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

Abstract

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, tRNA Pyl, which is aminoacylated with Pyl by pyrrolysyl-tRNA synthetase (PylRS). We compared the behavior of several archaeal and bacterial PylRS enzymes towards tRNA Pyl. Equilibrium binding analysis revealed that archaeal PylRS proteins bind tRNA Pyl with higher affinity ( K D = 0.1–1.0 μM) than D. hafniense PylRS ( K D = 5.3–6.9 μM). In aminoacylation the archaeal PylRS enzymes did not distinguish between archaeal and bacterial tRNA Pyl 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.