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Kinetics of d-Amino Acid Incorporation in Translation.
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2019-01-16 , DOI: 10.1021/acschembio.8b00952 Josefine Liljeruhm 1 , Jinfan Wang 1 , Marek Kwiatkowski 1 , Samudra Sabari 1 , Anthony C Forster 1
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2019-01-16 , DOI: 10.1021/acschembio.8b00952 Josefine Liljeruhm 1 , Jinfan Wang 1 , Marek Kwiatkowski 1 , Samudra Sabari 1 , Anthony C Forster 1
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Despite the stereospecificity of translation for l-amino acids (l-AAs) in vivo, synthetic biologists have enabled ribosomal incorporation of d-AAs in vitro toward encoding polypeptides with pharmacologically desirable properties. However, the steps in translation limiting d-AA incorporation need clarification. In this work, we compared d- and l-Phe incorporation in translation by quench-flow kinetics, measuring 250-fold slower incorporation into the dipeptide for the d isomer from a tRNAPhe-based adaptor (tRNAPheB). Incorporation was moderately hastened by tRNA body swaps and higher EF-Tu concentrations, indicating that binding by EF-Tu can be rate-limiting. However, from tRNAAlaB with a saturating concentration of EF-Tu, the slow d-Phe incorporation was unexpectedly very efficient in competition with incorporation of the l isomer, indicating fast binding to EF-Tu, fast binding of the resulting complex to the ribosome, and rate-limiting accommodation/peptide bond formation. Subsequent elongation with an l-AA was confirmed to be very slow and inefficient. This understanding helps rationalize incorporation efficiencies in vitro and stereospecific mechanisms in vivo and suggests approaches for improving incorporation.
中文翻译：
d-氨基酸并入翻译的动力学。
尽管体内l-氨基酸（l-AAs）的翻译具有立体特异性，但合成生物学家已使核糖体掺入d-AAs可以体外编码具有药理学所需特性的多肽。但是，限制d-AA掺入的翻译步骤需要澄清。在这项工作中，我们通过淬灭流动力学比较了翻译中的d-和1-Phe掺入，测量了基于tRNAPhe的衔接子（tRNAPheB）将d异构体的二肽掺入速度慢了250倍。通过tRNA体置换和较高的EF-Tu浓度可适度加快掺入，表明EF-Tu的结合可能是限速的。然而，从饱和浓度的EF-Tu的tRNAAlaB来看，缓慢的d-Phe掺入与l异构体的掺入竞争中出乎意料地非常有效，指示与EF-Tu的快速结合，所得复合物与核糖体的快速结合以及限速调节/肽键的形成。随后用1-AA的伸长被证实是非常缓慢和低效的。这种理解有助于合理化体外结合效率和体内立体定向机制，并提出了改善结合的方法。
更新日期：2019-01-16
中文翻译：
d-氨基酸并入翻译的动力学。
尽管体内l-氨基酸（l-AAs）的翻译具有立体特异性，但合成生物学家已使核糖体掺入d-AAs可以体外编码具有药理学所需特性的多肽。但是，限制d-AA掺入的翻译步骤需要澄清。在这项工作中，我们通过淬灭流动力学比较了翻译中的d-和1-Phe掺入，测量了基于tRNAPhe的衔接子（tRNAPheB）将d异构体的二肽掺入速度慢了250倍。通过tRNA体置换和较高的EF-Tu浓度可适度加快掺入，表明EF-Tu的结合可能是限速的。然而，从饱和浓度的EF-Tu的tRNAAlaB来看，缓慢的d-Phe掺入与l异构体的掺入竞争中出乎意料地非常有效，指示与EF-Tu的快速结合，所得复合物与核糖体的快速结合以及限速调节/肽键的形成。随后用1-AA的伸长被证实是非常缓慢和低效的。这种理解有助于合理化体外结合效率和体内立体定向机制，并提出了改善结合的方法。
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