Protein synthesis initiates with methionine, encoded by the initiator codon AUG, in all domains of life. Despite this conservation, methionine is not an essential component of translation initiation. Here we report a novel method to initiate in vivo protein synthesis with noncanonical amino acids (ncAAs), giving rise to proteins with an ncAA at their N-terminus. This method relies on a chimeric initiator tRNA (itRNATy2) derived from the Escherichia coli initiator tRNA (EctRNAfMet) and Methanocaldococcus jannaschii tyrosyine tRNA genes. We demonstrate that this chimeric tRNA is a good substrate for aminoacylation by the M. jannaschii tyrosyl-tRNA synthetase—an aminoacyl-tRNA synthetase (aaRS) that is widely used for genetic code expansion. Furthermore, we show that itRNATy2 is compatible with the E. coli initiation machinery and can initiate protein synthesis, in response to the UAG nonsense codon, with a variety of aromatic ncAAs bearing diverse sidechains. Although initiation with ncAAs was inefficient in wildtype E. coli, deleting redundant copies of the EctRNAfMet gene from the genome afforded an E. coli strain in which the efficiency of ncAA initiation was equivalent to elongation. Using this optimized system we produced proteins containing two distinct ncAAs encoded by reprogrammed initiator and elongating codons.

Initiating protein synthesis with noncanonical amino acids in vivo

ACS Spring Meeting 2021

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