Lewin Benjamin (ed.) Genes IX

MyoD

nta nsp nrg

Arg Lys Ala Ala Thr l\4et Arg

cln Arg

Arg Arg

ld

Arg

Leu

Pro

Ala Leu Leu

Asp Gln Glu Glu ValAsn Val Le-

MyoD

Leu Ser Lys :Valfuri

Gln Ala Phe cln Thr

Leu Lys

Arg

Cys

Thr

ld

LeuTyrAspMetAStclyCysTyr

SerArgtdtli:yfGtn Leuvat

Myo D

rye Vai c

tn,llii:

ler;l

nrg nsn

AiLir,ilr!

Arg

lii

r|e

crn

G ty

-Leu

Gtll

ld

Lis

Va!,:e

tn

lle'iie-u,

ctu His val

rli€t

Asp

li4ili4:Arg

Asp

lieu

Glu

Basic

region

Six conserved

residues

are absent

from ld

Helix

1

Conserved

residues are

Jound

in

both MyoD

and

ld

Helix 2

iiirijEi.

:i:,i:ir

At[ HLH

proteins

have

reg'ions corresponding to hetix

l

and

helix

2, which are

separated by a toop

of 10 to 24 residues. Basic HLH

proteins

have a

region with

conserved

positive

charges immediatety

adjacent to helix L.

are l5 to l6 amino

acids long, and

each con-

tains several conserved residues.

Ttvo exam-

ples

are compared in

ljL:;i,iiii

ill.ii{:.

The ability

to form dimers

resides

with these

amphipathic

helices and is common

to all HLH

proteins.

The

loop is

probably

important

only for allowing

the

freedom for the

two helical regions to inter-

act independently of one another.

Most HLH

proteins

contain a region adja-

cent to the HLH motif itself

that is highly basic,

and which

is needed for

binding to DNA. There

are

-6

conserved residues in

a stretch of

I

5

amino acids

(see

Figure 25.26).

Members of the

group

with such a

region

are called

bHLH

pro-

teins. A dimer in which both

subunits

have

the

basic

region can

bind to

DNA.

The HLH domains

probably

correctly orient the two

basic

regions

contributed by the individual

subunits.

The

bHLH

proteins

fall into two

general

groups.

Class A consists of

proteins

that

are ubiq-

uitously expressed,

including

mammalian

Ef2lE47. Class B consists

of

proteins

that are

expressed

in a tissue-specific

manner, includ-

ing mammalian MyoD, myogenin, and Myf-5

(a

group

of activators that are involved in myo-

genesis

[muscle

formation]

).

A common modus

operandi for a tissue-specific bHLH

protein

is to

form

a

heterodimer

with a ubiquitous

partner.

There is also a

group

of

gene

products

that spec-

ify development of the nervous system in

D. melanogaster

(where

Ac-S is the tissue-specific

component

and da is the

ubiquitous compo-

nenr). The Myc

proteins (which

are the cellu-

lar

counterparts

of oncogene

products

and

are

involved

in

growth

regulation)

form a separate

class of bHLH

proteins,

whose

partners

and tar-

gets

are different.

Dimers formed from bHLH

proteins

differ

in their abilities to bind to

DNA.

For example,

E47 homodimers, EI2-E47 heterodimers, and

MyoD-E47 heterodimers

all form

efficiently

and

bind strongly to

DNA; El2

homodimerizes

well

but

binds

DNA

poorly,

whereas

MyoD

homo-

dimerizes only

poorly. Thus both

dimer forma-

tion and DNA binding

may

represent

important

regulatory

points. At this

juncture,

it is

possible

to

define

groups

of

HLH

proteins

whose

mem-

bers form various

pairwise

combinations,

but

it is not

possible

to

predict

from the

sequences

the strengths of

dimer

formation

or DNAbind-

ing. All of the dimers

in this

group

that bind

DNA recognize the same

consensus

sequence,

but we do

not know

yet

whether

different

homodimers and

heterodimers

have

prefer-

ences

for

slightly

different

target

sites

that are

related to their

functions.

Differences

in DNA-binding

result

from

properties

of the

region

in or close

to the

HLH

motif; f or example,

E

I2 differs

fromB4T

in

pos-

sessing an inhibitory

region

just

by the basic

region, which

prevents DNA

binding by

homo-

dimers.

Some HLH

proteins lack the

basic

region

and/or contain

proline

residues

that

appear

to

disrupt its function.

The

example

of the

protein

Id is shown in

Figure

25.26. Proteins

o{ this

type

have the same

capacity

to dimerize

as bHLH

proteins,

but

a dimer

that contains

one

subunit

of this type

can

no longer

bind to

DNA specifi-

cally. This

is a forceful

demonstration

of the

importance

of doubling

the

DNA-binding

motif

in DNA-binding

proteins.

