Mistakes in translation: Reflections on mechanism.
Mistakes in translation: Reflections on mechanism.
复制标题
DOI：
10.1371/journal.pone.0180566
复制
发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Prestegard JH
中科院分区：
文献类型：
--
作者：
Liu Y;Sharp JS;Do DH;Kahn RA;Schwalbe H;Buhr F;Prestegard JH
信使RNA翻译成蛋白质的错误显然不利于用于生物物理研究或生物制药市场的纯蛋白质的重组生产。然而，它们也可以提供对翻译过程的机械细节的洞察。错误通常涉及将具有丰富密码子的氨基酸替换为具有稀有密码子的氨基酸，不同之处在于将G碱基替换为A碱基，如在赖氨酸（AAA）替换为精氨酸（阿加）的情况下。在这些情况下，人们预期取代频率取决于相应tRNA的相对丰度，因此，人们可能预期具有相同稀有密码子的所有位点的频率相似。在这里，我们证明，对于ADP-核糖基化因子从酵母表达在E。大肠杆菌中，赖氨酸取代精氨酸的频率是不相同的9个位点含有一个罕见的精氨酸密码子;错误掺入频率，而不是从小于1%至16%不等。我们认为，密码子发生的背景（罕见位点的聚集）可能是造成变异的原因。用于确定错误掺入频率的方法涉及分别在15 N和14 N富集培养基中平行表达野生型和密码子优化基因的产物的新型质谱分析。该方法的高灵敏度和低材料要求使其成为收集与其他错误相关的数据的有前途的技术。额外的数据可能是有价值的，在完善模型的核糖体翻译延伸过程。
Mistakes in translation of messenger RNA into protein are clearly a detriment to the recombinant production of pure proteins for biophysical study or the biopharmaceutical market. However, they may also provide insight into mechanistic details of the translation process. Mistakes often involve the substitution of an amino acid having an abundant codon for one having a rare codon, differing by substitution of a G base by an A base, as in the case of substitution of a lysine (AAA) for arginine (AGA). In these cases one expects the substitution frequency to depend on the relative abundances of the respective tRNAs, and thus, one might expect frequencies to be similar for all sites having the same rare codon. Here we demonstrate that, for the ADP-ribosylation factor from yeast expressed in E. coli, lysine for arginine substitutions frequencies are not the same at the 9 sites containing a rare arginine codon; mis-incorporation frequencies instead vary from less than 1 to 16%. We suggest that the context in which the codons occur (clustering of rare sites) may be responsible for the variation. The method employed to determine the frequency of mis-incorporation involves a novel mass spectrometric analysis of the products from the parallel expression of wild type and codon-optimized genes in 15N and 14N enriched media, respectively. The high sensitivity and low material requirements of the method make this a promising technology for the collection of data relevant to other mis-incorporations. The additional data could be of value in refining models for the ribosomal translation elongation process.