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9BB

the A site,

the assessment

of binding is being

made. The 1400

region can

be directly crosslinked

to

pepridyl-tRNA,

which

suggesrs that this region

is

a structural

component of the P

site.

The

basic conclusion

to be drawn from these

results

is that rRNA

has many interactions

with

both IRNA

and nRNA, and that

these inrerac-

tions recur in

each cycle

of

peptide

bond

formation.

@

23S rRNA Has

Peptidyl

Transferase

Activity

.

Peptidyt

transferase

activity resides

exctusivety in

the 235 rRNA.

The sites involved

in the functions

of 23S rRNA

are less

well identified

than those

of

l6S rRNA,

but the same

general pattern

is

observed: bases

at certain

positions

affect

specific functions.

Bases

at some

positions

in 23S rRNA

are affected

by the

conformation

of the A site

or

P

site. In

particular,

oligonucleotides

derived from

the 3'

CCA terminus

of IRNA

protect

a set of bases in

23S

rRNA

that essentially

are the

same as those

protected

by

peptidyl-IRNA.

This

suggests that

the major interaction

of 2lS rRNA

with

peptidyl-tRNA

in the P

site involves

the 3'end

of the IRNA.

The

IRNA makes

contacts with

the 23S

rRNA in

both the P

and A sites. At

the P site,

G2552

of 2I

S

rRNA

base

pairs

with C7 4

of the

peptidyl

IRNA. A muration

in the

G

in

the rRNA

prevents

interaction

with IRNA,

but interaction

is restored

by a compensating

mutation

in the

C

of the amino

acceptor end

of the IRNA. At

the A site.

G2553

of

the 23S

rRNA

base

pairs

with

C75 of the

aminoacyl-tRNA.

Thus there

is

a close role for

rRNA in

both the

tRNA-bind-

ing

sites. Indeed,

when

we have

a clearer struc-

tural

view

of the region,

we should

be able to

understand

the movements

of IRNA

between

the A and

P sites in terms

of making

and break-

ing

contacts with

rRNA.

Another

site that

binds IRNA

is the E

site,

which is localized

almost exclusively

on

the 50S

subunit.

Bases affected

by its

conformation

can

be identified

in 23S rRNA.

What

is the nature

of the site

on the 50S

subunit

that

provides

peptidyl

transferase func-

tion? The

involvement

of

rRNA

was

first indi-

cated

because

a region

of the 23S

rRNA is the

site of mutations

that confer resistance

to

antibi-

otics

that inhibit

peptidyl

transferase.

CHAPTER

8 Protein

Svnthesis

A long

search

for

ribosomal

proteins

that

might

possess

the catalytic activity

has been

unsuccessful. Recent results

suggest that the

ribosomal RNA

of

the large

subunit has

the cat-

alytic activity. Extraction

of almost all the

pro-

tein content

of

50S

subunits leaves

the 23S

rRNA associated largely

with fragments

of

pro-

teins,

amounting to

<5%

of the mass

of the ribo-

somal

proteins.

This

preparation

retains

peptidyl

transferase

activity. Tfeatments

that damage the

RNA abolish the

catalytic activity.

Following

from these results, 235

rRNApre-

pared

by transcription in vitro

can catalyze

the

formation

of a

peptide

bond between

Ac-Phe-

IRNA

and

Phe-tRNA.

The

yield

of Ac-Phe-Phe

is very low,

suggesting that the 23S

rRNA

requires

proteins

in order to function

at

a

high

efficiency. Given that

the

rRNA

has

the basic

catalytic activity,

though, the role

of the

pro-

teins must

be indirect, serving

to fold the rRNA

properly

or to

present

the

substrates

to

it.

The

reaction also

works, although less

effectively, if

the domains of 23S rRNA

are synthesized

sep-

arately and then

combined. In fact,

some activ-

ity is

shown by domain V alone,

which has

the

catalytic center. Activity

is abolished

by muta-

tions in

position

2252 of domain

V that lies

in

the P site.

The

crystal structure of an

archaeal 50S

sub-

unit

shows that the

peptidyl

transferase

site

basically

consists of 23S rRNA.

There is

no

pro-

tein

within 18 A of the active

site

where the

transfer

reaction occurs

between

peptidyl-tRNA

and aminoacyl-IRNA!

Peptide

bond

synthesis requires

an attack

by

the amino

group

of one amino

acid on

the car-

boxyl

group

of another

amino acid.

