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RNA-rich

FI6URI

S.36

The

30S subunit has

a

head

separated by a

neck from

the body.

with

a

protruding ptatform.

sites

exist

for

some

proteins.

As

each

protein

binds,

it induces

conformational changes in the

rRNA

that make it

possible

for other

proteins

to

bind. In E. coli, virtually all the 30S ribosomal

proteins

interact

(albeit

to varying degrees)

with

I65 rRNA. The binding sites

on the

proteins

show a wide variety

of structural features, sug-

gesting

that

protein-RNA

recognition

mecha-

nisms may

be diverse.

The 70S ribosome has

an asymmetric con-

struction.

FISIJftil

*,3& shows a

schematic of the

structure of the l0S

subunit, which is divided

into four regions:

the head, neck,

body, and

platform.

FIGUR{

*.3? shows a similar represen-

tation of the 50S

subunit, where two

promi-

nent

features

are the central

protuberance

(where

55 rRNA is located)

and the

stalk

(made

of multiple copies of

protein

L7). FI*Ufi{

s.3*

shows that the

platform

of the

small subunit

fits into the notch of the large

subunit. There is

a cavity

between the subunits that contains

some of the important

sites.

The

structure of the 30S

subunit follows

the organization of l65 rRNA,

with each struc-

tural feature

corresponding to a domain

of the

rRNA. The

body is based on the 5'domain,

the

platform

on the central

domain, and the head

on the

3'region.

F:fitJRC

S.3$ shows

that the 30S

subunit has an asymmetrical

distribution

of

RNA and

protein.

One important

feature is

that

the

platform

of

the l0S

subunit that

provides

the

interface

with the

50S

subunit is

composed

almost entirely of RNA. Only

two

proteins (a

small

part

of 57 and

possibly part

of SI2) Iie

near

the interface. This means

that the

associ-

ation and dissociation

of ribosomal

subunits

must

depend on interactions

with the I65 rRNA.

Subunit

association

is

affected by

a

mutation

in

a

loop

of l65 rRNA

(at

position

791) that is

located at

the subunit interface,

and other

nucleotides

in l65 rRNA have

been shown

to

be involved

by

modification/interference

exper-

iments.

This behavior

supports the idea

that the

evolutionary

origin of the ribosome

may have

been as a

particle

consisting

of RNA rather

than

protein.

The 50S subunit has

a

more

even

distribu-

tion of components than

the 30S,

with Iong

rods

of double-stranded

RNA crisscrossing

the

structure. The RNA

forms a mass

of tightly

packed

helices.

The exterior

surface largely

con-

sists of

protein,

except for the

peptidyl

trans-

ferase

center

(see

Section 8.19, 23S

rRNA Has

Peptidyl

Tlansferase Activity).

Almost

all seg-

ments

of the 23S rRNAinteractwithprotein,

but

many

of the

proteins

are relatively

unstructured.

FIfiUR[

$.3? The

50S subunit

has

a centraI

protuberance

where

55

rRNA

is located,

separated by a notch from

a

statk made

of copies of the

protein

17.

FiGriR[

8.3$ The

ptatform

ofthe

30S subunitfits into the

notch

of the

505 subunit to form

the 70S ribosome.

Fitu#Rg

*.3# The 30S ribosomal

subunit is

a

ribonucteo-

protein

pariicle.

Proteins

are in

yetlow.

Photo

coudesy of

V. Ramakrishnan.

MedicaI

Research

Councit

(UK).

CHAPTER

8

Protein

Synthesis

176

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:.:::;j-.:::

.::.r,,i

The

70S

ribosome

consists

of the 505 sub-

unit

(white)

and the 305 subunit

(purpte)

with

three tRNAs

located superficiatly:

yettow

in the A

site, btue

in

the

P site,

and

green

in the E site. Photo

courtesy of Harry

No[ter,

University of Catifornia,

Santa Cruz.

i:r.iii.ir

r1..l'

:

f[1gg tRNAs have

different

orientations on

the ribosome. mRNA

turns between

the P and A sites to

a[[ow

aminoacyltRNAs

to bind adjacent

codons.

Photo

courtesy

of Harry No[[er,

University of CaUfornia.

