Lewin Benjamin (ed.) Genes IX

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a 60 bp target of

08 M-I sec-r . The

actual for-

ward rate

constant for some

promoters

invitro,

however, appears to

-I08

M-l

sec-I, at or

above the diffusion limit. If

this value applies

in vivo, the time required

for random

cycles of

successive association and

dissociation at Ioose

binding sites is too

great

to account for

the way

RNA

polymerase

finds its

promoter.

RNA

polymerase

must

therefore

use some

other

means

to seek its binding

sites.

rril-iJ*{

li,i:.,r

shows that the

process

could

be speeded up if

the initial target for

RNA

polymerase

the

whole

genome,

not

just

a specific

promoter

sequence.

By increasing

the

target size, the rate

constant for diffusion

to DNA is correspond-

ingly increased

and is no longer limiting.

If this idea is correct,

a free RNA

polymerase

binds

DNA

and then remains in

contact with it.

How does the enzyme move

from a random

(loose)

binding site

DNA

to a

promoter?

The

most likely model

is to suppose

that the bound

sequence is directly

displaced by another

sequence.

Having

taken hold

of DNA, the

enzyme exchanges this

sequence with another

sequence

very

rapidly

and continues

exchange sequences

until a

promoter

is found.

The

enzyme then forms a stable.

open complex,

after which initiation

occurs. The search

process

becomes much faster because

association and

dissociation

are virtually simultaneous

and time

is not spent commuting

between sites. Direct

displacement can

give

"directed

walk," in

which the enzyme moves

preferentially

from

weak site to a stronger

site.

Another idea supposes that

the enzyme

slides along the DNA by a

one-dimensional ran-

domwalk, as shonmin i.i*i,iftF i:..;i+,

beinghalted

only

when it

encounters a

promoter.

There is,

however, no evidence that RNA

polymerase (or

other

DNA-binding

proteins)

can function in

this manner.

Sigma Factor

Contro[s

Binding

DNA

change

association between sigma factor and

holoenzyme changes

binding affinity for DNA so

that core enzyme can move

atong

DNA.

RNA

polymerase

encounters

a dilemma

in rec-

onciling

its needs for initiation

with those for

e I on

gatio

n. Initiation re

quire

s tighl

binding o

particular

sequences

(promoters),

whereas

elongation

requires

close association with 4//

sequences that the enzyme

encounters

during

transcription.

ii i-i

l: : L,;r' rii illustrates

how the

dilemma is solved by

the reversible

association

between

sigma

factor and core

enzyme.

As men-

tioned

previously

(see

Section

I 1.8, The

Asso-

ciation with Sigma Factor

Changes

at Initiation),

sigma

factor is

either

released

following

initia-

tion or changes its association

with

core enzyme

that it no longer

participates

DNA binding.

There are fewer molecules

of sigma

than of core

enzyme; thus the

utilization

of core enzyme

requires

that

sigma recycles.

This occurs

imme-

diately after

initiation

(as

shown

the

figure)

in about one third

of cases;

presumably

sigma

and core dissociate at

some

later

Doint

the

other cases.

ililli-tlt5;

li i

,l:-li RNA

polymerase

binds

very rapidty to

ran-

dom

DNA

sequences

and cou[d

find a

promoter

direct

displacement of

the bound

DNA sequence.

il].{;ii*t- ii

..1

RNA

po[ymerase

does

not s[ide along

DNA.

11.11 Sigma

Factor Controts

Binding to

DNA 277

rast

s'o'

{.............

Core

enzyme

stored on DNA

Sigma

factor

associates

with

core enzyme

Holoenzyme

moves Io

promoters

Core enzyme

synthesizes

RNA

Core enzyme

terminates

and

released

Very

tast

::i:":i,::=-:i

I :

-.::r

Sigma factor

and core enzyme recycte

at differ-

ent

points

transcription.

Irrespective

of the exact

timing of

its

release

from core

enzyme, sigma factor

is involved

only

in initiation.

It becomes

unnecessary

when

abortive initiation

concluded and RNA

syn-

thesis

has been

successfully initiated.

We do not

know

whether

the state

polymerase

changes

as a

consequence

of overcoming

abortive initi-

ation,

or whether

instead it is

the change in

state

that ends

abortive initiation

and allows

elongation

commence.

When

sigma factor is

released from

core

enzyme,

it becomes

immediately

available for

use by another

core

enzyme. Regardless

whether

sigma factor

is released

or remains

more loosely

associated

with

core enzyme, the

core

enzyme in

the ternary

complex is bound

very

tightly ro DNA.

Ir is essentially

"locked

in"

until

elongation has

been

completed. When

transcription

terminates,

the core enzyme is

released.

It is then

"stored"

binding to a loose

CHAPTER

Transcriotion

site on DNA. If it has lost its sigma factor, it must

find another sigma factor in

order to undertake

further

cycle of transcription.

Core enzyme has a high intrinsic

affinity

for DNA, which is increased

by the

presence

nascent RNA. Its

affinity

for

loose binding

sites

is, however, too high

to allow the enzyme

distinguish

promoters

efficiently from

other

sequences. By reducing the stability

of the loose

complexes, sigma factor allows

the

process

occur much more rapidly;

and by stabilizing

the

association at tight binding sites,

the

factor

drives

the reaction irreversibly into

the formation

open complexes. When the

enzyme releases

sigma factor

(or

changes its

association with it),

it reverts

to a

general

affinity for all DNA,

irre-

spective of sequence, that

suits

to continue

transcription.

