Kinetic Studies of Transcription Elongation

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DESCRIPTION (provided by applicant): The main goal of the proposed studies is to elucidate the detailed kinetic mechanisms that regulate and control transcription elongation. Transcription, the DNA directed synthesis of RNA, is the first step in the cascade of events that leads to gene expression. The central role of RNA polymerase (RNAP) in transcription is to synthesize the nascent RNA chain with high fidelity and at a reasonable rate. RNAP appears to have evolved such that it catalyzes multiple reactions and displays an unprecedented level of dynamic flexibility. In the past ten years, our understanding of the regulation of transcription at the level of elongation has evolved considerably; however, most of our understanding comes from studies either of static elongation complexes or at positions of regulatory events such as pause and termination sites. As such, the question as to the relevance of these studies to the mechanism of RNA synthesis remains unclear. To understand an enzyme that exhibits such conformational and functional diversity, it is essential to identify all steps in each of the pathways and to determine which step(s) might be rate-limiting and thus subject to regulation. Significantly, only transient-state kinetic methods can identify individual rate-limiting steps.In the previous grant period, using kinetics, we demonstrated that E. coli RNAP contains an allosteric binding site in addition to the catalytic site. Binding of the templated nucleoside triphosphate (NTP), but not non-templated NTPs, to this site increases the rate of nucleotide incorporation. The data suggest that RNA polymerase can exist in a state that catalyzes synthesis slowly (unactivated) and one that catalyzes synthesis rapidly (activated), with the transition from the slow to the fast state being induced by binding of the templated NTP to the allosteric site. We hypothesize that this conformational switch is paramount to the regulation of transcription elongation and termination.In the next grant period, we will test many predictions of this model and further investigate the role of NTP binding in regulating transcription elongation. In addition, to develop an integrated model of elongation, we also will characterize transcript cleavage which is important for maintaining accurate and processive synthesis. We will take advantage of the thermal stability and high cleavage activity of T. thermophilus RNAP for these latter studies. Finally, the recent publication of crystal structures of yeast RNAP Il and T. aquaticus RNAP core enzymes bring us into a new era in the study of transcription, providing an unprecedented opportunity to understand the mechanism of RNA synthesis at the atomic level. Accordingly, we will use this information to begin to understand, at the amino acid level, the role of NTP binding and conformational transitions in the regulation of elongation.