Single molecule analyses of RNA polymerase II elongation

RNA 聚合酶 II 延伸的单分子分析

• 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
• 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

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学诱捕技术通过染色质模板对真核RINA聚合酶II转录进行连续实时分析的方法。光学诱捕技术允许直接观察单个RNA聚合酶分子运动，从而对转录伸长率，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在常规人群分析过程中平均的动态相互作用，从而提供了先前无法访问的信息，即RNA聚合酶易位的动力学方面以及调节该活性的特定转录因子的机制。 该建议涉及专门针对RNA聚合酶伸长研究的单分子技术的系统开发。这些分析的基础是单个RNA聚合酶分子固定的新策略，而不会干扰天然的伸长活性。最初，该方法将使用易于操纵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延长单分子测定的模型。这些测定将首先用于确定裸体DNA模板上RNA聚合酶II转录的潜在特征，然后被利用，以详细研究RNA聚合酶II通过核小体屏障导航的机制。