What is responsible for

the ability of holoen-

zyme

to bind specifically to

promoters?

Sigma

factor has

domains that

recognize

the

promoter

DNA. As

independent

polypeptide,

sigma

factor does not

bind to

DNA,

but

when

holoen-

zyme forms

a tight binding complex,

o contacts

the DNA in

the

region

upstream

of the start-

point.

This difference is due to

a change in the

conformation of sigma factor

when it binds

core enzyme. The

N-terminal region

free

sigma

factor

suppresses the activity

of the DNA-

binding region; when

sigma binds to

core, this

inhibition is released,

and it becomes

able to

bind specifically to

promoter

sequences

(see

also Figure I I.35 in

Section

l.t7). The inabil-

ity of free sigma factor

recognize promoter

sequences may

be important: if

o could freely

bind to

promoters,

it might

block holoenzyme

from initiating

transcription.

Promoter

Recognition

Depends

on Consensus

Sequences

promoter

defined by the

presence

of short

consensus sequences

at specific locations.

The

promoter

consensus

sequences consist

of a

purine

at the startpoint,

the

hexamer

TATAAT

centered at

-10,

and another hexamer

centered

-35.

Individual

promoters

usually differ from

the

consensus at

one or

oositions.

As a sequence

of DNA whose function

to be

recognized

proteins,

promoter

differs from

sequences whose role

is to be

transcribed

272

translated. The information

for

promoter

func-

tion is

provided

directly

by the DNA

sequence:

its

structure

the signal. This is

a classic exam-

ple

a crs-acting

site, as defined

previously

Figure 2.16

and Figure 2.17.

By contrast,

expressed regions

gain

their meaning

only after

the

information

is transferred

into the form of

some other

nucleic

acid or

protein.

A key

question

in examining

the interaction

between an RNA

polymerase

and its

promoter

is how

the

protein

recognizes

a specific

pro-

moter sequence. Does the

enzyme have an active

site that distinguishes the chemical

structure of

particular

sequence

of bases in the DNA dou-

ble helix? How specific are its requirements?

One

way to

design a

promoter

would be

for

particular

sequence of DNA

to be recognized

by RNA

polymerase.

Every

promoter

would

consist of, or at least include,

this sequence.

the bacterial

genome,

the

minimum

length that

could

provide

an adequate signal is l2 bp.

(Any

shorter sequence

is likely

to occur-just by

chance-a sufficient number

of additional times

provide

false

signals.

The

minimum length

required for unique recognition increases

with

the size of

genome.)

The l2

bp sequence

need

not be contiguous. If a

specific number of base

pairs

separates

two

constant shorter sequences,

their combined

length

could be less than I2

bp,

because the distance of separation

itself

provides

part

of the signal

(even

if the intermediate

sequence

is itself irrelevant).

Attempts to identify

the features in DNA

that are necessary for RNA

polymerase

binding

started

by comparing

the sequences of differ-

ent

promoters.

Any essential nucleotide

sequence

should

present

in all the

promot-

ers. Such a sequence is said to

be conserved.

However, a conserved

sequence

need not nec-

essarily be conserved at every single

position;

some

variation

permitted.

How do we analyze

a sequence of

DNA

to determine whether

it is

sufficiently conserved to constitute a recogniz-

able signal?

Putative DNA recognition sites can be

defined

terms of an idealized sequence that

represents the base most often

present

at each

position.

consensus sequence is defined by

aligning

all known examples

so as

to maximize

their homology. For a sequence to be accepted

as a consensus, each

particular

base must be

reasonably

predominant

at its

position,

and

most of the actual examples must be related to

the consensus

only one or two substitutions.

The striking feature in the sequence of

pro-

moters

in E. coli is the lack of any extensive conser-

vation

of sequence

over the

60 bp associated

with

RNA

polymerase.

The sequence

of much

of the

binding site

is irrelevant.

Some short

stretches

within the

promoter

are conserved,

however,

and they are critical

for its

function. Conserva-

tion of only very short

consensus

sequences

is a typi-

cal

feature

of regulatory

sites

(such

promoters)

both

prokaryotic

and

eukaryotic

Aenlmes.

There are four

(perhaps five) conserved

fea-

tures

a bacterial

promoter:

the

startpoint,

the

-I0

sequence,

the

-35

sequence,

the separa-

tion

between

the

-10

and

-35

sequences,

and

(sometimes)

the

UP element:

The startpoint

is usually

(>90o/o

of the

time) a

purine. It is common

for the

startpoint

be the

central base

the sequence

CAT,

but the

conservation

of this triplet

not

great

enough

regard it as an

obligatory

signal.

Just upstream

the startpoint,

a 6-bp

region is

recognizable

in almost

all

pro-

moters.

The center of

the

hexamer

gen-

erally

is close

to l0 bp

upstream

of the

startpoint;

the distance

varies

in known

promoters from

Position

-18

-9.

