Ribosomal incorporation of negatively charged d-α- and N-methyl-l-α-amino acids enhanced by EF-Sep
Ribosomal incorporation of negatively charged d-α- and N-methyl-l-α-amino acids enhanced by EF-Sep
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DOI：
10.1098/rstb.2022.0038
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发表时间：
2023-02-27
影响因子：
6.3
通讯作者：
Suga, Hiroaki
中科院分区：
文献类型：
--
作者：
Katoh, Takayuki;Suga, Hiroaki
核糖体将带负电荷侧链（如d-Asp、d-Glu、（Me）Asp和（Me）Glu）的d- α -氨基酸（dAA）和n -甲基-l- α -氨基酸（Me）AA)掺入新生肽的效率远低于带中性或带正电荷侧链的核糖体。这是因为它们的氨基酰基转移RNA （tRNA）与热不稳定延伸因子（EF-Tu）的结合亲和力较低，这是一种负责将氨基酰基tRNA调节到核糖体上的翻译因子。众所周知，EF-Tu结合了氨基酰基trna的两个部分，氨基酸部分和t -茎；然而，携带Glu和Asp的EF-Tu的氨基酸结合袋对tRNA上带负电的氨基酸产生电排斥。为了避免这个问题，我们采用了两种策略：(i)使用EF-Tu变体，称为EF-Sep，其中EF-Tu中的Glu216和Asp217残基分别被Asn216和Gly217取代；（ii）使用人工开发的嵌合tRNA（Pro1E2）来增强t -茎的亲和力，其t -茎来源于大肠杆菌tRNA(Glu)，与EF-Tu具有高亲和力。因此，我们可以成功地提高d-Asp， d-Glu, (Me)Asp和（Me）Glu的掺入效率，并首次证明，据我们所知，核糖体合成含有多个d-Asp或（Me）Asp的大环肽。本文是“化学与合成生物学中的反应性及其机制”主题的一部分。
Ribosomal incorporation of d-alpha-amino acids (dAA) and N-methyl-l-alpha-amino acids ((Me)AA) with negatively charged sidechains, such as d-Asp, d-Glu, (Me)Asp and (Me)Glu, 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, tRNA(Pro1E2), whose T-stem is derived from Escherichia coli tRNA(Glu) that has high affinity to EF-Tu. Consequently, we could successfully enhance the incorporation efficiencies of d-Asp, d-Glu, (Me)Asp and (Me)Glu and demonstrated for the first time, to our knowledge, ribosomal synthesis of macrocyclic peptides containing multiple d-Asp or (Me)Asp.This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.