In bacteria, diverse regulatory RNAs mediate responses to signals and stress through gene regulation, and provide survival benefit during infections, biofilm formation, and antibiotic resistance. Small RNAs (sRNAs) represent a class of trans-acting regulatory RNAs. The most common strategy for sRNA to regulate gene expression is through base pairing with target mRNAs and thereby affecting translation efficiency and/or mRNA stability. Hfq is one essential RNA binding protein (RBP), and is largely required by many sRNAs. Hfq plays key roles in sRNA-mediated gene regulation in two ways: to increase the stability of sRNAs by protecting them from nuclease digestion, and to promote sRNAs binding to their cognate target mRNAs. sRNAs typically form a regulatory network, as many of them share the same chaperone, Hfq, and each of them can act on multiple mRNA targets, raising the question of how do RNAs compete for the same RBP, and how to mRNAs compete for the same sRNA. To address the question of how RNAs competes for binding to Hfq in vivo, we probed dynamic interactions between RNAs and Hfq using single-particular tracking (SPT). Our results uncover different mechanisms through which sRNAs bind mRNA-occupied Hfq, depending on their binding sites on Hfq. To address the question of how different target mRNAs compete for the same sRNA regulator, we combined quantitative imaging and computational modeling. We unexpectedly discovered that the tested sRNAs, previously thought to act post-transcriptionally (i.e., at the level of mRNA translation degradation), actually regulate some targets co-transcriptionally through Rho-dependent termination. This work has led to a new model wherein sRNA-mediated regulation could occur as soon as the sRNA binding site is transcribed, revealing important temporal organization of sRNA action. We have also characterized several important kinetic steps that contribute to the differential regulation of mRNA targets by an sRNA. In particular, the binding of an sRNA to its target mRNA dictate the regulatory hierarchy within an sRNA regulon.

Small RNA-mediated gene regulation in bacteria:  action under the light

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ACS Fall 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.