Lewin Benjamin (ed.) Genes IX

influences the

accuraqt

of translation:

translation

can

be

made more

or less accurate

by changing

the

struc-

ture

of

165

rRNA. The

combination

of

the effects

of the

Sl2

protein

and

streptomycin

on the

rRNA

structure

explains

the behavior

of differ-

ent

mutants in

S 12,

some

of which

even make

the ribosome

dependent

onthe

presence

of

strep-

tomycin for

correct

translation.

We

now know

from

the crystal

structure

of

the ribosome

that l65

rRNA

is in

a

position

ro

make contacts

with

aminoacyl-tRNA.

TWo bases

of l65 rRNA

can

contact

the minor

groove

of

the helix formed

by

pairing

between

the anti-

codon in

IRNA

with the first

two

bases

of the

codon in mRNA.

This

directly

stabilizes

the struc-

ture

when the

correct

codon-anticodon

con-

tacts are made

at the

first two

codon

positions,

but it does

not monitor

contacts

at the third

position.

The

stabilization

of correctly

paired

amino-

acyl-tRNA may

have two

effects.

By holding

the

aminoacyl-tRNA

in

the A site,

it

prevents

it from

escaping

before the

next stage

of

protein

syn-

thesis. The

conformational

change in

the rRNA

may help to

trigger the

reac-

tion,

which is the hydrolysis

of GTP

by EF-Tu.

Part of

the

proofreading

effect is determined

by timing.

An aminoacyl-tRNA

in

the A site

may

in effect

be trapped if

the next

stage

of

pro-

tein synthesis

occurs

while it

is there.

Thus a

delay between

entry into

the

A site and

pep-

tidyl

transfer may

give

more

opportunity

for a

mismatched

aminoacyl-tRNA

to dissociate. Mis-

matched

aminoacyl-tRNA

dissociates

more rap-

idly than

correctly matched

aminoacyl-tRNA,

probably

by a factor

of

-5x.

Its chance

of escap-

ing is therefore

increased

when

the

peptide

transfer

step is

slowed.

The

specificity

of decoding

has

been

assumed to reside

with the ribosome

itself, but

some recent results

suggest

that

translation fac-

tors influence

the

process

at

both the P

site and

A

site. An indication

that

EF-Tu is

involved in

maintaining

the reading

frame

is

provided

by

mutants

of

the

factor

that suppress

frameshift-

ing.

This implies

that EF-Tu

does not

merely

bring aminoacyl-tRNA

to the A

site, but also is

involved in

positioning

the incoming

amino-

acyl-IRNR relative

to the

pepridyl-tRNA

in the

P site.

A

striking case in which

factors

influence

meaning is found

at initiation.

Mutation

of the

AUG initiation

codon

to UUG in

the

yeast gene

Il1S4

prevents

initiation.

Extragenic

suppressor

mutations

can

be

found

that

allow

protein

syn-

thesis

to be initiated at

the mutant

UUG codon.

TWo

of these suppressors

prove

to be in

genes

coding for

the u and

p

subunits of eIF2,

the fac-

tor that

binds Met-tRNA to the P site.

The muta-

tion in eIFp2 resides in a

part

of the

protein

that

is almost

certainly

involved in

binding

nucleic

acid. It

seems likely that its target

is

either the

initiation

sequence of

mRNA as such or the

base-paired

association between the

mRNA

codon and tRNAlMet anticodon. This suggests

that

eIF2

participates

in the discrimination of

initiation

codons as well as bringing the

initia-

tor IRNA to the P site.

The

cost of

protein

synthesis

in terms of

high-energy

bonds may be

increased

by

proof

-

reading

processes.

An important

question

in

calculating the cost of

protein

synthesis

is

the

stage at

which the decision

is taken on whether

to accept

a IRNA.

If

a decision

occurs immedi-

ately to release an aminoacyl-tRNA-EF-TUGTP

complex,

there is

little

extra

cost for rejecting the

large

number of incorrect tRNAs that are

likely

(statistically)

to enter

the A site before the cor-

rect 1RNA is recognized. If, however, GTP is

hydrolyzed

before the

mismatched aminoacyl-

IRNA dissociates, the cost will be

greater.

