Lewin Benjamin (ed.) Genes IX

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30S-mBNA

comolex

tF_3

tF_1

lF2-GTP

joins

complex

GTP

Initiator

tRNA

joins

50S subunit

joins

and lF1-3

are released

tr.r

tF-1 tF-2

tF-3

cDP Pi

\r^\

r\.r'

Add nuclease

to digest all

unprotected

mRNA

lsolate

fragment

protected

mRNA

Determine

Shine-Dalgarno

0 bases

uostream of AUG

L----l--------l

Coding

region

::=i.;;ii.

.'i.::

IF-2

is needed

to bjnd fMet-tRNAlto

the 30S-

mRNA

complex. After

505 binding.

a[[ IF

fuctors are reteased

and GTP is cleaved.

l.i;iirr

ii. : ir Ribosome-bindjng sites on mRNA can be

recovered from initiation

complexes.

They include the

upstream Shine-Datgarno sequence and the

initiation codon.

The initiation sequences

protected

by bac-

terial ribosomes

are

-10

bases long. The ribo-

some-binding sites of different bacterial

mRNAs

display two common features:

The AUG

(or

less often, GUG or UUG)

initiation codon

is always included

within the

protected

sequence.

Within ten bases upstream

of the AUG

is a sequence that corresponds

part

or all of the

hexamer.

5, ,AGGAGG

.3,

This

polypurine

stretch

is known as the

Shine-Dalgarno sequence.

It is complemen-

tary to a highly conserved sequence

close to the

3'end

165 rRNA.

(The

extent of complemen-

tarity

differs with

individual mRNAs, and

may

extend

from a four-base

core sequence GAGG

nine-base

sequence extending

beyond each

end of the hexamer.

)

Written

in reverse direc-

tion,

the

rRNA

sequence

is the

hexamer:

3, .UCCUCC.

.5,

Does the Shine-Dalgarno

sequence

pair

with its

complement

rRNA during mRNA-

ribosome

binding? Mutations

of both

partners

in this reaction

demonstrate

its importance in

initiation.

Point mutations

in the Shine-

Dalgarno sequence can

prevent

an nRNA

from

Initiation

Involves

Base

Pairing

Between

mRNA

and

rRNA

An initiation

site on

bacterial mRNA

consists

the AUG initiation

codon

preceded

with

gap

-10

bases by the

Shine-Datgarno

potypurine

nexamer,

The rRNA

the 30S bacterial ribosomal

subunit

has a

complementary

sequence that

base

pairs

with the

Shine-Datgarno

sequence

during

initiation.

An nRNA

contains

many AUG

triplets:

How is

the initiation codon

recognized

providing

the

starting

point

for

translation?

The

sites on mRNA

where

protein

synthesis is

initiated

can be iden-

tified

by binding the ribosome

to mRNA

under

conditions

that block elongation.

Then

the ribo-

some remains

at the

initiation

site. When

ribonuclease

is added

to the

blocked initiation

complex, all

the regions

of mRNA

outside the

ribosome

are degraded.Those

actually

bound

to it are

protected,

though,

as illustrated

i:l.i.i*,tii:

i:]..i i.. The

protected

fragments

can

recovered and

characterized.

8.7

Initiation Involves

Base Pairing Between

mRNA and rRNA 16l

being translated.

addition,

the introduction

of mutations into the complementary

sequence

in rRNA is

deleterious

to the cell and changes

the

pattern

protein

synthesis.

The decisive

confirmation of the base-pairing

reaction

that

a mutation in the Shine-Dalgarno

sequence of

an nRNA can be suppressed by a

mutation in

the

rRNA

that

restores base

pairing.

The

sequence

at the 3'end of

rRNA is con-

served between

prokaryotes

and eukaryotes,

except that in all eukaryotes there

is a deletion

of the

five-base

sequence

CCUCC that

is the

principal

complement to

the

Shine-Dalgarno

sequence. There does not appear to be base

pair-

ing

between eukaryotic

mRNA and 18S rRNA.

