Determining selection free energetics from nucleotide pre-insertion to insertion in viral T7 RNA polymerase transcription fidelity control.
Nucleic Acids Res
; 47(9): 4721-4735, 2019 05 21.
Article
in En
| MEDLINE
| ID: mdl-30916310
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 â¼10-4 or lower; while no catalytic selection seems to be further needed against dATP to obtain an error rate â¼10-2.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Transcription, Genetic
/
Viral Proteins
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Virus Replication
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DNA-Directed RNA Polymerases
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Bacteriophage T7
Language:
En
Journal:
Nucleic Acids Res
Year:
2019
Type:
Article
Affiliation country:
China