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Title: Multiscale Simulation of mRNA Synthesis by RNA Polymerase II
Author: Zhang, Rui
Advisor: Salahub, Dennis
Keywords: Chemistry--Physical
Issue Date: 23-Dec-2013
Abstract: RNA polyermase II, a crucial enzyme for gene expression in eukaryotes, synthesizes messenger RNAs with high selectivity. Despite its importance, its selection and catalysis mechanism is not well understood. We first investigated, by a stochastic simulation algorithm, the entire nucleotide addition cycle based on an event-driven model. The results suggest that the discrimination of unmatched nucleotide mainly lies in its thermodynamic instability in the addition site, and the selectivity for the 2’-OH is from the catalytic reaction. To understand the stability of different nucleotides in the addition site on the atomistic level, we performed MD and free energy perturbation simulations, and found that mutating a cognate GTP to a non-cognate UTP in the active site costs ~16.8kcal/mol while mutating a cognate GTP to a 2’-deoxyGTP costs ~2kcal/mol. Since two binding sites exist in the enzyme, we conducted molecular dynamics and umbrella sampling calculations to simulate the entry of a cognate GTP from the entry site to the addition site. The results demonstrate that two key motifs, the trigger loop and the bridge helix, play important roles in this process. Facilitated by these two motifs, the NTP entry is a spontaneous process with an energy decrease of ~6kcal/mol. Simulation of the catalytic reaction requires a quantum mechanical/molecular mechanical (QM/MM) method to adequately describe the reaction centre and enzyme surroundings. Therefore, we reviewed QM/MM methods in the literature and implemented our own version using CHARMM and deMon2k. With this QM/MM implementation, we performed geometry optimization and MD simulations on the system at the level of DFT/MM. To speed up the calculations and cover more possible reaction pathways, we employed a specifically parametrized semiempirical method – AM1/d-PhoT for the reaction pathway search. The results reveal a proton-transfer-facilitated mechanism. While the acceptor of the initial proton transfer may vary depending on the particular conformation of the active site, all possible routes converge to the same destination. Comparison between different models shows that the role of Mg2+ (A) is more structural than catalytic.
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