Santa Cruz.

into

the A Site). EF-Tu

inirially

places

the

aminoacyl-IRNA

into the

small subunit, where

the

anticodon

pairs

with the

codon. Movement

of the

IRNA is required

to bring it fully

inro rhe

A

site, when its

3'end enters

the

peptidyl

trans-

ferase

center on the large

subunit. There

are

different

models

for how this

process

may

occur.

One calls

for the

entire 1RNA to

swivel, so that

the elbow

in the L-shaped

structure made

by

the D

and TYC

arms moves into

the ribosome,

enabling

the TYC

arm to

pair

with IRNA.

Protein

Svnthesis

Another calls for the internal structure

of the

IRNA to change, using the anticodon loop

as a

hinge, with the rest

of

the IRNA rotating from

a

position

in which it is stacked on the J'side

of the anticodon loop to

one

in

which it is

stacked on the 5'side. Following the

transition,

EF-Tu hydrolyzes

GTP,

allowing

peptide

syn-

thesis to

proceed.

Translocation involves large

movements in

the

positions

of the tRNAs within

the ribosome.

The

anticodon end of IRNA moves

-28

A from

the A site to the P site, and then

a further 20 A

from

the

P

site to the

E

site. As a result

of the

angle of each IRNA relative to the anticodon,

the

bulk of the IRNA moves much larger

distances:

40

A

from

A site to P site and 55 A from P

site

to E site. This

suggests

that

translocation requires

a major reorganization of structure.

For many

years,

it was

thought that translo-

cation

could occur only

in

the

presence

of

the

factor

EF-G. However, the antibiotic

spar-

somycin

(which

inhibits

the

peptidyl

transferase

activity) triggers translocation. This

suggests

that the energy

to drive translocation

actually

is

stored

in

the

ribosome

after

peptide

bond for-

mation

has occurred. Usually EF-G

acts on the

ribosome to release

this energy and enable it

to

drive translocation, but sparsomycin

can have

the

same

role.

Sparsomycin inhibits

peptidyl

transferase by binding to the

peptidyl-IRNA,

blocking its interaction with aminoacyl-IRNA.

It

probably

creates a conformation

that resem-

bles the usual

posttranslocation

conformation,

which

in

turn

promotes

movement

of the

pep-

tidyl-tRNA. The important

point

is that

translo-

cation is

an

intrinsic

property

of the ribosome.

The hybrid

states model

suggests that

translocation may take

place

in

two stages,

with one ribosomal

subunit moving relative

to the

other to create an intermediate

stage in

which there are hybrid tRNA-binding

sites

(50S

E/30S P

and 50SP/30S A)

(see

Figure

8.29).

Comparisons of the ribosome

structure

between

pre-

and

posttranslocation

states, and

comparisons in 165 rRNA

conformation

between free l0S

subunits and 70S ribosomes,

suggest that mobility of

structure is especially

marked in the head

and

platform

regions

of

the 30S subunit. An interesting

insight

on the

hybrid

states model is

cast by the fact

that many

bases in rRNA involved

in subunit

association

are close to bases involved in

interacting

with

IRNA. This

suggests that IRNA-binding

sites

are close to the interface

between

subunits,

and

carries the implication that

changes in

sub-

unit interaction

could be connected

with move-

ment of IRNA.

778

CHAPTER

8

Much

of the structure

of the ribosome

is

occupied by its

active centers.

The schematic

view of the ribosomal

sites in

'

,.

i

shows

they

comprise about two

thirds of

the

riboso-

mal structure. A IRNA

enters

the A site, is trans-

ferred

by translocation into

the P

site. and then

Ieaves the

(bacterial)

ribosome

by the E site.

The A and P

sites extend across

both ribosome

subunits; IRNA is

paired

with mRNA in

the 30S

subunit,

but

peptide

transfer

takes

place

in

the

50S subunit. The A

and P sites

are adjacent,

enabling translocation

to move

the IRNA from

one site into the other. The E

site is located near

the P

site

(representing

a

position

en route to

the surface of the 50S

subunit). The

peptidyl

transferase center is located

on the 50S

subunit,

close to the aminoacyl

ends of the

tRNAs

in

the

A and P sites

(see

Section

8.18, l65 rRNA Plays

an Active Role in Protein

Synthesis).

