Substantial Nucleotide Selection Prior to Full Insertion of the Nucleotide in T7 RNA Polymerase Elongation

Abstract

Nucleotide selection is essential for fidelity control in gene replication and transcription. Previous work on T7 RNA polymerase suggests that a small post-translocation free energy stabilizes Tyr639 in the active site to facilitate the nucleotide selection. Currently, we implement atomistic molecular dynamics simulations and demonstrate that Tyr639 is indeed stabilized by ∼ 2kBT favorable association with the end base pair of the DNA-RNA hybrid to serve for nucleotide ‘gating’ from pre-insertion to insertion. Upon the nucleotide pre-insertion, a relative binding free energy above thermal fluctuation level arises against the miscoded nucleotide, primarily due to electrostatic screening from charged residues that assist the nucleotide binding. Interestingly, the pre-insertion of a right nucleotide marginally destabilizes Tyr639, while a wrong nucleotide pre-insertion substantially stabilizes Tyr639 to hinder further nucleotide insertion. The activation barrier of the miscoded nucleotide insertion under an O-helix rotation rises significantly above that of the right nucleotide. The selection against deoxyribonucleotide can be even strong and arises essentially due to steric detection from Tyr639. Our studies suggest that substantial nucleotide selection in T7 RNAP happens upon the nucleotide pre-insertion and during the insertion, prior to full insertion of the nucleotide for base pairing and chemical addition.