The importance

of

the distinction

between

the

nonbasic HLH

and

bHLH

proteins is sug-

gested

by the

properties

of

two

pairs

of

HLH

proteins:

tine da-Ac-S/emcpair

and the

MyoD/Id

pair.

A model

for their

functions

in

forming

a

regulatory

network

is illustrated

in

i:i{;t"::-}* ;l*-ll;.

In D. melanlgaster,

the

gene emc

(extra'

macrochaetae)

is required

to establish

the

nor-

mal spatial

pattern

of

adult sensory

organs.

It

2 5. 15

HeLix- Loop-

Hetix

Protei

ns Interact

by Com

bi natoriaI

Association

661

binding to DNA when it is

sequestered by an

HLH

partner

such as

Id. It

can activate tran-

scription when bound to a bHLH

partner

such

as El2 orE47.It can also

act as a site-specific

repressor

when bound to another

partner;

the

bHLH

protein

MyoR forms a MyoD-MyoR

dimer

in

proliferating

myoblasts that represses

tran-

scription

(at

the same target loci

at which MyoD-

El2

lE47 activate transcription).

The behavior

of the

HLH

proteins

therefore

illustrates

two

general principles

of transcrip-

tional regulation. A

small

number

of

proteins

form combinatorial

associations. Particular

com-

binations have

different functions

with regard

to DNA binding and transcriptional

regulation.

Differentiation may

depend either

on the

pres-

ence or on the removal of

particular

partners.

ii,;:-i*.i: ili:.J."r

An HLH

dimer

in

which both

subunits are

ofthe

bHLH type can bind DNA,

but a dimer in which

one

subunit lacks

the basic reqion

cannot bind DNA.

functions

by suppressing the functions

of several

genes,

including

da

(daughterless)

and the achaete-

scute

(Ac-S)

complex. ,4c-S

and da

are

genes

of

the

bHLH type. The

suppressor

emc codes f.or an

HLH protein

that lacks the

basic region. We

sup-

pose

that, in the

absence of. emc function,

the

da

and Ac-S

proteins

form

dimers that activate

transcription

of appropriate

target

genes,

but

the

production

of emc

proLein

causes the for-

mation

of

heterodimers

that cannot

bind to

DNA. Thus production

of. emc

protein

in the

appropriate

cells is necessary

to suppress

the

function

oI Ac-S/da.

The

formation

of muscle

cells is triggered

by a change

in the

transcriptional

program

that

requires

several bHLH

proteins,

including

MyoD.

MyoD

is

produced

specifically

in myogenic

cells

and,

indeed,

overexpression

of MyoD

in cer-

tain

other cells

can induce them

to commence

a myogenic

program.

The trigger for

muscle

dif-

ferentiation

is

probably

a heterodimer

consist-

ing

of MyoD

-El2

or MyoD-847

rather

than a

MyoD

homodimer.

Before

myogenesis

begins,

a member

of

the nonbasic HLH

type,

the Id

pro-

tein,

may

bind to MyoD

and/or E l2

and E47 to

form

heterodimers

that

cannot

bind to DNA. It

binds

to EI2lE47

beter

than ro

MyoD, and

so

might

function

by sequestering

the ubiquitous

bHLH

partner.

Overexpression

of Id can

pre-

vent

myogenesis.

Thus

the removal

of Id

could

be the

trigger

that releases

MyoD

to

initiate

myogenesls.

AbHLH

activator

such

as MyoD

can be con-

trolled

in several

wavs. It is

prevented

from

Activating

Transcription

Leucine Zippers

Are Involved in

Dimer

Formation

.

The leucine zipper is

an amphipathic hetix

that

dimerizes.

r

The zipper

is adjacent to a basic region

that

binds

DNA.

r

Dimerization forms

the bZIP motif in which

the

two basic

regions

symmetricatty

bind inverted

repeats in DNA.

Interactions

between

proteins

are a common

theme

in

building a transcription

complex,

and

a motif found in

several activators

(and

other

proteins)

is involved

in both homo-

and het-

eromeric interactions.

The leucine

zipper is

a

stretch

of

amino

acids rich in leucine

residues

that

provides

a

dimerization motif.

Dimer for-

mation itself

has emerged as

a common

princi-

ple

in

the action

of

proteins

that

recognize

specific DNA

sequences,

and in the

case of the

leucine zipper,

its relationship

to DNA

binding

is

especially

clear, because

we can

see how

dimerization

juxtaposes

the DNA-binding

regions

of each subunit. The reaction

is

depicted

diagrammatically

in i:ftui,JFt* i:

*. Jis.

An

amphipathic

cx

helix

has

a structure

in

which

the hydrophobic

groups

(including

leucine)

face

one side while charged groups

face

the

other side. A leucine

zipper forms

an

amphi-

pathic

helix in

which the leucines

of the zipper

on one

protein

could

protrude

from

the cr-helix

and interdigitate

with the leucines

of the zipper

of another protein

in

parallel

to form

a coiled

coil. The

two right-handed

helices

wind

around

662

CHAPTER

25

each other, with 3.5 residues

per

turn, so the

pat-

tern

repeats integrally

every seven residues.

