Inefficient Delivery but Fast Peptide Bond Formation of Unnatural L-Aminoacyl-tRNAs in Translation
Inefficient Delivery but Fast Peptide Bond Formation of Unnatural L-Aminoacyl-tRNAs in Translation
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DOI：
10.1021/ja3063524
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发表时间：
2012-10-31
影响因子：
15
通讯作者：
Ehrenberg, Mans
中科院分区：
文献类型：
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作者：
Ieong, Ka-Weng;Pavlov, Michael Y.;Ehrenberg, Mans
非天然氨基酸（AAs）的翻译通常是低效的，并且它们从转移核糖核酸（tRNA）的翻译的动力学研究很少。在这里，小的和大的，非N-烷基化的，非天然的L-AAs到二肽的纯化与天然AAs使用猝灭流技术进行了比较。令人惊讶的是，所有的降解都发生在两个阶段：快速然后缓慢，非天然AA-tRNA的降解以与天然Phe-tRNA（Phe）相似的快速和缓慢阶段的速率进行。非天然AA-tRNA的慢相更为明显，这与它们已知的低效降解有关。重要的是，即使对于非天然的AA-tRNA，也可以通过使用高EF-Tu浓度和/或较低的反应温度使快速相占优势，这通常可用于改善纯化。此外，我们观察到的EF-Tu浓度对掺入的快相分数的影响使得能够直接测定翻译期间AA-tRNA对EF-Tu的亲和力。我们的未修饰的tRNA（Phe）衍生物衔接子装载了一个大的非天然AA，生物素基-赖氨酸，对EF-Tu：GTP的亲和力非常低，而同一tRNA体上的小的非天然AA对EF-Tu：GTP的亲和力与该tRNA上的天然AA基本相同，但仍比天然Phe-tRNA（Phe）低2倍。我们的结论是，非天然AA-tRNA降解的效率低下是由于EF-Tu向核糖体的传递效率低下，而不是核糖体上肽键形成缓慢。
Translations with unnatural amino acids (AAs) are generally inefficient, and kinetic studies of their incorporations from transfer ribonucleic acids (tRNAs) are few. Here, the incorporations of small and large, non-N-alkylated, unnatural L-AAs into dipeptides were compared with those of natural AAs using quench-flow techniques. Surprisingly, all incorporations occurred in two phases: fast then slow, and the incorporations of unnatural AA-tRNAs proceeded with rates of fast and slow phases similar to those for natural Phe-tRNA(Phe). The slow phases were much more pronounced with unnatural AA-tRNAs, correlating with their known inefficient incorporations. Importantly, even for unnatural AA-tRNAs the fast phases could be made dominant by using high EF-Tu concentrations and/or lower reaction temperature, which may be generally useful for improving incorporations. Also, our observed effects of EF-Tu concentration on the fraction of the fast phase of incorporation enabled direct assay of the affinities of the AA-tRNAs for EF-Tu during translation. Our unmodified tRNA(Phe) derivative adaptor charged with a large unnatural AA, biotinyl-lysine, had a very low affinity for EF-Tu:GTP, while the small unnatural AAs on the same tRNA body had essentially the same affinities to EF-Tu:GTP as natural AAs on this tRNA, but still 2-fold less than natural Phe-tRNA(Phe). We conclude that the inefficiencies of unnatural AA-tRNA incorporations were caused by inefficient delivery to the ribosome by EF-Tu, not slow peptide bond formation on the ribosome.