A mis-

matched

aminoacyl-tRNA

can be rejected either

before or after the cleavage of GTP,

although

we

do not know

yet

where on average

it is

rejected.

There is some evidence

that the use

of GTP in vivo is

greater

than the three

high-

energy bonds that are used

in adding every

(cor-

rect)

amino acid to the chain.

Recoding Changes

Codon

Meanings

.

Changes in codon meaning can

be caused by

mutant tRNAs or by tRNAs

with special

properLies.

.

The reading frame can be changed by

frameshifting or bypassing, both

of which

depend

on

properties

of the

mRNA.

The reading frame of a

messenger usually is

invariant.

Tlanslation starts

at an AUG codon

and continues in triplets to a

temination codon.

Reading

takes

no notice of sense:

insertion or

deletion of a base causes

a frameshift

mutation,

in

which

the reading frame

is changed beyond

the

site of

mutation. Ribosomes and

tRNAs con-

tinue ineluctably in triplets, synthesizing

an

entirely different series of amino

acids.

There are

some

exceptions

to the usual

pat-

tern

of

translation that enable

a reading frame

with an interruption of

some sort-such

as a

9.16 Recoding Changes Codon

Meanings ztt

is

caused by

mutated anticodon

Special

factor

+

IRNA recognizes codon

s+cvs\sg

Nil

8ilil

NNNNNUGANNNNNNNNNN

a:{i-jei

'-:.:l:l

A mutation in an individuat tRNA

(usuatty

in

the anticodon)

can suppress the usuaI meaning ofthat

codon. In a spec'iaI case, a specific IRNA

is

bound by an

unusual elongation

factor

to

recognize

a termination

codon adjacent to a

hairpin

[oop.

nonsense

codon or

frameshift-to

be translated

into a full-length

protein.

Recoding events are

responsible for making exceptions to the usual

rules,

and can involve several types of events.

Changing the meaning of a single codon

allows one amino acid to be substituted

in

place

of another, or for an amino acid to be inserted

at a termination codon.

f.i*tiFtE *"t:s

shows that

these changes rely on the

properties

of an

indi-

vidual IRNA

that

responds to the codon:

.

Suppression

involves recognition

of

a

codon by a

(mutant)

IRNA that usually

would respond to a

different codon

(see

Section

9.I2,

Suppressor tRNAs

Have

Mutated Anticodons That Read New

Codons).

.

Redefinition of the meaning of a codon

occurs when an aminoacyl-tRNA

is

modified

(see

Section 9.8,

Novel

Amino

Acids Can Be Inserted at

Certain

Stop

Codons).

Changing the reading frame

occurs

in two

types

of situations:

.

Frameshifting typically involves

chang-

ing

the

reading frame

when aminoacyl-

IRNA slips by one base, either

+l

forward

or

-l

backward

(see

the following sec-

tion, Frameshifting Occurs at Slippery

Sequences). The result

shown

in

a:*i,jt[

*.iF is that translation continues

past

a termination codon.

.

Bypassing involves

a

movement

of

the

ribosome

to change the codon that is

paired

with the

peptidyl-tRNA

in

the

Usinq

the Genetic Code

-1

frameshift in

HIV retrovirus

NNNNUUU GNNNNNNNN

Last codon

read in initial

reading frame

odon read

in new reading lrame

Reading

without f rameshift

NNNNUUUUUUAGGNNNNNNNN

Reading after

frameshift

NNNNUUU

F5*tjftil *.h-* A IRNA that stips one base

in

pairing

with

a codon causes

a frameshift that can suppress termina-

tion.

The

efficiency

is usua[[y

-5%.

agGti** 9"3* Bypassing occurs when the ribosome moves

atong

mRNA

so that

the

peptidyt-tRNA

in the P

site

is

reteased from

pairing

with its

codon

and

then

repairs with

another codon

farther

alonq.

P

site.

The sequence between

the two

codons fails to be represented in

pro-

tein. As shown

in F3fitiRF.

+.3{1, this

allows translation to continue

past

any

termination codons in the intervenins

region.