Named

for

its location,

the

hexamer

often called

the

-10

sequence.

Its con-

sensus

is TATAAT

and can

be summa-

rized in the

form

Teo Aqr

T+r Aoo

Aro Tso

where

the subscript

denotes

the

percent

occurrence

of the

most

frequently

found

base, which

varies

from

96%.

position

at which

there

discernible

preference

for any

base

would be

indi-

cated

by N.)

If the

frequency

of occur-

rence

indicates

likely

importance

binding

RNA

polymerase, we would

expect

the

initial

highly conserved

and the

final

almost

completely

con-

served

in the

-10

sequence

to be

the

most

important

bases.

Another

conserved

hexamer

is centered

-35

bp upstream

the startpoint.

This

called

the

-35

sequence.

The con-

sensus

is TTGACA;

inmore detailed

form,

the conservation

Tez

Ts+ Gzs

Aot Csa,

Aa,s-

The distance

separating

the

-35

and

-10

sites

is between

l6 and

I8 bp in

90"h oI

promoters; in the

exceptions,

it is as lit-

tle as

l5 bp or

great

2obp.

Although

the actual

sequence

in the

intentening

region

unimportant,

the distance

critical in

11.12

Promoter

Recognition

Depends

on Consensus

Sequences

273

Startpoint

-35

-10

TTGACA

16-19

bp TATAAT

5-9 bp

Promoter

Efficiencies

Can Be Increased

Decreased

Mutation

Down

mutations

to decrease

promoter

efficiency

usua[[y decrease

conformance

to the consensus

sequences, whereas

up mutations

have the

opposite effect.

Mutations

the

-35

sequence

usuatty affect

initiat

binding

of RNA

polymerase.

Mutations

in the

-10

sequence

usuatty affect the

metting reaction

that

converts a ctosed

to an open

compLex.

Mutations

are a major

source

information

about

promoter

function.

Mutations

promot-

ers

affect the level

of expression

the

gene(s)

they control

without

altering

the

gene

products

themselves.

Most

are identified

as bacterial

mutants

that

have lost,

or have

very much

reduced,

transcription

of the adjacent genes.

They

are

known

as down

mutations.

Less

often,

mutants

are found

in which

there is

increased

transcription from

the

promoter.

They

have

up mutations.

It is important

to remember

that

"up"

and

"down"

mutations

are defined

relative

to the

usual elliciency

with which

particular

pro-

moter functions.

This

varies widely.

Thus a

change

that is

recognized

as a

down mutation

Transcription

one

promoter

might never have

been iso-

Iated in another

(which

in its

wild-type state

could

be even

less

efficient than the mutant

form

of the first

promoter).

Information

gained

from studies in vivo

simply

identifies

the

over-

all direction of the

change caused by mutation.

Is the most

effective

promoter

one that has

the actual consensus sequences?

This expecta-

tion is

borne out by the simple rule

that up

mutations

usually

increase

homology

with one

of the consensus sequences

or bring the

dis-

tance between them

closer to l7 bp. Down

mutations

usually decrease the resemblance

either

site with the consensus or make

the dis-

tance between them more

distant from l7

bp.

Down mutations

tend to be concentrated

the

most highly conserved

positions,

which

con-

firms their

particular

importance

the main

determinant

promoter

efficiency.

There are,

however, occasional

exceptions to these

rules.

determine the absolute

effects

pro-

moter mutations, we must

measure

the affinity

of RNA

polymerase

for wild-type

and mutant

promoters

invitro. There is

-100-fold

variation

in the rate at

which

RNA

polymerase

binds to

different

promoters

in vitro,

which correlates

well with the frequencies

of transcription

when

their

genes

are expresse

d in vivo. Taking

this

analysis further, we

can investigate

the stage at

which a mutation influences

the capacity

of the

promoter.

Does it

change the affinity

of the

pro-

moter for

binding RNA

polyrnerase?

Does it leave

the enzyme able

to bind but unable

to initiate?

the

influence

of an ancillary

factor

altered?

By measuring

the

kinetic

constants

for for-

mation of a

closed complex and its

conversion

an open complex,

as defined in Figure

I 1.19,

we can dissect

the two stages

of the initiation

reaction:

Down mutations

in the

-35

sequence

reduce the rate

of closed

complex for-

mation

(they

reduce 1(s),

but they

not inhibit

the conversion

an open

complex.

Down

mutations in

the

-10

sequence

not affect

the

initial

formation

a closed

complex,

but they

slow its conversion

to the

open form

(they

reduce

k2).

These

results

suggest the model

shown

fI**R[ X1-f

;=. The

function

of the

-35

sequence

is to

provide

the

signal

for

recognition

by RNA

polymerase,

whereas the

-10

sequence

allows

the

complex to

convert from

closed

to open

form.

We might

view the

-35

sequence

as com-

prising

"recognition

domain,"

whereas the

-I0

sequence

comprises

"unwinding

domain"

of the

nromoter.

r:il*i:i

i i.:'*

typicaI

promoter

has

three components.

consisting of

consensus sequences at

-35. -10.

and the

sta

rtpoi

nt.

holding the two

sites at the

appropriate sep-

aration

for

the

geometry

RNA

polymerase.

