1、Protein-DNA Interactions Equilibria and free energies Estimate binding site occupancy Forces driving protein-DNA interactions Lambda repressor Methods to study protein-DNA interactions1AAAADDAAAADMH12AADMMajorA-TAA DMT-AMinorA-TT-AAAAAA A D HG-CC-GG-CC-GMajorH D A AAADAADMinorThe 4 Major Groove Patt

2、erns Are Distinct23AAAADDAAAADMH1Hyrdophobic Groups on Bases43Helix-Turn-Helix Motif34 A534125N-term346Specificity Determinants?Generic Interactions?5761041038Gln33 Networks with Gln44Ser G = 4.5 kcalLeu G = 6.2 kcal79Conclusion DNA binding specificity from major groove interactions Mutation in one

3、AA affects neighbors-Protein-DNA interfaces contain an intricate Network of hydrogen bonds10B DNA434 Complex94 bases dontcontact proteinBut very Important!11Filter Bindingvacuum% BoundProtein nM0 20 40 60 80 100100 50Kd = 20 nM1012Immunoprecipitaiton% BoundProtein nM0 20 40 60 80 100100 50Kd = 20 nM

4、10ASepharose13Gel-Shift% ShiftedProtein nM0 20 40 60 80 100100 50Kd = 20 nMProtein1114DNAse IFootprinting% ProtectedProtein nM0 20 40 60 80 100100 50Kd = 20 nMProtein+DNAse I1215Cooperative DNA binding By the repressor1316Interference FootprintingDNA + MMS + Saturating ProteinGel Shift1417Electron M

5、icroscopy1518YFPTilt+RollTilt-Roll19TiltRoll19Interaction of TBP (TATA Binding Protein) With DNAMajor GrooveMinor Groove1720Protein-Induced DNA Bending From Positive Roll185321Pyrimidine-Purine Steps Have Little Base Stacking5335CGAT19A (Lecture 1)22For advanced reading:Nucleic Acids:Structures, Properties, and Func