60 nucleotide bypass

in

phage

T4

gene

60

Last codon

in

original

reading frame

odon in new

reading frame

Reading

without frameshift

GAUGGAUGAC............AUUGGAUU

Reading

after

frameshift

GAUGGAUGAC...,......,.AUUGG

212 CHAPTER

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ypgl-1Ip;1de6

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The

sequence

of

the mRNA

triggers

the

blpass. The

important

features

are the

two

GGA

codons

for take-off

and landing,

the

spacing

between

them,

a stem-loop

structure

that

includes

the

take-off

codon,

and the

stop

codon

adjacent

to the

take-off

codon.

The

protein

under synthesis

is

also involved.

The

take-off

stage requires

the

peptidyl-tRNA

to unpair from

its

codon. This

is followed

by a

movement

of the

nRNA

that

prevents

it

from

re-pairing.

Then

the ribosome

scans

the mRNA

until the

peptidyl-tRNA

can repair with

the codon

in the landing

reaction.

This

is followed

by the

resumption

of

protein

synthesis when

amino-

acyl-tRNA

enters the A

site in

the

usual way.

Like frameshifting,

the bypass

reaction

depends

on a

pause

by

the ribosome.

The

prob-

ability that

peptidyl-tRNA

will dissociare

from

its

codon in the

P site is increased

by

delays in

the entry

of aminoacyl-tRNA

into

the A site.

Starvation

for an

amino

acid can

trigger

bypass-

ing

in bacterial

genes

because

of the

delay that

occurs

when there is

no aminoacyl-tRNA

avail-

able to enter

the A

site. In

phage

T4

gene

60,

one role

of mRNA

structure

may

be to reduce

the

efficiency

of termination,

thus creating

the

delay that is needed

for

the take-off

reaction.

@

Summary

The

sequence of mRNA

read

in triplets

5'

-+

3'

is related

by the

genetic

code to

the amino acid

sequence of

protein

read

from

N- to C-terminus.

Of the

sixty-four

triplets,

sixty-one

code for

amino acids

and three

provide

termination

sig-

nals.

Synonym

codons that

represent

the same

amino acids

are related,

often

by a change

in

the third

base of the

codon.

This third-base

degeneracy,

coupled

with a

pattern

in

which

tend to

be coded

by

the

effects of mutations.

The

genetic

code is universal

and

must have

been

established

very early

in evolution.

Changes in

nuclear

genomes

are rare,

but

some changes

have occurred

during mitochondrial

evolution.

Multiple

tRNAs

may respond

to

a

particu-

lar

codon. The

set of tRNAs

responding

to the

various

codons for

each amino

acid is

distinc-

tive for

each organism.

Codon-anticodon

recog-

nition involves

wobbling

at

the first

position

of

the anticodon

(third

position

of the codon),

which

allows some tRNAs

to recognize

multi-

ple

codons. AII tRNAs

have modified

bases,

introduced

by

enzymes that

recognize

targel

bases in the IRNA

structure.

Codon-anticodon

pairing

is influenced

by

modifications

of the

anticodon itself

and also by the context of adja-

cent

bases, especially

on the

3'side

of the anti-

codon.

Taking

advantage of codon-anticodon

wobble

allows vertebrate mitochondria to use

only twenty-two

tRNAs to

recognize

all codons,

compared

with the usual minimum of thirty-one

tRNAs; this is

assisted by the changes

in

the

mitochondrial

code.

Each

amino

acid

is recognized by a

partic-

ular aminoacyl-tnNl

synthetase, which also

recognizes

all of the tRNAs coding for that amino

acid. Aminoacyl-tRNA

synthetases

have

a

proof-

reading

function that scrutinizes the amino-

acyl-IRNA products

and

hydrolyzes incorrectly

joined

aminoacyl-tRNAs.

Aminoacyl-tRNA

synthetases vary widely,

but fall into

two

general

groups

according to the

structure

of the catalytic domain. Synthetases of

each

group

bind the IRNA from the side, making

contacts

principally

with the

extremities of the

acceptor

stem and the anticodon stem-loop; the

two types of

synthetases

bind tRNe from oppo-

site

sides. The relative importance attached to the

acceptor stem

and the anticodon

region for spe-

cific recognition

varies with

the individual IRNA.

