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of substrate

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potential

role in

peroxisomal

membrane

assembly. Proc Natl.

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2Lr6-2r2r.

South, S. T. and Gould,

S. J.

(I999).

Peroxisome

synthesis

in

the absence of

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somes. J. Cell Biol. 144,255-266.

Walton, P. A., Hill, P.

E., and Hill,

S.

(1995).

Import

of stably folded

proteins

into

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Mol. Biol. Cell 6, 675-683.

The

Sec System Transports Proteins

into

and Through

the

Inner

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Reviews

Lee,

C.

and Beckwith,

J.

(1986).

Cotranslational

and

posttranslational protein

translocation

in

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systems. Annu. Rev.

Cell Biol. 2,

)t5-)36.

Oliver, D.

(1985).

Protein

secretion inE. coli. Annu

Rey. Immunol

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Research

Beck, I(., Wu, L. F., Brunner,

J., and Muller, M.

(2000).

Discrimination

between SRP- and

SecA/SecB

-dependent

substrates involves

selective

recognition

of nascent

chains by SRP

and trigger factor. EMBO

J.19, 84-143.

Brundage, L. et al.

(1990).

The

purified

E coli inte-

gral

membrane

protein

SecY/E is sufficient for

reconstitution

of SecA-dependent

precursor

protein

translocation. Cell 62, 649-617.

Collier, D. N. et al.

(1988).

The

antifolding activity

of SecB

promotes

the export of the E coli

maltose-binding

protein.

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53, 27

)-28).

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(1988).

ProOmpA is

stabilized

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membrane translocation by either

purified

E. coli trigger Iactor or canine

signal

recogni-

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particle.

Cell 54, 1003-10l I

Valent,

Q.

A.,

Scotti,

P. A., High,

S., von Heijne, G.,

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W.,

Oudega,

B.,

and Luirink, J.

(1998).

The E.

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SecB targeting

pathways

converge at the

translocon.

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Yahr, T. L. and Wickner,

W. T.

(2000).

Evaluating

the oligomeric state of

SecYEG

in

preprotein

translocase.

EMBO J.

19, 439)-4401.

Sec-Independent

Transtocation

Systems

in E. coli

Reviews

Dalbey, R. E. and

I(uhn, A.

(2000).

Evolutionarily

related insertion

pathways of bacterial,

mito-

chondrial, and thylakoid

membrane

proteins.

Annu Rev. Cell Dev.

Biol. 16, 5l-87.

Dalbey,

R.

E

and

Robinson, C.

(1999).

Protein

translocation

into and across

the bacterial

plasma

membrane

and the

plant

thylakoid

membrane. Trends

Biochem. Sci.24,

17-22.

Resea rch

Beck, I(., Wu, L. F.,

Brunner, J.,

and Muller,

M.

(2000).

Discrimination

betwe

en SRP- and

SecA/SecB-dependent

substrates

involves

selective

recognition

of nascent

chains by SRP

and trigger

factor.

EMBO J.

19, 134-143.

Samuelson, J. C.,

Chen,

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Wiedmann,

M., I(uhn,

A., Phillips, G.

J., and

Dalbey, R. E.

(2000).

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mediates

mem-

brane

protein

insertion

in bacteria.

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406,6)7-641.

Scotti,

P A.,

Urbanus,

M. L., Brunner,

J., de Gier.

J. W., von

Heijne, G., van

der

Does, C.,

Driessen, A. J., Oudega,

B., and Luirink,

J.

(2000).

YidC, the

E. colihomologue

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chondrial Oxalp,

is

a

component

of the Sec

translocase.

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19,542-549.

Soekarjo,

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M.,

I(uhn, A., and

Vogel,

H.

(19961

.

Thermodynamics

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insertion

process

of the

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pro-

coat

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a

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35, 12)2-1241.

References

255

Transcription

CHAPTER

OUTLINE

Introduction

Transcription

Occurs

by Base Pairing in

a

"Bubb[e"

of Unoaired

DNA

r

RNA

potymerase

separates the

two strands of DNA in

a tran-

sient

"bubb[e"

and

uses one strand

as a template

to direct

synthesis

of

a complementary

sequence

of RNA.

.

The

length

of the

bubbte is

-i.2

to

14

bp, and

the length

of RNA-DNA hybrid

within

it is

-8

to 9 bp.

The Transcription

Reaction

Has Three

Stages

o

RNA

potymerase

initjates

transcription

after binding

to a

promoter

site

on DNA.

r

During

etongation

the

transcription

bubbte moves

atong

DNA

and the

RNA chain is

extended in

the 5'-3'direction.

r

When

transcription

stops,

the DNA

duptex

reforms

and

RNA

polymerase

dissociates

at a terminator

site.

