The long-range goal of this project is to define the interactions in the transcription complex that regulate pausing and termination by RNA polymerase. Nascent RNA hairpins are important regulatory signals in bacteria, where pausing and termination are major components of genetic regulatory mechanisms. Pausing and premature termination also affect expression of genes in mammalian cells and viruses, notably genes involved in the development of cancer and in growth of the AIDS virus, HIV- 1. In both bacteria and eukaryotes, specialized regulatory proteins modify the transcription complex to make it resistant to pausing and termination. Although significant progress has been made in understanding pausing, termination, and the regulatory proteins that control these events, two alternative models remain possible. In one view, called the allosteric model, pause signals, termination signals, and regulatory proteins primarily affect the conformation of RNA polymerase. In the other, these signals and proteins primarily affect translocation of a relatively rigid RNA polymerase on the RNA and DNA chains (the rigid-body model). Pausing and termination by E. coli RNA polymerase and their regulation by the NusA, NusG, and RfaH proteins, and pausing by human RNA polymerase II have been developed as model systems. A combination of biochemical, genetic, and biophysical approaches will be used to distinguish the allosteric and rigid-body models of transcriptional regulation, and to characterize the mechanisms of pausing, termination, and regulatory proteins that control them.
Specific aims will be to (i) characterize interactions of RNA polymerase's flap-tip helix with RNA, NusA, and sigma-70, and test how these interactions affect catalysis in the active site; (ii) determine the location of the RNA 3 about end in paused and nonpaused transcription elongation complexes; (iii) determine the kinetic mechanisms of elongation, pausing, and termination; (iv) map interactions between RNA polymerase and pause and terminator hairpins; and (v) determine the sites at which RfaH and NusG interact with RNA polymerase and the mechanisms by which they regulate transcript elongation.
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