Pyrrolysine is not hardwired for cotranslational insertion at UAG codons

Pyrrolysine (Pyl), the 22nd naturally encoded amino acid, gets acylated to its distinctive UAG suppressor tRNA(Pyl) by the cognate pyrrolysyl-tRNA synthetase (PyIRS). Here we determine the RNA elements required for recognition and aminoacylation of tRNA(Pyl) in vivo by using the Pyl analog N-epsilon-cyclopentyloxycarbonyl-L-lysine. Forty-two Methanosarcina barkeri tRNA(Pyl) variants were tested in Escherichia coli for suppression of the lac amber A24 mutation; then relevant tRNA(Pyl) mutants were selected to determine in vivo binding to M. barkeri PyIRS in a yeast three-hybrid system and to measure in vitro tRNAPyl aminoacylation. tRNA(Pyl) identity elements include the discriminator base, the first base pair of the acceptor stem, the T-stem base pair G51:C63, and the anticodon flanking nucleoticles U33 and A37. Transplantation of the tRNA(Pyl) identity elements into the mitochondrial bovine tRNA(Ser) scaffold yielded chimeric tRNAs active both in vitro and in vivo. Because the anticoclon is not important for PyIRS recognition, a tRNA(Pyl) variant could be constructed that efficiently suppressed the lac opal U4 mutation in E. coli. These data suggest that tRNA(Pyl) variants may decode numerous codons and that tRNA(Pyi):PYIRS is a fine orthogonal tRNA:synthetase pair that facilitated the late addition of Pyl to the genetic code.