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

这项研究将探讨大肠杆菌RNA聚合酶转录的两个基本方面，即它发生流产起始的倾向和它在转录延伸过程中的易位机制。为了澄清第一个问题，P.I.将在标准化的反应条件下，对一组全面的大肠杆菌启动子引发流产的程度和模式进行直接和定量的比较。这一测定的结果可能导致流产起始与每个启动子的初始转录序列的序列特异性关联。第二个问题旨在解决目前两种相互冲突的RNA聚合酶易位模型--一种是连续的，另一种是不连续的--随着RNA链的增长。实验上，DNA酶I、核酸外切酶III、羟基自由基和高锰酸钾的足迹分析将在一组连续的特定停止的拉长复合体上进行。RNA聚合酶是基因表达调控的中心酶。尽管进行了广泛的研究，但其催化机理的许多方面仍然存在争议和不清楚。这项工作应该会对易位是如何发生的形成一个更清晰的图景。该项目进一步推动了VPW项目的目标，为女性提供通过研究提升她们在科学或工程领域的职业生涯的机会，并通过调查员作为东道主校园榜样的提高知名度，鼓励其他女性在这些领域追求职业生涯。促进第二个目标的拟议活动包括：向非生物化学科学专业的学生教授生物化学入门课程的一部分；指导和指导学生；指导从事研究工作的本科生和研究生。

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.