Lewin Benjamin (ed.) Genes IX

The TtyC

arm is

named

for the

presence

of this

triplet rsequence. (y

stands for

pseudouridine,

a modified

base.)

The

anticodon

arm always

contains

the anticodon

tdplet in

the center

of the

loop.

The D

arm is.named

for

its content

of

the base dihydr:ouridine

(another

of the

modified

bases in

IRNA).

.

The

extra arm lies

between

the TyC

and anticodon

arms

and

varies

from

3

to 2 I

bases.

The numbering

system for

IRNA illustrates

the constancy

of the structure.

Positions

are

numbered

from 5'to

.l'according

to the most

common IRNA

structule,

which has 76 residues.

The

overall range of

IRNA lengths

is 74 to 95

bases. The variation

in length

is caused

by dif

-

ferences in

the D arm

and extra

arm.

The

base

pairing

that maintains

the second-

ary structure is

showrL in Figure

7.4. Within

a

given

IRNA,

most of the

base

pairings

are con-

ventional

partnerships

of

A-U

and

G-C. but

occasional G-U,

G-t+/, or A-y

pairs

are found.

The

additional

types of base

pairs

are less

sta-

ble than

the

regular pairs,

but

still allow a

double-helical

structure to form

in RNA.

When the

sequences

of tRNAs are

com-

pared,

the bases found

at some

positions

are

invariant

(or

conserved);

almost always

a

particular

base is founLd

at the

position.

Some

positions

are describecl

as semi-invariant

(or

semiconserved)

because

they are restricted

to

one type of base

(purine

versus

pyrimidine),

but either base

of that type may

be

present.

When

a IRNA is

charged with

the amino

acid corresponding

to its anticodon, it

is called

aminoacyl-tRNA.

The amino

acid is linked

by an ester bond from its

carboxyl

group

to the

2' or 3'hydroxyl

group

of the ribose

of the l'

terminal base of the

IRNA

(which

is

always ade-

nine). The

process

of charging

a IRNA is cat-

alyzed

by a specific enzyme,

aminoacyl-tRNA

synthetase. There are

(at

least) 20

aminoacyl-

IRNA synthetases.

Each recognizes

a single

amino acid and

all the tRNAs

on to which it can

legitimately

be

placed.

There is

at

least

rcne

IRNA

(but

usually

more) for

each amino acid. A

IRNA is named by

using the three-letter abbreviation

for the

amino

acid as a superscript.

If there is more

than one

IRNA for

the same amino acid.

subscript numer-

als are used to distinguish

them.

So

two

tRNAs

for tyrosine would

be clescribed as IRNAlrrr

3ni

tRNA2rvr. A IRNA carrying

an amino acid-that

is, an aminoacyl-tRNlr-is

indicated by a

pre-

i

:,,r,irl The meaning oftRNA

is determined by its anti-

codon and not by

its

amino acid.

fix

that

identifies the amino acid.

Ala-IRNA

describes tRy4ela carrying

its amino acid.

Does

the anticodon

sequence

alone allow

aminoacyl-tRNA to recognize the correct

codon?

A classic experiment to

test this

question

is illus-

trated in . Reductive

desulfuration

converts the amino

acid of cysteinyl-tRNA

into

alanine,

generating

alanyl-IRNAcv'.

The IRNA

has an anticodon that

responds to the

codon

UGU. Modification of

the amino acid does

not

influence

the specificity

of the

anticodon-codon

interaction,

so the

alanine residue

is incorporated

into

protein

in

place

of

cysteine. Once

a IRNA

has

been charged,

the amino

acid

plays

no

further

role

in its specificity,

which is determined

gaslusivpht

by the anticodon.

The

Acceptor Stem and

Anticodon

Are at

Ends

of

the

Tertiary Structure

o

The

ctoverleaf

forms an

L-shaped tertiary structure

with

the acceptor

arm at one

end and the

anticodon arm at

the other end.

