Cloning and expression of a mammalian peptide chain release factor with sequence similarity to tryptophanyl-tRNA synthetases.

The termination of protein synthesis is encoded by in-frame nonsense (stop) codons. Most organisms use three nonsense codons: UGA, UAG, and UAA. In contrast to sense codons, which are decoded by specific tRNAs, nonsense codons are decoded by proteins called release factors (RFs). Here we report the cloning of a mammalian RF cDNA by the use of monoclonal antibodies specific for rabbit RF. Functional studies showed that, when expressed in Escherichia coli, the protein encoded by this cDNA has in vitro biochemical characteristics similar to those of previously characterized mammalian RFs. DNA sequencing of this eukaryotic RF cDNA revealed a remarkable sequence similarity to bacterial and mitochondrial tryptophanyl-tRNA synthetases, with the greatest similarity confined to the synthetase active site, and no obvious similarity to bacterial RFs. IMAGES:

蛋白质合成的终止由读框内无义（终止）密码子（nonsense (stop) codons）所编码。绝大多数生物共使用三种无义密码子：UGA、UAG及UAA。与由特异性转运RNA（transfer RNA, tRNA）解码的有义密码子不同，无义密码子由被称为释放因子（release factors, RFs）的蛋白质所识别并解码。本研究通过使用靶向兔源释放因子的单克隆抗体，成功克隆得到一种哺乳动物源释放因子的互补脱氧核糖核酸（complementary DNA, cDNA）。功能实验结果显示，将该cDNA在大肠杆菌（Escherichia coli）中异源表达后，其所编码的蛋白质所具备的体外生化特性，与此前已被表征的哺乳动物源释放因子高度相似。对该真核生物释放因子cDNA的测序分析表明，其序列与细菌及线粒体色氨酰-tRNA合成酶（tryptophanyl-tRNA synthetase）存在显著的序列相似性，且最高的相似性集中于该合成酶的活性位点，而与细菌释放因子则无明显的序列相似性。图像：