Phage

T7

RNA Potymerase

Is

a Useful Model

System

o

T3

and T7

phage

RNA

polymerases

are singte

potypeptides

with minimal

activjties in recognizing

a sma[[ number

of

phage promoters.

o

Crysta[

structures

of T7

RNA

potymerase

with DNA identifo

the DNA-binding

region

and the active

site.

llt|

A Modet

for

Enzyme

Movement

Is

Suggested by

the

CrystaI

Structure

e

DNA moves

through

a

groove

in

yeast

RNA

potymerase

that

makes

a

sharp turn

at the

active site.

.

A

protein

bridge

changes

conformation

to control

the entry

of

nucleotides

to

the active

site.

Bacterial

RNA Potymerase

Consists

of Muttipte

Subunits

r

Bacterial

RNA

core

potymerases

are

-500

kD muttisubunit

comptexes

with

the

generaI

structure

o2Bp'.

.

DNA is

bound in

a channel

and is

contacted bv

both the

B

and

p'subunits.

RNA Potymerase

Consists

of

the Core Enzyme

and Sigma

Factor

r

BacteriaI

RNA

potymerase

can

be divjded into

the a2Bp'

core

enzyme

that catatyzes

transcription

and

the sigma

subunjt

that is required

onty

for

initiation.

r

Sigma factor

changes

the DNA-binding properties

of RNA

potymerase

so that its

affinity for

general

DNA is reduced

and its

affinity

for

promoters

is increased.

r

Binding constants of RNA

polymerase

for

different

promot-

ers vary over six orders

of

magnitude.

corresponding

to the

frequency

with which transcription is initiated

at

each

0romoter.

The

Association with

Sigma

Factor

Changes

at

Initiation

.

When RNA

potymerase

binds to a

promoter,

it

separates

the

DNA

strands to

form

a transcription bubble

and incorporates

up to nine nucleotides into

RNA.

.

There may

be a cycte of abortive initiations

before

the

enzyme

moves

to the next

phase.

r

Sigma factor may

be

released

from RNA

potymerase

when

the nascent

RNA chain reaches eiqht

to

nine

bases in

tength.

A

Stalted RNA Potymerase

Can Restart

r

An arrested RNA

potymerase

can restaft transcription

by

cleaving the RNA

transcript to

generate

a new

3'end.

How

Does RNA Potymerase Find

Promoter

Sequences?

o

The rate

at

which

RNA

potymerase

binds

to

promoters

is

too

fast to be accounted for

by random

diffusion.

o

RNA

polymerase

probabty

binds

to

random

sjtes

on

DNA

and

exchanges

them with other sequences very

rapidty

untiI a

Dromoter

is found.

Sigma Factor

Controls Binding

to

DNA

.

A

change

in

association

between sigma factor

and hotoen-

zyme changes

binding affinity for DNA

so that core

enzyme

can move atong DNA.

Promoter

Recognition Depends

on Consensus

Sequences

.

A

promoter

is

defined by the

presence

of short

consensus

sequences at specific

locations.

o

The

promoter

consensus sequences

consist of a

purine

at

the startpoint, the hexamer

TATAAT

centered at

-L0,

and

another hexamer

centered

at

-35.

.

IndividuaI

promoters

usuatly differ from

the consensus

at

one or more

positions.

Promoter Efficiencies

Can

Be Increased

or Decreased

by

Mutation

o

Down

mutations

to decrease

promoter

efficiency

usuatly

decrease conformance

to the consensus

sequences,

whereas

up mutations

have

the opposite effect.

o

Mutatjons

in

the

-35

sequence usually

affect initial

binding

of RNA

polymerase.

tlg

lltn

lttr,

256

SltEr

ll:A

ETEI

o

Mutations in

the

-10

sequence

usualty

affect

the metting

reaction

that

converts a

ctosed to an

open complex.

RNA Polymerase

Binds

to One Face

Of DNA

o

The

consensus

sequences

at

-35

and

-10

provide

most

of the

contact

points

for

RNA

potymerase

in

the

promoter.

r

The

points

of

contact lie

on one face

of

the DNA.

Supercoiting

Is

an

Important

Feature

of

Transcription

e

Negative

supercoiting

increases

the effi-

ciency

of some

promoters

by assisting

the

melting reaction.

.

