RUI: Mechanism of Promoter Escape by E. coli RNA Polymerase

在一轮转录中，RNA聚合酶要经过起始、延伸和终止三个阶段来合成全长转录物。启动子逃逸是调控高产基因表达的关键步骤，它构成了起始-延伸的转变。如果RNA聚合酶无法完成这一转变，就会导致重复合成流产RNA，这一过程被称为流产起始。流产起始和启动子逃逸的程度因启动子而异，并由每个启动子的初始转录序列（ITS）指导。虽然以前的研究已经导致了启动子逃逸过程的一般轮廓的描述，目前的研究将针对实现启动子逃逸的详细机制。这一目标将需要研究启动子逃逸的序列特异性，以了解ITS如何在单个启动子上指导独特的流产起始-启动子逃逸程序，并通过控制逃逸率和RNA聚合酶分配的程度来控制生产表达。启动子逃逸机制的证明还需要对处于逃逸边缘的初始转录复合物进行结构表征。本研究将采用的方法包括野生型和突变型启动子模板的体外定量转录分析，速率和平衡常数的动力学测定，以更好地了解转录复合物为生产性或非生产性构象的划分，以及表征转录复合物静态或动态结构的足迹技术。这两种方法的结合将揭示ITS序列如何指导RNA聚合酶在特定位置放弃启动子接触，并以确定的速率实现逃逸转变。从大肠杆菌RNA聚合酶的研究中获得的所有知识将有助于理解T7 RNA聚合酶和真核生物Pol II的启动子逃逸策略。更广泛的影响：作为一所女子学院的附属机构，首席研究员经常在研究和教学实验室与许多本科生一起工作。这项研究提供了许多独立的、精心设计的、尖端的项目，用于严谨和批判性的调查。研究项目的广泛影响导致对本科生，特别是不同种族的年轻妇女的培训，使她们为从事科学事业的研究做好准备。

In a round of transcription, RNA polymerase goes through three phases -- initiation, elongation, and termination -- to synthesize a full-length transcript. Promoter escape, which constitutes the initiation-elongation transition, is a key step in regulating productive gene expression. Failure of RNA polymerase to negotiate this transition results in the repetitive synthesis of abortive RNA, a process known as abortive initiation. The extent of abortive initiation vs. promoter escape differs from promoter to promoter and is guided by the initial transcribed sequence (ITS) at each promoter. While previous research has led to the description of a general outline of the promoter escape process, current research will be directed at achieving a detailed mechanism of promoter escape. This goal will require investigation into the sequence-specific nature of promoter escape to understand how an ITS directs a unique abortive initiation-promoter escape program at individual promoters, and controls productive expression by governing the rate of escape and the extent of RNA polymerase partitioning. The proof of a promoter escape mechanism will also require structural characterization of initial transcribing complexes poised on the brink of escape. The methods to be employed in this research will include quantitative in vitro transcription analysis of wild type and mutant promoter templates, kinetic determination of rate and equilibrium constants to understand better the partitioning of transcribing complexes into productive or unproductive conformation, and footprinting techniques to characterize the static or dynamic structure of transcribing complexes. The combined approaches will shed light on how the ITS sequence directs RNA polymerase to relinquish its promoter contacts at specific positions and bring about the escape transition at a defined rate. All of the knowledge gained from studying E. coli RNA polymerase will be of relevance to understanding the promoter escape strategies by T7 RNA polymerase and eukaryotic Pol II. Broader Impacts: Being affiliated with a women's college, the principal investigator routinely works with many undergraduate students in the research and teaching laboratories. The research provides many self-contained, well-designed, and cutting-edge projects for rigorous and critical investigation. A broad impact of the research projects leads to the training of undergraduates, especially young women and of diverse ethnicity, preparing them for research participation in a scientific career.