Baum M, Beier H
Institut für Biochemie, Bayerische Julius-Maximilians-Universität, Biozentrum, Am Hubland, D-97074 Würzburg, Germany.
Nucleic Acids Res. 1998 Mar 15;26(6):1390-5. doi: 10.1093/nar/26.6.1390.
Many RNA viruses express part of their genomic information by read-through over internal termination codons. We have recently characterized tobacco cytoplasmic (cyt) and chloroplast (chl) tRNACmCATrp and tRNAGCACys as natural suppressor tRNAs that are able to read the leaky UGA codon in RNA-1 of tobacco rattle virus, albeit with different efficiencies. Here we have identified a third natural UGA suppressor in plants. We have purified and sequenced four cyt tRNAArg isoacceptors with ICG, CCG, UCG and CCU anticodons from wheat germ. With the exception of tRNAICGArg, these are the first sequences of plant tRNAsArg. In order to study the potential suppressor activity of wheat tRNAsArg we have used in vitro synthesized mRNA transcripts in which different viral read-through regions had been placed. In vitro translation was carried out in a homologous wheat germ extract. We found that tRNAUCGArg is an efficient UGA suppressor in vitro, whereas the other three tRNAArg isoacceptors exhibit no or very low suppressor activity. Interaction of tRNAUCGArg with the UGA codon requires a G:U base pair at the third anticodon position. This is the first time that an arginine-accepting tRNA has been characterized as a natural UGA suppressor. A remarkable feature of cyt tRNAUCGArg is its ability to misread the UGA at the end of the coat protein cistron in RNA-1 of pea enation mosaic virus, which is not accomplished by cyt tRNACmCATrp or cyt tRNAGCACys, due to an unfavourable codon context.
许多RNA病毒通过通读内部终止密码子来表达其部分基因组信息。我们最近鉴定出烟草细胞质（cyt）和叶绿体（chl）的tRNACmCATrp和tRNAGCACys是天然抑制性tRNA，它们能够通读烟草脆裂病毒RNA-1中的渗漏UGA密码子，尽管效率不同。在这里，我们在植物中鉴定出了第三种天然UGA抑制子。我们用ICG、CCG、UCG和CCU反密码子从小麦胚芽中纯化并测序了四种细胞质tRNAArg同工受体。除了tRNAICGArg外，这些是植物tRNAArg的首批序列。为了研究小麦tRNAArg的潜在抑制活性，我们使用了体外合成的mRNA转录本，其中放置了不同的病毒通读区域。在同源小麦胚芽提取物中进行体外翻译。我们发现tRNAUCGArg在体外是一种高效的UGA抑制子，而其他三种tRNAArg同工受体则没有或只有非常低的抑制活性。tRNAUCGArg与UGA密码子的相互作用在反密码子的第三个位置需要一个G:U碱基对。这是首次将精氨酸接受性tRNA鉴定为天然UGA抑制子。细胞质tRNAUCGArg的一个显著特征是它能够错读豌豆耳突花叶病毒RNA-1中外壳蛋白顺反子末端的UGA，由于密码子上下文不利，细胞质tRNACmCATrp或细胞质tRNAGCACys无法做到这一点。
