Conformational dynamics and regulatory interactions in a bacteriophage RNA polymerase complex

RNA导向的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被调节：第一，通过在催化时间尺度上发生的构象转变，使酶达到能够RNA聚合的状态；第二，通过在多蛋白质RNA聚合酶复合体的背景下，调节与其他蛋白质的相互作用。这项拟议的研究将通过生物物理/结构研究和生化/功能分析相结合的方式，阐明噬菌体RdRp中这两个水平调控的性质和重要性。这项研究的多学科性质将为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、多族裔的研究和培训环境，其中包括在科学界代表性不足的群体，包括少数群体和妇女。为了加强来自代表性不足和经济困难背景的学生攻读STEM学科学位的渠道，PI将完善并将他的外联活动扩大到纽约地区的几所高中，包括位于全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，聚合酶活性部位的特定构象变化会影响核苷酸加成的速度。尽管已有几种病毒RdRp的晶体结构，但这些构象变化的性质仍不清楚。拟议的研究将通过将溶液核磁共振探测毫秒时间尺度上的动力学与核苷酸加成动力学的测量相结合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解释，将为RdRp催化的RNA病毒核酸聚合提供一个清晰的结构和动态视图。对两种远亲的酶(来自噬菌体Phi-12和脊髓灰质炎病毒)的比较将允许将其推广到广泛类别的病毒RdRp。除了动力学，聚合酶复合体中的蛋白质-蛋白质相互作用对其组成的RdRps的活性起到更高的控制作用。这些相互作用中的修饰会导致RdRp功能的改变，在极端情况下会导致非传染性病毒。拟议的研究将在PHI-12的背景下定义这些调控相互作用，PHI-12包含一个简单的4-蛋白质聚合酶复合体。所获得的结果将推广到更复杂的RNA病毒。该项目是由化学部生命过程化学项目共同资助的。

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.