This is a significant difference

the

mechanism

of initiation.

bacteria,

a l0S subunit binds directly

ribosome-binding

site.

As a result, the initia-

tion complex forms at a sequence surrounding

the

AUG initiation

codon.

When the mRNA

polycistronic,

each coding region starts

with

ribosome-binding site.

The nature

of bacterial

gene

expression

means that translation of a bacterial

nRNA

pro-

ceeds

sequentially

through its cistrons.

the

time when

ribosomes

attach to the

first coding

region, the subsequent coding regions

have not

yet

even been transcribed. By the time the sec-

ond ribosome site is available, translation is well

under way through the first cistron.

What happens between the coding

regions

depends on the individual mRNA. In most cases,

the ribosomes

probably

bind

independently at

the beginning of each cistron.

The most com-

mon

series of events

illustrated in

i;:,ij;ii

i:,

.'.

When

synthesis of the

first

protein

terminates,

the ribosomes leave

the

mRNA

and dissociate

into subunits. Then a new ribosome must assem-

:'l:..,i:rii

r,,

I .: Initiation

occurs

independentty

at each

cistron

polycistronic

mRNA. When the intercjstronic

region is

[onger than the span of the

ribosome.

dissocia-

tjon

at the termjnation site is foltowed by independent

reinitiation

at the

cistron.

CHAPTER

Protein

Svnthesis

ble at the

next coding

region and set out to trans-

Iate the

next cistron.

In some bacterial

mRNAs, translation

between

adjacent cistrons

directly

linked,

because

ribosomes

gain

access to the

initiation

codon of the second

cistron as they complete

translation of the

first cistron. This effect requires

the space

between the two

coding regions to be

small.

It may depend

on the high local density

of ribosomes, or

the

juxtaposition

of termina-

tion and initiation sites

could allow some of the

usual

intercistronic events to be bypassed.

ribosome

physically

spans

-30

bases of

nRNA.

that it could simultaneously

contact a termi-

nation codon and

the next initiation site if thev

are separated

by only a

few bases.

SmaL[ Subunits Scan

for Initiation Sites

Eukaryotic

mRNA

Eukaryotic 40S

ribosomal subunits bind to the 5'

end of mRNA and scan

the mRNA untiI they reach

an initiation site.

eukaryotic

initiation site consists of a ten-

nucteotide sequence that

includes

AUG

codon.

605

ribosomal subunits

join

the complex at the

initiation site.

Initiation of

protein

synthesis in eukaryotic

cytoplasm

resembles the

process

in bacteria,

but the order

of events is different and the num-

ber of accessory

factors is

greater.

Some of the

differences in initiation are

to a differ-

ence in the way that

bacterial 30S and eukary-

otic 40S subunits

find

their binding sites for

initiating

protein

synthesis on nRNA. In

eukaryotes, small subunits

first recognize

the

5'end of the

mRNA and then move to the ini-

tiation site, where

they are

joined

large

sub-

units.

(In prokaryotes,

small subunits bind

directly to the

initiation site.)

Virtually all eukaryotic mRNAs are mono-

cistronic, but

each nRNA usually is

substan-

tially

longer

than

necessary

just

to code

for

its

protein.

The average

mRNA in

eukaryotic cyto-

plasm

is 1000 to 2000 bases long, has a meth-

ylated

cap at the 5'terminus, and carries I00 to

200 bases of

poly(A)

at the 3'terminus.

The nontranslated 5'leader is relatively

short, usually

<I00

bases. The length of the cod-

ing region is

determined

by the size

of the

pro-

tein. The nontranslated 3'trailer is

often

rather

long,

at times

reaching lengths

of up to

-1000

bases.

The

first feature

be recognized

during

translation

eukaryotic

nRNA

the meth-

ylated

cap

that marks

the 5'

end.

Messengers

whose

caps have

been

removed

are not

trans-

lated efficiently

invitro.

Binding

of 40S subunits

mRNA requires

several initiation

factors,

including

proteins

that recognize

the structure

of the cap.