Some

promoters

have

an A-T-rich

sequence located farther

upstream. This

is called

the UP element. It interacts

with

the

cr subunit

of the RNA

polymerase.

It is typically

found in

promoters

that

are highly

expressed,

such as the

pro-

moters

for rRNA

genes.

The

optimal

promoter

is a

sequence con-

sisting of the

-3

5 hexamer,

separated by l7 bp

from

the

-10

hexamer,

lying 7

bp upstream of

the

startpoint.

The structure

of a

promoter,

showing

the

permitted

range

of variation from

this optimum,

is illustrated

ll{LJQfl

tt.fti.

274

CHAPTER 11

RNA

polymerase-promoter

complex

partially

attacked by

DNAase I

lsolate DNA and

denature

to single strands

jdq

rrr+

jd!"

y'"

/"a

li -/" 1q .r!

y'\

Sf{ita

r'*

{

/r /1 /r ,/\ /l

ttra

/,.

ELECTROPHORESIS

EXPERIMENTAL

GEL

Missing bands

identify

binding site

Farthest

from

labeled end

CONTBOL

GEL

DNA

not bound

polymerase

has bands at

positions

corresponding

to breakage

every bond

ffi

Nearest to

labeled end

DNA labeled at

one end of one strand

i:i'ii,i.i lti. :i l..a :i The

-3

5 sequence is

used

for i nitial recog-

nition

and the

-10

sequence

used for

the

metting reac-

tjon that converts a closed

comptex to an open comp[ex.

The consensus sequence

of the

-10

site con-

sists exclusively of A-T

base

pairs,

a configura-

tion that assists the initial melting

DNA into

single strands. The lower

energy needed to dis-

rupt A-T

pairs

compared with

G-C

pairs

means

that a stretch of A-T

pairs

demands the mini-

mum amount of energy for strand

separation.

The sequence immediately around

the start-

point

influences

the

initiation

event. The initial

transcribed region

(from

to +30) influences

the

rate

which RNA

polymerase

clears the

promoter

and therefore has

an effect upon

pro-

moter

strength.

Thus

the overall strength of a

promoter

cannot be

predicted

entirely from its

-15

and

-10

consensus sequences.

"typical"

promoter

relies

upon its

-35

and

-10

sequences to be recognized by RNA

poly-

merase,

but

one

or the other of these sequences

can be absent from some

(exceptional)

promot-

ers.

least

some of

these

cases, the

promoter

cannot be recognized by RNA

polymerase

alone;

the

reaction requires ancillary

proteins,

which

overcome the deficiency in intrinsic interaction

between RNA

polymerase

and the

promoter.

RNA

Pol.ymerase Binds

to One Face

DNA

The

consensus

sequences

-35

and

-L0

provide

most of the contact

points

for

RNA

potymerase

the

promoter.

The ooints of contact lie on one

face

of the DNA.

I.lr,;i.lft:ii

I i

.l:r

Footprinting

identifies

DNA-binding

sites

for

proteins

by their

protection

against

nicking.

The

ability of

RNA

polymerase

(or

indeed any

protein)

recognize

DNA can

be characterized

footprinting.

A sequence

DNA bound

the

protein

partially digested

with an

endonu-

clease

to attack

individual

phosphodiester bonds

within the

nucleic acid.

Under

appropriate

con-

ditions,

any

particular

phosphodiester bond

broken

some,

but

not in all,

DNA

molecules.

The

positions

that

are cleaved

are recognized

by using DNA

labeled on

one strand

at one

end

only.

The

principle is the same

that involved

in DNA

sequencing:

partial

cleavage

of an

end-

Iabeled

molecule at

a susceptible

site

creates

fragment of unique

length.

ii,i.ii:.i:

.;ii:.

shows,

following

the

nucle-

ase treatment,

the broken

DNA fragments

are

recovered and

electrophoresed

gel

that

separates

them according

length. Each

frag-

ment that retains

labeled

end

produces

radioactive

band.

The

position

of the

band

cor-

responds to the

number

bases

in the

11.14

RNA

Polvmerase

Binds to One

Face of

DNA

275

I l.l"lJlllll

AATXXXXA

T T

Coding strand

ilriri

ffi;;;l

ATA

Template stranc

TTAXXXX

Most

points

of contact lie

on one face of DNA

(on

the

nontemplate

strand)

-1

0 sequence

r rlll I

Modif

ications that

prevent

RNA

polymerase

from binding

Sites where RNA

polymerase

protects

against modification

Mutations

that

abolish or reduce

promoter

activtty

:=r...::r, :

0ne face

of the

promoter

contains the contact

points

for

RNA.

fragment.

The shortest fragments

move

the

fastest,

so distance from

the labeled

end is

counted

up from the bottom

of the

gel.

a free DNA,

every susceptible

bond

posi-

tion is broken in

one or another

molecule. When

the DNA

is complexed

with a

protein,

though,

the region

covered by the DNA-binding protein

is protected

in every molecule.

Thus

two

reac-

tions

are run in

parallel:

a control

DNA

alone,

and

an experimental

mixture

containing mol-

ecules

of DNA

bound to the

protein.