How is this structure related

to DNA bind-

ing? The region

adjacent to the leucine repeats

is highly basic in each of the zipper

proteins,

and could comprise a DNA-binding site. The

two leucine zippers in

effect

form

a

Y-shaped

structure, in which the zippers

comprise

the

stem and the two basic regions

stick out to

form

the arms that bind to DNA. This is known as

the bZIP structural motif. It

explains

why the

target sequences

for

such

proteins

are inverted

repeats with no separation.

Zippers may be used

to sponsor

formation

of

homodimers

or

heterodimers.

They are

lengthy motifs. Leucine

(or

another hydropho-

bic amino

acid)

occupies every seventh residue

in the

potential

zipper. There are four repeats

of the zipper

(Leu-X6)

in the

protein

C/EBP

(a

factor that binds as a dimer to

both

the CAAT

box and

the SV40

core enhancer) and

five

repeats in

the

factors

Jun and Fos

(which

form

the heterodimeric activator, APt

).

API

was

originally identified

by

its

binding

to a DNA sequence in the SV40 enhancer

(see

Figure 24.24it . The active

preparation

of API

includes

several

polypeptides.

A major

compo-

nent is Jun, the

product

of the

gene

c-jun,which

was

identified by its relationship

with the onco-

gene v-jun

carried by an avian sarcoma

virus.

The mouse

genome

contains a family of

c-jun

(the

original isolate),

junB,

and

junD

(identified

by sequence homology with

jun).

There are considerable sequence similarities

in

the three Jun

proteins;

they have leucine

zip-

pers

that

can interact to form homodimers or

heterodimers.

The other major component of API is the

product

of

another

gene

with an oncogenic

counterpart. The c-fos

gene

is the cellular

homolog to the oncogene

v-los

carried

by a

murine sarcoma virus. Expression of c-fos acri-

vates

genes

whose

promoters

or enhancers

pos-

sess an API target site. The c-los

product

is

a

nuclear

phosphoprotein

that is one of a

group

of

proteins.

The others are described as Fos-

related antigens

(FRA);

they

constitute

a fam-

ily

of

Fos-like

proteins.

Fos also

has a leucine zipper. Fos cannot

form homodimers, but can

form a heterodimer

with Jun.

A leucine zipper in

each

protein

is

required for the reaction. The ability to

form

dimers is a crucial

part

of the

interaction

of

these

factors with DNA. Fos cannot by itself bind

to

DNA,

possibly

because

of its failure to form a

dimer. The Jun-Fos

heterodimer

can,

however,

irlt.;iiliii: :::'r.;l:iri

The basic

regions of the

bZIP

motif are

held

together

by the dimerization

at the adjacent

zipper

region when

the

hydrophobic

faces of two leucine

zippers

'interact

in

oara[[eI

orientation.

bind

to DNA with

same

target specificity

as the

Jun-Jun

dimmer,

and this

heterodimer

binds

to the API site

with an

affinity

-10x

that of the

Jun

homodimer.

Summary

Transcription

factors

include

basal

factors,

acti-

vators,

and coactivators.

Basal

factors

interact

with RNA

polymerase at the

startpoint.

Activa-

tors bind

specific short

response

elements

(REs)

located

in

promoters or enhancers.

Activators

function by

making

protein-protein

interac-

tions

with the basal

apparatus.

Some

activators

interact directly

with the

basal

apparatus;

others

require coactivators

to

mediate

the

inter-

action.

Activators

often

have

a modular

con-

struction,

in which

there

are

independent

domains

responsible

for binding

to DNA

and

for activating

transcription.

The main

function

of the

DNA-binding

domain

may be

to tether

the

activating

domain

in the

vicinity

of

the initia-

tion complex.

Some

response

elements

are

pres-

ent

in many

genes and

are

recognized

by

ubiquitous

factors; others

are

present in a few

genes

and are

recognized

by tissue-specific

factors.

Promoters

for

RNA

polymerase II contain

a variety

of short

czs-acting

elements,

each

of

which

is recognized

by

a trans-acting

factor.

The

crs-acting elements

are

located

upstream

of

the

TATA box

and

may be

present

in

either

orien-

tation and

at a variety

of distances

with

regard

to

the startpoint.

The upstream

elements

are

recognized by

activators

that

interact

with the

@@@@

25.17 Summary

663

basal transcription

complex

to determine

the

efficiency with

which

the

promoter

is used.

Some activators

interact

directly with

compo-

nents

of the basal

apparatus;

others interact via

intermediaries

called coactivators.

The targets

in

the basal

apparatus are

the TAFs of TF11D,

or

TFnB

or TFnA.

