Lewin Benjamin (ed.) Genes IX

How do we

get

from

the 30 nm fiber to

the

specific structures displayed in mitotic

chromo-

somes? Is there any further

specificity in the

arrangement of

interphase

chromatin? Do

par-

ticular regions of 30 nm fibers

bear a fixed rela-

tionship to one another, or is

their arrangement

random?

Reproduction

of

Chromatin Requires

Assemb[y

of

Nucleosomes

o

Histone

octamers are

not

conserved during

reptication,

but

H2A-H28

dimers and H32-H4z

tetramers are conserved.

o

There are different

pathways

for

the assembly of

nucleosomes

during

replication

and independentty

of

reoUcation.

o

Accessory

proteins

are required to assist the

assembty of

nucleosomes.

.

CAF-1 is an assembly

protein

that

is

linked to the

PCNA subunit of the replisome; it is required for

deposition of H3z-H4z tetramers fotlowing

reptication.

r

A

different

assembty

protein

and a variant of

histone H3 may be used for replication-

independent assemb[y.

Replication separates the

strands of

DNA

and

therefore must inevitably disrupt the structure

of the

nucleosome. The transience

of the

repli-

cation event

is a major

difficulty in analyzing the

structure of a

particular

region while it is being

replicated. The structure

of

the replication fork

is distinctive.

It is

more resistant to micrococcal

nuclease and is digested into

bands

that differ

in

size

from nucleosomal DNA. The region

that

shows this altered structure

is

confined to the

immediate vicinity of the replication fork. This

suggests that a large

protein

complex is engaged

in replicating the

DNA,

but the nucleosomes

re-form more or less immediately behind it as

it moves along.

Reproduction of chromatin

does

not involve

any

protracted period

during

which

the DNA

is free of histones. Once DNA has been

repli-

cated,

nucleosomes are

quickly generated

on

both the duplicates.

This

point

is illustrated by

the electron micrograph of

ttri.{.i.iiii, ;lrri.!j*,

which

shows

a recently replicated stretch of DNA that

is already

packaged

into nucleosomes on both

daughter

duplex segments.

Both biochemical analysis and

visualiza-

tion

of the replication fork therefore suggest

that

the disruption of nucleosome structure

is

i:i s;

t

i

i;r

t

r:

r,1..,-l

ri:

Replicated

DNA is im

mediately i ncorporated

into nucteosomes. Photo

courtesy

ofSteven

L. McKnight,

UT Southwestern

MedicaI

Center

at Dat[as.

Iimited to a

short

region immediately

around

the

fork. Progress

of the

fork disrupts

nucleo-

somes, but they

form

very rapidly

on the

daugh-

ter duplexes as

the fork

moves

forward.

In fact,

the assembly

of

nucleosomes

is directly

linked

to the

replisome that

is replicating

DNA.

How do

histones associate

with

DNA to

gen-

erate

nucleosomes?

Do the

histones

preform

a

protein

octamer

around

which

the

DNA is sub-

sequently wrapped?

Or does

the

histone

octamer

assemble

on DNA

from

free histones?

iritiiririi: ,ri: .ii

shows

that two

pathways

can

be

used

ir vitr7 Io assemble

nucleosomes,

depend-

ing on the conditions

that are

employed.

In one

pathway,

a

preformed octamer

binds to

DNA.

In

the other

pathway,

a

tetramer

of.H3z-H4z

binds first,

and then

twoH2A-H2B

dimers

are

added. Both these

pathways

are

tions that

occur in

vivo

The

first reflects

the

capacity of chromatin

to be

remodeled

by mov-

ing histone octamers

along

DNA

(see

Sec-

tion

10.3, Chromatin

Remodeling

Is an

Active

Process).

The second

represents

the

pathway

that

is

used

in replication.

Accessory

proteins are

involved

in assist-

ing

histones to associate

with

DNA. candidates

for this

role can be

identified

by

using

extracts

that assemble

histones

and

exogenous

DNA

into

nucleosomes.