When a

bound

protein

blocks access of the

nuclease to DNA,

the

bonds in the

bound sequence

fail

to be broken

the experimental

mixture.

In the

control, every

bond is broken. This

generates

a series

of bands, with

one band rep-

resenting

each

base.

There

are thirty-one

bands

in the figure.

In the

protected

fragment,

bonds

cannot

be broken

in the region

bound by

the

protein,

so bands representing

fragments

the corresponding

sizes are not

generated.

The

absence

of bands 9-18 in

the figure identifies

protein-binding

site covering

the region located

9-18

bases from

the labeled

end of

the

DNA.

comparing

the control

and

experimental

lanes

with a

sequencing reaction

that is run in

parallel,

becomes

possible

"read

off" the

corresponding

sequence directly,

thus identify-

ing

the

nucleotide

sequence

of the binding

site.

described

previously

(see

Figure Il.20l,

RNA

polymerase

initially

binds the region

from

-50

to +20.

The

points

at which

RNA

polymerase

fi :'ff

ir'-:".Td;ili:fl

:lilililT.niff

:::

treat RNA

polymerase-promoter

complexes

CHAPTER

11 Transcription

with reagents that modify

particular

bases. We

can

perform

the experiment in two ways:

The

DNA can be modified

before it is

bound to RNA

polymerase.

If the mod-

ification

prevents

RNA

polymerase

from

binding, we have identified

a base

posi-

tion

where contact is essential.

The

RNA

polymerase-DNA

complex can

be modified. We then

compare the

pat-

tern of

protected

bands with that

of free

DNA

and the unmodified

complex.

Some

bands disappear, thus identifying

sites at which the enzyme

has

protected

the

promoter

against modification.

Other

bands increase

in intensity,

thus identi-

fying sites at which

the DNA must

held in

a conformation in

which it is

more exposed.

These changes

in sensitivity reveal

the

geometry

o{ the complex, as

summarized in

i:ltur.j*i: ri

:.!l'i

for a typical

promoter.

The regions

-35

and

-I0

contain most

of the contact

points

for the enzyme.

Within these regions,

the

same

sets of

positions

tend

both to

prevent

binding if

previously

modified,

and to

show increased

decreased susceptibility

to modification

after

binding. The

points

of contact

do not coincide

completely with sites

mutation;

however,

they occur in

the same limited region.

It is noteworthy

that the

same

positions

different

promoters

provide

the contact

points,

even

though a different

base is

present.

This

indicates

that there is

a common

mechanism

{or RNA

polymerase

binding, although

the reac-

tion

does not depend

on the

presence

partic-

ular bases

at some of the

points

of contact.

This

model

explains why

some of the

points

of con-

tact

are not sites of mutation.

addition, not

every mutation lies in

point

of contact; the

mutations may

influence the

neighborhood

without

actually being

touched

by the enzyme.

It is especially

significant

that the

experi-

ments

with

prior

modification

identify

only sites

in the

same region that is

protected

by the

enzyme

against subsequent

modification.

These

two

experiments measure

different

things. Prior

modification

identifies

all those

sites that

the

enzyme must recognize

in order

to bind

to DNA.

Protection

experiments recognize

all those

sites

that

actually make

contact in the

binary

com-

plex.

The

protected

sites

include

all the recog-

nition

sites and

also some additional positions,

which

suggests that

the enzyme first

recognizes

a set of bases

necessary for it

"touch

down"

and

then extends

its

points

of contact

to addi-

tional

bases.

276

The region

of DNA that is

unwound in the

binary complex can

identified

directly

chemical changes in its availability.

When the

strands

DNA

are separated, the

unpaired bases

become susceptible to reagents

that cannot reach

them in the double helix.

Such experiments

implicate

positions

between

-9

and

in the

initial melting reaction. The region

unwound

during

initiation

therefore includes the right

end of the

-10

sequence

and extends

just

past

the startpoint.

Viewed in three

dimensions, the

points

contact upstream of the

-10

sequence all lie on

one face of DNA. This can

be seen in the lower

drawing

in Figure I1.29,

in which the contact

points

are marked on a double helix

viewed

from

one side.

Most lie

on the coding strand.

These

bases are

probably

recognized in the ini-

tial formation of a closed binary

complex.

This

would make it

possible

for RNA

polymerase

approach DNA from one side and recognize

that

face

the DNA. As DNA

unwinding com-

mences,

further

sites that originally lay on the

other face of DNA can be recosnized

and bound.

(Negative

Transcribing Overwound

supercoils)

DNA

(Positive

supercoils)

Topoisomerase

Gyrase

relaxes negative

introduces

positive

supercoils

Duplex DNA

(10.4

bp/turn)

Supercoiling

Important Feature

Transcription

Negative

supercoiting

increases

the efficiency of

some

promoters

by assisting the melting reaction.

Transcription

generates positive

supercoi[s ahead

of the enzyme and

negative

supercoits behind

it,

and these

must

removed

gyrase

and

toooisomerase,

The

importance of strand separation in the

ini-

tiation reaction is emphasized by the effects of

supercoiling.

Both

prokaryotic

and eukaryotic

RNA

polymerases

can initiate transcription more

efficiently in vitro when the template is super-

coiled,

presumably

because

the supercoiled

structure requires Iess free energy for the ini-

tial

melting of DNA in the initiation complex.

