MINUS-STRAND INITIATION BY BROME MOSAIC-VIRUS REPLICASE WITHIN THE 3' TRANSFER RNA-LIKE STRUCTURE OF NATIVE AND MODIFIED RNA TEMPLATES
MINUS-STRAND INITIATION BY BROME MOSAIC-VIRUS REPLICASE WITHIN THE 3' TRANSFER RNA-LIKE STRUCTURE OF NATIVE AND MODIFIED RNA TEMPLATES
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DOI：
10.1016/0022-2836(86)90332-3
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发表时间：
1986-02-20
影响因子：
5.6
通讯作者：
HALL, TC
中科院分区：
文献类型：
--
作者：
MILLER, WA;BUJARSKI, JJ;HALL, TC
从感染雀麦花叶病毒的大麦叶片中提取的RNA依赖性RNA聚合酶（复制酶）已被证明可以启动（+）正义病毒体RNA合成（-）正义RNA。起始从头发生，如通过掺入[γ- [32 P]GTP进入产品。使用虫草素三磷酸在复制酶合成过程中终止（-）链进行测序，产生序列梯，证实复制是准确的，并且起始发生在非常接近3“末端的位置。通过在逐步去除3“-末端核苷酸后测试复制酶模板活性，进一步确定精确的起始位点。而去除末端A并不降低模板活性，去除下一个核苷酸（C-2）。因此，在体外，几乎可以肯定起始发生在倒数第二个3“-核苷酸（C-2）的对面。从雀麦花叶病毒感染的叶片中分离的RNA的双链复制形式的结构与体内发生的这种机制一致，因为它缺少在病毒体RNA上发现的3“-末端A。对于在3“末端添加多达15至30个A残基的RNA，保留了（-）链起始的特异性位点和正常的模板活性。然而，只有有限的寡核苷酸3“延伸可以存在于活性模板上。为了评估活性模板所需序列的5“范围，产生了对应于tRNA样结构的雀麦花叶病毒RNA的134个核苷酸长的片段。该RNA具有高模板活性，但较短的3“（85个核苷酸）片段无活性。具有5“至134位的各种异源序列的RNA也显示出高模板活性。因此，所有四种雀麦花叶病毒RNA共有的3“-末端tRNA样结构包含复制起始所需的所有信号，并且其5”端的序列在模板选择中不起作用。
An RNA-dependent RNA polymerase (replicase) extract from brome mosaic virus-infected barley leaves has been shown to initiate syntehsis of (-) sense RNA from (+) sense virion RNA. Initiation occurred de novo, as demonstrated by the incorporation of [.gamma.-32P]GTP into the product. Sequencing using cordycepin triphosphate to terminate (-) strands during their synthesis by the replicase generated sequence ladders that confirmed that copying was accurate, and that initiation occurred very close to the 3'' end. The precise site of initiation was further defined by testing the replicase template activity after stepwise removal of 3''-terminal nucleotides. Whereas removal of the terminal A did not decrease template activity, removal of the next nucleotide (C-2) did. Thus, initiation almost certainly occurs opposite the penultimate 3''-nucleotide (C-2) in vitro. The structure of the double-stranded replicative form of RNA isolated from brome mosaic virus-infected leaves was consistent with such a mechanism occurring in vivo, in that it lacked the 3''-terminal A found on virion RNAs. The specific site of (-) strand initiation and normal template activity were retained for RNAs with as many as 15 to 30 A residues added to the 3'' end. However, only limited oligonucleotide 3'' extensions can be present on active templates. In order to assess the 5'' extent of sequences required for an active template, a 134-nucleotide-long fragment of brome mosaic virus RNA, corresponding to the tRNA-like structure, was generated. This RNA had high template activity, but a shorter 3'' (85-nucleotide) fragment was inactive. RNAs with various heterologus sequences 5'' to position 134 also showed high template activity. Thus, the 3''-terminal tRNA-like structure common to all four brome mosaic virus virion RNAs contains all of the signals required for initiation of replication, and sequences 5'' to it do not play a role in template selection.