This project continues the elucidation of fundamental mechanisms that control transcription elongation and termination in bacteria and bacteriophages. Two broad areas of work on elongation control, seeking to achieve several major objectives by combined genetic and biochemical approaches, are proposed. The first area of study, a new direction for this grant, focuses on the function and the mechanism of action of two recently discovered elongation factors of E. coli, GreA and GreB, and the function of a recently discovered collaborator (or suppressor) gene, greC. GreA and GreB are essential for cell growth at elevated temperatures. GreC mutations bypass this requirement. In vitro, GreA and GreB promote transcript cleavage by RNA polymerase (RNAP), and, by virtue of this activity, may play multiple roles in transcription: suppression of abortive initiation (and, consequently, stimulation of productive elongation and new initiation), relief from elongation arrest, and enhancement of transcription fidelity. Whether this is true in vivo is to be determined, key target genes that are regulated by GreA, GreB and GreC are to be identified, and the molecular mechanisms involved in each regulatory event are to be dissected. The second area extends ongoing studies centering around the phage l N gene protein, the archetype antiterminator that binds to a unique structure in the nascent mRNA, captures RNAP and, in conjunction with cellular proteins, transforms that RNAP to a termination-resistant state. The critical hypothesis to be tested is that the N protein and its RNA target lock RNAP into the stable elongation state by masking or modifying sites in polymerase that are critical for termination and pausing. It is proposed to identify the subunits of RNAP, and localize the sites that contact N and its collaborators: the cis-acting RNA stem-loop structure and the host factors NusA, NusG and the ribosomal protein S10. It is also proposed to localize and dissect the domains of N that recognize A, RNAP and NusA, attempt to deduce the basic principles of RNA-protein and protein-protein interactions, and illuminate the potential allosteric modifications. It further proposed to uncover the mechanisms by which N and another host factor, NusG, accelerate RNAP through pause sites and stabilize the elongation conformation, phenomena that are likely to be quintessential for termination suppression. Additional positive and negative modulators of N that might play a role in lysis-lysogeny decisions of phage development are to be isolated. A vigorous genetic hunt for a cellular homologue of N and target genes activated by an N-like antitermination mechanism will be undertaken. It is asserted that these studies should provide fundamental insights into the relationships of RNA polymerase structures with functions, and mechanisms of gene regulation.
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