Mutations

may allow a

IRNA to read dif-

ferent

codons; the most common

form

of such

mutations

occurs

in

the anticodon

itself. Alter-

ation of its specificity may allow a IRNA to sup-

press

a mutation in a

gene

coding

for

protein.

A

1RNA that recognizes a termination codon

provides

a nonsense suppressor; one

that

changes the

amino acid

responding to a codon

is

a

missense

suppressor. Suppressors of

UAG

and UGA

codons

are more efficient than those

of UAA codons, which

is

explained by

the fact

that UAA is the most commonly used

natural

termination

codon.

The efficiency of all sup-

pressors,

however, depends on

the

context of

the

individual target

codon.

Frameshifts

of the

+l

type

may be caused

by aberrant tRNAs that read

"codons"

of

four

bases. Frameshifts

of

either

+l

or

-l

may

be

caused

by slippery sequences

in nRNA that

allow a

peptidyl-tRNA

to slip

from its codon to

an overlapping

sequence

that can also

pair

with

its anticodon. This frameshifting also requires

another sequence that causes

the ribosome to

delay. Frameshifts determined

by the nRNA

sequence may be required

for

expression

of nat-

ural

genes.

Bypassing

occurs when a ribosome

stops translation and

moves along mRNA with

its

peptidyl-tRNA

in the P site until the

peptidyl-tRNA pairs

with an

appropriate codon;

then translation resumes.

9.19 Summary 275

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The effect

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Wobbting

Resea rc h

Crick,

F. H

C.

(1966).

Codon-anticodonpairing:

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rxl\As Are

Processeo from ronger

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Review

Hopper, A. I(. and Phizicky,

E. M.

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IRNA

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IRNA Contains

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Reviews

Hopper, A. I(. and

Phizicky, E. M.

(2003).

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Dev. 17,

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ng

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Bjork, G. R.

(

I 987

)

. Tlansfer RNA modification.

Annu. Rev. Biochem.56,

26j-287

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There Are

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Code

Reviews

Fox, T. D.

(1987).

Natural

variation in the

genetic

code.

Annu. Rev.

Genet

21,

67-91.

Osawa, S et al.

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Recent

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229-264.

Novel Amino Acids

Can

Be Inserted

at Certain Stop Codons

Bock, A.

(1991

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Soll,

D.

(2004).

Aminoacyl-tRNAs:

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CHAPTER 9 Using

the Genetic Code

@

Reviews

Resea

rc h

Fagegaltier,

D.,

Hubert,

N.,

Yamada,

I(., Mizutani,

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Iftol, A.

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Characteri-

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tion

factor for selenoprotein

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EMBO J.19,4796-4805.

Hao, B., Gong, W.,

Ferguson,

T. I(.,

James, C.

M.,

ICzycki, J. A., and Chan,

M. K.

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A

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UAG-encoded

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Srinivasan,

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C. M., and

Iftzycki,

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A.

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Pyrrolysine

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Synthetases

Review

Schimmel,

P.

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Parameters

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molecular

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28,

27 47-27 59.

Aminoacyt-tRNA Synthetases

Fa[[

into Two Grouos

Review

Schimmel,

P.

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Aminoacyl-tRNA syn-

thetases:

general

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Rev. Biochem.56,

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Res ea rc h

Rould, M. A. et al.

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Structure of

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1p54cln and

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246, rr)5-1142.

Ruff

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M. et al.

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l99l

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Class

II

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yeast

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252, 1682-1689.

Synthetases

Use Proofreading to Improve

Accuracy

Review

Jakubowski,

H.

and

Goldman, E.

(19921.

Editing of

errors in selection of

amino acids for

protein

synthesis. Microbiol

Rev.

56,

412-429.

Resea rc h

Dock-Bregeon, A., Sankaranarayanan, R., Romby,

P., Caillet, J., Springer,

M., Rees,

B., Franck-

lyn,

C. S.,

Ehresmann, C., and Moras, D.

(2000).

Transfer

RNA-mediated

editing in

threonyl-tRNA synthetase.

The

class II solu-

tion to the double discrimination oroblem. Cel/

103,877-884.

