1、Protein-DNA Interactions Equilibria and free energies Estimate binding site occupancy Forces driving protein-DNA interactions Lambda repressor Methods to study protein-DNA interactionsAAAADDAAAADMH1AADMMajorA-TAA DMT-AMinorA-TT-AAAAAA A D HG-CC-GG-CC-GMajorH D A AAADAADMinorThe 4 Major Groove Patter
2、ns Are Distinct2AAAADDAAAADMH1Hyrdophobic Groups on Bases3Helix-Turn-Helix Motif34 A34125N-term34Specificity Determinants?Generic Interactions?56104103Gln33 Networks with Gln44Ser G=4.5 kcalLeu G=6.2 kcal7Conclusion DNA binding specificity from major groove interactions Mutation in one AA affects ne
3、ighbors-Protein-DNA interfaces contain an intricate Network of hydrogen bondsB DNA434 Complex94 bases dontcontact proteinBut very Important!Filter Bindingvacuum%BoundProtein nM0 20 40 60 80 100100 50Kd=20 nM10Immunoprecipitaiton%BoundProtein nM0 20 40 60 80 100100 50Kd=20 nM10ASepharoseGel-Shift%Shi
4、ftedProtein nM0 20 40 60 80 100100 50Kd=20 nMProtein11DNAse IFootprinting%ProtectedProtein nM0 20 40 60 80 100100 50Kd=20 nMProtein+DNAse I12Cooperative DNA binding By the repressor13Interference FootprintingDNA+MMS+Saturating ProteinGel Shift14Electron Microscopy15YFPTilt+RollTilt-Roll19TiltRollInt
5、eraction of TBP(TATA Binding Protein)With DNAMajor GrooveMinor Groove17Protein-Induced DNA Bending From Positive Roll1853Pyrimidine-Purine Steps Have Little Base Stacking5335CGAT19A(Lecture 1)For advanced reading:Nucleic Acids:Structures,Properties,and FunctionsV.A.BloomfieldD.M.CrothersI.Tinoco8Gln G=5.2 kcalLys G=4.1 kcal