The secondary structure

of each IRNA

folds into

a compact L-shaped tertiary

structure

in which

the 3' end that binds

the amino

acid is distant

from the anticodon

that binds

the mRNA.

AII

tRNAs have the same

general

tertiary

structure,

although they

are distinguished

by

individual

variations.

The base

paired

double-helical

stems of

the

secondary structure

are

maintained

in the ter-

tiary structure,

but their

arrangement

in three

dimensions essentially

creates

two double

helices at right angles

to

each other,

as

7

.4

lhe Acceptor Stem

and

Anticodon

Are at Ends of

the Tertiary Structure

131

Cloverleal

has four

arms

2D

projection

has two

perpendicular

duplexes

.'ii:1;:.::

.r.:--

Transfer RNA fotds into

a compact

L-shaped

tertiary

structure with the amino acid at one

end

and the

anticodon

at the other end.

i:,..:::r':

.'

A

space-fitting modeI

shows that the tRNAPhe

tertiary structure is

compact.

The

two views

of IRNA are

rotated

by 90". Photo

courtesy of Sung-Hou Kim,

Univer-

sity

of Ca[ifornia, Berke[ey.

illustrated in

iii;ijil,r. .:,i!. The

acceptor stem and

the TvC stem

form

one

continuous

double

helix

with a single

gap;

the

D

stem and anticodon

stem

form another continuous double helix,

also

with

a

gap.

The region between the dou-

ble helices, where

the turn in the L-shape is

made, contains the TyC

loop

and the D loop.

So the amino acid

resides at the

extremity of

one arm of the L-shape and the anticodon loop

forms the

other

end.

The tertiary structure

is

created by hydro-

gen

bonding, mostly

involving

bases that are

unpaired

in the

secondary

structure. Many

of

the invariant and semi-invariant bases are

involved in

these

H-bonds, which

explains their

conservation. Not every one of these interac-

tions

is universal, but

probably

they identify

the

general pattern

for establishing

IRNA

structure.

A molecular model of the

structure of

yeast

lpy4rhe

is

shown

in

i:ii:i.-:iii: ::.:.

The left

view

corresponds with the bottom

panel

in Figure 7 .6.

Differences in

the structure are

found

in other

tRNAs, thus accommodating the dilemma that

all tRNAs

must have

a similar shape.

yet

it must

be

possible

to recognize differences between

them.

For

example,

in tRNAe'p,

the angle

between the two axes

is

slightly

grearcr,

so the

molecule has a slightly more open conformation.

The structure suggests a

general

conclusion

about the

function

of IRNA:

Its

sites

for

exercising

particular

functions

are maximally separated. Th'e

amino acid is as far distant from

the anticodon

as

possible,

which

is consistent

with their roles

in

protein

synthesis.

Messenger

RNA Is

Translated

by Ribosomes

.

Ribosomes are characterized by

their

rate

of

sedimentation

(70S

for bacteriaI ribosomes

and

80S for eukaryotic ribosomes).

.

A ribosome

consists of

a

large subunit

(50S

or 605

for bacteria and eukaryotes)

and a smatl subunit

(30S

or a0S).

.

The ribosome

provides

the environment in which

aminoacyltRNAs add amino acids to the

growing potypeptide

chain in response

to the

corresponding triptet codons.

r

A

ribosome

moves alonq an mRNA from

5'to 3'.

Tlanslation of an mRNA into

a

polypeptide

chain

is catalyzed

by the

ribosome.

Ribosomes

are

traditionally

described in terms of their

(approx-

imate) rate of

sedimentation

(measured

in

Sved-

732

CHAPTER

7 Messenger

RNA

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different

soluble

proteins,

average

>1000

copies

of

bacterium.

there

must

be on

each

protein

in

a

The

Life

Cycle

of Bacterial

Messenger

RNA

r

Transcription

and transl,ation

occur simultaneousty

in bacteria,

as

ribosomes

begin translating

an

mRNA

before its synthesis

has been

completed.

r

Bacterial mRNA is

unstabte

and has a hatf-Life

of

only

a

few minutes.

r

A

bacterial mRNA may

be

potycistronic

in

having

severaI coding regions

that represent

different

genes.

