Lewin Benjamin (ed.) Genes IX

(see

Section

14.Il,

Repressor Uses a Helix-

T\rrn-Helix Motif to Bind DNA). Amino

acids

separated

by three

to

four

positions

lie on the

same face of an s-helix and are

therefore

in

a

position

to contact

adjacent base

pairs.

lril',l,jiil

5 i.:ri::

shows that amino acids lying along

one face of the 2.4 region

u-helix contact the

bases at

positions

-12

to

-10

of the

-10

pro-

moter sequence.

Region

2.3 resembles

proteins

that bind sin-

gle-stranded

nucleic acids

and

is

involved

in

the

melting reaction. Regions 2.1 and 2.2

(which

comprise

the most highly

conserved

part

of

sigma) are

involved

in the interaction with core

enzyme.

It is assumed

that all sigma factors bind

the same regions of the core

polymerase,

which

ensures that the reactions are competitive.

The

N-terminal

region

of o70 has

important

regulatory

functions. If

it is removed, the short-

ened

protein

becomes able to bind specifically

to

promoter

sequences. This suggests that the

N-terminal

region behaves

as an autoinhibition

domain.

It

occludes the DNA-binding domains

when o70 is free. Association with core enzyme

changes

the conformation

of sigma so

that the

inhibition

is released,

and the DNA-binding

domains can contact DNA.

i:,i.-:-!iiil:

t::. ::

j-

schematizes the conformational

change

in

sigma

factor

at open complex

forma-

tion. When sigma factor binds to the core

poly--

merase, the N-terminal domain swings

-2O

A

away

from the DNA-binding domains, and the

DNA-binding domains separate from one

another by

-15

A,

presumably

to acquire

a more

elongated conformation appropriate

for

con-

tacting DNA. Mutations in either the

-

I

0 or

-3

5

sequences

prevent

an

(N-terminal-deleted)

o70

from binding to

DNA,

which suggests that o70

contacts

both sequences simultaneously. This

implies that the sigma

factor must

have a

rather

elongated structure, extending over the

-68

A

of two turns

of DNA.

In the

free holoenzyme,

the N-terminal

domain

is located in the active

site

of the core

enzyme components, essentially mimicking the

location that DNA will occupy

when

a tran-

scription complex

is formed. When the holoen-

zyme

forms an

open complex on

DNA, the

N-terminal

sigma domain is displaced

from

the active

site. Its relationship

with

the rest of

the

protein

is therefore very flexible; the

rela-

tionship

changes when sigma factor binds to

core enzyme and again when the

holoenzyme

binds to

DNA.

Comparisons

of the crystal structures

of

the core enzyme

and holoenzyme show that

Protein

Position

-1T12-11-1H-a-7

ilii-iililir

:l i,lr:r: Amino

acids

in

the

2.4 cr-helix

of o70 con-

tact specjfic

bases

in the coding

strand

of the

-10

pro-

moter sequence.

i:iir,i"iiii-

1.l

"-i,:

The N-terminus

of sigma

blocks the

DNA-

binding

regions

from binding

to

DNA. When

an open

com-

ptex

forms, the

N-terminus

swings

20. A away,

and the

two

DNA-binding

regions separate

by

15

A.

sigma factor

lies

largely

on the

surface

of the

core enzyme.

ir

i:i,iia;i:a

i i.;itr

shows

that

it has

an elongated

structure

that

extends

past

the

DNA-binding

site.

This

places it in a

position

to contact

DNA

during

the

initial

binding.

The

DNA helix

has to

move

some

l6

A from the

initial

position in order

to enter

the

active

site.

iii.i.ri:.i:

::

r'i

illustrates

this

movement,

look-

ing in cross-section

down

the

helical

axis of

the

DNA.

An interesting

difference

in behavior

is

found with

the o5a

factor.

This

causes

RNA

poly-

merase to recognize

promoters that

have a dis-

tinct consensus

sequence,

with

a conserved

element at

-12

and

another

close

by at

-24

(given

in the

"-35"

column

of

Figure

11.32).

Thus the

geometry of the

polymerase-promoter

complex

is different

under

the direction

of this

sigma factor.

Another

diff

erence

in the

Nterminal region binds

11.17

Sigma

Factors

Directly

Contact

DNA

287

Sigma factor has

an elongated

structure

that

extends

atong the surface

of the

core subunits when

the hoLoenzvme

is formed.

,

DNA initiatty

contacts sigma factor (pink)

and core

enzyme

(gray).

