Single molecule analyses of RNA polymerase II elongation

描述（由申请人提供）： 该项目的基本目标是建立一种方法，用于连续，实时分析真核细胞RINA聚合酶II转录通过染色质模板使用单分子光捕获技术。光学捕获技术允许直接观察单个RNA聚合酶分子的运动，产生高分辨率的转录延伸率，暂停模式和暂停持续时间的测量。这种方法使我们能够专注于动态的相互作用，平均在传统的人口分析，提供以前无法访问的信息有关的动力学方面的易位由RNA聚合酶和机制，特定的转录因子调节这种活动。 这项建议涉及系统的发展单分子技术专门为RNA聚合酶延伸的研究。这些分析的基础是一种新的策略，用于固定单个RNA聚合酶分子而不干扰天然延伸活性。最初，该方法将使用易于操作的E. coli转录系统，为建立果蝇RNA聚合酶II延伸的单分子检测方法奠定了基础。这些试验将首先用于确定RNA聚合酶II转录的基本特征，然后利用裸露的DNA模板进行详细的研究，RNA聚合酶II通过核小体屏障导航的机制。

DESCRIPTION (provided by applicant): The fundamental goal of this project is to establish a method for continuous, real-time analysis of eukaryotic RINA polymerase II transcription through a chromatin template using single-molecule optical trapping techniques. Optical trapping technology permits direct observation of movement by individual RNA polymerase molecules, yielding high-resolution measurement of transcription elongation rates, pausing patterns, and pause durations. This approach allows us to focus on the dynamic interactions that are averaged out during conventional population analyses, providing previously inaccessible information concerning the kinetic aspects of translocation by RNA polymerase and the mechanism by which specific transcription factors regulate this activity. This proposal involves the systematic development of single-molecule techniques specifically tailored for the study of RNA polymerase elongation. Foundational for these analyses is a novel strategy for immobilization of single RNA polymerase molecules without disturbing native elongation activity. Initially, the method will be optimized using an easily manipulable E. coli transcription system, which will serve as a model for establishing single molecule assays of elongation by Drosophila RNA polymerase II. These assays will first be employed to determine the underlying characteristics of RNA polymerase II transcription on naked DNA templates before being exploited for a detailed investigation of the mechanism by which RNA polymerase II navigates through a nucleosomal barrier.