Catalysis

requires a

basic residue to

accept the hydrogen

atom that is released from

the amino

group,

as

shown in lg*i.ifig

Fi.+"{.

If rRNA

is the

catalyst it

must

provide

this residue,

but

we do not know

how

this happens. The

purine

and

pyrimidine

bases are not basic

at

physiological

pH.

A highly

conserved base

(at position

245I

in E.

colil had

been implicated,

but

appears now

neither

to

have

the right

properties

nor

to be

crucial for

peptidyl

transf erase activity.

Proteins

that are

bound to

the 23S rRNA

outside

of the

peptidyl

transfer region

are

almost

certainly required

to enable

the rRNA

to form

the

proper

structure invivo. The

idea

that rRNA

is

the catalytic

component is

consistent

with the

results

discussed in

Chapter 26, RNA

Splicing

and Processing,

which

identify

catalytic

prop-

erties in RNA

that are involved

with

several

RNA

processing

reactions.

It fits

with the notion

182

Aminoacyl{RNA

PeptidyltRNA

H-C-R

I

tRNA

peptide

chain

I

H-N

I

H-C.R

I

x"\^

VU

I

;

IRNA

I

V

o

Base

IRNA

,T

peptide

chain

I

H-N

I

H-C.R

Au

t\

I

H-C-R

I

t-l a

o

d"b

ll

IRNA

irt{,il,rFil

ii.+;::

Peptide bond formation requires

acid-base

catalysis in which an H atom

js

transferred to a basic

residue.

that

the ribosome evolved from

functions orig-

inally

possessed

by RNA.

thesis). More directly,

comparisons

of the

high

resolution crystal

structures

of the

individual

subunits with the

lower resolution

structure

of

the intact

ribosome suggests

the

existence of

significant differences.

These

ideas

have been

confirmed by

a crystal structure

of.the

E. coli

ribosome

ar

3.5 A, which

furthermore

identi-

fies two different

conformations

of the

ribo-

some,

possibly

representing

different

stages

in

protein

synthesis.

The crystal contains

two

ribosomes

per

unit,

each with a

different conformation.

The differ-

ences are due to changes

in the

positioning

of

domains

within each subunit,

the most

impor-

tant being that

in one conformation

the

head

of the small subunit

has swiveled

6' around

the

neck region toward

the

E site.

Also, a 6o

rota-

tion in the opposite

direction

is seen

in the

(low

resolution)

structures

of. Thermus

thermophilus

ribosomes that

are bound

to

nRNA and

have

tRNAs in both

A and P sites,

suggesting

that

the

head may

swivel

overall

by

l2' depending

on

the stage of

protein

synthesis.

The rotation

of

the

head follows the

path

of

tRNAs through

the

ribosome, raising

the

possibility that its swivel-

ing controls movement

of

nRNA

and IRNA.

The changes

in conformation

that occur

when subunits

join

together

are

much more

marked in the 30S

subunit

than

in the 50S

sub-

unit.

The

changes

are

probably

concerned

with

controlling

the

position and

movement

of

nRNA. The most significant

change

in the

50S

subunit concerns

the

peptidyl transferase

cen-

ter. 50S subunits

are

-1000x

less effective

in

catalyzing

peptide

bond

synthesis

than com-

plete

ribosomes;

the reason

may be a change

in

structure that

positions

the

substrate

more effec-

tively

in

the

active site

in the

complete

ribosome.

One of

the main

features

emerging

from

the structure

of the

complete

ribosome

is the

very high density

of

solvent

contacts

at

their

interface; this

may help

the

making and

break-

ing of contacts

that

is essential

for subunit

asso-

ciation and dissociation,

and

may also

be

involved in structural

changes

that occur

dur-

ing translocation.

Summary

Ribosomes

are

ribonucleoprotein

particles in

which a

majority

of the

mass

is

provided

by

rRNA. The shapes

of all

ribosomes

are

generally

similar,

but only

those

of

bacteria

(70S) have

been characterized

in detail.

The

small

(30S)

subunit

has a squashed

shape,

with

a

"body"

containing

about

two

thirds

of the

mass divided

RibosomaL

Structures

Change When the

Subunits Come

Together

.

The head of the 305 subunit swivels around the

neck when complete ribosomes

are

formed.

.

The

peptidyl

transferase active site of the 505

subunit

is more

active

in

complete

ribosomes

than

in individual.

50S subunits.

.

The

interface

between the 30S and 50S subunits

is

very rich

in

solvent contacts.

