Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, Japan.
Biochemistry. 2010 Mar 9;49(9):1809-13. doi: 10.1021/bi901805u.
During RNA replication mediated by Qbeta replicase, self-replicating RNAs (RQ RNAs) are amplified without the addition of template RNA. This undesired amplification makes the study of target RNA replication difficult, especially for long RNA such as genomic RNA of Qbeta phage. This perhaps is one of the reasons why the precise rate of genomic RNA replication in the presence of host factor Hfq has not been reported in vitro. Here, we report a method to repress RQ RNA amplification by compartmentalization of the reaction using a water-in-oil emulsion but maintaining the activity of Qbeta replicase. This method allowed us to amplify the phage Qbeta genome RNA exponentially without detectable amplification of RQ RNA. Furthermore, we found that the rate constant of genome RNA replication in the exponential phase at the optimum Hfq concentration was approximately 4.6 times larger than that of a previous report, close to in vivo data. This result indicates that the replication rate in vivo is largely explained by the presence of Hfq. This easy method paves the way for the study of genomic RNA replication without special care for the undesired RQ RNA amplification.
在 Qβ复制酶介导的 RNA 复制过程中，自我复制 RNA（RQ RNA）在不添加模板 RNA 的情况下扩增。这种不需要的扩增使得目标 RNA 复制的研究变得困难，特别是对于 Qβ噬菌体的基因组 RNA 等长 RNA 更是如此。这也许是为什么在体外尚未报道存在宿主因子 Hfq 时基因组 RNA 复制的确切速率的原因之一。在这里，我们报告了一种通过使用油水乳液分隔反应来抑制 RQ RNA 扩增的方法，但保持了 Qβ复制酶的活性。该方法允许我们指数扩增噬菌体 Qβ基因组 RNA，而 RQ RNA 的扩增可检测到。此外，我们发现，在最佳 Hfq 浓度下指数期基因组 RNA 复制的速率常数大约是先前报道的 4.6 倍，接近于体内数据。该结果表明，体内的复制速率在很大程度上由 Hfq 的存在决定。这种简单的方法为在无需特别注意不需要的 RQ RNA 扩增的情况下研究基因组 RNA 复制铺平了道路。