The efficiency of some

promoters

is influ-

enced by

the degree of supercoiling. The most

common

relationship is for transcription to be

aided by negative supercoiling. We understand

principle

how this assists the initiation reac-

tion. Why, though, should some

promoters

influenced by the extent of supercoiling whereas

others are not? One

possibility

is that the

dependence of a

promoter

on supercoiling

determined by

its

sequence.

This

would

predict

f,f {ii"5ii!. ::i ..li:i

Transcription

generates

more tightly

wound

(positivety

supercoi[ed)

DNA ahead

of RNA

polymerase,

white

the

DNA behind

becomes less

tightty

wound

(neg-

ativety supercoil"ed).

that some

promoters have sequences

that

are

easier

to meit

(and

are therefore

less dependent

on supercoiling),

whereas

others

have more

difficult sequences

(and

have a

greater need to

be supercoiled).

alternative

is that

the

loca-

tion of the

promoter might be

important

if dif-

ferent regions of

the bacterial

chromosome

have

different degrees

of supercoiling.

Supercoiling

also has

a continuing

involve-

ment with

transcription.

As RNA

polymerase

transcribes

DNA, unwinding

and

rewinding

occurs, as

illustrated

in Figure

I 1.4.

This requires

that either the entire

transcription

complex

rotates about

the

DNA or

the DNA

itself

must

rotate about its

helical axis.

The consequences

of the rotation

DNA

are illustrated

lj}{:i#fi

f: i. L. i.r

in t]ne

twin

domain

model

for tran-

scription.

As RNA

polymerase

pushes

forward

along the double

helix,

generates

positive

supercoils

(more

tightly

wound DNA)

ahead

and

leaves negative

supercoils

(partially

unwound DNA)

behind.

For

each

helical turn

traversed

by RNA

polymerase, +l turn

gen-

erated ahead

and

-l

turn

behind.

Transcription

therefore

has a

significant

effect on the

(local)

structure

DNA. As a

result,

the enzymes

gyrase

(introduces negative super-

coils)

and topoisomerase

(removes negative

supercoils)

are required

rectify

the situation

in front of and

behind

the

polymerase, respec-

tively. Blocking

the activities

gyrase

and topo-

isomerase

causes

major

changes

in the

supercoiling of

DNA.

For example,

yeast

lack-

ing

enzyme that

relaxes

negative

supercoils,

the density of

negative

supercoiling

doubles

a transcribed

region.

possible implication

these results

is that

transcription

responsible

11.15 Supercoiting

Is an

Important

Feature

Transcription

277

for

generating

a significant

proportion

of the

supercoiling

that occurs in the

cell.

similar situation occurs

in replication,

when DNA

must be

unwound at a moving repli-

cation fork

so that the individual

single strands

can be

used as templates to

synthesize daugh-

ter

strands.

(Solutions

for the topological

con-

straints associated

with

such reactions are

indicated

later,

in Figure 19.20.)

Substitution

of Sigma

Factors

May

Control

Initiation

coLi has several

sigma factors,

each of which

causes RNA

potymerase

to initiate

at a set of

promoters

defined by specific

-35

and

-10

sequences.

o70

used for

generaI

transcription.

and the other

sigma factors

are activated

by speciaI

conditions.

The

division

of labors

between a core

enzyme

that

undertakes chain

elongation

and a sigma

factor involved

site selection

immediately

raises

the

question

of whether

there is more

than

one type of

sigma factor,

each specific for

different

class of

promot".r.

;;i-:,riii: :

shows

the

principle

of a system in

which a

substitution

of the

sigma factor

changes

the choice

Dromoter.

::i1;1.;liir

i"l .:l i

The

sigma factor

associated

with core

enzyme

determines

the set of

promoters

at which transcrip-

tion is initiated.

CHAPTER 11

Transcription

E. coli

uses alternative sigma factors

respond

general

environmental

changes; they

are listed in

:ri*ljF.I

t:,1ii.

(They

are named either

by molecular weight

the

product

for

the

gene.)

The

general

factor, which is responsible

for

transcription of most

genes

under normal

conditions, is

o70.

The

alternative

sigma factors

6)2

oE, and o54 are activated

in response

environmental changes;

o28

used for

expres-

sion of flagellar

genes

during normal

growth,

but

its level

of expression responds

to changes

the environment.

AII the sigma

factors except

o5a belong to the same

protein

family and

func-

tion in the same

general

manner.

Temperature fluctuation

is a

common type

of environmental

challenge. Many

organisms,

both

prokaryotic

and

eukaryotic, respond

in a

similar way.

Upon an increase in

temperature,

synthesis of the

proteins

currently

being made

turned off or down, and

new

set of

proteins

synthesized. The new

proteins

are

the

prod-

ucts of the heat

shock

genes,

which

play

role in

protecting

the cell

against environmen-

tal stress. Heat

shock

genes

are

synthesized in

response

to conditions other

than heat

shock

as well. Several of the heat

shock

proteins

are

chaperones. In E. coli,

the expression

of seven-

teen heat

shock

proteins

is triggered

changes

at transcription. The

gene

rpoH is

a regulator

needed

to switch on the heat

shock response.

