Dynamics of Regulatory RNAs in Bacteria Small non-coding RNAs (sRNAs) in bacteria regulate cell growth and stress response, coordinating the levels of gene expression in response to diverse environmental conditions. Genes regulated by sRNAs control host interactions, virulence and toxin expression in many pathogenic bacteria, and thus have a direct impact on human health. How sRNAs recognize and base pair with their mRNA targets is not understood, in part because each RNA molecule must change structure in order for the two RNAs to pair with each other. Base pairing between sRNAs and mRNAs is facilitated by a protein chaperone Hfq, which is required for genetic regulation by sRNAs. The physical interactions between sRNAs, mRNAs and Hfq protein dictate how these regulatory switches operate and how different regulatory pathways intersect within the same bacterium. Biochemical, physical and genetic methods will used to study how bacterial non-coding sRNAs control gene expression, using the master stress-response regulator rpoS as a model. The 3D conformation of the Hfq-rpoS RNA complex will be determined by biochemical footprinting, mutagenesis and SAXS. The mechanism by which Hfq recruits sRNAs and stimulates pairing with mRNA targets will be investigated using fluorescence spectroscopy and protein engineering. The requirement for Hfq binding sites in mRNAs and sRNAs in vivo will be compared in negatively and positively regulated mRNAs, using rpoS-lacZ translational fusions in E. coli. Results of genetic experiments will be correlated with in vitro measurements of sRNA annealing and Hfq binding. The results will show how the structure or stability of Hfq-sRNA- mRNA complexes determines downstream regulatory outcomes, which is currently unknown. As Hfq is homologous to eukaryotic Sm proteins, the results will be also relevant to understanding how Sm proteins act in human mRNA turnover and pre-mRNA splicing.

Public Health Relevance

PROJECT DESCRIPTION Bacterial pathogens use small RNA molecules to control genes that help them survive in the human body and produce toxins after infection. The goal of this research project is to determine the physical shapes of these small RNAs, and how they come together with messenger RNAs and a RNA chaperone protein to turn genes on and off in bacterial cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM046686-20
Application #
8290547
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Preusch, Peter C
Project Start
1992-02-01
Project End
2015-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
20
Fiscal Year
2012
Total Cost
$330,024
Indirect Cost
$126,005
Name
Johns Hopkins University
Department
Physiology
Type
Schools of Arts and Sciences
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Peng, Yi; Soper, Toby J; Woodson, Sarah A (2014) Positional effects of AAN motifs in rpoS regulation by sRNAs and Hfq. J Mol Biol 426:275-85
Panja, Subrata; Schu, Daniel J; Woodson, Sarah A (2013) Conserved arginines on the rim of Hfq catalyze base pair formation and exchange. Nucleic Acids Res 41:7536-46
Panja, Subrata; Woodson, Sarah A (2012) Hexamer to monomer equilibrium of E. coli Hfq in solution and its impact on RNA annealing. J Mol Biol 417:406-12
Hopkins, Julia F; Panja, Subrata; Woodson, Sarah A (2011) Rapid binding and release of Hfq from ternary complexes during RNA annealing. Nucleic Acids Res 39:5193-202
Soper, Toby; Mandin, Pierre; Majdalani, Nadim et al. (2010) Positive regulation by small RNAs and the role of Hfq. Proc Natl Acad Sci U S A 107:9602-7
Hopkins, Julia F; Panja, Subrata; McNeil, Stephanie A N et al. (2009) Effect of salt and RNA structure on annealing and strand displacement by Hfq. Nucleic Acids Res 37:6205-13
Woodson, Sarah A; Koculi, Eda (2009) Analysis of RNA folding by native polyacrylamide gel electrophoresis. Methods Enzymol 469:189-208
Woodson, Sarah A (2008) RNA folding and ribosome assembly. Curr Opin Chem Biol 12:667-73
Soper, Toby J; Woodson, Sarah A (2008) The rpoS mRNA leader recruits Hfq to facilitate annealing with DsrA sRNA. RNA 14:1907-17
Koculi, Eda; Hyeon, Changbong; Thirumalai, D et al. (2007) Charge density of divalent metal cations determines RNA stability. J Am Chem Soc 129:2676-82

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