Identifying the targets of aminoacyl-tRNA synthetase inhibitors by primer extension inhibition

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 synth...

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Bibliographic Details
Published in:Nucleic acids research 2013-08, Vol.41 (14), p.e144-e144
Main Authors: Orelle, Cédric, Szal, Teresa, Klepacki, Dorota, Shaw, Karen J, Vázquez-Laslop, Nora, Mankin, Alexander S
Format: Article
Language:English
Subjects:
Amino Acyl-tRNA Synthetases - antagonists & inhibitors
Cell-Free System
DNA Primers
Drug Evaluation, Preclinical - methods
Enzyme Inhibitors - chemistry
Enzyme Inhibitors - pharmacology
Genes, Synthetic
Methods Online
Polymerase Chain Reaction - methods
Protein Biosynthesis
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Summary: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.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkt526