Small RNAs positively and negatively control transcription elongation through modulation of Rho utilization site accessibility

Bacteria use a multi-layered regulatory strategy to precisely and rapidly tune gene expression in response to environmental cues. Small RNAs (sRNAs) form an important layer of gene expression control and most act post-transcriptionally to control translation and stability of mRNAs. We have shown that at least five different sRNAs in Escherichia coli regulate the cyclopropane fatty acid synthase ( cfa ) mRNA. These sRNAs bind at different sites in the long 5’ untranslated region (UTR) of cfa mRNA and previous work suggested that they modulate RNase E-dependent mRNA turnover. Recently, the cfa 5’ UTR was identified as a site of Rho-dependent transcription termination, leading us to hypothesize that the sRNAs might also regulate cfa transcription elongation. In this study we find that a pyrimidine-rich region flanked by sRNA binding sites in the cfa 5’ UTR is required for premature Rho-dependent termination. We discovered that both the activating sRNA RydC and repressing sRNA CpxQ regulate cfa primarily by modulating Rho-dependent termination of cfa transcription, with only a minor effect on RNase E-mediated turnover of cfa mRNA. A stem-loop structure in the cfa 5’ UTR sequesters the pyrimidine-rich region required for Rho-dependent termination. CpxQ binding to the 5’ portion of the stem increases Rho-dependent termination whereas RydC binding downstream of the stem decreases termination. These results reveal the versatile mechanisms sRNAs use to regulate target gene expression at transcriptional and post-transcriptional levels and demonstrate that regulation by sRNAs in long UTRs can involve modulation of transcription elongation.