Transcript Document

Chapter 13
Regulatory RNA
13.1 Introduction
Key Concepts
 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 regulator that interacts with a specific sequence or
structure in DNA or mRNA at some stage prior to the synthesis of
protein.
 The stage of expression 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 regulator can be a protein or an RNA.
 Regulators may themselves be regulated, most typically in response
to small molecules whose supply responds to environmental
condition. Regulators may be controlled by other regulators to make
complex circuits.
 Regulation via RNA; intramolecular or intermolecular interaction.
 The most common role for intramolecular changes is for an RNA
molecule to assume alternative secondary structures by utilizing
different schemes for base pairing.
 In intermolecular interactions, an RNA regulator recognizes its
target by the familiar principle of complementary base pairing.
 Fig. 13.1: shows that the regulator is usually a small RNA molecule
with extensive secondary structure, but with a single-stranded
region(s) that is complementary to a single-stranded region in its
target.
Figure 13.1. A regulator RNA is a small RNA with a singlestranded region that can pair with a single-stranded region in a
target RNA.
13.2 Alternative Secondary Structures Control Attenuation
Key Concepts
 Termination of transcription can be attenuated by controlling formation
of the necessary hairpin structure in RNA.
 The most direct mechanisms for attenuation involve proteins that
either stabilize or destabilize the hairpin.
 RNA structure provides an opportunity for regulation in both
prokaryotes and eukaryotes.
 Its most common role occurs when an RNA molecule can take up
alternative secondary structure by utilizing different schemes for
intramolecular base pairing. This type of mechanism can be used to
regulate the termination of transcription, when the alternative
structures differ in whether they permit termination.
 Another means of controlling conformation is provided by the
cleavage of an RNA; by removing one segment of an RNA, the
conformation of the rest may be altered.
- Attenuation: describes the regulation of bacterial operons by
controlling termination of transcription at a site located before the
first structural gene.
- attenuator: is a terminator sequence at which attenuation occurs.
 The principle of attenuation is that some external event controls the
formation of the hairpin needed for intrinsic termination.
 If the hairpin is allowed to form, termination prevents RNA
polymerase from transcribing the structural genes. If the hairpin is
prevented from forming, RNA polymerase elongates through the
terminator and the genes are expressed.
 Fig. 13.2: shows an example in which a protein prevents formation
of the terminator hairpin.
Figure 13.2. Attenuation
occurs when a terminator
hairpin in RNA is prevented
from forming.
13.3 Termination of B. subtilis trp Genes Is Controlled by
Tryptophan and by tRNATrp
Key Concepts
 A terminator protein called TRAP is activated by tryptophan to prevent
transcription of trp genes.
 Activity of TRAP is (indirectly) inhibited by uncharged tRNATrp.
 The circuitry that controls transcription via termination can use both
direct and indirect means to respond to the level of small molecule
products or substrates.
 In B. subtilis, a protein called tryptophan RNA-binding attenuation
protein (TRAP) is activated by tryptophan (Trp) to bind to a
sequence in the leader of the nascent transcript. TRAP forms a
multimer of eleven subunits. Each subunit binds a single tryptophan
amino acid and a trinucleotide (GAG or UAG) of RNA. The RNA is
wound in a circle around the protein.
 Fig. 13.3: shows that the result is to ensure the availability of the
regions that are required to form the terminator hairpin.
Figure 13.3. TRAP is activated by tryptophan and binds to trp mRNA. This
allows the termination hairpin to form, with the result that RNA polymerase
terminates, and the genes are not expressed. In the absence of tryptophan, TRAP
does not bind, and the mRNA adopts a structure that prevents the terminator
hairpin from forming.
 The TRAP protein in turn, however, is also controlled by tRNATrp.
 Fig. 13.4: shows that uncharged tRNATrp binds to the mRNA for a
protein called antiTRAP (AT). This suppresses formation of a
termination hairpin in the mRNA.
 Expression of the B. subtilis trp genes is therefore controlled by
both tryptophan and tRNATrp.
Figure 13.4. Under normal
conditions (in the presence of
tryptophan) transcription terminates
before the anti-TRAP gene. When
tryptophan is absent, uncharged
tRNATrp base pairs with the antiTRAP mRNA, preventing formation
of the terminator hairpin, thus
causing expression of anti-TRAP.
