Insight into post-transcriptional regulatory mechanisms will be sought through the study of a novel paradigm in global regulation, the carbon storage regulatory (Csr) system of Escherichia coli. Csr includes CsrA, an RNA binding protein that regulates translation and/or modulates the stability of target mRNAs. CsrB and CsrC are non-coding regulatory RNAs that antagonize CsrA by sequestering this protein and CsrD is a protein that specifically targets CsrB and CsrC for degradation by RNase E. In E. coli, CsrA affects metabolism, physiology, motility and multicellular behavior on a broad scale, repressing certain genes expressed during the transition from exponential to stationary phase growth and activating various genes expressed during exponential phase. CsrA homologues are widely distributed among eubacteria and regulate the expression of virulence factors in both plant and animal pathogens. Thus, the proposed studies will also provide fundamental understanding of the regulation of bacterial physiology and pathogenesis, and may suggest novel therapeutic approaches for bacterial infections. The specifc aims of this proposal are: 1) Elucidate the molecular mechanisms by which CsrA activates or inhibits gene expression.
This aim will include an analysis of CsrA-mediated autoregulation, as well as an investigation into the factors that influence the stability of CsrA target transcripts. 2) Establish the Csr global regulon using a combination of bioinformatic, genomic, molecular genetic and biochemical approaches. 3) Stoichiometric and structural characterization of CsrA-RNA complexes.
This aim will include NMR structural determination of a model CsrA target RNA, as well as the structure of CsrA-RNA complexes by X-ray crystallography. 4) Elucidate the molecular mechanism of CsrD action. We will conduct experiments to elucidate the mechanism by which CsrD specifically targets CsrB and CsrC for degradation by RNase E. The long-range objectives of these studies are to fully understand the regulatory components, genetic circuitry, molecular mechanisms, and biological functions of the Csr system. Insight into post-transcriptional regulatory mechanisms will be sought through the study of a novel paradigm in global genetic regulation, the carbon storage regulatory (Csr) system of Escherichia coli. Csr controls bacterial metabolism, physiology, motility and biofilm development on a broad scale, and regulates the expression of virulence factors in both plant and animal pathogens. Thus, the proposed studies will provide fundamental understanding of the regulation of bacterial physiology and pathogenesis, and may suggest novel therapeutic approaches for bacterial infections.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059969-13
Application #
7791279
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Anderson, James J
Project Start
1999-08-01
Project End
2012-03-31
Budget Start
2010-04-01
Budget End
2011-03-31
Support Year
13
Fiscal Year
2010
Total Cost
$399,963
Indirect Cost
Name
University of Florida
Department
Microbiology/Immun/Virology
Type
Schools of Earth Sciences/Natur
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
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Vakulskas, Christopher A; Leng, Yuanyuan; Abe, Hazuki et al. (2016) Antagonistic control of the turnover pathway for the global regulatory sRNA CsrB by the CsrA and CsrD proteins. Nucleic Acids Res 44:7896-910
Mukherjee, Sampriti; Oshiro, Reid T; Yakhnin, Helen et al. (2016) FliW antagonizes CsrA RNA binding by a noncompetitive allosteric mechanism. Proc Natl Acad Sci U S A 113:9870-5
Leng, Yuanyuan; Vakulskas, Christopher A; Zere, Tesfalem R et al. (2016) Regulation of CsrB/C sRNA decay by EIIA(Glc) of the phosphoenolpyruvate: carbohydrate phosphotransferase system. Mol Microbiol 99:627-39

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