Identification of Attenuators of Transcriptional Termination: Implications for RNA Regulation in Escherichia coli

The regulatory function of many bacterial small RNAs (sRNAs) requires the binding of the RNA chaperone Hfq to the 3 ' portion of the sRNA intrinsic terminator, and therefore sRNA signaling might be regulated by modulating its terminator. Here, using a multicopy screen developed with the terminator of sRNA SgrS, we identified an sRNA gene (cyaR) and three protein-coding genes (cspD, ygjH, and rof) that attenuate SgrS termination in Escherichia coli. Analyses of CyaR and YgjH, a putative tRNA binding protein, suggested that the CyaR activity was indirect and the effect of YgjH was moderate. Overproduction of the protein attenuators CspD and Rof resulted in more frequent readthrough at terminators of SgrS and two other sRNAs, and regulation by SgrS of target mRNAs was reduced. The effect of Rof, a known inhibitor of Rho, was mimicked by bicyclomycin or by a rho mutant, suggesting an unexpected role for Rho in sRNA termination. CspD, a member of the cold shock protein family, bound both terminated and readthrough transcripts, stabilizing them and attenuating termination. By RNA sequencing analysis of the CspD overexpression strain, we found global effects of CspD on gene expression across some termination sites. We further demonstrated effects of endogenous CspD under slow growth conditions where cspD is highly expressed. These findings provided evidence of changes in the efficiency of intrinsic termination, confirming this as an additional layer of the regulation of sRNA signaling. IMPORTANCE Growing evidence suggests that the modulation of intrinsic termination and readthrough of transcription is more widespread than previously appreciated. For small RNAs, proper termination plays a critical role in their regulatory function. Here, we present a multicopy screen approach to identify factors that attenuate small RNA termination and therefore abrogate signaling dependent on the small RNA. This study highlights a new aspect of regulation of small RNA signaling as well as the modulation of intrinsic termination. Growing evidence suggests that the modulation of intrinsic termination and readthrough of transcription is more widespread than previously appreciated. For small RNAs, proper termination plays a critical role in their regulatory function.

Automated summary

The regulatory function of many bacterial small RNAs (sRNAs) is dependent on the RNA chaperone Hfq binding to the 3' portion of the sRNA intrinsic terminator. This study identified several genes, including an sRNA gene (cyaR) and three protein-coding genes (cspD, ygjH, and rof), that attenuate the termination of SgrS in Escherichia coli. Overexpression of the protein attenuators CspD and Rof resulted in increased readthrough at terminators of SgrS and other sRNAs, reducing their regulatory effects on target mRNAs. Further analysis revealed the unexpected role of Rho in sRNA termination and the stabilizing effect of CspD on both terminated and readthrough transcripts. This study provides evidence of the modulation of intrinsic termination as an additional layer of regulation in sRNA signaling.