Recognition of pyrrolysine tRNA by the Desulfitobacterium hafniense pyrrolysyl-tRNA synthetase

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Abstract
Pyrrolysine (Pyl), the 22nd co-translationally inserted amino acid, is incorporated in response to a UAG amber stop codon. Pyrrolysyl-tRNA synthetase (PylRS) attaches Pyl to its cognate tRNA, the special amber suppressor tRNAPyl. The genes for tRNAPyl (pylT) and PylRS (pylS) are found in all members of the archaeal family Methanosarcinaceae, and in Desulfitobacterium hafniense. The activation and aminoacylation properties of D. hafniense PylRS and the nature of the tRNAPyl identity elements were determined by measuring the ability of 24 mutant tRNAPyl species to be aminoacylated with the pyrrolysine analog N-ε-cyclopentyloxycarbonyl-l-lysine. The discriminator base G73 and the first base pair (G1·C72) in the acceptor stem were found to be major identity elements. Footprinting analysis showed that PylRS binds tRNAPyl predominantly along the phosphate backbone of the T-loop, the D-stem and the acceptor stem. Significant contacts with the anticodon arm were not observed. The tRNAPyl structure contains the highly conserved T-loop contact U54·A58 and position 57 is conserved as a purine, but the canonical T- to D-loop contact between positions 18 and 56 was not present. Unlike most tRNAs, the tRNAPyl anticodon was shown not to be important for recognition by bacterial PylRS.