Barrera I, Schuppli D, Sogo J M, Weber H
Institut für Molekularbiologie I, Universität Zürich, Switzerland.
J Mol Biol. 1993 Jul 20;232(2):512-21. doi: 10.1006/jmbi.1993.1407.
Our earlier work on the recognition of Q beta plus strand RNA by replicase had shown by RNase degradation and by electron microscopic techniques that specific binding interactions occurred at two internal sites, the S-site and the M-site, but not at the 3'-end, i.e. the site of initiation of synthesis. Using essentially similar methods, we have found now for binding complexes of replicase with the minus strand a completely different pattern, namely considerable terminal binding, whereas binding to internal sites was without detectable specificity. In the case of plus strand complexes, simultaneous binding at the two internal sites and at a terminal site could be demonstrated by electron microscopy after initiation of RNA synthesis in the presence of host factor, GTP and ATP. A variant plus strand RNA containing a 490 nucleotide duplication near the 5'-end resulted in similar double-looped complexes, however with an elongated free arm, showing that the protein-bound terminal site was the 3'-end of the RNA. Interestingly, the same two-looped structures were also found for complexes consisting of plus strand RNA and host factor without replicase. This suggests that the role of the host factor on the plus strand template is to bring the 3'-end into the proximity of the S-site/M-site domain, where replicase can initiate on it. In contrast, the 3'-end of the minus strand appears to be directly available to the enzyme.
我们早期关于复制酶对Qβ正链RNA识别的研究，通过核糖核酸酶降解和电子显微镜技术表明，特异性结合相互作用发生在两个内部位点，即S位点和M位点，而不是在3'末端，即合成起始位点。使用基本相似的方法，我们现在发现复制酶与负链的结合复合物呈现出完全不同的模式，即相当多的末端结合，而与内部位点的结合没有可检测到的特异性。对于正链复合物，在存在宿主因子、鸟苷三磷酸（GTP）和三磷酸腺苷（ATP）的情况下启动RNA合成后，通过电子显微镜可以证明在两个内部位点和一个末端位点同时存在结合。一种在5'末端附近含有490个核苷酸重复序列的变异正链RNA产生了类似的双环复合物，然而其游离臂伸长，表明与蛋白质结合的末端位点是RNA的3'末端。有趣的是，在由正链RNA和宿主因子组成的复合物（不含复制酶）中也发现了相同的双环结构。这表明宿主因子在正链模板上的作用是将3'末端带到S位点/M位点结构域附近，复制酶可以在此起始合成。相比之下，负链的3'末端似乎可直接被该酶利用。
