PDF(Pubmed)
Abstract：
Ribosomal incorporation of d-α-amino acids (dAA) and N-methyl-l-α-amino acids (MeAA) with negatively charged sidechains, such as d-Asp, d-Glu, MeAsp and MeGlu, into nascent peptides is far more inefficient compared to those with neutral or positively charged ones. This is because of low binding affinity of their aminoacyl-transfer RNA (tRNA) to elongation factor-thermo unstable (EF-Tu), a translation factor responsible for accommodation of aminoacyl-tRNA onto ribosome. It is well known that EF-Tu binds to two parts of aminoacyl-tRNA, the amino acid moiety and the T-stem; however, the amino acid binding pocket of EF-Tu bearing Glu and Asp causes electric repulsion against the negatively charged amino acid charged on tRNA. To circumvent this issue, here we adopted two strategies: (i) use of an EF-Tu variant, called EF-Sep, in which the Glu216 and Asp217 residues in EF-Tu are substituted with Asn216 and Gly217, respectively; and (ii) reinforcement of the T-stem affinity using an artificially developed chimeric tRNA, tRNAPro1E2, whose T-stem is derived from Escherichia coli tRNAGlu that has high affinity to EF-Tu. Consequently, we could successfully enhance the incorporation efficiencies of d-Asp, d-Glu, MeAsp and MeGlu and demonstrated for the first time, to our knowledge, ribosomal synthesis of macrocyclic peptides containing multiple d-Asp or MeAsp. This article is part of the theme issue \'Reactivity and mechanism in chemical and synthetic biology\'.
摘要：
带负电荷侧链的d-α-氨基酸（dAA）和N-甲基-1-α-氨基酸（MeAA）的核糖体掺入，比如d-Asp,d-Glu,MeAsp和MeGlu,与带有中性或带正电荷的肽相比，新生肽的效率要低得多。这是因为它们的氨酰基转移RNA（tRNA）与延伸因子热不稳定（EF-Tu）的结合亲和力低，一种翻译因子，负责将氨酰基tRNA调节到核糖体上。众所周知，EF-Tu与氨酰基-tRNA的两个部分结合，氨基酸部分和T-茎;然而,带有Glu和Asp的EF-Tu的氨基酸结合袋引起对tRNA上带负电荷的氨基酸的电排斥。为了避免这个问题，在这里，我们采用了两种策略：(I)使用EF-Tu变体，叫做EF-Sep,其中EF-Tu中的Glu216和Asp217残基分别被Asn216和Gly217取代;和（ii）使用人工开发的嵌合tRNA增强T-茎亲和力，tRNAPro1E2，其T-茎来自大肠杆菌tRNAGlu，对EF-Tu具有高亲和力。因此,我们可以成功地提高d-Asp的掺入效率，d-Glu,MeAsp和MeGlu第一次演示，根据我们的知识，含有多个d-Asp或MeAsp的大环肽的核糖体合成。本文是“化学和合成生物学中的反应性和机理”主题问题的一部分。