Hopfield, J. J.

(1974lr.I(ineticproofreading:

a new

mechanism

for reducing

errors

in

biosynthetic

processes

requiring high specificiry. Proc. Natl.

Acad. Sci. USA7l,

4lj5-41)9.

276

Suppressors

May

Compete

with WiLd-type

Frameshifting

0ccurs at SLippery

Jakubowski,

H.

(1990).

Proofreading

in

vivo:

edit-

ing

of homocysteine

by

methionyl-rRNA

syn-

thetase in

E. coli. Proc

Natl. Acad.

Sci

USA 87,

4504-4508.

Nomanbhoy, T. I(.,

Hendrickson,

T. L.,

and

Schim-

mel, P.

(1999).

Tiansfer

RNA-dependent

translocation

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misactivated

amino

acids to

prevent

errors

in

protein

synthesis.

Mol.

Cell 4,

519-528.

Nureki,

O. et al.

(1998).

Enzyme

structure with

two catalytic

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double

sieve

selection

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substrate.

Science 280,

578-58

l.

Silvian, L. F.,

Wang,

J., and

Sreitz, T.

A.

(1999).

Insights

into

editing from

an Ile-tRNA

syn-

thetase structure

with

tRNAIle

and mupirocin.

Science 285, lO7

4-1077.

Reading

of

the Code

Reviews

Atkins,

J. F.

(I991).

Towards

a

genetic

dissection

of

the basis

of triplet

decoding,

and its

natural

subversion:

programmed

reading

f rameshifts

and hops. Annu.

Rev.

Genet. 25, 2OI-228.

Beier, H.

and Grimm,

M.

(2001).

Misreading

of

termination

codons in

eukaryotes

by natural

nonsense

suppressor

tRNAs.

Nucleic Acids Res.

29,47674782.

Eggertsson,

G. and

Soll, D.

(

I 988

)

. Ttansfer

RNA-

mediated

suppression

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codons

in

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Rev.52,354-374.

Murgola,

E.

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IRNA, suppression,

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cod,e. Annu. Rev.

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57-80.

Normanly,

J. and Abelson,

J.

(

I 989).

Transfer

RNA

identity.

Annu Rev.

Biochem.

58, 1029-1049.

Resea

rch

Hirsh, D.

(197

ll . Tryptophan

rransfer RNA

as the

UGA suppressor.

J. Mol. Bi01.58,419-458.

Weiner,

A. M. and

Weber, K.

(1973).

A

single

UGA

codon functions

as a natural

termination

sig-

nal in the

coliphage

q

beta coat

protein

cistron.

J. Mol. Biol.80,

837-855.

The Ribosome

Inftuences

the Accuracv

of Translation

I(urland,

C. G.

(1992).

Translational

accuracy

and

the fitness

of bacteria. Annu.

Rev.

Genet.26.

29-50.

Ogle,

J.

M.,

and Ramakrishnan,

V.

(2005).

Sftuc-

tural

insights

into

translational

tidelity. Annu.

Rev.

Biochem. 7 4,

129-177.

Ramakrishnan,

V.

(2002).

Ribosome structure and

the mechanism

of translation. Cell

108.

557-572.

Resea rc h

Carter, A. P.,

Clemons, W.

M., Brodersen, D. E.,

Morgan-Warren, R.

J., Wimberly,

B. T.,

and

Ramakrishnan,

V.

(2000).

Functional

insights

from

the structure of

the

30S ribosomal

sub-

unit and its interactions

with antibiotics.

Nature

407,340-348.

Ogle, J. M., Brodersen, D. E., Clemons,

W.

M.,

Tarry

M. J.,

Carter,

A. P., and Ramakrishnan, V

(2001).

Recognition of cognate transfer RNA

by the 30S ribosomal

subunit. Science

292,

897-902.

Sequences

Reviews

Farabaugh,

P. J.

(1995).

Programmed translational

frameshifting . Microbiol. Rev. 60, lO3-134.

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P.

J. and

Bjorkk,

G.

R.

(1999).

How

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Gesteland, R. F.

and

Atkins,

J.

F.

(1996).

