Lewin Benjamin (ed.) Genes IX

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which a IRNA

responds,

without altering its

charging with amino acids.)

group

isoaccepting

tRNAs must be

charged only by the single aminoacyl-tRNA syn-

thetase specific

for

their amino acid. So isoac-

cepting tRNAs must share

some common

feature(s) enabling

the enzyme to distinguish

them from the other tRNAs. The

entire com-

plement

of tRNAs

divided into twenty isoac-

cepting

groups,

and each

group

able to

identify

itself to

its

particular

synthetase.

tRNAs are identified by their

synthetases

contacts that recognize

a small number of

bases, typically

from

one to five. Three types of

features

commonly are used:

Usually

(but

not always), at least one

base of the anticodon is recognized.

Sometimes all the

positions

the anti-

codon are

important.

Often one of the last three

base

pairs

the acceptor stem is recognized. An

extreme case is represented

alanine

IRNA, which is identified

single

unique base

pair

the acceptor stem.

The so-called discriminator base, which

Iies between the acceptor

stem and

the

CCA terminus, is always invariant

among isoacceptor

tRNAs.

No one of these

features

constitutes a unique

means of distinguishing twenty sets of tRNAs,

provides

sufficient specificity,

it appears

that recognition of tRNAs

is idiosyncratic,

with

each

following its own rules.

Several synthetases can specifically charge

"minihelix,"

which consists only of the accep-

tor and

Try C arms

(equivalent

to one arm of

the L-shaped

molecule)

with the correct amino

acid.

For certain tRNAs, specificity depends

exclusively

upon the acceptor stem. However,

it is

clear

that there are significant variations

between

tRNAs, and in some cases the anti-

codon region

is important. Mutations in

the

anticodon

can affect recognition by the class II

Phe-IRNA synthetase.

Multiple features may

involved; minihelices from the tRNAvuland

tRNAMet

(where

we know that the anticodon is

important

in vivol can react

specifically

with

their

synthetases.

Thus recognition depends on an interac-

tion between a

few

points

of contact

in the IRNA,

concentrated

at the extremities, and a few amino

acids constituting

the active site in the

protein.

The relative

importance

of the

roles

played

the acceptor stem and anticodon

different

for

each IRNA-synthetase

interaction.

Aminoacy[-tRNA

Synthetases

Fa[[

into

Two Groups

AminoacyltRNA synthetases

are divided

into

the

class I and class

groups

by sequence

and

structuraI similarities.

spite of

their common

function, synthetases

are a

rather diverse

group

proteins. The indi-

vidual subunits

vary

from

40 to 110

kD, and

the enzymes

may be

monomeric,

dimeric,

tetrameric. Homologies

between

them

are rare.

Of course,

the active

site

that

recognizes IRNA

comprises

a rather

small

part

of the

molecule.

It is interesting to compare

the active

sites

different synthetases.

Synthetases

have been

divided

into

two

general groups,

each

containing

ten

enzymes,

on the basis

of the structure

of the

domain

that

contains

the active

site.

general type of orga-

nization that

applies

to both

groups

repre-

sented in

:i,r'':':

i;.

The

catalytic

domain

includes the binding

sites

for

ATP and

amino

acid. It can be

recognized

a large

region that

is interrupted by

an insertion

of the domain

that binds

the acceptor

helix of the

IRNA.

This

places

the terminus

the IRNA

proximity to

the catalytic site.

A separate

domain

binds the

anticodon

region

of IRNA.

Those

synthetases

that are multimeric

also

possess

an oligomeriza-

tion domain.

Class

I synthetases

have an N-terminal

cat-

alytic domain

that

is identified

the

presence

of two short,

partly

conserved

sequences

amino acids,

sometimes

called

"signature

i.i,ir,.,iiiri:

.I,ir

An aminoacyt-tRNA

synthetase

contains

three

or four

regions with

different

functjons.

(0nty mut-

timeric

synthetases

possess

otigomerization

domain.)

9.10

Aminoacyt-tRNA

Synthetases

Fa[[ into

Two Groups

207

,r,:

:-:i:,r

,- CrystaI

structures

show that

class I and

ctass II aminoacV[-

tRNA

synthetases

bind the opposite

faces

oftheirIRNA

substrates. The IRNA

shown in red

and

the

protein

b[ue. Photo

courtesy

Dino

Moras, Insti-

tute

Genetics

and Motecu[ar

and Ce[[utar Biotogy.

7i';LiSrbase

Pair

Acceptor

stem lies in

deep

pocket

protein

ott

binds near

acceptor

stem

Anticodon looo is

distorted

U35-U36

Gln-tRNA

synthetase

ili.,r:iir:

:,, l

::,

class I

IRNA synthetase

contacts

IRNA

at the minor

groove

of the

acceptor

stem and at the

nticodon.

sequences."

