chromosomal

has

conversion the

of RNA

retroviral

DNA

DNA relatively

(set'

cDNA Section

21.5.3).

1.3 RNA transcription and gene expression

Jt the

2'

RNA

to

error

rJtes

during

or their

are generally mutation

which.

1.3.1 The flow of genetic information in cells is almost exclusively one way: DNA >RNA >protein

replication

replication

a result

a reverse franscr;ptase,

into

have an

as high as that encountered

(see Iilble 1.3; r:(~lIrc 1.10).As

using

polymerase high

Viruses have developed many different strategies to infect and subvert cells, and their genomes show extraordinary diversity

Among

R.NA'

RNA viruses in that they replicate in the nucleus in that the RNA replicates viJ a DNA inrcrme-

terlll

hereditary

viruses

suited

information.

normal

in the cytopbsm.

replicate

or cell.

;;,;;;aiJe.~

~;;'I?ef;(~/7"r;;r,;;di;;;;J?i(/

also

diJtc,

of an organism as the

and

RNA genome. RNA molecules can under 0 self-replicJtion, but the 2' 01 .{!rollp 011 rhe ri ose resi lies ~ RJ.\TA I~CS ~ eoxyri

~~~~

rlll~ are a group of known as retroviruses. Retroviruses are

viru>;cs

UI1USUJ)

by RNA

of genomes as the collective

to thinking

in the nucleus.

import-am exceptiC>ilSW this"geneTJ'l RNA

arc accustomed

reolicare

high quite

rates. Unlike

The expression of genetic largely a one-way system:

information in 311 cells IS very DNA specificli the synthesis of

RNA and then RNA specifies the synthesis or polypeptides (which subsequently form proteins). UeCJU"ie of its universality.

the

DNA

>RNA

information

dogma

of molecular biology. in all cellular organisms:

essential

has

>polypeptide

genetic

been

(protein)

deKribed Two

J'"

the

"iucces"iivl'

flow

of

central "itl'pS are

,

.

r'

~

CHAPTER ONE I DNA STRUCTURE AND GENE EXPRESSION

(A)

RNA

DNA

CIRCULAR

LINEAR SINGLESTRANDED

o

~(+) Parvoviruses

e.g. M13, Id, 11

CIRCULAR

LINEAR

o

~(+) but no DNA intermediate e.g. hepatitis

A

e.g. hepatitis delta virus

~(-) e.g. rhabdoviruses

~(+) Replicate

via DNA intermediate

e.g. retroviruses

DOUBLESTRANDED

11l11l11l11l!1I11111111111111111

Herpes virus, A

o

Papovaviruses baculoviruses

11111111111111111111111111111111

Reoviruses

..~-----~ 111I11I11I11I!1I1111111111111111.

Adenovirus (has covalently linked terminal protein.)

(11111111111111111111111111111111:)

Poxviruses (have closed ends)

(8)

Influenza virus (segmented negative strand RNA)

Figure 1.10: The extraordinary

Gemini virus (bipartite; 2 circular ds DNA molecules)

variety of viral genomes.

(A) Strandedness and topology of viral genomes. In the case of single-stranded viral genomes, the RNA used to make protein products may have the same sense as the genome which is therefore a positive-strand genome (+) or be the opposite sense (antisensel 01 the genome, which is described as a negative-strand genome (-I. Some single-stranded (+1 RNA viruses go through a DNA intermediate (retrovirusesl and some double-stranded DNA viruses such as hepatitis B go through a replicative RNA form. (B) Segmented and multipartite viral genomes. Segmented genomes have a variety of different nucleic acid molecules specifying monocistronic mRNAs, as in the case of influenza viruses which have eight different negative single-strand RNA molecules. Multipartite genomes are a subset of segmented genomes where each of the different molecules is packaged into a separate virus particle.

_____________________

1._3_1

RNA TRANSCRIPTION

AND GENE EXPRESSION

~

I MITOCHONDRION I

r

Chromosomal DNA

~NUCLEUS

I

I

Nuclear proteins

.•• Transcription

Transcription

~~Primary

t ~+-5~

I

Reverse transcription

.••

~Primary RNA trantri~t processing

..,,_._'J'__+snRNA

@ ! !

Other

'-A.J

'-A.J

transcript '-A.J

cDNA

Cytoplasmic ribosomes

i

!

~

rRNA

~,/ I'" .••

mRNA

tRNA

./' Translation ••••••.•.

"'

Small cytoplasmic RNA

Mitochondrial I ribosomes ••.

Translation

9

N-C

N-C

1

Post-translational processing

Ox. phospho

N-C

Proteins in other organelles + cytosol

Secreted proteins

Figure 1.11: Gene expression in an animal cell.

Note: Ii) very occasionally a small proportion of nuclear RNA molecules can be converted naturally to cDNA by virally encoded and cellular reverse transcriptases, and thereafter integrate into chromosomal DNA at diverse locations; liil the mitochondrion synthesizes its own rRNA and tRNA and a few proteins which are involved in the oxidative phosphorylation IDx.phosph.) system. However, the proteins of mitochondrial ribosomes and the majority of the proteins in the mitochondrial oxidative phosphoryiation system and other mitochondrial proteins are encoded by nuclear genes and translated on cytoplasmic ribosomes, before being imported into mitochondria.

I. transcription. This uses 0 DNA-directed RNA polymerase and occurs in the nucleus of eukaryotic cells and, to a limited extent, in mitochondria and chloroplasts, the only other organelles which hJve a genetic capacity in addition to the nucleus (see F(~l"e 1.11); 2. translation. RN A-protein

This

occurs

111

ribosomes,

large

c0l11plexes which are found in the cytoplasm and also in mitochondria and chloroplasts. The ItNA

molecules messenger

which specify polypeptide RNA (mRNA).

such as mcmbers of the llul11malian LINE-l repetitive DNA [,mily (see Section 9.5.2). Became some nonviral RNA sequences are known to act as templates for cellular DNA synthesis, the principle of unidirectionol flow of genetic information in cells is no longer strictly valid.

ore known

os

The expression of genetic infornurion follows a colinearity principle: the IiI/ear seq"el1c(' ofnucleotides in DNA is decoded in groups of three nucleotides at a tilne (base triplets) to give a lit/ear sequel/ce of nucleotides in RNA which can be decoded in turn in groups of threc nucleotides (codotls) to give a iiI/ear seqlle/1ce of amino acids in the polypeptide product. R.ecently it hos become c1eor thar eubryoric cells, including mammalian cells, contain nonviral chromosomal DNA sequences which encode cellular rcverse transcriptases,

1.3.2 Only a small fraction

organisms product

of the ONA in complex is expressed to give a protein or RNA

Only 0 snull proportion of oil the DNA in cells is ever tronscribed. According to their needs, different cells transcribe different segments of rhe DNA (transcription units) which are discrete units, spaced irregularly along the DNA sequence. RNA polymerases use transcription units as templates to synthesize initially equivalent sized ltNA nlolecules (primary transcripts) which are then modified to generate mature expression products. However, the great majority of the cellular DNA is never transcribed in any cell. Moreover, only

~

I

CHAPTER ONE

Table 1.3: Different

