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

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 tRNA(Pro) 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 tRNA(Pro1) and tRNA(GluE2), referred to as tRNA(Pro1E2), in which T-stem of tRNA(GluE2) was engineered into tRNA(Pro1). The combination of EF-P with tRNA(Pro1E2)(NNN) 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.