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Annu

Rev.

Biochem. 65, 7 41-7 68.

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Jacks,

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Power, M. D., Masiarz,F.R.,

Luciw, P. A.,

Barr,

P. J., and Varmus,

H. E.

(1988).

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280-28).

Bypassing Invotves Ribosome Movement

Review

Herr,

A. J., Atkins,

J.

F., and Gesteland,

R. F.

(2000).

Coupling

of open reading

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Annu. Rev. Biochem.

69,

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Resea rc h

Gallant,

J.

A.

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Lindsley, D.

(1998).

Ribosomes

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"hungry"

codons,

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protein

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elongation many

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Huang,

W. M.,

Ao,

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Reviews

References 2t7

Protein Localization

CHAPTER OUTLINE

Introduction

Passage

Across a Membrane Requires a SpeciaI

Apparatus

.

Proteins

pass

across

membranes

through specialized

protein

structures embedded in

the

membrane.

.

Substrate

proteins

interact

directty with the transport

appa-

ratus of the ER, mitochondria.

or chtoroplasts,

but require

carrier

proteins

to interact with

peroxisomes.

e

A

much

larger and comptex apparatus is required for trans-

oort

into

the nucleus.

tfaEf

Protein Translocation May Be Posttranslational

or CotranslationaI

.

Proteins

that are

imported into

cytoplasmic organettes are

synthesized on

free ribosomes

in the cytosot.

o

Proteins

that are

impoded

into the ER-Gotgi system are

synthesized

on

ribosomes

that are associated

with

the

ER.

.

Proteins associate

with

membranes

by

means

of specific

amino acid sequences

calted signaI sequences.

.

Signal sequences

are

most

often leaders that are located

at the N-terminus.

o

N-terminaI

signaI sequences are usuatly cleaved off the

protein

during the insertion

process.

tEE

Chaperones May Be Required for Protein Fotding

.

Protejns

that can acquire their conformation spontaneously

are said to self-assembte.

o

Proteins

can often assemble into

atternative structures.

.

A chaperone

directs a

protein

into

one

particular

pathway

by excluding atternative

pathways.

.

Chaperones

prevent

the formation

of

inconect structures

by interacting with

unfolded

prote'ins

to

prevent

them from

fotding incorrectty.

Chaperones Are Needed

by NewLy Synthesized and by

Denatured Proteins

.

Chaperones act

on

newly

synthesized

proteins, proteins

that

are

passing

through membranes,

or

proteins

that

have

been

denatured.

o

Hsp70

and some associated

proteins

form

a

major

class

of chaperones

that act

on

many

target

proteins.

r

Group I and

group

II

chaperonins are large oligomeric

assemblies that

act on target

proteins

they sequester in

internaI

cavities.

.

Hsp90

is a

speciatized chaperone that acts on

proteins

of

signaI transductjon

pathways.

278

The Hsp70 Famil.y Is Ubiquitous

o

Members of the

Hsp70 fami[y are

found in

the cytosol,

in

the

ER,

and

in mitochondria and

chloroplasts.

.

Hsp70 is a chaperone

that functions on target

proteins

in conjunction

with DnaJ and GrpE.

SignaI Sequences

Initiate

Transtocation

r

Proteins

associate

with the

ER system on[y cotranstationatty.

.

The signa[ sequence

of the substrate

protein

is responsibte

for membrane association.

The

Signal

Sequence

Interacts with the SRP

.

The signaI sequence binds

to the SRP

(signaI

recognition

paticte).

.

Signa[-SRP binding

causes

protein

synthesis to

pause.

r

Protein synthesis

resumes when the SRP binds to the SRP

receDtor in the membrane.

.

The

signal sequence

is cteaved

from

the translocating

pro-

tein by the signal

peptidase

located on the

"insjde"

face

of the

membrane.

The SRP

Interacts with the SRP Receptor

r

The SRP is a comptex of

75 RNA with six

proteins.

.

The bacteriaI equivalent

to the SRP

is

a comptex of 4.55

RNA

with two oroteins.

o

The

SRP

receptor is a dimer.

r

GTP

hydrolysis releases the SRP

from

the SRP

receptor

after

their interaction.

