A Critical Residue Selectively Recruits Nucleotides for T7 RNA Polymerase Transcription Fidelity Control

Abstract

Nucleotide selection is essential for fidelity control in gene replication and transcription. Recent work on T7 RNA polymerase suggested that a small posttranslocation free energy bias stabilizes Tyr639 in the active site to aid nucleotide selection. However, it was not clear exactly how Tyr639 assists the selection. Here we report a molecular-dynamics simulation study revealing atomistic detail of this critical selectivity. The study shows first that Tyr639 blocks the active site at posttranslocation by marginally stacking to the end basepair of the DNA-RNA hybrid. The study then demonstrates that at the nucleotide preinsertion state, a cognate RNA nucleotide does not affect the local Tyr639 stabilization, whereas a noncognate nucleotide substantially stabilizes Tyr639 so that Tyr639 keeps blocking the active site. As a result, further nucleotide insertion into the active site, which requires moving Tyr639 out of the site, would be hindered for the noncognate nucleotide, but not for the cognate nucleotide. In particular, we note that water molecules assist the ribose recognition in the RNA nucleotide preinsertion, and help Tyr639 stacking to the end basepair in the case of a DNA nucleotide. It was also seen that a base-mismatched nucleotide at preinsertion directly grabs Tyr639 for the active site stabilization. We also find that in a mutant polymerase Y639F the strong stabilization of residue 639 in the active site cannot establish upon the DNA nucleotide preinsertion. The finding explains the reduced differentiation between ribo- and deoxyribonucleotides that has been recorded experimentally for the mutant polymerase.