Single molecule analyses of RNA polymerase II elongation

描述(由申请人提供)： 这个项目的基本目标是建立一种利用单分子光学捕获技术通过染色质模板连续实时分析真核生物Rina聚合酶II转录的方法。光学捕获技术允许直接观察单个RNA聚合酶分子的运动，从而产生对转录延伸率、暂停模式和暂停持续时间的高分辨率测量。这种方法使我们能够专注于在传统的群体分析中平均出来的动态相互作用，提供了以前无法获得的关于RNA聚合酶易位的动力学方面的信息，以及特定转录因子调节这一活性的机制。 这项建议涉及系统地开发专门为研究RNA聚合酶延长而定制的单分子技术。这些分析的基础是一种新的策略，可以在不干扰自然延伸活性的情况下固定单个RNA聚合酶分子。最初，该方法将使用一个易于操作的大肠杆菌转录系统进行优化，该系统将作为建立果蝇RNA聚合酶II延伸的单分子检测的模型。这些检测将首先被用于确定RNA聚合酶II在裸露DNA模板上转录的潜在特征，然后被用于详细研究RNA聚合酶II通过核小体屏障的机制。

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.