Pre-Steady-State Kinetic Analysis of the Elongation Mode of Dengue Virus RNA Polymerase Domain

Abstract

Dengue viral RNA polymerase replicates its positive single-stranded RNA genome in a primer-independent manner. The slow and inefficient initiation during replication masks the elongation mode. The aim of this work was to further characterize the mechanism of elongation towards an increased understanding of how the enzyme selectively recognizes different nucleotides. Transient kinetic methods were used to measure the microscopic rates of the reaction pathway comprised of enzyme and RNA binding followed by nucleotide binding and incorporation. After extended pre-incubation of the enzyme with double stranded RNA (12-mer primer with a 26-mer template), addition of a correct nucleotide resulted in a burst of single nucleotide incorporation, and the amplitude of the reaction was used to monitor the time course of RNA binding. The kinetics of enzyme and RNA binding followed a two-step mechanism: an initial binding was followed by a conformational change. The enzyme and RNA binding proceeded to equilibrium about six times faster at 37 oC than at 30 oC, suggesting the conformational change involved in RNA binding is temperature sensitive. Following incubation of RNA and enzyme to form an active complex, we used chemical quench-flow methods to measure the ground state binding Kd and the incorporation rate of a correct nucleotide. The fidelity of the polymerase was determined by measurement of the incorporation of incorrect nucleotides. Using these methods, we characterized the Dengue polymerase in its elongation mode by measuring the kinetics of RNA and polymerase binding and the kinetics for nucleotide binding and incorporation. Based on these data, we built a working model for studying the selectivity of Dengue polymerase.