第一类肽链释放因子C端结构域参与调控终止密码子的识别过程

C-terminal Minidomain of eRF1 Modulates the Process of Stop Codon Decoding

真核生物蛋白质翻译终止过程中,第一类肽链释放因子(eukaryotic polypeptide release factor,eRF1)利用其N端结构域识别终止密码子。eRF1的N结构域中的GTS、NIKS和Yx Cxxx F模体对于终止密码子的识别发挥重要作用。但至目前为止,eRF1识别终止密码子的机制,尤其是对于终止密码子的选择性识别机制仍不清楚。我们构建了四膜虫(Tetrahymena thermophilia)eRF1的N端结构域与酿酒酵母(Saccharomyces cerevisiae)或裂殖酵母(Schizosaccharomyces pombe)eRF1的M和C结构域组成的杂合eRF1,即Tt/Sc eRF1和Tt/Sp eRF1。双荧光素酶检测结果证实,两种杂合eRF1在细胞中识别终止密码子的活性具有显著差异。Tt/Sc eRF1仅识别UGA密码子,与四膜虫eRF1一致,具有密码子识别特异性;而Tt/Sp eRF1可以识别3个终止密码子,无密码子识别特异性。为解释这一现象,将Sp eRF1的C结构域中的1个关键的小结构域中的氨基酸进行突变,与Sc eRF1相应位点的氨基酸一致。分析结果显示,突变体Tt/Sp eRF1识别密码子UAA和UAG的性质发生显著变化,说明第一类肽链释放因子的C端结构域参与了终止密码子的识别过程。这提示,四膜虫eRF1识别终止密码子的特异性可能依赖于eRF1分子内的结构域间相互作用。本研究结果为揭示肽链释放因子识别终止密码子的分子机制提供了数据支持。

Eukaryotie translation termination is performed by eRF1, which recognizes stop codons via its N-terminal domain. Conserved motifs, such as GTS, NIKS and YxCxxxF in N-domain of eRF1, were found to be important for stop codon recognition. However, the detailed decoding mechanism, especially the selective recognition of stop codon remains unclear. We characterized the C-terminal minidomain （329 -372 aa, human eRF1 numbering） which was involved in stop codon recognition. We revealed that the N-domain of Tetrahymena eRF1 showed different selectivity of stop codon recognition in Saccharomyces cerevisiae cells when fused with M- and C-domain of eRFls from S. cerevisiae and Schizosaccharomyces pombe, respectively. To explain this different function of hybrid Tt/Sp eRF1 and Tt/ Sc eRF1, we performed the mutagenesis in C-terminal minidomain by introducing the amino acids （L357A/D361A/A364Q /E367D） into Tt/Sp eRF1 from Tt/Sc eRF1. The readthrough efficiency indicated that specificity of stop codon decoding by mutants Tt/Sp eRF1 changed significantly at UAA and UAG, while not at UGA, suggesting that the C-terminal minidomain is possibly involved in modulating the process of the stop codon decoding cooperated with N-terminal domain. These results provide clues for elucidating the mechanism of stop codon recognition by polypeptide release factors.