The

catalytic

domain

takes the form

of a motif

called a nucleotide-binding

fold

(which

also found

other classes

of enzymes

that

bind nucleotides).

The

nucleotide

fold

consists

of alternating parallel

p-strands

and cr-helices;

the

signature

sequence

forms

part

of the ATP-

binding

site. The

insertion

that

contacts

the

acceptor

helix

of IRNA

differs

widely between

different

class I

enzymes.

The

C-terminal

domains

of the

class I

synthetases,

which include

the

IRNA

anticodon-binding

domain

and any

oligomerization

domain,

also are

quite

different

from

one

another.

Class

II enzymes

three rather

general

similarities

of sequence

their

catalytic

domains.

The

active site

contains

a large

antipar-

CHAPTER

Using

the

Genetic

Code

allel

B-sheet

surrounded by

c-helices. Again,

the acceptor helix-binding

domain that inter-

rupts

the catalytic domain has

a structure

that

depends

on the

individual

enzyme.

The

anti-

codon-binding domain

tends to be

N-terminal.

The location

of any oligomerization

domain

widely variable.

The lack

of any apparent

relationship

between the two

groups

of synthetases

is a

puz-

zle. Perhaps

they evolved independently

of one

another. This makes it

seem

possible

even that

an early form

of life could have

existed

with

proteins

that were made

up of

just

the

ten amino

acids coded by

one type or the

other.

general

model

for synthetase-IRNA

bind-

ing

suggests that the

protein

binds

the IRNA

along the

"side"

of the L-shaped

molecule.

The

same

general principle

applies for all

synthetase-

IRNA

binding: The IRNA is

bound

principally

its

two extremities,

and most

of the

IRNA

sequence is

not involved in

recognition

synthetase. However,

the detailed

nature

of the

interaction

is different

between

class I and

class

enzymes, as

can be seen from

the models

rli;iii:{

*.:.:i,

which are based

on crystal

struc-

tures. The

two types of

enzyme approach

the

IRNA from

opposite

sides, with

the result

that

the tRNA-protein

models look

almost

like mir-

ror

images

of one another.

A class I

enzyme

(Gln-tRNA

synthetase)

approaches

the D-loop

side of the

IRNA. Ir rec-

ognizes

the minor

groove

of the

acceptor

stem

one end of the

binding site, and

interacts

with

the anticodon

loop at the

other end. ***'

iFiil

-ir.

: n

a diagrammatic representation

of the

crystal

structure of

the tRNAcl"-synthetase

complex. A

revealing feature

of the

structure is

that

con-

tacts with

the enzyme

change the

structure

the

IRNA at two important points.

These

can

be seen

by comparing

the dotted

and solid lines

the anticodon loop

and acceptor

stem:

Bases

U35 and U36

in the

anticodon

loop

are

pulled

farther

out

of the IRNA

into

the

protein.

The

end

of the acceptor

stem is

seriously

distorted,

with

the

result

that base

pair-

ing

between

Ul and

A72 is

disrupred.

The single-stranded

end

of the

stem

pokes

into

a deep

pocket

the

syn-

thetase

protein,

which also

contains

the

binding

site for ATP.

This structure

explains

why

changes

in U35,

G73,

or the Ul-A72

base

pair

affect

the recog-

nition

of the

IRNA by its

synthetase.

Ar all

these

positions.

hydrogen

bonding

occurs

between

the

protein

and IRNA.

202

A class II enzyme

(Asp-tRNA

synthetase)

approaches the IRNA from

the other side; it rec-

ognizes both the

variable loop and

the major

groove

of the acceptor

stem, as drawn in

ri::liir;l

,,:.:,.

The

acceptor

stem remains in its

regular helical conformation.

ATP is

probably

bound near to the

terminal adenine. At

the

other end of the binding

site, there is a tight

contact with the

anticodon loop,

which

has

change in conformation

that allows the anti-

codon

to be

close contact

with the nrotein.

ctass

II aminoacyltRNA

synthetase con-

tacts IRNA at

the major

groove

of the

acceptor

hetix and

at the

anticodon looo.

that

only the

correct

amino acids

and

cognate

tRNAs

could

form a stable

attachment

at the

site.

Alternatively,

the

reaction

proceeds

through

some of

its stages,

after which

a decision

is reached

on whether

the

correct species

present. If it is not

pres-

ent, the

reaction

is reversed,

or a blpass

route is taken,

and

the wrong

member

is expelled.

This sort

postbinding

scrutiny

generally

described

proof-

reading. In the

example

of synthetases,

it would

require that

the charging

reac-

tion

proceeds through

certain

stages

even

if the wrong

IRNA

or amino

acid

present.

Synthetases

use

proofreading

mechanisms

to control the

recognition

of both

types

of sub-

strates.

