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.
Highlights
Transcription is the first step of gene expression in living organisms
In the end, based on the nucleotide selection free energetics derived from the MD simulation results, we show how we calculated the elongation error rates according to the chemical master equation (CME) approach, and inferred the selection free energetics during catalysis
We present the potential of mean forces (PMFs) for the non-cognate nucleotides rGTP and dATP, along with the key structures and movies, following either the on-path or off-path insertion process
Summary
Transcription is the first step of gene expression in living organisms. It is directed by RNA polymerases (RNAPs) that transcribe genetic information from DNA to RNA, based on the Watson–Crick (WC) base pairing between the synthesizing RNA and the template DNA strand. The transcription fidelity is controlled by nucleotide selections upon binding and incorporation along with proofreading during the RNAP transcription elongation [1,2,3,4,5,6,7,8]. The acceleration of polymerization along with nucleotide selection and proofreading conducted via an RNAP can quench the transcription error rate down to ∼10−4–10−7 [9]. The nucleotide selection in T7 RNAP plays a primary role in the viral transcription fidelity control, and it is of high interest to reveal how the nucleotide selection proceeds in structural and energetic detail
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