Controlling transcription termination upstream of a coding region is a common strategy to regulate gene expression in bacteria, including many with importance to human health. Such control mechanisms are collectively termed attenuation and antitermination. The proposed research will investigate the mechanisms by which RNA binding proteins recognize and bind to specific sites in RNA, and how these interactions regulate transcription. The mechanism by which protein-protein interactions can modulate the activity of an RNA-binding gene regulatory protein will also be studied. The model system of study is the TRAP protein, an RNA binding protein that regulates transcription attenuation of the tryptophan (trp) genes in Bacillus subtilis and related bacteria. In the presence of excess tryptophan, TRAP is activated to bind to the 5'leader region of the trp operon mRNA and induce formation of a transcription terminator, which halts expression of the genes. TRAP is a unique among characterized RNA binding proteins in that it consists of 11 identical subunits arranged in a ring structure, and in that it binds RNAs that contain up to 11 small (trinucleotide) repeated elements. Recent studies indicate that TRAP binds to RNA by a two-step mechanism. The hypothesis to be tested is that TRAP first binds to the 5'-end of the RNA and then scans until it encounters the 5-most repeats of the binding site, at which point an initiation complex is formed. This is followed by wrapping the remainder of the repeats around the outer perimeter of the protein ring. The detailed mechanism by which TRAP associates with its RNA target will be characterized using a combination of kinetic binding studies, as well as rapid-quench nuclease protection and fluorescence studies. A protein called anti-TRAP (AT) specifically binds to tryptophan-activated TRAP and inhibits it from binding to RNA. Studies will be performed to characterize the structure and function of AT, particularly the mechanism by which it recognizes and binds to TRAP.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM062750-08S1
Application #
7902750
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Tompkins, Laurie
Project Start
2009-08-14
Project End
2009-11-30
Budget Start
2009-08-14
Budget End
2009-11-30
Support Year
8
Fiscal Year
2009
Total Cost
$30,618
Indirect Cost
Name
State University of New York at Buffalo
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
038633251
City
Buffalo
State
NY
Country
United States
Zip Code
14260
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Potter, Kristine D; Merlino, Natalie M; Jacobs, Timothy et al. (2011) TRAP binding to the Bacillus subtilis trp leader region RNA causes efficient transcription termination at a weak intrinsic terminator. Nucleic Acids Res 39:2092-102
Shevtsov, Mikhail B; Chen, Yanling; Isupov, Michail N et al. (2010) Bacillus licheniformis Anti-TRAP can assemble into two types of dodecameric particles with the same symmetry but inverted orientation of trimers. J Struct Biol 170:127-33
Sachleben, Joseph R; McElroy, Craig A; Gollnick, Paul et al. (2010) Mechanism for pH-dependent gene regulation by amino-terminus-mediated homooligomerization of Bacillus subtilis anti-trp RNA-binding attenuation protein. Proc Natl Acad Sci U S A 107:15385-90
Chen, Yanling; Gollnick, Paul (2008) Alanine scanning mutagenesis of anti-TRAP (AT) reveals residues involved in binding to TRAP. J Mol Biol 377:1529-43
Barbolina, Maria V; Kristoforov, Roman; Manfredo, Amanda et al. (2007) The rate of TRAP binding to RNA is crucial for transcription attenuation control of the B. subtilis trp operon. J Mol Biol 370:925-38
Payal, Vandana; Gollnick, Paul (2006) Substitutions of Thr30 provide mechanistic insight into tryptophan-mediated activation of TRAP binding to RNA. Nucleic Acids Res 34:2933-42
McElroy, Craig A; Manfredo, Amanda; Gollnick, Paul et al. (2006) Thermodynamics of tryptophan-mediated activation of the trp RNA-binding attenuation protein. Biochemistry 45:7844-53
Shevtsov, Mikhail B; Chen, Yanling; Gollnick, Paul et al. (2005) Crystal structure of Bacillus subtilis anti-TRAP protein, an antagonist of TRAP/RNA interaction. Proc Natl Acad Sci U S A 102:17600-5
Barbolina, Maria V; Li, Xiufeng; Gollnick, Paul (2005) Bacillus subtilis TRAP binds to its RNA target by a 5' to 3' directional mechanism. J Mol Biol 345:667-79

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