The interaction

stimulates

assem-

bly

of the basal

apparatus.

Another

motif involved

in DNA-binding is

the zinc

finger, which is

found in

proteins

that

bind DNA

or RNA

(or

sometimes

both). A fin-

ger

has

cysteine

residues

that bind zinc.

One

type of finger

is found in

multiple repears

in

some

transcription

factors;

another is found in

single or

double repeats

in others.

Several

groups

of transcription

factors have

been

identified

by sequence

homologies.

The

homeodomain

is a 60-residue

sequence

found

in

genes

that regulates

development

in insects

and

worms and

in mammalian

transcription

factors.

It is

related to

the

prokaryotic

helix-

turn-helix

motif

and

provides

the motif

by

which

the factors

bind to lNA.

The leucine

zipper

contains a

stretch of amino

acids rich in

leucine

that are involved

in

dimer-

ization

of transcription

factors.

An

adjacent basic

region

is

responsible

for binding

to DNA.

Steroid receptors were

the first members

identified

of a

group

of transcription

factors in

which

the

protein

is

activated

by binding a

small

hydrophobic

hormone.

The

activated

factor

becomes localized

in

the nucleus

and

binds

to its

specific response

element,

where

it

activates

transcription.

The DNA-binding

domain

has

zinc fingers.

The receptors

are

homodimers

or heterodimers.

The

homodimers

all recognize palindromic

response

elements

with

the same

consensus

sequence;

the

differ-

ence between

the response

elements

is the

spacing

between

the inverted

repeats.

The het-

erodimers

recognize

direct

repeats,

again being

distinguished

by the

spacing

berween

the

repeats.

The DNA-binding

motif

of these recep-

tors

includes

two zinc

fingers;

the first

deter-

mines

which

consensus

sequence is

recognized,

and the

second

responds

to

the spacing

between

the repeats.

HLH

(helix-loop-helix)

proreins

have

amphipathic

helices

that are

responsible

for

dimerization,

which

are adjacent

to

basic

regions

that

bind

to DNA.

bHLH

proteins

have

a basic

region

that

binds to

DNA and

fall

into

two

groups:

ubiquitously

expressed

and

tissue-specific.

An active

protein

is usu-

ally

a heterodimer

between

two

subunits,

one from

each

group.

When a

dimer has

one

CHAPTER

25 Activating

Transcription

subunit that

does not have the

basic region

it fails

to bind DNA, so

such subunits

can

prevent

gene

expression. Combinatorial

as-

sociations of subunits f

orm regulatory

networks.

Many transcription

factors

function

as

dimers,

and

it

is common for

there to

be mul-

tiple members of a family

that form

homo-

dimers and heterodimers.

This

creates

tne

potential

for complex

combinations

to

govern

gene

expression. In

some cases, a family

includes

inhibitory members,

whose

participation

in

dimer formation

prevents

the

partner

from

acti-

vating transcription.

A?

Kererences

There Are

Several Types

of Transcription

Factors

Reviews

Lee,

T. I. and Young,

R. A.

(2000).

Ttanscriprion

of

eukaryotic

protein-coding

genes.

Annu. Rev.

GeneL )4,77-137.

Lemon,

B. and Tjian, R.

(2000).

Orchestrated

response:

a symphony

of transcription

factors

for

gene

control.

Genes

Dey.14,2551-2569.

Independent Domains

Bind DNA

and Activate Transcription

Guarente,

L.

(19871.

Regulatory proteins

in

yeast.

Annu.

Rev.

Genet

21,425-452.

Ptashne, M.

(

I 988). How

eukaryotic

transcrip-

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Protei n-

Protei n Interactions

Fields,

S. and Song,

O.

(1989).

A novel genetic

sys-

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protein-protein

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340,245-246.

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Interact

with

the Basal

Apparatus

Reviews

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B. and

Tjian, R.

(2000).

Orchestrated

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Goodbourn,

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(1987ll.

Regulation

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Chen, J.-L. et al.

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Assembly

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TFIID reveals

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Cell79,93-105.

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C.

X.,

Roeder, R. G., Myers,

L.

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Gustafsson,

C. M., Jiang, Y. W., Li, Y.,

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Dynlacht, B. D., Hoey, T., and Tjian, R.

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Li, Y.,

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Some

Promoter-Binding

Proteins

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Reoressors

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Goppelt, A., Stelzer, G., Lottspeich, F.,

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Meister-

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A mechanism

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of class

II

gene

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binding of NC2 to TBP-promoter

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EMBO

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Inostroza, J. A., Mermelstein, F. H., Ha, I.,Lane,

W.

S.,

and Reinberg, D.

(1992).

Drl, a

TATA-

binding

protein-associated phosphoprotein

and inhibitor of class II

gene

transcription. Cel/

70,477-489.

I(im, T. K., Zhao, Y.,

Ge,

H

,

Bernstein, R., and

Roeder, R.

