Lewin Benjamin (ed.) Genes IX
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Introduction
r
RNA
functions
as a regulator
by
forming
a
region
of secondary
structure
(either
inter-
or
intramolecular)
that changes
the
properties
of a
target sequence.
The
basic
principle
of regulation
in
bacteria is
that
gene
expression
is
controlled
by
a
regula-
tor
that interacts
with
a specific
sequence
or
structure
in DNA
or nRNA
at some
stage
prior
to the
synthesis
of
protein.
The
stage
of expres-
sion
that is
controlled
can
be transcription,
when
the
target
for regulation
is
DNA,
or it
can be at
translation,
when
the target
for regulation
is
RNA. When
control
is
during
transcription,
it
can be
at initiation
or at termination.
The reg-
ulator
can
be a
protein
or an
RNA.
"Controlled"
can
mean
that the regulator
turns
off
(represses)
the
target
or
that it turns
on
(activates)
the tar-
get.
Expression
of many
genes
can be
coordi-
nately
controlled
by
a single regulator gene
on
the
principle
that
each target
contains
a
copy
of
the sequence
or structure
that
the regulator
recognizes.
Regulators
may
themselves
be reg-
ulated,
most
typically in
response
to
small mol-
ecules
whose
supply
responds
to environmental
conditions.
Regulators
may
be
controlled
by
other
regulators
to make
complex
circuits.
Let's
compare
the ways
that
different
types
of regulators
work.
Protein
regulators
work on
the
principle
of
allostery.
The
protein
has
two
binding
sites-
one
for
a nucleic
acid
target,
the
other for
a
:r:',ii1;r:i li:.':
A regulator
RNA is
a smat[
RNA
with a sin-
gle-stranded
region
that
can
pair
with
a singte-stranded
region
in
a
target RNA.
small molecule.
Binding
of
the small
molecule
to
its
site
changes the
conformation
in
such a
way as
to alter the
affinity
of the other
site for
the
nucleic
acid. The
way in
which this
hap-
pens
is known
in
detail for
the Lac
repressor.
Protein
regulators
are
often multimeric,
with a
symmetrical
organization
that
allows
two sub-
units to
contact a
palindromic
target
on DNA.
This
can
generate
cooperative
binding
effects
that create
a more
sensitive
response
to
regulation.
Regulation
via RNA
uses
changes
in
sec-
ondary
structure
as the
guiding
principle.
The
ability
of an RNA
to shift
between
different
con-
formations
with regulatory
consequences
is
the
nucleic
acid's alternative
to the allosteric
changes
of
protein
conformation.
The
changes
in
struc-
ture may
result from
either
intramolecular
or
intermolecular
interactions.
The
most
common role
for
intramolecular
changes is
for an RNA
molecule
to
assume
alter-
native
secondary
structures
by utilizing
differ-
ent
schemes for
base
pairing.
The
properties
of
the
alternative
conformations
may be
different.
Changes in
secondary
structure
of an mRNA
can result in
a change
in its
ability
to be
trans-
lated.
Secondary
structure
also
is used
to regu-
late the
termination
of transcription,
when the
alternative
structures
differ
in
whether
they
permit
termination.
In intermolecular
interactions,
an RNA
reg-
ulator recognizes
its
target
by the
familiar
prin-
ciple
of complementary
base
pairing.
ti*ijfil
]i
"r".i
shows that
the regulator
is
usually
a small
RNA
molecule with
extensive
secondary
structure,
but with a
single-stranded
region(s)
that is
com-
plementary
to a
single-stranded
region
in its
target. The
formation
of
a double
helical
region
between
regulator
and
target
can have
two
t).pes
of consequence:
o
Formation
of the double
helical
struc-
ture
may itself
be
sufficient.
In
some
cases,
protein(s)
can
bind
only
to the
single-stranded
form
of the
target
sequence
and
are therefore prevented
from
acting
by
duplex
formation.
