Dynamic regulation of initiation and pausing of transcription by human RNA polymerase II

人类基因组包含大约30亿个碱基对的DNA，这些DNA为细胞对发育或环境提示做出反应提供必要的指令。反应的关键一步是将DNA中的信息转录成提供另一层信息的中间RNA分子。将DNA转录成RNA是一个高度受监管的过程，但它到底是如何控制的仍然是一个谜。这个项目的目标是通过汇集一支经验丰富的调查团队来破译这个问题，采取多方面的方法以新的方式催化发现。该研究计划将整合科罗拉多大学博尔德分校和加州大学洛杉矶分校的教育和推广项目，让高中生、大学生和博士后学者接触到高影响力的合作研究。将实施的工具、策略和方法将适用于其他生物过程和分子机制的研究。本项目所涉及的问题对于理解真核转录的分子调控具有重要意义。将使用最初通过分析细菌RNA聚合酶而开发的尖端方法详细研究人类RNA聚合酶II的转录机制。基因表达的高度调控阶段，包括转录启动和暂停，将是重点。这个合作项目将把基于溶液的单分子荧光技术与详细的功能分析结合起来，这是通过对整个4.5丙二醛人类转录装置进行生化重组而实现的。两个关键问题将被解决：RNA聚合酶II转录是如何由特定的蛋白质因子动态调节的？RNA聚合酶II回溯在调节启动和暂停中起什么作用？这项工作的一个重要方面将是用体外和基于细胞的方法测试和验证机械发现。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The human genome contains about 3 billion base pairs of DNA that provide essential instructions for cells to respond to developmental or environmental cues. A key step in the response is transcribing the information in DNA to intermediary RNA molecules that supply another layer of information. Transcribing DNA to RNA is a highly regulated process, but exactly how it is controlled remains an enigma. The goal of this project is to decipher this problem by bringing together an experienced team of investigators to take a multi-faceted approach to catalyze discovery in new ways. The research plan will integrate educational and outreach programs at University of Colorado-Boulder and University of California-Los Angeles to expose high school, college students, and postdoctoral scholars to high-impact collaborative research. The tools, strategies, and methodologies to be implemented will be applicable to the study of other biological processes and molecular machines.The questions addressed in this project are fundamentally important for understanding the molecular control of eukaryotic transcription. The human RNA polymerase II transcription machinery will be studied in detail using cutting-edge methods developed initially through analysis of bacterial RNA polymerase. Highly regulated stages in gene expression, including transcription initiation and pausing, will be the focus. This collaborative project will merge solution-based single-molecule fluorescence techniques with detailed functional assays enabled by biochemical reconstitution of the entire 4.5 MDa human transcription apparatus. Two key questions will be addressed: How is RNA polymerase II transcription dynamically regulated by specific protein factors? What role does RNA polymerase II backtracking play in regulating initiation and pausing? An important aspect of the work will be to test and validate mechanistic findings with in vitro and cell-based approaches.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.