DNA STRUCTURE AND GENE EXPRESSION

classes of genome

RNA Single-stranded (ss)

Double-stranded

Single-stranded

(ds)

(ss)

(ds)

Single circular molecule

Many bacteria and archaea; mitochondria; chloroplasts; some viruses

Some viruses

Single linear molecule

A very few bacteria e.g. Borrelia; some viruses

Some viruses

A few viruses

Some viruses

Multiple linear molecules

Eukaryotic nuclei; some segmented ds DNA viruses

Some segmented ss DNA viruses

Some segmented ds RNAviruses

Some segmented ss RNAviruses

Multiple circular molecules

Some bacteria; some

Mixed linear and circular

A very few bacteria e.g. Agrobacterium tumefaciens

bipartite and tripartite

is rr:lI1sbrt.'d into

transcriptioll IlIIif.' SpecU)1 lIollcodil/g Rl\A: the RNA product docs not encode polypeptides like IllRNA but has ;1 different function. In addition to the wdl-established ribosomal RNA (r1and
means normal orientation; < means reverse orientation. The glucocorticoid receptor gene is unusual in possessing 13 upstream GC boxes 110 in the normal orientation; three in the reverse orientation). The tRNA genes are transcribed by RNA polymerase III and have an internal bipartite promoter comprising element A lusually within the nucleotides numbered +8 to + 19 according to the standard tRNA nucleotide numbering system) and element B lusually between nucleotides +52 and +621. Specific transcription factors bind to these elements and then guide RNA polymerase III to start transcribing

at + 1.

is

the CAAT box, oftcn located at position -80. It is usually the strongest determinant of pronloter efficiency. Like the GC box, it appears to be able to function in either orientation (F({!//re 1,13). In addition to the above, more specific recognition elements are known to be recognized by tissue-restricted transcription factors (see 'fable '10.3).

cules and extensive acetylation of the four types of nucleosomal histones (i.e. histones H2A, H2B, H3 and H4; see Section 10.2.1). Additionally, the promoter regions of vertebrate genes are genera c aracterize by a sence of metl ated c tosines (see below). Transcription factors can displace nucleosomes, and so the open conformation of transcriptionally active chromatin can be distinguished experimentally because it also affords access to nucleases: at very low concentrations, the enzyme deoxyribonuclease I (DNasel) will digest long regions of nucleosome-free DNA. Although the regulatory regions may contain several sequence-specific binding proteins, the open chromatin structure is marked by the presence of DNase 1hypersellsitive sites (see Section 10.5.2).

~ enhancers comprise groups of cis-acting short sequence elements, which can enhance the transcriptional activity of specific eukaryotic genes. However, unlike promoter elements whose positions relative to the transcriptional initiation site are relatively constant (Figllre 1.13), enhancers are located a variable, and often considerable, distance from the transcriptional starr site, and their function is independent of their orientation ..I!l~ ~p£.e 10000 nucleotides

-

- < 20 nucieotides-

Exon

Figure 1.15: Consensus sequences atthe DNA level lor the splice donor, splice acceptor and branch sites in introns eukaryotes.

01 complex

Highlighted nucleotides are almost invariant (note: rare AT-AC introns also exist where the conserved splice donor dinucleotide GT is replaced by AT and where the conserved splice acceptor dinucleotide AG is replaced by AC; see text). Other nucleotides represent the majori nucleotid ound at this particular position, or equivalence between two nucleotides such as between C and T in the l/plypyrimidine tract djacent to the 3' end of the intron. Preferred motifs for the splice donor, branch site and splice acceptor in individual species (including human, Drosophila melanogaster, Caenorhabditis elegans and Saccharomyces cerevisiae) are listed by Um and Burge (2001). Some other exonic and intronic sequences are known to regulate splicing including splice enhancer and splice silencer sequences (see Berget et al., 19951.See also Fairbrother et al.120021for consensus sequences for exonic splicing enhancer sequences.

(A)

(B)

Splice E1

Branch site A

~r

Splice ac~orE2

E1

Splice donor .--.

Splice

Branch site

ac~orE2

~@bH 1. Cleavage at S' end !l) 2. Nucleophilic attack (,---) resulting in lariat fonmation

1

1

1. U1 SnRNP binds to splice donor site 2. U2 SnRNP binds to branch site E2

Lariat

E1

E2

1

Binding of U4/US/U6 SnRNP complex; US SnRNP binds to both splice donor + acceptor

1. Cleavage at 3' end 2. Ligation of exons

1 E1

E2

+ Figure 1.16: Mechanism 01 RNA splicing (GU-AG

introns).

(A) Mechanism. See Figure 1.15for consensus sequences of splice donor, splice acceptor and branch site. The nucleophilic attack involves the 2' -hydroxyl group attached to the conserved A at the branch site and the G of the conserved GU at the start of the intron and results in a new covaient bond linking these two nucleotides to give a branched structure lIariat). (B) Role of snRNPs. Small nuciear ribonucleoprotein particles {snRNPsl are part of the spliceosome. U1 snRNA has a terminal sequence complementary to the splice donor consensus and binds to it by RNA-RNA base pairing. After the U1 snRNP has bound, U2 snRNA recognizes the branch site by a similar base-pairing reaction. Interaction between the splice donor and splice acceptor junctions is stabilized by subsequent binding of a pre-formed multi-snRNP particle, containing U4, U5 and U6 snRNAs, with the U5 snRNP able to bind simultaneously to both splice donor and splice acceptor.

J

u,

~I

I DNA STRUCTURE

CHAPTER ONE

AND GENE EXPRESSION

before it). Howevn.

the order in which illtronic sequences are

removed

flanking

and their

cxonic

sequences

spliced

•

is not

governed by their linear ordt'r in the RNA transcript; instead, the conformation of the RNA is thought to influence the accessibility of 5' splice sitt's.

to RNA

'plicing.

RNA

polymerase

the transcript

(decapped

mRNA

II transcripts

to facilitate transport from the nuclcus to the cytoplasm;

~ ~

to facilitate

first 5' Ilucleotidt: phosphodiester

(m7G)

of the RNA bOlld. As thi,

transcript

bond

is linked

sites

carbon of the 1lJ7C; residue to the 5' carbon of the first nucleotide, the 51 l'nd is said to be blocked or capped (Fi.\?/IfC "/.17). Transcripts

of the snRNA

genes

are also capped.

role

in the

of the cytoplasmic

attachment

ribosomes

of the

but

their caps may undergo additional modification. The cap has been envisaged to have sevc:ral possible functions:

by both

RNA

polymerase

I and II I is known

transcription the

3'

ends

by

RNA

of mRNA

polymerase molecules

II is difficult

5'- 5' triphosphate linkage

