Initiating translation with D-amino acids
Initiating translation with D-amino acids
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DOI：
10.1261/rna.1020708
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发表时间：
2008-07-01
期刊：
影响因子：
4.5
通讯作者：
Suga, Hiroaki
中科院分区：
文献类型：
--
作者：
Goto, Yuki;Murakami, Hiroshi;Suga, Hiroaki
在这里，我们报告的实验证据表明，翻译起始装置接受D-氨基酸（（D）aa），而不是只有L-甲硫氨酸，作为引发剂。使用flexizyme技术将十九个（D）aa（作为其天然L-氨基酸的立体异构体）加载到起始物tRNA（CAU）（fMet）上，并测试在缺乏甲硫氨酸的重构大肠杆菌翻译系统中的起始，即。例如，起始子从甲硫氨酸重编程为（D）aa。值得注意的是，所有（D）aa都可以启动翻译，而启动效率取决于侧链的类型。以（D）aa起始的肽产物通常为非甲酰化形式，表明甲硫氨酰-tRNA甲酰转移酶对相应的（D）aa-（tRNA）（CAU）甲酰化较差。虽然（D）aa-tRNA（CAU）（fMet）的低效转录导致相应肽的适度表达，但（D）aa-tRNA（CAU）（fMet）的预乙酰化显著增加表达水平，这意味着转录效率是（D）aa的起始效率的关键决定因素之一。我们的研究结果不仅提供了翻译起始耐受Daa的实验证据，而且为mRNA指导合成N端为（D）aa或酰基-（D）aa的肽提供了新的手段。
Here we report experimental evidence that the translation initiation apparatus accepts D-amino acids ((D)aa), as opposed to only L-methionine, as initiators. Nineteen (D)aa, as the stereoisomers to their natural L-amino acids, were charged onto initiator tRNA(CAU)(fMet) using flexizyme technology and tested for initiation in a reconstituted Escherichia coli translation system lacking methionine, i. e., the initiator was reprogrammed from methionine to (D)aa. Remarkably, all (D)aa could initiate translation while the efficiency of initiation depends upon the type of side chain. The peptide product initiated with (D)aa was generally in a nonformylated form, indicating that methionyl-tRNA formyltransferase poorly formylated the corresponding (D)aa-(tRNA)(CAU). Although the inefficient formylation of (D)aa-tRNA(CAU)(fMet) resulted in modest expression of the corresponding peptide, preacetylation of (D)aa-tRNA(CAU)(fMet) dramatically increased expression level, implying that the formylation efficiency is one of the critical determinants of initiation efficiency with (D)aa. Our findings provide not only the experimental evidence that translation initiation tolerates Daa, but also a new means for the mRNA-directed synthesis of peptides capped with (D)aa or acyl-(D)aa at the N terminus.