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.
