Characterization of the stop codon readthrough signal of Colorado tick fever virus segment 9 RNA.
Characterization of the stop codon readthrough signal of Colorado tick fever virus segment 9 RNA.
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DOI：
10.1261/rna.030338.111
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发表时间：
2012-02
期刊：
影响因子：
4.5
通讯作者：
S. Napthine;Christina Yek;Michael L. Powell;T. Brown;I. Brierley
中科院分区：
文献类型：
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作者：
S. Napthine;Christina Yek;Michael L. Powell;T. Brown;I. Brierley
终止密码子通读被用作越来越多的病毒和细胞蛋白质的表达机制，但在许多情况下，促进通读的mRNA信号的特征很差。在这里，我们研究了科罗拉多蜱热病毒（CTFV）片段9 RNA（Seg-9）的通读信号。CTFV是呼肠孤病毒科Coltivirus属的模式种，是一种蜱传播的双链节段RNA病毒。Seg-9编码36-kDa蛋白质VP 9，并且通过UGA终止密码子的通读，编码65-kDa产物VP 9 '。使用一个报告系统，我们定义了最低的序列要求通读和确认的活动，在哺乳动物和昆虫无细胞翻译系统，并在转染的哺乳动物细胞。突变分析显示，通读是UGA特异性的，并且UGA周围的局部序列环境影响通读效率。通读也依赖于从UGA密码子下游8个碱基开始的稳定的RNA茎环结构。该茎环的突变分析揭示了茎区域的需要，但不需要环内鉴定的亚结构。出乎意料的是，我们无法检测到的CTFV信号的翻译过程中的核糖体暂停，这表明通读的机制，至少在这个网站上，是不太可能依赖于RNA二级结构诱导的核糖体暂停在重新编码的终止密码子。
Termination codon readthrough is utilized as a mechanism of expression of a growing number of viral and cellular proteins, but in many cases the mRNA signals that promote readthrough are poorly characterized. Here, we investigated the readthrough signal of Colorado tick fever virus (CTFV) segment 9 RNA (Seg-9). CTFV is the type-species of the genus Coltivirus within the family Reoviridae and is a tick-borne, double-stranded, segmented RNA virus. Seg-9 encodes a 36-kDa protein VP9, and by readthrough of a UGA stop codon, a 65-kDa product, VP9'. Using a reporter system, we defined the minimal sequence requirements for readthrough and confirmed activity in both mammalian and insect cell-free translation systems, and in transfected mammalian cells. Mutational analysis revealed that readthrough was UGA specific, and that the local sequence context around the UGA influenced readthrough efficiency. Readthrough was also dependent upon a stable RNA stem-loop structure beginning eight bases downstream from the UGA codon. Mutational analysis of this stem-loop revealed a requirement for the stem region but not for substructures identified within the loop. Unexpectedly, we were unable to detect a ribosomal pause during translation of the CTFV signal, suggesting that the mechanism of readthrough, at least at this site, is unlikely to be dependent upon RNA secondary-structure induced ribosomal pausing at the recoded stop codon.