Its

product

is o32, which functions

as an alter-

native

sigma factor

that causes

transcription

the heat

shock

genes.

The heat

shock response

is accomplished

increasing

the amount of

o32 when the

tem-

perature

increases

and

decreasing its

activity

when

the temperature

change is reversed.

The

basic

signal that induces

production

of ol2 is the

accumulation

unfolded

(partially

denatured)

proteins

that

results from increase

tempera-

ture.

The or2

protein

is unstable,

which is impor-

tant in allowing

its

quantity

to be increased

decreased rapidly. The

proteins

o70 and

ol2 can

compete for the

available core

enzyme,

so that

the

set of

genes

transcribed

during heat

shock

depends on the

balance between

them.Chang-

ing

sigma factors is

a serious

matter

that has

widespread

implications for

gene

expression

the

bacterium. It is

not surpdsing,

therefore,

that

the

production

of new

sigma factors

can

be the

target of many

regulatory

circuits.

The fac-

tor os is induced

when

bacteria make

the

tran-

sition from

growth

phase

stationary

phase

and also in

other stress

conditions.

It is

con-

trolled at

two levels. Translation

of. the rpoS

mRNA

is increased

by low

temperature

or high

Holoenzyme

with

o7o recognizes

one set of

oromoters

Substitution

of sigma factor

causes

enzyme to recognize

different set

promoters

278

osmolarity.

Proteolysis

of the

protein product

is inhibited by carbon starvation

(the

typical

signal of stationary

phase)

and by high

temperature.

Another

group

of heat-regulated

genes

controlled by the factor

oE.

It responds

to more

extreme temperature shifts than ol2 and is

induced by accumulation of

unfolded

proteins

the

periplasmic

space or outer membrane. It

is controlled by the intricate

circuit summa-

rized

r1i..

:r.

The factor oE binds to a

protein

(RseA)

that is located in the inner mem-

brane.

As a result, it cannot

activate

transcrip-

tion. The accumulation of unfolded

proteins

activates

protease

(DegS)

in the

periplasmic

space, which cleaves off the C-terminal end of

the RseA

protein.

This

cleavage activates

another

protein

in the inner membrane

(YaeL),

which cleaves the

N-terminal

region of RseA.

When this

happens,

the oE

factor is released

and can then activate transcription. The net

result is that the accumulation of unfolded

pro-

teins at the

periphery

of the bacterium is respon-

sible

for activating the set of

genes

controlled

by the sigma

factor.

This circuit has two interesting

parallels

with other

regulatory

circuits.

The response to

unfolded

proteins

in eukaryotic cells also uses

pathway

in which an unfolded

protein (within

the endoplasmic

reticulum) activates a mem-

brane

protein.

In this

case. the

membrane

pro-

tein

is an endonuclease that cleaves an RNA,

Ieading ultimately

to a change in

splicing

that

causes the

production

of a transcription

factor

(see

Section

26.17, The

Unfolded

Protein

Response

Is Related

to IRNA Splicing).

A more

direct

parallel is

with the

first

case to be discov-

ered,

which

cleavage of a membrane

protein

activates

a transcription {actor. In this case,

the

transcription

factor itself is

synthesized

as a

membrane

protein,

and the level

of sterols

in the

membrane controls the activation of

proteases

that

release the transcription factor

from the

cytosolic domain of

the

protein.

Another sigma

factor is

used under

condi-

tions

of nitrogen starvation. E coli cells

contain

a small amount of o5a,

which is activated when

ammonia

absent

from the medium. In these

conditions,

genes

are turned on to

allow uti-

lization of

alternative nitrogen sources. Coun-

terparts to this sigma

factor have

been

found in

a wide

range of bacteria, so it represents

response mechanism that

has

been conserved

in evolution.

Another case of evolutionary

conservation

of sigma

factors is

presented

by the

factor

oF,

which is

present

in small amounts

and

causes

RNA

polymerase

to transcribe

genes involved

chemotaxis

and

flagellar

structure.

Its coun-

terpart in Bacillus

subtilis

is oD,

which controls

flagellar and

motility

genes;

factors

with

the

same

promoter

specificity

are

present in many

species of bacteria.

Each sigma

factor causes

RNA

polymerase

to initiate at

particular

set

promoters. By

analyzingthe sequences

of these

promoters, we

can show that

each set

identified

by unique

sequence elements.

Indeed,

the sequence

each type of

promoter ensures

that

it is recog-

nized only by

RNA

polymerase directed

the

appropriate sigma

factor.

can deduce

the

general

rules for

promoter recognition

from

the

identification

of the

genes responding

to the

sigma

factors found

in E.

coli andthose

involved

irril.iirr

In addition

to oT0,

E. coll

has severat

sigma

factors that are

induced

particu[ar

environmentaI

con-

djtions.

(A number

the

name

of a

factor

indicates

its

mass. )

i'ii:,!.iirr,

RseA

is synthesized

protein in

the

inner

membrane.

Its cytoplasmic

domain

bjnds the

fac-

tor. RseA

cleaved

sequentialty

in the

periptasmic space

and then

in the cytopLasm.

