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The nucleotide addition cycle(NAC)of an elongating RNA polymerase(RNAP)along DNA usually consists of multiple kinetics steps,including from nucleotide binding or pre-insertion to the active-site insertion,catalytic reaction along with pyrophosphate(PPi)product releasing,and finally the RNAP translocation on the DNA.Correspondingly,there exists several kinetic checkpoints where non-cognate nucleotides can be selected against over a cognate one,according to the Watson-Crick(WC)base pairing between the incoming nucleotide and the template DNA.In this work,we conducted comprehensive free energetic calculations on various nucleotide insertions for viral T7 RNAP,employing all-atom molecular dynamics(MD)simulations and the umbrella sampling method.By comparing the insertion free energy profiles between the non-cognate nucleotide species(rGTP and dATP)and the cognate one(rATP),we obtained the selection free energetics from the nucleotide pre-insertion to the insertion checkpoints,and inferred the selection energetics down to catalytic checkpoint,according to the experimentally detected elongation error rates of T7 RNAP.Particularly,we find that the selection against the base-mismatch rGTP proceeds mainly through an off-path nucleotide-binding pathway,in which both the pre-insertion screening and the insertion inhibition play significant roles.In comparison,the selection against the sugar-deficiency dATP is found to go through an off-path pre-insertion screening as well as an on-path insertion inhibition.Interestingly,we noticed that two magnesium ions switched roles of leave and stay during the dATP on-path insertion simulation,which does not happen in other cases of the nucleotide insertion.Furthermore,we could infer from our calculations that substantial energetic is still required to catalytically select against rGTP over rATP,in order to achieve an elongation error rate~10-4; in contrast,no catalytic selection seems to be further needed against dATP over rATP,to allow an error rate of~10-2.