Lewin Benjamin (ed.) Genes IX

The

DNA-Binding

Form

of Repressor

Is

a Dimer

r

A repressor

monomer

has

two

distinct

domains.

o

The

N-terminaI

domain

contains

the DNA-bindinq

site.

r

The

C-terminal

domain

dimerizes.

o

Binding

to the

operator requires

the dimeric

form

so that

two DNA-binding

domains

can

contact the

operator

simultaneousty.

r

Cleavage of

the

repressor

between

the

two

domains reduces

the

affinitv for

the

operator and

induces

a lytic

cycte.

The

repressor

subunit

is a

polypeptide

of 27

kD

with

the two distinct

domains

summarized

in

!:?GLiR[ -!'.:.i.fi.

.

The

N-terminal

domain,

residues

l-92,

provides

the operator-binding

site.

.

The

C-terminal

domain,

residues

I)2-236, is

responsible

for

dimerization.

The two

domains

are

joined

by a connec-

tor of forty residues.

When repressor

is dige

sted

by

a

protease,

each

domain

is released

as a sep-

arate fragment.

Each

domain

can exercise

its

function

inde-

pendently

of the

other. The

C-terminal

frag-

ment

can form

oligomers.

The

N-terminal

fragment

can

bind the

operators,

although

with

a lower

affinity than

the intact

repressor.

Thus

the

information

for

specifically

contacting

DNA

is contained within

the

N-terminal

domain,

but

the efficiency

of the

process

is

enhanced

by the

attachment

of the

C-terminal

domain.

The

dimeric

structure

of the repressor

is crucial

inmaintaining

lysogeny

The induction

of a lyso-

genic prophage

to

enter the

lytic

cycle is caused

by cleavage

of the repressor

subunit in

the con-

nector

region,

between residues

I

I I and t

13.

(This

is a counterpart

to the allosteric

change

in conformation

that results

when a

small-mol-

ecule inducer

inactivates

the repressor

of a

bac-

terial

operon, a

capacity

that the lysogenic

repressor

does

not have.)

Induction

occurs

under certain

adverse

conditions,

such as

expo-

sure of lysogenic

bacteria

to UV irradiation,

which leads

to

proteolytic

inactivation

of the

repressor.

In the intact

state,

dimerization

of the C-

terminal

domains

ensures

that

when the repres-

sor binds

to DNA, its

two

N-terminal

domains

each contact DNA

simultaneously.

Note, how-

ever, that cleavage

releases

the

C-terminal

domains from

the N-terminal

domains.

As illus-

trated in

ri*',lRF

i.4"::.',

this

means

that the

ti;i;iJi{i:

,:

+.I

r

The

N-terminaI and C-terminat regions of

repressor

form

separate domains.

The

C-terminaI domains

associate

to

form

dimers; the N-terminal domains bind DNA.

Fli:t'!ll

:r.,.1; Repressor dimers bind to the operator.

The

affinity

of the N-terminaI domains

for DNA is controlted by

the dimerization

of the C-terminaI

domains.

N-terminal

domains

can no longer dimerize,

which

upsets the equilibrium

between monomers

and dimers. As

a

result, repressor dissociates

from

DNA,

which allows

lytic infection to start.

(Another

relevant

parameter is

the

loss of coop-

erative effects between adjacent

dimers.)

The balance between

lysogeny and the

Iytic

cycle depends on

the concentration of

14.10

The DNA-Binding Form of Repressor

Is

a

Dimer

361

repressor.

Intact repressor

is

present

in a

lyso-

genic

cell at a concentration

sufficient

to ensure

that the operators

are occupied.

If

the

repres-

sor

is cleaved, however,

this concentration

is

inadequate, because

of the lower affinity

of the

separate

N-terminal domain

for the operator.

A concentration

of repressor that

is too high

would

make it impossible to

induce the lytic

cycle in this way; a

level too low, of course,

would make it impossible to

maintain lysogeny.

A

repressor dimer

is the unit that binds

to DNA.

