The catalytic flexibility of tRNAIle-lysidine synthetase can generate alternative tRNA substrates for isoleucyl-tRNA synthetase.
Bacteria decode the isoleucine codon AUA using a tRNA species that is posttranscriptionally modified at the wobble position of the anticodon with a lysine-containing cytidine derivative called lysidine. The lysidine modification of tRNA(Ile2) is an essential identity determinant for proper aminoacylation by isoleucyl tRNA synthetase (IleRS) and codon recognition on the ribosome. The ATP- and lysine-dependent formation of lysidine is catalyzed by tRNA(Ile)-lysidine synthetase. Using the purified recombinant enzyme from Escherichia coli and an in vitro transcribed tRNA substrate, we have confirmed that lysidine modification is both necessary and sufficient to convert tRNA(Ile2) into a substrate for IleRS. A series of lysine analogs were tested as potential inhibitors during the mechanistic characterization of tRNA(Ile)-lysidine synthetase. Gel electrophoresis revealed that many of these analogs, including some simple alkyl amines, were alternative substrates. Incorporation of these amines into alternative tRNA products was confirmed by mass spectrometry. The availability of tRNA(Ile2) with differential modifications enabled an exploration of the structural requirements of the anticodon for aminoacylation by methionyl tRNA synthetase and IleRS. All of the modifications were effective at creating negative determinants for methionyl tRNA synthetase and positive determinants for IleRS, although the tolerance of IleRS differed between the enzymes from E. coli and Bacillus subtilis.
Salowe
Scott P
SP
Department of Infectious Disease Research, Merck Research Laboratories, Rahway, New Jersey 07065, USA. scott_salowe@merck.com
Wiltsie
Judyann
J
Hawkins
Julio C
JC
Sonatore
Lisa M
LM
D000255:Adenosine Triphosphate / Q000737:chemistry; D001412:Bacillus subtilis / Q000378:metabolism; D002384:Catalysis; D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D007533:Isoleucine-tRNA Ligase / Q000737:chemistry*; D007700:Kinetics; D008718:Methionine-tRNA Ligase / Q000737:chemistry; D008956:Models, Chemical; D016296:Mutagenesis; D012343:RNA, Transfer / Q000737:chemistry; D012355:RNA, Transfer, Ile / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity
D003062:Codon; D012355:RNA, Transfer, Ile; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D008718:Methionine-tRNA Ligase; D007533:Isoleucine-tRNA Ligase