13.4 The E. coli tryptophan Operon Is Controlled by Attenuation
Key Concepts
 An attenuator (intrinsic terminator) is located between the promoter
and the first gene of the trp cluster.
 The absence of tryptophan suppresses termination and results in a 10×
increase in transcription.
 A complex regulatory system is used in E. coli (where attenuation
was originally discovered). The changes in secondary structure that
control attenuation are determined by the position of the ribosome
on mRNA.
 Fig. 13.5: shows that termination requires that the ribosome can
translate a leader segment that precedes that trp genes in the mRNA.
When the ribosome translates the leader region, a termination
hairpin forms at terminator 1. When the ribosome is prevented from
translating the leader, though, the termination hairpin does not form,
and RNA polymerase transcribes the coding region.
 This mechanism of antitermination therefore depends upon the
ability of external circumstances to influence ribosome movement in
the leader region.
Figure 13.5. Termination can be controlled via changes in RNA
secondary structure that are determined by ribosome movement.
 The trp operon consists of 5 structural genes, which code for the 3
enzymes that convert chorismic acid to tryptophan.
 Fig. 13.6: shows that transcription starts at a promoter at the left end
of the cluster.
 Trp operon expression is controlled by two separate mechanisms; a
repressor protein (coded by the unlinked gene trpR) that binds to an
operator, and attenuation.
 An attenuator (intrinsic terminator) is located between the promoter
and the trpE gene. RNA polymerase terminates there to produce a
140-base transcript.
 Fig. 13.7: termination at the attenuator responds to the level of
tryptophan.
Figure 13.6. The trp operon consists of five contiguous structural
genes preceded by a control region that includes a promoter, operator,
leader peptide coding region, and attenuator.
Figure 13.7. An attenuator controls
the progression of RNA polymerase
into the trp genes. RNA polymerase
initiates at the promoter and then
proceeds to position 90, where it
pauses before proceeding to the
attenuator at position 140. In the
absence of tryptophan, the polymerase
continues into the structural genes
(trpE starts at +163). In the presence
of tryptophan there is ~90%
probability of termination to release
the 140-base leader RNA.
13.5 Attenuation Can Be Controlled by Translation
Key Concepts
 The leader region of the trp operon has a fourteen-codon open reading
frame that includes two codons for tryptophan.
 The structure of RNA at the attenuator depends on whether this
reading frame is translated.
 In the presence of tryptophan, the leader is translated, and the
attenuator is able to form the hairpin that causes termination.
 In the absence of tryptophan, the ribosome stalls at the tryptophan
codons and an alternative secondary structure prevents formation of
the hairpin, so that transcription continues.
 How can termination of transcription at the attenuator respond to the
level of tryptophan? The leader region has a short coding sequence
that could represent a leader peptide of 14 amino acids.
 Fig. 13.6: shows that it contains a ribosome binding site whose
AUG codon is followed by a short coding region that contains two
successive codons for tryptophan. When the cell runs out of
tryptophan, ribosomes initiate translation of the leader peptide but
stop when they reach the Trp codons. This ribosome stalling
influences termination at the attenuator..
- Leader peptide: is the product that would result from translation of
a short coding sequence used to regulate expression of the
tryptophan by controlling
- Ribosome stalling: describes the inhibition of movement that
occurs when a ribosome reaches a codon for which there is no
corresponding charged aminoacyl-tRNA.
 Fig. 13.8: The leader sequence can be written in alternative basepaired structure (Left: terminator hairpin).
 Fig. 13.9: shows that the position of the ribosome can determine
which structure is formed in such a way that termination is
attenuated only in the absence of tryptophan.
 Control by attenuation requires a precise timing of events.
Translation of the leader must occur at the same time when RNA
polymerase approaches the terminator site.
 Fig. 13.10: summarizes the role of Trp-tRNA in controlling
expression of the operon.
Figure 13.8. The trp leader region can exist in alternative 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 complementary to region 4. On
the left is the conformation produced when region 1 pairs with region 2, and region 3
pairs with region 4. On the right is the conformation when region 2 pairs with region 3,
leaving regions 1 and 4 unpaired.
Figure 13.9. The alternatives for
RNA polymerase at the attenuator
depend on the location of the
ribosome, which determines
whether regions 3 and 4 can pair to
form the terminator hairpin.