They improve

significantly

the intrin-

sic differences

among

amino

acids

or among

tRNAs, but,

consistent

with

the intrinsic

differ-

ences

in each

group, make

more mistakes

selecting amino

acids

(error rates are

l0-4 to

l0-5) than in selecting

tRNAs

(for

which

error

rates are

-10-6)

(see

Figure

8.8).

Transfer

RNA

binds to synthetase

by the

two-stage

reaction

depicted

':.

Cognate

tRNAs

have a

greater

intrinsic

affinity

for

the binding

site, so

they

are bound

rapidly

and dissociate

more slowly.

Following

binding,

the enzyme

scrutinizes

the IRNA

that

has been

bound.

the

correct IRNA

present,

binding

stabilized

by a

conformational

change

in the enzyme.

This

allows aminoacylation

occur

rapidly.

If the

Synthetases

Use

Proofreading

Improve

Accuracy

Specificity of recognition

of both amino acid and

IRNA is controtled

by aminoacy[-tRNA synthetases

proofreading

reactions

that reverse the

catatytic

reaction if

the wrong component has

been

incorporated.

Aminoacyl-IRNA

synthetases have a difficult

job.

Each

synthetase must

distinguish one out

of twenty amino acids,

and must differentiate

cognate tRNAs

(typically

one

to three)

from

the

total set

(perhaps

100 in all).

Many

amino acids are closely related to

one

another, and all amino acids are related

to the

metabolic intermediates in

their

particular

syn-

thetic

pathway.

is especially difficult to dis-

tinguish between two

amino acids that differ

only in the length ol the carbon

backbone

(that

is, by one CH2

group).

Intrinsic

discrimination

based on

relative

energies of binding two such

amino acids would be only

I / 5. The

synthetase

enzymes improve this ratio

-1000-fold.

Intrinsic discrimination between tRNAs is

better, because

the IRNA

offers a larger surface

with which to make more

contacts. It

still

true,

however,

that all tRNAs conform to the

same

general

structure, and

there

may

be a

quite

limited set of features that distinguish the cog-

nate tRNAs from the noncognate

tRNAs.

We can

imagine

two

general

ways in which

the enzyme

might

select its substrate:

The cycle of admittance, scrutiny, and

rejection/acceptance

could

represent a

single binding step that

precedes

all other

stages of whatever reaction is involved.

This is tantamount to saying that the

affinity of the binding site is sufficient to

control the entry of substrate.

the

case of synthetases, this would mean

9.11

Synthetases

Use

Proofreading

Improve

Accuracy

203

Cognate tRNA

associates

rapidly

dissociates

slowly

Cognate IRNA

triggers change

in conformation

Aminoacylation

without

change

conformation

occurs

slowly

Noncognate tRNA

associates

slowly

dissociates rapidly

Aminoacylation

occurs rapidly

.rNHz

RCH

cxa)co

AMP

IIS$R[

*.tr€ Recognition

of the correct

IRNA by syn-

thetaseis

controtted at two

steps. First,

the enzyme has

greater

affinity for its

cognate IRNA.

Second, the aminoa-

cylation

of the incorrect

IRNA is very

stow.

wrong

IRNA is

present,

the

conformational

change

does not occur. As

a result,

the reaction

proceeds

much more

slowly;

this increases the

chance

that

the IRNA will

dissociate from

the

enzyme

before it is

charged. This

type of con-

trol is

called kinetic

proofreading.

Specificity for

amino

acids varies

among

the

synthetases.

Some are highly

specific for

initially

binding

a single

amino

acid, whereas

others

can also activate

amino

acids closely

to the

proper

substrate.

The

analog amino

acid can

sometimes

be converted

to the aden-

ylate

form,

but in none

of these

cases is an incor-

rectly

activated

amino

acid actually

used to form

a stable

aminoacyl-IRNA.

The

presence

of the

cognate IRNA

usually

needed

to trigger

proofreading,

even if

the

reaction

occurs at the

stage before

formation

aminoacyl-adenylate. (An

exception

provided

by Met-tRNA

synthetase,

which

can reject

CHAPTER

9 Using

the

Genetic Code

Adenylation

Aminoacyl-adenylate

is hydrolyzed

Wrong

acid

&4nup

tRNA

charging

Aminoacyl-tRNA is

hydrolyzed

Wrong

mino

acid

>q* a

+Xo

Correct

amrno acro

NHz

RCH

)co

ftf Aminoacvl-tRNA

AMP

f,:*L€itt

*.tG When a synthetase

binds the incorrect

amino

acid,

proofreading

requires

binding ofthe

cognate

tRNA.

It may

take

ptace

either

by a conformation

change

that causes hydrotysis

of the

incorrect

aminoacyladeny-

late or by transfer of the

amino acid to IRNA. followed

hydrotysis.

noncognate

aminoacyl-adenylate

complexes

even in the

absence of IRNA.)