Accessory

proteins may

act as

"molecular

chaperones"

that

bind

to the

his-

tones

in order to

release

either

individual

his-

tones or complexes

(H32-H42

or

H2A-H2B)

to

the DNA in

a controlled

manner.

This could

be

necessary because

the

histones,

as basic

pro-

teins,

have a

general high affinity

for DNA. Szcft

interactions

allow

histones

to

form

nucleosomes

with-

out becoming

trapped

in

other

kinetic

intermediates

(that

is, other

complexes

resulting

from

indiscreet

binding of

histones

to DNA).

29.9

Reproduction

of Chromatin

Requires

Assembty of

Nucleosomes

Octamerassembles

Preformedoctamer

on DNA

binds

I

uzn-Hza

|

(underneath)

H2A-H2B

=*i"l*[

,?i].1.:

In vifro, DNA

can

either

interact

directty

with

an

jntact

(crossl.inked)

histone

octamer

or can assem-

bte with

the H32-H42

tetramer,

after which

two H2A-H2B

dimers

are added.

Attempts

to

produce

nucleosomes

in vitro

began

by

considering

a

process

of assembly

between

Iree

DNA

and histones.

Nucleosomes

Iorm

in vivo,

though,

only when

DNA is repli-

cated. A

system

that mimics

this requirement

has

been

developed

by using

extracts

of human

cells that

replicate

SV40 DNA

and assemble

the

products

into

chromatin. The

assembly

reac-

tion occurs preferentially

on replicating

DNA.

It requires

an ancillary factor,

chromatin

assem-

bly factor

(CAF)-

1,

that consisrs

of

>5

subunits,

with a

total mass

of 238 kD.

CAF- I is

recruited

to

the replication

fork

by

proliferating

cell

nuclear

antigen

(PCNA),

the

processivity

fac-

tor for DNA polymerase.

This provides

the link

between

replication

and nucleosome

assembly,

:l#

:13 iix

il,";:,:"j

:ffi::;".:

"'

-''I

e d a s

CAF-1

acts stoichiometrically,

and func-

tions

by binding

to newly

synrhesized

H3

and

FI4. This

suggests

that

new nucleosomes

form

by

assembling

first

the H32-H42

tetramer,

and

then adding

the H2A-H2B

dimers.

The nucle-

CHAPTER

29

Nucteosomes

F{filJltE tS.**

If histone

octamers were

conserved,

old

and new

octamers woutd

band at different

densities when

replication

of heavy

octamers occurs in

light

amino acids.

osomes that

are

formed

in vitro

have

a repeat

length

of 200

bp. They do not

have

any Hl his-

tone.

though, which

suggests that

proper

spac-

ing can

be accomplished

without HI.

When

chromatin

is reproduced,

a stretch

oIDNA already

associated with

nucleosomes

is repli-

cated,

giving

rise

to two

daughter

duplexes.

What happens

to the

preexisting

nucleosomes

at this

point?

Are the histone

octamers

dissoci-

ated into

free histones

for reuse,

or do they

remain

assembled?

The integrity

of the

octamer

can

be tested by crosslinking

the histones.

The

next two figures

compare

the

possible

outcomes

from an

experiment in

which

cells are

grown

in the

presence

of heavy amino

acids

to iden-

tify the histones

before replication.

Replication

is

then allowed

to occur in

the

presence

of light

amino acids.

At this

point

the histone

octamers

are crosslinked

and

centrifuged

on a density

gradient. fl3fiLlSil

JS.ffS

shows

that if

the original

octamers

have

been conserved,

they

will

be

found

at a

position

of high

density,

and new

octamers

will

occupy a low

density

position.

772

Histone

tetramer

is displaced

and

disassembles

Replication

fork

advances

toward

nucleosome

4. H2A-H2B

dimers

bind

3.

H3-H4

tetramers

bind

to daughter

duplexes

f,itlitre i*"

i:F

When

heavy octamers are

replicated

in

light

amino acids,

the

new

octamers

band diffuseLy

between

heavy and [ight

densities,

which suggests that

disassem-

bty and

reassembty has occurred.

This

does

not happen, though.