G.

(1995)

TATA-bindingprotein

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a

functional

interplay

between

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cofactor NC2

(Drl

)

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There Are Many Types

of

DNA-Binding

Domains

Harrison, S. C.

(1991).

A structural taxonomy of

DNA-binding

proteins.

Nature 35),

7 l5-7 19.

Pabo, C. T. and Sauer,

R. T.

(1992).

Tlanscription

factors:

structural

families

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principles

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DNA recognition.

Annu.

Rev. Biochem. 61,

1053-1095.

Resea rch

Miller, J. et al.

(1985).

Repetitive

zinc binding

domains in the

protein

transcription

factor

IIIA from Xenopus oocytes.

EMBO J. 4,

t609-16t4.

Murre,

C.,

McCaw, P. S.,

and Baltimore,

D.

(1989).

A

new DNA binding

and

dimerization

motif in

immunoglobulin

enhancer

binding, daughter-

less, MyoD, and

myc

proteins.

Cell 56,

777

-7

83.

A Zinc Finger

Motif

Is

a

DNA-Binding

Domain

Resea rc h

I(adonaga, J. et al.

(1987).

Isolation

of cDNA

encoding transcription

factor Spl and

func-

tional analysis

of the

DNA

binding domain.

Cell 51, 1079-1090.

Miller,

J. et

al.

(1985).

Repetitive

zinc binding

domains

in the

protein

transcription

factor

ILIAfrom Xenopus

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EMBO J.

4,

t609-t614.

Pavletich,

N.

P. and

Pabo, C. O.

(1991).

Zinc finger-

DNA

recognition: crystal

structure

oI a

Zif268'

DNA comolex

at 2I

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Science

252,809-81

7.

Steroid Receptors

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Reviews

Evans,

R. M.

(

1988) .

The steroid

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mone receptor

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Science

240,

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Mangelsdorf,

D. J. and

Evans,

R.

(1995). The RXR

heterodimers

and orphan

receptors. Cell

83,

841-850.

Steroid

Receptors

Have

Zinc Fingers

Review

Tsai,

M

J, and

O'Malley,

B. W.

(1994r. Molecular

mechanisms of action

of

steroid/thyroid

receptor superfamily

members.

Annu.

Rev.

Biochem 6),451-486.

Resea

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I(.

and

Evans,

R.

M.

(1989).

Determi-

nants of target

gene

specificity

for steroid/

thyroid

hormone

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Cell 57,

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Steroid Receptors

Recognize

Response

Etements by

a Combinatorial

Code

Reviews

Mangelsdorf, D. J.

and Evans,

R.

\1995).

The RXR

heterodimers

and orphan

receptors.

Cell 83'

84 l

-85

0.

Yamamoto,

K. R.

(1985).

Steroid

receptor

regu-

Iated transcription

of specific

genes

and

gene

networks.

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Rev. Genet.

19,209-252.

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References

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Resea rch

Hurlein, A.

J. et al.

(1995).

Ligand-independent

repression

by the thyroid hormone

receptor

mediated

by a nuclear receptor

corepressor.

Nature )77,

i97404.

Rastinejad,

F., Perlmann, T., Evans,

R. M., and

Sigler, P. B.

(1995).

Structural determinants of

nuclear

receptor assembly

on DNA direct

repeats.

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Umesono,

K., Murakami, K. K., Thompson,

C. C.,

and

Evans,

R. M.

(

l99l

)

. Direct repeats

as

selective response

elements for

the thyroid

hormone, retinoic

acid, and

vitamin D3 recep-

tors.

Cell 65, 1255-1266.

@

Homeodomains

Bind

Related Targets

in

DNA

Review

Gehring,

W.

J. et al.

(1994).

Homeodomain-DNA

recognition.

Cell

7

8, 2l l-223.

Resea rch

Han,

I(., Levine,

M. S., and Manley,

J. L.

(1989).

Synergistic activation

and repression

of tran-

scription by Drosophila

homeobox

proteins.

Cell 56, 571-58).

Wolberger,

C.

et al.

(199I).

Crystal

structure of a

MATc,2

homeodomain-operator

complex sug-

gests

a

general

model

for homeodomain-DNA

interactions.

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Weintraub,

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).

The MyoD

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nodal

point

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specification

of the muscle

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251,

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66.

Research

Benezra, R. et al.

(1990).

The

protein

Id: a nega-

tive regulator

of

helix-loop-helix

DNA-

binding

proteins.

Cell 61,49-59.

Davis, R. L. et al.

(1987

|

. Expression

of a single

transfected cDNA converts fibroblasts

to

myoblasts.

Cell 5L,

987-1000.

Davis, R. L.

et al.

(I990).

The

MyoD DNA

binding

domain contains a recognition code for

muscle-specific

gene

activation.

Cell 60,

7JJ-746.