There

are two

stages at

which proofread-

ing

of an incorrect

aminoacyl-adenylate

may

occur

during formation

of aminoacyl-IRNA.

F]"S{JRfl

S"i* shows that

both use

chemical

proofreading,

which the catalytic

reaction

is reversed. The

extent to which

one

pathway

or the other

predominates

varies

with the indi-

vidual

synthetase:

The noncognate

aminoacyl-adenylate

may

be hydrolyzed

when the

cognate

IRNA binds. This

mechanism

used

predominantly

by several

synthetases,

including

those for

methionine,

iso-

leucine,

and

valine.

(Usually,

the reac-

tion cannot

be seen invivo,

but it

can be

followed for

Met-tRNA

synthetase

when

the incorrectly

activated

amino acid is

homocysteine,

which lacks

the

methyl

group

of methionine).

Proofreading

releases

the amino

acid in

an altered

form,

as homocysteine

thiolactone.

fact,

homocysteine

thiolactone

pro-

204

duced

in E.

coli as

a by-product

of the

charging reaction

of Met-IRNA

syn-

thetase. This

shows that

continuous

proofreading

part

of the

process

charging

a IRNA with its

amino acid.

Some synthetases

use chemical

proof-

reading at a later

stage. The

wrong amino

acid is

actually transferred

to IRNA, is

then recognized as incorrect

by its struc-

ture in

the IRNA binding

site, and so is

hydrolyzed and released.

The

process

requires a continual

cycle

linkage

and

hydrolysis

until the correct

amino acid

is transferred to

the IRNA.

classic example in which

discrimination

between amino acids

depends on the

presence

of IRNA is

provided

by the Ile-IRNA

synthetase

of. E. coli. The enzyme can

charge valine

with

AMP, but hydrolyzes the valyl-adenylate

when

,ql.{4rle

added.

The

overall error rate

depends

on the specificities of

the

individual

steps, as

summarized

in i::i.i;c,i.$.lr

t;i.:i!.i. The

overall error

rate of I .5

I 0-5 is less than

the measured rate

at which

valine

is substituted for

isoleucine

(in

rabbit

globin),

which ranges

from 2 to 5

I04.

So mischarging

probably

provides

only a small

fraction

the

errors that actually

occur

pro-

tein synthesis.

Ile-IRNA synthetase uses

size as a basis for

discrimination among amino

acids.

i:ti.ijl:i,r

i-i

:,:.i

shows that it has two

active sites: the synthetic

(or

activation) site and the editing

(or

hydrolytic)

site. The crystal structure

of the enzyme shows

that the synthetic site is too small

to allow

leucine

close analog of isoleucine)

to enter. All amino

acids larger than isoleucine

are excluded from

activation because they cannot enter the

syn-

thetic site. An amino acid

that can enter the

synthetic site is

placed

on IRNA. Then

the

enzyme

tries to transfer it

to the editing site.

Isoleucine is

safe

from

editing because it is

too

large to enter the editing site. However,

valine

can

enter

this

site,

and

as a

result

an incorrect

Val-tRNAIle

hydrolyzed. Essentially

the

enzyme

provides

double

molecular

sieve, in

which size of the amino acid is used to discrim-

inate between closely related

species.

One interesting feature

Ile-IRNA

syn-

thetase is that the synthetic

and editing sites are

a considerable distance apafi,

-34

A. A crystal

structure

of the enzyme

complexed with an

edited analog of isoleucine shows

that the

amino

acid

transported

from

the synthetic site to the

editing site.

i';r,;i-:qi ;;.i.:

shows thatthis involves

a change in the conformation

of the IRNA.

The

amino acid acceptor stem of tRNAIl" can exist in

alternative

conformations.

It adoots an unusual

it;i.ri':i :.: r:ii The accuracy of

chargi

tRNAIte

by'its syn-

thetase depends on error control

at two stages.

irir: -i+i:r

i,'

r l Ite-tRNA synthetase

has two active

sites.

Amino

acids larger

than

Ile cannot be

activated because

they do

not fit in the synthetic

site.

Amino acids smalter

than

I[e

are

removed because

thev are

abte to enter

the

editinq site.

iii

iiiii:

r,l

iii:

amino

acid

transported

from the synthetic

site

to the editing site

of Ite-tRNA

synthetase

by a

change

the

conformation

of the amino

acceptor

stem

of IRNA.

Step

Frequency of Error

Activation

valine

11225

Val-AMPrre

Release of

Val-tRNA

11270

Overall rate of error

11225

11270

1/60,000

9.11

Synthetases

Use

Proofreading

Improve

Accuracy

hairpin in

order to be aminoacylated

amino

acid in the synthetic

site.

