An intrinsic curvature towards the minor groove in the cAMP-responsive element DNA found by combined NMR and molecular modelling studies

The cAMP-responsive element (CRE, 5'-TGACGTCA-3') is essential to the trans criptional function of numerous gene promoters in eukaryotic cells. We carr ied out NMR restrained molecular mechanics studies using two different forc e fields (Flex and <<AMBER94>>) on a hexadecanucleotide d(GAGATGACGTCATCTC) containing CRE. Results indicated that free CRE is a B-DNA that is intrins ically curved towards the minor groove. To our knowledge, NMR restraints ha ve not previously been useful in accounting for a global DNA curvature. In order to validate the bend in CRE, we applied a new strategy in which DNA s tructures displaying different curvatures were generated and then compared with NMR data. Conformations of CRE curved towards the minor groove provide d the best agreement with NMR data. Our results contrast with previous resu lts obtained from NMR restrained modelling and gel methods; these suggested conformations that were straight or curved towards the major groove, respe ctively. The curve in free CRE is spread along the DNA helix: several kinks are repeated in phase within the helical turn, although they are centred m ainly on CpG in between the TGA half-sites, thus slightly increasing their spacing within the major groove. Comparison with the crystal structure of C RE complexed to general control protein 4 showed that the curve orientation is reversed from the minor to the major groove upon protein binding, due t o a helix distortion concentrated mainly on CpG.