Little

material

is

found

at the

high density

position,

which

sug-

gests

that

histone octamers

are

not conserved.

The octamers

have an intermediate

density,

and

${{:;.:ft{: *+.:li.l shows that

this is the expected

result

if the

old histones

have been

released

and then

reassembled

with

newly synthesized

histones.

The

pattern

of

disassembly

and reassembly

has been difficult

to characterize

in detail,

but

a working

model

is illustrated

in

FEGiisf,

t*.3*.

The

replication

fork displaces

histone

octamers,

which

then

dissociate

into H32-H42

tetramers

and

H2A-H2B

dimers.

These

"old"

tetramers

and dimers

enter

a

Dool

that

also includes

"Dew"

F.i{;{Jlr.[

iiii,-t* RepLication

fork

passage

disptaces

histone

octamers

from

DNA.

Thev disassembl.e

into

H3-H4 tetramers

and

H2A-H2B

dimers.

Newty

synthesized

histones

are assembted

into

H3-H4

tetramers

and

H2A-H2B

dimers.

The otd

and

new tetramers

and

dimers

are

assembled

with

the

aid of

cAF-1

at

random

into

new

nucteosomes

immediatety

behind

the

reptication

fork.

tetramers

and dimers,

which

are assembled

from

newly

synthesized

histones.

Nucleosomes

assemble

-600

bp

behind

the

replication

fork.

Assembly

is

initiated

when

H32-H42

tetramers

bind

to each

of

the

daughter

duplexes,

assisted

by CAF-1.

TWo

H2A-H2B

dimers

then

bind

to

each

H32-H42

tetramer

to

complete

the

histone

octamer.

The assembly

of

tetramers

and

dimers

is random

with

respect

to

"old"

and

"new"

sub-

units, which

explains

the

results

of

Figure

29

.29

-

The

"old"

H32-H42tetramer

could

have

an abil-

ity

to be transiently

associated

with

a single

strand

of

DNA during

replication;

it

may

in fact

have an

increased

chance

of

remaining

with

the

Ieading strand

for

reuse.

It is

possible

that

nucleosomes

are

disrupted

and

reassembled

in

2g.g

Reproduction

of

chromatin

Requires

Assembly

of

Nucleosomes

773

a similar

way during

transcription (see

Sec-

tion

29.I l,

Are Tlanscribed

Genes

Organized

in

Nucleosomes?).

During

S

phase (the

period

of DNA

replica-

tion)

in

a eukaryotic

cell,

the duplication

of

chromatin

requires

synthesis

of sufficient

his-

tone proteins

to

package

an

entire

genome-

basically

the same

quantity

of

histones

must

be

synthesized

that are

already

contained

in nucle-

osomes.

The

synthesis

of histone

mRNAs is

con-

trolled

as

part

of

the cell

cycle,

and increases

enormously

in

S

phase.

The pathway

for

assem-

bling

chromatin

from

this

equal mix

of

old and

new

histones

during

S

phase

is called

the

replication-coupled

(RC

)

pathway.

Another pathway,

called

the replication-

independent

(RI)

pathway,

exists

for

assem-

bling

nucleosomes

during

other phases

of

cell

cycle,

when

DNA

is not

being

synthesized.

This

may

become

necessary

as the

result

of damage

to DNA

or because

nucleosomes

are displaced

during

transcription.

The

assembly process

must

necessarily

have

some

differences

from

the repli-

cation-coupled

pathway,

because

it

cannot

be

Iinked

to the

replication

apparatus.

One of

the

most interesting

features

of the replication-

independent

pathway

is that

it

uses

different

variants

of some

of the

histones

from

those

used

during

replicarion.

The

histone

H3.3

variant

differs

from

the

highly

conserved

H3 histone

at four

amino

acid

positions.

H3.3

slowly

replaces

Hl in

differen-

tiating

cells

that

do not

have replication

cycles.

This

happens

as

the result

of

assembly

of new

his-

tone

octamers

to replace

those

that have

been

displaced

from

DNA

for

whatever

reason.