Then it

returns

to the more common helical

structure in order

to move the

amino acid to the editing

site.

The

translocation

between sites is

the

rate-limiting

step in

proofreading.

Ile-tRNA

synthetase is a

class I

synthetase, but the double

sieve

mech-

anism is

used also by class II synthetases.

Wild type:

UUG codon

read by Leu-tRNA

AUG

XX.O

UAA

or\

Leu

Nonsense mutant: UAG codon terminates

@ffii:l'"

Suppressor mutation: changes Tyr-tRNA

anticodon

AUG

Xog

UAA

Suppressor

tRNAs

Have

Mutated Anticodons

That

Read New

Codons

A suppressor

IRNA typicatty has a mutation in

the

anticodon

that changes the codons

to which

respon0s.

When the new

anticodon corresoonds to a

termination

codon, an amino

acid

is inserted

and

the

potypeptide

chain is extended

beyond the

termination

codon. This resutts in nonsense

suppression

at a site of nonsense mutation,

or in

readthrough

at a naturaI

termination codon.

Missense

suppression occurs when

the IRNA

recognizes

a different codon from

usuat. so that

one amino acid is

substituted for another.

Isolation

of mutant

tRNAs has been

one of the

most

potent

tools for analyzing

the ability of a

IRNA

respond

to its

codon(s) in nRNA,

and

for determining

the

effects that different

parts

of the

IRNA molecule have

on codon-anticodon

recognition.

Mutant

tRNAs are isolated

by virtue of their

ability

to overcome

the effects

mutations

genes

coding

for

proteins.

general

genetic

terminology,

a mutation

that is

able to over-

come the

effects of another

mutation is

called

a suppressor.

IRNA

suppressor systems,

the

primary

mutation

changes a

codon in an mRNA

so that

the

protein

product

is no longer functional.

The

secondary

suppressor

mutation

changes the

anticodon

a IRNA, so that it

recognizes

the mutant

codon instead

(or

well as)

its

original

target

codon. The amino

acid that is

now

inserted

restores

protein

function. The

sup-

pressors

are described

as nonsense

suppres-

sors

missense

suppressors,

depending on

the nature

of the

original mutation.

a wild-type

cell, a nonsense

mutation

recognized

only by

release

factor,

which ter-

minates protein

synthesis. The

suppressor

muta-

tion

creates

an aminoacyl-tRNA

that

can

recognize

the termination

codon;

by inserting

an amino

acid, it allows

protein

synthesis

CHAPTER

Using

the

Genetic

Code

i1:rii:ri -j , rr Nonsense mutations

can be

suppressed by

a IRNA with a mutant anticodon, which

inserts

an amino

acid at the

mutant

codon,

producing

fu[[

length

protein

in which

the original Leu residue has

been reptaced

Tyr.

continue beyond the

site of

nonsense

mutation.

This new

capacity of the translation

system

allows a full-length

protein

to be

synthesized,

as illustrated in i:iriiiii

r:.i:i'r.

the amino

acid

inserted

by suppression is

different from

the

amino acid that was

originally

present

at this

site

the wild-type

protein,

the activity

of the

protein

may

be altered.

Missense mutations

change a codon

repre-

senting

one amino acid into a

codon represent-

ing

another amino acid-one

that

cannot

function

in the

protein

place

of the original

residue.

(Formally,

any substitution

of amino

acids

constitutes a missense

mutation,

but in

practice

it is detected

only if it changes

the activ-

ity

of the

protein.)

The mutation

can be sup-

pressed

by the insertion

either of the

original

amino acid

or of some other amino

acid

that is

acceptable

to the

protein.

206

i,ii:,i,lilil

'j":,i'ri

demonstrates

that missense

sup-

pression

can be accomplished in

the same

way

as nonsense suppression,

by mutating

the anti-

codon of a IRNA

carrying an acceptable

amino

acid so that it responds to

the mutant

codon.

So missense

suppression involves

a change in

the meaning of the

codon from

one amino acid

to another.

There

Are Nonsense

Suppressors for Each

Termination

Codon

Each type of nonsense

codon is suppressed

tRNAs with mutant anticodons.

Some

rare

suppressor tRNAs have

mutations in

other

parts

of the motecule.

Nonsense suppressors fall into

three classes, one

Ior each type of termination

codon.

l']iii"il1{:

i.l-lr:

describes the

properties

of some

of the best char-

acterized suppressors.

The easiest to characterize]i^ave

been amber

suppressors.

In E.

coli, at least

six tRNAs have

been mutated to recognize

UAG codons. AII

the amber suppressor tRNAs have

the anticodon

CUAe, in

each case derived from

wild type by

a single base change. The

site of mutation can

be any one of the three

bases of the anticodon,

seen

from

supD, supE, and srzpf Each

sup-

pressor

IRNA recognizes

only the UAG codon,

instead

its former

codon(s). The amino acids

inserted are serine,

glutamine,

or tyrosine, the

same as those carried by the corresponding

wild-

type tRNAs.

