TRANSIENT STATE KINETICS OF TRANSCRIPTION ELONGATION

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DESCRIPTION: The studies presented in this proposal are designed to elucidate the detailed kinetic mechanisms by which E. coii RNA polymerase catalyzes the synthesis of the nascent RNA transcript. The rationale for this effort lies in the choice of E. coli RNA polymerase as the best understood member of the class of enzymes which bind and process in a non-dissociative manner the production of RNA transcripts. As such, the E. coli enzyme serves as a prototype for the more complicated eukaryotic RNA polymerases. The enzyme functions to synthesize, under the direction of the DNA template, the formation of a nascent RNA chain with exceedingly high fidelity with catalytical effient turnover. The motion of the polymerase along the DNA template is uneven, however, and Dr. Erie defines this as a dwell time at a given template position which can range from 10 ms to several seconds. One of the main goals of the proposal is to define the sequence dependent differences in the transit rate of the polymerase along the DNA template and to determine the role of specific sequences in controlling the rate of polymerase polymerization. It is suggested that sequence-dependent transcription rates which result in pausing may be central features of a regulatory network, which plays an important role in the control of gene expression at the transcription level. As such, the proposal embarks on an ambitious project to identify and kinetically define all the steps in the transcription pathway and to determine which might be rate-limiting, and hence subject to regulation, for both correct and incorrect nucleotide incorporation and excision. The method of procedure centers on transient-state kinetic studies of the individual reaction steps in the polymerase cycle. Since RNA polymerase appears to be a multifunctional enzyme that catalyzes the endo- and exonucleolytic cleavage of the RNA transcript, it is essential to identify the rate-limiting steps to nucleotide incorporation and excision and to determine how sequence elements alter these steps. Specific steps to be investigated include, the kinetics of nucleotide incorporation with questions related to defining the rate limiting steps in correct nucleotide incorporation, the definition of the role of transcript position, sequence, and length on the kinetics of correct and incorrect nucleotide incorporation, the linkage of these kinetic steps to the manifestation of overall transcription fidelity, the rate-limiting steps in nucleotide excision by the polymerase, how the enzyme complex partitions between transcript cleavage and pyrophosphorolysis during nucleotide excision, and again the effect of transcript position, sequence, and length on the kinetics of nucleotide excision.