Messenger

RNA has

the same function

in all

cells, but there

are important

differences in

the

details

of the synthesis and

structure

of

prokary-

otic and eukaryotic n.RNA.

A major

differenr:e in

the

production

of

mRNA

depends on

the locations

where tran-

scription and translation

occur:

.

In bacteria,

mRNA is

transcribed and

translated

in the

single cellular compart-

ment; the two

processes

are so closely

linked

that they occur

simultaneously.

Ribosomes

attach to

bacterial mRNA

even before its transcription

has

been

completed, thr.rs

the

polysome

is likely

still to be attached

to DNA. Bacterial

nRNA

usually is unstable,

and is there-

fore

translated into

nroteins

for

onlv a

few minutes.

.

In

a eukaryotic cell,

synthesis and mat-

uration of nR.NA

occur exclusively in

the nucleus.

Only after these

events are

completed is the nRNA

exported to the

cytoplasm, where it

is translated by ribo-

somes.

Eukarl'otic

nRNA

is relatively

stable and continues

to be translated lor

several hours.

:

r,'

..

,,

rl

j

i

::

shows that transcription

and

translation are intimately related

in bacteria.

Transcription begins

when the enzyme RNA

polymerase

binds to DNA and

then moves along

making a copy of one

strand. As soon as tran-

scription begins, ribosomes

attach to the 5'end

of the nRNA and

start translation, even before

the

rest

of the

message

has been

synthesized. A

bunch of ribosomes rnoves

along the mRNA

while it is being

synthesized. The 3' end of the

mRNA is

generated

when transcription termi-

nates. Ribosomes

corntinue to translate the

nRNA

while

it

survives, but it is

degraded

in

Considering

E.

coLi

in terms of

its macromolecular

c0m

p0nenrs.

rriiiii*i:. ,', -j,: Overview:

mRNA is transcribed.

transtated,

and degraded

simuttaneously

in bacteria.

Component

Dry cell

Molecules

Different Copies of

mass

(%)

/cell

types

each type

mRNA

1 1,500 600

2-3

IRNA

3

200,000 60

>3,000

rRNA 16 38,000

2 19,000

Ribosomal

proteins

9

106 52

19,000

sotubte

proteins

46

? 9

"

19:

1

,850

>1

,ooo

Small

molecules 3

7.5 x 10'

8OO

7.7

The Life Cycte of

Bacterial

Messenger RNA

Transcription

units can be visuatized in

bacteria. Photo courtesv of Oscar

the overall 5'-+3'

direction

quite

rapidly. The

mRNA is

synthesized, translated

by the

ribo-

somes,

and degraded, all in rapid

succession.

An

individual molecule

of mRNA survives for

only a matter

of minutes at most.

Bacterial transcription

and translation take

place

at

similar

rates.

At 37o C, transcription

of

mRNA

occurs at

-40

nucleotides/second.

This

is very

close to the rate

of

protein

synthesis,

which

is roughly l5

amino acids/second. It

therefore takes

-2

minutes

to transcribe

and

translate

an mRNA of 5000

bp, corresponding

to 180

kD of

protein.

When expression

of

a new

ff

ili :5i *,ffi;:;:T*ift:+i[

:;li:m:

:

ding

protein

will

appear within

perhaps

another

0.5 minute.

Bacterial

translation

is very efficient,

and

most mRNAs

are translated

by a large number

of

tightly

packed

ribosomes.

In one

example

(/rp

mRNA),

about t5 initiations

of transcrip-

tion

occur every minute,

and each

of the

I5 mRNAs

probably

is translated

by

-30

ribo-

somes in

the interval

between its transcription

and

degradation.

The

instability

of most bacterial mRNAs is

striking.