All

of

the

GTP-binding

proteins

that func-

tion in

protein

synthesis

(EF-Tu,

EF-G, IF-2,

and RFl,

-2,

and

-3)

bind to

the same factor-

binding site

(sometimes

called the

GTPase cen-

ter), which

probably

triggers their hydrolysis

of

GTP.

This

site

is

located at the

base of the stalk

of the large subunit,

which

consists of the

pro-

teins L7 and Ll2.

(L7

is

a modification

oILl2

and

has

an acetyl

group

on the

N-terminus.) In

addition to this region,

the complex

of

protein

Ll l with a 58-base

stretch of 23S rRNA

pro-

vides

the binding site for some

antibiotics that

affect GTPase activity.

Neither of these riboso-

mal

structures actually

possesses

GTPase activ-

ity, but they are both necessary

for it. The role

of the

ribosome

is to trigger

GTP hydrolysis by

factors bound in the factor-binding

site.

Initial binding

of 30S subunits to nRNA

requires

protein

S l, which has a

strong affinity

for single-stranded nucleic

acid. It is responsi-

ble

for maintaining

the single-stranded

state in

nRNA that is bound to the

30S subunit. This

action is necessary to

prevent

the mRNA from

taking up a base-paired conformation

that

would be unsuitable for translation.

S

I

has an

extremely elongated structure and associates

with S18 and 52l. The three

proteins

consti-

tute a domain that is involved

in the initial bind-

ing

of

nRNA

and

in

binding initiator IRNA. This

locates

the

mRNA-binding

site in the

vicinity

of the cleft of the small subunit

(see

Figure 8.3).

The

3'end of

rRNA,

which

pairs

with the nRNA

initiation site, is located in this region.

The initiation factors bind in the

same

region

of

the ribosome. IF-l

can be crosslinked to the

3' end of

the rRNA,

as well as to several ribo-

somal

proteins,

including those

probably

involved in

binding

nRNA.

The role of IF-3

I

'

,

,.

The ribosome

has severa[ active centers.

It

may

be associated

with a

membrane. mRNA takes

a turn

as

it

passes

through

the A and

P sjtes, which are angted

with regard

to each other.

The

E

site

lies beyond the

P sjte.

The

peptidyt

transferase

site

(not

shown) stretches

across

the tops of the A and

P

sites.

Part of the site

bound by

EF-Iu/G ljes at the base

of the

A

and

P sites.

could be to stabilize

mRNA-30S subunit

bind-

ing;

then

it

would

be displaced

when

the 50S

subunit

joins.

The incorporation of

5S RNA

into 50S sub-

units that are assemble

d

in vitro depends on

the

ability of three

proteins-L5, L8, and L25-to

form a

stoichiometric

complex

with

it. The com-

plex

can bind to

2lS

rRNA, although

none of

the isolated components

can do so.

It lies in the

vicinity

of the

P and

A sites.

A nascent

protein

debouches

through

the

ribosome, away

from the active

sites,

into the

region in which ribosomes

may

be attached to

membranes

(see

Chapter

10, Protein

Localiza-

tion). A

polypeptide chain emerges

from the

ribosome

through

an exit

channel,

which leads

from the

peptidyl

transferase

site to the surface

of rhe 50S subunit.

The tunnel

is composed

mostly

of

rRNA. It is

quite

narrow-only

I to

2 nm wide-and

is

-10

nm

long. The

nascent

polypeptide

emerges

from the

ribosome

-I5

A

away from the

peptidyl transferase

site. The

tunnel can hold

-50

amino

acids, and

proba-

bly constrains

the

polypeptide chain so that

it

cannot fold until

it leaves

the exit

domain.

165 rRNA

Plays an

Active

Ro[e

in

Protein Synthesis

.

165 rRNA

ptays

an active

rote in the functions

of

the 30S subunit.

It

interacts directty

with mRNA,

with the 50S subunit,

and

with the anticodons

of

tRNAs in the

P

and

A sites.

