CsrA is a global regulatory RNA binding protein that has important roles in regulating carbon metabolism, motility, biofilm formation, and numerous other cellular processes. IraD functions as an antiadapter protein that inhibits RssB-mediated degradation of RpoS, the general stress response and stationary-phase sigma factor of Escherichia coli Here we identified a novel mechanism in which CsrA represses iraD translation via translational coupling. Expression studies with quantitative reverse transcriptase PCR, Western blotting, and lacZ fusions demonstrated that CsrA represses iraD expression. Gel mobility shift, footprint, and toeprint studies identified four CsrA binding sites in the iraD leader transcript, all of which are far upstream of the iraD ribosome binding site. Computational modeling and RNA structure mapping identified an RNA structure that sequesters the iraD Shine-Dalgarno (SD) sequence. Three open reading frames (ORFs), all of which are translated, were identified in the iraD leader region. Two of these ORFs do not affect iraD expression. However, the translation initiation region of the third ORF contains three of the CsrA binding sites, one of which overlaps its SD sequence. Furthermore, the ORF stop codon overlaps the iraD start codon, a sequence arrangement indicative of translational coupling. In vivo expression and in vitro translation studies with wild-type and mutant reporter fusions demonstrated that bound CsrA directly represses translation initiation of this ORF. We further established that CsrA-dependent repression of iraD translation occurs entirely via translational coupling with this ORF, leading to accelerated iraD mRNA decay.IMPORTANCE CsrA posttranscriptionally represses gene expression associated with stationary-phase bacterial growth, often in opposition to the transcriptional effects of the stationary-phase sigma factor RpoS. We show that CsrA employs a novel regulatory mechanism to repress translation of iraD, which encodes an antiadapter protein that protects RpoS against proteolysis. CsrA binds to four sites in the iraD leader transcript but does not directly occlude ribosome binding to the iraD SD sequence. Instead, CsrA represses translation of a short open reading frame encoded upstream of iraD, causing repression of iraD translation via translational coupling. This finding offers a novel mechanism of gene regulation by the global regulator CsrA, and since RpoS can activate csrA transcription, this also highlights a new negative-feedback loop within the complex Csr and RpoS circuitry.

In Escherichia coli, SdsR and RyeA, a unique pair of mutually cis-encoded small RNAs (sRNAs), act as toxin and antitoxin, respectively. SdsR and RyeA expression are reciprocally regulated; however, how each regulates the synthesis of the other remains unclear. Here, we characterized the biosynthesis of the two sRNAs during growth and investigated their coordinate regulation using sdsR and ryeA promoter mutant strains. We found that RyeA transcription occurred even upon entry of cells into the stationary phase, but its apparent expression was restricted to exponentially growing cells because of its degradation by SdsR. Likewise, the appearance of SdsR was delayed owing to its RyeA-mediated degradation. We also found that the sdsR promoter was primarily responsible for transcription of the downstream pphA gene encoding a phosphatase and that pphA mRNA was synthesized by transcriptional read-through over the sdsR terminator. Transcription from the σ70-dependent ryeA promoter inhibited transcription from the σS-dependent sdsR promoter through transcriptional interference. This transcriptional inhibition also downregulated pphA expression, but RyeA itself did not downregulate pphA expression.