PMID:12620834
| Citation |
Bergendahl, V, Heyduk, T and Burgess, RR (2003) Luminescence resonance energy transfer-based high-throughput screening assay for inhibitors of essential protein-protein interactions in bacterial RNA polymerase. Appl. Environ. Microbiol. 69:1492-8 |
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| 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. |
| Links | |
| Keywords |
Animals; Binding Sites; DNA-Directed RNA Polymerases/antagonists & inhibitors; DNA-Directed RNA Polymerases/metabolism; Energy Transfer; Escherichia coli/enzymology; Escherichia coli/metabolism; Lanthanoid Series Elements; Porifera/metabolism; Protein Binding/drug effects; Seawater; Sigma Factor/antagonists & inhibitors; Sigma Factor/metabolism; Transcription, Genetic |
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