Lewin Benjamin (ed.) Genes IX

of

great

viscosity. We

do not entirely under-

stand the

physiological

implications,

such as the

effect this

has

upon the ability

of

proteins

to

find

their binding sites on DNA.

The

packaging

of

chromatin is flexible; it

changes during the eukaryotic

cell cycle.

At

the time of division

(mitosis

or meiosis), the

genetic

material

becomes even more tightly

packaged,

and

individual

chromosomes

become recognizable.

The overall compression of the DNA can

be

described by

the

packing

ratio, which is the

Iength of the DNA divided

by the length of the

unit that contains it. For example, the smallest

human chromosome contains

-4.6

x

I07

bp of

DNA

(-10

times the

genome

size of the bac-

terium

E. coli). This is

equivalent to 14,000

pm

(=

1.4 cm) of extended DNA. At

the

most

con-

densed

moment

of

mitosis,

the chromosome is

-2

pm

long. Thus

the

packing

ratio of DNA in

the chromosome can be as

great

as 7000.

Packing ratios cannot

be established with

such certainty

for

the more amorphous overall

structures of

the

bacterial

nucleoid

or eukary-

otic chromatin. The usual reckoning, however,

is that mitotic chromosomes are likely to

be

five

to ten

times more tightly

packaged

than

inter-

phase

chromatin, which indicates a tlpical

pack-

ing ratio of 1000 to 2000.

A major unanswered

question

concerns the

specificity

of

packaging.

Is the DNA folded into

a

particular pattern,

or is it

different

in

each

indi-

vidual copy of the

genome?

How does the

pat-

tern of

packaging

change when a

segment

of

DNA is

renlicated

or transcribed?

@

Viral

Genomes

Are

Packaged into Their

Coats

o

The length of

DNA

that can be

incorporated into

a

virus

is

[imited by

the

structure of the

headshell.

r

Nucteic

acid

within

the

headshetl is

extremety

condensed.

r

Fitamentous RNA viruses

condense the RNA

genome

as they assembte the

headshetl

around

it.

.

SphericaI

DNA viruses insert the DNA into a

preassembted protein

shelt.

From the

perspective

of

packaging

the individ-

ual sequence,

there is an important difference

between

a cellular

genome

and a

virus.

The cel-

lular

genome

is essentially indefinite in size; the

number and location of individual sequences

can be

changed by duplication, deletion, and

rearrangement. Thus

it requires

a

generalized

method for

packaging

its DNA,

one that

is insen-

sitive to the total

content

or distribution

of

sequences.

By cc,ntrast,

two

restrictions define

the needs oI a virus.

The

amount of

nucleic acid

to be

packag

ed is

predeterminedby tl:,e size

of the

genome,

and

it nrust all

fit within

a coat assem-

bled from a

protein

or

proteins

coded

by the

viral

genes.

A virus

particle is deceptively

simple

in its

superficial

appealance.

The

nucleic acid

genome

is contained within

a capsid,

which

is a sym-

metrical or

quasisymmetrical

structure

assem-

bled from one or only

a

few

proteins.

Attached

to the capsid

(or

incorporated

into

it) are other

structures; these

structures

are

assembled

from

distinct

proteins

and are

necessary

for

infection

of the

host cell.

The virus

palticle is tightly

constructed.

The

internal volume

of the

capsid

is rarely

much

greater

than the

volume

of the

nucleic acid

it

must hold. The

,lifference

is usually

less than

twofold, and often

the

internal

volume is barely

larger than

the rLucleic

acid.

In its most

cxtreme

form,

the

restriction

that the capsid

rnust be

assembled

from

pro-

teins coded

by the

virus

means that

the entire

shell is constructed

from a single

type

of sub-

unit.

The rules

lor assembly

of

identical

sub-

units into closec

structures

restrict

the capsid

to one of two tylles.

For

the

first type, the

pro-

tein

subunits stack

sequentially

in a

helical

array to

f.orm a

fillmentlus

or rodlike

shape.

For

the second type,

they

form

a

pseudospherical

shell-a

type of structure

that

conforms

to a

polyhedron

with

icosahedral

symmetry.

Some viral

capsids

are

assembled

from

more

than a single

type

of

protein

subunit,

but

although this extends

the exact

types

of struc-

tures that

can br:

formed,

viral

capsids still

all

conform

to the

general

classes

of

quasicrys-

talline

filaments or

icosahedrons.