The

mechanism

that is

used

to ensure

the use

of H3.3

in

the replication-independent

pathway

is

dif-

ferent

in

two

cases

that have

been

investigated.

In the

protoz

oan

Tetrahymena,

histone

usage

is

determined

exclusively

by

availabiliry.

His-

tone

H3

is synthesized

only

during

the

cell cycle;

the

variant

replacement

histone

is

synthesized

only

in

nonreplicating

cells. ln

Drosophila,

how-

ever,

there

is an

active pathway

that

ensures

the

usage

of

H3.3

by

the

replication-

independent

pathway.

New nucleosomes

con-

taining

H3.3

assemble

at

sites

of transcription,

presumably

replacing

nucleosomes

that

were

displaced

by RNA polymerase.

The

assembly

process

discriminates

between

H3

and

H3.3

on

the

basis

of

their

sequences,

specifically

exclud-

ing

H3

from

being

utilized.

By

contrast,

repli-

cation-coupled

assembly

uses

both

types

of H3

(although

H3.3

is

available

at much

lower

lev-

els

than

Hl,

and

therefore

enters

only

a

small

proportion

of nucleosomes).

Nuc[eosomes

CAF-l

is

probably

not

involved

in

replication-independent

assembly. (There

also

are

organisms

such

as

yeast

and Arabidopsis

for

which

its

gene

is not

essential,

implying

that

alternative

assembly

processes

may

be used in

replication-coupled

assembly.)

A

protein

that

may be involved

in replication-independent

assembly

is

called HIRA.

Depletion

of HIRA

from

in vitro

systems for

nucleosome

assembly

inhibits

the formation

of nucleosomes

on nonreplicated

DNA,

but not

on replicating

DNA,

which

indi-

cates

that

the

pathways

do indeed

use

different

assembly

mechanisms.

HIRA

functions

as a

chap-

erone

to assist

the incorporation

of histones

into

nucleosomes.

This

pathway

appears

to be

gen-

erally responsible

for replication-independent

assembly;

for

example,

HIRA

is required

for

the

decondensation

of the

sperm

nucleus,

when

protamines

are replaced

by histones,

in

order

to

generate

chromatin

that

is competent

to

be repli-

cated

following

fertilization.

Assembly

of nucleosomes

containing

an

alternative

to H3

also occurs

at

centromeres

(see

Section

31.3,

Heterochromatin

Depends

on

Interactions

with Histones).

Centromeric

DNA

replicates

early during

the

replication phase

of

the

cell cycle

(in

contrast

with

the surrounding

heterochromatic

sequences

that

replicate

later;

see Section

15.7,

Each Eukaryotic

Chromosome

Contains

Many

Replicons).

The

incorporation

of H3 at

the centromeres

is inhibited,

and instead

a

protein

called

CENP-A

is incorporated

in higher

eukaryotic

cells

(in

Drosophila

it

is

called

Cid,

and

in

yeast

it is

called

Cse4). This

occurs

by the

replication-independent

assembly

pathway,

apparently

because

the replication-coupled

path-

way is inhibited

for

a brief

period

while

cen-

tromeric

DNA

replicates.

Do

Nucleosomes

Lie

at

Specific

Positions?

r

Nucteosomes

may

form

at specific

positions

as the

resutt

either

of

the [oca[

structure

of DNA

or

of

proteins

that interact

with

specific

sequences.

o

The

most

common

cause

of nucteosome

positioning

is

when

proteins

binding

to DNA

estab[ish

a boundary.

r

Positioning

may

affect

which regions

of DNA

are in

the

linker

and which

face

of DNA

is

exposed

on the

nucleosome

surface.

We know

that nucleosomes

can

be reconsti-

tuled

in vitro

wilhout

regard

to DNA

sequence,

but

this

does not

mean

that

their

formation

in

tzivo is

independent

of

sequence.

Does

a

partic-

774

CHAPTER

29

ular

DNA sequence

always lie in a certain

posi-

tion in

vivo w\th regard to the topography

of the

nucleosome?