Transcription generates

positive

supercoils

ahead of

the enzyme

and negative

super-

coits

behind

it,

and

these must

be

removed

by

gyrase

and topoisomerase.

Substitution

of Sigma Factors

May

ControI Initiation

t

E.

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.

.

070

js

used for

generaI

transcription,

and

the other

sigma factors

are activated

by

speciaI

conditions.

Sigma Factors

Directty

Contact DNA

.

o70 changes its

structure

to retease its

DNA-binding

regions

when

it associates

with

core enzyme.

.

o7o binds

both the

-35

and

-i.0

sequences.

Sigma Factors May

Be

0rganized into

Cascades

o

A

cascade of

sigma factors is

created when

one

sigma factor is required

to transcribe

the

gene

coding for

the next

sigma

factor.

o

The

early

genes

of

phage

SP01

are tran-

scribed

by host

RNA

potymerase.

r

One of the

early

genes

codes for a sigma

factor

that

causes RNA

polymerase

to

transcribe

the midd[e

genes.

o

Two

of the middte

genes

code

for

subunits

of a sigma factor

that causes

RNA

poly-

merase

to transcribe

the

late

genes.

Sporulation Is

Controtted

by Sigma

Factors

.

Sporulation

divides

a bacterium into

a

mother

ce[[ that is tysed

and a spore

that

is

released.

r

Each comoartment advances to the

new sigma factor that displaces the

previ-

ous sigma factor.

o

Communication between

the two compart-

ments

coordinates the timing

of sigma

factor

substitutions.

BacteriaI

RNA

Potymerase Terminates at

Discrete

Sites

o

Termination may require both recognition

of the terminator

sequence

in

DNA and the

formation

of a

hairoin structure in the

RNA oroduct.

There Are Two Types of

Terminators in

E.

coli

r

Intrinsic terminators consist of a G-C-rich

hairpin in the RNA

product

followed

by a

U-rich region

jn

which termination occurs.

How Does Rho

Factor

Work?

o

Rho

factor is a terminator

orotein

that

binds to a

/uf site on nascent RNA and

tracks along

the RNA to

retease it from

the

RNA-DNA hybrid structure at the

RNA

poLymerase.

Antitermination Is a Regutatory

Event

o

Termination is

prevented

when antitermi-

nation

proteins

act on RNA

polymerase

to

cause

it

to

read through a specific termi-

nator

or terminators.

r

Phage [ambda has two antitermination

proteins, pN

and

pQ.

that act on different

transcription units.

Antitermination

Reouires Sites

That Are

Indeoendent of the

Terminators

o

The

site

where an antiterminator

protein

acts is uostream of

the terminator site

in

the transcription unit.

.

The

location

of the antiterminator site

varies in different cases

and can be in the

promoter

or within

the transcription unit.

Termination and

AntiTermination

Factors

Interact with RNA

Potymerase

o

Severa[

bacterial

proteins

are required

for

lambda oN to

interact with RNA

poLymerase.

.

These

proteins

are atso

involved in

antitermination

in the

rn

operons

of the

host

bacterium.

o

The tambda antiterminator

pQ

has

a

differ-

ent mode of

interaction that

invotves

binding to

DNA at the

promoter.

Summarv

CHAPTER

11

Transcription 257

Coding

strand

Template strand

RNA

sequence

is

TRANSCRIPTION

comptementarytotemplatestrand

identical to coding strand

RNA transcript

-35-10-1+1

+10

i ii:j*i' i :.:

The function of RNA

polymerase

is to copy one strand of duptex

DNA

jnto

RNA.

i:i.iitti

:

i

-:

A transcription unjt is a sequence of DNA

transcribed

into

a sing[e RNA, starting at the

promoter

and

endino at the terminator.

@

Introduction

Tfanscription involves

synthesis of an

RNA

chain

representing

one strand of a DNA duplex. When

we say

"representing,"

we mean that the RNA

is

identical in sequence with

one strand

of the

DNA, which is

called the coding strand.It

is

clmplementary to tlrre

other strand.

which

pro-

vides the

template strand for its synthesis.

i::+:-liii: : i. i

recapitulates

the relationship

between

double-stranded DNA and

its

single-

stranded

RNA transcript.

RNA synthesis is

catalyzed by the enzyme

RNA

polymerase.

Transcription starts when

RNA

polymerase

binds to

a special

region,

the

promoter,

at the start

of the

gene.

The

pro-

moter

surrounds the first

base

pair

that

is

tran-

scribed into RNA,

the startpoint. From this

point,

RNA

polymerase

moves

along the tem-

plate,

synthesizing RNA, until it reaches

a ter-

minator

(/)

sequence. This action

defines

a

transcription

unit that extends from the

pro-

CHAPTER 11 Transcriotion

moter to the terminator.