It recognizes

a sequence

of I

7

bp displaying

par-

tial symmetry

about

an axis through the

cen-

tral

base

pair. tir**ft{ :'""}* shows an example

of a binding

site.

The

sequence

on each side

of

the

central base

pair

is sometimes

called a

"half

-

site."

Each individual

N-terminal

region

con-

tacts a

half-site. Several

DNA-binding

proteins

that

regulate bacterial

transcription share

a sim-

ilar

mode of holding

DNA, in which the active

domain

contains

two short

regions of o-helix

that contact

DNA.

(Some

transcription

factors

in eukaryotic cells

use a similar

motif; see Sec-

tion25.I4,

Homeodomains

Bind

Related Thrgets

in DNA.)

The

N-terminal

domain of lambda

repres-

sor contains

several stretches of

u-helix, which

are

arranged as

illustrated diagrammatically

in

ri'"iit'il

li:i.iii. Two of

the helical regions are

responsible

for binding

DNA. The helix-turn-

helix model

for contact

is illustrated in

rr:r:t-iFl

':+"fti.

Looking at

a single monomer, o-

helix-3 consists of

nine amino acids, each of

which

lies at an angle to the

preceding

region

of

seven amino

acids that forms u-helix-2.

In

the

dimer, the two

apposed helix-3 regions

lie

34 A. apart,

enabling them

to fit into successive

major

grooves

of

DNA. The helix-2 regions

lie

at an angle that

would

place

them across

the

groove.

The symmetrical

binding of dimer

to

the site means that

each N-terminal domain of

the

dimer contacts

a similar set of bases in

its

half

-site.

i.i{j

i"J

fi i ii.

i"r.,

iii.i In the two-hetix model for DNA bi ndi n

g.

hetix-

3 of each

monomer

lies

in

the

wide

groove

on the same

face

of DNA, and helix-Z lies across the

qroove.

@

Repressor

Uses a

Helix-

Turn-Helix Motif to

Bind DNA

Each

DNA-binding region in the repressor contacts

a half-site in the DNA.

The DNA-binding site of the

repressor inctudes two

short a,-hetical

regions that

fit

into the successive

turns of the

major

groove

of DNA.

A DNA-binding site

is

a

(partial.ty)

pa[indromic

sequence of 17 bp.

:i:-

-..:i l:,. ..:.

The

operatorisa

L7-bpsequence with an

axis of symmetry through the centraI base

pair.

Each ha[f-

s'ite is marked in tight b[ue. Base

pairs

that are

identicaI

in each operator hatf are

in

dark btue.

iii.i1-rrilii ;.i :ii Lambda repressor's N-terminaI domain

contains five stretches ofo-hetix;

hetices 2

and 3 bind

DNA.

CHAPTER 14 Phage

Strategies

N-terminal

domain

consists of

five

s-helices

362

The

Recognition

Helix

Determines

Specificity

for

DNA

.

The

amino

acid

sequence

of the recognition

helix

makes

contacts with

particutar

bases in

the

operator

sequence

that it recognizes.

Related forms

of the

o,-helical

motifs

employed

in the

helix-turn-helix

of the

lambda

repressor

are found

in

several DNA-binding

proteins,

including

cyclic

AMP receptor protein

(

CRp),

rhe

/ac

repressor,

and

several

other

phage

repressors.

By

comparing

the abilities

of these

proteins

to

bind DNA,

we can

define

the roles

of

each helix:

o

Contacts

between

helix-l

and DNA rely

on hydrogen

bonds

between

the

amino

acid side

chains

and the

exposed posi-

tions

of the

base

pairs.

This

helix

is

responsible

for recognizing

the

specific

target DNA

sequence

and is

therefore

also known

as the

recognition

helix.

.

Contacts

from

helix-2

to

the DNA

take

the form

of hydrogen

bonds connect-

ing with

the

phosphate

backbone.

These

interactions

are necessary

for binding,

but do not

control

the

specificity

of tar-

get

recognition.

In

addition

to these

con-

tacts,

a large

part

of

the overall

energy

of interaction

with DNA

is

provided

by

ionic

interactions

with

the

phosphate

backbone.

