Direct measurement of the poliovirus RNA polymerase error frequency in vitro

TLDR

The study purified poliovirus RNA polymerase and measured its error frequency by copying homopolymeric RNA templates (poly(A), poly(C), poly(I)) in vitro, quantifying errors as the proportion of noncomplementary nucleotides incorporated. The polymerase exhibited high error rates (7 × 10⁻⁴–5.4 × 10⁻³), with no substrate or template bias, and a fivefold increase in error frequency when Mg²⁺ concentration and pH were raised, mirroring an eightfold rise in elongation rate.

Abstract

The fidelity of RNA replication by the poliovirus-RNA-dependent RNA polymerase was examined by copying homopolymeric RNA templates in vitro. The poliovirus RNA polymerase was extensively purified and used to copy poly(A), poly(C), or poly(I) templates with equimolar concentrations of noncomplementary and complementary ribonucleotides. The error frequency was expressed as the amount of a noncomplementary nucleotide incorporated divided by the total amount of complementary and noncomplementary nucleotide incorporated. The polymerase error frequencies were very high and ranged from 7 x 10(-4) to 5.4 x 10(-3), depending on the specific reaction conditions. There were no significant differences among the error frequencies obtained with different noncomplementary nucleotide substrates on a given template or between the values determined on two different templates for a specific noncomplementary substrate. The activity of the polymerase on poly(U) and poly(G) was too low to measure error frequencies on these templates. A fivefold increase in the error frequency was observed when the reaction conditions were changed from 3.0 mM Mg2+ (pH 7.0) to 7.0 mM Mg2+ (pH 8.0). This increase in the error frequency correlates with an eightfold increase in the elongation rate that was observed under the same conditions in a previous study.