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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. NEW! Q: When does 4 x $17 = $48? A: When Professors, Mentors and University Department Chairs purchase 4 or more student memberships for their undergraduate and graduate scholars! Buy 4 Student Memberships and receive a discount of $5 per membership! 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