It moves

deeper into

the core

enzyme

to make

contacts

at the

-10

sequence.

When sigma

is

released,

the width

of

the

passage

containing DNA

increases.

Reproduced

from Vassylye4

D.

G., et al. Nature.

2002.

41.7: 71,2-71.9.

Photo

courtesy

of Shigeyuki

Yokoyama,

The

University

of

Tokyo.

mechanism

of regulation

is

that high-level

tran-

scription

directed

by

otn requires

other

activa-

tors

to bind

to sites

that are

quite

distant from

the

promoter.

This

contrasts

with

the other

types

of

bacterial

promoter,

for which

the regulator

sites

are always

in

close

proximity

to

the

pro-

moter.

The

behavior

of o5a itself

is different

from

other

sigma

factors,

most notably

in its

ability

to bind

to DNA

independently

of

core

poly-

merase.

In

this regard,

o5a is more

like

the

eukaryotic

regulators

we discuss

in

Chapter 24,

Promoters

and

Enhancers,

than the

typical

prokaryotic

regulators

discussed

in

Chapter I 2,

Ihe

Operon.

CHAPTER

L1 Transcriotion

Sigma Factors

May Be

0rganized into

Cascades

.

A cascade of

sigma

factors is

created

when one

sigma factor is required

to transcribe

the

gene

coding for

the

factor.

.

The earty

genes

of

phage

SP01

are transcribed

by

host RNA

polymerase.

.

0ne of the earty

genes

codes for

a sigma factor

that

causes RNA

potymerase

to transcribe

the

midd[e

genes.

r

Two

of the midd[e

genes

code for

subunits of a

sigma

factor

that

causes RNA

polymerase

to

transcribe the [ate

genes.

Sigma factors are

used extensively

to

control

initiation

of transcription

in the

bacterium

B.

subtilis, for which

-10

different

o factors

are

known.

Some

are

present

in

vegetative

cells;

others are

produced

only in

the special

circum-

stances

of

phage

infection

or the

change from

vegetative growth

to sporulation.

The major RNA

polymerase

found in

B. sub-

tilis cells engaged

in normal

vegetative

growth

has the

same structure as

that of. E.

coli, szFF'o.

Its

sigma factor

(described

as oa3

or oA) recog-

nizes

promoters

with the

same

consensus

sequences

used

by the E. coli

enzyme

under

direction from

o70. Several

variants

of the RNA

polymerase

that contain

other sigma factors

are

found in

much smaller

amounts. The

variant

enzymes

recognize

different

promoters

on the

basis of consensus

sequences

at-35

and

-I0.

Transitions

from

expression

of one

set of

genes

to

expression of another

set

are a com-

mon feature

of bacteriophage

infection.

In

all

but the

very simplest

cases, the

development

of the

phage

involves

shifts in

the

pattern

of

transcription

during the infective

cycle. These

shifts

may be accomplished

by the synthesis

of

a

phage-encoded

RNA

polymerase

or by

the

efforts of

phage-encoded

ancillary

factors

that

control

the bacterial RNA

polymerase.

A

well-

characterized

example of

control

via

the

pro-

duction of new

sigma factors

occurs

during

infection

of. B.

subtilis by

phage

SPOL

The infective

cycle

of SPOl

passes

through

three

stages

of

gene

expression.

Immediately

on

infection,

the early

genes

of

the

phage

are

transcribed.

After four

to five minutes,

the early

genes

cease

transcription

and the middle genes

are transcribed.

At

eight to

twelve minutes,

mid-

dle

gene

transcription

is

replaced

by transcrip-

tion

of late

genes.

The

early

genes

are transcribed

by the

holoenzyme

of

the host bacterium.

They

are

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DNA replicates

w

r:

,,':

.

. Sporutation invotves

the differentiation

of

a

vegetative

bacterium into

a mother

cetl that is

tysed

and

a sDore

that is reteased.

cell is

eventually lysed,

and

the spore that is

released

has

an entirely

different

structure from

the original

bacterium.

Sporulation

involves

a

drastic change in

the

biosynthetic

activities

of the bacterium,

in

which

many

genes

are involved.

The

basic level

of con-

trol lies

at transcription.

Some of

the

genes

that

functioned

in

the

vegetative

phase

are turned

off

during

sporulation,

but most

continue to

be

expressed.

In

addition,

the

genes

specific for

sporulation

are expressed

only

during this

period.

At

the end

of sporulation,

-40%

of the

bacterial

mRNA

is

sporulation

specific.

