Lewin Benjamin (ed.) Genes IX

Intron

inserted in

codon 10

I

Translation

B

galactosidase

I

V

Blue

plaques

generated

by staining for-

,'

:

*

B

galactosidase o

@

Catalytic

RNA has a

guanosine-binding

site and

substrate-binding site

Guanosine

binding site

-

3',

First transfer G-OF occupies

G-binding

site; 5'

exon

occupies substrate-binding

site

5'

,'

CUCUCU

Substrate bindino

site

-

uuuAuu

q.oH

\

s', eilffiJr/

GGGAGG

UUUACCU

I

lncludes

416 un6

g2o

::

i;.il : il i;

it

.r

.

r;

Ptaci ng

the

Tetrahy

m e na intron within the

B-gatactosidase

coding

sequence creates an assay for se[f-

spticing

in E. coLi. Synthesis

of

B

galactosidase

can be

tested by

adding a compound

that

is

turned blue by the

enzyme.

The

sequence

js

carried by a bacteriophage, so

the

presence

of blue

plaques (containing

infected bacte-

ria) indicates successfuI

splicing.

similar

effects. A mutation in

one consensus

sequence

may

be

reverted

by a mutation in the

complementary consensus sequence to restore

pairing;

for example, mutations in the R con-

sensus

can be compensated

by

mutations in the

S consensus.

Together these results

suggest that the

group

I splicing reaction depends on the formation of

secondary structure between

pairs

of consensus

sequences

within the intron. The

principle

estab-

lished

by

this work is

that sequences distant

from

the splice

junctions

themselves are required to

form

the active site that makes self-splicing

possible.

@

Ribozymes

Have

Various

Catalytic

Activities

By changing the substrate binding-site of

a

group

I

intron. it is

possible

to introduce alternative

sequences

that

jnteract

with the reactive G.

The

reactions foltow

ctassicaI enzyme

kinetics with

a low catalytic

rate.

Reactions using

2'-0H

bonds coutd

have been the

basis

for

evotving the

original catalytic activitjes

in RNA.

Second transfer G't1a

is in G-binding

site;

5' exon

is in

substrate-binding

s;ite

G

/:

GGGAGG

5"..,cucucu

@3'

Third transfer Gala

is in G-binding

site;

5' end

of intron

is

in substrate-bincling

site

lil{:i.jltt i:,r',ir.

Excision of the

group

I intron

in Tetrahymena

rRNA occurs by successive

reactions

between

the occupants

of

the

guanosine-binding

site

and

the substrate-binding

site.

The

[eft exon

is

pink,

and the

right

exon

is

purpte.

The catalytic

activity

of

group I introns was

dis-

covered by virtue

of their

ability

to autosplice,

but

they are abl,:

to undertake

other

catalytic

reactions invitro.

All of

these

reactions

are based

on transesterific;rtions.

We analyze

these

reac-

tions

in

terms

of their

relationship

to the

splic-

ing reaction

itself.

The catalytic

activity

of a

group I intron

is

conferred

by its

ability

to

generate

particular

secondary

and lertiary

structures

that

create

active sites that

are equivalent

to the

active

sites

of a conventional

(proteinaceous)

enzyme.

l:;i;iji:l{:

l:-i.,:-

illustrates

the splicing

reaction

in

terms of

these sites

(this

is the

same

series

of

reactions

shown

previously

in Figue

27

.2).

u

27.4 Ribozymes

Have Various

Catatytic

Activities

7lt

Contacts found before

substrate bindino

Contacts

tound after substrate

bindino

F:GUftt 27.? The

posjtion

ofthe IGS in the

tertiary struc-

ture

changes when P1 is

formed

by substrate binding.

The substrate-binding

site is formed

from

the PI helix,

in which

the J' end

of the first

intron

base

pairs

with

the IGS in

an

intermol-

ecular reaction.

A

guanosine-binding

site is

formed

by sequences

in P7. This

site may be

occupied

either

by a free

guanosine

nucleotide

or by the

G residue in

position

4l4.In

the first

transfer reaction,

it is

used by free

guanosine

nucleotide;

it is subsequently

occupied by

(+t+.

The

second transfer

releases

the

joined

exons.

The

third transfer

creates

the circular

intron.

