Translation initiation with exotic amino acids using EF-P-responsive artificial initiator tRNA.

Takayuki Katoh, Hiroaki Suga

Nucleic acids research 2023 Aug 25

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Translation initiation using noncanonical initiator substrates with poor peptidyl donor activities, such as N-acetyl-l-proline (AcPro), induces the N-terminal drop-off-reinitiation event. Thereby, the initiator tRNA drops-off from the ribosome and the translation reinitiates from the second amino acid to yield a truncated peptide lacking the N-terminal initiator substrate. In order to suppress this event for the synthesis of full-length peptides, here we have devised a chimeric initiator tRNA, referred to as tRNAiniP, whose D-arm comprises a recognition motif for EF-P, an elongation factor that accelerates peptide bond formation. We have shown that the use of tRNAiniP and EF-P enhances the incorporation of not only AcPro but also d-amino, β-amino and γ-amino acids at the N-terminus. By optimizing the translation conditions, e.g. concentrations of translation factors, codon sequence and Shine-Dalgarno sequence, we could achieve complete suppression of the N-terminal drop-off-reinitiation for the exotic amino acids and enhance the expression level of full-length peptide up to 1000-fold compared with the use of the ordinary translation conditions. © The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.

Citation

Takayuki Katoh, Hiroaki Suga. Translation initiation with exotic amino acids using EF-P-responsive artificial initiator tRNA. Nucleic acids research. 2023 Aug 25;51(15):8169-8180