Much indirect evidence suggests that the struc-

tures of the individual subunits change signif-

icantly when they

join

together to

form

a

complete ribosome. Differences in the suscep-

tibilities of the rRNAs to outside agents are one

of

the strongest indicators

(see

Section 8.18,

I6S rRNA Plays an Active Role in Protein Syn-

8.21

Summary

183

from the

"head"

by a

cleft. The large

(50S)

sub-

unit

is

more

spherical, with a

prominent

"stalk"

on the right

and a

"central

protuberance."

Loca-

tions

of all

proteins

are known approximately

in the

small

subunit.

Each subunit

contains

a single major rRNA,

I 65 and 2

35 in

prokaryotes,

and I 8S

and

28S in

eukaryotic

cytosol. There are

also minor rRNAs,

most notably

5S rRNA

in the large

subunit. Both

major

rRNAs have

extensive

base

pairing,

mostly

in

the form

of short, imperfectly

paired

duplex

stems with

single-stranded loops.

Conserved fea-

tures in

the rRNA

can be identified

by compar-

ing

sequences

and the secondary

structures that

can

be drawn for rRNA

of a variety

of organisms.

The l65

rRNA has

four distinct

domains; the 2lS

rRNA

has

six distinct domains. Eukaryotic

rRNAs

have

additional

domains.

The

crystal

structure

shows that the 30S

subunit

has an asymmetrical

distribution

of

RNA

and

protein.

RNA is

concentrated at the

interface with

the 50S

subunit. The 50S

sub-

unit has a

surface of

protein,

with long rods

of

double-stranded

RNA

crisscrossing

the struc-

ture.

loSto-50S

joining

involves

contacts

between l65

rRNA and 235

rRNA. The

inter-

face

between the

subunits is

very rich in con-

tacts

for

solvent. Structural

changes

occur in

both subunits

when

they

join

to form

a complete

ribosome.

Each

subunit

has several

active

centers,

which

are

concentrated

in the translational

domain

of the ribosome

where

proteins

are syn-

thesized. Proteins

leave

the ribosome

through

the exit

domain,

which can

associate with a

membrane.

The

major active

sites are

the P and

A

sites, the

E site, the

EF-Tu and

EF-G binding

sites,

peptidyl

transferase,

and the mRNA-bind-

ing

site. Ribosome

conformation

may change

at

stages during

protein

synthesis;

differences in

the accessibility

of

particular

regions

of the major

rRNAs

have

been

detected.

The

tRNAs in

the A

and P sites

are

parallel

to

one another.

The

anticodon loops

are

bound

to mRNA in

a

groove

on the

30S subunit.

The

rest

of each

IRNA is

bound

to the 50S

subunit.

A

conformational

shift of

IRNA within

the A

site is required

to bring its

aminoacyl

end into

illr:

T

:'i,".1 ffirJffi$

:l i*,::::'i*

:iil*

links

the

P- and

A-binding

sires is

made

of

23S

rRNA,

which has

the

peptidyl

transferase

cat-

i'JJ;:3',-i:';#,iTlJr1"i,:,T:,Xllo'oouo''

An

active

role for

the rRNAs

in

protein

syn-

thesis

is indicated

by mutations

that affect

ribo-

CHAPTER

8

Protein

Synthesis

somal function, interactions

with mRNA

or

IRNA that

can be detected by chemical

crosslink-

ing,

and the requirement to maintain

individ-

ual base

pairing

interactions

with the IRNA

or

mRNA. The 3'terminal region

of the rRNA

base

pairs

with

mRNA

at initiation. Internal

regions

make individual contacts

with the tRNAs

in

both

the

P

and A sites. Ribosomal

RNA is the tar-

get

for some antibiotics

or other

agents that

inhibit

protein

synthesis

A

codon

in

nRNA is recognized

by

an

aminoacyl-tRNA, which has an

anticodon

com-

plementary

to the

codon and carries

the amino

acid corresponding to the

codon. A special ini-

tiator IRNA

(fMet-tRNAr

in

prokaryotes

or Met-

tRNAi in

eukaryotes) recognizes

the AUG

codon,

which is used to start all

coding sequences.

In

prokaryotes,

GUG is also used.

Only the termi-

nation

(nonsense)

codons,

UAA,

UAG, and

UGA, are not recognized

by aminoacyl-tRNAs.

