Affiliation:
1. Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Abstract
Ribosomal incorporation of
d
-α-amino acids (
d
AA) and
N
-methyl-
l
-α-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’.
Funder
Japan Society for the Promotion of Science
Subject
General Agricultural and Biological Sciences,General Biochemistry, Genetics and Molecular Biology
Cited by
1 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献
1. Reactivity and mechanism in chemical and synthetic biology;Philosophical Transactions of the Royal Society B: Biological Sciences;2023-01-11