ESCHERICHIA-COLI CAMP RECEPTOR PROTEIN-DNA COMPLEXES - 2 - STRUCTURALASYMMETRY OF DNA BENDING

The effect of DNA sequence variability and the degree of cyclic AMP re ceptor protein (CRP)induced bending of the flanking ends of fluorescen tly labeled DNA were investigated by steady-state fluorescence and dif ferential phase polarization studies in the presence and absence of CR P. Six sequences, including the primary CRP binding sites of lac P1 (c lass I) and gal P1 (class II), were studied. Excitation and emission s pectra of CPM-DNA upon binding CRP were observed to be qualitatively s imilar to one another, regardless of the CRP binding site sequence exa mined or the location of the probe. This result implies that the probe is not interacting with the protein. However, the magnitude of the ch anges in the fluorescence intensities of sensitized emission spectra o f CPM-DNA is apparently dependent on the DNA sequence, indicating that the environments of the flanking ends of DNA may be different from on e another in the protein-DNA complex. Differential phase polarization results were qualitatively consistent with the fluorescence energy tra nsfer measurements. The implication of this study supports the idea th at the DNA is bent symmetrically in the lac-CRP complex but is asymmet rically bent in the gal-CRP complex. The sequence in the half-site in conjunction with the flanking sequence defines the geometry of the ben t DNA. It appears that the CRP-induced bend in the DNA may also be cla ss dependent. This may be an important feature used by the system to r egulate transcription at different promoter sites.