Single molecule analyses of RNA polymerase II elongation

RNA 聚合酶 II 延伸的单分子分析

基本信息

批准号：
6640558
负责人：
Karen L Adelman
金额：
$ 4.99万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-08-01 至 2005-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6640558
关键词：
DNA directed RNA polymerase chromatin enzyme activity eukaryote genetic transcription molecular dynamics nanotechnology optics technology /technique development transcription factor

项目摘要

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.

描述（由申请人提供）：该项目的基本目标是建立一种使用单分子光捕获技术通过染色质模板连续、实时分析真核 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，从而对转录延伸率、暂停模式和暂停持续时间进行高分辨率测量。这种方法使我们能够专注于传统群体分析期间平均的动态相互作用，提供以前无法获得的有关 RNA 聚合酶易位动力学方面以及特定转录因子调节这种活动的机制的信息。该提案涉及专门为 RNA 聚合酶延伸研究而定制的单分子技术的系统开发。这些分析的基础是一种固定单个 RNA 聚合酶分子而不干扰天然延伸活性的新策略。最初，该方法将使用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 伸长的单分子测定的模型。这些检测方法将首先用于确定 RNA 聚合酶 II 在裸 DNA 模板上转录的基本特征，然后再用于详细研究 RNA 聚合酶 II 穿过核小体屏障的机制。