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

智力优势：RNA聚合酶II（也称为pol II）是催化DNA转录为信使RNA（以及大多数其他形式的RNA）的基本酶，是基因表达的重要第一步。该项目将阐明pol II的预起始复合物（Pre-Initiation Complex， PIC）组装的关键结构和功能方面，以及转录起始机制，即启动新基因转录的机械步骤。复杂的基于溶液的单分子荧光技术，结合微流体，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）组装以及它如何执行其催化功能。由于单分子方法消除了集合和时间平均，关于组装顺序和动力学的信息将被解开。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技术的出现，为研究定义生命的过程提供了巨大的机会。其中一个过程就是转录，这是“生物学中心法则”的基础。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。要开发的工具和方法，以及所采用的一般方法，如果分发给接受和创新的年轻科学家的广泛受众，将产生最大的影响。通过教育和推广项目，该项目将使年轻的观众（高中生和大学生）以及更成熟的科学家接触到新的研究工具和方法，这些工具和方法可以用于研究许多其他生物过程和分子机器。

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.