The power of FRET to study molecular complexes is expanded by the use of two or more donor/acceptor pairs. A general theoretical framework for distance measurements in three-chromophore systems is presented. Three energy transfer schemes 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 multiple energy transfer steps are derived for both donor quenching and sensitized emission measurements. The theory is supported by experimental data on model systems of known structure using steady-state donor quenching, lifetime quenching, and sensitized emission. The distances measured in the three-chromophore systems agree with those in two-chromophore systems and molecular models. Finally, labeling requirements for diagnosis of the energy transfer scheme and subsequent distance measurements are discussed.
Strand separation in promoter DNA induced by Escherichia coli RNA polymerase is likely initiated at a conserved A residue at position -11 of the nontemplate strand. Here we describe the use of fluorescence techniques to study the interaction of RNA polymerase with the -11 base. Forked DNA templates were employed, containing the fluorescent base, 2-aminopurine (2AP), substituted at the -11 position in a single-stranded tail comprising the nucleotides on the nontemplate strand at which base pairing is disrupted in an RNA polymerase-promoter complex. We demonstrate that the presence of 2AP instead of an A at position -11 has no major effect on the accessibility of DNA to DNase I or KMnO(4) in the presence or absence of RNA polymerase, thus justifying the use of templates containing the 2AP substitution in the fluorescence studies. A blue shift of the 2AP fluorescence emission maximum is observed in the presence of RNA polymerase. The results of fluorescence anisotropy decay studies indicate that about 60% of the 2AP residues at -11 are immobilized in an RNA polymerase complex. This value is in good agreement with the fraction of 2AP-substituted templates determined to be in a stable, heparin-resistant complex with RNA polymerase. These results are consistent with the residue at -11 being tightly bound in a hydrophobic pocket of the enzyme.
The N-terminal region of EcoRI endonuclease is essential for cleavage yet is invisible in the 2.5 A crystal structure of endonuclease-DNA complex [Kim, Y., Grable, J. C., Love, R., Greene, P. J., Rosenberg, J. M. (1990) Science 249, 1307-1309]. We used site-directed fluorescence spectroscopy and chemical cross-linking to locate the N-terminal region and assess its flexibility in the absence and presence of DNA substrate. The second amino acid in each subunit of the homodimer was replaced with cysteine and labeled with pyrene or reacted with bifunctional cross-linkers. The broad absorption spectra and characteristic excimer emission bands of pyrene-labeled muteins indicated stacking of the two pyrene rings in the homodimer. Proximity of N-terminal cysteines was confirmed by disulfide bond formation and chemical cross-linking. The dynamics of the N-terminal region were determined from time-resolved emission anisotropy measurements. The anisotropy decay had two components: a fast component with rotational correlation time of 0.3-3 ns representing probe internal motions and a slow component with 50-100 ns correlation time representing overall tumbling of the protein conjugate. We conclude that the N-termini are close together at the dimer interface with limited flexibility. Binding of Mg2+ cofactor or DNA substrate did not affect the location or flexibility of the N-terminal region as sensed by pyrene fluorescence and cross-linking, indicating that substrate binding is not accompanied by folding or unfolding of the N-terminus.
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