o I

Pu

4·

1·

sibilitie,

- UAA.

UAG.

AGA

Fi~/I"(, 1.22;

andAGG:see

redefinition oj codons. Signals

illcluding J fcw typc~ lead to rcddinitioll of somc varicty

Iluclcar-t"ncoded as a 21 'I amino

of ccll~ (including IllRNAs

CJn

which

rransbrion reactions,

prodllct~ invoking

arc artJched

covalcndy

ofrcll undergo a variety of modithe addition of chemical group,,; to the polypeptide

chain

Jt the

translation:J! and post-translational levels. This can involve ~il11pk chcmical modification (hydroxylation. pho~phorylation. ete.) of the side chains of single J1llino ;lcid~ or till' :ldditiOIl of ditTerent

ill ";Ollie

of Iluckar-cllcodl'd codons. For cxample,

types of carbohydrate

or lipid group'" (set'

Table I. 7). Protein modification

by addition of carbohydrate

groups

hUIll~H1 cells), SOllle

altcrnatively

acid, sdetlocystei"e,

Primary ficarion

interpret

and UAG

UGA

call bl: alter-

Glycoproteins lently

attached

contain

oligosaccharidcs

to the side chains

of certain

which

;lrc cova-

;lJ1lino :lCids. Fcw

1.5

proteins

TRANSLATION, POST-TRANSLATIONAL

are ,-I(/y(osylated.

in the cytosol

carbohydrate,

and those

.\--acerylglucosamillc. nine

that arc carry

covalently

Uy contrast.

residue.

frol11 cells or exported

that

proteins

to lysosomes,

attached

sligar

residue.

to a serine or threo-

linked

those

is have

a single that

are sc:crcrcd

rhe Go\gi

or

apparatus

the plasma membrane are glycosylated. Oligosaccharide components of glycoproteins are largely preformed and added ell hlor to polypeptides. Two major types of glycosylation are recognized: ~

N-glycosylation: a CDIlllllon NH2

or an of

Proteoglycans

arc protein,

proteins,

the addition serve

::IS

within

the

endo-

with

difTcrL'nr

apparatus;

membrane

JttJchcd

to a glycine

t'llables

rhe modified

The

most

anchors.

proteins.

types

cysteine

serves

which

of fatty

at the extreme (0 interact

by typically

acyl groups

N terminus,

with

of the membrane.

of modification

as a membrane

becomes

anchor

attached

is the

residues.

groups typically become attached to cysteine n:siducs close to the C terminus, and include farnesyl (CIS) groups and geranylgeranyl (C20) groups. Many proteins that participate in signal transduction and protein targeting C0I1(3111either a farnesyl or J. gcr~lI1ylger;:l11yl unit at their C terminlls. Anchoring

of a protein

(0

the

outer

layer

of the

lIses a different

unit

to the C terminus

of the protl:in.

The entire protein except for the GPI anchor is located in the t:xtr:1cdlular space. cleavage

and

a IlH.'lllbranc

Anorhn

fatty

The primary translation product may also undergo inrerllal ck'avagl' to gt:llcrare a smaller mature product. Occasiol1:llly rht.' initiating methionine is cle;:lVed from the primary translation product. as during the synthesis of r3-globin (F(el/rc 1.19). More substantial polypeptide cleavage is observed in thL' case of the maturation proteins, polypeptide f:lctors. ete. As described sequences

are often

of many

proteills,

including

used to tag proteins

th;tt arc destined

Phosphorylation IPO.-!

Tyrosine, serine, threonine

Achieved by specific kinases. May be reversed by phosphatases

Methylation ICH3)

lysine

Achieved by methylases and undone by demethylases

Hydroxylation IOH!

Proline, lysine, aspartic acid

Hydroxyproline and hydroxylysine are particularly common in collagens

lysine

Achieved by an acetylase and undone by deacetylase

Glutamate

Achieved by 'V-carboxylase

Carboxylation ICOOH!

to be

of polypeptides Comments

'1 Acetylation ICH3CO)

plasma

hormones, nL'uropeptioes, growth in the next SL'ctioll, cleavable sign;.!

Target amino acids

ICH3CO!

plasma

meciunisl1l: the attachment of a glycosylphosphatidyl inositol (GPI) group. This complex glycolipid group contains a fatty acyl group that serves as the 11l1'Illbrane :1I1chor which is linked slIccc'ssively to a glyccrophosphate unit. an oligosaccharide unit and finally through a Ill\:mbranc

Type of modification (group added)

1Acetylation

Cl6

to the S atom of

Prell)'1

Post-translational

are modified

groups

Examples

protcin

group, which

m:Hrix.

group which is a C I.lipid that is found n:siduc

which

palmitoyl

27

units containing wcll-charanL'rized

of the extracellular

Illembrane

or the lipid bilayer

1.7: Major

,\tIYC(lS(/llIiIIOJ!IY((/l/s

repeating

by addition of lipid groups

notably

the myristoyl

Table

chain

acyl group

phosphol'thanolamine

of fatty acyl or prenyl

include

receptor

side

I. 7). Subsequent

with attached

arc componcnts

Protein modification Some

to the

replacement

disaccharide

or galacros:ll11ine.

protL'oglycJIl'\

of

see Table 1.7.

lIsually cont:lin

glllcO~a11lillc

and

Table

occlirs in the Golgi

~ O-glycosylation: which

residue

(ER; see

n:sidut's

mOllosaccharides

sequence

asparagine

reticulum

trimllling

in most cases, initial transfer

oligosaccharick

group

plasmic

involves,

PROCESSING AND PROTEIN STRUCTURE

lysine

Achieved by an acetylase and undone by deacetylase

N-glycosylation Icomplex carbohydrate!

Asparagine, usually in the sequence: Asn-X-(Ser(Thrl

Takes place initially in the endoplasmic reticulum; X is any amino acid other than proline

O-glycosylation (complex carbohydrate!

Serine, threonine, hydroxylysine

Takes place in the Golgi apparatus; less common than N-glycosylation

GPllglycolipid)

Aspartate at C terminus

Serves to anchor protein to auterlayer of plasma membrane

Myristoylation IC" fatty acyl group)

Glycine at N terminus Isee text!

Serves as membrane anchor

Palmitoylation IC'6fatty acyl group)

Cysteine to form S-palmitoyllink

Serves as membrane anchor

Farnesylation ICls prenyl group)

Cysteine at C terminus Isee text!

Serves as membrane anchor

Geranylgeranylation IC20prenyl group) Cysteine at Cterminus Isee text!

Serves as membrane anchor

~I

CHAPTER ONE

I DNA STRUCTURE

AND GENE EXPRESSION

hormones and other intt'rcellular signaling molecules) be exported to specific intracellular compartments,

sent to a specific location. Additionally, in some cases, a single mRNA molecule may specify more than one functional polypeptide precursor

chain as a result of proteolytic polypeptide

as the

cleavage of a large

nucleus

DNA

and

RNA

1.5.4 Protein secretion and intracellular export is controlled by specific localization signals or by chemical modifications

To do this,

a specific

localization

signal

to be

Exon 3

Intron 2

Exon 2

110

*

G GT

MG

AG

needs

embedded in the structure of the polypeptide so that it can be sent to the correct addn:ss. Usually. the localization signal takes the form of a shorr peptide sequence. Often, but not always. this constitutes a so-called signal sequence (or leader sequence) which is removed from the protein by a specialized signal peptidase once the sorting process has been achieved.

