Cells must change the activity of their genes in response to environmental cues, such as occur when a bacterium infects a person or when neurons connect with each other in the developing brain. Many controlling RNAs function as small switches, that instruct the cell to respond to signals such as a change in pH or a chemical secreted by nearby cells. The operation of a """"""""genetic switch"""""""" often involves a change in the 3D shape of one RNA or base pairing between two antisense RNAs. The main goals of this research are to understand (1) the physical forces that control the structures of RNA molecules and (2) how changes in RNA structure can switch gene expression on and off.The outcome of this research will improve human health, by providing basic knowledge of how bacteria adapt to the environment of their human host and enabling the rational design of artificial RNA sensors and switches for therapeutic applications. Two problems that will be addressed by this proposal are (1) how helix switching is coupled to RNA tertiary structure and (2) how proteins promote RNA strand exchange and the hybridization of natural antisense RNAs with their targets. The study of the folding pathway of the Tetrahymena ribozyme by ourselves and others has revealed general principles of RNA folding mechanisms. These principles and the experimental methods developed for the study of RNA folding reactions will be applied to the problem of conformational switching during RNA regulation.
In aim 1, stopped-flow fluorescence spectroscopy will be used to investigate the coupling of tertiary and secondary structure during folding of the PSabc domain of the Tetrahymena intron. These studies will provide basic information about RNA folding transition states.
Aim 2 will determine RNA secondary and tertiary interactions required for regulation of E. coli rpoS mRNA by the small regulatory RNA DsrA and the bacterial Sm-like protein Hfq.
In aim 3, fluorescent 'molecular beacons'and small RNA substrates will be used to investigate how Hfq protein facilitates RNA annealing and strand exchange reactions. Hfq regulates mRNA stability in bactria and is necessary for the function of more than 20 natural antisense RNAs. The results of this study will be directly relevant to the function of human Sm and Lsm proteins, which are homologous to Hfq, and which are vital to many stages of pre-mRNA splicing and mRNA expression.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM046686-18
Application #
7743801
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Preusch, Peter C
Project Start
1992-02-01
Project End
2011-06-30
Budget Start
2009-12-01
Budget End
2011-06-30
Support Year
18
Fiscal Year
2010
Total Cost
$280,081
Indirect Cost
Name
Johns Hopkins University
Department
Physiology
Type
Schools of Arts and Sciences
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Zheng, Amy; Panja, Subrata; Woodson, Sarah A (2016) Arginine Patch Predicts the RNA Annealing Activity of Hfq from Gram-Negative and Gram-Positive Bacteria. J Mol Biol 428:2259-64
Santiago-Frangos, Andrew; Kavita, Kumari; Schu, Daniel J et al. (2016) C-terminal domain of the RNA chaperone Hfq drives sRNA competition and release of target RNA. Proc Natl Acad Sci U S A 113:E6089-E6096
Panja, Subrata; Paul, Rakesh; Greenberg, Marc M et al. (2015) Light-Triggered RNA Annealing by an RNA Chaperone. Angew Chem Int Ed Engl 54:7281-4
Panja, Subrata; Woodson, Sarah A (2015) Fluorescence reporters for Hfq oligomerization and RNA annealing. Methods Mol Biol 1259:369-83
Panja, Subrata; Santiago-Frangos, Andrew; Schu, Daniel J et al. (2015) Acidic Residues in the Hfq Chaperone Increase the Selectivity of sRNA Binding and Annealing. J Mol Biol 427:3491-3500
Peng, Yi; Curtis, Joseph E; Fang, Xianyang et al. (2014) Structural model of an mRNA in complex with the bacterial chaperone Hfq. Proc Natl Acad Sci U S A 111:17134-9
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
Peng, Yi; Soper, Toby J; Woodson, Sarah A (2012) RNase footprinting of protein binding sites on an mRNA target of small RNAs. Methods Mol Biol 905:213-24

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