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 transcriptional function of numerous gene promoters in eukaryotic cells. We carried out NMR restrained molecular mechanics studies using two different force fields (Flex and "AMBER94") on a hexadecanucleotide d(GAGATGACGTCATCTC) containing CRE. Results indicated that free CRE is a B-DNA that is intrinsically curved towards the minor groove. To our knowledge, NMR restraints have 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 structures displaying different curvatures were generated and then compared with NMR data. Conformations of CRE curved towards the minor groove provided the best agreement with NMR data. Our results contrast with previous results obtained from NMR restrained modelling and gel methods; these suggested conformations that were straight or curved towards the major groove, respectively. 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 mainly on CpG in between the TGA half-sites, thus slightly increasing their spacing within the major groove. Comparison with the crystal structure of CRE complexed to general control protein 4 showed that the curve orientation is reversed from the minor to the major groove upon protein binding, due to a helix distortion concentrated mainly on CpG.
Chaoui
M
M
Département de Physicochimie et Pharmacologie des Macromolécules Biologiques, Institut Gustave Roussy, Villejuif, France.
Derreumaux
S
S
Mauffret
O
O
Fermandjian
S
S
D000242:Cyclic AMP / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008968:Molecular Conformation; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D011494:Protein Kinases / Q000737:chemistry; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D012984:Software
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D000242:Cyclic AMP; D011494:Protein Kinases