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: -prolyl-lysine) and N-e-cyclopentyloxycarbonyl-L L-lysine (Cyc). Escherichia coli, transformed with the tRNA Pyl and PylRS genes, suppressed a lacZ amber mutant dependent on the presence of D D-prolyl-lysine or Cyc in the medium, implying that the E. coli translation machinery is able to use Cyc-tRNA Pyl and D D-prolyl-lysine-tRNA Pyl as substrates during protein synthesis. Furthermore, the formation of active b-galactosidase shows that a specialized mRNA motif is not essential for stop-codon recoding, unlike for selenocysteine incorporation.

Pyrrolysine is not hardwired for cotranslational insertion at UAG codons

“…Although the insertion of Sec at UGA requires a specific mRNA context provided by the presence of the SECIS stem/loop structure as well the selenocysteine-specific elongation factor SelB (6), Pyl can be efficiently inserted into proteins in an anonymous context and appears not to depend on the presence of additional proteins. This characteristic is well demonstrated by the fact that Pyl or Cyc could be efficiently incorporated into E. coli reporter proteins lacking any specific RNA structures in the vicinity of the UAG codon when tested in Methanosarcina acetivorans (22) or in E. coli (12). A stem/loop structure located just downstream of the UAG codon and therefore termed PYLIS by analogy to the established SECIS element, was proposed to stimulate Pyl incorporation (22,23).…”

“…Therefore, we transformed E. coli strain XAC/ A24 with plasmid-borne copies of M. barkeri pylS and 42 mutant pylT genes and grew the transformants in the presence of the Pyl analog N--cyclopentyloxycarbonyl-L-lysine (Cyc), because Pyl is not commercially available (12). Suppression was quantitated by measuring ␤-galactosidase activity ( Table 1).…”

“…To screen a large number of M. barkeri Fusaro tRNA Pyl variants generated by mutagenesis, we made use of this tRNA's ability to suppress the lac amber mutation A24 in a lacI-lacZ fusion system (12,13). Therefore, we transformed E. coli strain XAC/ A24 with plasmid-borne copies of M. barkeri pylS and 42 mutant pylT genes and grew the transformants in the presence of the Pyl analog N--cyclopentyloxycarbonyl-L-lysine (Cyc), because Pyl is not commercially available (12).…”

“…eukaryotic proteins (1,3). Because Pyl-tRNA Pyl formation is independent of the tRNA anticodon sequence, because the PylRS structure is being solved (27), because tRNA Pyl is a strong suppressor (at least as tested for UAG and UAA) in E. coli (10,12), and because additional RNA or protein factors are not required for Pyl or Cyc incorporation, the potential use of the PylRS:tRNA Pyl orthogonal pair, optimized through natural or man-made evolutionary processes, is particularly appealing.…”

“…The Methanosarcinaceae contain a devoted UAG-recognizing suppressor tRNA Pyl (8) and a pyrrolysyltRNA synthetase (PylRS) dedicated to forming Pyl-tRNA Pyl (9,10). Initial studies indicated that Lys-tRNA Pyl can be recognized by bacterial EF-Tu (11) and that in Escherichia coli tRNA Pyl acts like an amber suppressor (10,12).…”