Download RNA Molecules: More than Mere Information Intermediaries

RNA Molecules: More than Mere
Information Intermediaries
Low-molecular-weight RNA molecules help to regulate
gene expression in many types of bacteria
Jörgen Johansson
ntil recently, RNA was overlooked
compared to DNA or proteins—
consigned to a mere assistant role in
the flow of information from genes
to functioning molecules in living
cells. Few except hardcore fans expected RNA
to perform more interesting roles, such as regulating gene expression. But this status is changing as new functions are being identified for
RNA molecules, particularly as they help to
control essential events in both pathogenic and
non-pathogenic bacteria. Through their unique
features, these RNA molecules add some new
wrinkles of complexity to our understanding of
how cells regulate gene expression.
U
Direct Sensing by RNA Structures
Bacterial pathogens sense their environments,
somehow recognizing and responding appropriately when they encounter susceptible hosts.
Different pathogens use different methods, but
• Low-molecular-weight RNA molecules help to
regulate gene expression in many types of bacteria, including pathogens.
• RNA molecules directly sense and respond to
changes in temperature and to other stimuli,
including metabolites.
• Noncoding-RNA-controlled gene expression
systems are found among several distinct kinds
of pathogens, providing such cells with a versatile means for controlling gene expression without requiring translation of genes into proteins.
many of them depend on the temperature transition to 37°C when entering mammalian hosts.
For example, the DNA conformation of a
specific regulatory region along the genome of
the pathogen Shigella flexneri changes when the
temperature increases to 37°C, thereby preventing a repressor protein from binding to this
segment and enabling expression of virulence
genes. Similarly, when Salmonella enterica serovar Typhimurium is exposed to higher temperatures, one of its transcriptional repressors no
longer forms multimers and thus binds DNA
only poorly, if at all, leading to increased virulence gene expression.
However, among other pathogens, RNA molecules rather than repressor proteins directly
sense temperature changes. For example, thermal control over lcrF, the main transcriptional
virulence regulator in Yersinia pestis, appears to
be exerted directly through the 5⬘-untranslated
part of the lcrF messenger. Below 37°C, the
Shine-Dalgarno (SD) ribosomal binding site is
believed to be hidden by a hairpin structure, and
translation cannot initiate.
A similar phenomenon occurs in Listeria
monocytogenes. The messenger encoding
the transcriptional regulator of virulence,
PrfA, contains a 5⬘-untranslated region that
functions as a thermosensor (Fig. 1A). In
this case, base substitution mutations that
destabilize the region around the SD site
allow PrfA to be translated at low, nonpermissive temperatures. Inserting this untranslated region ahead of the gene encoding green fluorescence protein (GFP) in
Escherichia coli makes GFP expression temperature dependent, suggesting that this
thermosensor can function ectopically.
Jörgen Johansson
is a Junior Researcher at the Department of Molecular Biology at
Umeå University in
Umeå, Sweden.
Volume 71, Number 11, 2005 / ASM News Y 515
FIGURE 1
(A) Model of thermosensing by the prfA-messenger. The prfA-untranslated RNA forms an occluding secondary structure preventing binding
of the ribosome (blue) at low temperatures. At higher temperatures, this secondary structure is partially melted and translation can proceed.
(B) Riboswitch-mediated regulation. Binding of a metabolite (light green) to the untranslated RNA region preceding the downstream mRNA
causes the formation of a terminator structure. In the absence of the metabolite, an antiterminator structure is formed that allows
transcription of the downstream gene.
Other similar thermosensor mechanisms for
controlling gene expression include heat shock
genes in rhizobia species as well as Escherichia
coli heat shock sigma factor ␴32. Also, bacteriophage ␭ is more likely to enter its lytic cycle
when temperatures increase, due to a temperature-sensitive RNA switching mechanism.
RNA molecules can also directly sense and
respond to stimuli other than changes in temperature. For instance, riboswitches, which are untranslated RNA elements, control expression of
downstream mRNA by binding metabolites.
Those downstream genes typically encode proteins involved in synthesizing vitamins or amino
acids, and the RNA-binding metabolite often is
an intermediate or final product involved in
those particular biosynthetic pathways. In many
516 Y ASM News / Volume 71, Number 11, 2005
cases, when the molecule binds to the riboswitch, it shifts conformation and forms a termination structure, blocking downstream mRNA
from being synthesized. In the absence of the
metabolite, however, the conformation permits
synthesis of the downstream mRNA (Fig. 1B).