Binding

to the substrate

involves

a change

of

conformation.

Before

substrate

binding, the

5'

end of

the IGS is

close to P2

and P8; after

binding,

when

it forms

the Pl helix,

it is close

to

conserved

bases that

lie between

P4 and P5.

The

reaction

is visualized

by contacts

that

are

detected

in the

secondary

structure in

FiSlJftg

t?.7.

In the

tertiary structure,

the

two

sites

alternatively

conracted

by Pl

are 37 A apart,

which implies

a substantial

movement

in

the

position

of Pl.

The

L- l9 RNA

is

generated

by opening the

circular intron

(shown

as

the last

stage of the

intramolecular

rearrangements

in Figure

2 7.

3

)

.

lt still retains

enzymatic

abilities,

which resem-

ble the activities

involved

in

the

original splic-

ing

reaction.

We may

consider

ribozyme

lunction

in

terms of

the ability

to bind

an

intramolecular

sequence

complementary

to the

CHAPTER

27 Catalvtic

RNA

Cs

pairs

with

IGS site near

5'

end of RNA

G-OH attacks

CpC bond

C is transferred

to

3'-G;

Ca

is

released

Another

C, binds;

transfer

reaction

is reversed

C5

is released,

regenerating

L-19

RNA

FtGtlSt

??.& The L-19

linear RNA

can bind C in

tne

substrate-binding

site;

the

reactive

G-0H

3'end

js

located

in

the G-binding

site, and catalyzes

transfer reactions

that

conved two C5

otigo-nucleotides into

a Ca and a

C6 otigonu-

cteotide.

IGS in the

substrate-binding

site while

binding

either

the terminal

G414 or a free

G-nucleotide

in

the G-binding

site.

FI6U*f

?7.8 illustrates

the mechanism

by

which

the oligonucleotide

C5 is extended

to

generate

a C6 chain.

The C5

oligonucleotide

binds in

the substrate-binding

site, whereas

Gala

occupies the

G-binding site.

By transesterifica-

tion reactions,

a

C

is

transferred

from

C5 to

the

3'-terminal

G, and

then back

to a new

C5 mol-

ecule.

Further

transfer reactions

lead

to

the

accumulation

of longer

cytosine

oligonu-

cleotides.

The reaction

is a true

catalysis,

because

the

L- l9 RNA remains

unchanged

and is

avail-

able

to catalyze

multiple

cycles.

The ribozyme

is

behaving

as a nucleotidyl

transferase.

772

Sequence-specific endoribonuclease

3',

G-OH

5' 3',

-.;5'*QgCU-OH

5'G

@3'

GGGAGG

5'

RNA ligase

5'@Y*"3'

+G

Phosphatase

\r-un

v

5' UCUp

3'

uuuAuu 3

+ s', UCU-OH 3',

il,:+LlitL

:i1".:.1

Catatytic reactions

of the

ribozyme involve

transesterifications

between

a

group

in

the substrate-

binding site and a

group

in the

G-binding site.

Some

further

enzymatic reactions are char-

acterized in

:rTi:,qiiti

-::,!.t.

The ribozyme can func-

tion as a sequence-specific endoribonuclease

by utilizing the ability of the IGS to bind com-

plementary

sequences.

In this

example,

it binds

an external substrate containing the sequence

CUCU, instead of binding the analogous

sequence

that is usually contained

at

the end of

the left exon. A

guanine-containing

nucleotide

is

present

in

the G-binding site, and attacks

the

CUCU sequence

in

precisely

the

same

way that

the exon

is usually attacked in the first trans-

fer reaction.

This

cleaves the target sequence

into a 5'molecule that

resembles

the

left

exon,

and a l' molecule that bears a terminal G

residue.

By mutating the IGS element, it is

pos-

sible

to change the specificity of the

ribozyme

so that

it recognizes sequences complementary

to the

new

sequence

at the IGS region.

Altering

the IGS so that the specificity of

the substrate-binding

site

is

changed

to enable

other RNA targets

to enter can be used to

gen-

19-base virusoid

L-19 lntron

RNAase

P RNA

RNAase P completr:

RNAase T1

B

galactosidase

24-base

RNA 0.0006

0.5

cccccc

0.04

1.7

pre-tRNA

0.00003

0.4

pre{RNA

0.00003

29

GpA

0 05

5,700

lactose

4.0 12,500

+rISLiiqil

,ill:.1i]

Reac.:ions

catatyzed

by

RNA have the

same

features as those

catatyzed

by

proteins,

although

the rate

is

stower.

