REGULATION OF THE TRYTOPHAN GENES IN BACILLUS

点击翻译按钮获取中文摘要

Controlling transcription termination prior to the coding region is a commonly used 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 attenuation. The model system of study is the TRAP protein (trp RNA-binding Attenuation Protein), an RNA binding protein that regulates transcription attenuation of the tryptophan biosynthetic genes in Bacillus subtilis and related Bacilli. In the presence of excess tryptophan, TRAP is activated to bind to a series of 11 GAG or UAG repeats in the 5' leader region of the trp operon. This binding induces formation of a transcription terminator, which halts expression of the genes. TRAP is an 11 subunit protein that forms a symmetric ring. RNA binds to TRAP by wrapping 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 equilibrium binding studies, nucleoside analogs, and rapid-quench stopped-flow studies. TRAP is activated to bind RNA by binding 11 molecules of L-tryptophan. Crystallography, genetics and biochemical approaches will be used to determine the mechanism by which tryptophan binding activates TRAP. The third objective is to develop a more detailed understanding of the mechanism of TRAP mediated transcription attenuation. Key elements of the TRAP binding site in the trp leader will be altered and the effects of these changes on attenuation studied in vivo using a trpE''-'lacZ gene fusion. These studies will be guided by the information learned from in vitro studies of the TRAP/RNA interaction.