The amino-terminal domain of pyrrolysyl-tRNA synthetase is dispensable in vitro but required for in vivo activity

Pyrrolysine (Pyl) is co-translationally inserted into a subset of proteins in the Methanosarcinaceae and in Desulfitobacterium hafniense programmed by an in-frame UAG stop codon. Suppression of this UAG codon is mediated by the Pyl amber suppressor tRNA, tRNAPyl, which is aminoacylated with Pyl by pyrrolysyl-tRNA synthetase (PylRS). We compared the behavior of several archaeal and bacterial PylRS enzymes towards tRNAPyl. Equilibrium binding analysis revealed that archaeal PylRS proteins bind tRNAPyl with higher affinity (KD = 0.1–1.0 μM) than D. hafniense PylRS (KD = 5.3–6.9 μM). In aminoacylation the archaeal PylRS enzymes did not distinguish between archaeal and bacterial tRNAPyl species, while the bacterial PylRS displays a clear preference for the homologous cognate tRNA. We also show that the amino-terminal extension present in archaeal PylRSs is dispensable for in vitro activity, but required for PylRS function in vivo.