Figure 13.10. In the presence of
tryptophan tRNA, ribosomes
translate the leader peptide and
are released. This allows hairpin
formation, so that RNA
polymerase terminates. In the
absence of tryptophan tRNA, the
ribosome is blocked, the
termination hairpin cannot form,
and RNA polymerase continues.
13.6 Antisense RNA Can Be Used to Inactivate Gene Expression
Key Concepts
 Antisense genes block expression of their targets when introduced into
eukaryotic cells.
 There are many cases in both prokaryotes and eukaryotes where a
(usually rather short) single-stranded RNA base pairs with a
complementary region of an RNA, and as a result it prevents
expression of the mRNA.
- antisense gene: codes for an (antisense) RNA that has a
complementary sequence to an RNA that is its target.
 Fig. 13.11: antisense genes are constructed by reversing the
orientation of a gene with regard to its promoter, so that the
“antisense” strand is transcribed. An antisense RNA is in effect a
synthetic RNA regulator.
Figure 13.11. Antisense RNA can be generated by reversing the
orientation of a gene with respect to its promoter, and can anneal with
the wild-type transcript to form duplex RNA.
13.7 Small RNA Molecules Can Regulate Translation
Key Concepts
 A regulator RNA functions by forming a duplex region with a target
RNA.
 The duplex may block initiation of translation, cause termination of
transcription, or create a target for an endonuclease.
 Like a protein regulator, a small regulator RNA is an independently
synthesized molecule that diffuses to a target site consisting of a
specific nucleotide sequence.
 The regulator RNA functions by complementarity with its target, at
which it can form a double-stranded region.
 Two general mechanisms for the action of a regulator RNA: (1)
Formation of a duplex region with the target nucleic acid directly
prevents its ability to function by forming or sequestering a specific
site. (Fig. 13.12; Fig. 13.13); (2) Formation of a duplex region in
one part of the target molecule changes the conformation of another
region, thus indirectly affecting its function (Fig. 13.14)
 Fig. 13.15: riboswitch (ribozyme) provides an exception.
 Another regulatory mechanism that involves transcription of a
noncoding RNA works indirectly. Initiation at a target promoter can
be suppressed by transcription from another promoter upstream
from it (Fig. 13.16)
Figure 13.12. A protein that binds to a single-stranded region in a
target RNA could be excluded by a regulator RNA that forms a
duplex in this region.
Figure 13.13. By binding to a target RNA to form a
duplex region, a regulator RNA may create a site that is
attacked by a nuclease.
Figure 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 regulator RNA. In this example, the ability of the 3′ end of the RNA to pair
with the 5′ end is prevented by the regulator.
Figure 13.15. The 5’
untranslated region of the
mRNA for the enzyme that
synthesizes GlcN6P
(glucosamine-6-phosphate)
contains a ribozyme that is
activated by the metabolic
product. The ribozyme
inactivates the mRNA by
cleaving it.
Figure 13.16. Transcription from an upstream promoter may inhibit initiation at a
promoter downstream from it, because reading through the downstream promoter
prevents the necessary transcription factors from binding to it. The RNA transcribed
from the upstream promoter has no coding function.
13.8 Bacteria Contain Regulator RNAs
Key Concepts
 Bacterial regulator RNAs are called sRNAs.
 Several of the sRNAs are bound by the protein Hfq, which increases
their effectiveness.
 The OxyS sRNA activates or represses expression of >10 loci at the
post-transcriptional level.
 In bacteria, regulator RNAs are short molecules that are collectively
known as sRNAs; E. coli contains at least 17 different sRNAs.
Some of the sRNAs are general regulators that affect many target
genes.
- sRNA: is a small bacterial RNA that functions as a regulator of gene
expression.
 Oxidative stress provides an interesting example of a general control
system in which RNA is the regulator. When exposed to reactive
oxygen species, bacteria respond by inducing antioxidant defense
genes.
 Hydrogen peroxide activates the transcription activator OxyR which
controls the expression of several inducible genes, One of these
genes is oxyS, which codes for a small RNA (Fig. 13.17).
 The OxyS RNA is a short sequence (109 nt) that does not code for
protein. It is a trans-acting regulator that affects gene expression at
posttranscriptional levels. It has >10 target loci; at some of them, it
activates expression; at others it represses expression. Fig. 13.18
shows the mechanism of repression of one target, the FlhA mRNA.
