Pyrrolysine (symbol Pyl or O;[2] encoded by the 'amber' stop codon UAG) is an α-amino acid that is used in the biosynthesis of proteins in some methanogenic archaea and bacteria;[3][4] it is not present in humans.

[6] Unlike posttranslational modifications of lysine such as hydroxylysine, methyllysine, and hypusine, pyrrolysine is incorporated during translation (protein synthesis) as directed by the genetic code, just like the standard amino acids.

[12] (See Expanded genetic code) It was originally proposed that a specific downstream sequence "PYLIS", forming a stem-loop in the mRNA, forced the incorporation of pyrrolysine instead of terminating translation in methanogenic archaea.

[14] The pylT (tRNA) and pylS (aa-tRNA synthase) genes are part of an operon of Methanosarcina barkeri, with homologues in other sequenced members of the Methanosarcinaceae family: M. acetivorans, M. mazei, and M. thermophila.

The current (2022) view, given available sequences for tRNA and Pyl-tRNA (PylRS) synthase genes, is that:[17] Earlier evolutionary scenarios were limited by the taxonomic range of known synthases: The tRNA(CUA) can be charged with lysine in vitro by the concerted action of the M. barkeri Class I and Class II lysyl-tRNA synthetases, which do not recognize pyrrolysine.

Charging a tRNA(CUA) with lysine was originally hypothesized to be the first step in translating UAG amber codons as pyrrolysine, a mechanism analogous to that used for selenocysteine.

More recent data favor direct charging of pyrrolysine on to the tRNA(CUA) by the protein product of the pylS gene, leading to the suggestion that the LysRS1:LysRS2 complex may participate in a parallel pathway designed to ensure that proteins containing the UAG codon can be fully translated using lysine as a substitute amino acid in the event of pyrrolysine deficiency.