Key Laboratory of Chemical Biology and Molecular Engineering of The Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, People's Republic of China.
Biosci Rep. 2010 Dec;30(6):425-31. doi: 10.1042/BSR20090154.
In higher eukaryotes, RF-I (class I release factor) [eRF1 (eukaryotic release factor 1)] is responsible for stop codon recognition and promotes nascent polypeptide release from the ribosome. Interestingly, two class I RFs, eRF1a and eRF1b, have been identified among the ciliates Euplotes, which are variant code organisms. In the present study, we analysed the comparative expression of eRF1a and eRF1b in Euplotes cells, demonstrating that the expression of eRF1b was higher than that of eRF1a. An interaction between eRF1b and eRF3 was confirmed, suggesting that an eRF1b function is facilitated by eRF3. Co-localization of both eRF1s indicated that they function in the same subcellular location in Euplotes cells. We also analysed the characteristics of stop codon discrimination by eRF1b. Like eRF1a, eRF1b recognized UAA and UAG as stop codons, but not UGA. This finding disagreed with the deduced characteristics of eRF1a/eRF1b from the classic hypothesis of 'anticodon-mimicry' proposed by Muramatsu et al. [Muramatsu, Heckmann, Kitanaka and Kuchino (2001) FEBS Lett. 488, 105-109]. Mutagenesis experiments indicated that the absolutely conserved amino acid motif 'G31T32' (numbered as for human eRF1) in eRF1b was the key to efficient stop codon recognition by eRF1b. In conclusion, these findings support and improve the 'cavity model' of stop codon discrimination by eRF1 proposed by Bertram et al. [Bertram, Bell, Ritchie, Fullerton and Stansfield (2000) RNA 6, 1236-1247] and Inagaki et al. [Inagaki, Blouin, Doolittle and Roger (2002) Nucleic Acids Res. 30, 532-544].
在高等真核生物中，RF-I（I 类释放因子）[eRF1（真核释放因子 1）]负责识别终止密码子，并促进新生多肽从核糖体上释放。有趣的是，在变异密码生物体纤毛虫 Euplotes 中，已经鉴定出两种 I 类 RF，eRF1a 和 eRF1b。在本研究中，我们分析了 Euplotes 细胞中 eRF1a 和 eRF1b 的比较表达，证明 eRF1b 的表达高于 eRF1a。证实了 eRF1b 与 eRF3 之间的相互作用，表明 eRF1b 的功能是由 eRF3 促进的。两种 eRF1 的共定位表明它们在 Euplotes 细胞中具有相同的亚细胞定位。我们还分析了 eRF1b 对终止密码子的识别特征。与 eRF1a 一样，eRF1b 识别 UAA 和 UAG 为终止密码子，但不识别 UGA。这一发现与 Muramatsu 等人提出的经典“反密码子模拟”假设所推断的 eRF1a/eRF1b 特征不一致。[Muramatsu、Heckmann、Kitanaka 和 Kuchino（2001）FEBS Lett. 488, 105-109]。突变实验表明，eRF1b 中绝对保守的氨基酸基序“G31T32”（编号为人类 eRF1）是 eRF1b 有效识别终止密码子的关键。总之，这些发现支持并改进了 Bertram 等人提出的 eRF1 终止密码子识别的“腔模型”[Bertram、Bell、Ritchie、Fullerton 和 Stansfield（2000）RNA 6, 1236-1247]和 Inagaki 等人。[Inagaki、Blouin、Doolittle 和 Roger（2002）Nucleic Acids Res. 30, 532-544]。
