Abstract:

RNA polymerases are the core of transcription for all organisms. While viruses and humans differ greatly, in the design of polymerase-targeting antivirals, off-target toxicity of our polymerases has been a major concern. The single-subunit human mitochondrial DNA directed RNA polymerase (POLRMT) is of particular concern since it is virus-like relative to nuclear polymerases.  This dissertation utilizes all-atom molecular dynamics (MD) as a computational microscope to contextualize nucleotide fidelity between on-target SARS-CoV-2 RNA directed RNA polymerase (CoV-2 RdRp) and off-target POLRMT. It then delves further into the POLRMT translocation dynamics along DNA to further distinguish POLRMT from viral RNA polymerases. Additionally, to provide biophysical insight, these systems are leveraged to investigate geometric collective variables (CV) for enhanced MD simulation and abstract CV for simulation analysis and kinetic prediction. First, using umbrella sampling MD and a CV known a priori, free energetics of nucleotide fidelity of CoV-2 RdRp nucleotide insertion and pre-insertional conformational trapping was explored for non-cognates GTP and dATP and compared to previous work on cognates. Second, POLRMT equilibrium dynamics of cognate ATP and non-cognates dATP, GTP, and antiviral Remdesivir-TP pre-insertion and pre-catalysis are explored with two observed non-cognate dynamical changes: pre-insertion rejection and pre-catalytic instability. Third, translocation dynamics and kinetics of POLRMT are explored with extensive equilibrium MD, and abstract CV method time-lagged independent component analysis (tICA) was compared to the neural network enhanced variational approach to Markov processes (VAMP/VAMPnets). Here two potential mechanisms are examined: a non-coupled fast path where conformational opening of POLRMT is unsuccessful during translocation; and translocation-conformational opening coupled slow path. Overall, by applying multiple MD simulation and analysis techniques, this thesis provides comparison of a viral RNA polymerase example (CoV-2) and virus-like human mitochondrial (POLRMT) RNA polymerase nucleotide fidelity providing insight for antiviral design, comparison of collective variables for future data analysis, and for POLRMT, a potential mechanism for translocation.