The

critical

feature of

the transcription

unit, depicted in

Fi{ili*?H I i..ll,

is

that

it

constitutes

a stretch of

DNA

expressed

vi a the

p

ro du ction of a sing

le RN A

mo

le cule. A tr an-

scription unit

may include

more

than one

gene.

Sequences

prior

to the startpoint are

described as upstream

of it; those after the

startpoint

(within

the transcribed sequence) are

downstream of

it. Sequences are convention-

ally written

so that transcription

proceeds

from

left

(upstream)

to right

(downstream).

This

corresponds to writing

the mRNA in the usual

5'

-+

3' direction.

The DNA sequence

often is

written to

show

only

the coding strand, which has the same

sequence as the

RNA. Base

positions

are num-

bered

in

both directions

away from the start-

point,

which

is

assigned

the value +l; numbers

increase as they

go

downstream. The base before

the startpoint

is numbered

-1,

and the nega-

tive numbers increase

going

upstream.

(There

is no

base

assigned the number 0.)

The immediate

product

of transcription

is

called the

prirnary

transcript. It

consists of

an RNA extending

from

the

promoter

to the

terminator

and

possesses

the original 5' and 3'

ends. The

primary

transcript is, however, almost

always unstable.

In

prokaryotes,

it is rapidly

degraded

(nRNA)

or

cleaved to

give

mature

products (rRNA

and

tnNR;. In

eukaryotes,

it is

modified at the ends

(mRNA)

and/or cleaved

to

give

mature

products (all

RNA).

Tfanscription is the first stage in

gene

expres-

sion and the

principal

step

at

which it is con-

trolled.

Regulatory

proteins

determine whether

a

particular gene

is

available to be transcribed

by RNA

polymerase.

The initial

(and

often the

only) step

in regulation is

the decision on

whether or not to transcribe a

gene.

Most reg-

ulatory events occur at the initiation of tran-

scription, although subsequent

stages

in

258

transcription

(or

other

stages

of

gene

expres-

sion) are

sometimes

regulated.

Within this

context,

there

are

two basic

questions

in

gene

expression:

.

How

does

RNA

polymerase

find

pro-

moters

on DNA?

This

is a

particular

example

of a more general

question:

How

do

proteins

distinguish

their spe-

cific binding

sites in

DNA

from other

sequences?

.

How

do regulatory proteins

interact

with

RNA

polyrnerase

(and

with one another)

to activate

or to repress

specific steps in

the initiation,

elongation,

or termina-

tion of transcription?

In this chapter,

we

analyze

the interactions

of bacterial

RNA

polymerase

with DNA

from

its

initial contact

with a

gene,

through

the

act

of transcription,

and

then finally

its release

when

the transcript has

been

completed.

Chapter 12,

The Operon,

describes

the

various

means

by

which regulatory proteins

can

assist

or

prevent

bacterial

RNA

polymerase

from recognizing

a

particular

gene

for

transcription.

Chapter 13,

Regulatory

RNA,

discusses

other

means of reg-

ulation. including

the

use of

small RNAs,

and

considers

how these interactions

can

be con-

nected

into larger

regulatory

networks.

In Chap-

ter 14, Phage

Strategies,

we consider

how

individual

regulatory

interactions

can be con-

nected into

more complex

networks.

In

Chap-

ter

24,

Promoters

and Enhancers,

and

Chapter

25,

Activating Transcription,

we

consider the

analogous

reactions

between

eukaryotic RNA

polymerases

and

their templates.

DNA is melted

RNA chain

is

extended

t;i"i;r,iil:ri:

j

i..i

DNA

strands separate

to

form

a transcription

bubbte.

RNA

is

synthesized by

comp[ementary base

pairing

with

one of the

DNA

strands.

into its single strands and the

template strand

is used to

direct synthesis

of the

RNA strand.

The RNA chain is synthesized

from the 5'

end toward the 3'end. The 3'-OH

group

of the

last nucleotide

added

to the chain

reacts

with

an incoming nucleoside

5'triphosphate.

The

incoming nucleotide loses

its terminal two

phos-

phate groups (y

and

B);

its a

group

is used in

the

phosphodiester

bond

linking it to the chain.

The

overall

reaction rate is

-40

nucleotides/sec-

ond at 37" C

(for

the bacterial

RNA

polymerase);

this is

about the same

as the

rate

of

translation

(i

5

amino acids/sec), but

much slower

than the

rate

of DNA replication

(800

bp/sec).