Modification

the 5'end

occurs

to almost

all

cellular

viral

mRNAs

and

is essential

for

their translation

eukaryotic

cytoplasm

(although

it is

not needed

organelles).

The

sole exception

to this

rule is

provided

by a few

viral mRNAs

(such

poliovirus)

that are not

capped;

only these

exceptional

viral mRNAs

can be translated

in vitro

without

caps. They

use

an alternative pathway

that bypasses

the need

for the

cap.

Some viruses

take

advantage

of this

differ-

ence. Poliovirus

infection

inhibits

the transla-

tion

hosr

mRNAs.

This is

accomplished

interfering

with

the cap-binding proteins

rhat

are needed for

initiation

cellular mRNAs,

but

that

are superfluous

for

the

noncapped

poliovirus

nRNA.

We have

dealt with

the

process

of initiation

as though

the ribosome-binding

site is always

freely

available.

However,

its availability

may

be impeded

by secondary

structure. The

recog-

nition of nRNA

requires

several

additional fac-

tors; an important part

of their

function is

remove

any secondary

structure

the nRNA

(see

Figure

8.20).

Sometimes the

AUG initiation

codon lies

within 40

bases of the 5'terminus

of the nRNA,

so that both the

cap and AUG

lie

within the

span of ribosome

binding.

In many

mRNAs,

however, the

cap and AUG

are farther

apart-

extreme cases, they

can

be as much

1000

bases away from

each

other. Yet the

presence

of the

cap still is necessary

for

a stable complex

to be formed

at the initiation

codon.

How can

the ribosome

rely on two

sites

so far apart?

which

supposes that

the 40S

subunit initially

recognizes

the 5'cap

and then

"migrates"

along

the mRNA. Scanning

from

the 5'end is

lin-

ear

process.

When 40S

subunits

scan the leader

region, they

can melt secondary

structure

hair-

pins

with stabilities <-30

kcal,

but hairpins of

greater

stability impede

prevent

migration.

Migration

stops when

the 40S

subunit

encounters the AUG initiation

codon. Usually,

although

not always,

the first

AUG triplet

sequence to be encountered

will be the initia-

tion

codon. However.

the AUG triplet

by itself

is not sufficient

to halt migration;

it is recog-

nized

efficiently as an

initiation

codon

only

when it

is in the right context.

The most impor-

tant determinants of context are the bases in

positions

--4

and

+1. An initiation codon may be

recognized

in the sequence NNNPuNN,AUGG.

The

purine

or G) 3 bases

before the AUG

codon, and the G immediately

following it.

can

influence

the efficiency

of translation by l0x.

When

the leader sequence

is long, further 40S

subunits can recognize t};^e 5' end before the

first has left

the

initiation

site,

creating a

queue

of subunits

proceeding

along the

leader

the

initiation

site.

It is

probably

true that

the initiation codon

is the

first AUG to be encountered

in the most

efficiently translated mRNAs. What

happens,

though,

when there

is an AUG triplet in the

5'nontranslated region? There are two

pos-

sible

escape mechanisms

for a ribosome that

starts scanning at the 5'end.

The most com-

mon is that

scanning

is leaky, that

is, a ribo-

some may

continue

past

a noninitiation

AUG

because it is not in the

right

context.

In the

rare case

that

does

recognize Lhe

AUG, it

may initiate

translation

but terminate before

the

proper

initiation codon,

after which

resumes

scanning.

The

vast

majority of eukaryotic

initiation

events involve

scanning

from the 5' cap, but

Eukaryotic

ribosomes

migrate from the 5'

end of

mRNA

to the

ribosome bindinq site,

which

inctudes

AUG initiation

codon.

8.8 smatl Subunits

scan

for Initiation Sites

on Eukarvotic

mRNA 163

there is an alternative means of initiation,

used

especially by certain

viral RNAs, in which a 40S

subunit associates directly with an internal site

called an

IRES.