In
other
cases, the
duplex
region
becomes
a target
for
binding;
for
example,
by
nucleases
that
degrade
the
RNA
and
therefore prevent
its
expression.
.
Duplex
formation
may
be important
because it
sequesters
a region
of the
tar-
get
RNA
that
would
otherwise
partici-
pate
in
some
alternative
secondary
structure.
332
CHAPTER
13
Regutatory
RNA
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UT:
UU
U.A
GI CU
GU
CA
GU
AU
UA
uc
A
U
u
U
A
Regions3&4
ALTERNATIVESTRUCTURES
Regions2&3pair;
pair
to form the
Region
2 is
complementary
to
1
& 3
terminator
region
terminator hairpin
Region 3 is complementary
to 2 and
4
is single-stranded
ili*iJRil 1.lt-ii The
trp leader
region can exist
in
atternative
base-paired
conformations.
The
center shows the
four regions that can base
pair.
Region 1 is complementary
to
region
2, which
is
complementary
to region 3, which
is
comptementary
to region
4. 0n
the left
is the
confor-
mation
produced
when
region 1
pairs
with region
2
and
region 3
pairs
with
region 4.0n
the
rightis the conformation when
region 2
pairs
with
region 3, [eaving
regions
1 and
4 unpaired.
4
generates
the hairpin that
precedes
the Ug
sequence:
this
is the essential signal for
intrin-
sic termination.
It is likely that the
RNA would
take
up this structure
in lieu of any outside
intervention.
A dilferent structure
is formed if region
I
is
prevented
from
pairing
with
region 2. In this
case, region
2 is free to
pair
with region
3. Region
4 then
has no available
pairing partner,
so it
is
compelled
to
remain single-stranded.
Thus the
terminator
hairpin cannot be
formed.
fi{:ijRg
ri"*
shows that the
position
of the
ribosome can
determine which structure
is
formed
in such a way
that termination
is
atten-
uated only
in the absence of tryptophan.
The
crucial
feature is the
position
of
the Ttp codons
in the
leader
peptide
coding
sequence.
When
try?tophan is
present,
ribosomes are
able to synthesize
the leader
peptide.
They con-
tinue
along the
leader
section
of the
nRNA to
the UGA
codon, which
lies
between
regions
I
and
2. As shown
in the lower
part
of
the figure,
by
progressing
to this
point,
the ribosomes
extend over
region
2
and
prevent
it
from base
pairing. The result is that
region 3 is available
to base
pair
with
region 4, which
generates
the
terminator
hairpin. Under these
conditions,
therefore,
RNA
polymerase
terminates
at the
attenuator.
When
there is no tryptophan
ribosomes
stall
at the
Trp codons, which are
part
of region
l, as shown
in
the
upper
part
of the
figure. Thus
F;*iJfr.fl
i.*.$ The atternatives
for
RNA
potymerase at the
attenuator
depend
on the
locatjon
ofthe
ribosome,
which
determines
whether
regions 3
and
4 can
pair
to
form
the
terminator
hairpin.
U
U
Ribosome
halts at
Trp codons
TRYPTOPHAN
PRESENT
Ribosome
movement
disrupts
2:3
pairing
3:4
pairing
forms
terminator
hairPin
13.5
Attentuation
Can
Be Controlted
by
Transtation
337
i
:i.i:iiL
:
:,
ti.
In
the
presence
oftryptophan
IRNA, ribo-
somes
translate
the leader
peptide
and are reteased.
This
altows
hairpin formatjon,
so that
RNA
potymerase
termi-
nates.
In
the absence
of tryptophan
IRNA, the ribosome
is
btocked,
the termination
hairpin
cannot form,
and RNA
polymerase
continues.
region
I is
sequestered
within
the ribosome
and
cannot
base
pair
with region
2. This
means
that
regions
2 and
3 become
base
paired
before
region
4 has
been
transcribed.
This
compels
region
4 to remain
in
a single-stranded
form.