What

happens if

we

manipulate

the

cod-

ing

sequence

to construct

a new

protein

by sub-

stituting

the recognition

helix

in

one repressor

with

the corresponding

sequence

from

a closely

related repressor?

The

specificity

of the hybrid

protein

is that

of its new

recognition

helix.

The

amino acid

sequence

of this short

region

determines

the

sequence specificities

of the individual

proteins

and is able to

act in conjunction

with

the rest

of the

nnlvnpntiip rhnin

i.

,

,.

r ,

shows

the details

of the

bind-

ing

to DNA

of two

proteins

that

bind

similar

DNA

sequences. Both

lambda

repressor

and

Cro

protein

have

a similar

organization

of

the helix-

turn-helix

motif, although

their individual

speci-

ficities

for DNA

are not identical:

.

Each

protein

uses similar

interactions

between hydrophobic

amino

acids to

maintain

the relationship

between

helix-2

and helix-3:

repressor

has

an

Ala-Val

connection,

whereas

Cro has an

Ala-Ile

association.

.

Amino

acids

in

helix-3 of the repressor

make contacts with

specific bases

in

the

operator. Three amino acids in repres-

sor recognize

three bases

in

DNA; the

amino

acids at these

positions,

and also

at additional

positions

in

Cro,

recognize

five

(or possibly

six) bases in DNA.

TWo

of the amino acids involved in specific

recognition

are identical in repressor

and Cro

(Gln

and

Ser at the N-terminal end of the helix),

whereas

the other

contacts

are

different

(Ala

in

repressor versus

Lys and the additional Asn in

Cro). In addition,

a

Thr in helix-2

of Cro directly

contacts DNA.

The

interactions shown in Figure 14.21 rep-

resent

binding to the DNA sequence that each

protein

recognizes most tightly. The sequences

shown at

the bottom of the

figure

with the con-

tact

points

in color differ at three of the nine

base

pairs.

The use of overlapping, but not iden-

tical, contacts

between amino acids and bases

shows

how related recognitioq helices confer

recognition

of

related DNA sequences. This

enables repressor

and Cro to recognize the same

set of sequences,

but

with different relative

affinities for

particular

members

of the

group.

The

bases contacted by helix-3 of repressor

or Cro lie

on one

face

of

DNA, as can be

seen

from

the

positions

indicated on the helical dia-

gram

in Figure

14.21

.

Note,

however,

that

repressor

makes an additional contact with the

other

face of DNA. Removing the last six

N-terminal

amino acids

(which protrude

from

:

rr,itrrrr

r

r,

, r

Two

proteins

that use the two-hetix arrange-

ment

to contact DNA recognize [ambda

operators with affini-

ties determjned by the amino acjd sequence

of heLix-3.

L4.1.2

Ihe Recognition

Helix Determines Specificity

for DNA

363

Repressor

Dimers Bind

CooperativeLy

to the 0perator

Repressor binding to one operator increases the

affinity for binding a second

repressor

dimer to the

adjacent operator.

The affinity is 10x

greater

for 0r1 and 0*1 than

other operators, so they are bound first.

Cooperativity

attows

repressor to

bind

the 07/02

sites at [ower concentrations.

Each operator

contains

three

repressor-binding

sites.

As can be

seen from

FI6i",Rf 14-33,

no two

of the six

individual

repressor-binding

sites are

identical,

but they all

conform with

a

consensus

sequence.

The binding sites

within each opera-

tor are

separated by

spacers of

I

to

7 bp that are

rich in

A-T base

pairs.

The sites at each operator

are

numbered so that

O* consists of the series

of

binding

sites O*1-O*2-OR3,

whereas O, consists

of the

series OLI-OL2-OL3.

In each case,

site I lies

closest to

the startpoint

for transcription

in the

promoter,

and sites

2 and3lie

farther upstream.

Faced

with the triplication

of binding sites

at each

operator,

how does

repressor decide

where to start

binding?

At each operator, site

I

has a

greater

affinity

(roughly

tenfold) than the

other

sites for the

repressor.

