Sporadic distribution of a new archaeal genetic code with all TAG codons as pyrrolysine

Abstract Numerous genetic codes developed during the evolution of Eukaryotes and three are known in Bacteria, yet no alternative genetic code has been established for Archaea. Some bacterial and archaeal proteins include selenocysteine or pyrrolysine, the 21 st and 22 nd amino acids, but no evidence establishes the adoption of a genetic code in which a stop codon universally encodes either amino acid. Here, we used proteomics to confirm the prediction that certain Archaea consistently incorporate pyrrolysine at TAG codons, supporting a new archaeal genetic code which we designate Genetic Code 34. This genetic code has 62 sense codons encoding 21 amino acids, and only two stop codons. In contrast with monophyletic genetic code distributions in bacteria, Code 34 occurs sporadically. This, combined with evidence for lateral gene transfer of the code change machinery and anticipated barriers to code reversal, suggests Code 34 arose independently in multiple lineages. TAG codon distribution patterns in Code 34 genomes imply a wide range in time since code switch. We identified many new enzymes containing Pyl residues, raising questions about potential roles of this amino acid in protein structure and function. We used five new PylRS/tRNA Pyl pairs from Code 34 archaea to introduce new-to-nature pyrrolysine analogs into proteins in E. coli , demonstrating their utility for genetic code expansion. Graphical abstract