Fluorescence spectroscopy as a tool for probing DNA-protein interactions

Fluorescence spectroscopy is a powerful technique that can provide structural, thermodynamic, and kinetic information. By exploiting the high sensitivity and small sample volume requirements, fluorescence has become a popular technique to study DNA-protein interactions. In this work fluorescence spectroscopy was used to investigate two DNA-protein systems: EcoRI restriction endonuclease and E. coli RNA Polymerase. In addition, this work has significantly expanded the application of one particular fluorescence technique, Forster resonance energy transfer (FRET), by including a third fluorophore in the traditional one-step donor/acceptor FRET system. A general theoretical framework for distance measurements in three-chromophore systems is presented. Three arrangements of chromophores applicable to many diverse situations are considered: (I) two-step FRET relay with FRET between the first and second chromophores and between the second and third, (II) FRET from a single donor to two different acceptors, and (III) two-step FRET relay with FRET also between the first and third chromophores. Equations for the efficiencies involving single or multiple energy transfer steps are derived for both donor quenching and sensitized emission experiments. The theory is supported by experimental data on model systems of known structure using steady-state donor quenching, lifetime quenching, and sensitized emission measurements. The distances obtained for the three-chromophore systems agree with those of two-chromophore systems and molecular models. Finally, labeling requirements for diagnosis of the energy transfer scheme and subsequent distance measurements are discussed.