Degradation

of

mRNA

closely follows

its

translation

and likely begins

within one

minute

of the start of transcription.

The 5'end

of

the mRNA

starts to decay

before the 3' end

has

been

synthesized

or translated. Degrada-

tion seems

to follow

the last ribosome

of the

ffi.'."ffi

liffi

',HffiY;.T'i;,i,":T:J"ffi",1;

the speed

of transcription

or translation.

The stability

of mRNA has

a major influ-

ence

on the amount

of

protein

that is

produced.

CHAPTER

7

Messenqer

RNA

It

is

usually expressed

in terms

of the half-life.

The mRNA representing any

particular

gene

has

a characteristic

half-life, but

the average is

-2

minutes in bacteria.

This series of events is only

possible,

of

course, because

transcription, translation,

and

degradation all occur in the same direction. The

dynamics of

gene

expression have

been caught

in

flagrante

delicto in the electron micrograph

of

ir.i.:t,,ii,ti:

.,

i.:. In

these

(unknown)

transcription

units, several mRNAs are under synthesis simul-

taneously,

and

each

carries many ribosomes

engaged in translation.

(This

corresponds

to the

stage shown

in

the second

panel

in Figure

7.13.

)

An RNA whose synthesis

has

not

yet

been com-

pleted

is often called a

nascent

RNA.

Bacterial mRNAs vary

greatly

in the num-

ber of

proteins

for which

they code. Some

mRNAs represent only a single

gene:

they are

monocistronic.

Others

(the

majority)

carry

sequences coding

for

several

proteins:

they are

polycistronic.

In these cases, a single mRNA

is transcribed from a

group

of

adjacent

genes.

(Such

a cluster of

genes

constitutes an

operon

that is controlled as a single

genetic

unit; see

Chapter I2, The Operon.)

All mRNAs contain two types

of

region. The

coding region consists of a series

of codons rep-

resenting the

amino acid sequence of the

pro-

tein, starting

(usually)

with AUG and

ending

with a termination codon. The nRNA

is always

Ionger

than the coding

region,

though, as

extra

regions

are

present

at both ends. An additional

sequence at the 5' end,

preceding

the

start of

the coding region, is described as

the leader or

5'UTR

(untranslated

region).

An additional

sequence

following

the termination

signal, form-

ing

the 3' end,

is

called the trailer

or

f'

UTR.

Although

part

of the transcription

unit, these

sequences are

not

used to code for

protein.

A

polycistronic

mRNA also

contains inter-

cistronic regions,

as illustrated in irii-;l"it';i:

.r.:i:ri.

They vary

greatly

in

size:

They

may be

as

long

as 30 nucleotides in

bacterial mRNAs

(and

even

longer in

phage

RNAs).

or they may

be very

short, with as few as

one or two nucleotioes

separating

the termination codon

for one

pro-

tein from the initiation

codon for

the next. In

an

extreme case, two

genes

actually overlap,

so

that the last base

of one coding region

is also

the first

base of the next coding region.

The number

of ribosomes

engaged in trans-

Iating a

particular

cistron depends

on the

effi-

ciency

of

its initiation

site. The initiation

site for

the first cistron

becomes available

as soon

as

the

5'end of the nRNA is

synthesized.

How are

136

subsequent

cistrons translated?

Are

the several

coding regions in

a

polycistronic

mRNA

trans-

lated

independently

or is their

expression

con-

nected? Is

the mechanism

of initiation

the same

for all

cistrons, or is it different

for the first

cistron

and the

internal

cistrons?

T?anslation

of a

bilcterial mRNA

proceeds

sequentially through

its cistrons.

At the

time

when ribosomes

attach

to the first coding region,

the subsequent

codirLg regions

have not

yet

even been transcribed.

By the

time the second

ribosome

site is available,

translation is

well

under way through

ttre first

cistron. Tlpically

ribosomes terminate

translation

at the end of the

first

cistron

(and

dissociate into

subunits),

and

a new ribosome

assembles independently

at the

start of the next coding region.

