Chai Baofeng, Li Cui, Yu Jingfei, Hao Yanrong, Guo Ping, Shen Quan
Key Laboratory of Chemical Biology and Molecular Engineering, Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, China.
Faculty of Environment and Economics, Shanxi University of Finance and Economics, Taiyuan 030006, China.
Mol Biochem Parasitol. 2015 Jul;202(1):15-21. doi: 10.1016/j.molbiopara.2015.08.004. Epub 2015 Aug 24.
Two classes of polypeptide release factors (RFs) are responsible for maintaining accuracy in translation termination; however, their detailed mechanism of action and evolutionary history of these factors remain elusive. The structure and function of RFs vary in bacteria and eukaryotes, a fact that is suggestive of evolutionary changes in the translation termination system. Giardia lamblia (Diplomonada) and Trichomonas vaginalis (Parabasalia) are considered as early-diverged eukaryotes. The class II release factor, eRF3, of Giardia (Gl-eRF3) appears to have only one domain that corresponds to EF-1α and lacks the N-terminal domain, similar to that of eRF3 of other organisms. In the present study, we show that the chimeric molecules Gl/Sc eRF1 and Tv/Sc eRF1, which are composed of the N-terminal domain of Gl-eRF1 or Tv-eRF1, fused to the core domain (M and C domain) of Saccharomyces cerevisiae eRF1 (Sc-eRF1), resulting in loss of the RF properties of the N-terminal domain. This suggests that the conformation of eRF1 for stop codon recognition in Giardia and Trichomonas varies from the eRF1s of other eukaryotes, including ciliates and yeast. Further studies using intra-N-terminal chimeras of eRF1 indicated that the combination of the GTS loop and NIKS motif from Gl-eRF1 and the Y-C-F motif from Sc-eRF1within the N terminal domain of hybrid eRF1 could restore UGA, but not UAG and UGA recognition. In contrast, the combination of the GTS loop and the NIKS motif of Sc-eRF1 and the Y-C-F motif of Gl-eRF1 could restore UAG and UAA recognition, but not UGA recognition. Thus, these results confirm the findings of previous studies that three motifs in eRF1 are necessary for discrimination of the three bases of stop codons. The NIKS motif is responsible for recognition of the first two bases of UAA and UAG, and the Y-C-F motif identifies the second base of UGA by Gl-eRF1. Amino acid residue substitutions in Gl/Sc-eRF1 by corresponding residues of Sc-eRF1 could change and even restore RF activity, further suggesting different conformation of eRF1 are used for stop codon recognition in Giardia and in Saccharomyces.
两类多肽释放因子（RFs）负责维持翻译终止的准确性；然而，它们详细的作用机制以及这些因子的进化史仍不清楚。RFs的结构和功能在细菌和真核生物中有所不同，这一事实表明翻译终止系统发生了进化变化。贾第虫（双滴虫目）和阴道毛滴虫（毛滴虫目）被认为是早期分化的真核生物。贾第虫的II类释放因子eRF3（Gl-eRF3）似乎只有一个与EF-1α对应的结构域，并且缺少N端结构域，这与其他生物的eRF3类似。在本研究中，我们表明嵌合分子Gl/Sc eRF1和Tv/Sc eRF1，它们由Gl-eRF1或Tv-eRF1的N端结构域与酿酒酵母eRF1（Sc-eRF1）的核心结构域（M和C结构域）融合而成，导致N端结构域丧失RF特性。这表明贾第虫和阴道毛滴虫中用于识别终止密码子的eRF1构象与其他真核生物（包括纤毛虫和酵母）的eRF1不同。使用eRF1的N端内部嵌合体的进一步研究表明，来自Gl-eRF1的GTS环和NIKS基序与来自Sc-eRF1的Y-C-F基序在杂交eRF1的N端结构域内的组合可以恢复对UGA的识别，但不能恢复对UAG和UAA的识别。相反，Sc-eRF1的GTS环和NIKS基序与Gl-eRF1的Y-C-F基序的组合可以恢复对UAG和UAA的识别，但不能恢复对UGA的识别。因此，这些结果证实了先前研究的发现——eRF1中的三个基序对于区分终止密码子的三个碱基是必要的。NIKS基序负责识别UAA和UAG的前两个碱基，而Y-C-F基序通过Gl-eRF1识别UGA的第二个碱基。用Sc-eRF1的相应残基替换Gl/Sc-eRF1中的氨基酸残基可以改变甚至恢复RF活性，这进一步表明在贾第虫和酿酒酵母中，eRF1用于识别终止密码子的构象不同。
