Kinetic analysis of the entire RNA amplification process by Qβ replicase

Abstract

The kinetics of the RNA replication reaction by Qβ replicase were investigated. Qβ replicase is an RNA-dependent RNA polymerase responsible for replicating the RNA genome of coliphage Qβ and plays a key role in the life cycle of the Qβ phage. Although the RNA replication reaction using this enzyme has long been studied, a kinetic model that can describe the entire RNA amplification process has yet to be determined. In this study, we propose a kinetic model that is able to account for the entire RNA amplification process. The key to our proposed kinetic model is the consideration of nonproductive binding (i.e. binding of an enzyme to the RNA where the enzyme cannot initiate the reaction). By considering nonproductive binding and the notable enzyme inactivation we observed, the previous observations that remained unresolved could also be explained. Moreover, based on the kinetic model and the experimental results, we determined rate and equilibrium constants using template RNAs of various lengths. The proposed model and the obtained constants provide important information both for understanding the basis of Qβ phage amplification and the applications using Qβ replicase. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.