Or are

nucleosomes arranged ran-

domly

on DNA, so

that a

particular

sequence

may occur

at any location,

for

example,

in the

core region

in one copy of the

genome

and in

the

linker region

in

another?

To

investigate this

question,

it is necessary

to

use a defined

sequence of DNA;

more

pre-

cisely, we

need to determine

the

position rela-

tive to the

nucleosome of a defined

point

in the

DNA.

:-;rl,r:tii

,i1:.'r l

illustrates the

principle

of a

procedure

used to achieve

this.

Suppose that

the DNA sequence

is orga-

nized

into nucleosomes

in

only

one

particular

configuration,

so that

each site on the

DNA

always

is

located at a

particular position

on the

nucleosome.

This type of organization

is called

nucleosome

positioning

(or

sometimes

nucleosome

phasing).In

a series of

positioned

nucleosomes,

the linker regions of

DNA com-

prise

unique

sites.

Consider

the consequences

for

just

a sin-

gle

nucleosome.

Cleavage

with micrococcal

nuclease

generates

a monomeric

fragment that

constitutes

a specific sequence

If. the DNA

is iso-

lated and

cleaved with a

restriction enzyme

that

has only

one target

site in this

fragment,

it

should be

cut at a unique

point. This

produces

two fragments,

each of

unique size.

The

products

of the

micrococcal/restriction

double

digest are separated

by

gel

electrophore-

sis.

A

probe

representing the sequence

on one

side

of the

restriction site

is used to

identify

the corresponding

fragment in the

double

digest.

This

technique

is

called

indirect

end

labeling.

Reversing

the argument,

the identification

of

a single

sharp band

demonstrates

that the

position

of the

restriction site

is

uniquely

defined

with

respect to the

end of the nucleosomal

DNA

(as

defined

by the

micrococcal nuclease

cut).

Thus

the nucleosome

has a unique

sequence

of

DNA.

What

happens if the

nucleosomes do

rollie

at a single

position?

Now the

linkers consist

of

different

DNA

sequences

in each copy

of the

genome. Thus

the restriction site

lies at

a differ-

ent

position

each time;

in fact,

it lies at all

pos-

sible

locations

relative to the

ends

of the

mOnomeriC

nuCIeOSOmal

DNA.

.i;ji,i,flil,

,r1ir,

jrl:

shows

that the double

cleavage

then

generates

a broad

smear,

ranging from the

smallest

detectable

fragment

(-20

bases)

to the

length ot

the monomeric

DNA.

In discussing

these

experiments,

we

have

treated

micrococcal

nuclease as

an enzyme

that

f:iili.iiii:

;,lil.

I

Nucteosome

positioning

ptaces

restriction

sites

at unique

positions retative

to the

[inker

sites

cteaved

by

micro-

coccaI

nuctease.

cleaves

DNA at

the

exposed

linker

regions

with-

out

any sort

of sequence

specificity.

The

enzyme

actually

does

have

some

sequence

specificity,

though,

which is

biased

toward

selection

of

A-T-rich

sequences.

Thus

we

cannot

assume

that

the existence

of

a specific

band

in the

indi-

rect

end-labeling

technique

represents

the

dis-

tance

from a

restriction

cut

to the

linker

region.

It could

instead

represent

the

distance

from

the

restriction

cut to

a

preferred

micrococcal

nucle-

ase cleavaqe

site!

Positioning

places

target sequence

(red)

at

unique

position

Restriction enzyme

cleaves

at

target

sequence

I

i

Fragment

has

restriction

cut

at one

end,

micrococcal

cut

at other

end;

electrophoresis

gives

unique

band

Micrococcal

nuclease

releases

monomers

29.L0

Do Nucteosomes

Lie at

Specific

Positions?

775

.!"

.'

In

the absence

of nucleosome

Dosition-

ing,

a restriction

site

[ies at

at[

possibte

Locations

in

djf-

ferent

copies

of the

genome.

Fragments

of

at[

possib[e

sizes are

produced

when

a restriction

enzyme

cuts

at a

target

site

(red)

and

micrococcaI

nuctease

cuts at

the

junctions

between

nucleosomes (green).

