Cargando…
Determining selection free energetics from nucleotide pre-insertion to insertion in viral T7 RNA polymerase transcription fidelity control
An elongation cycle of a transcribing RNA polymerase (RNAP) usually consists of multiple kinetics steps, so there exist multiple kinetic checkpoints where non-cognate nucleotides can be selected against. We conducted comprehensive free energy calculations on various nucleotide insertions for viral T...
Autores principales: | , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2019
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511863/ https://www.ncbi.nlm.nih.gov/pubmed/30916310 http://dx.doi.org/10.1093/nar/gkz213 |
Sumario: | An elongation cycle of a transcribing RNA polymerase (RNAP) usually consists of multiple kinetics steps, so there exist multiple kinetic checkpoints where non-cognate nucleotides can be selected against. We conducted comprehensive free energy calculations on various nucleotide insertions for viral T7 RNAP employing all-atom molecular dynamics simulations. By comparing insertion free energy profiles between the non-cognate nucleotide species (rGTP and dATP) and a cognate one (rATP), we obtained selection free energetics from the nucleotide pre-insertion to the insertion checkpoints, and further inferred the selection energetics down to the catalytic stage. We find that the insertion of base mismatch rGTP proceeds mainly through an off-path along which both pre-insertion screening and insertion inhibition play significant roles. In comparison, the selection against dATP is found to go through an off-path pre-insertion screening along with an on-path insertion inhibition. Interestingly, we notice that two magnesium ions switch roles of leave and stay during the dATP on-path insertion. Finally, we infer that substantial selection energetic is still required to catalytically inhibit the mismatched rGTP to achieve an elongation error rate ∼10(−4) or lower; while no catalytic selection seems to be further needed against dATP to obtain an error rate ∼10(−2). |
---|