In the last decade, the important role of small non-coding RNAs in regulation have been recognized and begun to be studied. Our laboratory, in collaboration with others, have undertaken two global searches for non-coding RNAs in E. coli, contributing significantly to the more than 80 regulatory RNAs that are now identified. A large number of these RNAs bind tightly to the RNA chaperone Hfq. We and others have shown that every RNA that binds tightly to Hfq acts by pairing with target mRNAs, regulating stability and translation of the mRNA, either positively or negatively. Our lab has studied a number of these small RNAs in detail. We have found that expression of each small RNA is regulated by different stress conditions, and that the small RNA plays an important role in adapting to stress. The best-studied of these is RyhB. RyhB transcription is repressed by the Fur iron-dependent repressor, and the small RNA is therefore made in high quantities when intracellular iron is limiting. When it is made, it targets mRNAs that encode iron-binding proteins for degradation. Therefore, this small RNA, which is also found in VibrioSalmonella, Klebsiella, and Yersinia, reprograms iron use in the cells and may be an important component of virulence for some pathogens. Two other small RNAs, now called OmrA and OmrB, have been found to regulate a number of outer membrane proteins; these small RNAs are made at high osmolarity as part of the OmpR/EnvZ regulon, previously known for its regulation of major outer membrane porins. Studies on the regulation of RybB, another Hfq-binding RNA, have demonstrated that it is dependent on an alternative sigma factor, Sigma E, for transcription. Sigma E becomes active when misfolded outer membrane proteins accumulate in the periplasm of the cell. RybB autoregulates sigma E, possibly at both the level of synthesis and activity. The demonstration of the activity of RybB suggests that trafficking to the outer membrane may be even more tightly regulated than previously known. These RNAs are characteristic of a growing family of regulatory RNAs that regulate the cell surface, possibly important during infection. Another small RNA, now named SgrS, is made when cells accumulate glucose-6-phosphate or a phosphorylated glucose analog, and down-regulates the mRNA for the glucose-specific transporter, encoded by ptsG. SgrS induction depends on a novel transcriptional regulator, encoded by the divergent gene, named by us sgrR. The SgrR protein, which is the first studied member of a conserved family of transcriptional regulators (previously mis-annotated), directly binds DNA, negatively autoregulates, and may directly sense the accumulation of sugar phosphate. When either the small RNA or the transcriptional regulator are mutant, cells are unable to recover from glucose-phosphate accumulation. Previous studies had demonstrated the roles of two small RNAs, DsrA and RprA, in positively regulating translation of the stress sigma factor RpoS. More recently, we have shown that RprA also has a number of other mRNA targets, which are negatively regulated; these targets differ from those for DsrA. The new RprA targets expand the likely role of RprA and its regulators, RcsC, RcsD, and RcsB, in controlling biofilm formation by this bacteria. Studies on the mechanism of action of DsrA and RprA suggest that they increase both the stability and translation of rpoS mRNA, protecting it from degradation by RNAse E. All of these Hfq-binding sRNAs pair with target mRNAs, but the regions of pairing are often short, interrupted, and therefore difficult to identify. In addition, even when pairing can be predicted, regulation is not always seen. Examination of the details of pairing by the OmrA/B RNAs demonstrate that the 5 end of the RNA is required for pairing to all targets. It is possible that flexibility at the 5 end allows more efficient pairing. In other experiments, we have chosen genes with no known small RNA regulators, created translational fusions to them, and used genetic screens to identify sRNA translational regulators. Our work, combined with work from other labs on this family of regulators, suggests that a large number of genes in bacteria will be subject to this post-transcriptional regulation. RpoS is subject to control at the level of protein turnover as well. RpoS is rapidly degraded during active growth, in a process that requires the energy-dependent ClpXP protease and the adaptor protein RssB, a phosphorylatable protein that presents RpoS to the protease. RpoS becomes stable after various stress or starvation treatments; the mode of stabilization has been a mystery. Recent studies in our labs and others demonstrated significant regulation of degradation in the absence of phosphorylation. A genetic screen for regulators of RpoS degradation led to discovery of a small, previously uncharacterized protein, YaiB, now renamed IraP. Mutants of iraP have somewhat decreased stability of RpoS under normal growth conditions and totally abolish the stabilization of RpoS after phosphate starvation. IraP blocks RpoS turnover in a purified in vitro system, and directly interacts with RssB. In E. coli, phosphate starvation is sensed by an increase in the levels of the small molecule ppGpp, and the iraP promoter is positively regulated by ppGpp. In Salmonella, expression of this gene is also induced in response to starvation for magnesium. This anti-adaptor is only the first of these proteins. We have now identified two other small proteins that also act to stabilize RpoS in a purified in vitro system, YcgW (renamed IraM), and YjiD (renamed IraD). IraM is made in response to magnesium starvation; IraD is important after DNA damage. At least one other anti-adaptor is likely to exist, since we have conditions that lead to RpoS stabilization when all the known anti-adaptors are mutant. Thus, the anti-adaptors define a new level of regulatory control, interacting with the RssB adaptor protein and blocking its ability to act; environmental signals regulate RpoS turnover by regulating expression of different antiadaptors
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