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Abstract Bacteria contain a diverse set of RNAs to provide tight regulation of gene expression in response to environmental stimuli. Bacterial small RNAs (sRNAs) work in conjunction with protein cofactors to bind complementary mRNA sequences in the cell, leading to up‐ or downregulation of protein synthesis.In vivoimaging of sRNAs can aid in understanding their spatiotemporal dynamics in real time, which inspires new ways to manipulate these systems for a variety of applications including synthetic biology and therapeutics. Current methods for sRNA imaging are quite limitedin vivoand do not provide real‐time information about fluctuations in sRNA levels. Herein, we describe our efforts toward the development of an RNA‐based fluorescent biosensor for bacterial sRNA bothin vitroandin vivo. We validated these sensors for three different bacterial sRNAs inEscherichia coliand demonstrated that the designs provide a bright, sequence‐specific signal output in response to exogenous and endogenous RNA targets.
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Determinants of target prioritization and regulatory hierarchy for the bacterial small RNA SgrS
Summary Small RNA (sRNA) regulators promote efficient responses to stress, but the mechanisms for prioritizing target mRNA regulation remain poorly understood. This study examines mechanisms underlying hierarchical regulation by the sRNA SgrS, found in enteric bacteria and produced under conditions of metabolic stress. SgrS posttranscriptionally coordinates a nine‐gene regulon to restore growth and homeostasis. Anin vivoreporter system quantified SgrS‐dependent regulation of target genes and established that SgrS exhibits a clear target preference. Regulation of some targets is efficient even at low SgrS levels, whereas higher SgrS concentrations are required to regulate other targets.In vivoandin vitroanalyses revealed that RNA structure and the number and position of base pairing sites relative to the start of translation impact the efficiency of regulation of SgrS targets. The RNA chaperone Hfq uses distinct modes of binding to different SgrS mRNA targets, which differentially influences positive and negative regulation. The RNA degradosome plays a larger role in regulation of some SgrS targets compared to others. Collectively, our results suggest that sRNA selection of target mRNAs and regulatory hierarchy are influenced by several molecular features and that the combination of these features precisely tunes the efficiency of regulation of multi‐target sRNA regulons.
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- Award ID(s):
- 1430124
- PAR ID:
- 10460014
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Molecular Microbiology
- Volume:
- 112
- Issue:
- 4
- ISSN:
- 0950-382X
- Page Range / eLocation ID:
- p. 1199-1218
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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