Mechanism of Positive Regulation by DsrA and RprA Small Noncoding RNAs: Pairing Increases Translation and Protects rpoS mRNA from Degradation

ABSTRACT Small noncoding RNAs (sRNAs) regulate gene expression in Escherichia coli by base pairing with mRNAs and modulating translation and mRNA stability. The sRNAs DsrA and RprA stimulate the translation of the stress response transcription factor RpoS by base pairing with the 5′ untranslated region of the rpoS mRNA. In the present study, we found that the rpoS mRNA was unstable in the absence of DsrA and RprA and that expression of these sRNAs increased both the accumulation and the half-life of the rpoS mRNA. Mutations in dsrA, rprA, or rpoS that disrupt the predicted pairing sequences and reduce translation of RpoS also destabilize the rpoS mRNA. We found that the rpoS mRNA accumulates in an RNase E mutant strain in the absence of sRNA expression and, therefore, is degraded by an RNase E-mediated mechanism. DsrA expression is required, however, for maximal translation even when rpoS mRNA is abundant. This suggests that DsrA protects rpoS mRNA from degradation by RNase E and that DsrA has a further activity in stimulating RpoS protein synthesis. rpoS mRNA is subject to degradation by an additional pathway, mediated by RNase III, which, in contrast to the RNase E-mediated pathway, occurs in the presence and absence of DsrA or RprA. rpoS mRNA and RpoS protein levels are increased in an RNase III mutant strain with or without the sRNAs, suggesting that the role of RNase III in this context is to reduce the translation of RpoS even when the sRNAs are acting to stimulate translation.

Controlled induction of the RpoS regulon inEscherichia coli, using an RpoS-expressing plasmid

RpoS, an alternative sigma factor produced by many Gram-negative bacteria, primarily controls genes that are expressed in stationary phase in response to nutrient deprivation. To test the idea that induction of RpoS in the exponential phase, when RpoS is not normally expressed, increases RpoS-dependent gene expression, we constructed a plasmid carrying the rpoS gene under the control of an IPTG (isopropyl-β-D-thiogalactopyranoside)-inducible T7lac promoter. Northern and Western analyses revealed that levels of RpoS mRNA and protein, respectively, increased in response to the inducer IPTG. Assays of changes in RpoS-dependent functions (catalase activity and glycogen accumulation), confirmed that induced RpoS was functional in exponential phase and was sufficient for the expression of RpoS-dependent functions. Controlled expression of RpoS and RpoS-dependent genes by plasmid-encoded rpoS may thus offer a useful tool for the study of RpoS-dependent gene expression.Key words: RpoS, regulon, gene expression, Escherichia coli.

Signal Transduction and Regulatory Mechanisms Involved in Control of the σS (RpoS) Subunit of RNA Polymerase

SUMMARY The σS (RpoS) subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli and related bacteria. While rapidly growing cells contain very little σS, exposure to many different stress conditions results in rapid and strong σS induction. Consequently, transcription of numerous σS-dependent genes is activated, many of which encode gene products with stress-protective functions. Multiple signal integration in the control of the cellular σS level is achieved by rpoS transcriptional and translational control as well as by regulated σS proteolysis, with various stress conditions differentially affecting these levels of σS control. Thus, a reduced growth rate results in increased rpoS transcription whereas high osmolarity, low temperature, acidic pH, and some late-log-phase signals stimulate the translation of already present rpoS mRNA. In addition, carbon starvation, high osmolarity, acidic pH, and high temperature result in stabilization of σS, which, under nonstress conditions, is degraded with a half-life of one to several minutes. Important cis-regulatory determinants as well as trans-acting regulatory factors involved at all levels of σS regulation have been identified. rpoS translation is controlled by several proteins (Hfq and HU) and small regulatory RNAs that probably affect the secondary structure of rpoS mRNA. For σS proteolysis, the response regulator RssB is essential. RssB is a specific direct σS recognition factor, whose affinity for σS is modulated by phosphorylation of its receiver domain. RssB delivers σS to the ClpXP protease, where σS is unfolded and completely degraded. This review summarizes our current knowledge about the molecular functions and interactions of these components and tries to establish a framework for further research on the mode of multiple signal input into this complex regulatory system.