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Riboswitch regulates RNA

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Science  22 Aug 2014:
Vol. 345, Issue 6199, pp. 876-877
DOI: 10.1126/science.1258494

Bacteria are expert at adapting to various ecological niches, including the human gut, amid multiple other competing organisms. Their success in these many environments means that they have perfected the ability to use what is available, to not waste resources making RNA and protein for unnecessary processes, and to sense and respond when conditions and requirements change. It has become increasingly clear that RNA-based regulation plays an important part in empowering bacteria to respond to environmental changes. On pages 937 and 940 of this issue, DebRoy et al. (1) and Mellin et al. (2), respectively, report a new example of adaptation in which type of RNA-based regulation is controlled by another, allowing the integrated sensing of two different nutrients. The finding points to an unexpected role for riboswitches, RNA binding proteins, and noncoding RNAs in controlling gene expression by joining forces.

DebRoy et al. and Mellin et al. investigated the regulated utilization of ethanolamine as a carbon and nitrogen source in the pathogens Enterococcus faecalis and Listeria monocytogenes. The genes necessary for ethanolamine catabolism are clustered together in the bacterial genome, with most of them under the control of a single promoter. This unit, called the eut operon, is present in a wide spectrum of bacterial species, and in all studied cases, the metabolic genes are well expressed only when two nutrients are present—the substrate ethanolamine and vitamin B12, an essential cofactor for enzymes in the catabolic pathway. In the pathogen Salmonella enterica, this response is mediated by the transcriptional activator EutR. EutR is activated upon binding to both ethanolamine and vitamin B12 (3). Now, DebRoy et al. and Mellin et al. have uncovered a more sophisticated RNA-based circuitry for controlling ethanolamine metabolism in response to both nutrient signals.

Ethanolamine is directly sensed by EutW, a histidine kinase. EutW, in turn, phosphorylates and thereby activates EutV, an RNA binding protein (4). EutV binds to RNA hairpin motifs and act as an “antiterminator”—that is, the dimer blocks the formation of RNA hairpins that terminate further transcription of the eut operons. Therefore, when EutV is active and available, ethanolamine metabolism genes are expressed. The surprise arises from how this system uses a riboswitch to integrate a vitamin B12 signal into this control system—it regulates the availability of active EutV.

A riboswitch is a metabolite-sensing RNA element predominantly present in the 5′ untranslated region of mRNA. Ligand binding to the aptamer domain of a riboswitch causes structural changes downstream in the RNA, usually resulting in inhibition of translation initiation or premature transcription termination (5). Riboswitches that recognize vitamin B12 and lead to the down-regulation of vitamin B12 synthesis genes were among the first to be studied (5, 6). For these, vitamin B12 acts as an OFF switch—when vitamin B12 is abundant, more vitamin B12 synthesis is not needed. DebRoy et al. and Mellin et al. report a vitamin B12-binding riboswitch (see the first figure) that regulates transcription termination in the same manner as a classical vitamin B12 switch. However, the RNA made in the absence of vitamin B12 has a unique role. Instead of encoding a protein, the transcript produced in the absence of vitamin B12 is a noncoding RNA that sequesters the activated EutV protein. This prevents EutV from stimulating the synthesis of ethanolamine metabolic genes (see the second figure). Thus, this vitamin B12 riboswitch acts as an ON switch for the catabolism of a nutrient by blocking the formation of a negatively acting RNA.

The secondary structure of the vitamin B12 riboswitch. CREDIT: WADE C. WINKLER/UNIVERSITY OF MARYLAND, COLLEGE PARK
Riboswitch-based regulation.

The eut operon contains genes involved in ethanolamine metabolism in bacteria. (A) EutV is phosphorylated and activated by EutW in response to ethanolamine. Active EutV forms a dimer that binds to adjacent RNA hairpins in the target transcript. Binding at these sites leads to antitermination; thus, transcription of the operon is turned ON, but this can only happen in the presence of vitamin B12. (B) In the absence of vitamin B12, a noncoding RNA is generated that sequesters EutV; thus, transcription of the operon is turned OFF. (C) In the presence of vitamin B12, the riboswitch blocks transcription of the noncoding RNA through a structural change that produces a terminating hairpin RNA (T). Active EutV protein is then free to bind to the target transcript and promote expression of ethanolamine metabolic genes.

ILLUSTRATION: V. ALTOUNIAN/SCIENCE

The finding of DebRoy et al. and Mellin et al. extends the ways by which bacteria exploit riboswitches to modulate RNA structure and function in response to specific ligands. Loh et al. found that two S-adenosylmethionine riboswitches control expression of the virulence regulator PrfA by acting as noncoding RNAs (7). Another Listeria vitamin B12 riboswitch controls the metabolism of the nutrient propanediol (a carbon source). Vitamin B12 is a cofactor of enzymes for this catabolic process as well. Here, the vitamin B12 riboswitch controls transcript length—and therefore the activity—of the antisense RNA AspocR. AspocR is a negative regulator of the propanediol-activated transcription factor PocR. In the absence of vitamin B12, the long isoform antisense RNA inhibits PocR expression; binding of vitamin B12 to the riboswitch leads to early transcription termination of the antisense RNA, allowing PocR expression and activation of propanediol catabolism (8). Thus, for both ethanolamine and propanediol, the vitamin B12 riboswitch ensures expression of the metabolic genes only when both the substrate and vitamin B12 are present. It seems likely that in addition to downstream protein-coding genes, many other regulatory RNAs will be found downstream of riboswitches.

EutV is a member of the AmiR and NasR transcriptional antiterminator regulator domain (ANTAR) family of RNA binding proteins. It has an N-terminal response regulator domain that is separated by a coiled-coil structure from the RNA-binding ANTAR domain, the most common architecture for this family of proteins (9). When activated, ANTAR proteins dimerize and bind to two short RNA stem-loops in the target transcript (9). Few ANTAR proteins have been studied, and although they all control antitermination, each is regulated differently. The ANTAR protein AmiR is controlled by an inhibitor protein, AmiC, that itself is regulated by a ligand. ANTAR protein NasR is controlled by direct binding of a small molecule to the regulatory domain (9). One would expect activation of the ANTAR proteins to be fully reversible; certainly, most response regulatory domains can be rapidly dephosphorylated when the inducing signal is absent, although this has not been studied for EutV.

DebRoy et al. and Mellin et al. demonstrate an additional level of negative control: the sequestration of ANTAR proteins by an RNA that mimics the ANTAR binding site of the target RNA. This theme of controlling RNA binding proteins by regulatory RNAs that titrate their availability has been well studied for the RNA translational regulatory proteins of the bacterial CsrA family (10, 11). For regulation to work effectively, the titrating RNA must be abundant and/or have a higher affinity than the target RNAs for the regulatory protein, yet must also have some way of being removed when no longer needed. Instability of the titrating RNA would be sufficient, but a cascade of titrators is another possibility: antisense RNAs, for example, which bind to and remove the primary titrating RNAs.

The world of RNA regulators is intricately interlinked, with no clear boundaries separating trans-acting regulatory RNAs, cis-acting sites for RNA binding proteins, and riboswitches. Every combination is possible and will probably be found, and each of the many regulatory RNA binding proteins is probably subject to multiple levels of regulation by, not surprisingly, RNAs.

References and Notes

  1. Acknowledgments: Supported by the Intramural Research Program of the National Cancer Institute, Center for Cancer Research. We thank G. Storz, K. Ramamurthi, M. M. Gottesman, and S. Gottesman lab members for comments.
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