The purpose of this research is to elucidate the molecular mechanism for the regulation of pyrimidine gene expression in Salmonella typhimurium. The six pyr genes encoding the pyrimidine biosynthetic enzymes are unlinked and noncoordinately expressed. Their regulation is complex: three genes controlled by a uridine compound and three controlled primarily by a cytidine compound. The exact identity of the pyrimidine regulatory effectors is unknown as is the control mechanism in which they function. In this project biochemical, physiological and genetic experiments are designed to identify essential regulatory elements including small molecule effectors and to test possible control mechanisms (e.g., coupled transcription-translation control and corepressor-repressor protein mediated control). The pyrimidine effectors will be identified using an in vitro DNA dependent coupled transcription-translation system using cloned pyr genes as a source of DNA. The effects of other putative small molecule effectors (e.g., ppGpp) on pyr gene expression will be measured. The in vitro system will also be used to probe the mechanism of regulation with metabolic inhibitors and pyrimidine analogs. Regulatory mutants will be isolated employing strains in which the promoter-regulatory region of a pyr gene has been fused to the structural genes of the E. coli lactose operon. These strains allow the use of mutant selection techniques developed for the lac operon. These mutants will be characterized to identify the regulatory defects. Pyrimidine regulation will be studied in vivo using a variety of specific metabolic inhibitors, antibiotics and mutations. The promoter-regulatory region of one pyr gene will be cloned and sequenced to inspect for unique features that may help identify the mechanism of regulation. Hopefully, these experiments will add to our understanding of the control of gene expression in procaryotes.
NIH ExPorter
NIH ExPorter