New forms

of the RNA polymerase

become

::l'ff

;trJ:ll&Hlf

J:""',J?1',1*"Jft::

ent

proteins

in

place

of the

vegetative

o4l. The

changes

in transcriptional

specificity

are sum-

CHAPTER

1.1 Transcription

marized

in ijrli,ljiii I

j.r,.l'.

The

principle

is

that

in

each compartment

the existing

sigma factor is

successively displaced by a new factor

that

causes

transcription of a different

set of

genes.

Com-

munication between the

compartments

occurs

in order to

coordinate the timing

of the changes

in

the forespore and mother

cell.

The

sporulation cascade is

initiated

when

environmental

conditions trigger

a

phospho-

relay, in which

a

phosphate

group

is

passed

along

a series of

proteins

until it

reaches

SpoOA.

(Several gene

products

are involved

in this

process,

whose

complexity may

reflect the need

to avoid mistakes in

triggering

sporulation

unnecessarily.)

SpoOA is a transcriptional

reg-

ulator whose activity is

affected by

phosphory-

lation. In the

phosphorylated

form. it

activates

transcription

of two operons,

each of which

is

transcribed by a different form

of

the host RNA

polymerase.

Under the direction

of

phosphor-

ylated

SpoOA, host

enzyme utilizing

the

gen-

eral dl transcribes

the

gene

coding

for the factor

oF, and host enzyme

under the

direction

of a

minor factor,

oH, transcribes

the

gene

coding

for

the factor

pro-oE.

Both

of these

new sigma

factors

are

produced

before septum

formation,

but become

active later.

Factor

oF is the first

one to become

active

in

the forespore

compartment. It is inhibited

by

an antisigma factor

that binds to it;

in the fore-

spore,

an anti-antisigma factor

removes

the

inhibitor.

This reaction

is controlled

by

a series

of

phosphorylation/dephosphorylation

evenrs.

The initial

determinant is

a

phosphatase

(SpotrE)

that is an integral membrane protein

that

accu-

mulates at

the

pole,

with the result

that its

phos-

phatase

domain

becomes more

concentrated

in

the forespore.

It dephosphorylates,

and thereby

activates,

SpoIIAA, which

in turn

displaces

the

antisigma factor

SpoIIAB from

the

complex

of

SpoIIAB-oF.

Release

of oF activates

it.

Activation

of oF

is

the start

of sporulation.

Under

the direction

of oF, RNA

polymerase

tran-

scribes the first

set of sporulation genes

instead

of the vegetative genes

it

was

previously

tran-

scribing. The

replacement

reaction probably

affects

only

part

of the

RNA

polymerase

popu-

Iation,

because

oF

is

produced

only in

small

amounts.

Some vegetative

enzyme

remains

present

during

sporulation.

The displaced

oar

is not

destroyed;

it can

be recovered

from

extracts

of sporulating

cells.

TWo

regulatory

events follow

from

the activ-

ity

of

oF, as detailed in

i i

i;l,iii

!.'i

:t

i!ti..

In the fore-

spore itself,

another

factor,

oG. is the

product

of

one

of the early

sporulation

genes.

Factor

oc

284

982

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sp

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tsnu

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sleubrs fq

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uoqelntod5

It'i.l :*{:*ij

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aulAzueololl

ltSC HlHlOt/!

BacteriaI

RNA

Polymerase

Terminates

at Discrete

Sites

r

Termination

may require both recognition

of the

terminator sequence in DNA

and the formation

of a

hairpin

structure in the RNA

product.

Once RNA

polymerase

has started

transcrip-

tion,

the enzyme moves along

the

template,

synthesizing RNA,

until it meets

a terminator

sequence.

At this

point,

the

enzyme

stops adding

nucleotides

to the

growing

RNA

chain, releases

the completed

product,

and

dissociates from

the

DNA

template. Termination

requires

that

all

hydrogen

bonds holding the RNA-DNA

hybrid

together

must be broken,

after

which the DNA

duplex reforms.

It is difficult

to define the

terminarion

point

of an RNA

molecule that has

been

synthesized

in

the living cell. It is

always

possible

rhat

the

3' end of

the molecule has

been

generated

by

cleavage of the

primary

transcript,

and therefore

does not represent

the actual

site at

which RNA

polymerase

terminated.

