Logical engineering of D-arm and T-stem of tRNA that enhances d-amino acid incorporation

Abstract

A bacterial translation factor EF-P alleviates ribosomal stalling caused by polyproline sequence by accelerating Pro-Pro formation. EF-P recognizes a specific D-arm motif found in tRNAPro isoacceptors, 9-nt D-loop closed by a stable D-stem sequence, for Pro-selective peptidyl-transfer acceleration. It is also known that the T-stem sequence on aminoacyl-tRNAs modulates strength of the interaction with EF-Tu, giving enhanced incorporation of non-proteinogenic amino acids such as some N-methyl amino acids. Based on the above knowledge, we logically engineered tRNA’s D-arm and T-stem sequences to investigate a series of tRNAs for the improvement of consecutive incorporation of d-amino acids and an α, α-disubstituted amino acid. We have devised a chimera of tRNAPro1 and tRNAGluE2, referred to as tRNAPro1E2, in which T-stem of tRNAGluE2 was engineered into tRNAPro1. The combination of EF-P with tRNAPro1E2NNN pre-charged with d-Phe, d-Ser, d-Ala, and/or d-Cys has drastically enhanced expression level of not only linear peptides but also a thioether-macrocyclic peptide consisting of the four consecutive d-amino acids over the previous method using orthogonal tRNAs.

Ribosomal Incorporation of Aromatic Oligoamides as Peptide Sidechain Appendages

“…Multiple directions exist to further improve the breadth of these reactions including, but not limited to, the optimization of tRNA units. [2,25,26] Interest in increasing the diversity of mRNA-encoded chemical entities is driven by the possibility to implement selection from very large libraries. Non-peptidic features may enhance resistance to protease degradation, cell penetration or, in the case of foldamers, conformational control.…”

Cell-Free Approach for Non-canonical Amino Acids Incorporation Into Polypeptides

“…In some cases, a group of substrates, including N-methylated, D-amino acids and β-amino acids, based on the canonical ones (labeled as single letter) are tested and divided into groups linked with ">" indicating the decrease of suppression efficiency. 2018) and 2-aminoisobutyric acid (Figure 6, compound 57-58, 59-61, and 68) (Katoh et al, 2017b). The design of tRNA ProE2 is inspired by the recent breakthrough in discovery of the recognition elements in tRNA for recruiting EF-P for enhanced peptide bond formation (Katoh et al, 2016).…”

Ribosomal Incorporation of Aromatic Oligoamides as Peptide Sidechain Appendages

“…The sidechain of Amf(‐Gly‐QPQ‐Ac), which spans a full foldamer helix turn, is by far the largest and most structurally defined ever incorporated within a peptide sequence by the ribosome. Multiple directions exist to further improve the breadth of these reactions including, but not limited to, the optimization of tRNA units . Interest in increasing the diversity of mRNA‐encoded chemical entities is driven by the possibility to implement selection from very large libraries.…”