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Applied and Environmental Microbiology, March 2003, p. 1492-1498, Vol. 69, No. 3
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.3.1492-1498.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Luminescence Resonance Energy Transfer-Based High-Throughput Screening Assay for Inhibitors of Essential Protein-Protein Interactions in Bacterial RNA Polymerase

Veit Bergendahl,1 Tomasz Heyduk,2 and Richard R. Burgess1*

McArdle Laboratory for Cancer Research, University of Wisconsin—Madison, Madison, Wisconsin 53706,1 Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Medical School, St. Louis, Missouri 631042

Received 23 September 2002/ Accepted 18 December 2002

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 {sigma}70 and an IC5-labeled fragment of the ß' 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.

* Corresponding author. Mailing address: McArdle Laboratory for Cancer Research, University of Wisconsin—Madison, 1400 University Ave., Madison, WI 53706. Phone: (608) 263-2635. Fax: (608) 262-2824. E-mail: burgess{at}oncology.wisc.edu.

Applied and Environmental Microbiology, March 2003, p. 1492-1498, Vol. 69, No. 3
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.3.1492-1498.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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