An archaeal genetic code with all TAG codons as pyrrolysine.

Multiple genetic codes developed during the evolution of eukaryotes and bacteria, yet no alternative genetic code is known for archaea. We used proteomics to confirm our prediction that certain archaea consistently incorporate pyrrolysine (Pyl) at TAG codons, supporting an alternative archaeal genetic code that we designate the Pyl code. This genetic code has 62 sense codons encoding 21 amino acids. In contrast to monophyletic genetic code distributions in bacteria, the archaeal Pyl code occurs sporadically, indicating that it arose independently in multiple lineages. We discovered that more than 1800 archaeal proteins contain Pyl, increasing the number of such proteins by two orders of magnitude. Additionally, five Pyl transfer RNA (tRNA) pyrrolysyl–tRNA synthetase pairs from Pyl-code archaea were used to introduce Pyl analogs into proteins in Escherichia coli. Editor's summary: The universal triple-nucleotide genetic code is common to all living organisms on Earth, but it varies in small and subtle ways across the tree of life. Kivenson et al. explored the reassignment of TAG codons from stop to pyrrolysine (Pyl) among diverse archaea and showed that this is not a rare event restricted to a few proteins; rather, it appears to represent a distinct genetic code that arose multiple independent times in different lineages. Proteomics data support the incorporation of Pyl into many proteins, and engineering experiments successfully assimilated it into Escherichia coli proteins. TAG occurs sporadically across the Archaea and seems to be more easily gained than lost, suggesting that a mobile element may have been instrumental in its evolution. —Caroline Ash INTRODUCTION: Although the genetic code was originally thought to be universal, alternative codon assignments have since been documented in bacteria and eukaryotes. In archaea, no alternative genetic code is known. However, a few archaeal genes use the standard stop codon TAG to encode pyrrolysine (Pyl), the 22nd proteinogenic amino acid. Additionally, the machinery for Pyl insertion has also become widely used for genetic code expansion, enabling precise incorporation of diverse noncanonical amino acids into proteins. Although Pyl was discovered in archaea more than two decades ago, it remains unknown whether the TAG codon has a dual encoding (stop/Pyl) or could be fully reassigned in some archaea. RATIONALE: Pyl-encoding archaeal genomes are thought to restrict TAG codon use and contain downstream stop codons after TAG to mitigate potential effects of readthrough. However, it remains unknown how TAG codons are interpreted throughout the genome. To systematically assess how TAG is used and search for Pyl-containing proteins, we analyzed codon usage patterns across archaeal genomes, revealing evidence for genome-wide reassignment of TAG from stop to Pyl in a subset of Pyl-encoding archaea. We tested these predictions experimentally through the cultivation of human gut and marine isolates followed by global and targeted proteomics. RESULTS: Bioinformatically, we predicted the presence of Pyl in >1800 proteins of various function. Proteomic data show that select archaea have genome-wide incorporation of Pyl at TAG codons. These archaea use an alternative genetic code, that we named the Pyl code, where the TAG stop codon is fully repurposed as a sense codon. The Pyl code is the only genetic code with 62 dedicated sense codons encoding 21 amino acids. Pyl is only conserved in the methylamine methyltransferases, suggesting that a functional role for Pyl is limited to these proteins. Additionally, Pyl tRNA synthetase pairs derived from Pyl-code archaea identified in this study enabled the incorporation of Pyl analogs into target proteins of Escherichia coli. CONCLUSION: We uncovered archaeal lineages in which the standard stop codon TAG has been reassigned genome-wide to encode Pyl, and the need to retain Pyl insertion in methylamine methyltransferases likely drives the adoption of this alternative genetic code. The sporadic distribution of the Pyl code and the presence of Pyl in essential proteins suggest that this genetic code may be more easily gained than lost. Finally, Pyl systems occurring in these archaea can be used to add Pyl analogs into proteins in E. coli, underscoring their applications for genetic code expansion and biotechnology. Discovery of a new genetic code in archaea.: The interpretation of TAG codons in Pyl-encoding archaeal genomes was unknown. We predicted genome-wide TAG codon reassignment in silico and then tested these predictions in Methanomethylophilus alvi and Methanococcoides burtonii isolates using targeted and global proteomics. We confirmed that the TAG codon is encoded as Pyl genome-wide. The Pyl code has 62 sense codons and only two stop codons and has a sporadic distribution among archaea. [ABSTRACT FROM AUTHOR]