Ochre suppressors also

arise by mutations

in the anticodon. The

best known are supC and

supG, which insert tyrosine

lysine

in response

to both ochre

(UAA)

and amber

(UAG)

codons.

This conforms with the

prediction

of the wob-

ble

hypothesis that

UAA cannot be recognized

alone.

A UGA suppressor has an

unexpected

prop-

erty. It is derived from

tRNArrp, but its only

mutation

the substitution of A in

place

of G

position

24. This change replaces

a G-U

pair

in the D stem with an A-U

pair,

increasing the

stability of the helix. The

sequence of the anti-

codon remains the same as the

wild

type, CCAe.

So the mutation in the D stem must in

some

way alter the conformation of

the anticodon

loop, allowing CCAe to

pair

with UGA

unusual wobble

pairing

of C with

A. The

611-i:r,:1.:

il.:.l.i

Missense suppression

occurs

when the anti-

codon of tRNA

is mutated so that

it responds

to the wrong

codon.

The

suppression

onLy

partial

because

both the

witd-type

IRNA

and the suppressor

IRNA

can respond

AGA.

l:+ijiti.

r,i,r.ri

Nonsense suppressor

tRNAs are

generated

mutations in the anticodon.

Locus

IRNA Wild

Type SuPPressor

Codon/Anti Anti/Codon

supD

(su1)

Ser

UCG

CGA

CUA UAG

supE

(su2)

Gln

CAG

CUG

CUA UAG

supF

(su3)

Tyr

UAg

cUA

UAG

supC(su4)

Tyr

UAg GUA

UUA

UAA

supG

(sus)

Lys

AAA

UUU

UUA UAA

supU

(su7)

Trp

UGG

CCA

UCA

9.13

There Are

Nonsense Suppressors

for

Each Termination

Codon

207

suppressor

IRNA continues to recognize its

usual

codon, UGG.

A related

response is seen with

a eukaryotic

IRNA. Bovine liver

contains a tRNAser with the

anticodon

-CCA'.

The wobble rules

predict

that

this IRNA should respond

to the trypto-

phan

codon UGG, but in fact it responds

to the

termination

codon UGA. So it is

possible

that

UGA

suppressed naturally in

this situation.

The

general

importance

of these observa-

tions lies in

the demonstration that

codon-anti-

codon recognition

of either wild-type

mutant

IRNA cannot

predicted

entirely from the rel-

evant

triplet sequences, but is influenced

other features

of the

molecule.

There is an interesting

difference between the

usual recognition of a codon

by its

proper

aminoacyl-IRNA and the situation in

which

mutation allows a suppressor IRNA

to recog-

nize a new codon. In the wild-type

cell, only

one

meaning

can be attributed to a

given

codon,

which represents either a

particular

amino acid

or a

signal

for

termination.

a cell carrying

suppressor mutation, though, the mutant

codon

has the alternatives of being recognized

by the

suppressor IRNA or of being

read

with its

usual

meanrng.

A nonsense suppressor

IRNA must com-

pete

with the release factors that recognize

the

termination

codon(s).

A missense

suppressor

IRNA

must

compete with the tRNAs

that

respond

properly

to its new

codon. The extent

of competition influences

the efficiency

of sup-

pression;

thus the effectiveness

of a

particular

suppressor depends not

only on the

affinity

between its anticodon and the target

codon, but

also on its concentration in

the cell, and

on the

parameters governing

the competing

termina-

tion or

insertion

reactions.

The efficiency

with

which any

particular

codon

is read

is influenced by its location.

Thus

the extent of nonsense

suppression

by a

given

IRNA

can vary

quite

widely, depending

on the

context of the codon. We

do not understand

the effect that neighboring

bases in nRNA have

codon-anticodon recognition,

but the con-

text can change the frequency

with

which a

codon is recognized

by a

particular

IRNA by

more than an

order of

magnitude.

The

base on

the 3'side

of a codon appears

have

partic-

ularly strong effect.

A nonsense

suppressor is isolated

by its

abil-

ity

to respond to a mutant nonsense

codon.

The

same triplet

sequence, however,

constitutes one

of the normal

termination signals

of the cell!

The mutant

IRNA that suppresses

the nonsense

mutation

must in

principle

be able to

suppress

natural termination

at the end of

any

gene

that

uses this codon.

iriiiJfri: 5.:+ shows

that this

readthrough results in

the synthesis

of a longer

protein,

with additional

C-terminal

material.

The

extended

protein

will end ar

the next ter-

mination

triplet sequence found

in the

phase

of the reading frame.

