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

RNA聚合酶在进行一轮启动子特异性转录时，会经历起始、延伸和终止三个阶段。酶进行起始-延伸过渡的能力，称为启动子逃逸，是决定起始效率的一个重要因素。在这一转变过程中，RNA聚合酶形成被称为初始转录复合体的高度应激和短暂的中间产物，这些中间产物可能会或可能不会克服伴随启动子逃逸转变的构象变化。无法进行这种转换导致RNA合成失败，并减少全长转录本的合成。大肠杆菌启动子的逃逸效率由嵌入在启动子中的序列信号控制——共识启动子元件诱导RNA聚合酶结合形成催化活性复合物，而初始转录序列（ITS）调节结合酶的启动子逃逸过程。本研究旨在阐明初始转录序列影响启动子逃逸的机制，利用野生型和突变型RNA聚合酶和重要辅助因子，通过定量体外转录反应、电泳迁移量转移和足迹分析来研究多种突变ITS启动子的逃逸特性。在智力上，这个项目的优点将导致对初始转录复合物进行彻底的生化表征，并阐明启动子逃逸的机制步骤。这项活动将在培养本科生，特别是妇女方面产生广泛的影响，使她们为从事科学事业的研究做好准备。

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.