title: Identifying the targets of aminoacyl-tRNA synthetase inhibitors by primer extension inhibition.
abstract: Aminoacyl-transfer RNA (tRNA) synthetases (RS) are essential components of the cellular translation machinery and can be exploited for antibiotic discovery. Because cells have many different RS, usually one for each amino acid, identification of the specific enzyme targeted by a new natural or synthetic inhibitor can be cumbersome. We describe the use of the primer extension technique in conjunction with specifically designed synthetic genes to identify the RS targeted by an inhibitor. Suppression of a synthetase activity reduces the amount of the cognate aminoacyl-tRNA in a cell-free translation system resulting in arrest of translation when the corresponding codon enters the decoding center of the ribosome. The utility of the technique is demonstrated by identifying a switch in target specificity of some synthetic inhibitors of threonyl-tRNA synthetase.
authors.affiliation: Center for Pharmaceutical Biotechnology, University of Illinois, Chicago, IL 60607, USA and Trius Therapeutics, Inc., San Diego, CA 92121, USA.
authors.forename: Cédric
authors.initials: C
authors.lastname: Orelle
authors.forename: Teresa
authors.initials: T
authors.lastname: Szal
authors.forename: Dorota
authors.initials: D
authors.lastname: Klepacki
authors.forename: Karen J
authors.initials: KJ
authors.lastname: Shaw
authors.forename: Nora
authors.initials: N
authors.lastname: Vázquez-Laslop
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Mankin
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000037:antagonists & inhibitors*; D002474:Cell-Free System; D017931:DNA Primers; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005813:Genes, Synthetic; D016133:Polymerase Chain Reaction / Q000379:methods; D014176:Protein Biosynthesis
chemical_list: D017931:DNA Primers; D004791:Enzyme Inhibitors; D000604:Amino Acyl-tRNA Synthetases