Thus the repressor

always binds

first to Orl

and o*I.

Lambda repressor

binds to subsequent

sites within

each operator

in a cooperative

manner. The

pres-

ence of a dimer

at site

I

greatly

increases the

affinity

with

which

a second dimer can bind

to

site 2. When

both sites

I and 2 are occupied,

this

interaction does

not extend farther,

to

site

3. At the concentrations

of

repressor usually

found in a lysogen,

both sites I and2 are

filled

at each

operator, but

site 3 is not occupied.

If

site

I is inactive

(because

of mutation),

then repressor

binds cooperatively to sites

2 and

3. That is, binding

at site 2 assists another dimer

to bind at site

3. This

interaction

occurs

directly

between

repressor

dimers and not via confor-

mational

change

in DNA. The C-terminal

domain

is responsible for the cooperative

inter-

action between dimers,

as well as for the dimer

formation

between subunits.

FISURS 14-24

shows

that it

involves both subunits of each dimer,

that is, each subunit

contacts its counterpart in

the

other dimer,

forming a tetrameric structure.

A result of cooperative binding

is to increase

the effective

affinity of

repressor for

the oper-

ator

at

physiological

concentrations.

This enables

a lower concentration

of repressor to achieve

occupancy

of the operator.

This is

an

important

consideration

in a system

in

which

release

of

repression

has irreversible consequences.

In

an

operon coding

for metabolic enzymes, after all,

failure of repression

will merely allow

unnec-

essary synthesis of

enzymes. Failure to repress

Iambda

prophage,

however, will

lead

to

induc-

tion of

phage

and

lysis

of the cell.

From the sequences shown

in

Figure 14.23,

we see

that O.l and O^l lie more or less in the

center of the

RNA

polymerase

binding sites of

P,

and

P*, respectively. Occupancy of. OrI-Or2

and O*

I

-O*2

thus

physically

blocks access of RNA

polymerase

to the corresponding

promoters.

lIGuftt 14.2t

A view

from the back shows that

the bul.k

of the

repressor

contacts

one face of

DNA, but

its

N-terminal arms

reach around to the other

face.

helix-l)

eliminates

some of the contacts.

This

observation

provides

the basis for the

idea that

the bulk of the N-terminal

domain contacts

one

face of DNA, whereas the

last six

N-terminal

amino acids

form an

"

arm" extending

around

the back.

FIGURE 14.22

shows

the view

from the

back.

Lysine residues in the arm

make contacts

with G residues in the

major

groove

and also

with the

phosphate

backbone.

The interaction

between the arm

and DNA contributes

heavily

to DNA binding; the affinity of

the armless

repressor for DNA is reduced by

-1000-fold.

Bases that are

not

contacted

directly by

repressor

protein

may

have

an

important effect

on

binding. The related

phage

434 repressor

binds

DNA

via a

helix-turn-helix motif, and

the

crystal structure shows that

helix-3 is

positioned

at

each

half-site so that it contacts the

five out-

ermost base

pairs,

but

not the inner two. Oper-

ators with A-T base

pairs

at the

inner

positions,

however,

bind434

repressor more strongly

than

operators with G-C

base

pairs.

The

reason is

that

434 repressor

binding slightly

twists

DNA

at the center of the operator, which

widens the

angle between the two

half

-sites

of DNA by

-3'.

This is

probably

needed to allow each monomer

of the repressor dimer

to make

optimal

contacts

with DNA. A-T base

pairs

allow this twist more

readily than G-C

pairs,

thus affecting the

affin-

ity of the operator

for repressor.

364 CHAPTER 14 Phage

Strategies

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I

ANf90SAl

tor

protein

that is necessary

for transcription

of

the clgene

(see

Section 14.14,

Repressor at Oo2

Interacts with RNA Polymerase

at P**1 . The

represslr is

the

producl

o/ cI; thus this interaction

creates a

positive

autogenous circuit, in which

the

presence

of repressor is necessary

to support its own

continued

synthesis.

The nature oI this control

circuit explains

the biological

features

of lysogenic existence.