Proteins synthesized on mitochondrial ribosomes are required to fUllction within the mitochondria. However, the numerous protcins that are synthesized 011 cytoplasmic ribosomes havt' diverse fUllctions which may require them to be secreted from the cdl where they were synthesized (as with

Gene

polymerases.

transcription factors, RNA-processing proteins, etc.). the mitochondrion (mitochondrial ribosomal proteins. many respiratory chain components. etc.). peroxisomes. and so on.

(Figl/re 1.23).

Intron 1

(histones.

or to such

mRNA I

I I I I I I I

Preproinsulin

: 1 @IMet

I I I I I I

110 :

24 ~ 25 Alai Phe

Asnl@

UTR

24 Leader seq.1 Met

Alai 1 I Phe

30 ~31

65 ~ 66

Leu I Glu

Argl Gly

I Proinsulin

1

35

Key:

I Glu

Algi

C=:J Untranslated

Connecting 30 l-P-h-e--Leu--I + Insulin B chain

Figure 1.23: Insulin synthesis involves multiple post-translational

86 Asn

region

peptide 1 I Gly

21 Asn I

~ Cleavage site

Insulin A chain

cleavages 01 polypeptide precursors.

The lirst intron interrupts the 5' untranslated region; the second intron interrupts base positions 1 and 2 of codon 63 and is classified as a phase I intron (see Box 12.2,and also Figure I. 1910r other intron phases). The primary translation product, preproinsulin. has a leader sequence of 24 amino acids which is required for the protein to cross the cell membrane, and is discarded thereafter. The proinsulin precursor contains a central segment, the connecting peptide, which is thought to be important in maintaining the conformation of the A and B chain segments so that they can form disulfide bridges (see Figure 1.25).

______

'_.5_1

TRANSLATION,

POST-TRANSLATIONAL

Signals for export to the ER and extracellular space In the case of secreted

proteins

the first 20 or so amino

acids at the N-tcfminal

includes

a substantial

h,ble 'I. 8).The

signal sequence particle

consisting growing

polypeptide

The

I~NA

SRP

rough

reticulum

endoplasmic

can pass into

export

the cell, unless

(RER)

RNA,

both

and directs

the thelll

side surface

membrane.

the lumen there

7SL

binds

of the

Thereafter

a

of the Ell.., destined

are additional

Proteins which

are composed

for

hydrophobic

which NAD+)

cally binds),

to traverse

the mitochondrial

c1e~lved. Typicdly, addition

membranes,

a mitochondrial

to mallY hydrophobic

is n.:quircd

and is subsequently

signal

peptide

has,

al11ino acids, several

organization

(see'lid}le

f.9).

Within

ill

positively

the side chains. of

surf.1ce and hydrophobic and P(~lIrc L24A). Nuclear consist acids,

amino

within

the

of J stretch together

bctween

acids on one

acids on another

signals polypeptide neighboring

seguence

and charged

proline

arc

JUSt abollt

positively

some

by about

nuclear

10 amino

prott:ins

but are transported

from other

nuclear

proteins

localization

into the nucleus which

atom

fOllr amino

the

usually

chains

that

can

structure

coils

are found

keratin fibers blood clot;

sequences ~

the

units

in the trans-compartment

(often

and is recognized of the Golgi.

rOllnd

really

anriparallel)

chain

other

011

This

factors

An amphi-

one surface

to form Long

features

hydrogen bonds

segments

of

(see Figllre 1.2~). J3-plcated

aside

coiled

as the cx-

sllch

nails or fibrinogen

peptide

;:l11d

J particular

rod-like

proteins,

the core of most, but not all globular

the

1.24). Note

of nonpolar

coil.

fibrolls

opposed

with

(P(~IIYI' f.24). Identical

a coiled

sheet.

between

IO.2A).

residues

of skin, hair Jnd

bonding

bond

Figllre

(see

Section

each

cylinder.

of a pc.:ptide bond

arrangement

in many

rigid

of transcription

on another

called

J3-pleated

by a recepror

localization

coil

stable

polypeptide

of protein

group

hydrogen

nitrogen

away

(see

a repeating

of the Golgi

1.8: Examples

a-helical

with

the cis-colllpartment

Table

of

of the NH

of a peptide

donlJins

helices

formation

protein

acids

DNA-binding

Lysosomal proteins are targeted to the lysosome by the addition of a lllannOSt' fi-phosphate residue which is added in apparatus

for

(CO)

of:l

by

of the amino

Often,

signals.

scope

irrespective

structural

formation

oxygen

ones

with assistance

do have appropriate

for proteins

carbonyl

Fundamcntal

dominated

hydrophobic

acids (see Table '1.8). Nott'

lack any Iluclear

themselves

is

Jlllino

however, the signal is bipartite, and the positively c113rged amino acids are found in two blocks of two to four residues, separated

bond's

pathic a-helix has charged

typiGdly

residues.

of a peptide

to the hydrogen

the carbonyl

located

(see below

can be located

of four to eight

with

amino

on the levels of

is ample

residues;

bond.

structure

dut

localization

anywhere

charged

bond

peptide

there

different

~ the a-helix. This involves

hydrogen

with

the oxygen

distinguished

chain,

between

influences

four different

defined by hydrogen bonding between closely neighboring :Imino Jcid residues of a single polypeptide include:

cx-j,rlix,

structure

have been

can hydrogen

:Inotht'r

The

helical

At kast

a single polypeptide bonding

charged amino acids, usually spaced at intervals of about four amino acids. This structure is thought to form an alllpj,ipatj,ic J

each of

enzyme activity, e.g. which a protein specifi-

can be powerful

of the protein.

structural

group signal sequence

for functional (any molecule

each of which

conformation

Other signals N-terminal

~

to post-trans!ationalmodification.They

3rc required or ligands

hydrogen

JIl

STRUCTURE

of one or mon.' polypeptides,

can be subject

segments which stop the transfer process, as in the else of transmclnbrane proteins.

Like the ER '\ignal.

PROTEIN

Gin interact with specific co-factors (for example, divalent cations such as Ca2+, Fe2+, Cu2+, Zn2+ or small molecules

complex

species,

complex

on the cytosolic

polypeptide

acids (see

to the EI~ by a signal

chain and the ribosome protein

comprises

amino

AND

1.5.5 Protein structure is highly varied and not easily predicted from the amino acid sequence

end and Jlways

an RNA-protein

cytoplasmic

proteins.

to an SRJ> receptor

from

is guided

(SRP),

of a small

peptide

ofhyclrophobic

number

recognition and six specific

the signal

PROCESSING

of the

bond

in parallel the

same

sheets

form

prott'ills;

sequences

Destination of protein

Location and fonn of signal

Examples

Endoplasmic reticulum and secretion from cell

N-terminal peptide of 20 or so amino acids; very hydrophobic

