RUI: Abortive Initiation and Promoter Escape by E. coli RNA Polymerase

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RNA polymerase goes through three phases -- initiation, elongation, and termination -- as it performs a round of promoter-specified transcription. The ability of the enzyme to undergo the initiation-elongation transition, called promoter escape, is an important factor in determining the efficiency of initiation. During this transition, RNA polymerase forms highly stressed and short-lived intermediates called initial transcribing complexes that may or may not surmount the conformational changes that accompany the promoter escape transition. The inability to negotiate this transition results in the synthesis of abortive RNA and reduces the synthesis of full-length transcripts. The efficiency of escape at E. coli promoters is governed by sequence signals embedded in the promoter -- the consensus promoter elements entice the binding of RNA polymerase to form catalytically active complexes, while the initial transcribed sequence (ITS) modulates the promoter escape process of the bound enzyme. This research aims to elucidate the mechanism(s) by which the initial transcribed sequence affects promoter escape, by investigating the escape properties of a wide variety of mutant ITS promoters through quantitative in vitro transcription reactions, electrophoretic mobility shift assays, and footprinting analysis, using wild type and mutant RNA polymerases with important accessory factors. Intellectually, the merit of this project will lead to a thorough biochemical characterization of the initial transcribing complexes and shed light on the mechanistic steps involved in promoter escape. This activity will reap a broad impact in the training of undergraduate students, especially women, preparing them for research participation in a scientific career.