UC Irvine

Here, we studied the complete process of a viral T7 RNA polymerase (RNAP) translocation on DNA during transcription elongation by implementing extensive all-atom molecular dynamics (MD) simulations to construct a Markov state model (MSM). Our studies show that translocation proceeds in a Brownian motion, and the RNAP thermally transits among multiple metastable states. We observed non-synchronized backbone movements of the nucleic acid (NA) chains with the RNA translocation accomplished first, while the template DNA lagged. Notably, both the O-helix and Y-helix on the fingers domain play key roles in facilitating NA translocation through the helix opening. The helix opening allows a key residue Tyr639 to become inserted into the active site, which pushes the RNA-DNA hybrid forward. Another key residue, Phe644, coordinates the downstream template DNA motions by stacking and un-stacking with a transition nucleotide (TN) and its adjacent nucleotide. Moreover, the O-helix opening at pre-translocation (pre-trans) likely resists backtracking. To test this hypothesis, we computationally designed mutants of T7 RNAP by replacing the amino acids on the O-helix with counterpart residues from a mitochondrial RNAP that is capable of backtracking. The current experimental results support the hypothesis.