The KM

gives

the

concentration

of substrate

required for half-maximum

vetocity;

this

is

an

inverse meas-

ure of the affinity

of t he enzyme

for substrate.

The turnover

number

gives

the number

of substrate

molecules trans-

formed in unit time lly

a sing[e catatytic

site.

erate

a ligase activity.

An

RNA terminating

in a

l'-OH is bound

in the

substrate

site,

and an

RNA terminating

in a 5'-G

residue

is bound

in

the G-binding

sit.e.

An attack

by the

hydroxyl

on the

phosphat,:

bond

connects

the

two RNA

molecules, with

the

loss of the

G residue.

The

phosphltase reaction

is not directly

related to the str'licing

transfer

reactions.

An

oligonucleotide

sequence

that

is complemen-

tary to the

IGS and

terminates

in

a 3'-phos-

phate

can be

lttacked

by

the G4r4.

The

phosphate

is transferred

to the

Gaia, and

an

oligonucleotide

',,vith

a free

3'-OH

end

is

then

released. The

phc,sphate can then

be transferred

either to an

oli;lonucleotide

terminating

in

3'-OH

(effective

ly

reversing

the

reaction)

or

indeed to water

(releasing

inorganic

phosphate

and completing,

an

authentic

phosphatase

reaction).

The

reactions catalyzed

by

RNA can be

char-

acterized in the

sirme

way as

classical

enzymatic

reactions in terrrLs

of

Michaelis-Menten

kinet-

ics.

trli.;Lil'dt: ;f

,:

i

ii

analyzes

the

reactions

catalyzed

by RNA. The

I(y'ralues

for

RNA-catalyzed

reac-

tions are

low, anrl

therefore

imply that

the

RNA

can bind

its subs:rate

with

high specificity.

The

turnover

numbers

are

low, which

reflects a

low

catalytic

rate.

Lt effect,

the

RNA

molecules

behave

in the s,rme

general manner

as tradi-

tionally defined

Ior enzymes,

although

they are

relatively slow

<:ompared

to

protein

catalysts

(where

a

typical

range

of turnover

numbers

is

103 ro 106).

A

powerful

extension

of the

activities

of

ribozymes

has been

made

with

the

discovery

that

they can be

regulated

by

ligands

(see

Sec-

tion 13.7, Small

RNA

Molecules

Can

Regulate

Enzyme

Substrate

Ky

Turnover

(mM)

(/min)

27.4 Ribozymes

Have Various

Catalytic

Activities

773

::iti;&i

i:..1

t

A ribozyme

is

contained within

the 5'untranstated region

of the mRNA

coding for

the

enzyme

that

produces

gtucosamine-6-phosphate.

When

G[c-6-P binds to the ribozyme,

it cteaves

offthe

5'end ofthe mRNA,

thereby

inactivating it

and

preventing

further

production

ofthe enzyme.

Translation).

ij'L:ij$ii

i:,::

summarizes

the reg-

ulation

of a riboswitch.

The

small

metabolite

GlcN6P

binds

to a ribozyme

and

activates its

ability to

cleave the

RNA in

an intramolecular

reaction.

The

purpose

of the system is

to regu-

late

production

of

GlcN6P;

the ribozyme is

located

in

the 5' untranslated

region

of the

nRNA

that

codes for

the enzyme

involved in

producing

GlcN6P,

and the

cleavage

prevents

translation.

How

does RNA

provide

a catalytic

center?

Its ability

seems

reasonable

if

we think

of an

active

center

as a surface

that exposes

a series

of active

groups

in

a fixed relationship.

In a

pro-

tein, the

active

groups

are

provided

by the

side

chains

of the amino

acids,

which have

appre-

ciable

variety, including positive

and negative

ionic groups

and hydrophobic groups.

In

an

RNA,

the available

moieties

are more

restricted,

consisting primarily

of the exposed groups

of

bases.

Short

regions

are held

in

a

particular

**.1.'dffitril*,':1"i{ii;:rrur

in

which

bonds

can

be broken

and made

in

another

molecule.