RNA

polymerase

creates

the transcription

bubble

when

it binds to a

promoter. Ijt{.it-ll't{ 1 i

"q

shows that as

RNA

polymerase moves along the

DNA, the bubble moves

with

it and

the RNA

chain

grows

longer.

The

process

of base

pairing

and

base addition

within the

bubble is catalyzed

and scrutinized by the

enzyme.

The structure of the

bubble within

RNA

polymerase

is shown

in the expanded

view of

F.{i;l-Jfii;

l.j.l,. As RNA

polymerase moves along the

DNA template,

it

unwinds

the duplex

at the

11.2 Transcription Occurs

by Base

Pairing in a

"Bubbte"

of Unpaired

DNA

Transcription

0ccurs

by

Base

Pairing

in

a

"BubbLe"

of

Unpaired DNA

o

RNA

polymerase

separates the

two strands

of

DNA

in

a transient

"bubbte"

and uses

one strand as a

temptate to direct synthesis

of a

comptementary

sequence of RNA.

o

The length of the

bubble is

-12

to 14 bp,

and the

length of RNA-DNA

hybrid within it is

-8

to

9 bp.

Ttanscription

takes

place

by the

usual

process

of complementary

base

pairing.

iri*Li$*

t1".i illus-

trates the

general

principle

of transcription.

RNA

synthesis takes

place

within

a

"transcription

bubble," in which DNA

is transiently

separated

259

with RNA at any

given

moment. Certainly the

RNA-DNA

hybrid is short and transient.

As the

enzyme moves on,

the DNA duplex reforms,

and the

RNA is displaced as a

free

polynucleotide

chain.

Roughly the last twenty-five

ribonu-

cleotides added to

a

growing

chain are com-

plexed

with DNA and/or

enzyme at any

moment.

i!ai-iiii i

i.i

Transcription

takes

p[ace

in

a bubbl.e,

in which

RNA is synthesized

by base

pairing

with

one

strand of DNA

in

the transiently

unwound

region. As

the bubbte

progresses,

the DNA duptex reforms

behind

it,

displacing

the RNA in the

form of a singte

potynucteotide

chain.

l-i::iirii

i,:.: During transcription,

the bubbl.e

is main-

tained within bacterjal RNA

potymerase,

which

unwinds

and

rewinds

DNA and

synthesizes

RNA.

front

of the bubble

(the

unwinding

point),

and

rewinds

the DNA at the back

(the

rewinding

point).

The length

of the transcription bubble

is

-12

to

l4bp,

but the length of the RNA-DNA

hybrid region

within it is shorter.

There is

a

major

change in the topology of

DNA

extending over

-l

turn,

but

it is not

clear

how much

of this region is actually

base

paired

The Transcription

Reaction

Has Three

Stages

o

RNA

potymerase

initiates transcription after

binding to a

promoter

site on DNA.

r

During etongation the transcription bubble

moves

aLong DNA and the

RNA

chain

is

extended

in the

5'-3'direction.

o

When transcription

stops, the DNA duplex reforms

and

RNA

polymerase

dissociates at a terminator

site.

The transcription

reaction

can be divided

into

the stages

illustrated in

ri$tlftl:

1 :i

"{-i,

in which a

bubble

is created, RNA synthesis begins, the

bubble

moves along the DNA, and finally the

bubble

is

terminated:

.

Template

recognition begins with the bind-

ing of RNA

polymerase

to the double-

stranded DNA at a

promoter

to form a

"closed

complex."

The strands

of

DNA

are then separated to

form

the

"open

complex" thatmakes the template strand

available

for

base

pairing

with

ribonu-

cleotides.

The transcription

bubble

is

created by a

local

unwinding that begins

at the site bound by RNA

polymerase.

.

Initiation describes the synthesis of the

first nucleotide bonds in RNA. The

enzyme

remains at the

promoter

while

it synthesizes the first

-9

nucleotide

bonds.

The initiation

phase

is

protracted

by the occurrence of abortive events, in

which the enzyme makes short tran-

scripts, releases them, and then starts

synthesis of RNA again. The initiation

phase

ends when the enzyme succeeds

in

extending the chain and clears the

promoter.

The sequence of DNA needed

for

RNApolymerase to bind to the template

and

accomplish the initiation reaction defines the

prlmlter.

Abortive initiation

probably

involves synthesizing an RNA

chain that

RNA binding site

CHAPTER lL Transcription

Lewin Benjamin (ed.) Genes IX

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