Lysogeny is stable because

the control circuit

ensures that as

long

as the level of repressor is

adequate, there is continued

expression of the

clgene. The

result is

that O. and O* remain

occu-

pied

indefinitely. By repressing

the entire lytic

cascade, this action maintains the

prophage

in

its inert form.

i:il..iii-ili

r,,.,:1

i

In the [ysogenic

state,

the

repressors bound

at 0r1

and 0r2 interact

with those bound

at

0*1 and 0R2.

RNA

potymerase

is bound at P*,

(which

overtaps

with 0*3)

and

interacts

with the

repressor bound at 0*2.

lii;'iiii1 i;,.i.;i'r 0r3 and

0*3 are

brought

into

proximity

by

formation

of the

repressor octamer,

ind

an increase

in

repressor concentration

attows dimers

to bind at

these sites

and to

interact.

The DNA between

the O,

and Oo

sites

(that

is. the

gene

c1)

forms alarge

loop,

which

is held

together by

the repressor

octamer.

The octamer

brings

the sites Or3

and

O*3

into

proximity. As

a result, two

repressor

dimers

can bind

to

these

sites and interact

with

one

another,

as shown

in

i:ii:l.irii i

;

:,::.

The occupation

of O*3

prevents

RNA

polymerase from binding

to

P** and

there-

fore turns off expression

of

repressor.

This

shows

us

how the

expression

of the

cI

gene

becomes

exquisitely

sensitive

to

repres-

sor

concentration.

At the

lowest

concentrations,

it forms the octamer

and

activates

RNA

poly-

merase in a

positive

autogenous

regulation.

An

increase in concentration

allows

binding

to Ot3

and O*3 and

turns

off transcription

in a nega-

tive autogenous

regulation.

The threshold

lev-

els of repressor

that

are

required

for each

of

these events

is reduced

by the

cooperative

inter-

actions,

which

makes the

overall

regulatory

sys-

tem

much more

sensitive.

Any

change

in

repressor level

triggers

the

appropriate

regula-

tory

response to

restore

the

lysogenic

level.

14.16

cooperative

Interactions

Increase

the sensitivity

of Regutation

367

Cooperative

Interactio ns

Increase

the Sensitivity

of ReguLation

o

Repressor dimers bound

at

0r1

and 0r2

interact

with dimers bound at 0*1 and 0*2 to

form

0cla

mers.

r

Octamer formation brings 0,3 ctose to 0*3,

a[[owing

interactions

between dimers bound there.

o

These cooperative interactions increase the

sensitivity of reg utation.

Cooperative

interactions

between

repressor

dimers

occur at both the left and right opera-

tors, so that

their normal condition

when

occu-

pied

by repressor

is

to

have

dimers at both the

I

and

2 binding sites. In effect, each operator

has a tetramer of

repressor.

This is not the end

of the story,

though. The two dimers interact

with one another

to form

an octamer.

The

interaction occurs between the C-terminal

domains,

which can form an octamer as a crys-

tal structure.

:

ii:.i:!i.

.

r

,

shows the distribution of

repressors at the operator sites that

are

occu-

pied

in a lysogen. Repressors are occupying O.1,

OL2,

ORI, and O*2, and the repressor at the

last

of these sites

is interacting with RNA

poly-

merase, which

is initiating

transcription

at Pono.

The interaction between the two

operators

has

several

consequences. It stabilizes

repres-

sor binding,

thereby making it

possible

for

repressor

to occupy operators at Iower concen-

trations. Binding

at

OR2 stabilizes

RNA

poly-

merase binding

at P*r, which enables low

concentrations

of repressor to autogenously

stimulate

their own

production.

oL3

ot2

or_1

J+

t'nnf

f

.F

f;f,

State of c/

gene:

OFF

The

overall level

of

repressor

has

been

reduced

(about

threefold from

the level

that

would

be required if

there were no

cooperative

effects),

and

as a result

there is less repressor

that has

to be eliminated

when it

becomes nec-

essary

to induce

the

phage.

This

increases

the

efficiency

of

induction.

