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Mutations

in the

Lac repressor

identified

the

existence

of different

domains

even

before

the

structure

was

known.

We

can now

explain

the

nature

of the mutations

more

fully

by reference

to

the structure,

as

summarized

in

,

lr,l:':r

: L

.,.

Recessive

mutations

of the

lacl-

Iype

can

occur

anl.where

in the

bulk

of the

protein.

Basi-

cally, any mutation

that inactivates

the

protein

will have

this

phenotype.

The

more

detailed

mapping

of mutations

on to the

crystal

struc-

ture in

Figure 12.13

identifies

specific

impair-

ments

for

some of

these mutations,

for example,

those

that affect

oligomerization.

The

special

class of

dominant-negalive

lacla

mutations

lie

in the

DNA-binding

site

of the

repressor

subunit

(see

Section 12.9,

Multimeric

Proteins

Have

Special

Genetic

Properties).

This

explains their

ability

to

prevent

mixed

tetramers

from

binding to the

operator;

a reduction

in

the

number

of

binding

sites reduces

the specific

affinity for

the operator.

The

role

of the

N-ter-

minal

region

in specifically

binding

DNA is

shown

also by its location

as the

site of

occur-

rence

of

"tight

binding"

mutations.

These

increase

the

affinity

of the

repressor

for

the

operator,

sometimes

so much

that it

cannot

be

released

by inducer.

They

are rare.

Uninducible

/acls

mutations

map largely

in

a

region

of the

core

domain I

extending

from

the inducer-binding

site to

the hinge.

One

group

Iies in

amino acids

that

contact

the inducer,

and

these mutations

function

by

preventing

bind-

ing

of inducer.

The remaining

mutations

lie at

sites that

must be involved

in transmitting

the

allosteric

change in

conformation

to the hinge

when inducer

binds.

Repressor

Protein

Binds

to the

Operator

o

Repressor

protein

binds to

the doubte-stranded

DNA

sequence of

the operator.

r

The

operator is

a

palindromic

sequence

of 26

bp.

o

Each inverted repeat

of the operator

binds

to the

DNA-binding

site of one repressor

subunit.

The repressor

was isolated

originally

by

purify-

ing the

component

able to

bind the

gratuitous

inducer

IPTG.

(The

amount

of repressor

in

the

cell is so small

that in

order to

obtain enough

material, it

was necessary

to

use a

promoter-

up mutation

to increase

lacl

Iranscription

and

to

place

this /acllocus

on

a DNA molecule

pres-

'

,l,l-li::, 1,,

The

[ocations of three type

of mutations

in

lactose repressor

are

mapped on the domain structure

ofthe

protein.

Recessive

lacf-

mutants

that cannot

repress

can map anywhere in

the

protein.

Dominant negative lacli

mutants

that

cannot

repress map to the DNA-binding

domain. Dominant

locf'

mutants that cannot induce

because

they do

not

bind

inducer

or cannot undergo the

allosteric

change

map

to core domain

1.

ent in many copies

per

cell. This results in an

overall overproduction of 100 to 1000-fold.)

The repressor

binds to double-stranded

DNA

containing the

sequence

of the wild-type lac

operator. The repressor

does

not bind DNA from

an OC mutant. The addition of IPTG

releases

the

repressor

from

operator

DNA in vitro. The in

vitro reacion

between

repressor

protein

and

operator DNA therefore displays the character-

istics

of control inferred

in vivo;thus it can be

used to establish the basis for

repression.

How

does the

repressor recognize the spe-

cific

sequence of operator

DNA? The operator

has

a feature common to many

recognition

sites

for bacterial regulator

proteins:

it is a

palin-

drome. The inverted repeats are

highlighted

in

i

ri

r , . Each repeat can be

regarded

as

a half-site

of the operator.

We can use the same

approaches to define

the

points

that the repressor

contacts in the

operator that we used for analyzing the

poly-

merase-promoter

interaction

(see

Section I l. 14,

RNA Polymerase Binds to One

Face

of

DNA).