(We

discuss the

processes

of initiation

and termination in

Chan-

ter 8, Protein Synthesis.)

@

Eukaryotic

mRNA

Is

Modified During

or

after

Its

Transcription

r

A eukaryotic mRNA

transcript is modified

in the

nucteus

during or shortty

after transcription.

o

The modifications inctude

the addition

of a

methylated

cap at the Ii'end

and a sequence of

poty(A)

at the

3'end.

.

The mRNA is

exported from

the

nucleus

to the

cytoplasm on[y after ail modifications

have been

com

pteted.

The

production

of eukaryotic

mRNA involves

additional stages

after transcription. Tfanscrip-

tion occurs

in

the usual way, initiating

a tran-

script with a 5'triphosphate

end. However,

the

3' end

is

generated

by

cleaving the transcript,

rather

than by terminating

transcription at a

fixed

site.

Those

RNA.s

that are derived from

interrupted

genes

require

splicing to remove

the introns,

generatirLg

a smaller nRNA

that

contains an

intact

coding sequence.

;''r.:,..ri:

,: ::

Shows

that both ends of the

transcript

are

modifierC

by additions

of

further

nucleotides

(involving

additional

enzyme sys-

tems). The 5' end of the RNA is

modified by the

addition of a

"

cap" virtually as

soon

as it

appears. This replaces the

triphosphate of the

initial transcript

with a nucleotide in reverse

(3'-+5'\

orientation, thus

"sealing"

the end.

The 3'

end

is modifierl

by addition of a

series

of adenylic acid

nucleotides

[polyadenylic

acid

or

poly(A)l

immediately after its

cleavage.

ilr,;..1',.,1, BacterialmRNAincludesuntranslatedaswetlastranstatedregions.

Each coding region

has its own

initiation and termination signats.

A typicat

mRNA

may have severaI coding

regions.

i

:i,1r1r;,,

1. : r

Eukaryotic

mRNA

is modified by the addi-

tion ofa cap to the 5'end

and

poty(A)

to the 3'end.

Only after the

completion

of all

modification

and

processing

events

can the

nRNA be

exported from the

nucleus to

the cytoplasm.

The

average

delay in

leaving

for

the

cytoplasm

is

-20

minutes. Once

the

nRNA has entered

the cytoplasm,

it is

recognized by

ribosomes

and translated.

j

r"

r;.,r

l

shows

that

the life cycle

of

eukaryotic

nRNA

is more

protracted

than that

of bacterial nRNA.

Tfanscription

in animal

cells

occurs at about

the same

speed as

in bacteria,

-40

nucleotides

per

second.

Many eukaryotic

genes

are

large; a

gene

of

10,000 bp takes

-5

minutes to transcribe.

Ttanscription

of

nRNA

is

not terminated

by the

release of enzyme

from

the DNA; instead

the enzyme

continues

past

the

end

of the

gene.

A coordinated

series

of

events

generates

the 3' end

of the

nRNA by

cleavage, and adds

a length

of

poly(A)

to the

newly

generated

3'end.

Eukaryotic mRNA

constitutes

only a small

proportion

of the

total cellular

RNA

(-3%

ol

the

mass). Half-lives are

relatively

short

in

yeast,

ranging from

l-60 minutes.

There is a substan-

tial

increase in stability

in

higher eukaryotes;

animal cell

nRNA

is relatively

stable, with

half-

lives ranging

from 4-24hours.

7.8

Eukaryotic

mRNA

Is Modified

During or after

Its Transcription

137

i:iill:iii:

.'. i;i

Overview: Expression

of mRNA in animaI

ce[[s requires

transcription, modification.

processing,

nucleo-cytoplasmic

transport, and

translation.

i-i+i-litl

i. !

il

The

cap

blocks the 5'

end of mRNA and may

be methylated

at sev-

eraI

positions.

