Kinetic Analysis of the Entire RNA Amplification Process by Qβ Replicase

Kazufumi Hosoda,Norikazu Ichihashi,Hiroshi Kita,Tomoaki Matsuura,Koji Tsukada,Tetsuya Yomo

doi:10.1074/jbc.m700307200

Kazufumi Hosoda, Norikazu Ichihashi + Show 4 more

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https://doi.org/10.1074/jbc.m700307200

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Abstract

The kinetics of the RNA replication reaction by Qbeta replicase were investigated. Qbeta replicase is an RNA-dependent RNA polymerase responsible for replicating the RNA genome of coliphage Qbeta and plays a key role in the life cycle of the Qbeta 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 Qbeta phage amplification and the applications using Qbeta replicase.

Highlights

Q␤ replicase is an RNA-dependent RNA polymerase responsible for replicating the RNA genome of coliphage Q␤ (1) that plays a key role in the life cycle of the Q␤ phage (2, 3)
Kinetic models to explain the entire amplification process have been studied mainly by computer simulation (5, 11, 25), and these studies have not resolved the following three observations: (i) the replication of the phage genome RNA is somehow inhibited by the genomic RNA itself (26, 27), (ii) the reaction velocity decreases in the late phase of the reaction (5, 28), and (iii) the catalytic constant seems to differ between the earlier and later phases of the amplification process (5)
We analyzed the entire process of RNA amplification by Q␤ replicase based on a simple kinetic model, which considers nonproductive binding

Summary

Introduction

Q␤ replicase is an RNA-dependent RNA polymerase responsible for replicating the RNA genome of coliphage Q␤ (1) that plays a key role in the life cycle of the Q␤ phage (2, 3). Kinetic models to explain the entire amplification process have been studied mainly by computer simulation (5, 11, 25), and these studies have not resolved the following three observations: (i) the replication of the phage genome RNA is somehow inhibited by the genomic RNA itself (26, 27), (ii) the reaction velocity decreases in the late phase of the reaction (5, 28), and (iii) the catalytic constant seems to differ between the earlier and later phases of the amplification process (5) The bases of these observations are still unclear. The key in our proposed kinetic model is the VOLUME 282 NUMBER 21 MAY 25, 2007

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