Human insulin - 24 amino acid, highly hydrophobic signal peptide: N-Met-Ala-Leu·Trp-Met-Arg-Leu-Leu-Pro-Leu-Leu-Ala-Leu-Leu-Ala-Leu-TrpGly-Pro-Asp·Pro-Ala -Ala-Ala

Mitochondria

N-terminal peptide; ,,·helix with positively charged residues on one face and hydrophobic ones on the other

Human mitochondrial aldehyde dehydrogenase N-terminal17 amino acids: N-M et -Leu-Arg-Ala -Ala-Ala-Arg-Phe·G Iy-Pro-Arg -Leu-GIy-Arg-Arg- Leu-Leu

Nucleus

Internal sequence of amino acids; often a string of basic amino acids plus pralines; may be bipartite

SV40 T antigen - continuous: Pro-Pro-Lys-lys-lys-Arg-lys-Val p53 - bipartite: lys·Arg-Ala-Leu-Pro-Asn-Asn- Thr-Ser-Ser-SerPro-Gln- Pro-lys-lys-lys

Lysosome

Addition of mannose 6-phosphate residues

~I

CHAPTER ONE

I DNA STRUCTURE AND GENE EXPRESSION

Table 1.9: Levels of protein structure

level

Definition

Comment

Primary

The linear sequence of amino acids in a polypeptide

Canvary enormously in length from a small peptide to thousands of amino acids long

Secondary

The path that a polypeptide backbone follows

May vary locally e.g. as a-helix or l3-pleatedsheet

Tertiary

The overall three dimensional structure of a polypeptide

Canvary enormously e.g.globular, rod-like, tube, coil, sheet etc.

Quaternary

The overall structure of a multimeric protein I = a combination of protein subunits)

Qftenstabilized by disulfide bridges and by binding to ligands etc.

(B)

......... H

0

I

II

N,~ ..... C...•.

H

0

R

H

0

I

II

I

I

II

/N,~

I R

........ ®Arg

"'Arg

I

I

H

R

I II I I II I I C ~'N C ~ N C ~ N C ~ N,C ~'N II I I II I I II I

Ala Leu

I R

.•.....•...•..•......

---~-~------'-----'----_ .. R 0 H R 0 H R

....

Phe Met Leu Ala

/N,~ ..... C,

C'N ~'

.'

o

H

R

H

0

RHO

I

II

I

.

0

H

I

II

R

0

H

I

II

.' ....·····

Gly Leu

RHO

I

N,CH C N/CH...•....... N...•. H C...•....... C...•....... N...•. H C C C NHC C

I

RHO

I

II

I

I

RHO

II

I

R

Figure 1.24:Regions of secondary structure in polypeptides are often dominated by intrachain hydrogen bonding. (A) Structure of en a-helix. Left: only the backbone of the polypeptide is shown for clarity. The carbonyllCOI oxygen of each peptide bond is hydrogen bonded to the hydrogen on the peptide bond amide group INH) of the fourth amino acid away, so that the helix has 3.6 amino acids per turn. Note: for clarification purposes some bonds have been omitted. The side chains of each amino acid are located on the outside of the helix and there is almost no free space within the helix. Right: charged amino acids and hydrophobic amino acids are located on different surfaces in an amphipathic a-helix. The sequence shown is that for the 17-amino acid long signal peptide sequence for the mitochondrial aldehyde dehydrogenase Isee Tab/e 1.8).(B) Structure of a (3-p/eated sheet. Note:that hydrogen bonding occurs between the COoxygen and NH hydrogen atoms of peptide bonds on adjacent parallel segments of the polypeptide backbone. The example shows a case of bonding between antiparallel segments of the polypeptide backbone lantiparallel Ii-sheet) and enforced abrupt change of direction between antiparallel segments is olten accomplished using J3-turnsIsee text). Arrows mark the direction from N terminus to C terminus. Note: parallel J3-pleated sheets with the adjacent segments running in the same direction are also commonly found.

------------

1

o

H3N-G

[§,ill

[§,ill

I V EQCCTSICS

Y C N -coo

~

~

H3N- F V N Q H L C G 5 H L V E A L Y L V C G ERG

0

H3N-G

IV

~-~ CS L Y Q LEN

EQC

iTS

READING

~

A chain

)~

~

o

o

L Y Q LEN

FURTHER

I

0

H3N- F V NQHLCGSHLVEA

0

F F Y T P K T -coo

B chain

0

Y C N -coo

t

LYLVCGERGF

0

F Y T P K T -coo

Figure 1.25: Intrachain and interchain disulfide bridges in human insulin, The disulfide bridges (-S-S-I are formed by a condensation reaction between opposed sulfhydryll-SHI 11 of the A chain or between indicated residues of the different chains.

~ the J3-turn. Hydrogen bonding between the peptide bond CO group of amino acid TL'sidue 11 of a polypeptide with the peptide bond NH group of residue 11+3 results ill :l hairpin turn. By permitting the polypeptide to reverse direction abruptly, compact globular shapes can be achieved. p-TlIrns arc so named because they also of tell COllllect :lIltiparallel strands in J3-pleated sheets (see Fixl/rc 1.24B). Mon.' complex structural J1lori( 4n). Triploidy (3n) is less common became triploids have problems with meiosis (see below). The diploid cells of our body are all derived ultimately from a single diploid cell. the Z)'gote. by repeated rounds of mitotic cell division.

Each

roulld

are diploid

call be sUIllmarized

as one

turn

2C

Figure 2.1: Human chromosomal DNA content during the cell cycle. Interphase comprises Gl + S + G2. Chromosomes contain one DNA double helix from anaphase of mitosis right through until the DNA has duplicated in S phase. Fromthis stage until the end of metaphase of mitosis, the chromosome consists of two chromatids each containing a DNA duplex, making two double helices per chromosome. The DNA content of a diploid cell before S phase is 2C (twice the DNA content of a haploid cell), while between S phase and mitosis it is 4C.

of

the cell cycle (F(~/lf(' 2. 1).This comprises a short stage of cell division. the M phase (mitosis: see Fig/lre 2.7) and a long intervening

interphase.

Interphase

can

be

divided

into

S phase (D A ,ynthesis), G 1 phase (gap berween M phase and 5 phase) and G2 phase (gap between 5 phase and M phase). From anaphase of mitosis right through until D A duplication in 5 phase, a chromosome of a diploid cell contains a single DNA double helix and the total DNA content

is 2C. G I is the normal

state of a cell, and the long-

term end-state of non-dividing if they are committed

to mitosis;

modified G I sta e, sometimes cycle diagram action

cells. Cells enter 5 phase only nondividin

caIJed the GO phase.

can give the impression

happens

in Sand

cells remain

M phases

ill a

he cel

46,XX or 46.XY (F(~I/rc 2.2). The other cells of apart

cells. Human lack::t nucleus

p/oid. rounds

~ but this is an illusioll.

2.2 Structure

textbooks

(n

=

haploid

gametes

23) each gamett'

mosomcs) mosomc

can

(sperm

divide

pluli onL' liex chromosome. the zygote

c~ C.

r r{,\'

1' ••••• " ••

(

is diploid

.s.- ""I'

" •....

~~

by meiosis

to

and egg). In humans

22 autosomes

is always an X; in sperm

fertilizatioll

Y\ O.

that

contains

as somatic

and function

of

chromosomes Chromosomes

testis

are knowll

A

cells in the

and

thc germ-linc,

3.1.4).

does most of its work.

ovary

from

somatic cells are usually diploid but some cells and any chromosomes and are said to be I",/li-