This

possibility

is

controlled

by treating

the

naked

DNA in

exactly

the

same

way

as the

chro-

matin.

If

there

are

preferred

sites

for micrococ-

cal nuclease

in the

particular

region,

specific

bands

are found.

This pattern

of bands

can

then be

com-

pared

with

the

pattem

generated

from

chromatin.

A

difference

between

the

control

DNA

band

pattern

and

the

chromatin pattern

provides

evi-

dence

for

nucleosome

positioning.

Some

of the

bands

present

in

the

control

DNA

digest

may

disappear

from

the nucleosome

digest,

indicat-

ing

that preferentially

cleaved positions

are

unavailable.

New bands

may

appear

in the

nucleosome

digest

when

new

sites

are rendered

preferentially

accessible

by the

nucleosomal

organization.

Nucleosome

positioning

might

be accom-

plished

in

either

of

two

ways:

.

It is

intrinsic:

Every

nucleosome

is deposited

specifically

at a

particular

DNA

sequence.

This

modifies

our

view

of the

nucleo-

some

as a

subunit

able

to form

between

any

sequence

of DNA

and

a histone

octamer.

CHAPTER

29

Nucteosomes

.

It is

extrinsic:

The

ftrst

nucleosome

in a

region

is

preferentially

assembled

at a

particular

site. A

preferential

starting point

for

nucleosome

positioning

results

from

the

presence

of a

region

from

which nucle-

osomes

are excluded.

The

excluded

region provides

a boundary

that

restricts

the

positions

available

to the

adjacent

nucleosome.

A series

of nucleosornes

may

then be

assembled

sequentially,

with a

defined repeat

length.

It is now

clear

that the

deposition

of his-

tone

octamers

on DNA is

not random

with

regard

to sequence.

The

pattern

is intrinsic

in

some

cases, in

which it

is determined

by

struc-

tural

features

in DNA.

It is

extrinsic

in

other

cases.

in which

it results

from

the interactions

of other

proteins

with the

DNA

and/or

histones.

Certain

structural features

of DNA

affect

placement

of histone

octamers.

DNA

has intrin-

sic

tendencies

to

bend in

one direction

ratner

than

another;

thus A-T-rich

regions

locate

so

that the

minor

groove

faces

in toward

the

octamer,

whereas

G-C-rich

regions

are arranged

so

that the

minor

groove

points

out. Long

runs

of dA-dT

(>8

bp)

avoid

positioning

in

rhe

cen-

tral superhelical

turn

of the core.

It is

not

yet pos-

sible

to sum

all

of the relevant

structural

effects

and

thus entirely predict

the

location

of a

par-

ticular

DNA

sequence with

regard

to the

nucle-

osome.

Sequences

that

cause

DNA

to

take up

more

extreme

structures

may

have

effects

such

as

the exclusion

of nucleosomes,

and

thus

could

cause

boundary

effects.

Positioning

of nucleosomes

near

bound-

aries is

common.

If

there is

some

variability

in

the

construction

of nucleosomes-for

exam-

ple,

if

the length

of

the linker

can vary

by,

say,

l0

bp-the

specificity

of locarion

would

decline

proceeding

away from

the first,

defined

nucle-

osome

at the

boundary.

In

this

case,

we might

expect

the

positioning

to be

maintained

rigor-

ously

only

relatively

near

the

boundary.

The location

of DNA

on nucleosomes

can

be described

in

two

ways.

l.iL,;tii'.

::t;i,i.:

shows

that

translational positioning

describes

the

position

of DNA

with regard

to

the

boundaries

of

the nucleosome.

In

particular,

it

determines

which

sequences

are found

in

the linker

regions.

Shifting

the

DNA

by l0

bp brings

the nexr

turn

into

a linker

region.

Thus

translational

position-

ing

determines

which

regions

are

more

acces-

sible

(at

least

as

judged

by

sensitivity

ro

micrococcal

nuclease

)

.

DNA

lies

on the

outside

of

the historre

octamer.

