Conformational dynamics and regulatory interactions in a bacteriophage RNA polymerase complex

RNA定向RNA聚合酶（RdRp）在RNA病毒的生命周期中起着核心作用。这些酶的催化活性在两个水平上受到调节：首先，通过在催化时间尺度上发生的构象转变，使酶达到能够进行RNA聚合的状态；第二，在多蛋白RNA聚合酶复合体的背景下，通过与其他蛋白质的调节相互作用。拟议的研究，通过结合生物物理/结构研究和生化/功能分析，将阐明这两个水平的调控在噬菌体RdRp中的性质和重要性。该研究的多学科性质将为来自不同背景的学生在其专业准备的多个阶段提供培训。这些努力将符合纽约城市学院的总体使命，即提供一个多元文化、多民族的研究和培训环境，包括在科学领域代表性不足的群体，包括少数民族和妇女。为了加强来自代表性不足和经济弱势背景的学生攻读STEM学科学位的渠道，PI将改进和扩大他在纽约地区的几所高中的外展活动，其中包括位于该国最贫穷的国会选区之一的一所高中。对病毒RdRps的生化研究表明，在RNA聚合的延伸阶段，聚合酶活性位点的特定构象变化影响核苷酸加成的速率。尽管有几种病毒RdRps的晶体结构，但这些构象变化的性质仍然未知。提出的研究将通过结合溶液核磁共振在毫秒时间尺度上探测核苷酸加成动力学的测量来解决这个长期存在的问题。这些互补的数据来源，核磁共振动力学和快速动力学，在现有晶体结构和其他有待确定的框架内进行解释，将为RNA病毒中rdrp催化的核酸聚合提供清晰的结构和动态视图。比较两种亲缘关系较远的酶（来自噬菌体phi-12和脊髓灰质炎病毒）将有助于归纳出一类广泛的病毒RdRps。除了动力学之外，聚合酶复合体内的蛋白质-蛋白质相互作用对其组成RdRps的活性施加更高层次的控制。这些相互作用的改变导致RdRp功能的改变，在极端情况下导致非传染性病毒的改变。拟议的研究将在包含简单4蛋白聚合酶复合物的pi -12的背景下定义这些调节相互作用。所得结果可推广到更复杂的RNA病毒。本项目由化学学部生命过程化学项目共同资助。

RNA directed RNA polymerases (RdRp) play a central role in the life cycle of RNA viruses. The catalytic activity of these enzymes is regulated at two levels: first, through conformational transitions occurring on the catalytic timescale allowing the enzyme to attain a state capable of RNA polymerization; second, through regulatory interactions with other proteins in the context of a multi-protein RNA polymerase complex. The proposed research, through a combination of biophysical/structural studies and biochemical/functional assays, will elucidate the nature and importance of these two levels of regulation in a bacteriophage RdRp. The multidisciplinary nature of the research will provide training for students from various backgrounds at multiple stages of their professional preparation. These efforts will be in line with the overarching mission of the City College of New York to provide a multi-cultural, multi-ethnic research and training environment that is inclusive of groups underrepresented in the sciences including minorities and women. In order to strengthen the pipeline of students from underrepresented and economically disadvantaged backgrounds pursuing degrees in the STEM disciplines, the PI will refine and expand his outreach activities to several New York area high schools including one situated in one of the poorest congressional districts in the country. Biochemical studies on viral RdRps have indicated that specific conformational changes at the polymerase active site influence the rate of nucleotide addition during the elongation stage of RNA polymerization. The nature of these conformational changes remains unknown despite the availability of crystal structures of several viral RdRps. The proposed research will resolve this longstanding problem by combining solution NMR to probe dynamics on the millisecond timescale with measurements of nucleotide addition kinetics. These complimentary sources of data, NMR dynamics and fast kinetics, interpreted within the framework of the available crystal structures and others to be determined, will provide a clear structural and dynamic view of RdRp-catalyzed nucleic acid polymerization in RNA viruses. Comparison of two distantly related enzymes (from bacteriophage phi-12 and poliovirus) will allow generalization to a broad class of viral RdRps. In addition to dynamics, protein-protein interactions within polymerase complexes exert higher order control over the activity of their constituent RdRps. Modifications in these interactions lead to altered RdRp function, and in extreme cases to non-infectious viruses. The proposed studies will define these regulatory interactions in the context of phi-12 that contains a simple 4-protein polymerase complex. The results obtained will be generalizable to more complex RNA viruses.This project was co-funded by the Chemistry of Life Processes program in the Division of Chemistry.