Methanogenic archaea encoding Pyrrolysine maintain ambiguous amber codon usage

Natural genetic code expansion is a phenomenon wherein an additional amino acid is encoded by a stop codon. These nonstandard amino acids are beneficial as they facilitate novel biochemical reactions. However, code expansion leads to ambiguity at the recoded stop codon, which can either be read-through or terminated. Pyrrolysine (Pyl) is encoded by the amber codon (TAG/UAG) and is widespread in archaea, where it is required for methylamine-mediated methanogenesis, an environmentally important metabolism. Mechanisms to conditionally suppress the amber stop codon for Pyl installation during protein synthesis have not been identified. In the model methanogen, <i>Methanosarcina acetivorans,</i> we demonstrate that the UAG codon encodes dual meaning as stop and Pyl. Our data suggest that expression of Pyl biosynthesis and incorporation genes is tuned to the cellular demand for Pyl, which might allow these archaea to navigate ambiguous stop decoding in response to environmental cues.