There are

two types

of solution

to the

prob-

lem of how to

construct

a capsid

that

contains

nucleic acid:

.

The

protein

shell

can

be

assembled

around

the

nucleic

acid,

thereby

con-

densing

the

DNA or

RNA bY

Protein-

nucleic acid

interactions

dudng

the

process

c'f assembly.

.

The capsid

can

be constructed

from

its

componr:nt(s)

in the

form

of an empty

shell,

into which

the

nucleic

acid

must

be insertt:d,

being

condensed

as it enters.

The capsid

is assembled

around

the

genome

for single-stranded

RNA

viruses.

The

principle

28.2 Viral

Genomes

Are

Packaged

into

Their Coats

731

:

,.':-ii

.:

A hetical

path

for TMV

RNA is

created by the

stacking

of

protein

subunjts in the virion.

of assembly

is that the

position

of the RNA

within

the

capsid is determined

directly

by its binding to

the

proteins

of the

shell. The best

characterized

exam-

ple

is TMV

(tobacco

mosaic virus).

Assembly

starts

at a

duplex hairpin

that lies within

the

RNA

sequence.

From

this nucleation

center,

it

proceeds

bidirectionally

along

the RNA until

it reaches

the ends. The

unit

of the capsid is

a

two-layer

disk,

with each layer

containing 17

identical protein

subunits.

The disk is

a circu-

lar structure,

which

forms

a

helix

as it interacts

with

the RNA.

At the nucleation

center, the

RNA

hairpin

inserts into

the central

hole in the

disk, and

the disk changes

conformation

into

a

helical

structure that

surrounds

the RNA. Addi-

tional

disks

are added,

with each new

disk

pulling

a new

stretch

of RNA into

its central

hole.

The RNA

becomes

coiled in

a helical array

on the inside

of the

protein

shell.

as illustrated

in itr,l.,,r',l::',

,.

The

spherical

capsids

of DNA viruses

are

assembled

in

a different

way,

as best character-

ized for

the

phages

lambda

and T4. In

each case,

an

empty headshell

is

assembled from

a

small

set

of

proteins.

The duplex

genlme

then is inserted

into the

head, accompanied

by a structural

change

in the

capsid.

;

i .i.:.::

,

i.

-r

Summarizes

the assembly

of

lambda.

It starts

with a

small headshell

that

con-

tains

a

protein "core."

This

is

converted

to an

empty headshell

of more

distinct

shape. At

this

point

the DNA

packaging

begins,

the headshell

expands

in

size though

remaining

the

same

shape,

and

finally

the full head

is sealed

by the

addition

of the

tail.

CHAPTER

28 Chromosomes

r:'i;i,Jir!

;$.:t

Maturation

of

phage

[ambda

passes

through

several

stages.

The

empty head

changes shape

and expands

when it

becomes fitted with DNA. The

etectron

micrographs

show

the

partictes

at the start

and the end

of the matura-

tion

pathway.

Top

photo

reproduced from

Cue, D.

and Feiss,

M. 1993. Proc. Natl.

Acad. Sci. USA.90:9290-9294.

Copy-

right

1993 National Academy

ofScience,

U.S.A. Photo

cour-

tesy of Michael G. Feiss,

University of Iowa.

Bottom

photo

courtesy of Robert Duda,

University

of Pittsburgh.

A double-stranded

DNA

that spans

short dis-

tances is a fairly

rigid rod,

yet

it must

be com-

pressed

into

a compact structure

to fit

within

the

capsid. We should like

to know

whether

pack-

aging involves

a

smooth coiling

of the DNA

into

the head

or whether it requires

abrupt

bends.

Inserting

DNA into

a

phage

head

involves

two types

of reaction:

translocation

and

con-

densation.

Both are energetically

unfavorable.

Tlanslocation

is

an active

process

in

which

the DNA is

driven into

the head

by an

ATP-

dependent

mechanism.

A common

mechanism

is used

for many

viruses that replicate

by a rolling

circle

mechanism

to

generate

long

tails

that con-

tain multimers

of the

viral

genome.

The

best

char-

acterized

example

is

phage

lambda.

The genome

is

packaged

into the

empty capsid

by the

termi-

nase

enz).rne. il{illlit

i:rii.i summarizes

the

process.

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,EL

recombination reactions.