Deletions

of

material

on

either side define the

end

points

of the

region; constitutive

point

mutations

identify individual

base

pairs

that

must

be crucial. Experiments

in which DNA

bound to repressor is compared with

unbound

12.14 Reoressor

Protein Binds to the 0perator 313

MRNA

TGTTGTGTG

GAATT

ACAACACACCTTA

-10 -5

+1

of the operator

makes

the same

pattern

of

con-

tacts with

a repressor

monomer. This is shown

by symmetry

in the contacts

that repressor

makes with the operator

(the pattern

between

+l and

+6 is identical with

that between

+21

and

+16) and by

matching constitutive

muta-

tions in each

inverted

repeat.

(The

operator

is

not

perfectly

symmetrical,

though; the left side

binds

more strongly

than the

right

side

to the

repressor.

A

stronger

operator

is created by a

perfect

inverted duplication

of the left side and

eliminated

of

the central base

pair.)

iii:r.ji.:i

:.j. i

.r

The

loc operator

has a symmetricaI sequence.

The sequence

is num-

bered

retative to

the startpoint

for transcription at

+L, The

pink

arrows to the

teft

and to the right identify the two dyad repeats.

The

green

blocks

indjcate the

posi-

tions of

identity.

i:liri.ii1li.

-i,i"

l:;ri Bases that

contact

the repressor can be identified bv

chemicaI

cross[inking or by experiments to see whether modification

prevents

binding.

They

identify

positions

on both strands of DNA extending

from +1

to

+23. Constitutive

mutations

occur at eight

positions

in

the operator between

+5

and +17.

DNA for its

susceptibility

to methylation or UV

crosslinking

identify bases that are either

protected

or more susceptible

when associated

with the

protein.

iii".iiiii:

::j.:ii

shows that the

region of DNA

protected

from nucleases

by bound

repressor

lies within the region of symmetry, comprising

the 26 bp region from

-5

to

+2

l. The area

iden-

tified

by constitutive

mutations is even smaller.

Within a

central

region extending over the l3 bp

from +5

Io

+I7

,

there are eight sites at

which sin-

+[iffi

Jffi::1T:"':nfi

,ff ni:Htx

promoter

mutations

summarized earlier

in Fig-

ure I I.29. A

small

number of essential specific con-

tacts within

a larger

region

can be

responsible

for

sequence-specific association of DNA with

protein

The

symmetry of the DNA sequence reflects

the symmetry in the

protein.

Each

of

the iden-

tical subunits in a repressor tetramer has a DNA-

binding site. TWo of these sites contact the

operator in such a way that each inverted repeat

CHAPTER 12 The

0oeron

CT

GA

t+

ACAACACACCT T AACACT

tttt

-

Protected

by

-10 -5

+1 +5

+

lltt+

AJAAc_44If

rc49494

t tfttf t

repressor

+

+15 +2O +25

Binding of

Inducer

Releases

Repressor

from

the

0perator

r

Inducer binding

causes a change

in repressor

conformation

that reduces

its affinity for DNA anc

releases it from the operator.

Various

inducers cause characteristic

reductions

in the affinity of

the repressor

for

the operator

invitro.

These changes

correlate with the effec-

tiveness of

the inducers in

vivo. This

suggests

that

induction results

from a reduction in the

attraction between

operator and

repressor. Thus

when inducer

enters the cell,

it

binds to

free

repressors and

in effect

prevents

them

from

finding their operators.

Consider, though, a

repressor tetramer

that is already bound tightly

to the operator.

How does

inducer cause this

repressor to be

released?

TWo models

for renressor action are illus-

trated

in

FI"";iisi.

:.;:.:*s:

.

The equilibrium

model

(left)

calls for

repressor bound to

DNA

to be

in rapid

equilibrium with

free repressor. Inducer

would bind

to the free form of repres-

sor and thus

unbalance the equilibrium

by

preventing

reassociation with DNA.

.

The rate of dissociation of

the repressor

from

the operator,

however, is much too

slow

to be compatible with this model

(the

half-life in vitro in the absence of

inducer is

>I5

min). This means that

instead the inducer must bind directly to

represslr

protein

complexed with

the

opera-

/or As indicated in the model on the right,

inducer binding must

produce

a change

in the

repressor that makes it release the

operator.