Eukaryotic

polysomes

are reasonably

stable.

The modifications

at both ends of the mRNA

contribute to their stabilitv.

The

5'

End of Eukaryotic

mRNA Is Capped

.

A 5'cap is formed by adding a G to the

terminal

base of the transcript vta a 5'-5' link.

One to three

methyl

groups

are added to the base or

ribose

of

the

new

terminaI

guanosine.

Tlanscription starts with a nucleoside

triphos-

phate (usually

a

purine,

A or G). The first

nucleotide retains its 5'triphosphate

group

and

makes

the usual

phosphodiester

bond from its

3'

position

to the 5'

position

of the next

nucleotide. The initial

sequence of the tran-

script can be

represented

as:

5'pppA/6pNpNpNp .

When the mature mRNA is

treated in vitro

with enzymes that should degrade it into indi-

vidual nucleotides, however, the 5'

end does

not

give

rise to the expected nucleoside

triphos-

phate.

Instead it

contains two

nucleotides,

con-

nected

by a 5'-5' triphosphate linkage

and also

bearing methyl

groups.

The terminal

base is

always a

guanine

that

is

added to the

original

RNA molecule

after transcription.

Addition of the 5'terminal

G

is

catalyzed

by a nuclear enzyme,

guanylyl

transferase. The

reaction

occurs so soon after transcription

has

started that it is not

possible

to detect more

than

trace

amounts of the original 5'triphosphate

end in the nuclear RNA. The

overall reaction

can

be

represented

as a condensation

between

GTP and the original 5'triphosphate

terminus

of the RNA. Thus

-, -,

))

Gppp

+

pppApNpNp

.

v

)-)

GpppApNpNp.

. .+pp+p

The

new G residue added

to the end

of the

RNA

is in the reverse

orientation from

all the

other nucleotides.

This structure is

called a cap. It is

a sub-

strate for

several methylation events.

F5*iitri:.;.tS

shows the full structure

of a cap after

all

possi-

ble methyl

groups

have

been added.

\pes

of

caps are

distinguished by how

many of these

methylations

have occurred:

Present in

all caps

CHAPTER

7 Messenqer

RNA

.

The first

methylation

occurs

in all

eukaryotes,

and consists

of the addition

of a

methyl

group

to the 7

position

of

the terminal

guanine.

A

cap that

pos-

sesses this single methyl

group

is known

as a

cap 0. This is as far

as the reaction

proceeds

in

unicellular eukaryotes.

The

enzyme responsible

for

this modification

is called

guanine-7-methyltransferase.

.

The next

step

is

to add another

methyl

group,

to the 2'-O

position

of the

penul-

timate

base

(which

was

actually the orig-

inal first

base of the

transcripr before

any modifications

were made). This

reaction is

catalyzed by

another enzyme

(2'-O-methyl-transferase).

A

cap with

the two methl'l

groups

is

called cap l.

This is the

predominant

type

of cap

in

all eukaryotes

except

unicellular or-

ganrsms.

o

In

a small minority

of cases in higher

eukaryotes,

another methyl

group

is

added to the second

base. This hap-

pens

only when the

position

is

occu-

pied

by adenine; the reaction involves

addition of a methyl

group

at the

N6

position.

The

enzyme responsible acts

only on an adenosine

substrate that

already has

the

methyl

group

in the

2'-O

position.

.

In some species,

a

methyl

group

is added

to

the third

base of the capped mRNA.

The substrate for this reaction is

the cap

I mRNA

that already

possesses

two

methyl

groups.

The

third-base modifica-

tion is always

a2'-O

ribose

methylation.

This creates

the cap 2 type. This cap usu-

ally

represents

less than I0%-I 5o/o

of.

the total capped

population.

In

a

population

of eukaryotic mRNAs,

every molecule is capped. The

proportions

of

the different types of cap are characteristic for

a

particular

organism.

We do

not know

whether the structure

of a

particular

mRNA

is invariant or can have more than

one type

of

cap.