@

The

cII and

cIII

Genes

Are

Needed

to Establish

o

The

detayed

early

gene products

cII

and cIII are

necessary for

RNA

potymerase

to

initiate

transcription

at

the

promoter

P*r.

o

cII

acts direct

at the

promoter

and cIII

protects

cII from

degradation.

o

Transcription

from

P*, [eads

to synthesis

of

repressor

and

atso blocks the

transcription

of cro.

The

control

circuit for maintaining

lysogeny

presents

a

paradox.

The

presence

of repressor

pro-

tein

is necessary

for

its

own synthesis. This

explains

how

the lysogenic

condition

is

perpetuated.

How,

though,

is the

synthesis

of

repressor

estab-

Iished in

the first

place?

When

a

lambda

DNA

enters a new

host cell,

RNA

polymerase

cannot

transcribe

c1 because

there

is no

repressor

present

to

aid its binding

at P*r. This

same

absence

of

repressor,

how-

ever,

means

that P*

and P, are

available. Thus

the first

event after

lambda

DNA infects

a bac-

terium

is when

genes

Nand

crl are transcribed.

After

this,

pN

allows transcription

to

be extended

t

T

F\

Cll

protein

FiGlJRt

14.29

Repressor

synthesis is

estabHshed

by the

action

of cII

and RNA

polymerase

at P*, to

jnitiate

transciiption

that

extends from

the

antisense

strand of

cro throuqh

the c/

qene.

farther. This allows

cIII

(and

other

genes)

to be

transcribed

on the

left,

whereas cI1

(and

other

genes)

are transcribed

on the right

(see

Figure 14.14).

The

c1land cIII

genes

share

with clthe

prop-

erty that mutations in

them cause

clear

plaques.

There is, however,

a difference. The

clmutants

can neither establish nor

maintain lysogeny.

The c11

or cIII mutants have

some difficulty

in

establishing lysogeny,

but once it is

established

they are able

to maintain it by

the c1 autoge-

nous circuit.

This

implicates the

cII andclllgenes

as

pos-

itive regulators

whose

products

are needed for

an

alternative system for repressor

synthesis.

The

system

is

needed only

to

initiate

the

expres-

sion of clin order

to circumvent

the inability

of

the autogenous

circuit to engage in

de novo

syn-

thesis. They

are not needed

for

continued

expresslon.

The

cII

protein

acts directly

on

gene

expres-

sion. Between the cro

and c1I

genes

is another

promoter,

called P*..

(The

subscript

"RE"

stands

for repressor

establishment.) This

promoter

can

be recognized

by RNA

polymerase

only in

the

presence

of

cII, whose action

is illustrated

in

FIGURE 14-29.

The cII

protein

is extremely

unstable

invivo,

because it

is degraded as

the result

of the activ-

ity

of a

host

protein

called

HflA. The

role of

cIII

is to

protect

cII against

this degradation.

Tlanscription

from P*u

promotes

lysogeny

in

two ways. Its

direct effect is

that cI is

trans-

Iated into repressor

protein.

An indirect

effect

is that transcription proceeds

through

the

to

gene

in

the

"wrong"

direction.

Thus

the 5'

part

of the RNA

corresponds

to an antisense

transcript

of cro; in

fact, it

hybridizes

to

authentic

cro mRNA,

which inhibits

its

trans-

lation.

This is important

because

cro expres-

sion is needed

to enter

the lytic

cycle

(see

Section 14.20,

The

cro Repressor

Is

Needed

for Lytic Infection).

The

cI coding

region

on the P*,

transcript

is very

efficiently

translated,

in contrast

with

the weak translation

of the P*,

transcript.

In

fact,

repressor

is synthesized

approximately

seven to

eight times more

effectively

via

expres-

sion from P*.

than from

P*n,,.

This reflects

the

fact that

the P*u transcript

has

an

efficient ribo-

some-binding

site,

whereas

the P*,

transcript

has no

ribosome-binding

site and

actually

starts

with the AUG

initiation

codon.

Lysogeny

368

CHAPTER

14 Phage

Strategies

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