Indeed, addition of IPTG causes

an

immediate destabilization of the

repressor-operator complex in vitro.

314

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ers

(see

Figure 11.22

and Figure I1.23).

The

same solution

is likely:

movement could

be

accomplished by reducing the

dimensionality

of the search by sliding along DNA

or by direct

displacement from site

to site

(as

indicated in Fig-

ure 12.241. A displacement reaction might

be

aided by the

presence

of more binding sites

per

tetramer

(four)

than are actually needed

to con-

tact DNA at any one time

(two).

The

parameters

involved

in finding a high-

affinity operator in the face

of competition from

many low-affinity sites

pose

a dilemma for

repressor. Under conditions

of

repression,

there

must

be

high

specificity

for

the operator. Under

conditions of induction, however,

this speci-

ficity must

be

relieved.

Suppose, for example,

thatthere were 1000 molecules

of

repressorper

cell: only

0.04oh

of operators would be free

under conditions of repression. Upon induc-

tion, though, only 40o/o

of operators would

become

free. We therefore

see an inverse cor-

relation between the ability to achieve com-

plete

repression

and the ability to relieve

repression effectively. We assume that the num-

ber of

repressors

synthesized in

vivo

has been

subject to selective

forces

that balance these

demands.

The

difference

in

expression of the lactose

operon

between its induced and repressed

states

in wvo isactually

I

03x.

In

other

words,

even

when

inducer is absent, there is a basal level of expres-

sion of

-O.l%

of the induced level. This

would

be reduced if there were more repressor

protein

present

and

increased if there

were

less. Thus it

could be impossible to establish tight repression

if

there were

fewer repressors

than the ten

found

per

cell, and

it might

become difficult to

induce

the operon if there were too many. It is

possible

to

introduce t};re lac operator-repressor system

into the mouse. When Lhe lac operator is con-

nected to a tyrosinase

reporter

gene,

the enzyme

is induced by the addition of IPTG. This means

that the

repressor is finding its

target

in a

genome

l0r times

larger

than that of

E

coli.Induction

occurs

at approximately the same concentration

of

IPTG

as

in bacteria. We do not. however. know

the concentration of

Lac repressor

and how effec-

tively

the target is induced.

In

order

to extrapolate invivo fromthe affin-

ity of a DNA-protein

interaction

in vitro, we

need

to

know the effective concentration of DNA ln

vivo. T];re

"effective

concentration" differs

frt-rm

the mass/volume because of several

factors. The

effective concentration

is increased, for exam-

ple,

by molecular crowding, which occurs

when

polyvalent

cations neutralize

-90%

of

the

charges on DNA, and

the

nucleic acid collapses

into

condensed

structures.

The

major force

that

decreases the effective

concentration

is the inac-

cessibility of

DNA that

results

from occlusion

or sequestration by

DNA-binding

proteins.

One way to

determine

the effective

con-

centration

is to compare

the

rate of a

reaction

in

vitro

and

in vivo thar depends

on DNA concen-

tration. This

has been done

using

intermolec-

ular recombination

between

two

DNA

molecules. To

provide

a

control, the

same

reac-

tion is followed as an

intramolecular

recombi-

nation, that

is, the two

recombining

sites

are

presented

on the

same

DNA

molecule.

We

assume

that concentration

is the same

in

vivo

and in vitro for t]ne

intramolecular

reaction,

and

therefore

any difference

in the ratio

of inter-

molecular/

intramolecular

recombination

rates

can

be attributed

to a change

in the effective

concentrationinvivo.

The

results of such

a com-

parison

suggest

that the

effective

concentration

of DNA is reduced

>10-fold invivo.

This could

affect

the rates

of reactions

that

depend on DNA concentration,

including

DNA

recombination

and

protein-DNA binding.

It

emphasizes

the

problem

encountered

by all

DNA-binding

proteins in finding

their

targets

with sufficient

speed

and

reinforces

the conclu-

sion that diffusion

is

not adequate

(see

Fig-

ure I L22).

Repression

Can 0ccur

at

Multiple

Loc'

o

A repressor will act

on a[[ loci

that

have a copy

of

its

target

operator

sequence,

The lac repressor acts

only

on the

operator

of

tine lacZYA cluster.

