Published online 30 September 2004
Nucleic Acids Research, Vol. 32 No. 17 © Oxford University Press 2004; all rights reserved
DNA-binding domain of GCN4 induces bending of both the ATF/CREB and AP-1 binding sites of DNA
Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA
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Received July 4, 2004; Revised August 9, 2004; Accepted September 9, 2004
The interaction of proteins with DNA results, in some cases, in DNA bending, and this might have functional importance. However, when the protein-induced bending of DNA is small, its measurement presents a problem. It is shown that the fluorescence resonance energy transfer between fluorophores placed on the ends of the specially designed U-shaped DNA, which contains the DNA-binding sites at its central part, can be successfully used for this purpose. The lever effect of the arms of such U-shaped DNA ensures that the distance between the fluorophores is very sensitive to bending of the central part. Using this technique, it was shown that (i) the AP-1 and ATF/CREB binding sites of GCN4 transcription factor are pre-bent to the same extent (
12° toward the major groove) and (ii) binding of the GCN4 DNA-binding domain (GCN4-bZIP) results in additional bending of both these target sites but to a greater extent at the ATF/CREB site. In total, in the complex with GCN4-bZIP, the ATF/CREB site is bent by (25 ± 2)° and the AP-1 site by (20 ± 2)° toward the minor groove.