In the
absence
of
the terminator
hairpin,
RNA
polymerase
continues
transcription past
the
attenuator.
Control
by
attenuation
requires
a
precise
timing
of
events.
For ribosome
movement
to
determine
formation
of
alternative
secondary
structures
that control
termination,
translation
of the
leader
must
occur at the
same
time when
RNA
polymerase
approaches
the terminator
site. A
crili-
cal
event in
controlling
the timing
is
the
pres-
ence
of a
site that
causes
the RNA polymerase
to
pause
at base 90
along
the leader.
The
RNA
polymerase
remains paused
until a ribosome
translates
the leader peptide.
The
polymerase
is
then
released
and
move
s off
toward
the atten-
uation
site. By
the time
it arrives
there,
second-
ary structure
of the
attenuation
region
has
been
determined.
iri:,::.ii:ii:
;-;
i:ii
s1111t
utires
the
role
of
Trp_tRNA
in
controlling
expression
of the
operon.
By
pro-
viding
amechanismto
sense the
inadequaqt
of the sup-
ply
of Trp-tRNA,
attenuation
responds
directly
to the
need of
the cell
for
tryptophan
in
protein
synthesis.
CHAPTER
13
Regutatory
RNA
How
widespread is
the use
of attenuation
as a control
mechanism
for bacterial
operons?
It is
used in
at
least
six operons
that
code for
enzymes concerned
with
the biosynthesis
of
amino acids.
Thus a feedback
from
the level
of
the
amino acid
available for
protein
synthesis
(as
represented
by the
availability
of aminoacyl-
IRNA)
to the
production
of the
enzymes
may
be common.
The
use of the ribosome
to
control RNA
sec-
ondary
structure in response
to the
availability
of an aminoacyl-IRNA
establishes
an inverse
relationship
between the
presence
of amino-
acyl-tnNA
and the
transcription
of the
operon,
which
is equivalent
to a
situation
in
which
aminoacyl-IRNA
functions
as a
corepressor
of
transcription.
The regulatory
mechanism
is
mediated
by changes in
the formation
of
duplex
regions;
thus attenuation provides
a striking
example of the
importance
of secondary
struc-
ture in the
termination
event
and of its
use in
regulation.
E.
coli and B.
subtilis, therefore,
use the
same
types of mechanisms,
which involve
control
of
nRNA
structure
in response
to
the
presence
or
absence
of a IRNA,
but they have
combined
the
individual
interactions
in
different
ways.
The
end result is
the same:
to inhibit
production
of
the enzymes
when
there is
an excess
supply
of
the amino
acid.
and to activate
production
when
a
shortage is
indicated
by the
accumulation
of
uncharged
tRNAftp.
Antisense
RNA
Can
Be
Used
to
Inactivate
Gene Expression
r
Antisense
genes
btock
expression
of
their targets
when introduced
into
eukarvotic
cetts.
Base
pairing
offers
a
powerful
means
for
one
RNA
to control
the
activity
of another.
There
are
many
cases in
both
prokaryotes
and
eukary-
otes
where a
(usually
rather
short)
single-
stranded
RNA
base
pairs
with a
complementary
region
of an mRNA,
and as
a
result
it
prevents
expression
of the nRNA.
One of
the early
illus-
trations
of
this effect
was
provided
by an
artifi-
cial situation
in
which antisense genes
were
introduced
into
eukaryotic
cells.
Antisense genes
are
constructed
by revers-
ing
the
orientation
of
a
gene
with regard
to its
promoter,
so that
the
"antisense"
strand
is tran-
338
scribed,
as illustrated
in
f:
I
l'
1.;
ii
r:1
i
1
i i
. Synthesis
of antisense
RNA can inactivate a target RNA in
either
prokaryotic
or eukaryotic cells.
An
anti-
sense RNA
is in
effect a synthetic
RNA regula-
tor. An antisense
thymidine kinase
gene
inhibits
synthesis of thymidine
kinase from the endoge-
nous
gene.
