Bacterial small RNAs (here referred to as sRNAs) are important regulators for gene expression, especially those associated with stress responses and virulence. sRNAs function by base pairing with their target mRNAs and affecting their translation and stability. The ability of each sRNA to regulate multiple targets in its regulon creates a complex network for shaping gene expression and phenotype, which ultimately leads to global adaptation of bacteria. How do the kinetic properties of sRNA-mediated regulation establish an ordered pattern of gene expression? What is the global impact of sRNA-mediated regulation on bacterial phenotypes? These are two important fundamental questions yet to be addressed. In this proposal, we will tackle these questions. One of the bottlenecks so far that has precluded the building a complete model to describe sRNA regulatory networks is the lack of kinetic measurements inside bacterial cells. Therefore, we first propose to develop a super-resolution imaging and analysis platform allowing direct visualization and characterization of sRNA and target mRNAs at the single-cell level with single copy sensitivity. Applying this imaging and analysis platform to a model sRNA system, SgrS, in E. coli, we will fully dissect kinetic mechanisms of sRNA regulation on individual targets in Aim1, and further explore the molecular mechanism that governs the regulation selectivity among multiple targets in the regulon in Aim 2. In order to understand the global impact of sRNA regulation on the bacterial phenotype, in Aim 3, we will integrate high-throughput transcriptomic data and single cell imaging data with genome scale flux balance models of E. coli metabolism and identify differential metabolic pathway usage as a result of sRNA regulation. The proposed study by the combination of multi-level experimental characterization and computational simulation will provide the most systematic description of sRNA regulation to date and will establish a novel framework for sRNA analysis that can be generalized to other bacterial and eukaryotic sRNAs.

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Bacterial small RNAs (sRNAs) play important regulatory roles in gene expression. We propose to directly characterize the kinetics of sRNA-mediate regulation inside the cell with a super-resolution imaging and analysis platform and correlate sRNA regulon to the cellular metabolic network by computational modeling. The systematic description of sRNA-mediate regulation provided by our study will shed light on sRNA- associated virulence in many pathogenic bacteria.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Modeling and Analysis of Biological Systems Study Section (MABS)
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Brazhnik, Paul
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Johns Hopkins University
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Singh, Digvijay; Wang, Yanbo; Mallon, John et al. (2018) Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis. Nat Struct Mol Biol 25:347-354
Hellenkamp, Björn; Schmid, Sonja; Doroshenko, Olga et al. (2018) Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study. Nat Methods 15:669-676
Hua, Boyang; Panja, Subrata; Wang, Yanbo et al. (2018) Mimicking Co-Transcriptional RNA Folding Using a Superhelicase. J Am Chem Soc 140:10067-10070
Earnest, Tyler M; Cole, John A; Luthey-Schulten, Zaida (2018) Simulating biological processes: stochastic physics from whole cells to colonies. Rep Prog Phys 81:052601
Singh, Digvijay; Mallon, John; Poddar, Anustup et al. (2018) Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a). Proc Natl Acad Sci U S A 115:5444-5449
Peterson, Joseph R; Cole, John A; Luthey-Schulten, Zaida (2017) Parametric studies of metabolic cooperativity in Escherichia coli colonies: Strain and geometric confinement effects. PLoS One 12:e0182570
Earnest, Tyler M; Watanabe, Reika; Stone, John E et al. (2017) Challenges of Integrating Stochastic Dynamics and Cryo-Electron Tomograms in Whole-Cell Simulations. J Phys Chem B 121:3871-3881
Earnest, Tyler M; Cole, John A; Peterson, Joseph R et al. (2016) Ribosome biogenesis in replicating cells: Integration of experiment and theory. Biopolymers 105:735-751
Bobrovskyy, Maksym; Vanderpool, Carin K (2016) Diverse mechanisms of post-transcriptional repression by the small RNA regulator of glucose-phosphate stress. Mol Microbiol 99:254-73
Singh, Digvijay; Sternberg, Samuel H; Fei, Jingyi et al. (2016) Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9. Nat Commun 7:12778

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