Determining selection free energetics from nucleotide pre-insertion to insertion in viral T7 RNA polymerase transcription fidelity control.

Abstract:

: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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Long C,E C,Da LT,Yu J

doi

10.1093/nar/gkz213

subject

Has Abstract

pub_date

2019-05-21 00:00:00

pages

4721-4735

issue

9

eissn

0305-1048

issn

1362-4962

pii

5420531

journal_volume

47

pub_type

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