四膜虫eRF1识别终止密码子的功能结构域

The Functional Domains of Tetrahymena eRF1 for Stop Codon Recognition

原生动物的一些纤毛虫中终止密码子发生重分配现象,将1个或2个终止密码子翻译为氨基酸.目前对这一现象的发生机制仍无合理解释.近年来,对蛋白质合成终止过程中肽链释放因子(eukaryotic polypeptide release factor,eRF)结构和功能的深入研究,为揭示终止密码子的重分配机制提供了重要的线索.本实验以具有终止密码子识别特异性的四膜虫Tt-eRF1为研究对象,将其中与密码子识别有关的GTx、NIKS和Y-C-F关键模体(motif)引入识别通用终止密码子的酵母Sc-eRF1中,构建成各种嵌合体eRF1.利用双荧光素酶报告系统和细胞活性实验,分析关键模体及其周边的氨基酸对eRF1识别终止密码子性质的影响.结果表明,GTx和NIKS模体一定程度上决定eRF1识别终止密码子第1位碱基U和第2位碱基A;Y-C-F模体决定eRF1识别终止密码子UGA的第2位碱基G.模体内及其相邻氨基酸定点突变分析进一步支持以上结果.本研究推测,eRF1在进化过程中一些关键模体结构的改变决定其识别终止密码子的特异性,只能识别3个终止密码子中的1个或2个.随后,由于tRNA基因的突变产生阻抑性tRNA,促成终止密码子在原生动物纤毛虫中的重新分配.

How one or two stop codons may encode amino acids in protozoan ciliates remained unclear to date. Studies of eukaryotic polypeptide release factors （eRF） have provided clues for elucidating this phenomenon. When the GTx, NIKS, and Y-C-F motifs on Tetrahymena thermophilia eRF1 introduced into Saccharomyces cerevisiae eRF1, it allowed the formed chimeric eRFls to recognize UGA as the sole stop eodon. The characteristics of these chimeric eRFls and their site-directed mutants were analyzed using a dual luciferase read-through report system in viability assays. Our results indicated that the GTx and NIKS motifs contributed to the discrimination of the U and second-place A in UAA and UAG stop codons. The Y-C-F motif recognized the G in UGA. Other residues in or near the motifs were critically involved in direct or indirect recognition of the three stop codons. Our findings suggested that the key motifs in eRF1 from ciliates endow its recognition specificity to stop codons. Evolutionally, additional mutations in tRNA might also be responsible for the reassignment of stop codons in ciliates.