Native replication intermediates of the yeast 20 S RNA virus have a single-stranded RNA backbone
Native replication intermediates of the yeast 20 S RNA virus have a single-stranded RNA backbone
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DOI：
10.1074/jbc.m412048200
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发表时间：
2005-02-25
影响因子：
4.8
通讯作者：
Esteban, R
中科院分区：
文献类型：
--
作者：
Fujimura, T;Solórzano, A;Esteban, R
S核糖核酸病毒是在酿酒酵母中发现的一种正链核糖核酸病毒。病毒基因组(2.5kb)只编码其RNA聚合酶(P91)，在体内与P91形成核糖核蛋白复合体。从20个S核糖核酸诱导的细胞中提取的裂解物显示出合成病毒基因组正链的核糖核酸聚合酶活性。当苯酚提取后的体外产物在一段时间内被分析时，放射性核苷酸首先被掺入双链RNA(DsRNA)中间体，然后被追逐到最终的单链RNA产品。这些dsRNA中间体中的正链和负链是非共价结合的，从中间体释放正链产物需要净RNA合成。然而，我们发现这些dsRNA中间体是苯酚提取引起的产物。由RNase敏感性实验判断，天然复制中间产物具有单链RNA骨架，并且它们在非变性凝胶中明显地从dsRNA形式迁移。当RNA合成完成后，正链RNA产物和负链模板从复制中间产物中释放出来。这些结果表明，天然复制中间体由一个小于单位长度的正链和一个松散结合的负链模板组成，可能是通过RNA聚合酶P91。因此，W是20 S RNA的双链RNA形式，在37℃培养的酵母细胞中积累，不是20 S RNA复制周期的中间产物，而是副产品。
20 S RNA virus is a positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome (2.5 kb) only encodes its RNA polymerase (p91) and forms a ribonucleoprotein complex with p91 in vivo. A lysate prepared from 20 S RNA-induced cells showed an RNA polymerase activity that synthesized the positive strands of viral genome. When in vitro products, after phenol extraction, were analyzed in a time course, radioactive nucleotides were first incorporated into double-stranded RNA (dsRNA) intermediates and then chased out to the final single-stranded RNA products. The positive and negative strands in these dsRNA intermediates were non-covalently associated, and the release of the positive strand products from the intermediates required a net RNA synthesis. We found, however, that these dsRNA intermediates were an artifact caused by phenol extraction. Native replication intermediates had a single-stranded RNA backbone as judged by RNase sensitivity experiments, and they migrated distinctly from a dsRNA form in non-denaturing gels. Upon completion of RNA synthesis, positive strand RNA products as well as negative strand templates were released from replication intermediates. These results indicate that the native replication intermediates consist of a positive strand of less than unit length and a negative strand template loosely associated, probably through the RNA polymerase p91. Therefore, W, a dsRNA form of 20 S RNA that accumulates in yeast cells grown at 37 degreesC, is not an intermediate in the 20 S RNA replication cycle, but a by-product.