Lassar, A. B.

et al.

(1991).

Functional

activity of

myogenic HLH

proteins

requires hetero-

oligomerization with El2 /E47

-like

proteins

in vitro.

Cell 66, 305-315.

Murre,

C.,

McCaw

P. S., and Baltimore,

D.

(1989).

A new

DNA binding and dimerization

motif in

immunoglobulin

enhancer binding,

daughter-

less,

MyoD, and myc

proteins.

Cell 56,

777-783.

Leucine Zippers Are Involved

in Dimer Formation

Review

Vinson,

C.

R.,

Siglea P. B., and McKnight,

S. L.

(1989).

Scissors-grip model for DNA

recogni-

tion

by a

family

of leucine zipper

proteins.

Science

246, 9lI-916.

Research

Landschulz,

W. H., Johnson, P. F.,

and McKnight,

S. L.

(1988).

The leucine

zipper: a hypotheti-

cal structure common

to a new class

of DNA

binding

proteins.

Science

240,

1759-1764.

@

Review

666

CHAPTER

25

Activating

Transcription

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3NI]INO

U]IdVH3

o

?EM

?frd

?r13ar

Spticing

Is

Connected to Export of mRNA

.

The REF

proteins

bind to splicing

junc-

tions

by associating with the

spliceosome.

.

After spLicing,

they

remain

attached to

the RNA at the exon-exon

junction.

.

They interact with

the transport

protein

TAP/Mex

that exports the RNA

through

the

nuctear

oore.

Group Ii Introns Autosptice

via Larjat

Formation

.

GrouD II introns excise

themselves

from

RNA by an autocatalytic

spticing event.

.

The

splice

junctions

and mechanism

of

spticing of

group

II introns

are similar to

spticing

of

nuctear

introns.

r

A

group

II intron folds into

a secondary

structure that

generates

a

catalytic site

resemb[ing

the structure

of U6-U2-nuctear

intron.

Atternative

Spticing Invotves DifferentiaI

Use of Sptice Junctions

.

Specific

exons

may

be exctuded or

inctuded

jn

the RNA

product

by using or

faiting

to use a

pair

of spticing

junctions.

o

Exons may

be extended by changing

one

of

the sptice

junctions

to

use an alterna-

tive

junction.

o

Sex determination in Drosophila

involves

a series of atternative

sp[ic'ing events in

genes

coding for successive

products

of a

pathway.

.

P

etements of Drosophila

show

germ[ine-

specifi c atternative spticing.

frans-Splicing

Reactions Use

Smatl

RNAs

.

Spticing reactions

usuatly

occur onty

in

crs

between

splice

junctions

on

the same

molecule

of RNA.

e

trons-spticing

occurs in

trypanosomes and

worms where

a short

sequence

(SL

RNA) is

spliced to the

5'ends of many

precursor

mRNAs.

.

SL RNA has

a structure resemb[ing

the

Sm-binding

site of U snRNAs

and

may

ptay

an anatogous role in

the reaction.

Yeast

IRNA

Spticing Involves

Cutting and

Rejoining

o

IRNA

spl.icing occurs by

successive cteav-

age and Ligation reactions.

The

Spl.icing Endonuclease

Recognizes

tRNA

o

An

endonuclease

cteaves the IRNA

orecur-

sors

at both ends

of the intron.

e

The

yeast

endonuctease is

a hetero-

tetramer with

two

(retated)

catatytic

subunits.

.

It

uses a

measuring

mechanism

to deter-

mine

the sites of cleavage

by their

posi-

tions retative to a

point

in

the IRNA

structu

re.

.

The archaeal nuctease has

a simpter struc-

ture and recognizes a buLge-helix-bulge

structural motif in the substrate.

IRNA Cleavage and

Ligation

Are

Separate

Reactions

o

Release

of the

intron

generates

two

half-

tRNAs that

pair

to

form

the mature

structure.

o

The hatves

have the unusual

ends

5' hydroxyl and 2'-3' cyctic

phosphate.

r

The

5'-0H end

is

phosphorytated

by a

polynucleotide

kinase,

the cyctic

phos-

phate group

is

opened by

phosphodi-

esterase

to

generate

a 2'-phosphate

terminus

and 3'-0H

group,

exon ends

are

joined

by an RNA

ligase, and the 2'-

phosphate

is removed

by a

phosphatase.

The

Unfotded

Protein

Response

Is

Related to IRNA Spticing

.

Irelp is an inner nuclear

membrane

pro-

tein with its N-terminal

domain in the ER

lumen, and its C-terminal

domain in the

nucteus.

o

Binding

of an unfolded

protein

to the

N-terminal

domain activates

the C-terminaI

nuctease

by autophosphorytation.

o

The activated nuclease

cteaves Hac].

mRNA to release an intron

and

generate

exons that are ligated by

a IRNA tigase.

.

