The objective of the present proposal is to understand the mechanisms regulating pyrimidine gene expression in Escherichia coli. In this bacterium, the de novo synthesis of UMP, the precursor of all pyrimidine nucleotides is catalyzed by six enzymes encoded by six unlinked genes and small operons: carAB, pyrBI, pyrC, pyrD, pyrE, and pyrF. The expression of these genes and operons is noncoordinately regulated by the intracellular levels of uridine or cytidine nucleotides. Recent studies have shown that pyrBI and pyrE expression is regulated primarily by an attenuation control mechanism in which transcriptional termination at a Rho-independent terminator (attenuator) immediately preceding the pyr structural gene(s) is regulated by the relative rates of UTP- sensitive transcription and coupled translation within a leader region upstream of the attenuator. At present, little is known about the mechanisms regulating the expression of the other pyrimidine genes. In this study experiments are designed to identify new regulatory elements and to test possible models for the regulation of pyrimidine gene expression. The proposed research will focus on the pyrBI operon and the pyrC and pyrF genes. Experiments are planned to complete the genetic and biochemical characterization of key regulatory elements involved in attenuation control of pyrBI expression, including nucleotide (UTP and GTP)specific transcriptional pausing. Strains exhibiting altered attenuation control will be isolated and should provide a variety of interesting mutations, including rpo mutations which will be used to characterize fundamental properties of RNA polymerase. Attenuator-independent regulation of pyrBI expression, which significantly contributes to the pyrimidine-mediated control of this operon, also will be characterized. Of particular interest is the possible involvement of UTP-sensitive abortive transcriptional initiation in attenuator-independent regulation. Recent results suggest that pyrC expression is regulated by a novel attenuation control mechanism in which transcriptional termination at a Rho-dependent erminator within the pyrC structural gene is controlled by the extent of coupled transcription and translation. Experiments are planned to test the effect of rho mutations and mutations that alter the rate of pyrC translational initiation on the regulation of pyrC expression. In addition, the possible role of the purine repressor, encoded by the purR gene, in the regulation of pyrC, pyrD, and car AB expression will be examined. The remaining experiments outlined will examine the mechanism regulating pyrF expression. The possible regulatory roles of trans- acting factors, abortive initiation, and the orF gene (the second gene in the pyrF-orfF operon) will be explored.
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