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首页> 外文期刊>Virology >BIOCHEMICAL AND GENETIC ANALYSES OF THE INTERACTION BETWEEN THE HELICASE-LIKE AND POLYMERASE-LIKE PROTEINS OF THE BROME MOSAIC VIRUS
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BIOCHEMICAL AND GENETIC ANALYSES OF THE INTERACTION BETWEEN THE HELICASE-LIKE AND POLYMERASE-LIKE PROTEINS OF THE BROME MOSAIC VIRUS

机译：布鲁姆病毒的类Helicase和Polymerase类蛋白相互作用的生物化学和遗传分析

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Replication of the three positive-strand genomic RNAs of brome mosaic virus requires the activities of the helicase-like la and the polymerase-like 2a proteins. One hundred fifteen amino acids of the 2a N-terminus and the 1a helicase-like region of over 50 kDa are both necessary and sufficient for 1a-2a interaction. Requirement of the large size of the la helicase-like domain suggests that higher order structures might be necessary for the protein's interaction with 2a. To explore the structural properties of la, we used limited proteolysis of in vitro-translated la protein. Treatment of 1a and its deletion derivatives with papain or trypsin revealed that the C-terminal helicase-like segment of approximately 50-60 kDa is highly resistant under our assay conditions to proteolysis, while the N-terminus is rapidly degraded. All tested mutations in the helicase-like region that renders this region protease-sensitive have previously been found to be defective for RNA replication in vivo. To complement the in vitro studies, we examined the interaction of the 1a helicase-like domain and the 2a N-terminus in yeast using the two-hybrid system. Mutations previously known to disrupt 1a-2a interaction also prevented interaction in yeast. Furthermore, results from two-hybrid analysis suggest that the structural domain mapped in vitro is important for 1a-2a interaction. Finally, we found that the helicase-like proteins of three other tripartite RNA Viruses also contain equivalently located protease-resistant domains. (C) 1995 Academic Press, Inc.
机译：溴化花叶病毒的三个正链基因组RNA的复制需要解旋酶样1a和聚合酶样2a蛋白的活性。超过50 kDa的2a N末端和1a解旋酶样区域的115个氨基酸都是1a-2a相互作用所必需的。对1a解旋酶样结构域的大尺寸的要求表明，更高级别的结构对于蛋白质与2a的相互作用可能是必需的。为了探索la的结构特性，我们使用了体外翻译的la蛋白的有限蛋白水解作用。用木瓜蛋白酶或胰蛋白酶处理1a及其缺失衍生物表明，在我们的测定条件下，约50-60 kDa的C端解旋酶样片段对蛋白水解具有高度抗性，而N端则迅速降解。先前已经发现使该区域对蛋白酶敏感的解旋酶样区域中的所有测试突变对于体内RNA复制都是有缺陷的。为了补充体外研究，我们使用双杂交系统检查了酵母中1a解旋酶样结构域和2a N末端的相互作用。先前已知破坏1a-2a相互作用的突变也阻止了酵母中的相互作用。此外，两次杂交分析的结果表明，体外绘制的结构域对于1a-2a相互作用很重要。最后，我们发现其他三种三方RNA病毒的解旋酶样蛋白也含有同等定位的蛋白酶抗性结构域。 （C）1995 Academic Press，Inc.

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