The

Translocon Forms a Pore

e

The Sec61 trimeric

complex

provides

the channel

for

proteins

to

pass

through a

membrane.

o

A transtocating

protein passes

directly

from

the

ribosome

to the translocon

without exposure to the cytosot.

Transtocation Requires

Insertion into the Translocon and

(Sometimes)

a Ratchet in the

ER

o

The ribosome,

SRP, and

SRP receptor are sufficient to insert

a

nascent

protein

into a transtocon.

.

Proteins

that

are inserted

posttranstationatty

require addi-

tionaI components

in the cytosol and Bip in the ER.

.

Bip is a ratchet that

prevents

a

protein

from

stipping

backward.

Reverse Transtocation

Sends

Proteins

to the Cytosol

for Degradation

.

Sec61 transtocons

can be used for

reverse

translocation

of

proteins

from

the ER into

the cytoso[.

Proteins

Reside in

Membranes

bv Means

of

Hydrophobic

Regions

r

Group I

proteins

have

the N-terminus

on

the

far

sjde

of the membrane; group

II

proteins

have

the opposite

orientation.

.

Some

proteins

have

multipte

membrane-

spanning

domains.

Anchor

Sequences

Determine

Protein

0rientation

o

An anchor

sequence

halts

the

passage

of

a

protein

through

the

transtocon.

Typi-

catty this is

located

at the C-terminaI

end

and resutts

in

a

group

I

orientation in

which

the

N-terminus

has

passed

through

the membrane.

r

A combined

signat-anchor

sequence

can

be

used to insert

a

protein

into

the

mem-

brane and anchor

the

site of insertion.

Typicatty

this

is

internaI

and results

in a

group

II orientation

in which

the

N-

terminus is

cytoso[ic.

How Do Proteins

Insert

into

Membranes?

r

Transfer

of transmembrane

domains from

the transtocon

into

the tipid

bitayer is

triggered

by the interactjon

of the

trans-

membrane

region

with

the translocon.

PosttranslationaI

Membrane

Insertion

Depends

on Leader

Sequences

o

N-termina[

leader

sequences

provide

the

information

that altows

proteins

to associ-

ate with mitochondriaI

or

chtoroolast

mem

branes.

A

Hierarchy

of Sequences

Determines

Location within

0rganelles

e

The N-terminal

part

of a leader

sequence

targets

a

protein

to the mitochondrial

matrix

or chloroptast

[umen.

o

An adjacent

sequence

can control further

targeting

to a

membrane

or the intermem-

0rane

sDaces.

r

The

sequences

are cleaved

successivety

from

the

protein.

Inner and

0uter Mitochondrial

Membranes

Have Different

Translocons

o

Transpoft

through

the

outer and inner

mitochondriaI

membranes

uses different

receptor

comptexes.

e

The TOM

(outer

membrane)

complex

is

a

large

comptex

in which

substrate

proteins

are directed to the

Tom40

channel by one

of two subcomptexes.

r

Different TIM

(inner

membrane)

complexes

are used depending on whether the

sub-

strate

protein

is targeted to the

jnner

membrane or to the lumen.

r

Proteins

pass

direct[y

from

the

TOM

to the

TIM comp[ex.

Peroxisomes

Employ Another Type of

Translocation

System

o

Proteins are imported into

peroxisomes

jn

their futly fotded state.

o

They have

either a

PTS1

sequence at the

C-terminus or a

PTS2

seouence at the

N-termi nus.

e

The receptor Pex5p binds the PTS1

sequence and the receptor PexTp binds

the

PTSZ

seouence.

o

The receptors

are cytosolic

proteins

that

shuttle

into

the

peroxisome

carrying a

substrate

protein

and then

return

to the

cytosoL.

Bacteria Use Both CotranstationaI

and PosttranslationaI Translocation

.

Bacterial

proteins

that

are

expoded to or

through

membranes

use both

posttransta-

tionaI and cotranslationaI

mechanisms.

The

Sec System

Transports Proteins into

and Through the Inner Membrane

.

The

bacteriat SecYEG

translocon in the

inner membrane is retated to the eukary-

otic

Sec61

translocon.

r

Various chaoerones are

invotved in

direct-

ing

secreted

proteins

to the trans[ocon.