It seems

inevitable

that

the

CHAPTER

27

CataLytic

RNA

interaction

between the RNA

catalyst

and the

RNA substrate

will rely on

base

pairing

to cre-

ate the environment.

Divalent

cations

(typically

Mg2+)

play

an important

role in

structure,

typ-

ically

being

present

at the

active site

where they

coordinate

the

positions

of the various groups.

They

play

a direct role in

the

endonucleolytic

activity

of virusoid ribozymes

(see

Section 27.9,

Viroids Have

Catalytic Activity).

The

evolutionary

implications

of

these dis-

coveries are intriguing.

The

split

personality

of

the

genetic

apparatus-in

which

RNA is

pres-

ent in

all components.

but

proteins

undertake

catalytic reactions-has

always

been

puzzling.

It seems

unlikely

that the very

first replicating

systems

could have

contained

both nucleic

acid

and

protein.

Suppose,

though, that

the first

systems

con-

tained

only

a self-replicating

nucleic

acid

with

primitive

catalytic activities-just

those

needed

to make

and break

phosphodiesrer

bonds.

If we

suppose

that the involvement

of.2'-OH

bonds

in

current

splicing reactions

is

derived

from

these

primitive

catalytic activities,

we may argue

that the original

nucleic

acid

was RNA,

because

DNA lacks

the

2'-OH

group

and therefore

could

714

not undertake such reactions.

Proteins

could

have

been added for their ability

to stabilize the

RNA

structure.

The

greater

versatility

of

pro-

teins then could have

allowed them to take

over

catalytic reactions, leading

eventually

to the

complex and sophisticated apparatus

of modern

gene

expression.

Some Group I Introns

Code

for Endonucleases

That

Sponsor

MobiLity

o

Mobite

introns

are able to insert themselves into

new

sites.

.

Mobile

group

I introns code for

an endonuctease

that makes a doubte-strand break

at a target site.

o

The intron

transposes

into

the site of the doubte-

strand break by a

DNA-mediated

replicative

mechanism.

Certain introns of both the

group

I

and

group

II

classes contain open reading frames that are

translated into

proteins.

Expression

of the

pro-

teins allows the

intron

(either

in its original

DNA form or as a DNA copy

of the

RNA)

to be

mobile:It is able to insert itself

into a new

genomic

site.

Introns

of both

groups

I and II are

extremely widespread,

being

found in

both

prokaryotes

and eukaryotes. Group I in-

trons migrate by DNA-mediated mechanisms,

whereas

group

II introns migrate

by

RNA-medi-

ated

mechanisms.

Intron mobility was first detected by crosses

in which the alleles

for

the relevant

gene

differ

with

regard to their

possession

of the

intron.

Polymorphisms

for

the

presence

or absence of

introns are common in fungal mitochondria.

This is

consistent with the

view

that these

introns originated by

insertion

into the

gene.

Some

light on the

process

that

could be

involved

is cast by an analysis of

recombination in

crosses

involving the

large

rRNA

gene

of the

yeast

mitochondrion.

This

gene

has a

group

I intron

that

contains

a coding sequence.

The intron is

present

in

some

strains

of

yeast

(called

ol+) but absent in others

(trl-).

Genetic crosses between ro+ and

al- are

polar:The progeny

are usually <rl+.

If we think of the cl+ strain as a donor and

the ol- strain

as a recipient, we form the

view

that

in rrl+

x

o- crosses a new copy of the intron

is

generated

in the rrl-

genome.

As a result, the

progeny

are

all rrr+.

1111i;i:ir li

.;'.:f An

intron codes

for an endonuctease

that

makes a

double-strand

break

in DNA.

The sequence ofthe

intron

is duplicated

and then inserted at

the break.

Mutations

can occur

in either

parent

to

abolish the

polarLty. Mutants

show

normal seg-

regation, with eq

ual numbers

of

o+ and o-

prog-

eny.

The mutations

indicate

the

nature of the

process.

Mutations

in the

ro- strain occur

close

to the site where

the

intron would

be

inserted.

