An intermediate state of T7 RNA polymerase provides another pathway of nucleotide selection

Abstract

Phage T7 RNA polymerase is a single-subunit transcription enzyme, transcribing template DNA to RNA. Nucleoside triphosphate (NTP) selection and translocation are two critical steps of the transcription elongation. Here, using all-atom molecular dynamics simulations, we found that between pre-and post-translocation states of T7 RNA polymerase an intermediate state exists, where the O helix C-terminal residue tyrosine 639, which plays important roles in translocation, locates between its pre-and post-translocation positions and the side chain of the next template DNA nucleotide has moved into the active site. NTP selection in this intermediate state was studied, revealing that the selection in the intermediate state can be achieved relying on the effect of Watson-Crick interaction between NTP and template DNA nucleotide, effect of stability of the components near the active site such as the nascent DNA-RNA hybrid and role of tyrosine 639. This indicates that another NTP-selection pathway can also exist besides the main pathway where NTP selection begins at the post-translocation state upon the entry of NTP.

Preorganized Internal Electric Field Promotes a Double-Displacement Mechanism for the Adenine Excision Reaction by Adenine DNA Glycosylase

“…The PMF was constructed with the weighted histogram analysis method (WHAM), , using the CV data from the production simulations. Good convergence has been achieved since the final PMF has nearly no change when it is recalculated with the first 80% of the data, indicating that the production simulation time is long enough to ensure sufficient overlap between two adjacent windows and further elongation is not needed. , …”

“…Good convergence has been achieved since the final PMF has nearly no change when it is recalculated with the first 80% of the data, indicating that the production simulation time is long enough to ensure sufficient overlap between two adjacent windows and further elongation is not needed. 50,51 Quantum Mechanical/Molecular Mechanical (QM/ MM) Calculations. The last snapshot of the restraint simulation of system I was used as the input of QM/MM calculations to investigate the first displacement reaction and the water-attack pathway.…”

Preorganized Internal Electric Field Promotes a Double-displacement Mechanism for the Adenine Excision Reaction by Adenine DNA Glycosylase

“…Good convergence has been achieved since the final PMF has nearly no change when it is recalculated with the first 80% of the data, indicating that the production simulation time is long enough to ensure sufficient overlap between two adjacent windows and further elongation is not needed. [49][50] Quantum Mechanical/Molecular Mechanical (QM/MM) Calculation. The last snapshot of the restraint simulation of system I was used as the input of QM/MM calculations to investigate the first displacement reaction and the water-attack pathway, and a representative snapshot extracted from the MD simulation trajectory of system III was used as the input structure to investigate the second displacement reaction.…”