and others are /lal/lrally polyp/oid as a result of multiple of 0 A replication without cdl division (see Section

A subset of the diploid body cells constitute the germline (see F(~/lre 2.9). These give rise to specialized diploid produce

(\

body,

that all the interesting

cell spends most of its life in GO or G 1 phase, and that is where the genome

constitution thc

(nonliex

chro-

In eggs thc liCX chro-

1It1"s/lalstate

phase

(2n) with

an

set

(/1(

'l

Jlt.4.1

,/

",

L

=3.6/,))

which

the microscope

misleading

occurs

brit;.t7y

becallse

and illustrated thc)'

in

represent

in the cell cycle. during

3n a part

of the M phase as cells prepare to undergo cell division (metaphase). Metaphase chromosomes and prometa-

it may be an X or a Y. Aftcr the chromosome

as seen under are rather

chromosomes

11I1/1."lIal tlI1o-chrol/latid

are in a /ll:l!hly ({)l1dellSed stnt((lIre

because

stare and

have

at thi" stage the 1)

A

1.5

proteins

TRANSLATION, POST-TRANSLATIONAL

are ,-I(/y(osylated.

in the cytosol

carbohydrate,

and those

.\--acerylglucosamillc. nine

that arc carry

covalently

Uy contrast.

residue.

frol11 cells or exported

that

proteins

to lysosomes,

attached

sligar

residue.

to a serine or threo-

linked

those

is have

a single that

are sc:crcrcd

rhe Go\gi

or

apparatus

the plasma membrane are glycosylated. Oligosaccharide components of glycoproteins are largely preformed and added ell hlor to polypeptides. Two major types of glycosylation are recognized: ~

N-glycosylation: a CDIlllllon NH2

or an of

Proteoglycans

arc protein,

proteins,

the addition serve

::IS

within

the

endo-

with

difTcrL'nr

apparatus;

membrane

JttJchcd

to a glycine

t'llables

rhe modified

The

most

anchors.

proteins.

types

cysteine

serves

which

of fatty

at the extreme (0 interact

by typically

acyl groups

N terminus,

with

of the membrane.

of modification

as a membrane

becomes

anchor

attached

is the

residues.

groups typically become attached to cysteine n:siducs close to the C terminus, and include farnesyl (CIS) groups and geranylgeranyl (C20) groups. Many proteins that participate in signal transduction and protein targeting C0I1(3111either a farnesyl or J. gcr~lI1ylger;:l11yl unit at their C terminlls. Anchoring

of a protein

(0

the

outer

layer

of the

lIses a different

unit

to the C terminus

of the protl:in.

The entire protein except for the GPI anchor is located in the t:xtr:1cdlular space. cleavage

and

a IlH.'lllbranc

Anorhn

fatty

The primary translation product may also undergo inrerllal ck'avagl' to gt:llcrare a smaller mature product. Occasiol1:llly rht.' initiating methionine is cle;:lVed from the primary translation product. as during the synthesis of r3-globin (F(el/rc 1.19). More substantial polypeptide cleavage is observed in thL' case of the maturation proteins, polypeptide f:lctors. ete. As described sequences

are often

of many

proteills,

including

used to tag proteins

th;tt arc destined

Phosphorylation IPO.-!

Tyrosine, serine, threonine

Achieved by specific kinases. May be reversed by phosphatases

Methylation ICH3)

lysine

Achieved by methylases and undone by demethylases

Hydroxylation IOH!

Proline, lysine, aspartic acid

Hydroxyproline and hydroxylysine are particularly common in collagens

lysine

Achieved by an acetylase and undone by deacetylase

Glutamate

Achieved by 'V-carboxylase

'1 Acetylation ICH3CO)

to be

of polypeptides Comments

Carboxylation ICOOH!

plasma

hormones, nL'uropeptioes, growth in the next SL'ctioll, cleavable sign;.!

Target amino acids

ICH3CO!

plasma

meciunisl1l: the attachment of a glycosylphosphatidyl inositol (GPI) group. This complex glycolipid group contains a fatty acyl group that serves as the 11l1'Illbrane :1I1chor which is linked slIccc'ssively to a glyccrophosphate unit. an oligosaccharide unit and finally through a Ill\:mbranc

Type of modification (group added)

1Acetylation

Cl6

to the S atom of

Prell)'1

Post-translational

are modified

groups

Examples

protcin

group, which

m:Hrix.

group which is a C I.lipid that is found n:siduc

which

palmitoyl

27

units containing wcll-charanL'rized

of the extracellular

Illembrane

or the lipid bilayer

1.7: Major

,\tIYC(lS(/llIiIIOJ!IY((/l/s

repeating

by addition of lipid groups

notably

the myristoyl

Table

chain

acyl group

phosphol'thanolamine

of fatty acyl or prenyl

include

receptor

side

I. 7). Subsequent

with attached

arc componcnts

Protein modification Some

to the

replacement

disaccharide

or galacros:ll11ine.

protL'oglycJIl'\

of

see Table 1.7.

lIsually cont:lin

glllcO~a11lillc

and

Table

occlirs in the Golgi

~ O-glycosylation: which

residue

(ER; see

n:sidut's

mOllosaccharides

sequence

asparagine

reticulum

trimllling

in most cases, initial transfer

oligosaccharick

group

plasmic

involves,

PROCESSING AND PROTEIN STRUCTURE

lysine

Achieved by an acetylase and undone by deacetylase

N-glycosylation Icomplex carbohydrate!

Asparagine, usually in the sequence: Asn-X-(Ser(Thrl

Takes place initially in the endoplasmic reticulum; X is any amino acid other than proline

O-glycosylation (complex carbohydrate!

Serine, threonine, hydroxylysine

Takes place in the Golgi apparatus; less common than N-glycosylation

GPllglycolipid)

Aspartate at C terminus

Serves to anchor protein to auterlayer of plasma membrane

Myristoylation IC" fatty acyl group)

Glycine at N terminus Isee text!

Serves as membrane anchor

Palmitoylation IC'6fatty acyl group)

Cysteine to form S-palmitoyllink

Serves as membrane anchor

Farnesylation ICls prenyl group)

Cysteine at C terminus Isee text!

Serves as membrane anchor

Geranylgeranylation IC20prenyl group) Cysteine at Cterminus Isee text!

Serves as membrane anchor

~l

I

CHAPTER TWO

CHROMOSOME

STRUCTURE AND FUNCTION

functions in a contexL Human D A is structured into and chromosomes function within cells which are derived by cell di"ision from other cells. The process of cell division ili. of course. a small component of the cell cycle, rhe process in which chromosomes and (heir constituent DNA molecules need {Q make perfect copies of themselves and then segregate into daughter cells. This needs to be very carefully orchestrated so that the daughter cells receive the correct set of choll1osomcs. In the usual form of eelJ division, mitosis, each daughter cell h" the same number and types of chromosome as the parent cell. In addition, a specialized form of DNA

chromosomes

cell division,

meiosis,

occurs

in certain

Total of 92

46 chromosomes

chromosomes

U divided

into two daughter cells

(condensed)

, 4C

,, ,

46 chromosomes (extended), two DNA double helices /"" per chromosome

/

,,

~

cells of the testis ;md