As

a result,

one

face

of anv

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i.,r:'ii i: ;ll:i

I

:i"i:l

uor6er relut;

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t-g

sulnl

ii*l-iFi

I*"-l=

The isolated

nucleolar

genes

rDNA

tran-

scription

unjts

atternate with nontranscribed

DNA

seg-

ments.

Reproduced

from

Mitter,

0. L.

and Beatty, B.

R. 1969.

Science. 1.64:955-957.

Photo

courtesv

of 0scar

Mitter.

iISliFI

f$.s+

An

SV40 minichromosome

can be

tran-

scribed.

Reproduced

from

J. Mol.

Bio.,

vol.131.

GarigLio,

P.,

et at., The

template

of

the isoLated

. . .

,

p.131.

Copy-

right1.979,

with

permission

from

Elsevier.

photo

courtesv

of Pierre

Chambon.

Attempts

to visualize genes

during

transcrip-

tion

have produced

conflicting

results.

The

figures

show

each extreme.

Heavily

transcribed

chromatin

can

be seen

to

be rather

extended

(too

extended

to

be covered

in

nucleosomes).

In

the intensively

transcribed

genes

coding

for

rRNA

shown

in

FItil.tfr$:

t'"+.jt$,

the

extreme

packing

of RNA

polymerases

makes

it

hard

to

see the

DNA.

We cannot

directly

measure

the lengths

of

the rRNA

transcripts

because

the

Nucteosomes

RNA is

compacted

by

proteins,

but

we know

(from

the

sequence

of the rRNA)

how long

the transcript

must

be. The

length

of the transcribed

DNA

seg-

ment.

which is measured

by

the length

of the

axis

of the

"Christmas

tree," is

-85%

of the length

of

the rRNA.

This means

that the

DNAis

almost

com-

pletely

extended.

On the

other hand,

transcription

complexes

of SV40

minichromosomes

can

be extracted

from

infected

cells. They

contain

the usual

com-

plement

of histones

and display

a

beaded

struc-

ture. Chains

of RNA

can be seen

to extend

from

the minichromosome,

as in

the

example

of

Fi{;|ifiil

t*"}*.

This

argues that

transcription

can

proceed

while

the SV40 DNA

is

organized

into

nucleosomes.

Of course,

the

SV40 minichro-

mosome

is transcribed

less intensively

than

the

rRNA

genes.

Transcription

involves

the

unwinding

of

DNA,

and

may require

the fiber

ro

unfold

in

restricted

regions

of chromatin.

A simplistic

view

suggests

that some

"elbow-room"

must

be needed for

the

process.

The

features

of

poly-

tene and

lampbrush

chromosomes

described

in

Chapter

28,

Chromosomes,

offer

hints

that

a

more

expansive

structural

organization

is asso-

ciated with

gene

expression.

In

thinking

about

transcription,

we must

bear in mind

the relative

sizes

of RNA

poly-

merase

and

the nucleosome.

The

eukaryotic

enzymes

are large multisubunit

proteins,

typi-

cally

>500

kD. Compare

this

wirh

the

-260

kD

of the nucleosome.

$t{#ftf,

t1;.}?

illustrates

the

approach

of RNA

polymerase

to nucleosomal

DNA.

Even

without

detailed

knowledge

of the

interaction,

it is

evident

that it

involves

the

approach

of two comparable

bodies.

Consider

the two

turns

that DNA

makes

around

the nucleosome.

Would

RNApolynerase

have

sufficient

access

to DNA

if

the nucleic

acid

were

confined

to this

path?

During

transcription,

as RNA

polymerase

moves

along

the

template,

it

binds

tightly

to a region

of

-50

bp,

including

a

Iocally

unwound

segmentol-l2bp.

The

need

to

unwind

DNA

makes it

seem

unlikely

that the

segment

engaged

by RNA

polymerase

could

remain

on the

surface

of the histone

octamer.

It therefore

seems inevitable

that

transcrip-

tion

must

involve

a structural

change.

Thus

the

first

question

to

ask about

the

structure

of active

genes

is

whether

DNA

being transcribed

remains

organized

in

nucleosomes.