In addition to the methylation involved in

capping, a Iow

frequency

of internal methyla-

tion occurs

in

the mRNA only of higher eukary-

otes. This is accomplished by

the

generation

of

N6

methyladenine residues

at a frequency of

about one modification

per

1000

bases.

There

are one

or two methyladenines

in a typical

higher eukaryotic mRNA, although

their

pres-

ence

is not obligatory; some mRNAs

do

not

have any.

The 3'Terminus

.

A

length of

poty(A)

-200

nucleotides long

is added

to a

nuctear

transcript

after transcription.

.

The

poty(A)

is bound by

a specific

protein (PABP).

.

the

poty(A)

stabilizes

the

mRNA against

degradation.

The 3'terminal stretch

of

A residues

is

often

described as the

poly(A)

tail;

mRNA with this

feature is denoted

poly(A)+.

The

poly(A)

sequence

is not coded

in the

DNA,

but

rather

is

added

to the

RNA

in

the

nucleus after transcription.

The addition

of

poly(A)

is catalyzed

by the enzyme

poly(A)

polymerase,

which adds

-200

A residues to

the

free

3'-OH

end of

the mRNA.

The

poly(A)

tract of both

nuclear RNA

and mRNA

is associ-

atedwith a

protein,

the

poly(A)-binding

pro-

tein

(PABP).

protein

are

found in many eukaryotes.

One

PABP monomer

of.

-70

kD is bound every

l0 to

20 bases of

the

poly(A)

tail.

Thus a common

feature in

many

or most eukaryotes

is that

the

3'

end

of the

nRNA consists of a stretch

of

poly(A)

bound

to

a large mass of

protein.

Addition

of

poly(A)

occurs as

part

of a

reaction

in which the

3'end

of the mRNA

is

generated

and

modified by

a

complex of enzymes

(see

Section

26.I9, The 3'

Ends

of

mRNAs Are Generated

by Cleavage

and

Polyadenylation).

Binding of the

PABP to the

initiation

factor

eIF4G

generates

a closed

loop,

in which the

5'

and3'ends of

the mRNA

find themselves

held

in the same

protein

complex

(see

Figure

8.20

in Section 8.9,

Eukaryotes

Use a Complex

of

Many Initiation

Factors).

The

formation of

this

complex

may be

responsible

for some

of the

effects of

poly(A)

on

the

properties

of

nRNA.

Poly(A) usually stabilizes

mRNA.

The ability

of

the

poly(A)

to

protect

mRNA

against

degrada-

tion requires binding

of

the

PABP.

Removal of

poly(A) inhibits

the initiation

of translation

in vitro,

and depletion

of PABP

has the same

effect

in

yeast invivo.

These effects

could depend

on the

binding

of PABP to

the

ini-

tiation complex

at the

5' end

of nRNA.

There

are many examples

in early

embryonic

devel-

opment where

polyadenylation of a

particular

mRNA is correlated

with

its translation.

In some

cases, mRNAs

are stored

in a nonpolyadeny-

Iated form,

and

poly(A) is added when

their

translation

is required;

in other

cases,

poly(A)+

Is Polyadenylated

7.10

The 3'Terminus

Is Potyadenylated

139

Most

oJ RNA

population

mRNA

with

poly(A)

is

is rRNA

that lacks

poly(A)

small

proportion

of RNA

oligo(dT)

-

Sepharose

AA

-

POIY(A)+

RNA

sticks 10 column

The availability of a cloned DNA makes it

easy to isolate the corresponding

mRNA by

hybridization

techniques.

Even

mRNAs that are

present

in

only very

few

copies

per

cell

can be

isolated

by this approach. Indeed, only mRNAs

that are

present

in relatively large

amounts can

be isolated directly without using

a cloning step.

Almost all

cellular

mRNAs

possess

poly(A).