Any extensive

suppres-

sion

of termination is likely

to be deleterious

the cell by

producing

extended

proteins

whose

functions

are thereby

altered.

Amber

suppressors

tend to be relatively

effi-

cient, usually

in the range

of l0o/o to 50oh,

depending

on the system. This

efficiency

pos-

sible

because amber codons

are used relatively

Suppressors May

Compete

with

Wil.d-Type

Reading

of the Code

Suppressor tRNAs compete with

witd-type tRNAs

that have

the same anticodon

read

the

corresponding

codon(s).

Efficient

suppression is

deteterious because it

resutts

in readthrough

past

normaI termination

co00ns.

The

UGA codon is leaky

and

is misread

by Trp-tRNA

at 1%

to 3olo

frequency.

riiliiiri;

i,i.iili

Nonsense

suppressors

atso read

through

:*ili!::'Jl'#li'l

il|3.'''

svnthesi zi ns

protei

ns th at

CHAPTER

Usinq the

Genetic

Code

Wild-type

translation

AUG

Release

factor terminates

synthesis

at stop codon

UAG

AUG

Suppressor tRNA

reads

UAG codon

and

protein

is extended

next stop

codon

UAG

AUC

/'f

ry..

Tyr

208

infrequently

to terminate

protein

synthesis in

coli.

Ochre suppressors

are

difficult

to isolate.

They

are always

much

less

efficient,

usually

with activities

below

I0%.

AII

ochre suppres-

sors

grow

rather

poorly,

which

indicates

that

suppression

of both

UAA and

UAG

is damaging

Io E. clli,

probably

because

the

ochre codon

used most

frequently

a natural

termination

signal.

UGA is the least

efficient

of the rermina-

tion

codons in its natural

function;

it is misread

by ftp-tRNA

as frequently

3 % in wild-

type

situations. In

spite

of this deficiency,

how-

ever, it is used more

commonly

than the

amber

triplet

to terminate

bacterial

genes.

One

gene's

missense

suppressor

is likely

be another

gene's

mutator.

A suppressor

cor-

rects

mutation

by substituting

one

amino acid

for

another at the

mutant

site. In

other loca-

tions, though,

the same

substitution

will replace

the wild-type

amino

acid

with a new

amino

acid. The change

may inhibit

normal protein

function.

This

poses

a dilemma

for the

cell: it must

suppress what

is a mutant

codon

one

loca-

tion

while failing

to change

too extensively

its

normal

meaning

at other locations.

The absence

of any

strong missense

suppressors

therefore

explained

by the damaging

effects

that would

be caused

by a

general

and

efficient

substitu-

tion

of amino acids.

A mutation

that creates

a suppressor

IRNA

can have

two consequences.

First,

it ailows

the

IRNA to recognize

a new

codon.

Second, it

sometimes

prevents

the IRNA

from recogniz-

ing

the codons to

which it

previously

responded.

It is significant

that all

the high-efficiency

amber

suppressors are derived

by mutation

of one copy

of a redundant

IRNA set.

In these

cases, the

cell

has

several tRNAs

able to respond

to the

codon

originally recognized

by the wild-type

IRNA.

Thus the mutation

does not

abolish

recognition

the old codons, which

continue

to be

served

adequately by

the tRNAs

of the

set. In the

unusual

situation in which

there is

only a sin-

gle

IRNA that responds

to a

particular

codon,

any

mutation

that

prevents

the response

is lethal.

Suppression is most

often

considered in

the

context of a mutation

that

changes the reading

of a codon. There

are, however,

some

situations

which a stop codon

is read

as an amino acid

at a low frequency in

the

wild-type

situation.

The

first example to

be discovered was

the coat

protein gene

of the RNA

phage

Q0.

The forma-

tion

infective

particles

requires

that the

stop codon at the

end

of this

gene

is suppressed

at a low frequency

generate

a small

propor-

tion of coat

proteins

with a C-terminal exten-

sion. In

effect, this stop codon

is leaky.

The

reason

is that Tfp-IRNA recognizes the codon at

a low frequency.

Readthrough

past

stop codons also occurs

eukaryotes, where it is employed most often

by RNA viruses. This may involve the suppres-

sion

of UAG/UAA by

Tyr-tRNA,

Gln-tRNA, or

Leu-tRNA,

or the suppression

of UGA by Trp-

IRNA or Arg-tRNA. The extent of

partial

sup-

pression

dictated by the context

surrounding

the

codon.

209

The Ribosome

Influences

the

Accuracy of

TransLation

The

structure of the

165 rRNA at the P and A sites

the

ribosome

influences the

accuracv of

transtation.

The lack

of detectable

variation when the

sequence of a

protein

analyzed

demonstrates

that

protein

synthesis

must be extremely accu-

rate.

Very few mistakes are

apparent in the form

of substitutions of one amino acid

for another.