The best identification

of termination

sites

is

provided

by systems in

which RNA

poll.rnerase

terminates

invitro. The

ability

of the enzyme

to

terminate is

strongly influenced

by

parameters

such

as the ionic strength;

as a result,

its termi-

nation

at a

particular

point

in vitro

does

not

prove

that this same

point

is a

natural

termi-

nator.

We

can, however,

identify

authentic

J'

ends

when the same

end is

generated

in

vitro

and in vivo.

fi*LFR*

1.1-:i$

summarizes the

two

types of

features

found in

bacterial terminators.

.

Terminators

in

bacteria

and their

phages

have

been identified

as sequences

that

are needed for

the termination

reaction

(in

vitro

or in vivo).

The

sequences

ar

prokaryotic

terminators

show no

sim-

ilarities

beyond

the

point

at which

the

last base is

added

to the RNA.

The

responsibility

for

termination

lies

with

the sequences

already transcribedby

RNA

polymerase.

Thus

termination

relies

on

scrutiny of the

template

or

product

that

the

polymerase

is currently

transcribing.

.

Many

terminators

require

a

hairpin

to

form in

the secondary

structure

of the

RNA being

transcribed.

This indicates

that

termination

depends

on the RNA

product

and

is not determined

simply

by

scrutiny of

the DNA

sequence

during transcription.

ii*-*i

:

:.'t-:

oF triggers synthesis

of the next

sigma

factor

in the forespore (oG)

and turns on

SpoIIR, which

causes SpoI-

IGA to

cleave

oro-oE.

a-ILiii:s

::.ilr;

The

coordinates

timing

forespore.

crisscross

regutation

of sporulation

of events in

the mother

ce[[ and

compartment

are successively

activated,

each

directing

the synthesis

of a

particular

set of

genes.

-i!Liq{

::..++

outlines how

the

two cas-

cades

are

connected

by the

transmission

of sig-

nals

from

one compartment

to the

other. As

new sigma

factors

become

active,

old sigma fac-

tors

are displaced,

so that transitions

in

sigma

factors

turn

genes

off as

well as

on. The incor-

poration

of

each factor into

RNA

polymerase

dictates

when its

set of

target

genes

is expressed,

and the

amount

of factor

available

influences

the

level

of

gene

expression.

More

than

one sigma

factor

may be

active at

any time,

and the

speci-

ficities

of some

of the

sigma factors

overlap.

We

do not

know

what is responsible

for

the ability

of each

sigma factor

to replace

its

predecessor.

CHAPTER

11 Transcription

MOTHER

':

FORESPORE

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286

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sroleurural

very

end of the

unit that is rich

in U residues.

Both features

are needed for

termination. The

hairpin

usually

contains

a G-C-rich region

near

the

base of the

stem. The

typical distance

between

the hairpin

and

the U-rich region

is

seven to nine

bases. There

are

-l

100

sequences

in

the E.

coli

genome

that fit these

criteria, which

suggests that

about half

of the

genes

have

intrin-

sic

terminators.

Point

mutations

that

prevent

termination

occur

within the

stem region

of the hairpin.

What is

the effect

of a hairpin

on transcription?

It is likely

that

all

hairpins

that

form in

the RNA

product

cause

the

polyme

rase

to

slow

(and per-

haps

to

pause)

in

RNA synthesis.

Pausing

creates

an opportunity

for termina-

tion to

occur. Pausing

occurs

at sites

that

resem-

ble

terminators

but have

an increased

separation

(typically

ten

to eleven

bases)

between the hair-

and

the U-run.

If the

pause

site

does not

correspond

to a terminator,

though,

the enzyme

usually

moves

on again

to continue

transcrip-

tion.

The length

of the

pause

varies,

but

at a

typical

terminator

lasts

-60

seconds.

A downstream

U-rich region

destabilizes

the RNA-DNA

hybrid

when RNA

polymerase

pauses

at the hairpin.

The

rU-dA RNA-DNA

hybrid

has an

unusually

weak base-paired

struc-

ture;

it requires

the

least

energy

of any

RNA-DNA

hybrid to

break

the association

between

the two

strands.

When

the

polymerase

pauses,

the RNA-DNA

hybrid

unravels from

the

weakly

bonded

rU-dA

terminal region.

Often,

the actual

termination

event

takes

place

at any

one of

several

positions

toward

or at

the

end

of the U-rich

region,

as though

the enzyme

"stutters"

during

termination.

The

U-rich region

in

RNA

corresponds

to

an A-T-rich

region in

DNA,

so we

see that A-T-rich

regions

are impor-

tant

in intrinsic

termination

as well

as initiation.

