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, 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 microM) than D. hafniense PylRS (K(D)=5.3-6.9 microM). 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.
Herring
Stephanie
S
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
Ambrogelly
Alexandre
A
Gundllapalli
Sarath
S
O'Donoghue
Patrick
P
Polycarpo
Carla R
CR
Söll
Dieter
D
D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001483:Base Sequence; D045858:Desulfitobacterium / Q000201:enzymology / Q000235:genetics; D004789:Enzyme Activation; D014644:Genetic Variation; D008239:Lysine / Q000031:analogs & derivatives* / Q000378:metabolism; D017020:Methanosarcina / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017434:Protein Structure, Tertiary / Q000502:physiology; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
D000604:Amino Acyl-tRNA Synthetases; C456839:pyrrolysine; D008239:Lysine