Whether riboswitch structures are involved in
regulating virulence gene expression is not known.
However, one can imagine riboswitch structures
of pathogens binding host metabolites—for example, found in airway passages or the intestine—and then triggering expression of virulence genes. Examining untranslated RNA regions
preceding virulence genes could help determine
whether this scenario is ever realized. Another
question is whether riboswitches exert broader
control functions over mRNA or protein targets.
FIGURE 2
(A) Schematic drawing of the chromosomal context where ncRNAs might be found. (B) The ribosome binding site (S.D., green box), of the
target mRNA is kept in an hairpin structure, preventingtranslation. Binding of ncRNA (red) to the upstream region of the target mRNA frees
the ribosome binding site, ribosome (blue) binds and translation occurs. (C) The ribosome binding site is free in the absence of the ncRNA,
and translation occurs. Binding of ncRNA to the ribosome bindingsite prevents the ribosome from binding and translation is repressed. (D)
Interaction between the ncRNA (red) and target RNA (black) allows degradation of the complex by RNases. The interaction between the
ncRNA and the target mRNA might be stabilized by Hfq (green).
Noncoding RNA Molecules Are Widely
Distributed
Cells of many eukaryotes and microorganisms
contain plenty of short, noncoding RNA
(ncRNA) molecules that appear to affect a variety of functions, including mRNA stability,
translation efficiency, and protein stability. Before these RNA molecules were identified, researchers were aware of other RNA molecules
with regulatory functions, including antisense
RNAs that help to control the replication of
plasmids such as ColE1. However, each of these
latter regulatory RNA species has only one RNA
target, to which it is complementary. When
these pairs of molecules bind one another, the
regulatory RNA molecule typically renders its
target RNA inactive.
By the end of 2000, investigators had identified several ncRNAs encoded at a distance from
their targets (trans position). These trans-functioning ncRNAs typically are located between
open reading frames in the genome, and each
has its own promoter and terminator ensuring
expression as an independent RNA species (Fig.
2A).
During 2001, several research groups, using
different search criteria (such as homology,
structure conservation, and base composition),
identified several separate novel ncRNAs within
intergenic regions in Escherichia coli. Each of
Volume 71, Number 11, 2005 / ASM News Y 517
those ncRNAs appears to act by an antisense
function, binding target mRNA molecules because of their complementarity. These ncRNA
molecules differ from cis-acting RNA molecules
involved in plasmid regulation by having several
targets scattered throughout the chromosome.
These more recently identified ncRNA molecules also show a relatively low level of complementarity to their mRNA targets.
A few antisense ncRNAs function either by
stimulating or repressing translation of their
target. For some of these mRNA targets, the SD
region forms an inaccessible secondary structure
with the upstream untranslated RNA. If an
ncRNA complements the upstream RNA region, the SD-region opens to allow translation
(Fig. 2B). In contrast, some ncRNAs complement the SD region, blocking binding to the
ribosome and, hence, repressing translation (Fig.
2C).
However, most antisense RNAs studied so far
form ncRNA-mRNA hybrids that RNase nucleases degrade efficiently (Fig. 2D). In such cases,
both the ncRNA and the target mRNA are degraded. For many of these interactions, the RNA
binding protein Hfq is a critical component,
apparently stabilizing the pairing between the
two RNA molecules, particularly in cases where
the level of complementarity between them is
low.
Some ncRNAs bind specific proteins (RNAPs)
that typically have very high affinity for specific
RNA sequences. For example, in stationary
phase, 6S RNA binds the vegetative RNAPholoenzyme that is responsible for about 95%
of total transcription in Escherichia coli. This
association inactivates all RNA polymerases
carrying the vegetative sigma factor, thereby
promoting the activity of RNA polymerases harboring the stationary-phase sigma factor.
Versatile ncRNAs Are Involved in
Controlling Virulence Genes
In parallel to E. coli, many ncRNAs are found in
other bacteria, including several pathogens. In
Staphylococcus aureus, for instance, several virulence genes are controlled by the accessory
gene regulator (agr) system. It consists of a classical two-component signal transduction system, namely an auto-inducing peptide (AIP) and
a protein responsible for secreting AIP.