(This

entirely bypasses any

AUG

codons that

may

in the 5' nontranslated

region.) There are

few

sequence

homologies

between

known IRES elements. We can distin-

guish

three types on the basis of their

interac-

tion with the 40S subunit:

One type of IRES

includes

the

AUG ini-

tiation

codon

at its upstream boundary.

The 40S subunit binds directly

to it,

using a subset

of the same factors that

are required for

initiation

5'ends.

Another is located as much as

I00

nucleotides upstream of the AUG,

requiring

40S

subunit to

migrate, again

probably

by a scanning mechanism.

An exceptional type of

IRES in hepati-

tis C

virus

can bind a 40S subunit

directly.

without requiring any initia-

tion

factors. The

order

of events is

different from all other eukaryotic

ini-

tiation. Following 40S-mRNA binding,

a complex containing

initiator factors

and the initiator IRNA binds.

Use

the IRES is especially important in

picornavirus

infection,

where

was

first dis-

covered, because the

virus

inhibits host

protein

synthesis by

destroying

cap

structures

and

inhibiting

the initiation

factors

that bind them

(see

Section 8.9, Eukaryotes Use a Complex of

Many Initiation Factors).

Binding is stabilized at the initiation site.

When

the

40S

subunit

joined

by a 605 sub-

unit, the intact ribosome is located at the site

identified

by the

protection

assay. A 40S subunit

protects

a region of up to 60 bases; when the 605

subunits

join

the complex, the

protected

region

contracts to about the same length of 30 to 40

bases seen in

prokaryotes.

Eukaryotes

Use a Complex

Many Initiation Factors

In'itiation factors

are

required

for a[[ stages of

initiation, inctuding

binding the

initiator

IRNA.

40S

subunit attachment to mRNA, movement

along the mRNA,

and

joining

of the 605 subunit.

Eukaryotic initiator

IRNA

Met-tRNA

that

different from the Met-tRNA

used

elongation,

but the methionine is not formutated.

eIF2 binds

the

initiator Met-tRNA1

and GTP, and

the comptex binds to the 40S subunit

before

associates with mRNA.

Protein

Synthesis

Initiation

in eukaryotes

has the same

general

features as in bacteria

in using a specific

initia-

tion codon

and initiator IRNA.

Initiation in

eukaryotic cytoplasm

uses

AUG

as the

initiator.

The initiator 1RNA

is a distinct species, but

its

methionine does not become

formylated. It is

called tllNAiM.t.

Thus the difference between

the

initiating and elongating

Met-tRNAs lies solely

in the

IRNA moiety, with

Met-tRNAi used for

initiation

and Met-tRNA. used

for

elongation.

At least two

features are unique to the

ini-

tiator IRNA

Met

yeast:

it has an unusual

rerriary structure, and

it is modified by

phos-

phorylation

the 2'ribose

position

on base

(if

this

modification is

prevented,

the initiator

can be used in elongation).

Thus the

principle

of a distinction

between

initiator

and elongator

Met-tRNAs

is maintained in eukaryotes, but

its

structural basis

is different

from

that

bacte-

ria

(for

comparison see

Figure 8.13).

Eukaryotic cells have

more initiation fac-

tors than

bacteria-the current

list includes l2

factors that are directly

or indirectly required

for initiation. The factors are

named

similarly

those

bacteria,

sometimes by analogy with

the bacterial

factors, and are

given

the

prefix

"e"

indicate their eukaryotic origin. They act

at all stages of the

process,

including:

forming an initiation complex with the

5'end

nRNA;

forming a complex with

Met-tRNAi;

binding the mRNA-factor complex to

the Met-IRNA1-factor complex;

enabling

the ribosome to scan mRNA

from the 5'end to the

first AUG;

detecting binding of initiator IRNA to

AUG

at the

start site; and

mediating

joining

of the 605 subunit.

i:l{,.ili;i:

i:..: rl summarizes the stages of initi-

ation and shows

which

initiation factors are

involved at each stage: eIF2 and eIF3 bind to

the 40S ribosome subunit; eIF4A, eIF4B, and

eIF4F bind to the

mRNA;

and eIFl and eIFIA

bind to the ribosome subunit-mRNA complex.

