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

RUI：大肠杆菌 RNA 聚合酶启动子逃逸机制

• 批准号: 0841452
• 负责人: Lilian Hsu
• 金额: $ 46.5万
• 依托单位: Mount Holyoke College
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0841452&HistoricalAwards=false
• 关键词: RUI; Mechanism; Promoter; Escape; E.

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.

在一轮转录中，RNA聚合酶经历三个阶段——起始、延伸和终止——以合成全长转录物。启动子逃逸构成起始-延伸转变，是调节生产性基因表达的关键步骤。 RNA 聚合酶无法协调这一转变，导致 RNA 的重复合成失败，这一过程称为失败起始。失败起始与启动子逃逸的程度因启动子而异，并且由每个启动子处的初始转录序列（ITS）引导。虽然之前的研究已经描述了启动子逃逸过程的总体轮廓，但当前的研究将致力于实现启动子逃逸的详细机制。这一目标需要研究启动子逃逸的序列特异性，以了解 ITS 如何在各个启动子上引导独特的失败启动子逃逸程序，并通过控制逃逸速率和 RNA 聚合酶分配的程度来控制生产性表达。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。本研究中使用的方法将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划分为生产性或非生产性构象，以及足迹技术以表征转录复合物的静态或动态结构。组合方法将揭示 ITS 序列如何引导 RNA 聚合酶放弃其在特定位置的启动子接触并以定义的速率实现逃逸转变。 通过研究大肠杆菌 RNA 聚合酶获得的所有知识都将有助于理解 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合作。该研究提供了许多独立的、精心设计的、前沿的项目，用于严格和批判性的调查。研究项目的广泛影响导致对本科生，特别是年轻女性和不同种族的本科生的培训，为他们参与科学职业的研究做好准备。