Activation of the pyrrolysine suppressor tRNA requires formation of a ternary complex with class I and class II lysyl-tRNA synthetases

Monomethylamine methyltransferase of the archaeon Methanosarcina barkeri contains a rare amino acid, pyrrolysine, encoded by the termination codon UAG. Translation of this UAG requires the aminoacylation of the corresponding amber suppressor tRNA(Pyl). Previous studies reported that tRNA(Pyl) could be amino acylated by the synthetase-like protein PyIS. We now show that tRNA(Pyl) is efficiently aminoacylated in the presence of both the class I LysRS and class II LysRS of M. barkeri, but not by either enzyme acting alone or by PyIS. In vitro studies show that both the class I and II LysRS enzymes must bind tRNA(Pyl) in order for the aminoacylation reaction to proceed. Structural modeling and selective inhibition experiments indicate that the class I and II LysRSs form a ternary complex with tRNA(Pyl), with the aminoacylation activity residing in the class II enzyme.