i'.ii.:il*i: :;"i:i:

shows the

group

of factors that

bind to the 5'end of mRNA. The factor eIF4F

is a

protein

complex that contains three

of the

initiation factors. It is not clear whether it

pre-

assembles as a complex before binding to mRNA

or whether the individual subunits are added

individually to form the complex

mRNA.

includes the cap-binding subunit

eIF4E, the

helicase

eIF4A,

and the

"scaffolding"

subunit

eIF4G. Aiter eIF4E binds the cap, eIF4A unwinds

any secondary structure that exists in the first

bases of the mRNA. Energy for the unwind-

ing is

provided

hydrolysis

ATP.

Unwind-

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dBVd

of eIF2. Phosphorylation

prevents

eIF2B

from

regenerating

the active

form.

This limits

the

action

of eIF2B to

one

cycle of initiation,

and

thereby inhibits protein

synthesis.

Etongation

Factor

Loads

Aminoacyl-tRNA

into

the A

Site

EF-Tu is a monomeric

protein

whose

active form

(bound

GTP) binds

aminoacy[-tRNA.

The EF-Tu-GTP-aminoacyt-tRNA

complex

binds to

the

ribosome

site.

Once the

complete ribosome

is formed

at the

initiation

codon,

the stage

is set for

a cycle in

which aminoacyl-tRNA

enters

the A

site of a

ribosome

whose P

site is

occupied

peptidyl-

IRNA. Any

aminoacyl-rRNA

excepr the initia-

tor can

enter the A

site. Its

entry is mediated

elongation factor

(EF-Tu

bacteria). The

process

is similar

in eukaryotes.

EF-Tu

is a highly

conserved

protein

throughout

bacteria

and

mitochondria

and is homologous

to its eukary-

otic counterpart.

Just like its

counterpart

in initiation

(IF-2),

EF-Tu is associated

with

the ribosome

only

dur-

ing the

process

of aminoacyl-tRNA

entry.

Once

the aminoacyl-tRNA

is in

place.

EF-Tu leaves

the ribosome,

to work

again

with another

aminoaryl-tRNA.

Thus it

displays

the cyclic asso-

ciation

with, and

dissociation

from, the ribo-

some that is the

hallmark

of the

accessory

factors.

The

pathway

for aminoacyl-tnXA

entry to

the A

site

is iilustrated

ii,,.,r;':, i: ..

. EF-Tu car-

ries

guanine

nucleotide.

The factor is

monomeric

protein

whose

activity is

con-

trolled by the

state of the

guanine

nucleotide:

When GTP is

present,

the factor is in its

actrve state.

When

the GTP is hydrolyzed

to GDp,

the factor becomes

inactive.

Activity is restored

when

the GDP is

replaced

GTP.

The binary

complex

EF-Tu-GTP

binds

aminoacyl-IRNA

to form

a ternary

complex

aminoacyl-tRNA-EF-Tu-

GTP. The ternary

com-

plex

binds

only to the A

site of ribosomes

whose

P site is

already occupied

peptidyl-rRNA.

This

the critical reaction

ensuring that

the

aminoacyl-IRNA

and

peptidyl-rRNA

are

cor-

rectly

positioned

for

peptide

bond

formation.

Aminoacyl-tRNA

is loaded

into the

A site

two stages. First, the

anticodon

end binds to

ir'irr

'r:

EF-Tu-GTP

ptaces

aminoacyltRNA on the

ribosome

and then

is reteased

as EF-Tu-GDP. EF-Ts

is required to mediate the replacement of

GDP by

GTP.

The reaction

consumes

GTP and reteases GDP. The onty aminoa-

cyltRNA

that cannot be

recognized

EF-Tu-GTP is fMet-tRNAr, whose fajt-

ure

to bind

prevents

it from

responding to internal AUG or GUG codons.

the A

site of the l0S subunit.

