The purpose of this research is to elucidate the mechanisms regulating pyrimidine gene expression in Escherichia coli and Salmonella typhimurium. In these bacteria, the de novo synthesis of UMP, the precursor of all pyrimidine nucleotides, is catalyzed by six enzymes encoded by six unlinked genes and operons. These genes and operons are expressed noncoordinately and are subject to negative regulation by either a uridine or cytidine nucleotide. The exact identities of the nucleotide effectors for all the genes except the pyrBI operon are unknown. Experiments in this study are designed to identify regulatory elements and to test possible control mechanisms of pyr gene expression. Of particular interest is the pyrBI operon of E. coli, which encodes the subunits of the pyrimidine biosynthetic enzyme aspartate transcarbamylase. The expression of this operon is negatively regulated by UTP. Initial studies have revealed an attenuator immediately preceding the structural genes at which site transcription initiated upstream at either of two pyrBI promoters is efficiently terminated. Additional features identified suggest a model for regulation in which the relative rates of UTP-dependent transcription within the pyrBI leader region and coupled translation of the leader transcript control transcriptional termination at the attenuator. This model will be tested by in vitro site-directed mutagenesis to alter DNA sequences apparently involved in attenuation control. The effects of these mutations on regulation will be studied in vivo. Additional pyrBI regulatory mutations will be isolated in vivo and characterized. Physiological and genetic factors influencing the frequency of attenuated and readthrough transcription will be examined. Also included are studies of pyrC and pyrF expression in E. coli. Preliminary studies indicate that pyrF expression, which also is negatively regulated by a uridine nucleotide, may not be controlled by an attenuation mechanism. These studies will be extended. The possible involvement of a regulatory DNA binding factor will be explored. The pyrC gene is included as a representative of the pyr genes that are regulated by a cytidine nucleotide. Studies are described to identify features of attenuation control and other types of control mechanisms. The effect of UTP, CTP, and ppGpp on pyrC and pyrF expression will be measured in an in vitro DNA-dependent, coupled transcription-translation system.
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