Mutations in the

ot+ strain

lie

in the reading

frame of the

intron and

prevent

production

of

the

protein. Tris suggests

the model

of

]'iii.riti:,r

i'. i"r,

in

v,zhich the

protein

coded by

the

intron in

an

o+ sl

rain recognizes

the

site where

the intron should

be

inserted

in an

co- strain

and causes

it to be

preferentially

inherited.

What is the

action of

the

protein? The

prod-

uct of the

o intron

is an endonu

clease that

rec-

ognizes the o-

ger'e

as

a target

for

a

double-strand

break.

The endonuclease

recognizes

an

18 bp

target sequence

that

contains

the

site where

the

intron is inserted

The

target sequence

is cleaved

on

each strand of

DNA

two bases

to the

l' side

of the

insertion site.

Thus

the cleavage

sites

are

4

bp

apart and

generate overhanging

single

strands.

This type

of

rleavage

is related

to the cleav-

age characteristic

of transposons

when

they

migrate to

new sites

(see

Chapter

21,

Trans-

posons).

The

dorLble-strand

break

probably ini-

tiates a

gene

colrversion

process in which

the

sequence

of the

rrt+

gene

is copied

to

replace the

sequence

of the

o-

gene.

The

reaction

involves

transposition

by

a duplicative

mechanism,

and

occurs solely

at

the

level of

DNA.

Insertion

of

27.5

Some

Group

I Introns

Code

for

Endonucleases

That sponsor

Mobil.ity

775

The

introrr is replicated

and then

inserted

the intron interrupts

the

sequence recognized

by the

endonuclease,

thus ensuring stability.

Many group

I introns

code for

endonucle-

ases that

make them mobile.

Several different

families

of endonucleases

are found;

one com-

mon

feature is

the

presence

of the

amino acid

sequence LAGLIDADG

near the

active site. Sim-

ilar introns

often carry

quite

different

endonu-

cleases.

There

are differences in

the details of

insertion; for

example,

the endonuclease

coded

by the

phage

T4

td

intron

cleaves

a target site

that is 24

bp upstream of the

site at which the

intron

is itself

inserted. The

dissociation between

the intron

sequence

and the

endonuclease

sequence is

emphasized by

the fact that the

same

endonuclease

sequences are found in

inteins

(sequences

that code for

sel{-splicing

proteins;

see

Section 27.12, Protein

Splicing Is

Autocatalytic).

i-1.i.:i.tl.li:

;ir'.

ii:

Reverse

transcriptase

coded by an intron

allows

a copy of

the

RNA

to be inserted

at a target

site

generated

by a doubte-strand

break.

CHAPTER

27

Catatytic

RNA

The

variation

in

the endonucleases

means

that there is no homology

between

the

sequences of their target

sites. The target

sites

are among the Iongest and therefore

the most

specific known for any

endonucleases

(with

a

range of 14to 40

bp).

The

specificity

ensures

that the intron

perpetuates

itself

only by inser-

tion into a single target

site and not elsewhere

in the

genome.

This

is called intron

homing.

Introns carrying

sequences that

code for

endonucleases

are found in a

variety of bac-

teria and lower eukaryotes.

These results

strengthen the view that introns

carrying

cod-

ing sequences

originated as independent

elements.

Group

II

Introns May

Code

for Multifunction

Proteins

Group II introns

can autosptice in

vitro, but

are

usua[[y

assisted by

protein

activities

coded within

the intron.

A single

coding frame specifies

a

protein

with

reverse

tra

nscri

ptase

a ctivity, maturase

activity,

DNA-binding

motif, and a DNA

endonuctease.

The reverse

transcriptase

generates

a DNA copy

of

the RNA sequence that transposes

by a retroposon-

[ike mechanism.

The

endonuctease cteaves

target DNA

to altow

insertion

of the transposon

at a new

site.

The

best characterized

mobile

group

II introns

code for a single

protein

in a region

of the intron

beyond its

catalytic core. The

typical

protein

contains an

N-terminal reverse

transcriptase

activity,

a central

domain associated

with an

ancillary

activity that assists folding

of the intron

into its

active structure

(called

the maturase;

see Section

27 .7

,

Some Autosplicing

Introns

Require

Maturases),

a DNA-binding

domain,

and

a C-terminal

endonuclease

domain.

The

endonuclease initiates

the transposi-

tion reaction,

and

plays

the

same role

in hom-

ing as its

counterpart in

a

group

I

intron.

