Collaborative Research: Elucidating Pre-initiation Complex Assembly and Transcription Initiation by Pol-II Using Advanced Single Molecule and Microfluidic Methods

合作研究：利用先进的单分子和微流体方法阐明 Pol-II 的预启动复合物组装和转录启动

• 批准号: 1244098
• 负责人: Shimon Weiss
• 金额: $ 90万
• 依托单位: University of California-Los Angeles
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-15 至 2019-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1244098&HistoricalAwards=false
• 关键词: Collaborative; Research; Elucidating; Pre; initiation

Intellectual Merit: RNA polymerase II (also called pol II) is an essential enzyme that catalyzes the transcription of DNA into messenger RNA (and most other forms of RNA), an important first step in gene expression. This project will elucidate key structural and functional aspects of the assembly of the Pre-Initiation Complex (PIC) of pol II and the mechanism of transcription initiation, i.e. the mechanistic steps involved in starting the transcription of a new gene. Sophisticated solution-based single-molecule fluorescence techniques, combined with microfluidics, will be applied to individual complexes in order to study how pol II assembles at a promoter (gene start site) and how it performs its catalytic function. Because single-molecule approaches remove ensemble and temporal averaging, information about the order and the kinetics of assembly will be unraveled. Broader Impacts: The "nano-biotechnology revolution" and the emergence of single molecule biophysics and super-resolution imaging have opened up tremendous opportunities in the investigation of processes that define life. One such process, fundamental to the "Central Dogma of Biology," is transcription. The approach for studying this basic process to be taken in this research could break conceptual and practical barriers that have hindered progress, and offers potential to catalyze discovery in many new ways. The tools and methods to be developed, as well as the general approaches pursued, will have greatest impact if distributed to a broad audience of receptive and innovative young scientists. Through educational and outreach programs, the project will expose a young audience (high school and college students), as well as more mature scientists, to novel research tools and methodologies that could be used to study many other biological processes and molecular machines.

智力优点：RNA 聚合酶 II（也称为 pol II）是一种重要酶，可催化 DNA 转录为信使 RNA（以及大多数其他形式的 RNA），这是基因表达的重要第一步。该项目将阐明 pol II 预起始复合物 (PIC) 组装的关键结构和功能方面以及转录起始机制，即启动新基因转录所涉及的机械步骤。复杂的基于溶液的单分子荧光技术与微流体技术相结合，将应用于单个复合物，以研究 pol II 如何在启动子（基因起始位点）处组装以及它如何发挥其催化功能。由于单分子方法消除了整体和时间平均，因此有关组装顺序和动力学的信息将被阐明。更广泛的影响：“纳米生物技术革命”以及单分子生物物理学和超分辨率成像的出现为研究定义生命的过程提供了巨大的机会。 “生物学中心法则”的基础过程之一就是转录。本研究中采用的研究这一基本过程的方法可以打破阻碍进展的概念和实践障碍，并提供以许多新方式催化发现的潜力。待开发的工具和方法，以及所追求的一般方法，如果分发给广泛的接受和创新的年轻科学家，将产生最大的影响。通过教育和推广计划，该项目将使年轻观众（高中生和大学生）以及更成熟的科学家接触到新颖的研究工具和方法，这些工具和方法可用于研究许多其他生物过程和分子机器。