Regulation of the tryptophan genes in Bacillus

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DESCRIPTION (provided by applicant): 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.