How does the agr locus control other genes?
518 Y ASM News / Volume 71, Number 11, 2005
The key is RNAIII, a 512-nucleotide transcript
that can regulate expression of various virulence
genes. RNAIII is highly structured, containing
14 hairpins that control the expression of different genes. In general, the RNAIII molecule activates genes that encode secreted proteins and
represses genes that encode surface proteins. For
instance, the 5⬘ end of RNAIII positively controls translation of hla, which encodes ␣-hemolysin, by competing with an inhibitory intramolecular RNA structure in the hla transcript (Fig.
2C).
RNAIII also encodes a small peptide, ␦-hemolysin, on its 5⬘ end, whereas its 3⬘ end seems to
repress translation of ␦-hemolysin. The 3⬘ end of
RNAIII also represses expression of spa, which
encodes protein A, an immunoglobulin G (IgG)binding protein. In this case, RNAIII is believed
to function on the posttranscriptional level by
mediating RNaseIII-dependent degradation.
Thus, RNAIII of S. aureus provides an example
of a relatively small RNA molecule that controls
expression of various genes by several different
mechanisms.
Clostridium perfringens contains a system
called VR-RNA that resembles RNAIII. A small
untranslated RNA is the effector molecule of the
two-component VR-RNA system, and it controls virulence gene expression, apparently
mainly through the 3⬘ end of the regulatory RNA.
Another gram-positive bacterium, Streptococcus pyogenes, also contains an RNAIII-like
regulatory system, partly situated in the Fas
operon. S. pyogenes also harbors a 459-nucleotide RNA, called pel for pleiotropic effect locus,
that regulates expression of several virulence
genes. As with RNAIII, pel encodes a peptide,
SagA, that is not involved in virulence gene
regulation. So far, pel appears to control target
gene expression on both the transcriptional and
posttranscriptional levels. Expression of pel is
growth phase regulated, showing maximal expression in stationary phase, and is also induced
by spent medium, suggesting involvement of a
quorum-sensing mechanism.
Additional examples of ncRNA control gene
expression systems are found in several Vibrio
species. For instance, Vibrio cholerae encode
three different quorum-sensing systems to control virulence and biofilm formation. In this
species, four ncRNAs regulate the stability of
the messenger encoding the quorum-sensing
master regulator, HapR. This posttranscrip-
tional regulation is probably mediFIGURE 3
ated by an antisense mechanism and
requires the RNA-binding protein
Hfq. All four ncRNAs need to be
absent before quorum sensing occurs, suggesting a tightly controlled
system to prevent expression of
HapR under inappropriate conditions.
Similar ncRNAs can be found in
other Vibrio species. For instance,
the fish pathogen Vibrio anguillarum
contains an ncRNA that functions
by means of an antisense mechanism. Although this bacterium requires iron for virulence, an excess is
harmful. V. anguillarum uses two
Virulence gene regulation by RsmA/RsmB⬘. The RsmA protein (green) binds to and induces
the degradation of target mRNAs involved in virulence. Binding of RsmA to RsmB⬘ (light
different systems to control the iron
blue) depletes the free pool of RsmA, and causes anincrease in virulence gene expression.
uptake system, which is encoded by
the fat operon. One of them is
RNA␣, a 650-nucleotide ncRNA
molecule that is expressed on the opposite
stresses, and this expression is controlled mainly
strand of fatB, one of the iron uptake genes.
by sigma factors. More importantly, some short
Excess iron increases the stability of RNA␣,
ncRNA molecules directly bind metabolites, as
leading to increased interaction between RNA␣
riboswitches do, permitting instant regulation
and the fatB mRNA. This interaction promotes
of the activity and level of those ncRNAs. Some
degradation of the fat messenger, lowering the
ncRNAs involved in virulence might bind meuptake of iron. The stability of RNA␣ decreases
tabolites encountered inside specific host organs
with lowered levels of iron, again allowing eleand tissues, becoming “activated” and thereby
vated expression of iron-uptake genes.
controlling pathogenesis through antisense or
The plant pathogen Erwinia carotovora ssp.
protein-binding mechanisms.
