Different modes of stop codon restriction by the stylonychia and paramecium eRF1 translation termination factors
Different modes of stop codon restriction by the stylonychia and paramecium eRF1 translation termination factors
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DOI：
10.1073/pnas.0703887104
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发表时间：
2007-06-26
影响因子：
11.1
通讯作者：
Kisselev, Lev
中科院分区：
文献类型：
--
作者：
Lekomtsev, Sergey;Kolosov, Petr;Kisselev, Lev
在真核生物中，一个单一的翻译终止因子，真核生物第一类多肽释放因子(ERF1)，解码三个终止密码子：UAA、UAG和UGA。在一些纤毛虫中，如柱头甲属和草履虫，eRF1仅表现出UGA的译码特异性，而UAG和UAA被重新指定为正义密码子。由于变异编码纤毛虫可能是从通用编码祖先(S)进化而来的，纤毛虫eRF1中应该存在限制其终止密码子识别的结构特征。在全能的eRF1中，终止密码子识别与蛋白质的N-末端结构域有关。通过体外和体内试验，我们发现由柱甲eRF1的N-末端结构域与人eRF1的核心区(MC结构域)融合而成的嵌合分子对UGA具有特异性；这明确地将eRF1终止密码子的特异性与其N-末端结构域的性质联系起来。通过将纤毛虫N-末端结构域序列与人蛋白中的匹配序列互换而构建的eRF1嵌合体的功能分析表明，三肽QFM在限制柱头甲eRF1对UGA的特异性方面发挥了关键作用。通过定点突变，我们发现草履虫eRF1对UGA的特异性位于NIKS(61-氨基酸)和YxCxxxF(第124-131氨基酸)基序中。因此，我们建立了来自两种不同纤毛虫的eRF1依赖于不同的分子机制来实现对UGA终止密码子的特异性。这一发现表明，eRF1对UGA的特异性限制可能是纤毛虫进化历史中发生在独立实例中的早期事件，可能有助于将UAG和UAA重新分配到正义密码子。
In universal-code eukaryotes, a single-translation termination factor, eukaryote class-1 polypeptide release factor (eRF1), decodes the three stop codons: UAA, UAG, and UGA. In some ciliates, like Stylonychia and Paramecium, eRF1s exhibit UGA-only decoding specificity, whereas UAG and UAA are reassigned as sense codons. Because variant-code ciliates may have evolved from universal-code ancestor(s), structural features should exist in ciliate eRF1s that restrict their stop codon recognition. In omnipotent eRF1s, stop codon recognition is associated with the N-terminal domain of the protein. Using both in vitro and in vivo assays, we show here that chimeric molecules composed of the N-terminal domain of Stylonychia eRF1 fused to the core domain (MC domain) of human eRF1 retained specificity toward UGA; this unambiguously associates eRF1 stop codon specificity to the nature of its N-terminal domain. Functional analysis of eRF1 chimeras constructed by swapping ciliate N-terminal domain sequences with the matching ones from the human protein highlighted the crucial role of the tripeptide QFM in restricting Stylonychia eRF1 specificity toward UGA. Using the site-directed mutagenesis, we show that Paramecium eRF1 specificity toward UGA resides within the NIKS (amino acids 61-64) and YxCxxxF (amino acids 124-131) motifs. Thus, we establish that eRF1 from two different ciliates relies on different molecular mechanisms to achieve specificity toward the UGA stop codon. This finding suggests that eRF1 restriction of specificity to only UGA might have been an early event occurring in independent instances in ciliate evolutionary history, possibly facilitating the reassignment of UAG and UAA to sense codons.