Null

mutations

in

either of the

genes

coding

for the

subunits of

HU

(hupA

and

-B)

have little

effect, but loss of

both functions causes

a cold-sensitive

pheno-

type and some loss

of superhelicity

in DNA.

These results raise

the

possibility

that HU

plays

some

general

role in nucleoid

condensation.

Protein HI

(also

known

as

H-NS)

binds

DNA,

interacting

preferentially

with sequences

that are bent. Mutations

in its

gene

have

turned

up

in

a variety of

guises

(osmZ,

bglY,

pilG),

each

of which is identified

as an apparent regulator

of a different system. These

results

probably

reflect the effect

that Hl has on

the

local

topol-

ogy of DNA, with effects

upon

gene

expression

that depend upon the

particular promoter.

We

might

expect that the absence

of a

pro-

tein required for nucleoid

structure would have

serious effects

upon viability. Why, then,

are

the effects of deletions in the

genes

for

proteins

HU and Hl relatively

restricted? One explana-

tion

is

that these

proteins

are redundant,

and

that any one can substitute for

the others so

that deletions of. all

of them would be neces-

sary to

interfere

seriously with nucleoid

struc-

ture. Another

possibility

is

that we have

yet

to

identify

the

proteins

responsible

for the major

features of nucleoid integrity.

The nucleoid

can be isolated

directly

in

the

form of a very rapidly sedimenting

complex,

which consists

of

-80%

DNA by mass.

(The

analogous complexes in eukaryotes have

-507o

DNA

by

mass;

see Section 28.4, Thre Bacterial

Genome

Is

Supercoiled.) It can be

unfolded

by

treatment with reagents that

act on

RNA

or on

protein.

The

possible

role

of

proteins

in stabi-

lizing its

structure is evident. The role of RNA

has

been

quite

refractory

to analysis.

@

The Bacterial

Genome

Is

SupercoiLed

The nucteoid has

-100

independent negativety

suoerco'ited domains.

The average density of supercoi[ing is

-1

turn/100 bo.

The DNA of the bacterial nucleoid isolated in

vitro

behaves

as a

closed duplex structure, as

judged

by

its response

to ethidium bromide.

This

small

molecule intercalates

between

base

pairs

to

generate positive

superhelical turns in

"closed"

circular DNA molecules,

that

is, mol-

ecules in which both strands have covalent

integrity.

(In

"op:n"

circular

molecules,

which

contain a nick

in

r)ne

strdr-Id, or

with linear

mol-

ecules, the

DNA can

rotate freely

in response to

the intercalation, thus

relieving

the tension.)

In

a

natural closed

DNA that

is negatively

supercoiled,

the . ntercalation

of ethidium

bro-

mide first

remov,3s the

negative supercoils

and

then introduces

positive

supercoils.

The amount

of ethidium brornide

needed to

achieve zero

supercoiling

is

a

rneasure of

the original

density

of negative supelcoils.

Some nicks

occur

in the compact

nucleoid

during

its isolation; they

can also

be

generated

by limited treatnent

with

DNAase.

This does

not, however, abolish

the

ability of ethidium

bromide

to introduce

positive supercoils.

This

capacity of the

gr

nome to

retain

its response to

ethidium bromide

in the

face of nicking

means

that

it must havc many

independent

chromo-

somal domains,

and that

the supercoiling

in each

domqin

is not affeaed by

events

in

the

other domains.

This autonolny

suggests

that

the structure

of the bacterial

r:hromosome has the

general

organization de

picted

diagrammatically

in

iii,,ii:ii:

,r::r

.'

.

puar,

domain

consists

of a

loop of

DNA, the ends c,f

which are

secured

in some

(unknown)

way

that does

not allow

rotational

events to

prop,igate

from one domain

to

another.

Early data

suggested

that each

domain

con-

sists of

-40

kb of

DNA, but

more

recent analy-

sis suggests

that the domains

may be smaller,

at

-10

kb each.

This would

correspond

to

-400

;

I

11:,::

'r'i

:

The

Lacterial

genome consists of

a large

number of loops of

luplex

DNA

(in

the

form of a fiber),

each of which

is

secured

at

the base

to form

an independ-

ent structuraI domain.

28.4

The Bacterial

Genome

Is Supercoited

735

Duplex

DNA

Unconstrained

path

is

supercoiled

in

soace and

creates tension

Constrained

path

is

supercoiled

around

protein

but

creates no tension

fIGURf

28.8

An

unrestrained supercoil in

the

DNA

path

creates

tension,

but

no

tension is transmitted

along

DNA

when

a supercoiI is restrained

by

protein

binding.

domains

in the E.

coli

genome.

