Multiple Small RNAs Modulate Rho-Dependent Termination in the Cyclopropane Fatty Acid Synthase mRNA 5' Untranslated Region

Abstract

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. ImportanceBacteria respond to stress by rapidly regulating gene expression. Regulation can occur through control of messenger RNA (mRNA) production (transcription elongation), stability of mRNAs, or translation of mRNAs. Bacteria can use small RNAs (sRNAs) to regulate gene expression at each of these steps, but we often do not understand how this works at a molecular level. In this study, we find that sRNAs in Escherichia coli regulate gene expression at the level of transcription elongation by promoting or inhibiting transcription termination by a protein called Rho. These results help us understand new molecular mechanisms of gene expression regulation in bacteria.