Ribosomes are released

from

protein

syn-

thesis to enter a

pool

of

free

ribosomes

that are

in equilibrium

with separate small

and large

subunits. Small subunits

bind to mRNA

and

then are

joined

by large

subunits

to

generate

an intact ribosome

that undertakes

protein

syn-

thesis. Recognition

of a

prokaryotic

initiation

site involves

binding of a sequence

at the J'end

of

rRNA

to the Shine-Dalgarno

motif,

which

precedes

the AUG

(or

GUG)

codon in the

mRNA. Recognition

of a eukaryotic

mRNA

involves

binding to the 5'

cap; the

small sub-

unit then migrates

to the initiation

site by

scan-

ning

for AUG codons.

When it recognizes

an

appropriate AUG

codon

(usually,

but not always,

the first it

encounters), it is

joined

by a large

subunit.

A

ribosome

can

carry two aminoacyl-tRNAs

simultaneously: its

P site is

occupied

by a

pollpeptidyl-tRNA,

which carries

the

polypep

-

tide chain synthesized

so far, whereas

the A

site

is used for

entry by an aminoacyl-tRNA

carry-

ing

the next amino

acid to be added

to

the chain.

Bacterial ribosomes

also have

an E

site, through

which

deacylated

IRNA

passes

before it

is

released

after

being used in

protein

synthesis.

The

polypeptide

chain in the P

site is transferred

to the aminoacyl-tRNA

in

the A site,

creating

a

deacylated

IRNA in

the P site

and a

peptidyl-

IRNA in the

A site.

Following

peptide

bond synthesis,

the ribo-

some

translocates

one codon

along the nRNA,

moving

deacylated

IRNA into

the E

site and

peptidyl

tRNA from

the A

site into

the P site.

Tlanslocation

is

catalyzed

by the elongation

fac-

tor EF-G and,

like several

other

stages of ribo-

some function. requires

hydrolysis

of GTP. Dur-

ing

translocation, the ribosome

passes

through

a hybrid

stage

in

which the 50S

subunit

moves

relative to the 30S subunit.

Protein synthesis is an

expensive

process.

ATP is

used

to

provide

energy at

several stages,

including

the charging of IRNA

with

its

amino

acid and the unwinding

of

mRNA.

It has been

estimated

that

up to

90o/"

of all the ATP mole-

cules synthesized in a rapidly

growing

bacterium

are consumed in assemblins

amino acids into

protein!

Additional factors

are required at each stage

of

protein

synthesis. They are

defined by their

cyclic association with,

and dissociation from,

the ribosome.

IF

factors are involved in

prokary-

otic

initiation. IF-l is

needed for 30S subunits

to bind to mRNA, and also is responsible for

maintaining the l0S subunit in

a

free form. IF-

2 is needed for fMet-tRNAl to

bind to the 30S

subunit and

is responsible

for excluding other

aminoacyl-tRNAs from the initiation reaction.

GTP

is hydrolyzed

after the initiator IRNA has

been bound to the initiation complex. The ini-

tiation factors must be released in

order to allow

a

large

subunit to

join

the initiation complex.

Eukaryotic initiation involves

a

greater

number of factors. Some of them

are

involved

in

the

initial

binding of the 40S subunit to the

capped 5'end of the mRNA, at

which

point

the

initiator IRNA is bound by another

group

of fac-

tors. After this initial binding, the

small subunit

scans the

nRNA

until it recognizes the correct

AUG codon. At this

point,

initiation factors are

released and the 60S subunit

joins

the complex.

Prokaryotic EF factors

are

involved in

elon-

gation.

EF-Tir

binds aminoacyl-tRNA to the

70S

ribosome. GTP is hydrolyzed when EF-Tu is

released, and EF-Ts is required

to

regenerate

the active form of EF-Tu. EF-G is required for

translocation.

Binding

of the EF-Tu and

EF-G

factors to ribosomes

is

mutually exclusive, which

ensures that each step must be completed before

the

Termination occurs at any one of the three

special codons,

UAA, UAG, and

UGA.

Class I RF

factors that specifically

recognize

the termina-

tion codons activate the ribosome to

hydrolyze

the

peptidyl-tRNA.

A class 2 RF factor is required

to

release the class I RF factor from the ribo-

some. The GTP-binding factors IF-2,

EF-Tu,

EF-G, and

RFI all have similar

structures,

with

the

latter two mimicking the RNA-protein struc-

ture of the first two when they are bound to

IRNA.They

all bind to the same ribosomal site,

the G-factor

binding site.

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68r

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