Recent developments of engineered translational machineries for the incorporation of non-canonical amino acids into polypeptides.

Naohiro Terasaka,Anna-Skrollan Geiermann,Yuki Goto,Yoshihiko Iwane,Hiroaki Suga

doi:10.3390/ijms16036513

Abstract

Genetic code expansion and reprogramming methodologies allow us to incorporate non-canonical amino acids (ncAAs) bearing various functional groups, such as fluorescent groups, bioorthogonal functional groups, and post-translational modifications, into a desired position or multiple positions in polypeptides both in vitro and in vivo. In order to efficiently incorporate a wide range of ncAAs, several methodologies have been developed, such as orthogonal aminoacyl-tRNA-synthetase (AARS)–tRNA pairs, aminoacylation ribozymes, frame-shift suppression of quadruplet codons, and engineered ribosomes. More recently, it has been reported that an engineered translation system specifically utilizes an artificially built genetic code and functions orthogonally to naturally occurring counterpart. In this review we summarize recent advances in the field of ribosomal polypeptide synthesis containing ncAAs.

Highlights

Assignment of 20 canonical amino acids to trinucleotides, so-called codons, is achieved by specific acylation of tRNA with cognate amino acid catalyzed by aminoacyl-tRNA synthetase (AARS)
Since each tRNA has a trinucleotide that pairs with the codon, the codons on mRNA can be decoded by the cognate aminoacyl-tRNAs (AA-tRNAs) according to the genetic code; and ribosome is able to catalyze the formation of peptide bond along the mRNA
While 22 amino acids are used in the native translation system, it has been demonstrated that hundreds of different non-canonical amino acids can be incorporated into nascent polypeptide chain by engineering of the genetic code; for instance, those containing post-translational modified sidechains found in nature, but artificially designed fluorescent or bioorthogonal functional groups (Figure 1) [7]

Summary

Introduction

Assignment of 20 canonical (or proteinogenic) amino acids to trinucleotides, so-called codons, is achieved by specific acylation of tRNA with cognate amino acid catalyzed by aminoacyl-tRNA synthetase (AARS). While 22 amino acids are used in the native translation system, it has been demonstrated that hundreds of different non-canonical amino acids (ncAAs) can be incorporated into nascent polypeptide chain by engineering of the genetic code; for instance, those containing post-translational modified sidechains found in nature, but artificially designed fluorescent or bioorthogonal functional groups (Figure 1) [7]. For ncAA incorporation via initiation, (2i) formation of initiation complex with the ncAA-tRNAfMet assisted by MTF (Methionyl-tRNA formyltransferase) and IFs (initiation factors) and (3i) the efficient PT reaction between ncAA-tRNAfMet at the P site and AA-tRNA at the A site are required These engineered events are normally competed with canonical AA-tRNAs and/or release factors (RFs), which can potentially read the codon artificially assigned to ncAA. Position-1 bases are found in all cytoplasmic mature tRNAHisGUG from the three biological domains

Engineering of Translation Components for the Improvement of ncAAs

Conclusions and Perspectives

Methods