The

ends of the

domains

appear

to be randomly

distributed

instead

of located

at

predetermined

sites on the

chromosome.

The

existence

of separate

domains could

permit

different degrees

of supercoiling

to be

maintained

in

different regions

of the

genome.

This could

be relevant in

considering the

dif-

ferent

susceptibilities

of

particular

bacterial

pro-

moters

to supercoiling

(see

Section ll.l5,

Supercoiling

Is an Important

Feature

of

Transcription).

As

shown in

FI$URI 2&.*,

supercoiling

in the

genome

can in

principle

take

either of two

forms:

.

If

a supercoiled DNA

is free its

path

is

unconstrained,

and negative

supercoils

generate

a state

of torsional

tension that

is

transmitted freely

along

the DNA

within

a domain. It

can be relieved

by unwinding

the double

helix, as

described in

Section 19.12,

Supercoil-

ing Affects

the

Structure of DNA.

The

DNA

is in

a dynamic

equilibrium

between

the states

of tension

and

unwinding.

.

Supercoiling

can be

constrained if

pro-

teins

are bound to

the DNA

to

hold

it in

a

pafiicular

three-dimensional

config-

uration.

In this

case, the

supercoils are

represenred

by the

path

the DNA fol-

lows in

its fixed

association

with the

pro-

teins.

The energy

of interaction

between

the

proteins

and the

supercoiled DNA

stabilizes

the nucleic

acid,

so that no ten-

sion is

transmitted

along

the molecule.

Are

the supercoils

in E. coli DNA

constrained

in vivo

or is

the double helix

subject

to the tor-

sional

tension

characteristic

of free DNA?

Mea-

surements

of supercoiling

in vitro

encounter

the

CHAPTER 28

Chromosomes

difficulty

that constraining

proteins

may have

been

lost

during isolation. Various approaches

suggest that DNA is under torsional

slress invivo.

One approach is to measure

the effect

of

nicking

the DNA. Unconstrained

supercoils are

released by nicking, whereas

constrained super-

coils are unaffected.

Nicking releases

-5oo/o

of

the

overall supercoiling. This suggests

that about

half of the supercoiling is

transmitted as

ten-

sion along DNA, with the other half

beino

absorbed

by

protein

binding.

Another

approach uses the

crosslinking

reagent

psoralen,

which

binds more readily

to

DNA

when

it is

under torsional tension.

The

reaction

of

psoralen

with E. coli DNA

in

vivo

cor-

responds to an

average density of one negative

superhelical turn/200 bp

(o

=

-0.05).

We can also examine

the ability of

cells to

form alternative DNA

structures; for

example,

to

generate

cruciforms at

palindromic

sequences.

From

the change in linking

number

that is

required

to drive such reactions, it

is

possible

to calculate the

original supercoiling

density.

This

approach suggests an average

density

of o

=

-0.025

,

or one negative

superhelical

turn/ I 00

base

pairs.

Thus

supercoils /o create

torsional tension in

vivo. There may

be variation about

an average

Ievel,

and the

precise

range of

densities is difficult

to measure. It is,

however, clear

that the level is

sufficient

to exert significant effects

on DNA

struc-

ture-for example, in

assisting melting in

partic-

ular regions

such as origins

or

promoters.

Many of the important

features

of the struc-

ture

of the compact nucleoid remain

to be

estab-

Iished.

What is the specificity

with

which

domains are

constructed? Do

the same

sequences always lie at

the same relative

loca-

tions, or can the

contents of individual

domains

shift? How

is the integrity

of the domain

main-

tained? Biochemical

analysis

by

itself

is

unable

to answer

these

questions

fully,

but if it

is

pos-

sible to

devise suitable selective

techniques,

the

properties

of structural mutants

should

lead to

a

molecular

analysis

of nucleoid

construction.

Eukaryotic

DNA

Has

Loops

and

Domains

Attached

to a Scaffold

e

DNA ofinterphase

chromatin is

negativety

supercoi[ed into independent

domains of

-85

kb.

r

Metaphase

chromosomes

have a

protein

scaffotd

to

which the

loops of supercoited

DNA are

attached.

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Lewin Benjamin (ed.) Genes IX

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