The cytop[asmic

cteavage

reteases o'.

rpoD o7o

general

rpoS os

stress

rpoH o32

heat shock

rpoE oE

heat shock

rpoN o54

nitrogen

starvation

fliA

(ot)

flagellar sYnthesis

11.16

Substitution

ofsigma

Factors

May Control

Initiation

279

Gene Factor

-35

Sequence

Separation

-10

Sequence

rpoD

o7o

TTGAoA

16-18

bp TATAAT

rpoH

o32

cccrrcAA

13-15

bp ccccATNT

rpoN

o54

CTGGNA

6 bp TTGoA

fliA

o28

(oF)

CTAAA

15 bp

ccccATAA

sigH

AGGANPUPu

11-12

bp GCTGAATCA

r-t:-.i-,il::

.: :.:..=

coli sigma factors

recognize

promoters

with

differ-

ent c0nsensus

sequences.

Core

enzyme binding

_ l!J"'T!lys_*_?qq-____-""

100 _

i90*

1.1

1.2

2.12.22.4

4.14.2

llgYgntt

Interactions

with

promoter

DNA-bindins

I I

:.....

...

J.......Y..

-10

-35

€Gene

TAATAT.ACAGTT-

i:...i-;ii ::

A map

of the E.

coli o70 factor identifies

conserved

regions.

Regions 2.1

and 2.2

contact core

poty-

merase,

2.3 is required

for metting,

and 2.4 and

4.2 con-

#T*:',-;',

XJT

:.iJ.il'# i

lli;'ff J;l' il'Ji

sporulation

in B

subtilis

(see

Section

I1.19,

Sporulation

Controlled

by Sigma Factors).

significant

feature

of the

promoters

for

each enzyme

that they have

the

same size and

Iocation

relative

to the

startpoint,

and they

show con-

serued

sequences

only around

the usual

centers

of-35

and-10.

(The

factor

o5a

an exception

for

which

the

consensus

sequences

are

closer

together and

are

positione

-24

and

-l

2; see

Section I L

I 7,

Sigma Factors

Directly

Conract

DNA.) As

sum-

marized

in ilii=i:r

.::",-:+,

the

consensus

sequences

for

each

set of

promoters

are different

from

one

another

either

or both

of the

-35

and

-10

positions.

This means

that

an enzyme

contain-

ing

particular

sigma

factor

can recognize

only

its

own

set of

promoters,

so that

transcription

of the

different groups

is mutually

exclusive.

Substitution

of one

sigma factor

by another

there-

fore

turns

off

transcription

of the

old

set of

genes

as well

turning

on transcription

of a new

set

genes.

(Some

genes

are expressed

by RNA

polymerases

with

different

sigma factors

because

they

have

more than

one

promoter,

each

with

a different

set

of consensus

sequences.)

CHAPTER

1.1 Transcription

Sigma

Factors

Directly

Contact DNA

o70 changes its

structure to retease its

DNA-

binding regions

when it associates

with

core

enzyme.

o70 binds both the

-35

and

-10

sequences.

The

definition of a

series of different

consensus

sequences recognized

-35

and

-10

by holoen-

zymes

containing

different sigma

factors

(see

Figure I1.34)

carries the immediate

implica-

tion

that the sigma factor

subunit

must itself

contact DNA in

these regions.

This suggests

the

general principle

that there

is a

common

type

relationship

between sigma

factor

and core

enzyme, in

which the

sigma factor is

positioned

such a way as to make

critical

contacts

with

the

promoter

sequences

in the

vicinity

-35

and

-10.

Direct

evidence

that sigma

factor

contacts

the

promoter

directly

at both

the

-35

and

-10

consensus

sequences is

provided

by mutations

in sigma factor

that

suppress mutations

the

consensus

sequences.

When

a mutation

at a

particular

position

in the

promoter

prevents

recognition

by RNA

polymerase,

and

a com-

pensating

mutation in

sigma factor

allows

the

polymerase

to use the mutant promoter,

the

most likely

explanation is

that the relevant

base

pair

in DNA

is contacted

by the

amino acid

that

has

been substituted.

Comparisons

of the

sequences

several

bacterial sigma factors

identify

regions

that have

been

conserved. Their locations

in E. coli

o70 are

summarized

F{{itJFti

t:i.:i;.

The

crystal

struc-

ture

of a sigma factor fragment

from

the bac-

terium

Thermus

aquaticus

shows

that

these

regions fold

into three independent

domains in

the

protein:

domain

02 contains

\.2-2.4,63

Con-

tains 3.0-1.3,

and

oa contains 4.14.2.

Figure I

I .3 5 shows

that two

short

parts

regions

and4

(named

2.4

and4.2)

are involved

in contacting

bases in

the

-10

and

-l5

elements,

respectively.

Both of these

regions

form

short

stretches

of s-helix

in the

protein.

Experiments

with heteroduplexes

show that

o70 makes

con-

tacts

with

bases

principally

the coding

strand,

and it continues

to hold

these

contacts

after the

DNA has

been

unwound in

this region.

This

suggests

that sigma factor

could

be important

the melting

reaction.

The

use

of a,-helical

motifs in

proteins

recognize

duplex DNA

sequences

common

280