Abstract：
The nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 3\' contexts that are unfavorable for translation termination have been described; however, the exact molecular mechanism that mediates their effects remains unknown. In this study, we used a reconstituted mammalian translation system to examine the efficiency of stop codons in different contexts, including several previously described weak 3\' stop codon contexts. We developed an approach to estimate the level of stop codon readthrough in the absence of eukaryotic release factors (eRFs). In this system, the stop codon is recognized by the suppressor or near-cognate tRNAs. We observed that in the absence of eRFs, readthrough occurs in a 3\' nucleotide context-dependent manner, and the main factors determining readthrough efficiency were the type of stop codon and the sequence of the 3\' nucleotides. Moreover, the efficiency of translation termination in weak 3\' contexts was almost equal to that in the tested standard context. Therefore, the ability of eRFs to recognize stop codons and induce peptide release is not affected by mRNA context. We propose that ribosomes or other participants of the elongation cycle can independently recognize certain contexts and increase the readthrough of stop codons. Thus, the efficiency of translation termination is regulated by the 3\' nucleotide context following the stop codon and depends on the concentrations of eRFs and suppressor/near-cognate tRNAs.
摘要：
终止密码子周围的核苷酸环境显著影响翻译终止的效率。在真核生物中,描述了各种不利于翻译终止的3个上下文；然而，介导其作用的确切分子机制仍然未知。在这项研究中,我们使用重组的哺乳动物翻译系统来检查不同情况下停止密码子的效率，包括几个先前描述的弱3'终止密码子上下文。我们开发了一种在不存在真核释放因子（eRF）的情况下估计终止密码子连读水平的方法。在这个系统中,终止密码子被抑制子或接近同源的tRNA识别。我们观察到，在没有eRF的情况下，读取以3'核苷酸上下文依赖的方式发生，决定通读效率的主要因素是终止密码子的类型和3个核苷酸的序列。此外，弱3上下文中翻译终止的效率几乎等于测试标准上下文中的效率。因此,eRF识别终止密码子和诱导肽释放的能力不受mRNA环境的影响。我们建议核糖体或延伸周期的其他参与者可以独立识别某些上下文并增加终止密码子的读入。因此，翻译终止的效率受终止密码子后3个核苷酸的调节,并取决于eRF和抑制/近同源tRNA的浓度。
