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 aminoacylated by the synthetase-like protein PylS. 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 PylS. 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.