Structural and Dynamic NMR Studies of RNA Polymerase

智力价值：本提案的目的是使用溶液NMR光谱学研究RNAP在不同的转录阶段。DNA依赖性RNA聚合酶（RNAP）是所有细胞生物中基因表达和调节的主要酶。RNAP是一种显著的蛋白质机制，能够（i）特异性结合到DNA沿着的启动子位点，（ii）熔化双链DNA（dsDNA）以形成转录“泡”，和（iii）使用核苷三磷酸（NTP）底物合成与DNA模板链互补的RNA链。从细菌到人类，细菌RNAP的基本核心组分（亚基组成α 2、β、β ′和ω）在进化上是保守的。序列保守指向结构和功能同源性，使得更简单的细菌RNAP成为理解所有细胞RNAP工作的基本原理的极好模型系统。RNAP的结构，生物化学和生物物理特性的最新进展突出了这种酶作为一个复杂的，多功能的蛋白质机器，通过使用一个复杂的平衡结构和动态变化的功能。该项目的目标是通过研究全球和微妙的结构变化以及功能运动的幅度和时间尺度，获得支持RNAP功能的复杂机制的综合结构和动态信息。为了实现这一目标，该项目将（1）开发策略和方法，以克服RNAP的大尺寸和复杂性，（2）通过应用综合NMR方法获取RNAP的结构和动态数据，该方法涉及针对以下方面量身定制的实验：（i）获得长程结构信息，（ii）检测瞬时填充构象状态，以及（iii）确定快速和慢速时间尺度运动并评估它们的重要性。 更广泛的影响：实现这些目标将对各个领域产生巨大影响。首先，它将提供RNAP在溶液中作用过程中的位点特异性结构和动态信息，从而为这一重要酶的功能机制提供前所未有的见解。其次，它将提供一个模型研究如何核磁共振可以应用于获得集成的结构和动力学信息的超分子生物系统。第三，通过补充X射线晶体学提供的静态结构，它将确立NMR作为动态表征大型复杂蛋白质机器的有力工具。除了解决基本的生物学问题，该项目将用于培养学生在结构生物学，生物物理学和分子生物学，这些领域正在迅速成为21世纪世纪科学整合。博士后，研究生和本科生将有机会参与一个多学科项目，该项目旨在开发突破性的方法，以通过高分辨率NMR光谱表征超分子蛋白质复合物。这将使项目的参与者能够从多学科和互动的角度处理问题，从而亲身体验将最先进的方法应用于重要生物问题的实用性。两名研究生将在培训赠款和助教奖学金的支持下就该项目撰写论文。他们将参与开发新的标记协议，并将先进的NMR方法应用于RNAP的动态和结构表征。结合结构，动力学，热力学和动力学方法来研究复杂蛋白质系统的范例将被纳入PI目前设计的新课程，以强调使用跨学科和定量方法来回答科学和生物医学重要性问题的价值。本课程面向罗格斯大学分子生物科学，化学化学生物学，生物医学工程和BIOMAPS课程的研究生和高级本科生组成的大型，多样化的观众。

Intellectual merit: The objective of this proposal is to use solution NMR spectroscopy to study RNAP in various transcriptional stages. The DNA-dependent RNA polymerase (RNAP) is the principal enzyme of gene expression and regulation in all cellular organisms. RNAP is a remarkable protein machinery capable of (i) specifically binding to promoter sites along the DNA, (ii) melting the double-stranded DNA (dsDNA) to form the transcription "bubble", and (iii) synthesizing the RNA chain complementary to the DNA template strand using nucleoside triphosphate (NTP) substrates. The essential core component of the bacterial RNAP (subunit composition alpha 2, beta, beta prime and omega) has been evolutionarily conserved from bacteria to humans. Sequence conservation points to structural and functional homologies, rendering the simpler bacterial RNAPs excellent model systems for understanding the basic principles at work for all cellular RNAPs. Recent progress in the structural, biochemical and biophysical characterization of RNAP has highlighted this enzyme as a complex, multifunctional protein machinery that functions by using an intricate balance of structural and dynamic changes. The goal of this project is to obtain integrated structural and dynamic information of the intricate mechanisms that underpin RNAP functionality by studying global and subtle structural changes as well as the amplitude and the time scale of functional motions. Towards this goal, the project will (1) develop strategies and methodologies to overcome the large size and complexity of RNAP, and (2) acquire structural and dynamic data on RNAP by applying an integrated NMR approach involving experiments tailored for (i) obtaining long-range structural information, (ii) detecting transiently populated conformational states, and (iii) determining both fast and slow time-scale motions and assessing their significance. Broader impact: Accomplishment of these objectives will have a tremendous impact on various fields. First, it will provide site-specific structural and dynamic information of RNAP during its action in solution, thereby offering unprecedented insight into the functional mechanisms of this important enzyme. Second, it will provide a model study about how NMR can be applied to obtain integrated structural and dynamic information on supramolecular biological systems. Third, it will establish NMR as a powerful tool for the dynamic characterization of large, intricate protein machineries by complementing static structures offered by X-ray crystallography. In addition to addressing fundamental biological questions, this project will be used to train students in structural biology, biophysics, and molecular biology, areas that are rapidly becoming integrated in 21st century science. Postdocs, graduate and undergraduate students will have the opportunity to be involved in a multi-disciplinary project that aims at the development of groundbreaking methodologies to enable characterization of supramolecular protein complexes by high resolution NMR spectroscopy. This will enable participants in the project to approach problems from a multidisciplinary and interactive perspective, thus experiencing first hand the utility of applying state-of-the-art methodologies to important biological problems. Two graduate students, supported by training grants and teaching assistantships, will do their theses on this project. They will be involved in the development of new labeling protocols and in the application of advanced NMR methodologies towards the dynamic and structural characterization of RNAP. The paradigm of combining structural, dynamic, thermodynamic and kinetic approaches to study complex protein systems will be included in a new course, currently designed by the PI, to exemplify the value of using an interdisciplinary and quantitative approach to answer questions of scientific and biomedical importance. The course is intended for a large, diverse audience consisting of graduate and advanced undergraduate students in the programs of Molecular Biosciences, Chemistry & Chemical Biology, Biomedical Engineering and BIOMAPS at Rutgers University.