The spliced Hacl mRNA

codes for a

tran-

scription factor that

activates

genes

cod-

ing for

chaperones that hetp

to fotd

unfotded

proteins.

The 3' Ends

of

pof

and

polIII

Transcripts

Are

Generated by Termination

r

RNA

potymerase

I terminates

transcrip-

tion at an L8-base

terminator

seouence.

o

RNA

poLymerase

III

terminates

transcrip-

tion in

poty(U)a

seQU€nce

embedded in

a

G-C-rich

seouence.

The

3' Ends of mRNAs Are

Generated

by

Cteavage and

Potyadenytation

r

The sequence AAUAAA

is a

signaI for

cleavage to

generate

a 3'end

of mRNA

that

is

pol.yadenylated.

.

The reaction

requires

a

protein

complex

that contains a specificity factor,

an

endonuclease, and

poty(A)

polymerase.

r

The specificity factor

and

endonuctease

cteave RNA

downstream

of

AAUAAA.

.

The

specificity factor

and

poty(A)

poty-

merase

add

-200

A residues

processivety

to the 3'end.

?frtill

668

CHAPTER 26 RNA

Spticing

and Processing

o

A-U-rich

sequences in

the 3'taiI control

cytoplasmic

potyadenytation

or deadeny[a-

tion during Xenopus

embryonic

deve[ooment.

Cteavage ofthe 3'End

of

Histone mRNA

May Require

a Smatl RNA

.

Histone mRNAs are not

poLyadenylated;

their

3'ends are

generated

by a cteavage reaction that

depends on the structure

of the

mRNA.

.

The

cteavage

reaction requires

the

SLBP to bind to

a stem-Loop structure and the

U7 snRNA to

pair

with an adjacent singte-stranded region.

Production

of rRNA

Requires Cleavage

Events

o

The [arge and smat[

rRNAs

are released

by cteavage

from

a common orecursor RNA.

Smatt

RNAs

Are Required

for rRNA

Processi

ng

.

The

C/D

group

of snoRNAs

is required

for modifying

the

2'

position

of ribose

with a

methyl

group.

.

The H/ACA

group

of snoRNAs

is required

for

converting uridine

to

pseudouridine.

o

In

each case the

snoRNA

base

pairs

with a

sequence of

rRNA that contains

the target

base to

generate

a typicaI structure

that

is the substrate

for

modification.

Summarv

Introduction

Interrupted

genes

are found in

all classes of

organisms. They represent a minor

proportion

of the

genes

of the

very

lowest

eukaryotes, but

the vast

majority

of

genes

in higher eukaryotic

genomes.

Genes vary widely

according to the

numbers and lengths

of introns, but a typical

mammalian

gene

has seven

to eight exons

spread out over

-

I 6 kb. The

exons are relatively

short

(-100

to

200

bp) and the introns are rel-

atively long

(>l

kb)

(see

Section

3.6, Genes

Show a Wide

Distribution

of Sizes).

The discrepancy between

the

interrupted

organization of the

gene

and

the uninterrupted

organization

of its nRNA requires

processing

of the

primary

transcription

product.

The

pri-

mary transcript has the

same organization as

the

gene

and

is

sometimes called the

pre-

mRNA. Removal

of the

introns

from

pre-

mRNA leaves a typical messenger of

-2.2

kb.

The

process

by which the introns are removed

is called RNA splicing. Removal

of

introns is

a major

part

of the

production

of RNA in all

eukaryotes.

(Although

interrupted

genes

are

relatively rare

in lower

eukaryotes such as

yeast,

the overall

proportion

underestimates the

importance of introns because most of the

genes

that are

interrupted

code

for relatively

abun-

dant

proteins.

Splicing

is

therefore involved

in

the

production

of a

greater proportion

of total

nRNA than would be apparent

from

analysis

of the

genome, perhaps

as much as 50%.)

One of the first clues about the nature of

the discrepancy

in size between nuclear

genes

and their

products

in higher

eukaryotes was

provided

by the

properties

of nuclear

RNA. Its

average size

is much larger

than

nRNA, it is

very unstable, and

it has

a

much

greater

sequence complexity.

Taking its name from its

r

.,,

:

,

'

hnRNA exists

as a

ribonucteoprotein

particte

oroanized as a series

of beads.

broad size distribution.

it was called

heteroge-

neous nuclear

RNA

(hnRNA). It includes

pre-mRNA,

but could

also

include

other tran-

scripts

(that

is, transcripts

that

are not ultimately

processed

to

nRNA).

The

physical form of

hnRNA

is a ribonu-

cleoprotein

particle

(hnRNP), in which

the

hnRNA is bound

by

proteins. As characterized

in vitro, an hnRNP

particle takes

the

form of

beads connected

by a

fiber.

The structure

is sum-

marized

in

t

i'.

r,;lrr

,;{

i.

