Initiation of Protein Synthesis with Non-Canonical Amino Acids In Vivo.

By transplanting identity elements into E. coli tRNA ^fMet , we have engineered an orthogonal initiator tRNA (itRNA ^Ty2 ) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNA ^Ty2 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 tRNA ^fMet 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.
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