The

reverse

transcriptase generates

a DNA

copy

of

the intron

that is inserted

at the

homing

site.

The

endonuclease

also cleaves

target

sites that

resemble,

but are not identical

to, the

homing

site

at much lower

frequency,

leading

to inser-

tion

of the intron

at new locations.

t:; ii:

i.i

l;:

h ir

;i . :i :1

illustrates

the transposition

reac-

tion for

a typical

group

II intron.

The

endonu-

clease makes

a

double-strand

break at

the target

776

site.

The

endonuclease activity

requires both

the

domain of the

protein

and the intron

RNA. The

protein

domain cleaves

the antisense

strand of

DNA,

and the intron RNA

actually cleaves the

sense strand. This reaction

directly inserts

the

intron into the DNA

target site.

The intron RNA

provides

the

template

for

the synthesis of cDNA. Almost

all

group

II

introns have a reverse

transcriptase activity

that

is

specific

for

the

intron.

The reverse

transcrip-

tase

generates

a DNA

copy of the intron. the

result being the insertion of

the

intron

into the

target site as a duplex DNA. The

mechanism

resembles the transposition

of retroviruses, in

which the

RNA is

an obligatory intermediate

(see

Section 22.2, Tlrre Retrovirus

Life Cycle

Involves Transposition-Like

Events). The type

of retrotransposition involved in

this case resem-

bles that of a

group

of retroposons that lack

LIRs, and which

generate

the 3'-OH needed

for

priming

by making

a

nick

in the target

(see

Figure 22.20 in

Section 22.I2, LINES Use an

Endonuclease to Generate

a

Priming

End).

Some

AutospLicing

Introns Require Maturases

.

Autospticing introns may require maturase

activities encoded within the intron

to assist

fotding

into

the active catalytic structure.

Although

group

I

and

group

II introns both have

the capacity to autosplice in vitro,

under

physi-

ological conditions they usually require assis-

tance

from

proteins.

Both

types of intron may

code for maturase activities that are required

to assist the splicing

reaction.

The maturase activity is

part

of the single

open reading frame coded by the intron. In the

example

of introns that

code

for homing

endonucleases,

the

single

protein product

has

both endonuclease and maturase activity. Muta-

tional

analysis shows that the two activities are

independent.

Structural analysis shows that the endonu-

clease

and maturase activities are

provided

by

different

active sites in the

protein,

each

coded

by a separate domain.

The

endonuclease site

binds

to DNA, but the maturase site binds to

the intron

RNA. ,'..r,.'.

:

,'

shows the struc-

ture of one such

protein

bound to DNA.

A

char-

acteristic

feature

of the endonuclease

is the

presence

of

parallel

cr

helices,

which

contain

the hallmark

LAGLIDADG

sequences,

Ieading

to the two cataly.ic

amino

acids.

The maturase

activity is locaterl some

distance

away on the

surface of the

protein.

Introns that code

for maturases

may be

unable to splice

themselves

effectively

in the

absence of the

prctein activity.

The maturase

is

in effect a splicinl;

Iactor that

is required

specif

-

ically for splicing of the

sequence

that codes

for

it. It functions tc, assist

the folding

of the cat-

alytic core to

forrn an active

site.

The

coexistence

of

endonuclease

and

mat-

urase activities

in the same

protein

suggests

a route for tht:

evolution

of the

intron.

irirli:iir

,.;' I

suggests

that the

intron originated

in

an

independent autosplicing

element.

The

insertion into th

s element

of a sequence

cod-

ing for

an endonuclease

gave

it mobility.

The

insertion,

howe ver,

might well

disrupt the

ability

of

the R|trA sequence

to

fold into the

active structure,

This would

create

pressure

for assistance

from

proteins

that

could restore

folding ability.

'fhe

incorporation

of such a

sequence

into tlLe

intron would

maintain

its

independence.

Some

group

II introns

that

do not code

for

maturase activit es

may

use comparable

pro-

teins that are

coled by

sequences

in the host

genome.

This sul;gests

a

possible route for the

evolution of

gent:ral

splicing

factors. The

factor

may

have

originirted

as a

maturase that

specif-

ically assisted

the splicing

of

a

particular intron.