Both

the sequence

of

the hairpin

and

the

length

of

the U-run

influence

the

efficiency

of

termination.

Termination

efficiency

in vitro,

however, varies

from 2o/o

lo 90oh

and

does not

correlate

in any

simple

way with

the constitu-

tion

of the hairpin

or

the number

of

U

residues

in

the

U-rich region.

The

hairpin

and

U-region

are therefore

necessary,

but not

sufficient,

and

additional

parameters

influence

the interaction

with

RNA

polymerase.

In

particular,

the

sequences

both

upstream

and

downstream

of

the intrinsic

terminator

influence

its

efficiency.

Less is

known

about

the signals

and

ancil-

lary

factors

involved

in termination

for eukary-

otic

polymerases.

Each

class

of

polymerase

uses

a different

mechanism

(see

Chapter

26,

RNA

Splicing

and Processing).

Transcri

ption

How

Does

Rho Factor

Work?

o

Rho factor is

a terminator

protein

that

binds to a

ruf

site on nascent

RNA and tracks

atong

the RNA

to

retease

it from

the RNA-DNA hvbrid

structure

ar

the RNA

potymerase.

Rho

factor is an

essential

protein

in E.

coli that

functions

solely

at the stage

of termination.

It

acts at rho-dependent

terminators,

which

account

for about half

of E. coli

Lerminators.

FIGURE x t

-4? shows how

rho functions.

First

it

binds to

a sequence within

the

transcript

upstream

of the

site of termination.

This

sequence

is called

a rut site

(an

acronym

for

rho

utilization).

The rho then

tracks

along

the

RNA

until it catches

up ro RNA

polymerase.

When the RNA

polymerase

reaches

the

termi-

nation

site, rho

acts on the RNA-DNA

hybrid

in

the enzyme

to cause release

of the

RNA. Paus-

ing

by the

polymerase

at

the site

of termination

allows

time for rho factor

to translocate

to the

hybrid

stretch

and is an important

feature

of

termination.

We

see

an

important general

principle

here.

When

we

know

the

site on DNA

at which

some

protein

exercises its

effect, we

cannot

assume

that

this coincides

with the DNA

sequence

rhat

it

initially recognizes.

They

can

be separate,

and

there

need not

be a fixed relationship

between

them. In f.act,

rut sites in

different

transcription

units are found

at varying

distances preceding

the sites

of termination.

A similar

distinction

is

made

by antitermination

factors (see

Sec-

tion 11.24, Antitermination

Requires

Sites That

Are Independent

of the Terminators).

The

common feature

of. rut

sites is

that

the

sequence is

rich in

C

residues

and

poor

in

G

residues

and has no

secondary

structure.

An

example

is

given

in FSGttRil

it.4S.

C is by far

the

most

common

base

(4lo/o)

and

G is

the least

common

base

(Ia%|.

rut

sites vary in

length.

As

a

general

rule,

the efficiency

of

a rutsile

increases

with

the length

of the

C-rich/G-poor

region.

Rho is a member

of

the family

of hexameric

ATP-dependent

helicases.

The

subunit

has

an

RNA-binding

domain

and an

ATP hydrolysis

domain. The

hexamer

functions

by

passing

nucleic

acid through

the

hole in

the middle

of

the

assembly

formed from

the RNA-binding

domains

of

the subunits.

f,cfruRg

11.4s

shows

that

the structure

of rho

gives

some indications

of how

it functions.

It

winds

RNA from

the

3'

end

around

the

exterior

of the

N-terminal

CHAPTER

11

domains, and

pushes

the 5'

end of the bound

region

into

the

interior,

where it is bound by a

secondary

RNA-binding

domain in the C-termi-

nal domains. The initial form

of

rho is

a

gapped

ring,

but binding of the

RNA

converts it to a

closed ring.

After binding to

the

rut

site, rho uses

its

helicase activity, driven by ATP hydrolysis, to

translocate

along RNA

until

it reaches

the

RNA-DNA hybrid stretch in RNA

polymerase.

It then unwinds the duplex structure. We do

not know whether this action is

sufficient

to

release the transcript or whether rho

also

inter-

acts with

RNApolymerase

to help release

RNA.

Rho functions as an ancillary factor for RNA

polymerase;

typically its maximum aclivity in

vitro is displayed when it is

present

at

-l0o/o

of

the concentration of the RNA

polymerase.

Rho needs to translocate along RNA from

thre rut site to the actual

point

of termination.