Then, codon-

anticodon recognition triggers a change

in the

conformation

of the

ribosome. This stabilizes

tRNA binding and causes

EF-Tu to hydrolyze

its

GTP.

The

CCA end

of the IRNA now moves

into

the A site on the

50S

subunit.

The binary

complex EF-Tu-GDP is released.

This form of

EF-Tu is inactive

and does

not bind aminoacyl-

IRNA effectively.

Another factor, EF-Ts, mediates the

regen-

eration of the used

form. EF-TIr-GDP. into the

active

form EF-Tu-GTP.

First, EF-Ts displaces

the GDP from EF-Tu, forming the

combined fac-

tor EF-Tu-EF-Ts. Then the EF-Ts is

turn dis-

placed

by GTP,

reforming EF-Tu-GTP.

The active

binary complex binds aminoacyl-tRNA,

and the

released EF-Ts

can

recycle.

There are

-70,000

molecules of

EF-Tu

per

bacterium

(-5%

of. the total bacterial

protein),

which

approaches the

number of aminoacyl-

IRNA molecules. This implies that

most aminoacyl-

tRNAs

are

likely to be

present

in ternary

complexes. There are only

-10,000

molecules

EF-Ts

per

cell

(about

the same

as the

number of

ribosomes). The kinetics of

the interaction

between EF-TU and EF-Ts suggest

that the EF-

Tu-EF-Ts

complex exists

only transiently, so

that

the EF-Tu is

very

rapidly converted

to the GTP-

bound form, and then

to a ternary complex.

aa-tRNA enters

A site on 30S

CCA end

moves

inlo A

site on 50S

Tu-GDP

8.10

Elongation

Factor Tu Loads

Aminoacyt-tRNA

into

the

Site

767

The role of GTP

the

ternary complex

has

been studied by substituting an analog

that can-

not

hydrolyzed. The compound GMP-PCP

has a methylene bridge

place

the oxygen

that

links

the

and

yphosphates

in GTP.

In the

presence

o{ GMP-PCP,

a ternary complex can be

formed that binds aminoacyl-tRNA

to the ribo-

some. The

peptide

bond cannot be

formed,

though. so the

presence

of GTP is needed

for

aminoacyl-IRNA to be bound

the

A site. The

hydrolysis is not required until later.

Kirromycin

antibiotic that inhibits

the function of

EF-Tu. When EF-Tir is bound by

kirromycin, it remains able to bind aminoacyl-

IRNA to the

site.

But the EF-Tu-GDP com-

plex

cannot be released

from

the

ribosome. Its

continued

presence prevents

formation of the

peptide

bond between the

peptidyl-tRNA

and

the aminoacyl-tRNA.

result,

the

ribosome

becomes

"stalled"

on mRNA, bringing

protein

synthesis to a halt.

This effect of kirromycin demonstrates

that

inhibiting

one step

protein

synthesis blocks

the

step.

The reason is that the continued

presence

of EF-Tu

prevents

the aminoacyl

end

of aminoacyl-IRNA from entering the A site on

the 50S subunit

(see

Figure

8.3I).

Thus the

release of EF-Tu-GDP is needed for the ribo-

some to undertake

peptide

bond

formation. The

same

principle

seen at other stages of

protein

${'*l"JSil

;$.I*

Peptide

bond

formation

takes

ptace

reac-

tion between the

potypeptide

peptidyt-tRNA

in the

site and the amino acid of aminoacvt-tRNA in

the

A site.

CHAPTER

Protein

Svnthesis

synthesis: one

reaction

must

be completed

prop-

erly before the

next can occur.

The interaction with

EF-Tu also

plays

role in

quality

control. Aminoacyl-tRNAs are

brought

into the

site without

knowing

whether

their

anticodons will

fit

the codon.

The hydrolysis

of EF-Tu-GTP is relatively slow:

it takes longer

than the time required for an

incorrect

aminoacyl-IRNA to dissociate

from

the A site, therefore

most incorrect species are

removed

at this stage.

The release

EF-

Tu-GDP after

hydrolysis also is slow, so any

surviving

incorrect

aminoacyl-tRNAs may dis-

sociate

at this stage.