There are two

general

stages

protein

synthe-

sis at which

errors

might be made

(see

Figure 8.8

Section 8.3, Special

Mechanisms Control the

Accuracy

Protein Synthesis):

Charging a IRNA

only with its correct

amino acid clearly

critical.

This is a

function

of the

aminoacyl-tRNA syn-

thetase. The error

rate

probably

varies

with the

particular

enzyme, but

gen-

eral mistakes occur

<1/105

amino-

acylations.

The specificity of codon-anticodon

recognition is crucial, but

puzzling.

Although binding

constants vary with

the individual codon-anticodon

reac-

tion, the specificity

always

much too

Iow to

provide

error rate of

<10-5.

When free in solution,

tRNAs bind to

their trinucleotide

codon sequences

only

rather weakly.

Related, but erroneous,

triplets

(with

two

correct bases out of

three)

are recognized

l0-I to l0-2 times

as efficiently as

the correct triplets.

Codon-anticodon

base

pairing

therefore

seems to be a weak

point in

the

accuracy of

9.15

The Ribosome

Influences the

Accuracv of Transtation

translation. The ribosome has an

important role

controlling the

specificity of this interaction:

It functions directly or

indirectly as a

"proof-

reader" in order to distinguish

correct and incor-

rect codon-anticodon

pairs,

thus amplifying

the

rather modest intrinsic difference by

-I000x.

In addition to the

role

of the

ribosome itself,

the

factors

that

place

initiator- and aminoacyl-

tRNAs

the

ribosome also may influence the

pairing

reaction.

There must be some mechanism

for

stabi-

Iizing

the correct aminoacyl-tRNA,

allowing

its

amino acid to be accepted as a substrate

for

receipt

of the

polypeptide

chain; contacts with

an incorrect aminoacyl-tRNA

must

rapidly

broken, so that the complex leaves without

reacting. Suppose that there is no specificity

the

initial

collision between

the aminoacyl-

The correct IRNA interacts with rRNA

lnteraction

An incorrect IRNA

diffuses out

FI6URE 9.27

Any aminoacyltRNA

can be

placed

the

site

(by

EF-Tu), but onty one that

pairs

with

the anticodon

can

make

stabilizing contacts with rRNA. In

the absence

of these contacts, the aminoacv[-tRNA

diffuses out of tne

A site.

CHAPTER

9 Using

the Genetic Code

IRNA-EF-Tu-GTP

complex and the ribosome.

If any complex,

irrespective of its IRNA, can

enter the

site,

the number of

incorrect entries

must far exceed

the number of correct entries.

There are two

basic models for how the

ribosome

might discriminate

between correctly

and

incorrectly

paired

aminoacyl-tRNAs. The

actual situation

incorporates

elements of both

models.

The direct

recognition model supposes

that

the structure of

the ribosome is

designed

to recognize aminoacyl-tRNAs

that are correctly

paired.

This

would

mean that

the correct

pairing

results in

some

small change

in the

conformation

of the

aminoacyl-tRNA that the

ribo-

some can

recognize. Discrimination

occurs before

any further reaction

occurs.

The kinetic

proofreading

model

pro-

poses

that there are at

least

two stages

in the

process,

so that the aminoacyl-

IRNA has multiple opportunities to

disengage.

An incorrectly

paired

aminoacyl-tRNA

may

pass

through

some stages

of the reaction before it is

rejected. Overall

selectivity can in

prin-

ciple be the

product

of the selectivities

at each stage.

FIfr

URE 9.27 illustrates diagrammatically what

happens to correctly and

incorrectly

paired

aminoacyl-tRNAs.

A correctly

paired

amino-

acyl-tRNA is able to

make

stabilizing contacts

with

rRNA. An incorrectly

paired

aminoacyl-

IRNA does

not make these contacts, and there-

fore is able to diffuse out of the

site.

The

path

to discovering these

interactions

started with investigations of the effects of the

antibiotic streptomycin

in the I960s.

Strepto-

mycin inhibits

protein

synthesis by binding to

i65 rRNA and inhibiting the ability of EF-G to

catalyze translocation.

also

increases

the level

misreading

the

pyrimidines

and

(usu-

ally one is mistaken

for

the other, occasionally

for A) . The site at which streptomycin acts is

influenced by the S I2

protein;

the sequence of

this

protein

is altered in resistant mutants. Ribo-

somes with an S

protein

derived from resist-

ant bacteria show

a reduction in

the

level

misreading compared with wild-type ribosomes.

In effect, S l2 controls the

level

of misreading.

When

is mutated to decrease misreading, it

suppresses the effect of streptomycin.

I 2

stabilizes the structure of I 6 S rRNA in

the region that is bound by streptomycin. The

important

point

to note here is that the P/A site region

2to