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‐ε‐d ‐prolyl‐l ‐lysine (d ‐prolyl‐lysine) and N‐ε‐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 β‐galactosidase shows that a specialized mRNA motif is not essential for stop‐codon recoding, unlike for selenocysteine incorporation.