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重要作用。这些酶的催化活性在两个水平上受到调节：第一，通过在催化时间尺度上发生的构象转变，允许酶达到能够进行RNA聚合的状态;第二，通过在多蛋白RNA聚合酶复合物的背景下与其他蛋白质的调节相互作用。拟议的研究，通过生物物理/结构研究和生物化学/功能测定相结合，将阐明这两个层次的监管在噬菌体RdRp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，即提供一个多文化、多种族的研究和培训环境，包括少数民族和妇女等在科学领域代表性不足的群体。为了加强来自代表性不足和经济弱势背景的学生在STEM学科攻读学位的管道，PI将完善和扩大他的外联活动，以几个纽约地区的高中，包括一个位于该国最贫穷的国会选区之一。对病毒RdRps的生物化学研究表明，在聚合酶活性位点的特定构象变化影响RNA聚合的延伸阶段期间核苷酸添加的速率。这些构象变化的性质仍然未知，尽管几种病毒RdRps的晶体结构的可用性。拟议的研究将解决这个长期存在的问题相结合的解决方案核磁共振探测动力学的毫秒时间尺度与测量核苷酸添加动力学。这些免费的数据，NMR动力学和快速动力学的来源，在现有的晶体结构和其他待确定的框架内解释，将提供一个清晰的结构和动态的RdRp催化的RNA病毒中的核酸聚合。比较两种远亲的酶（来自噬菌体phi-12和脊髓灰质炎病毒）将允许推广到广泛的一类病毒RdRps。除了动力学，聚合酶复合物内的蛋白质-蛋白质相互作用对其组成RdRps的活性施加更高阶的控制。这些相互作用中的修饰导致RdRp功能改变，并且在极端情况下导致非感染性病毒。拟议的研究将在含有简单的4-蛋白聚合酶复合物的phi-12的背景下定义这些调节相互作用。该研究结果将被推广到更复杂的RNA病毒。该项目由化学系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.