The DNA binding domain of the yeast transcription factor Mbp1 is a winged helix-turn-helix structure, with an extended DNA binding site involving C-terminal "tail" residues. The thermodynamics of the interaction of the DNA binding domain with its target DNA sequence have been determined using fluorescence anisotropy and calorimetry. The dissociation constant was determined as a function of pH and ionic strength in assessing the relative importance of specific and nonspecific ionic interactions. Mutational analysis of the residues in the binding site was used to determine their contributions to binding. The three tail histidine residues and His 63 in the recognition helix accounted for most of the pH dependence of the DNA binding. The tail histidine residues, along with two previously identified lysine residues, account for a major part of the polyelectrolyte contribution to binding and for the nonspecific affinity of Mbp1 for DNA. Gln 67 was shown to be a very important residue, which interacts in the minor groove of the target DNA. Systematic mutations of the DNA consensus binding sites showed that the CGCG core contributes most to recognition. Isothermal titration calorimetry revealed a strong temperature-dependent enthalpy change, with a Delta C p of -1.3 kJ mol (-1) K (-1), consistent with a specific binding mode and burial of surface area. Parsing the free energy contributions demonstrates that polyelectrolyte effects account for half of the total free energy at the physiological pH and salt concentration. We present a model for the origin of the sequence specificity and overall affinity of the protein that accounts for the observed thermodynamics.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|