VPW: Mechanistic Investigation of Transcription Initiation and Elongation by E. coli RNA Polymerase (Biochemistry)

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The proposed research will address two fundamental aspects of transcription by Escherichia coli RNA polymerase, namely, its tendency to undergo abortive initiation and its mechanism of translocation during transcription elongation. To clarify the first question, the P.I. will perform direct and quantitative comparisons, under a standardized reaction condition, of the extent and pattern of abortive initiation by a comprehensive set of E. coli promoters. The results of this determination may lead to sequence-specific correlations of abortive initiation with the initial transcribed sequence of each promoter. The second question is aimed at resolving two currently conflicting models of RNA polymerase translocation- one continuous and the other discontinuous-with the growth of the RNA chain. Experimentally, footprinting analysis with DNase I, exonuclease III, hydroxyl radical, and potassium permanganate will be performed on a contiguous set of specifically-halted elongating complexes. RNA polymerase is a central enzyme in the regulation of gene expression. Despite extensive investigation, many aspects of its catalytic mechanisms remain controversial and unclear. This work should forge a clearer picture of how translocation occurs. The project furthers VPW program objectives to provide opportunities for women to advance their careers in science or engineering through research, and to encourage other women to pursue careers in these areas through the investigator's enhanced visibility as a role model on the host campus. The proposed activities which contribute to the second objective include: teaching part of the introductory Biochemistry course to non-biochemistry science majors; mentoring and advising students; and directing undergraduate and graduate students working on research.