Fidelity Control of a Non-Proofreading Polymerase Studied from Kinetics to Molecular Dynamics

Abstract

Polymerases catalyze template-based polymerization in gene replication and transcription. It is essential for the polymerase to achieve sufficiently high fidelity at sufficiently high speed. We investigated kinetically how polymerases select nucleotides efficiently under energy constraints. We noticed that initial screening is indispensable for lowering error rates without lowering much the polymerization speed. Still, multiple checkpoints seem to be necessary for the fidelity requirement overall. To see how exactly the nucleotide selection proceeds, we studied a single-subunit T7 RNA polymerase in molecular dynamics details. We found that substantial nucleotide selection happens prior to full insertion of the nucleotide or formation of the Watson-Crick base pairing. The initial selection replies primarily on electrostatic screening to destabilize a wrong nucleotide relative to the right one at the pre-insertion site. A highly conserved tyrosine can detect the nucleotide identity upon the pre-insertion, and assists the selection through 'gating' during the nucleotide insertion. In particular, the tyrosine residue differentiates dNTP from rNTP by favorably associating with dNTP but not rNTP. The mutation of this residue to phenylalanine loses this differentiation. That explains why the mutant was experimentally detected as not only an RNA polymerase, but also a DNA polymerase.