Sec-Independent

Tra nstocation

Systems

in

E. coLi

t

E.

coli and organetles

have retated systems

for

protein

transtocation.

.

One system attows certain

proteins

to

insert into membranes without a trans[o-

cation apparatus.

r

YidC is homologous

to

a mitochondrial

system for transferring

proteins

into

the

inner membrane.

r

The

tat system transfers

proteins

with

a

twin arginine

motif into the

periplasmic

sDace.

Summarv

CHAPTER 10

Protein Localization 219

@

Introduction

Proteins

are synthesized

in

two

tlpes of location:

.

The vast majority of

proteins

are syn-

thesized by

ribosomes in the cytosol.

.

A

small

minority are sl,nthesized

by

ribo-

somes within organelles

(mitochondria

or chloroplasts).

Proteins synthesized

in

the

cytosol can be

divided into two

general

classes with

regard to

localization: those that are

not associated

with

membranes, and

those that are associated

with

membranes.

i-i:;ir::i ::J:

:

maps the cell in terms

of the

possible

ultimate

destinations

for a newly

synthesized

protein

and the systems that

trans-

port

it:

.

Cytosolic

(or

"soluble")

proteins

are not

localized in any

particular

organelle.

J;.::

r.::::

i i-t

:

Overview: Proteins

that are

locatized

posttranslationalty

are

released

into

the cytosoI after synthesis on free ribosomes. Some

have

sig-

nals

for targeting

to organeltes such as the nucteus or mjtochondria.

Pro-

teins

that are locatized cotranslatjonalty associate with the ER membrane

during

synthesis,

so their

ribosomes

are

"membrane

bound."

The

proteins

pass

into

the endoplasmic reticutum,

a[ong to the Gotgi apparatus,

and then

through the

plasma

membrane. untess they have signats that cause

reten-

tion

at one ofthe steps on the

pathway.

They may

also be

directed to other

organe[[es, such

as endosomes or lysosomes.

CHAPTER 10 Protein Localization

They are synthesized

in

the cytosol,

and

remain there,

where they

function as

individual

catalytic centers, acting on

metabolites

that are in solution

in

the

cytosol.

Macromolecular

structures may be

Iocated at

particular

sites

in

the cyto-

plasm;

for example,

centrioles are asso-

ciated

with the

regions that become the

poles

of

the mitotic spindle.

Nuclear

proteins

must be transported

from their

site of synthesis in the cytosol

through the

nuclear envelope into the

nucleus.

Most of

the

proteins

in cytoplasmic

organelles

are synthesized

in the cytosol

and transported

specifically to

(and

through) the organelle

membrane, for

example,

to the mitochondrion or

per-

oxisome or

(in plant

cells) to the chlo-

roplast.

(Those

proteins

that are

synthesized

within the organelle remain

within

it.)

The cytoplasm

contains a series of mem-

branous

bodies,

including

endoplasmic

reticulum

(ER),

Golgi apparatus, endo-

somes,

andlysosomes. This is sometimes

referred to as the

"reticuloendothelial

system."

Proteins that reside within these

compartments

are inserted into ER

membranes and then are directed to

their

particular

locations by the transport

system of

the Golgi apparatus.

Proteins that are secreted from the cell

are transported

to the

plasma

membrane

and then must

pass

through

it

to the exte-

rior. They start their s)'nthesis in the same

way

as

proteins

associated with the retic-

uloendothelial

system, but

pass

entirely

through the system

instead

of

halting

at

some

particular

point

within

it.

a

Coated vesicle transDort

Posttranslational

transDod

Passage Across

Membrane Requires

SpeciaI

Apparatus

r

Proteins

pass

across

membranes through

specialized

protein

structures embedded in

the membrane.

.

Substrate

proteins

interact directty with

the

transport apparatus

of the ER, mitochondria,

or

chtoroptasts,

but require carrier

proteins

to

interact

with

peroxisomes.

r

A much

larger and comptex apparatus

is required

for transport

into

the

nucteus.

220

Lewin Benjamin (ed.) Genes IX

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