Determinants of target prioritization and regulatory hierarchy for the bacterial small RNA SgrS

Abstract

The mechanisms by which small RNA (sRNA) regulators select and prioritize target mRNAs remain poorly understood, but serve to promote efficient responses to environmental cues and stresses. We sought to uncover mechanisms that establish regulatory hierarchy for a model sRNA, SgrS, found in enteric bacteria and produced under conditions of metabolic stress when sugar transport and metabolism are unbalanced. SgrS post-transcriptionally controls a nine-gene regulon to restore growth and homeostasis under stress conditions. An in vivo reporter system was used to quantify SgrS-dependent regulation of target genes and established that SgrS exhibits a clear preference for certain targets, and regulates those targets efficiently even at low SgrS levels. Higher SgrS concentrations are required to regulate other targets. The position of targets in the regulatory hierarchy is not well-correlated with the predicted thermodynamic stability of SgrS-mRNA interactions or the SgrS-mRNA binding affinity as measured in vitro. Detailed analyses of SgrS interaction with asd mRNA demonstrate that SgrS binds cooperatively to two sites and remodels asd mRNA secondary structure. SgrS binding at both sites increases the efficiency of asd mRNA regulation compared to mutants that have only a single SgrS binding site. Our results suggest that sRNA selection of target mRNAs and regulatory hierarchy are influenced by several molecular features. The sRNA-mRNA interaction, including the number and position of sRNA binding sites on the mRNA and cofactors like the RNA chaperone Hfq, seem to tune the efficiency of regulation of specific mRNA targets.\n\nIMPORTANCETo survive, bacteria must respond rapidly to stress and simultaneously maintain metabolic homeostasis. The small RNA (sRNA) SgrS mediates the response to stress arising from imbalanced sugar transport and metabolism. To coordinate the stress response, SgrS regulates genes involved in sugar uptake and metabolism. Intrinsic properties of sRNAs such as SgrS allow them to regulate extensive networks of genes. To date, sRNA regulation of targets has largely been studied in the context of \"one sRNA-one target\", and little is known about coordination of multi-gene regulons and sRNA regulatory network structure. Here, we explore the molecular basis for regulatory hierarchy in sRNA regulons. Our results reveal a complex interplay of factors that influence the outcome of sRNA regulation. The number and location of sRNA binding sites on mRNA targets and the participation of an RNA chaperone dictate prioritized regulation of targets to promote an efficient response to stress.