Some

repressors,

however,

control dispersed

structural

genes

by binding

at

more than one

operator.

An example

is lhe

trp

repressor, which

controls

three

unlinked

sets

of

genes:

.

An

operator

at the

cluster

of structural

genes

trpEDBC,

controls

coordinate

s1m-

thesis

of the

enzymes

that synthesize

tryptophan

from

chorismic

acid.

.

An operator

at another

locus controls

tine aroH

gene, which

codes

for one

of

the

three enzymes

thatcatalyze

the ini-

tial

reaction

in the

common

pathway

of

aromatic

amino

acid

biosynthesis.

o

The trpRregulator

gene

is repressed

by

its own

product,

tlne

trp

repressor'

Thus

12.19

Repression

Can

Occur

at

Multipte

Loci 319

€ Operator region

+

mRNA

trp

AATCI'i''

.i:Arrr.:l

:1,i',

'

:

::

11,tl ]

.'r.i.:t'i

;!i,GCA

mRNA

frpF

ffiATCGTACTCTTTAGCGAGTAC*4@

mRNA

ri*iJftf

i?.t5 The

frp repressor recognizes

operators at three

loci. Conserved bases

are shown in

red. The

[ocation

of the startpoint

and

mRNA

varies, as indicated

bv the white arrows.

Startpoint

'-Promoter.i}'

3 Operat6y lq66lisns-r>

fiGiJtf 1i.I6

0perators may

lie at various

positions

rel

ative to the

promoter.

the repressor

protein

acts to reduce its

own synthesis.

This

circuit is an

exam-

ple

of autogenous

control.

Such cir-

cuits are

quite

common

in regulatory

genes

and may be

either negative

or

pos-

itive (see

Section 12.23,

r-Protein

Syn-

thesis Is

Controlled

by Autogenous

Regulation

and

Section l4.lr,

Repres-

sor Maintains

an Autogenous

Circuit).

A related

2i bp operator

sequence is

pres-

ent at each

of the

three loci

at which

the trp

repressor

acts. The

conservation

of sequence

is

indicated

in

FIiliJR[

:.?..J5.

Each

operator

con-

tains

appreciable (but

not identical)

dyad sym-

metry.

The

features

conserved

at all three

operators

include

the important

points

of con-

tact

for /rp

repressor.

This

explains how

one

CHAPTER

12 The

0oeron

repressor

protein

acts on several loci:

each locus

has a copy of a specific DNA-binding

sequence reclg-

nized by the repressor

(just

as each

promoter

shares

consensus

sequences with

other

promoters).

FtilLiRE

X?"?S

summarizes

the

variety

of rela-

tionships between

operators and

promoters.

A

notable feature

of the dispersed

operators

rec-

ognized by TrpR is

their

presence

at different

Iocations

within the

promoter

in

each locus. In

trpRlh.e

operator lies between

positions

-I2

and

+9,

whereas inthe trp

operon it

occupies

posi-

tions

-23

to

-3.In

the aroH locus it

lies farther

upstream,

between

-49

and-29.In

other

cases,

the

operator lies downstream

from

the

pro-

moter

(as

in lac)

,

or apparently

just

upstream

of

the

promoter (asingal,

for which

the nature

of

the repressive

effect is not

quite

clear).

The abil-

ity

of the repressors

to act at operators

whose

positions

are

different in each

target

promoter

suggests

that there

could be differences

in

the

exact mode

of repression,

the common

feature

being

that RNA

polymerase

is

prevented

from

initiating

transcription

at the

promoter.

Cyclic AMP

Is

an Effector

That Activates

CRP

to Act

at

Many

Operons

o

CRP is

an activator

protein

that binds

to a

target

sequence at

a

promoter.

r

A dimer

of CRP is

activated by a

single motecute

of

cyclic AMP.

Thus far

we have dealt

with the

promoter

as a

DNA

sequence

that is competent

to

bind RNA

polymerase,

which then initiates

transcription.

There

are, however,

some

promoters

at

which

320

Lewin Benjamin (ed.) Genes IX

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