Pyrrolysine analogues as substrates for pyrrolysyl-tRNA synthetase.
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.
Polycarpo
Carla R
CR
Department of Chemistry, Yale University, New Haven, CT 06520-8114, USA.
Herring
Stephanie
S
Bérubé
Amélie
A
Wood
John L
JL
Söll
Dieter
D
Ambrogelly
Alexandre
A
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
D001426:Bacterial Proteins; D012333:RNA, Messenger; D001616:beta-Galactosidase; D014367:Tryptophan Synthase; D000604:Amino Acyl-tRNA Synthetases; C456839:pyrrolysine; D008239:Lysine