The tRNA discriminator base defines the mutual orthogonality of two distinct pyrrolysyl-tRNA synthetase/tRNA(Pyl) pairs in the same organism

Site-specific incorporation of distinct non-canonical amino acids into proteins via genetic code expansion requires mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Pyrrolysyl-tRNA synthetase (PylRS)/tRNA(Pyl) pairs are ideal for genetic code expansion and have been extensively engineered for developing mutually orthogonal pairs. Here, we identify two novel wild-type PylRS/tRNA(Pyl) pairs simultaneously present in the deep-rooted extremely halophilic euryarchaeal methanogen Candidatus Methanohalarchaeum thermophilum HMET1, and show that both pairs are functional in the model halophilic archaeon Haloferax volcanii. These pairs consist of two different PylRS enzymes and two distinct tRNAs with dissimilar discriminator bases. Surprisingly, these two PylRS/tRNA(Pyl) pairs display mutual orthogonality enabled by two unique features, the A73 discriminator base of tRNA(Pyl)2 and a shorter motif 2 loop in PylRS2. In vivo translation experiments show that tRNA(Pyl)2 charging by PylRS2 is defined by the enzyme's shortened motif 2 loop. Finally, we demonstrate that the two HMET1 PylRS/tRNA(Pyl) pairs can simultaneously decode UAG and UAA codons for incorporation of two distinct noncanonical amino acids into protein. This example of a single base change in a tRNA leading to additional coding capacity suggests that the growth of the genetic code is not yet limited by the number of identity elements fitting into the tRNA structure.