Ribosomal Incorporation of Negatively Charged D-α- and -methyl-l-α-amino Acids Enhanced by EF-Sep

Ribosomal incorporation of d-α-amino acids (dAA) and -methyl-l-α-amino acids (AA) with negatively charged sidechains, such as d-Asp, d-Glu, Asp and 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, whose T-stem is derived from tRNA that has high affinity to EF-Tu. Consequently, we could successfully enhance the incorporation efficiencies of d-Asp, d-Glu, Asp and Glu and demonstrated for the first time, to our knowledge, ribosomal synthesis of macrocyclic peptides containing multiple d-Asp or Asp. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.