A

significant exception

is

provided

by the

mRNAs

that code for the histone

proteins (a

major structural component

of chromosomal

material). These mRNAs

comprise most

or all

of the

poly(A)-

fraction. The

significance of

the absence

of

poly(A)

from histone mRNAs

is not

clear, and there is no

particular

aspect of

their function for

which this appears to

be

necessary.

:'rr'

::.

: Poty(A)+

RNA can

be separated from other

RNAs by fractionation

on Sepharose-otigo(dT).

mRNAs

are

de-adenylated, and

their transla-

tion is reduced.

The

presence

of

poly(A)

has an important

practical

consequence. The

poly(A)

region of

mRNA

can base

pair

with

oligo(U) or

oligo(dT),

and this reaction

can be used to isolate

poly(A)+

mRNA. The

most convenient

technique is to

immobilize

the oligo

(U

or dT) on solid

support

material.

Then,

when an RNA

population

is

applied to

the colurnn, as illustrated

in

\

ir

.i

jitlj

.r: ,

: |i,

only

the

poly(A)+

RNA

is retained. It

can be

retrieved

by treating

the column with

a solution

that

breaks the

bonding to release

the

RNA.

The

only

drawback to this

procedure

is that

it isolates

all the RNA that

contains

poly(A).

If

RNA

of the whole

cell is used, for

example, both

nuclear

and

cytoplasmic

poly(A)+

RNA will be

retained.

If

preparations

of

polysomes

are used

(a

common

procedure),

most

of the isolated

poly(A)+

RNA will

be active nRNA.

However,

in

addition to mRNA

in

polysomes,

there

are

also ribonucleoprotein particles

in

the cytosol

that

contain

poly(A)+

nRNA,

but which are not

translated.

This

RNA may

be

"stored"

for

use at

some

other

time. Isolation

of total

poly(A)+

nRNA

therefore

does not

correspond

exactly

with the

active nRNA

population.

The

"cloning"

approach

for

purifying

nRNA

uses a

procedure

in

which the mRNA

is copied to

make a

complementary

DNA

strand

(known

as

cDNA). Then

the cDNA

can be

used as a template

to synthesize

a DNA

strand that is

identical

with

the original

nRNA

sequence. The

product

of these

reactions

is a double-stranded

DNA

correspon-

ding to

the sequence

of the nRNA.

This DNA

can

be reproduced

in large

amounts.

CHAPTER

7

Messenger

RNA

BacteriaI mRNA

Degradation

Involves

MuLtiple

Enzymes

r

The

overall direction of degradation

of bacterial

mRNA is 5'+3'.

r

Degradation resutts from

the combination

of

endonucteolytic

cteavages

foltowed

by

exonucleolytic

degradation of the fragment

rrom J

-+5

.

Bacterial mRNA is

constantly

degraded by a

combination

of endonucleases

and exonucle-

ases. Endonucleases

cleave an RNA

at an inter-

nal

site. Exonucleases are involved

in trimming

reactions

in which the

extra

residues

are

whit-

tled

away, base by base, from

the end. Bacter-

ial

exonucleases that act on

single-stranded

RNA

proceed

along the nucleic

acid chain from

the

3'end.

The

way the two types

of enzymes

work

together to

degrade an mRNA is

shown in

irii.iil:i-

.,,iri.

Degradation

of a

bacterial nRNA

is initiated

by an endonucleolytic

attack.

Sev-

eral 3'

ends may be

generated

by endonucleo-

lytic

cleavages

within the mRNA.

The

overall

direction

of degradation

(as

measured

by loss

of

ability to

synthesize

proteins)

is 5'-+3'.

This

probably

results

from a succession

of endonu-

cleolytic cleavages

following

the last ribosome.

Degradation

of the released fragments

of nRNA

into nucleotides

then

proceeds

by

exonucle-

olytic

attack from the free

3'-OH

end toward

the 5'terminus

(that

is, in

the opposite

direc-

tion from transcription).

Endonucleolytic

attack

Lewin Benjamin (ed.) Genes IX

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