The coding sequcnce

became

isolated

from the

intron in the

host

genome, and

then

it

evolved

to

function with ir wider

range of substrates

that

the original

intron sequence.

The catalytic

core

of the

intron could

have evolved

into an snRNA.

:

i';.1rt'1 .

'.

i A homing

intron

codes

for an endonucte-

ase ofthe

LAGLIDADTi

famity that

atso

has

maturase activ-

ity. The LAGLIDADG

sequences

are

paft

ofthe

two a

helices

that terminate

in

tl-e

catalytic

amjno

acids close

to the

DNA duplex.

The maturase

active

site'is

identified

by an

arginine residue

etstrwhere

on

the surface

of the

protein.

27.7

Some

Autospticing

Introns

Require

Maturases

777

\/\t/\t\f\r'

\/\/\t\/\t\/

I

v

InIron

Endonuclease

gene

is

inserted

into intron

Intron

carries

endonuclease

Maturase

gene

is inserted

into intron

lntron

carries

endonuclease

and maturase

component

superfluous, though. The RNA

alone

can catalyze the reaction! Analyzing

the results as

though the RNA

were an enzyme,

each

"enzyme"

catalyzes the cleavage

of multiple

substrates. Although the catalytic

activity resides

in the RNA, the

protein

component

greatly

increases

the speed of the reaction,

as

seen in

the increase in turnover

number

(see

Fig-

ure 27.I0).

Mutations in

either the

gene

for

the RNA

or the

gene

for

protein

can inactivate

RNAase

P invivo,

so we know that both

components

are

necessary for natural

enzyme

activity. Odgi-

nally

it had been assumed

that the

protein

pro-

vided the catalytic

activity, whereas

the RNA

filled some

subsidiary

role-for

example,

assist-

ing in the

binding of substrate

(it

has some

short

sequences complementary

to exposed

regions

of IRNA). These

roles, however,

are reversed!

Viroids Have

Catalytic

Activity

Viroids and virusoids

form a hammerhead

structure

that has

a self-cleaving activity.

Simitar

structures can be

generated

by

pairing

a

substrate strand that is

cteaved by an

enzyme

stra nd.

When an enzyme strand is introduced

into

a cet[, it

can

pair

with a substrate

strand target

that is then

cteaved.

Another

example

of the ability

of RNA ro func-

tion as

an endonuclease is

provided

by some

small

plant

RNAs

(-350

bases)

that undeftake

a

self-cleavage reaction.

As with

the case of

the

Tetrahymena

group

I intron,

however,

it is

pos-

sible to

engineer constructs

that

can function

on external substrates.

These

small

plant

RNAs

fall into

two

gen-

eral

groups:

viroids and

virusoids. The

viroids

are infectious

RNA molecules

that function

in-

dependently

without encapsidation

by any

protein

coat. The virusoids

are

similar

in

organizalion

but

are encapsidated

by

plant

viruses,

being

packaged

together

with

a viral

genome.

The

virusoids cannot

replicate

inde-

pendently.

but require assistance

from

the

virus.

The

virusoids

are sometimes

called

satellite

RNAs.

Viroids

and virusoids

both

replicate

via

rolling

circles

(see

Figure

I6.6). The

srrand

of

RNA

that is

packaged

into

the virus is

called the

plus

strand. The

complementary

strand,

gen-

ii';U*[

ti.15

The intron

originated

as

independent

sequence coding

for a self-splicing

RNA. The insertjon

of

the

endonuclease

sequence

created a homing intron

that

was mobi[e.

The insertion

of the maturase

sequence

then

enhanced

the abitity

of the

intron

sequences to fold into

the active

structure for splicing.

@

The

CataLytic Activity

of

RNAase P

Is Due

to RNA

o

Ribonuctease

P is

a ribonucteoprote'in

in

which the

RNA has

catalytic

activity.

One of the

first demonstrations

of the

capabil-

ities

of RNA

was

provided

by the dissection

of

ribonuclease

P, an E.

coli

tRNA-processing

endonuclease.

Ribonuclease

P can

be dissoci-

ated into

its

two components

,

the

37 5 base RNA

and

the 20 kD

polypeptide.

Under

the condi-

tions initially

used to

characterize

the enzyme

activity

in vitro,

both components

were neces-

sary to

cleave the

IRNA

substrate.

