Hosoda Kazufumi, Matsuura Tomoaki, Kita Hiroshi, Ichihashi Norikazu, Tsukada Koji, Yomo Tetsuya
Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan.
J Biol Chem. 2007 May 25;282(21):15516-27. doi: 10.1074/jbc.M700307200. Epub 2007 Apr 5.
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.
研究了Qβ复制酶催化的RNA复制反应动力学。Qβ复制酶是一种依赖RNA的RNA聚合酶，负责复制大肠杆菌噬菌体Qβ的RNA基因组，在Qβ噬菌体的生命周期中起关键作用。尽管使用这种酶的RNA复制反应早已被研究，但尚未确定一个能够描述整个RNA扩增过程的动力学模型。在本研究中，我们提出了一个能够解释整个RNA扩增过程的动力学模型。我们提出的动力学模型的关键在于考虑非生产性结合（即酶与RNA的结合，在此情况下酶无法启动反应）。通过考虑非生产性结合以及我们观察到的显著的酶失活现象，之前未解决的观察结果也能够得到解释。此外，基于动力学模型和实验结果，我们使用不同长度的模板RNA确定了速率常数和平衡常数。所提出的模型和获得的常数为理解Qβ噬菌体扩增的基础以及使用Qβ复制酶的应用提供了重要信息。