The

basic

principle

is that

the reactions

involving EF-Tu occur slowly

enough

to allow incorrect aminoacyl-tRNAs

to dissociate before

they become trapped in

protein

synthesis.

In eukaryotes, the

factor

eEFlcx

is respon-

sible for bringing

aminoacyl-tRNA to the ribo-

some, again

in a reaction that

involves

cleavage

of a high-energy

bond in GTP. Like its

prokary-

otic

homolog

(EF-Tu),

it is

an abundant

pro-

tein.

After hydrolysis of GTP, the active form

regenerated by the factor eEFlBy, a counter-

part

to EF-Ts.

The

PoLypeptide

Chain

Is Transferred

Ami noacyL-tRNA

The 50S subunit

has

peptidyt

transferase activity.

The

nascent

poLypeptide

chain

transferred

from

peptidyltRNA

the

site to aminoacyt-tRNA

the A site.

Peptide bond synthesis

generates

deacytated IRNA

in the P site and

peptidyt-tRNA

in the A site.

The ribosome remains in

place

while the

polypeptide

chain

is elongated

by transferring

the

polypeptide

attached to the IRNA in the P

site

to the aminoacyl-tRNA in the A

site.

The

reaction is shown in

Fi*SftS

*.f b. The activity

responsible for synthesis of the

peptide

bond is

called

peptidyl

transferase.

The nature

the

transfer reaction is

revealed by the ability of the antibiotic

puromycin

inhibit

protein

synthesis.

Puromycin resembles an amino acid attached

the terminal adenosine of IRNA. l-:GtlR$ &.f F

shows that

puromycin

has

an N instead of the

O that

joins

an amino acid to IRNA. The

antibi-

otic is treated

by the

ribosome

as though

were

incoming

aminoacyl-tRNA, after which the

168

polypeptide

attached

pepridyl-rRNA

is rrans-

ferred

the NH2

group

of the

puromycin.

The

puromycin

moiety

is not

anchored

the A

site of the ribosome,

and

a result

the

pollpeptidyl-puromycin

adduct

released from

the ribosome

the

form

polypeptidyl-

puromycin.

This

premature

termination

pro-

tein synthesis

is responsible

for the

lethal

action

of the

antibiotic.

Peptidyl

transferase

a function

of the

large

(50S

or 605) ribosomal

subunit.

The

reacrion

is triggered

when

EF-Tu releases

the aminoacyl

end of its IRNA.

The

aminoacyl

end

then swings

into

location

close to

the end

of the

peptidyl-

IRNA. This

site has

peptidyl

transferase

activ-

ity that

essentially

ensures

a rapid

transfer

the

peptide

chain

to the

aminoacyl-rRNA.

Borh

rRNA

and 50S

subunit

proteins

are necessary

for

this

activity, but

the actual

act

of catalysis

is a

property

of the ribosomal

RNA

of the 50S

sub-

unit

(see

Section 8.19,235

rRNA

Has Peptidyl

Tlansferase

Activitv).

r:iili,ii:ii: i:;

,,.:.- Puromycin

mimics

aminoacy[-tRNA

because

resembles

an aromatic

amino acid

[inked to a

suqar-

base moiety.

Trans[ocation Moves

the Ribosome

Ribosomal transtocation moves the mRNA

through

the

ribosome

by three bases.

Translocation

moves deacytated IRNA

into

the

site and

peptidyt-tRNA

into the P site, and

empties

the

site.

The hybrid

state model

proposes

that transtocation

occurs in

two stages, in which the 50S moves

relative

to the 30S. and then the 30S moves along

mRNA

to restore

the

oriqinal conformation.

