We have focused our studies on two types of post-transcriptional regulation of gene expression, regulation of mRNA degradation and translation by small regulatory RNAs and regulation of protein stability by energy-dependent proteases. RpoS, a central stress response regulator in Escherichia coli, is subject to both of these levels of control. Degradation of RpoS requires the energy-dependent ClpXP protease and RssB, a protein that presents RpoS to the protease. Degradation is in part signaled by phosphorylation of RssB. However, recent studies in our labs and others demonstrated significant regulation of degradation in the absence of phosphorylation, suggesting a novel method of regulation that needs to be understood. A genetic screen for regulators of RpoS degradation led to discovery of a small, previously uncharacterized protein, YaiB. When overproduced, YaiB stabilizes RpoS; mutants of yaiB have somewhat decreased stability of RpoS under normal growth conditions and totally abolish the stabilization of RpoS after phosphate starvation. YaiB blocks RpoS turnover in a purified in vitro system, most likely by directly interacting with RssB. The identification and characterization of YaiB suggests the presence of a novel family of modulators of protein turnover that act by competing for adaptor proteins.? ? RpoS translation is positively regulated by multiple small RNAs. The message upstream of the RpoS translation start folds into a hairpin that occludes ribosome binding and therefore translation. The small regulatory RNAs, DsrA and RprA, compete for the inhibitory stem of the hairpin, disrupting the secondary structure of the RpoS leader, allowing translation. The action of DsrA and RprA in positive regulation of RpoS translation is rather unique and raised the question whether pairing simply opens a hairpin or has effects on the processing or stability of the target mRNA and the small RNA. Using previously tested mutations and compensating mutations in the small RNAs and the target messages to provide specificity, we can show that small RNA pairing with the rpoS message results in higher levels of the message, and also seems to stabilize the small RNAs. Further studies on this should increase our understanding of the mechanism of positive action by small RNAs. The existence of multiple small RNAs that regulate a single target suggests that each RNA may be made under unique growth conditions. The promoter of dsrAis regulated by temperature. RprA, identified as a multicopy suppressor of dsrAmutants, is regulated by the RcsC, RcsD, RcsB phosphorelay regulatory system. These regulators also act to turn up capsular polysaccharide synthesis and to up regulate a cell division protein as well as many other genes, some involved in biofilm formation; they are activated by cell surface stress. Genetic studies have demonstrated that sensing to activate the phosphorelay is via two distinct pathways; one pathway acts by modification of the activity or structure of RcsF, a lipoprotein that acts upstream of the membrane sensor, RcsC. The other pathway is independent of RcsF. Another small RNA has now been found to positively regulate RpoS; synthesis of this RNA is regulated by the sigma E sigma factor, a regulator that is involved in the response to periplasmic stress. Finally, some novel levels of regulation of translation of RpoS have been found that are independent of small RNAs. Both tmRNA, an RNA that is used at stalled ribosomes to tag the peptide for degradation and relieve the stalling, and the miaA-directed RNA modification are necessary for high levels of RpoS translation. Thus, a variety of cellular systems affect RpoS levels. ? ?
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