There are three objectives in the present proposal. The first is to continue efforts to identify and characterize regulatory elements and mechanisms involved in controlling pyrimidine gene expression in Escherichia coli. Control mechanisms of particular interest are (i) regulation during transcriptional initiation at the pyrBI promoter through UTP-induced reiterative RNA synthesis, (ii) attenuation control of pyrBI expression in which UTP-sensitive transcriptional pausing in the pyrBI leader region regulates Rho-independent transcriptional termination at an attenuator preceding the pyrB gene, and (iii) translational control of pyrC and pyrD expression by CTP/GTP-sensitive selection of alternative transcriptional start sites that results in the synthesis of multiple transcripts with different potentials for translation.
Specific aims i nclude the characterization by mutational analysis of pyrBI promoter sequences required for reiterative RNA synthesis and the physical state of RNA polymerase during this process. Additional aims include the identification of sequences and structures that cause nucleotide-sensitive transcriptional pausing in the pyrBI leader region, the isolation and characterization of cis- and trans-acting mutations that alter UTP- sensitive regulation of pyrBI expression, and the investigation of the possible role of endonucleolytic processing of pyrC transcripts in translational control of pyrC expression. The second objective is to use the pyrimidine genes as tools to probe fundamental steps in gene expression. The pyrBI leader region will be used to examine various aspects of Rho-independent transcriptional termination. We will examine the uniqueness of RNA terminator hairpins, the role of terminator- specified long runs of uridine residues, possible regulation of termination activity by UTP levels, and upstream sequences that modulate transcriptional readthrough at terminators. In a related experiment, we will examine the effects of DNA sequence in the template and nontemplate strands of a Rho-independent terminator on transcriptional termination. The third objective is to investigate the possibility that a number of other genes are regulated by intracellular nucleotide pool sizes using control mechanisms similar to those described for the pyrimidine genes, namely, regulation by UTP-induced reiterative RNA synthesis during transcriptional initiation and translational control mediated by nucleotide-sensitive selection of alternative transcriptional start sites. These simple but powerful mechanisms rely only on the subtle modulation of the basic elements of transcription and translation. Candidate genes containing putative regulatory sequences include other pyrimidine genes and genes involved in nucleotide metabolism. The overall goal of this project is to provide new information about the mechanisms controlling gene expression in bacteria and about key steps in transcription and translation.
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