The

cycle

of addition of amino

acids to the

grow-

ing

polypeptide

chain

completed by translo-

cation,

when the ribosome advances three

nucleotides

along

the nRNA. l:ir,rtii;h i:::.;lE shows

that translocation

expels

the uncharged IRNA

from

the P site, so that the new

peptidyl-tRNA

can enter. The ribosome then has an empty A

site ready

for entry of the aminoacyl-tRNA cor-

responding

to the

next codon. As the figure

shows, in

bacteria the discharged

IRNA is trans-

ferred from

the

site to

the E site

(from

which

it is

then expelled into the cytoplasm).

eukaryotes it is

expelled

directly into the cytosol.

The A

and P sites straddle both

the large and

small subunits;

the

E site

(in

bacteria) is

located

largely

on the 50S subunit,

but has some con-

tacts in

the l0S subunit.

Most thinking about translocation

follows

the hybrid

state model, which

proposes

that

translocation occurs in two stages.

i::l.i:!-iiiL

ij.i:i.t

shows that first

there

is a shift of the 50S sub-

unit relative

to the 30S subunit,

followed by

second shift that occurs

when the 30S sub-

unit moves along nRNA to

restore the

origi-

nal conformation. The

basis

for this model was

the observation that the

pattern

of contacts

that IRNA

makes with

the ribosome

(meas-

ured

by chemical footprinting)

changes in two

stages. When

puromycin is added to a ribo-

some that has

aminoacylated IRNA

in the

P site, the

contacts of

IRNA on the 50S subunit

change from the P site to

the E site, but the

contacts on the l0S subunit do

not change.

This

suggests that the 50S subunit

has moved

to a

posttransfer

state,

but the 30S

subunit has

not changed.

The

interpretation of

these results

that

first the aminoacyl ends of the

tRNAs

(located

the 50S subunit) move

into the new sites

(while

the anticodon ends

remain bound to

their anticodons in the

30S subunit). At this

stage, the

tRNAs

are effectively

bound in

8.12

Translocation

Moves the Ribosome 169

'r.,

:, A

bacterial

ribosome has three tRNA-bind-

ing sites. AminoacyltRNA enters the A site of a ribosome

that has

peptidyt-tRNA

in the P site. Peptide bond syn-

thesis deacylates the P sjte tRNA and

generates peptidyt-

IRNA in the A site. Translocation moves the deacyl"ated IRNA

into

the E sjte and moves

peptidyt-tRNA

into

the

site.

hybrid

sites, consisting of the

50SE/30S P and

rhe 50SP/l0S A sites. Then movement is

:; :",x1".i :: rff :,il*l ilf ,,"i. li : :liil ?i:

right

site. The most likely means of creating the

hybrid

state is by a movement of one

riboso-

mal subunit relative to the other, so that

translocation in

effect involves two stages,

with the normal structure of the ribosome

being restored by the second stage.

The ribosome faces an interesting

dilemma

:: :lHlTi

li: i.

iiffi

t.'.".: l"J :,i: il L:'":

ment. At

the same time. however.

it must

;ffi

1T.::il'ff

,i"ffi i":Tt*T.'"T,:T;

CHAPTER

Protein

Svnthesis

ir:i1tii.ri:

i:,,,iir

Models fortranstocation

invotve

two stages.

First. at

peptide

bond

formation the aminoacyl end ofthe

IRNA

the

site

becomes

retocated in

the

site. Sec-

ond. the anticodon

end of the IRNA becomes

retocated in

the

site.

IRNA only

at the right moment). One

possibil-

ity is that the

ribosome switches between alter-

native, discrete

conformations. The switch

could

consist of changes

in rRNA

base

pairing.

The accuracy of

translation is influenced by

certain mutations

that influence alternative

base

pairing

arrangements. The most

likely

interpretation

is that the effect

is mediated

the tightness of

binding to IRNA of the alter-

native conlormalions.

Elongation

Factors

Bind Alternate[y

to the

Ribosome

Transtocation requires EF-G, whose structure

resem btes the aminoacy[-tRNA-EF-Tu-GTP com

ptex.

Binding

EF-Tu and EF-G to the ribosome is

mutualty exctusive.

Transtocation requires

GTP

hydrotysis, which

triggers a change in EF-G, which in turn triggers

a chanqe

in ribosome

structure.

170