Pyrrolysine is not hardwired for cotranslational insertion at UAG codons

Abstract

Pyrrolysine (Pyl), the 22nd naturally encoded amino acid, gets acylated to its distinctive UAG suppressor tRNA(Pyl) by the cognate pyrrolysyl-tRNA synthetase (PylRS). 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 PylRS in a yeast three-hybrid system and to measure in vitro tRNA(Pyl) 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 nucleotides 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 anticodon is not important for PylRS 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(Pyl):PylRS is a fine orthogonal tRNA:synthetase pair that facilitated the late addition of Pyl to the genetic code.

Fig. 1.

M. barkeri tRNA^Pyl and bovine mitochondrial tRNA^Ser L-shape structures and scheme of tRNA numbering. The circled nucleotides indicate tRNA positions that, upon mutation, resulted in a significant decrease in in vivo suppression activity, in vivo binding, and in vitro aminoacylation. The boxed nucleotides indicate tRNA positions that, upon mutation, only affected in vivo suppression activity.

Fig. 2.

Transplantation of M. barkeri tRNA^Pyl identity elements into the bovine mitochondrial tRNA^Ser scaffold. L-shape structures of stabilized tRNA^Ser (see Materials and Methods) and of two tRNA^Ser/Pyl chimeric molecules (see Results). Filled circles refer to nucleotides different in the stabilized tRNA^Ser and tRNA^Pyl; circled nucleotides refer to positions mutated in the stabilized tRNA^Ser required to obtain Cyc accepting activity. The other indicated nucleotides are common to the stabilized tRNA^Ser and tRNA^Pyl species.