Quantitation
of the effect
is not
entirely
reliable,
but
it
seems
that an excess
(perhaps
a considerable excess) of the antisense
RNA may be
necessary.
At what level does the antisense RNA
inhibit
expression?
It could in
principle prevent
tran-
scription
of the authentic
gene. processing
of
its
RNA
product,
or translation o{ the
messen-
ger.
Results with different systems show
that
the inhibition
depends on formation of
RNA-RNA
duplex molecules, but this can occur
either in the
nucleus or in the cytoplasm.
In
the
case of an antisense
gene
stably carried by a cul-
tured
cell, sense-antisense
RNA duplexes
form
in the
nucleus,
preventing
normal
processing
and/or
transport of
the
sense
RNA. In another
case,
injection of antisense RNA into the
cyto-
plasm
inhibits translation
by forming duplex
RNA
in the 5'region of the
nRNA.
This technique
offers a
powerful
approach
for turning
off
genes
at will;
for
example,
the
function of a
regulatory
gene
can be
investi-
gated
by
introducing an antisense
version. An
extension
of this technique
is
to
place
the anti-
sense
gene
under
the control of a
promoter
that
is
itself subject to
regulation. The target
gene
can then be
turned off and on by
regulating the
production
of antisense
RNA. This technique
allows
investigation
of the importance of
the
timing
of expression of
the target
gene.
SmaLL
RNA
Motecules Can
ReguLate
Translation
o
A regulator
RNA
functions
by
forming a duplex
region
with a target RNA.
.
The duptex
may
block
in'itiation of transtation,
cause
termination of
transcription, or create a
target
for an endonuctease.
Repressors
and activators are
trans-acting
pro-
teins,
yet
the
formal circuitry of a
regulatory
network
could equally
well
be
constructed by
using
an RNA as
regulator. In fact, the
original
model
for the operon
left open the
question
of
whether
the regulator
might be RNA or
pro-
tein. Indeed,
the construction of
synthetic anti-
sense
RNAs
turns out to
mimic
a
class of
RNA
l
lfiliiii,
.i:: li
.
Antisense
RNA can
be
generated
by
reversing
the orienta-
tion of a
gene
with
respect to
its
promoter
and
can anneaI
with
the witd-
type transcript
to
form duplex
RNA.
regulators that
is becoming
increasingly
important.
Like a
protein regulator,
a small
regulator
RNA
is
an
independently
synthesized
molecule
that diffuses
to a
target
site
consisting
of a
spe-
cific
nucleotide
sequence.
The target
for a
reg-
ulator
RNA
is a single-stranded
nucleic
acid
sequence.
The regulator
RNA
functions
by
com-
plementarity
with
its
target,
at which
it can form
a double-stranded
region.
We can
imagine
two
general
mechanisms
for
the
action
of a
regulator
RNA:
.
Formation
of
a duplex
region with
the
target
nucleic
acid
directly
prevents
its
ability
to
function
by
forming
or seques-
tering
a specific
site.
!'i{;iililir
i i'i";:
illus-
trates the
situation
in
which
a
protein
that
binds
to single-stranded
RNA is
pre-
vented
from acting
by formation
of a
duplex.
',
irri,ji-li- li..r
:irlr
shows
the oppo-
site
type
of
relationship,
in which
the
formation
of a
double-stranded
region
creates
a target
site
for an
endonucle-
ase that
destroYs
the
RNA
target.
'
Formation
of
a duplex
region
in one
part
of the
target
molecule
changes
the
con-
formation
of
another
region,
thus
indi-
rectly
affecting
its
function.
i ll'i-.:fiS"
,1 :i.'i
"1
shows
an
examPle.
The
mechanism
is essentially
similar
to
the use
of sec-
ondary
structure
in
attenuation
(see
Promoter
Wild-type
gene
I-v
s'
s'
Transcript
I
V
Promoter
Antisense
gene
s'
s'
Antisense
transcriPt
3',
3',
RNA-RNA
duPlex
13.7
Smatt
RNA
Molecutes
Can
Regutate
Translation
339
Protein
binds
single-stranded
region
in
target
cannot
bind to target
Fl*tlR[
13.1?
