Initiation of Protein Synthesis with Non‐Canonical Amino Acids In Vivo

Abstract

By transplanting identity elements into E. coli tRNAfMet, we have engineered an orthogonal initiator tRNA (itRNATy2) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNATy2 can initiate translation in vivo with aromatic non‐canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNAfMet from the genome afforded an E. coli strain in which the efficiency of non‐canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo.

Geared Toward Applications: A Perspective on Functional Sequence-Controlled Polymers

“…One particularly noteworthy direction that holds great promise to generate protein-like synthetic polymers is to engineer the ribosome as a polymer synthesis machine. This formidable task may require many iterations of mutagenesis and structural optimization to the natural ribosome- t RNA machinery, but the seminal work by Fahnestock and Rich and recent reports by Suga, − Schepartz, − and Jewett have shown important advances in engineering the ribosome- t RNA machinery to incorporate non-natural β- and γ-amino acids in polypeptides as well as α-hydroxyacids in polyesters.…”

The Potential of a Protein Model Synthesized Absent of Methionine

“…In E. coli, substitutions of the methionine initiating anticodon CAU with alternative anti-codons have shown an ability to initiate protein synthesis using valine, phenylalanine, isoleucine, tryptophan, and glutamine [98][99][100][101]. In a recent review [102] and subsequent experiments [103,104], the initiation of protein synthesis This takes place in the endoplasmic reticulum of the cell and is mediated by the protein disulfide isomerase (PDI). Upon disulfide bridge formation, PDI is reduced and must transfer its electrons to the disulfide oxidase enzyme endoplasmic reticulum oxireductin 1 (Ero1) in order to return to its active state.…”

“…In E. coli, substitutions of the methionine initiating anti-codon CAU with alternative anti-codons have shown an ability to initiate protein synthesis using valine, phenylalanine, isoleucine, tryptophan, and glutamine [98][99][100][101]. In a recent review [102] and subsequent experiments [103,104], the initiation of protein synthesis using non-canonical amino acids with mis-acetylated formyl methionine tRNAs both in vivo and in vitro is shown to be possible. These findings are very new; however, they display incredible plasticity and adaptability of translation machinery.…”