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

Abstract

Bacterial small RNAs (sRNAs) have been commonly characterized as regulators of post-transcriptional steps of gene expression including translation and stability of mRNA targets. Previous work revealed that the Escherichia coli cyclopropane fatty acid synthase (cfa) mRNA is regulated by at least five different sRNAs by a proposed mechanism involving regulated cfa mRNA turnover by the RNase E degradosome. However, recent work identified the long 5' untranslated region (UTR) of cfa mRNA as a potential target for Rho-dependent transcription termination, leading us to question whether sRNAs might regulate cfa gene expression at the level of transcription elongation. In this study we report evidence for premature Rho-dependent termination within the long 5' UTR of cfa, and demonstrate that a pyrimidine-only tract within the 5' UTR is required for efficient Rho-dependent regulation of cfa. Our data suggest that all of the sequence determinants required for efficient Rho-mediated termination are harbored within the cfa long mRNA 5' UTR. Finally, 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 degradosome-dependent turnover of cfa mRNA. These results illustrate the versatile mechanisms sRNAs use to regulate target gene expression at transcriptional and post-transcriptional levels and suggest that regulation by sRNAs in long UTRs can involve modulation of transcription elongation.