Logical engineering of D-arm and T-stem of tRNA that enhances d-amino acid incorporation.
Nucleic Acids Res
; 45(22): 12601-12610, 2017 Dec 15.
Article
in En
| MEDLINE
| ID: mdl-29155943
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
DNA, Recombinant
/
RNA, Transfer
/
RNA, Transfer, Amino Acyl
/
Amino Acids
Language:
En
Journal:
Nucleic Acids Res
Year:
2017
Document type:
Article
Affiliation country:
Japan