A
protein
that
binds to a
singte-stranded
region
in
a target
RNA coul.d
be excluded
by a regulator
RNA that
forms
a duptex in
this
reoion.
=ffitr
04
FIfiIJR[
13.1]
By
binding
to a target
RNA to form
a duptex
region,
a
regulator
RNA may
create
a site that is
attacked
bv a nuclease.
'j'l
'i
j
Secondary
slructure
forms
in
absence
of regulator
r:6URt
13.14
The
secondary
structure
formed
by
base
pairing
between
two regions
of the
target RNA
may
be
prevented
from
forming
by base
pairing
with a regutator
RNA. In
this
example.
the abitity
of the
3' end
of the RNA
to
pair
with
the
5' end is
prevented
by the regulator.
CHAPTER
13
Regutatory
RNA
Section
I 3.2, Alternative
Secondary
Structures
Control
Attenuation),
except
that
the interacting
regions
are
on dif-
ferent
RNA molecules
instead
of being
part
of
the same RNA
molecule.
The
feature
clmmln
to both
types
of RNA-
mediated
regulation
is that
changes
in secondary
structure
ofthe target control
its activity.
A
small RNA regulator
typically
can
be
turned
on by
controlling
transcription
of its
gene
or turned
off by
an enzyme
that degrades
the
RNA
regulator
product.
Usually
it is not
possi-
ble otherwise
to regulate
the
activity
of an RNA
regulator.
In fact,
it used
to be thought
that it
would
not
be
possible
for
an
RNA to
have
allosteric
propefiies;
unlike repressor proteins
that
control
operons, an
RNA
usually
cannot
respond
to small molecules
by
changing
its
abil-
ity
to recognize
its target.
The
discovery of
the riboswitch provides
an exception
to this rule. FISURE
g3.Ii
summa-
rizes
the regulation
of the
system
that
produces
the metabolite
GlcN6P.
The
gene
glms
codes for
an
enzyme
that synthesizes
GlcN6P
(Glu-
cosamine-6-phosphate)
from
fructose-6-
phosphate
and
glutamine.
The
mRNA
contains
a long,
5'untranslated
region (UTR)
before
the
coding frame.
Within
the UTR
is a ribozyme-
a sequence
of RNA
that has
a catalytic
activity
(see
Section 27.4,Rlbozymes
Have Various
Cat-
alytic Activities).
In
this case,
the
catalytic
activ-
ity is an
endonuclease
that
cleaves its
own
RNA.
It is
activated
by binding
of the metabolite prod-
uct, GlcN6P,
to the ribozyme.
The
consequence
is that
accumulation
of GlcN6P
activates
the
ribozyme,
which
cleaves the
mRNA,
which
in
turn
prevents
further
translation.
This
is an
exact
parallel
to allosteric
control
of a repressor
protein
by the
end
product
of a
metabolic
path-
way. There
are
several examples
of
such
riboswitches
in
bacteria.
Another
regulatory
mechanism
that in-
volves
transcription
of a noncoding
RNA
works
indirectly.
Initiation
at
a target
promoter
can
be suppressed
by transcription
from
another
promoter
upstream
from
it,
as shown
in
Fi$ljftE
13.36.
The cause
of the
inhibition
is that
the RNA
polymerase
initiating
at the
upstream
promoter
reads
through
the
downstream
pro-
moter,
which
prevents
transcription
factors
and
RNA polymerase
from
binding
ro it.
This
type
of
effect has
been
demonstrated
in eukaryotic
systems
and
may depend
on
the
disruption
of
chromosomal
structure
at
the target
promoter.
The RNA
that
is transcribed
from
the upstream
(regulatory)
promoter
has
no
coding function.
340