title: Simultaneous screening of multiple bacterial tRNA synthetases using an Escherichia coli S30-based transcription and translation assay.
abstract: The search for novel antibiotics to combat the growing threat of resistance has led researchers to screen libraries with coupled transcription and translation systems. In these systems, a bacterial cell lysate supplies the proteins necessary for transcription and translation, a plasmid encoding a reporter protein is added as a template, and a complex mixture of amino acids and cofactors is added to supply building blocks and energy to the assay. Firefly luciferase is typically used as the reporter protein in high-throughput screens because the luminescent signal is strong and, since bacterial lysates contain no luciferase, the background is negligible. The typical coupled transcription and translation assay is sensitive to inhibitors of RNA polymerase and to compounds that bind tightly to the ribosome. We have found a way to increase the information content of the screen by making the assay more sensitive to inhibitors of tRNA synthetases. Restricting the concentration of amino acids added to the reaction mixture allows the simultaneous screening of multiple tRNA synthetase enzymes along with the classic transcription and translation targets. In addition, this assay can be used as a convenient way to determine if an antibacterial compound of unknown mechanism inhibits translation through inhibition of a tRNA synthetase, and to identify which synthetase is the target.
authors.affiliation: Pfizer Global Research and Development, Michigan Laboratories, Arbor, MI 48105, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Dermyer
authors.forename: Scott C
authors.initials: SC
authors.lastname: Wise
authors.forename: Timothy
authors.initials: T
authors.lastname: Braden
authors.forename: Tod P
authors.initials: TP
authors.lastname: Holler
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000037:antagonists & inhibitors*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000235:genetics; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D014158:Transcription, Genetic
chemical_list: D000596:Amino Acids; D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D008156:Luciferases; D012321:DNA-Directed RNA Polymerases; D000604:Amino Acyl-tRNA Synthetases