This requires the

factor

to move faster than RNA

polymerase.

The enzl.rne

pauses

when

it reaches

a terminator, and termination occurs if rho

catches

it there. Pausing is therefore important

in

rho-dependent termination,

just

as in intrin-

sic termination, because

it

gives

time

for

the

other

necessary events to occur.

The idea that

rho moves

along

RNA leads

to an

important

prediction

about

the

relation-

ship

between transcription and translation. Rho

must first have access to a binding sequence on

RNA and then

must be able to move along the

RNA. Either or both of these conditions

may

be

prevented

if ribosomes are translating an RNA.

Thus

the

ability of rho factor to reach

RNA

poly-

merase

at

a terminator depends on what is hap-

pening

in translation.

This model explains apluzzlir'g

phenome-

non. In some

cases, a nonsense mutation in one

gene

of

a transcription unit

prevents

the expres-

sion of subsequent

genes

in

the unit.

This effect

is called

polarity.

A

common cause

is the

iI+i.il:l i i.ri.j Rho

factor binds

to

RNA at a

ruf site and

trans[ocates

atong

RNA untjt

it

reaches the

RNA-DNA

hybrid in RNA

poLymerase,

where

it

reteases the

RNA

from

the

DNA.

AU * UA U

AUAU

*

A U U

AAA.*

UA.]

U

*A AfiAAA**C*U..U

UU

€ Deletion

prevents

termination +

l:$liFi:li.+S

ArufsitehasasequencerichinCandpoorinGprecedingtheactuaIsite(s)

ofter-

mination. The seouence corresoonds

to the 3'end of the

RNA.

RNA

polymerase

transcribes

DNA

Rho attaches

to ruf site on

RNA

I

V

Rho lrans

es

along BNA

RNA

polymerase

pauses

at

hairpin and

Rho unwinds

DNA-FINA

hybrid

Termination: all components

released

17.22

How Does

Rho

Factor

Work?

Rho monomer

has two

domains

domain

Hexameric

ring

binds

RNA

iiiL:il

: :.+"s

Rho has an N-terminaL

RNA-binding

domain

and

a C-terminaI ATPase

domain. A

hexamer in

the form of

a

gapped

ring

binds RNA along

the exterior

of the N-ter-

mjnal

domains. The

5'end

ofthe RNA is

bound by a sec-

ondary

binding

site in the

jnterior

of

the hexamer.

absence

of the nRNA corresponding

to the

sub-

sequent

(distal) pafis

of the unit.

Suppose

that there are rho-dependent

ter-

minators

within the transcription

unit,

that is,

before the terminator

that usually

is used. The

consequences

are illustrated

in

n:{*{t*

11,5*.

Normally these earlier

terminators

are not

used,

because the ribosomes

prevent

rho

from reach-

ing RNA

polymerase.

A nonsense

mutation,

however, releases

the ribosomes,

so

that rho is

free

to attach

to and/or move

along the mRNA,

enabling

it to act on RNA

polymerase

at the ter-

minator. As

a result, the enzyme

is released,

and the distal regions

of the transcription

unit

are never expressed.

(Why

should

there

be

inter-

nal terminators?

Perhaps

they are

simply

sequences

that by coincidence

mimic

the usual

rho-dependent

terminator.)

Some stable

RNAs

that have

extensive

secondary structure

are

pre-

served from

polar

effects,

presumably

because

the structure impedes

rho

attachment

or

movement.

rho mutations

show wide

variations

in their

influence

on termination.

The

basic nature

of

the effect is

a

failure

to terminate.

The

magni-

tude

of the

failure,

however,

as seen

in the

per-

cent

of

readthrough

in

vivo,

depends

on the

particular

target locus.

Similarly,

the need

for rho

WILD TYPE

NONSENSE

MUTANT

Ribosomes

pack

mRNA

behind

RNA

polymerase

Ribosomes

impede

rho

attachment

and/or

movemenl

Ribosomes

dissociate

at mutation

Rho attaches

I

Rho obtains

but ribosomes

I

access to

impede

its

I

RNA

movement

I

polymerase

Transcription

lTranscription

continues

I

terminates

prematurely

fl;#lJfti:'i'1.li#

Theactionofrhofactormaycreatealinkbetweentranscriptionandtranstationwhen

a rho-dependent

terminator

lies

soon after a nonsense

mutation.

290

CHAPTER

11 Transcription

Lewin Benjamin (ed.) Genes IX

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