The

most abundant

pro-

teins

in the

particle

are

the core

proteins,

but

other

proteins

are

present

at

lower

stoichiom-

etry, making

a total of

-20

proteins. The

proteins

typically are

present

at

-

I 08

copies

per

nucleus,

compared with

-l

06 molecules

of hnRNA.

Some

of the

proteins may have

a structural

role in

packaging

the hnRNA;

several

are known

to

shuttle

between the

nucleus

and

cytoplasm

and

play

roles in exporting

the

RNA or otherwise

controlling

its activity.

26.1

Introduction

669

Cap at

5' end

Exon

1

lntron

1 Exon

2 Intron 2 Exon

3 Intron

3

Exon4

lntron4 Exon 5

I

f

Transcription

is

controlled. What ensures

that the ends

of

each intron

are

recognized

in

pairs

so that the

correct sequence is removed from

the RNA?

Are introns excised from

a

precursor

in

a

par-

ticular order? Is the maturation

of RNA

used to

regulate

gene

expression by discriminating

among the available

precursors

or by changing

the

pattern

of splicing?

We can identify

several types of

splicing

systems:

.

Introns

are removed from

the nuclear

pre-mRNAs

of higher

eukaryotes

by a

system that

recognizes

only shclrt con-

sensus sequences

conserved

at exon-

intron

boundaries and within

the intron.

This reaction

requires

a

large

splicing

apparatus, which takes

the form

of an

array of

proteins

and ribonucleoproteins

that functions

as a large

particulate

com-

plex (the

spliceosome). The

mechanism

of splicing involves transesterifications,

and the catalytic

center includes

RNA

as well as

proleins.

.

Certain RNAs have

the ability

to excise

their introns autonomously.

Introns

of

this

type

fall

into two

groups,

as

distin-

guished

by secondary/tertiary

structure.

Both

groups

use

transesterificaticln

reac-

tions in

which the RNA is

the catalytic

agent

(see

Chapter 27,

Catalytic RNA).

.

The

removal of introns

from

yeast

nuclear

IRNA

precursors

involves

enzy-

matic

activities that handle

the

substrate

in

a way resembling

the IRNA

process-

ing

enzymes, in

which a critical

feature

is

the conformation

of the

IRNA

precur-

sor. These

splicing reactions

are

accom-

plished

by enzymes that

use cleavage

and ligation.

Nuclear

Splice

Junctions

Are

Short

Sequences

.

Splice

sites are the sequences

immediatety

surrounding the

exon-intron boundaries.

They

are

named

for their

positions

relative

to the intron.

r

The

5'splice

site at the 5'(teft)

end of

the'intron

jnctudes

the consensus

sequence

GU.

r

The

3' spLice site

at the 3'

(right)

end

of the intron

includes

the consensus

sequence AG.

r

The

GU-AG ruLe (origina[Ly

catled

the GT-AG ru[e

in

terms of DNA

sequence) describes

the requirement

for

these constant

dinucteotides

at the first

two

and

last two

positions

of introns in

pre-mRNAs.

I End

modification

PolY(A)

al 3' end

V

tAzoo

I ^ ..

.

+

5pilong

exon intron

exon

I

Exon-intron

tunctrons

are

broken

I

eron. are

joined

tu=

I

^200

Transporl

NUCLEUS

CYTOPLASM

Azoo

RNA

is modified

in

the

nucLeus

by additions to the 5'and

3'ends and by

splicing

to remove

the introns. The

splicing

event requires

breakage

of the exon-jntron

junctions

and

joining

of the

ends of the

exons. Mature mRNA is

transported

tnrougn

nuc[ear

pores

to the

cytop[asm,

where it is

trans[ated.

Splicing

occurs in

the

nucleus,

together

with

the other modifications

that are made

to newly

synthesized

RNAs. The

process

of expressing an

interrupted gene

is

reviewed in

. The

transcript is

capped

at the 5'end

(see

Section 7.9,

The 5'End

of Eukaryotic

nRNA Is

Capped), has

the introns

removed,

and is

polyadenylated

at

the l' end

(see

Section

7. 10, The

J'

Terminus

Is

Polyadenylated).

The RNA is

then transported

through nuclear pores

to the

cyroplasm,

where

it is

available

to be translated.

With regard

to the various

processing

reac-

tions

that occur in

the nucleus.

we

should like

to kncrw

at what

point

splicing occurs

vis-a-vis

the

other modifications

of RNA. Does

splicing

occur

at a

particular

location

in the nucleus,

and is

it connected

with

other events-for

exam-

ple,

nucleocytoplasmic

transport? Does

the lack

of splicing

make

an important

difference

in the

expression

of

uninterrupted genes?

With regard

to the

splicing reaction

itself,

one

of the

main

questions

is how

its specificity

670

CHAPTER

26 RNA

Spticing

and

Processing

Lewin Benjamin (ed.) Genes IX

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