Cyclic AMP receptor protein is involved in the regulation of more than 20 genes. A step in the mechanism of activation of transcription is to induce a significant bending of the DNA upon complex formation between specific DNA and the protein. The induced DNA bending and a structure of the protein-DNA complex were studied by fluorescence energy transfer in 50 mM Tris, 1 mM EDTA, and 50 mM KCl at pH 7.8 and 20°C. The symmetry of the DNA bend was estimated by measuring the efficiency of transfer between the protein and a label on either the upstream or the downstream end of a lac DNA fragment. The results show that the bend, despite the asymmetry in the DNA sequence, is symmetrical, for the fragments which length ranges from 26 to 40 bp. Using fluorescence energy transfer, the extent of DNA bending was estimated by measuring the end-to-end distance of the DNA fragment which was labeled with a donor-acceptor pair on two opposite ends. Both steady-state and time-resolved measurements showed that in a 26 bp lac DNA fragment complexed with cyclic AMP receptor protein, the end-to-end distance is about 77 Å which corresponds to a bending angle of 80° or 100°, depending on the actual contour length between the fluorophores in the free DNA fragment. The results using longer DNA fragments show no measurable amount of energy transfer; thus, it is very unlikely that the DNA completely wraps around the CRP molecule. This study shows that the approach of fluorescence energy transfer has proven to be a versatile technique to provide useful structural information on the DNA-protein complex in potentially any solution conditions.
|Original language||English (US)|
|Number of pages||7|
|State||Published - 1992|
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