Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame.

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 Here we identified a novel mechanism in which CsrA represses translation via translational coupling. Expression studies with quantitative reverse transcriptase PCR, Western blotting, and fusions demonstrated that CsrA represses expression. Gel mobility shift, footprint, and toeprint studies identified four CsrA binding sites in the leader transcript, all of which are far upstream of the ribosome binding site. Computational modeling and RNA structure mapping identified an RNA structure that sequesters the Shine-Dalgarno (SD) sequence. Three open reading frames (ORFs), all of which are translated, were identified in the leader region. Two of these ORFs do not affect 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 start codon, a sequence arrangement indicative of translational coupling. expression and 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 translation occurs entirely via translational coupling with this ORF, leading to accelerated mRNA decay. 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 , which encodes an antiadapter protein that protects RpoS against proteolysis. CsrA binds to four sites in the leader transcript but does not directly occlude ribosome binding to the SD sequence. Instead, CsrA represses translation of a short open reading frame encoded upstream of , causing repression of translation via translational coupling. This finding offers a novel mechanism of gene regulation by the global regulator CsrA, and since RpoS can activate transcription, this also highlights a new negative-feedback loop within the complex Csr and RpoS circuitry.