carotovora (ECC) causes soft-rot disease in
I expect additional ncRNAs and untranslated
plants. The bacterium harbors the RNA-binding
regions of messenger RNA molecules to be idenprotein RsmA, which promotes degradation of
tified and characterized. As early as 1961, Frantarget mRNA, and a 259-nucleotide untranscois Jacob and Jacques Monod asserted the imlated RNA, RsmB⬘. Together, these two compoportance of RNA molecules in regulatory
nents control expression of several virulence
cellular functions, writing: “The operator tends
factors, including cellulases, proteases, and pecto combine (by virtue of possessing a particular
tolytic enzymes. Binding of the RsmA protein to
base sequence) specifically and reversibly with a
the target mRNA destabilizes the latter. Howcertain (RNA) fraction possessing the proper
ever, target mRNA degradation can be counter(complementary) sequence. This combination
acted when RsmB⬘ is present to sequester and
blocks the initiation of cytoplasmic transcripbind up to 18 RsmA molecules (Fig. 3).
tion and therefore the formation of the messenger by the structural genes in the whole operon.
The specific ‘repressor’ (RNA?), acting with a
Conclusions and Perspective
given operator, is synthesized by a regulator
ncRNA molecules provide cells with a versatile
gene.”
means for controlling gene expression without
Even so, several decades elapsed before small
requiring translation. In many cases, ncRNAs
regulatory RNA molecules were discovered.
control expression level of regulatory proteins,
Now, with so many genomes being sequenced, it
placing them atop regulatory cascades.
will be relatively straightforward to uncover
What is controlling the ncRNAs? Many
other small regulatory RNAs, based on homolncRNAs are expressed after exposure to various
ogies between different species. Also, the knowl-
Volume 71, Number 11, 2005 / ASM News Y 519
edge that such RNA molecules exist will induce
researchers to learn whether such small regulatory RNA regulate their favorite operons. Indeed, many researchers likely are storing mu-
tants whose lesions map to intergenic regions,
perhaps pointing to just such regulators. I advise
reexaming those mutants. Some great surprises
lie ahead!
ACKNOWLEDGMENTS
I thank F. Repoila, P. Mandin and E. Charpentier for valuable comments and suggestions.
My work is supported by the Wenner-Gren Foundation, the Swedish Research Council grant 15144, and by Umeå University.
SUGGESTED READING
Argaman, L., R. Hershberg, J. Vogel, G. Bejerano, E. G. Wagner, H. Margalit, and S. Altuvia. 2001. Novel small
RNA-encoding genes in the intergenic regions of Escherichia coli. Curr. Biol. 11:941–950.
Jacob, F., and J. Monod. 1961. Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3:318 –356.
Johansson, J., and P. Cossart. 2003. RNA-mediated control of virulence gene expression in bacterial pathogens. Trends
Microbiol. 11:280 –285.
Johansson, J., P. Mandin, A. Renzoni, C. Chiaruttini, M. Springer, and P. Cossart. 2002. An RNA thermosensor controls
expression of virulence genes in Listeria monocytogenes. Cell 110:551–561.
Lenz, D. H., K. C. Mok, B. N. Lilley, R. V. Kulkarni, N. S. Wingreen, and B. L. Bassler. 2004. The small RNA chaperone Hfq
and multiple small RNAs control quorum sensing in Vibrio harveyi and Vibrio cholerae. Cell 118:69 – 82.
Mironov, A. S., I. Gusarov, R. Rafikov, L. E. Lopez, K. Shatalin, R. A. Kreneva, D. A. Perumov, and E. Nudler. 2002. Sensing
small molecules by nascent RNA: a mechanism to control transcription in bacteria. Cell 111:747–756.
Rivas, E., R. J. Klein, T. A. Jones, and S. R. Eddy. 2001. Computational identification of noncoding RNAs in E. coli by
comparative genomics. Curr. Biol. 11:1369 –1373.
Wassarman, K. M., F. Repoila, C. Rosenow, G. Storz, and S. Gottesman. 2001. Identification of novel small RNAs using
comparative genomics and microarrays. Genes Dev. 15:1637–1651.
Winkler, W., A. Nahvi, and R. R. Breaker. 2002. Thiamine derivatives bind messenger RNAs directly to regulate bacterial
gene expression. Nature 419:952–956.
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