The ribosome was

originally

viewed

as a col-

lection of

proteins with various

catalytic

8.18

165

rRNA

Ptays an Active

Rote

in Protein Synthesis

179

r:i:-:.i:

.:

,i::

Some sites in 165 rRNA

are

orotected

from

chemical orobes

when

50S subunits

join

30S subunits

or when aminoacyltRNA

binds to the

A

site. 0thers

are the sites of mutations

that affect

protein

synthesis.

TERM

suppression

sites may

affect termination

at some or several

termination

codons. The

[arge colored

btocks

indicate

the four

domains ofthe rRNA.

activities

held together

by

protein-protein

inrer-

actions

and

by binding to rRNA. The

discovery

of

RNA

molecules

with catalytic activities

(see

Chapter 26, RNA

Splicing

and Processing)

immediately

suggests, however,

that rRNA

might

play

a

more

active role in ribosome

func-

tion. There

is now evidence

that rRNA inter-

acts with

nRNA

or tRNA at each

stage of

translation,

and that

the

proteins

are necessary

to maintain

the rRNA in a

structure in which it

can

perform

the catalytic functions.

Several

interactions

involve

specific regions

of rRNA:

.

The

3' terminus of the rRNA

interacts

directly

with mRNA at initiation.

.

Specific regions

of l6S rRNA

interact

directly with the

anticodon regions

of

tRNAs in

both the A site

and the P site.

Similarly, 23S IRNA

interacts

with the

CCA

terminus

of

peptidyl-tRNA

in

both

the P

site and A site.

CHAPTER

8 Protein

Synthesis

.

Subunit

interaction involves

interac-

tions between

I65

and 23S rRNAs

(see

Section 8.16,

Ribosomal

RNA Pervades

Both Ribosomal Subunits).

Much information about

the

individual

steps of bacterial

protein

synthesis has

been

obtained

by using

antibiotics

that inhibit the

process

at

particular

stages. The target for

the

antibiotic can be identified by the

component

in which resistant mutations

occur. Some antibi-

otics act

on

individual ribosomal

proteins,

but

several act on rRNA, which suggests

that the

rRNA is involved

with

many

or even all

of the

functions

of the

ribosome.

The functions of rRNA have

been investi-

gated

by two types

of

approach.

Structural stud-

ies

show that

particular

regions

of

rRNA

are

located in important

sites of the ribosome,

and

that chemical modifications

of

these

bases

impede

particular

ribosomal functions.

In addi-

tion, mutations identify

bases in rRNA that

are

required for

particular

ribosomal functions.

Fg*tJftil $.4*

summarizes the

sites in l6S rRNA

that have

been

identified

by these means.

An indication of the importance

of the 3'

end of I65 rRNA is

given

by

its

susceptibility

to

the lethal

agent colicin E3. Produced

by some

bacteria, the colicin cleaves

-50

nucleotides

from

the 3'end

of the

I65 rRNA

of E. coli.The

cleav-

age entirely abolishes initiation

of

protein

syn-

thesis. Several important functions

require

the

region that is

cleaved: binding

the factor

IF-3, recognition

of

mRNA,

andbinding

of IRNA.

The

3' end of the 165 rRNA

is directly

involved in the initiation

reaction

by

pairing

with the Shine-Dalgarno sequence

in the ribo-

some-binding site of mRNA

(see

Figure

8.16).

Another direct role for

the 3'end of 165

rRNA

in

protein

synthesis is

shown by the

properties

of kasugamycin-resistant

mutants,

which lack

certain modifications in l65

rRNA.

I(asug-

amycin

blocks initiation of

protein

synthesis.

Resistant

mutants of the

type ksgAlacka

meth-

ylase

enzyme

that

introduces

four

methyl

groups

into two

adjacent adenines

at a site near

the

3'terminus of the 165 rRNA.

The

methy-

lation

generates

the highly

conserved

sequence

G-m26A-m26A,

found in

both

prokaryotic

and

eukaryotic small rRNA.

The methylated

sequence is involved in

the

joining

of the 30S

and

50S

subunits,

which in turn is

connected

also with the retention

of initiator

IRNA

in the

complete ribosome. I(asugamycin

causes fMet-

tRNAl to

be

released

from

the sensitive

(meth-

ylated)

ribosomes,

but the resistant

ribosomes

are able to retain

the initiator.

TERM

suppressron

180

Lewin Benjamin (ed.) Genes IX

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