If

the

histone

octamers

are

displaced,

do they

remain

attached

in

some

way to

the transcribed

DNA?

One experimental

approach

is

to digest

chromatin

with

micrococcal

nuclease,

and

then

to

use a

probe

to

some

specific

gene

or

genes

to

778

CHAPTER

29

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RNA

polymerase

Torsion

ahead

of RNA

polymerase

displaces

octamer, which

reinserts

behind

polymerase

DNA is

displaced from

octamer

and forms

closed looo

l':iil"!*l

:i;.-i-i: RNA

polymerase

disptaces

DNA from

the

histone

octamer as it

advances. The

DNA

loons

back and

attaches (to polymerase

or to

the octamer)

to form

a

closed

[oop. As

the

pol.ymerase

proceeds,

it

generates

pos-

itive

supercoiling

ahead. This

disp[aces

the

octamer, whjch

keeps

contact

with

DNA and/or

polymerase.

and is inserted

behjnd

the RNA

polymerase.

nucleosome

(the

be added

are

essentially

at the

axis of

dyad symmetry), paus-

rng

ceases,

and

the

polymerase

advances

rapidly.

This

suggests

that

the midpoint

of the nucleo-

some

marks

the

point

at which

the

octamer is

displaced

(possibly

because positive

supercoil-

ing

has reached

some

critical level

that

expels

the

octamer

from

DNA).

This releases

tension

ahead

of the

polymerase

and allows

it to

pro-

ceed.

The

octamer

then

binds

to the DNA

behind

the

polymerase

and no

longer

presents

an

obsta-

cle

to

progress.

It

is likely

that

the

octamer

changes position

without

ever

completely

los-

rng

contact

with

the DNA.

Is

the

octamer

released

as an intact

unit?

Crosslinking

the

octamer's

proteins

does not

create

an

obstacle

to transcription.

Transcrip-

tion

can continue

even

when

crosslinking

is

extensive

enough

to

ensure

that

the central

regions

of

the core histones

have

been linked.

Ihis

implies

that transcription

does

not require

Cissociation

of

the octamer

into

its

component

CHAPTER

29 Nucteosomes

FT$#fii:,

tL;-+*

URl3

gene

sequences

are fused

to a regu-

lated GALL

promoter

and

to a

ribosomaI

DNA

sequence. The

UR43 has transitionatty

positioned

nucleosomes

before tran-

scription. When

transcription is

induced

under

the control

of an

inducibte promoter,

nucleosome

positions

are random-

ized.

When transcription

is repressed,

the nucteosomes

resume

their

paticu[ar positions.

Reproduced

from

Suter. B.,

et at. L997.

EMBO

J.

1.6:21,50-21,60.

Copyright

O 0xford Uni-

versity Press.

Photos

courtesy

of

Fritz

Thomas,

ETH

Zi.irich.

histones,

nor

is it likely

to require

any major

unfolding

of the

central structure.

The

addition

of histone

Hl

to this

system, however,

causes

a rapid

decline in

transcription.

This

suggests

two

conclusions:

The histone

octamer

(whether

remaining present

or displaced)

functions

as

an

intact

unit, and

it may

be necessary

to remove

Hl from

active

chromatin

or to

modify

its inter-

actions in

some

way.

Thus

a small RNA

polymerase

can

displace

a single

nucleosome,

which

reforms

behind

it,

during

transcription.

Of course,

the

situation

is

more

complex in

a eukaryotic

nucleus.

RNA

polymerase

is

much larger,

and

the impediment

to

progress

is

a string

of connected

nucleosomes.

Overcoming

this obstacle

requires

additional

factors

that

act on

chromatin (see

Chapter

30,

Controlling

Chromatin

Structure).

The

organization

of nucleosomes

may

be

changed

by

transcription.

i1i*i:fiil

?+.4t]

shows

what

happens

to the

yeast

URA3

gene

when it

is

transcribed

under the

control

of an

inducible

780

Lewin Benjamin (ed.) Genes IX

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