Reduced Amino Acid Specificity of Mammalian Tyrosyl-tRNA Synthetase Is Associated with Elevated Mistranslation of Tyr Codons
Reduced Amino Acid Specificity of Mammalian Tyrosyl-tRNA Synthetase Is Associated with Elevated Mistranslation of Tyr Codons
复制标题
DOI：
10.1074/jbc.m114.564609
复制
发表时间：
2014-06-20
影响因子：
4.8
通讯作者：
Ibba, Michael
中科院分区：
文献类型：
--
作者：
Raina, Medha;Moghal, Adil;Ibba, Michael
质量控制在翻译的不同步骤进行，以将错误限制在mRNA指导的蛋白质合成期间每10，000个密码子中约一个误译密码子。最近的研究表明，在不同的生长条件下，翻译过程中的错误率实际上可能会有很大的差异。在这里，我们研究了在酪氨酸限制的中国仓鼠卵巢（CHO）细胞中产生的重组抗体合成过程中，Phe在Tyr密码子处的错误掺入。先前发现Tyr至Phe的置换在整个抗体中以高达0.7%的比率发生，而与翻译的Tyr密码子的身份或背景无关。尽管这种相对较高的误译率，没有观察到细胞活力的显着变化。Phe和Tyr水平的监测显示，错误率的变化与氨基酸库的变化相关，这表明酪氨酰-tRNA合成酶将tRNA（Tyr）与非同源的Phe误充电是造成误译的原因。CHO细胞质酪氨酰-tRNA合成酶的稳态动力学分析显示，与先前表征的细菌酶相比，Tyr对Phe的特异性低25倍，这与酪氨酸限制期间观察到的翻译错误率增加一致。哺乳动物和细菌酪氨酰-tRNA合成酶的功能比较显示，负责氨基酸识别的残基的关键差异，突出了翻译质量控制的进化限制的差异。
Quality control operates at different steps in translation to limit errors to approximately one mistranslated codon per 10,000 codons during mRNA-directed protein synthesis. Recent studies have suggested that error rates may actually vary considerably during translation under different growth conditions. Here we examined the misincorporation of Phe at Tyr codons during synthesis of a recombinant antibody produced in tyrosine-limited Chinese hamster ovary (CHO) cells. Tyr to Phe replacements were previously found to occur throughout the antibody at a rate of up to 0.7% irrespective of the identity or context of the Tyr codon translated. Despite this comparatively high mistranslation rate, no significant change in cellular viability was observed. Monitoring of Phe and Tyr levels revealed that changes in error rates correlated with changes in amino acid pools, suggesting that mischarging of tRNA(Tyr) with noncognate Phe by tyrosyl-tRNA synthetase was responsible for mistranslation. Steady-state kinetic analyses of CHO cytoplasmic tyrosyl-tRNA synthetase revealed a 25-fold lower specificity for Tyr over Phe as compared with previously characterized bacterial enzymes, consistent with the observed increase in translation error rates during tyrosine limitation. Functional comparisons of mammalian and bacterial tyrosyl-tRNA synthetase revealed key differences at residues responsible for amino acid recognition, highlighting differences in evolutionary constraints for translation quality control.