ovary and gives rise respectively to sperm and egg cells.

2. t

Ploidy and the cell cycle

The number of diffcrcm chromosomes in any nucleated cell. the chrolnosolne set. and the associated D A content are designated

=

C

nand

C respectively.

in the I1lllllber of copies Sperm

MOlit human

chromoliome

cells,

set and

a single

23 and

howt'\-er.

carry

are diploid

set (the

chromosome

(n chromosomes;

0

two

set

A coment,

copies

of the

46 chromosomes (extended), one DNA double helix

DNA

per chromosome

(2n chromosomes:

= 2C). AlmoSt all mammals

content

=

n

they havl: of the chromosome

and t'gg cells carry

and are liaid to be haploid C).

humans

3.5 pg (3.5 x 10-12 g). Cells may differ

approximately

ploidy).

For

DNA

synthesis

(the red .~~1£b~r~ ili an lIIlllSUa exception - see Gallarc 0 ct al., 1999), bllt all~e are many examples of species that are normally haploid, tetraploid (4n) or polyploid (> 4n). Triploidy (3n) is less common became triploids have problems with meiosis (see below). The diploid cells of our body are all derived ultimately from a single diploid cell. the Z)'gote. by repeated rounds of mitotic cell division.

Each

roulld

are diploid

call be sUIllmarized

as one

turn

2C

Figure 2.1: Human chromosomal DNA content during the cell cycle. Interphase comprises Gl + S + G2. Chromosomes contain one DNA double helix from anaphase of mitosis right through until the DNA has duplicated in S phase. Fromthis stage until the end of metaphase of mitosis, the chromosome consists of two chromatids each containing a DNA duplex, making two double helices per chromosome. The DNA content of a diploid cell before S phase is 2C (twice the DNA content of a haploid cell), while between S phase and mitosis it is 4C.

of

the cell cycle (F(~/lf(' 2. 1).This comprises a short stage of cell division. the M phase (mitosis: see Fig/lre 2.7) and a long intervening

interphase.

Interphase

can

be

divided

into

S phase (D A ,ynthesis), G 1 phase (gap berween M phase and 5 phase) and G2 phase (gap between 5 phase and M phase). From anaphase of mitosis right through until D A duplication in 5 phase, a chromosome of a diploid cell contains a single DNA double helix and the total DNA content

is 2C. G I is the normal

state of a cell, and the long-

term end-state of non-dividing if they are committed

to mitosis;

modified G I sta e, sometimes cycle diagram action

cells. Cells enter 5 phase only nondividin

caIJed the GO phase.

can give the impression

happens

in Sand

cells remain

M phases

ill a

he cel

46,XX or 46.XY (F(~I/rc 2.2). The other cells of apart

cells. Human lack::t nucleus

p/oid. rounds

~ but this is an illusioll.

2.2 Structure

textbooks

(n

=

haploid

gametes

23) each gamett'

mosomcs) mosomc

can

(sperm

divide

pluli onL' liex chromosome. the zygote

c~ C.

r r{,\'

1' ••••• " ••

(

is diploid

.s.- ""I'

" •....

~~

by meiosis

to

and egg). In humans

22 autosomes

is always an X; in sperm

fertilizatioll

Y\ O.

that

contains

as somatic

and function

of

chromosomes Chromosomes

testis

are knowll

A

cells in the

and

thc germ-linc,

3.1.4).

does most of its work.

ovary

from

somatic cells are usually diploid but some cells and any chromosomes and are said to be I",/li-

and others are /lal/lrally polyp/oid as a result of multiple of 0 A replication without cdl division (see Section

A subset of the diploid body cells constitute the germline (see F(~/lre 2.9). These give rise to specialized diploid produce

(\

body,

that all the interesting

cell spends most of its life in GO or G 1 phase, and that is where the genome

constitution thc

(nonliex

chro-

In eggs thc liCX chro-

1It1"s/lalstate

phase

(2n) with

an

set

(/1(

'l

Jlt.4.1

,/

",

L

=3.6/,))

which

the microscope

misleading

occurs

brit;.t7y

becallse

and illustrated thc)'

in

represent

in the cell cycle. during

3n a part

of the M phase as cells prepare to undergo cell division (metaphase). Metaphase chromosomes and prometa-

it may be an X or a Y. Aftcr the chromosome

as seen under are rather

chromosomes

11I1/1."lIal tlI1o-chrol/latid

are in a /ll:l!hly ({)l1dellSed stnt((lIre

because

stare and

have

at thi" stage the 1)

A

___________________

2_._2_1

STRUCTURE AND FUNCTION OF CHROMOSOMES

~

Box 2.1: The mitotic spindle and its components. The mitotic spindle Isee Figure below) is formed from microtubules (polymers made from a repeating heterodimer of ntubulin and IHubulin) and microtubule-associated proteins. Each of the two soindle Doles is defined by a centrosome, the major microtubule organizing center. The centrosome seeds the outward growth of oolar microtubule fibers, with a H end at the centrosome end and the distal growing end defined as the (+) end. Each centrosome is composed of a fibrous matrix Iconsisting of about 50 copies of a '1-tubulin ring complex in which is embedded a pair of centrioles (see panel A in figure belowl. Centrioles are short cylindrical structures composed of microtubules and associated proteins, and the two centrioles of the pair are always arranged at right angles to each other, forming an L shape Isee panel A of the figure). At a certain point in the Gl stage the two centrioles in a pair separate and during S phase a daughter centriole begins to grow at the base of each mother centriole and at right angles to it until fully formed by G2. The two centriole pairs remain close together in a single (A)

centrosomal complex until the beginning of M phase when the complex splits in two and the two halves begin to separate. Each centrosome now develops its own radial array of microtubules lasteli and begin to migrate to opposite ends of the cell where they will form the spindle poles Isee panel C in figurel. Three different forms of microtubule fiber are found in the spindle (see panel C in Figure belowl: ~ polar fibers extend from the two poles of the spindle towards the equator. They develop at prophase while the nuclear membrane is still intact. Note:for the sake of clarity only a pair of such overlapping fibers is shown; ~ kinetochore fibers do not develop until prometaphase. These fibers attach to the kinetochore, a large multiprotein structure attached to the centromere of each chromatid (panel 81 and extend in the direction of the spindle poles; ~ astral fibers form around each centrosome and extend to the periphery.

(B)

+

(e) Centromere

Centrosome matrix Kinetochore

+

mi~Ules

+ +

{

Metaphase } plate

+

+ o

= y-tubulin

Astral microtubules

ring complexes

association; Ie) mitotic spindle structure.

(A) Centrosome structure: (B) kinetochore-centromere

