Rules of UGA-N decoding by near-cognate tRNAs and analysis of readthrough on short uORFs in yeast.
Rules of UGA-N decoding by near-cognate tRNAs and analysis of readthrough on short uORFs in yeast.
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DOI：
10.1261/rna.054452.115
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发表时间：
2016-03
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通讯作者：
Valášek LS
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作者：
Beznosková P;Gunišová S;Valášek LS
终止密码子识别的分子机制的释放因子eRF 1与eRF 3复合物已被描述得非常详细，然而，我们的理解是什么决定了不同的终止密码子四核苷酸之间的终止效率的差异，以及如何近同源（NC）的tRNA重新编码终止密码子在酿酒酵母程序通读仍然是穷人。在这里，我们表明，UGA-C作为所有四种可能的组合中唯一的四核苷酸，大大加剧了eRF 1中终止密码子缺失突变体的通读表型。由于同样的情况也适用于UAA-C和UAG-C，我们提出，所有三个终止密码子在胞嘧啶之后显示的异常高的通读水平部分是由eRF 1的受损采样能力引起的，eRF 1特异性地在+4位置感测胞嘧啶。其余三种UGA-N四核苷酸之间终止效率的差异则由它们对nc-tRNA的不同偏好给出。特别是，UGA-A允许增加Trp-tRNA的掺入，而UGA-G和UGA-C有利于Cys-tRNA。因此，我们的研究结果扩展了一般解码规则的剧目，显示+4碱基决定了nc-tRNA的优先选择，在胞嘧啶的情况下，它也与eRF 1遗传相互作用。最后，使用一个例子的GCN 4的翻译控制由四个短的uORF，我们还展示了如何处理不可取的通读这些uORF的演变作为关键的翻译重新启动促进功能的GCN 4的监管，因为这两个否则抵消活动，通读与重新启动，是由eIF 3介导的。
The molecular mechanism of stop codon recognition by the release factor eRF1 in complex with eRF3 has been described in great detail; however, our understanding of what determines the difference in termination efficiencies among various stop codon tetranucleotides and how near-cognate (nc) tRNAs recode stop codons during programmed readthrough in Saccharomyces cerevisiae is still poor. Here, we show that UGA-C as the only tetranucleotide of all four possible combinations dramatically exacerbated the readthrough phenotype of the stop codon recognition-deficient mutants in eRF1. Since the same is true also for UAA-C and UAG-C, we propose that the exceptionally high readthrough levels that all three stop codons display when followed by cytosine are partially caused by the compromised sampling ability of eRF1, which specifically senses cytosine at the +4 position. The difference in termination efficiencies among the remaining three UGA-N tetranucleotides is then given by their varying preferences for nc-tRNAs. In particular, UGA-A allows increased incorporation of Trp-tRNA whereas UGA-G and UGA-C favor Cys-tRNA. Our findings thus expand the repertoire of general decoding rules by showing that the +4 base determines the preferred selection of nc-tRNAs and, in the case of cytosine, it also genetically interacts with eRF1. Finally, using an example of the GCN4 translational control governed by four short uORFs, we also show how the evolution of this mechanism dealt with undesirable readthrough on those uORFs that serve as the key translation reinitiation promoting features of the GCN4 regulation, as both of these otherwise counteracting activities, readthrough versus reinitiation, are mediated by eIF3.