Luminescence resonance energy transfer analysis of RNA polymerase complexes.
Resonance energy transfer allows measurement of atomic-scale distances under a variety of solution conditions. Luminescence resonance energy transfer (LRET) is a variant of energy transfer measurement in which lanthanide chelates are used as the probes. The unusual properties of lanthanide emission, in particular their long microsecond-scale lifetimes, offer several advantages for energy transfer measurements with biological samples. One of the unique features of LRET is the ability to measure energy transfer under conditions where severe heterogeneity of labeled macromolecules exists. This feature of LRET is the special emphasis of this article. We describe here LRET methodology with a particular attention to using sensitized acceptor emission to determine efficiency of energy transfer. Although we employed this technique in the characterization of Escherichia coli RNA polymerase complexes it is readily compatible with the study of essentially any protein.
Heyduk
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E. A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, Missouri 63104, USA. heydukt@slu.edu
D002614:Chelating Agents; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology; D005470:Fluorometry / Q000295:instrumentation / Q000379:methods*; D028581:Lanthanoid Series Elements; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding
D002614:Chelating Agents; D028581:Lanthanoid Series Elements; D008164:Luminescent Proteins; D012321:DNA-Directed RNA Polymerases