nor becol1le attached ro the mitotic spindle (see Box 2. J and r:~l!.lIr(' 2. 7). and so fail to be included in rhe nuclei of either of the daughter cells. During late prophase of mitosis, a pair oflarge l11ultiprorein cOl11pkxcs known as killetocllOres, forms at CJch centrolllere,

ol1e att:lched to each sister chromatid. Microtubules

attach to

Spt.'cific DNA sequences presulllJbly specify the structure :lIld function of centromeres. In silllpk eukaryotcs. the sequcnces that For cxample, centromere

specify centromere function in the yeast S(/cc//(mJ/II}'(cs

clement

of two highly

(CEN)

conserved

t":lch kinetochore. linking the centromere of a chromosome and the two spindle poles (see Box 2."1 and Figure 2.7). At

and a central AT-rich 2.5). The centromeres

anaphase. chromatids

a CEN fragment derived Cln repbce the centromere consequence.

the kinetochore toward opposite

Kinetochores assembly through driving

playa

central

and disassembly

microtllbules pull the two sister poles of the spindle (Figlfre 2.7). role in this process, of the attached

the presenCi..' of motor chronlOsonH.' movement.

by controlling

microtubuks

molecules.

and.

by ultimJtely

In Illalllmals, of hundreds Illosome-specific

the

is about

nanking

are very

(crcvi.,·i(/c

110 bp long,

elements

of9

short. thc

consisting

bp and II bp

segment of JbOlIt 80-YO bp (SL'l.' Figurc of such cells Jre interc!ullgt.'ablc from one of another

DNA

of individual

of kilobases

of rcpctitiw

Jnd sOllle nonspecific.

yeast chro111osome with no Jpparellt ccntrOllleres DNA.

some

consists c1Ho-

A J11ajor component

~

CHAPTER TWO

I

CHROMOSOME STRUCTURE AND FUNCTION

80-90 bp >90% (A+T)

TGATTTCCGAA ACTAAAGGCTT

II

III

Telomere Tandem repeats based on the general formula (TG),-3 TG2-:1C2-3A(CA)'-3

e.g.

I Autonomous

S·

TGTGTGGGTGTGGTGTGTGTGG

3'

3·

ACACACCCACACCACACACACC

S·

replicating

sequence

I

Contains an 11-bp core consensus that is AT-rich, plus some imperfect copies of this sequence spanning an approximately SO-bp region of DNA Figure 2.4: Individual chromosomes occupy distinct chromosome territories in the interphase nucleus.

I

I

The picture shows an example of chromosome painting (see Section 2.4.3) with two chromosome paints. one specific for HSA18 (= human chromosome 18 - green signal. peripheral locationl and one for HSA19 (= human chromosome 19- red signal. internal nuclear location) within an interphase nucleus. The nucleus appears blue as a result of counterstaining with DAPI. a fluorescent DNA-binding dye. Image kindly provided by Dr. Wendy Bickmore. MRC Human Genetics Unit. Edinburgh.

Cl.'lltrolllcric J) A is a-satellite DNA. a complex family of tandemly repeated DNA ba,ed on a 171 bp monOTllcr - set: Section 9.-+.1 for;] fuller description of hUlllan ceJltrolllcric ,equences). A variety of different proteins are known to as'iociate with human centromere'i, including CE P-13 which bind, directly to ci of cell division, frol11 cleavage of the zygote to death of the persall. III the lifetime of a human there may be something like 1017 mitotic divisions (Section i i ,2, I), The M phase of the cell cycle (F(~III'l'2.1) collsists of the v:lriotls stages of nuclear division (prophase. prollletaphase. I1ll't3phase. anaphase and telophase of mitosis). and cell division (cytokinesis), which ovcrbps the final stages of mitosis (Figl/re 2. 7). In preparation for cl'il divisioll. the previously highly extended chromosollles contract and condense so that. by metaphase of mitosis, rhey are readily visible under the microscope. Even though tht' DNA was replicated sOllle time previously, it is only :It prollll'taplust.' that individual chromosomes CJn be st:l'n to COlliprist' t\Vo sister chromatids, attached at the centrOllll'rt'. The interaction between the differcnt spindit' fibers (see Box 2.1) pulls the chromosollles towards the center, and by Illet:lphase each chromosollle is :lligned all tilt' equJtoriJI plane (nletaphase plate). Note: during mitosis each chro1Il0some in the diploid set bduvcs illdt:pcndcnrly and paren/a! (/l1d I/lafcma! 1/(H1lOlogs do 110/ (/s.wrillfC 11( ,,/I. At anaphasl' the Ct'ntrollleres divide leading to physical separ:ltion of what wert' previously sister chrolllJtids, and tht' pull by the spindle fibers t'l1sures that the separated sister chroll1:1tids go to opposite poles (FigHres 2.7 and 2'{)). The DNA of the two sister chromatids is identical. bJrring any errors ill DNA rcplication. Thus the effect of mitosis is to gent'rate daughter cells that contain precisely the SJme set of DNA scquellcc.:s.

______________

2_.3_1

MITOSIS AND MEIOSIS ARE THE TWO TYPES OF CELL DIVISION ~

IINTERPHASE I • Nuclear envelope intact • No chromosomes

visible

Prophase • Chromosomes condense and become visible • Bipolar spindle develops

• Nuclear envelope dissolves .Chromosomes begin to migrate to equatorial plane (metaphase plate) and are seen to contain two chromatids

Pole

---~-----

soi9~

Pole

Metaphase .Chromosomes fully condensed and located at metaphase plate

'~.:." P'T g [8 Metaphase

Prometaphase

Equatorial plane = metaphase plate

Anaphase .Each

centromere splits

• The two chromatids of each chromosome are pulled to opposite poles

Telophase .Chromosomes reach poles and start to decondense .Nuclear membrane reforms .Cytoplasm

starts to divide

Cytokinesis .Cytoplasm division completed to give two daughter cells

Figure 2.7: Cell division by mitosis: an outline.

2.3.2 Meiosis is a specialized form of cell division giving rise to sperm and egg cells I,

Primordial germ cells migrate into the enlbryonic gonad and engage in repeated rounds of mitosis to form oogonia in females and spermatogonia in males (Note: this involves many more mitoses in males than in females - see Box J 1. 4 - which Illay be a significant factor in explaining sex differences in mutation rate). Further growth and differentiation produces primary oocytes in the ovary and primary spermatocytes in the testis. These specialized diploid cells can undergo meiosis (F(gure 2.9). Meiosis involves two sllccessive cell divisions but only one round of DNA replication. so the products ate haploid. In males. the produC{ is four spermJwzoa; in females. however,

there is as}",u1Jetric cell division because the cytoplasm divides unequally at each stage: the products of meiosis I (the first meiotic division) are a large secondary oocyte and a small celi (polar body), During meiosis 11 (the second meiotic division) the secondary oocyte then gives rise to the large matllte egg celi and a second polar body. There are two crucial differences betwl:en mitosis and meiosis (Table 2,2): ~ the products of mitosis are diploid; the products of meiosis are haploid; •

rill: products of mitosis are genetically identical, products of meiosis are genetically different. tl

the

Mitosis involves a single turn of the cell cycle (F(