Phan L D, Perentesis J P, Bodley J W
Department of Biochemistry, University of Minnesota, Minneapolis 55455.
J Biol Chem. 1993 Apr 25;268(12):8665-8.
Protein synthesis elongation factor 2 (EF-2) is the target of the ADP-ribosylating activity of diphtheria toxin which is responsible for cell killing. Diphthamide, an unique post-translationally modified histidine residue, is both required for and the site of this ADP-ribosylation. Although present in the EF-2 of all eukaryotes and archaebacteria, the function of diphthamide is unknown. Here we describe the site-specific mutagenesis of the histidine precursor of diphthamide, histidine 699, in yeast EF-2. Plasmid-borne EFT was randomly mutagenized at the histidine 699 codon, and the technique of plasmid shuffling was utilized to select strains that were maintained by the mutant EFT. These mutants were screened for diphtheria toxin resistance. Sequence analysis of the EFT in 49 toxin-resistant isolates showed that histidine 699 had been replaced by 1 of 4 amino acids: asparagine, glutamine, leucine, or methionine. All 11 of the possible codons corresponding to these 4 amino acids were found. The growth rates of cells sustained by the mutant forms of EF-2 were slightly slower than those of isogenic wild-type cells. We conclude that despite its strict conservation and universal post-translational modification, the histidine precursor of diphthamide is not essential to the function of yeast EF-2 in protein synthesis.
蛋白质合成延伸因子2(EF-2)是白喉毒素ADP核糖基化活性的作用靶点,该活性导致细胞死亡。白喉酰胺是一种独特的翻译后修饰的组氨酸残基,既是这种ADP核糖基化所必需的,也是其发生位点。尽管在所有真核生物和古细菌的EF-2中都存在,但白喉酰胺的功能尚不清楚。在此,我们描述了酵母EF-2中白喉酰胺的组氨酸前体——组氨酸699的位点特异性诱变。携带质粒的EFT在组氨酸699密码子处进行随机诱变,并利用质粒洗牌技术筛选由突变EFT维持的菌株。对这些突变体进行白喉毒素抗性筛选。对49个毒素抗性分离株中的EFT进行序列分析,结果显示组氨酸699已被4种氨基酸之一取代:天冬酰胺、谷氨酰胺、亮氨酸或甲硫氨酸。发现了与这4种氨基酸对应的所有11种可能的密码子。由EF-2突变形式维持的细胞的生长速率略低于同基因野生型细胞。我们得出结论,尽管白喉酰胺的组氨酸前体具有严格的保守性和普遍的翻译后修饰,但它对酵母EF-2在蛋白质合成中的功能并非必不可少。