title: Pyrrolysine analogues as substrates for pyrrolysyl-tRNA synthetase.
abstract: In certain methanogenic archaea a new amino acid, pyrrolysine (Pyl), is inserted at in-frame UAG codons in the mRNAs of some methyltransferases. Pyl is directly acylated onto a suppressor tRNA(Pyl) by pyrrolysyl-tRNA synthetase (PylRS). Due to the lack of a readily available Pyl source, we looked for structural analogues that could be aminoacylated by PylRS onto tRNA(Pyl). We report here the in vitro aminoacylation of tRNA(Pyl) by PylRS with two Pyl analogues: N-epsilon-d-prolyl-l-lysine (d-prolyl-lysine) and N-epsilon-cyclopentyloxycarbonyl-l-lysine (Cyc). Escherichia coli, transformed with the tRNA(Pyl) and PylRS genes, suppressed a lacZ amber mutant dependent on the presence of d-prolyl-lysine or Cyc in the medium, implying that the E. coli translation machinery is able to use Cyc-tRNA(Pyl) and d-prolyl-lysine-tRNA(Pyl) as substrates during protein synthesis. Furthermore, the formation of active beta-galactosidase shows that a specialized mRNA motif is not essential for stop-codon recoding, unlike for selenocysteine incorporation.
authors.affiliation: Department of Chemistry, Yale University, New Haven, CT 06520-8114, USA.
authors.forename: Carla R
authors.initials: CR
authors.lastname: Polycarpo
authors.forename: Stephanie
authors.initials: S
authors.lastname: Herring
authors.forename: Amélie
authors.initials: A
authors.lastname: Bérubé
authors.forename: John L
authors.initials: JL
authors.lastname: Wood
authors.forename: Dieter
authors.initials: D
authors.lastname: Söll
authors.forename: Alexandre
authors.initials: A
authors.lastname: Ambrogelly
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D008239:Lysine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger; D013379:Substrate Specificity; D013997:Time Factors; D014367:Tryptophan Synthase / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D012333:RNA, Messenger; D001616:beta-Galactosidase; D014367:Tryptophan Synthase; D000604:Amino Acyl-tRNA Synthetases; C456839:pyrrolysine; D008239:Lysine