The objective of this application is to elucidate new mechanisms of gene regulation in bacteria and to characterize the molecular interactions involved in the regulatory process. In the proposed research the investigator will attempt to identify operons in E. coli that are regulated by reiterative transcription during transcriptional initiation. This reaction involves the repetitive addition of a particular nucleotide (usually U or A) to the 3' end of the nascent transcript due to slippage between a homopolymeric sequence in the transcript and a complementary sequence in the DNA template. The resulting transcripts are unlikely to be productively extended to include downstream sequences, thus their synthesis results in reduced gene expression. Previously, Dr. Turnbough identified several pyrimidine operons that are regulated by UTP-dependent reiterative transcription, namely the pyrBI, carAB, codBA and upp operons, which encode pyrimidine metabolic enzymes. In these examples the mechanism employed to modulate reiterative transcription were found to be quite different. He identified a number of other operons with promoters whose sequences contain likely sites for UTP-dependent reiterative transcription.
In Aim 1, he will determine whether reiterative transcription occurs at these promoters and whether it is involved in regulation, which presumably is mediated by changes in the intracellular levels of UTP. These operons include cya (adenylate cyclase), metY-nusA-infB (tRNAfMet, NusA, and IF2), gal (galactose catabolism), udk (uridine kinase), atp (ATP synthetase subunits), pheST (phenylalanyl-tRNA synthetase), purMN (purine biosynthesis), and proS (prolyl-tRNA synthetase).
In Aim 2, he will continue the analysis of UTP-dependent reiterative transcription (and associated gene regulation) using the promoters of the pyrBI, carAB, codBA, and upp operons. He will identify required promoter sequences, examine the effects of transcriptional start site position (relative to the -10 region), explore the role of RNA transcript-DNA template hybrid stability, and examine the involvement of transcript cleavage (Gre) factors. In addition, he will determine the effect on reiterative transcription of tethering the 5' end of the nascent transcript to the active site of RNA polymerase (RNAP), attempt to measure the RNAP footprint before and during reiterative transcription, and determine the fate of sigma factor during reiterative transcription.
In Aim 3, he will examine the purHD (purine biosynthesis) and glnLG (NtrB and NtrC nitrogen regulators) operons for regulation involving ATP-dependent reiterative transcription since their promoters are predicted to contain sequences appropriate for this reaction. The investigator will determine whether reiterative transcription occurs at these promoters and is used for purine (ATP and/or GTP)-mediated regulation of operon expression.
In Aim 4, he will continue to isolate and characterize mutations that alter UTP-sensitive regulation of pyrBI expression. Mutations that act in cis may affect sequences required for UTP-dependent reiterative transcription, transcriptional elongation/pausing, or intrinsic transcriptional termination. Mutations that act in trans may affect factors that influence the foregoing activities, RNAP, components of the translational machinery, and elements that control the rate of translation. Each mutation (and affected gene product) will be identified, and the step in pyrBI expression affected by the mutation will be determined. The results should provide important new information about each step in the transcription cycle and also about factors controlling translation.
In Aim 5, he will determine the ranges of intracellular UTP concentrations detected by each of the multiple control mechanisms of the pyrBI and carAB operons and by the apparently single, reiterative transcription control mechanisms of the codBA and upp operons.
In Aim 6, he will continue their studies on intrinsic transcriptional termination by examining the in vivo effects of mutations located in the required thymidine tract of the pyrBI attenuator. The investigator will also examine the role of DNA sequence (template or nontemplate strand) and the requirements for particular types of RNA hairpins in intrinsic termination. Taken together, these studies should provide new examples of general types of gene regulation and further define critical steps in gene expression. Although these studies are done with E. coli, the work is likely to be applicable to the study of gene expression and regulation in all bacteria and probably in eukaryotes, as well.
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