A

change in ionic

conditions

(an

increase

in

the

concentration

of Mg2+) renders

the

protein

CHAPTER

27

Catatytic

RNA

778

6r.L

flrnrpy

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oDEAEOIS

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Luels

saseq

peruesuoc pue

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ooJr,.ll o^eq speaqreuruJPLl

snsuosuoc

s', 3',

GC

Stem3!

A

t/u

GA

UA

.

,'.:i..

:

-'

-.

i

:

A

hammerhead

ribozyme

forms a V-

shaped

tertiary

structure in which

stem 2 is

stacked upon

stem

3.

The

catatytic

center

lies between

stems 2 and

3

and stem

1. It

contains a maqnesium

ion

that

initiates

the

hydroLytic

reaction.

reaction

to occur in vivo.

A ribozyme

designed

in

this

way essentially provides

a

highly

spe-

cific

restriction-like

activity

directed against

an

RNA

target.

By

placing

the ribozyme

under con-

trol

of a regulated promoter,

it can

be used in

the same

way as

(for

example)

antisense

con-

structs

specifically

to

turn off

expression

of a

target

gene

under

defined

circumstances.

RNA Editing

Occurs

at IndividuaL

Bases

.

Apotipoprotein-B

and

glutamate

receptors

have

site-specific

deamjnations

catalyzed

by cytidine

and

adenosine

deaminases

that

change the

coding

sequence.

A

prime

axiom

of molecular

biology

is that

the

sequence

of an nRNA

can

only represent

what

is coded

in

the DNA.

The

central

dogma

envis-

aged

a linear

relationship

in

which

a continu-

ous

sequence

of DNA

is

transcribed

into

a

sequence

of mRNA

that is

in turn

directly

trans-

lated

into protein.

The

occurrence

of

interrupted

genes

and

the removal

of introns

by RNA

splic-

ing

introduces

an additional

step into

the

process

of

gene

expression:

The

coding

sequences

(exons)

in

DNA

must

be

reconnected

in

RNA.

The

process

remains

one

of information

trans-

CHAPTER

27

Catatytic

RNA

fer,

though, in which

the actual

coding sequence

in DNA

remains unchanged.

Changes in the information

coded by DNA

occur in

some exceptional

circumstances,

most

notably

in the

generation

of new

sequences

cod-

ing for immunoglobulins

in

mammals

and birds.

These

changes

occur specifically in

the

somatic

cells

(B

lymphocytes)

in which

immunoglobu-

Iins are

synthesized

(see

Chapter 23,

Immune

Diversity).

New

information

is

generated

in

the

DNA of an individual

during

the

process

of

reconstructing

an immunoglobulin gene,

and

information

coded in the DNA

is changed

by

somatic mutation. The

information

in DNA

con-

tinues to

be

faithfully

transcribed

into

RNA.

RNA

editing is

a

process

in

which informa-

tion changes

at the level of

mRN,4. It is revealed

by

situations

in which

the coding

sequence

in an

RNA differs from

the sequence

of

DNA

from

which it

was transcribed.

RNA

editing occurs

in two

different situations,

each

with different

causes. In mammalian

cells

there

are cases in

which

a substitution

occurs in

an individual

base in mRNA,

causing a change

in

the sequence

of the

protein

that is

coded. In

trypanosome

mitochondria,

more

widespread

changes

occur

in

transcripts

of several

genes.

when

bases are

systematically

added

or deleted.

t t;;;i3 i:S ;'

r-. i ij

summarizes

the

sequences

of

the apolipoprotein-B gene

and mRNA

in mam-

i:l{i,i!'ii.'

r i.li

i* The

sequence

of the

apo-B

gene

is the

same in

intestine

and Uver,

but the

sequence

ofthe mRNA

is

modjfied

by a base

change

that creates

a terminatjon

codon in

intestine.

Apolipoprotein

B

gene

has 29

exons

UAA

I

v

oftry

Intestine

mRNA

has

UAA codon

that

terminates

synthesis

at

2153

I

Y

CAA

I

V

ofrfufury

Spliced mRNA

in liver

codes

tor

protein

of

4563

residues

720

Lewin Benjamin (ed.) Genes IX

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