This research will study the regulation of bacterial genes by an important class of mechanisms called attenuation or antitermination. Such mechanisms control gene expression by regulating transcription termination signals that lie between the start of transcription (mRNA synthesis) and the coding regions of the genes being regulated. They are very widespread in bacteria. Two such systems have been discovered in the Switzer laboratory; these will be biochemically characterized in detail. In the first system the attenuation regulatory protein PyrR regulates pyrimidine biosynthetic (pyr) genes by binding to specific sites on pyr mRNA. A detailed study of PyrR-RNA interaction will be undertaken by binding studies using surface plasmon resonance and by high-resolution x-ray crystallography of PyrR-RNA complexes. The role of transcriptional pausing in PyrR action will be studied by genetic methods. The integrated regulation of the B. subtilis pyr operon by PyrR at three termination sites in the operon will be characterized by quantitative measurements of pyr RNA species in vivo. In the second system direct regulation of termination in the 5' leader of B. subtilis pyrG RNA (encoding CTP synthetase) by CTP without involvement of a regulatory protein has been demonstrated. The mechanism of this regulation will be studied by biochemical analysis of pyrG transcription in vitro. Comparative genomics demonstrates that the regulation of pyr genes by PyrR-dependent processes and pyrG antitermination similar to B. subtilis are found in many diverse bacterial genera, including many disease-causing bacteria in which antibiotic resistance is a growing clinical problem and others that are important in fermentation and biotechnology. A final objective of this research is to characterize the regulation of pyr genes in Mycobacteria, in which PyrR appears to act as an inhibitor of protein synthesis. It is planned to use nonpathogenic species for these investigations, but Mycobacteria are the agents of tuberculosis and leprosy.