Figure 13.17. The gels on the left
show that oxyS RNA is induced in
an oxyR constitutive mutant. The
gels on the right show that oxyS
RNA is induced within 1 minute
of adding hydrogen peroxide to a
wild-type culture.
Figure 13.18. oxyS RNA inhibits translation of flhA mRNA by base
pairing with a sequence just upstream of the AUG initiation codon.
13.9 MicroRNAs Are Regulators in Many Eukaryotes
Key Concepts
 Animal and plant genomes code for many short (~22 base) RNA
molecules, called microRNAs.
 MicroRNAs regulate gene expression by base pairing with
complementary sequences in target mRNAs.
 Very small RNAs are gene regulators in many eukaryotes.
 The first example was discovered in the nematode C. elegans as the
result of the interaction between the regulator gene lin4 and its
target gene, lin14 (Fig. 13.19).
 The lin4 RNA is an example of a microRNA (miRNA). There are
~80 genes in the C. elegans genome coding for miRNAs that are 21
to 24 nucleotides long.
- MicroRNAs are very short RNAs that may regulate gene
expression.
Figure 13.19. lin4 RNA
regulates expression of lin14 by
binding to the 3′ nontranslated
region.
 Many of the C. elegans miRNAs have homologs in mammals, so
the mechanism may be widespread. They are also found in plants.
 The mechanism of production of the miRNAs is also widely
conserved. In the example of lin4, the gene is transcribed into a
transcript that forms a double-stranded region that becomes a target
for a nuclease called Dicer. This has an N-terminal helicase activity,
which enables it to unwind the double-stranded region, and two
nuclease domains that are related to the bacterial ribonuclease III.
Cleavage of the initial transcript generates the active miRNA.
13.10 RNA Interference Is Related to Gene Silencing
Key Concepts
 RNA interference triggers degradation of mRNAs complementary to
either strand of a short dsRNA.
 dsRNA may cause silencing of host genes.
 The regulation of mRNAs by miRNAs is mimicked by the
phenomenon of RNA interference (RNAi).
- RNA interference (RNAi): describes situations in which antisense
and sense RNAs apparently are equally effective in inhibiting
expression of a target gene. It is caused by the ability of doublestranded sequences to cause degradation of sequences that are
complementary to them.
 The dsRNA is degraded by ATP-dependent cleavage to give
oligonuclotides of 21 to 23 bases. The short RNA is sometimes
called siRNA (short interfering RNA).
 Fig. 13.20: shows that the mechanism of cleavage involves making
breaks relative to each 3’ end of a long dsRNA to generate siRNA
fragments with short (two base) protruding 3’ ends. The same
enzyme (Dicer) that generates miRNAs is responsible for the
cleavage.
 RNAi occurs posttranscriptionally when an siRNA induces
degradation of a complementary mRNA (Fig. 13.21).
 The siRNA directs cleavage of the mRNA in the middle of the
paired segment. These reactions occur within a ribonucleoprotein
complex called RISC (RNA-induced silencing complex).
 Fig. 13.22: dsRNA has both general and specific effects.
- RNA silencing: describes the ability of a dsRNA to suppress
expression of the corresponding gene systemically in a plant.
- Cosuppression: describes the ability of a transgene (usually in
plants) to inhibit expression of the corresponding endogenous gene.
Figure 13.20. siRNA that mediates RNA interference is generated by
cleaving dsRNA into smaller fragments. The cleavage reaction occurs 2123 nucleotides from a 3′ end. The siRNA product has protruding bases on
its 3′ ends.
Figure 13.21. RNAi
occurs when a dsRNA is
cleaved into fragments
that direct cleavage of
the corresponding
mRNA.
Figure 13.22. dsRNA inhibits protein synthesis and triggers
degradation of all mRNA in mammalian cells as well as having
sequence-specific effects.
General mechanism of RNA-induced gene silencing
dsRNA
dsRNA
RDRP
Dicer
AAAA
Complex
miRNAs
siRNAs
AGO
RISC effector complex
AAAA
DNA/Histone Methylation
TGS
mRNA Degradation
RNAi/PTGS/VIGS
AAAA
Translation Block
Developmental Processes