An aminoacyl-tRNA synthetase that specifically activates pyrrolysine

<jats:p> Pyrrolysine, the 22nd cotranslationally inserted amino acid, was found in the <jats:italic>Methanosarcina barkeri</jats:italic> monomethylamine methyltransferase protein in a position that is encoded by an in-frame UAG stop codon in the mRNA. <jats:italic>M. barkeri</jats:italic> encodes a special amber suppressor tRNA (tRNA <jats:sup>Pyl</jats:sup> ) that presumably recognizes this UAG codon. It was reported that Lys-tRNA <jats:sup>Pyl</jats:sup> can be formed by the aminoacyl-tRNA synthetase-like <jats:italic>M. barkeri</jats:italic> protein PylS [Srinivasan, G., James, C. M. & Krzycki, J. A. (2002) <jats:italic>Science</jats:italic> 296, 1459–1462], whereas a later article showed that Lys-tRNA <jats:sup>Pyl</jats:sup> is synthesized by the combined action of LysRS1 and LysRS2, the two different <jats:italic>M. barkeri</jats:italic> lysyl-tRNA synthetases. Pyrrolysyl-tRNA <jats:sup>Pyl</jats:sup> formation was presumed to result from subsequent modification of lysine attached to tRNA <jats:sup>Pyl</jats:sup> . To investigate whether pyrrolysine can be directly attached to tRNA <jats:sup>Pyl</jats:sup> we chemically synthesized pyrrolysine. We show that PylS is a specialized aminoacyl-tRNA synthetase for charging pyrrolysine to tRNA <jats:sup>Pyl</jats:sup> ; lysine and tRNA <jats:sup>Lys</jats:sup> are not substrates of the enzyme. In view of the properties of PylS we propose to name this enzyme pyrrolysyl-tRNA synthetase. In contrast, the LysRS1:LysRS2 complex does not recognize pyrrolysine and charges tRNA <jats:sup>Pyl</jats:sup> with lysine. These <jats:italic>in vitro</jats:italic> data suggest that <jats:italic>Methanosarcina</jats:italic> cells have two pathways for acylating the suppressor tRNA <jats:sup>Pyl</jats:sup> . This would ensure efficient translation of the in-frame UAG codon in case of pyrrolysine deficiency and safeguard the biosynthesis of the proteins whose genes contain this special codon. </jats:p>