title: Luminescence resonance energy transfer-based high-throughput screening assay for inhibitors of essential protein-protein interactions in bacterial RNA polymerase.
abstract: The binding of sigma factors to core RNA polymerase is essential for the specific initiation of transcription in eubacteria and is thus critical for cell growth. Since the responsible protein-binding regions are highly conserved among all eubacteria but differ significantly from eukaryotic RNA polymerases, sigma factor binding is a promising target for drug discovery. A homogeneous assay for sigma binding to RNA polymerase (Escherichia coli) based on luminescence resonance energy transfer (LRET) was developed by using a europium-labeled sigma70 and an IC5-labeled fragment of the beta' subunit of RNA polymerase (amino acid residues 100 through 309). Inhibition of sigma binding was measured by the loss of LRET through a decrease in IC5 emission. The technical advances offered by LRET resulted in a very robust assay suitable for high-throughput screening, and LRET was successfully used to screen a crude natural-product library. We illustrate this method as a powerful tool to investigate any essential protein-protein interaction for basic research and drug discovery.
authors.affiliation: McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, 1400 University Avenue, Madison, WI 53706, USA.
authors.forename: Veit
authors.initials: V
authors.lastname: Bergendahl
authors.forename: Tomasz
authors.initials: T
authors.lastname: Heyduk
authors.forename: Richard R
authors.initials: RR
authors.lastname: Burgess
mesh_terms: D000818:Animals; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D028581:Lanthanoid Series Elements; D011161:Porifera / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D012623:Seawater; D012808:Sigma Factor / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D028581:Lanthanoid Series Elements; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit