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

摘要

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 alpha, alpha-disubstituted amino acid. We have devised a chimera of tRNA(Pro1) and tRNA(GluE2), referred to as tRNA(Pro1E2), inwhich 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 thioethermacrocyclic peptide consisting of the four consecutive D-amino acids over the previous method using orthogonal tRNAs.