哈佛分子生物学--蛋白质和DNA的关系.ppt

Protein-DNA Interactions,Equilibria and free energies Estimate binding site occupancy Forces driving protein-DNA interactions Lambda repressor Methods to study protein-DNA interactions,A,A,A,A,D,D,H,1,A,A,D,M,Major,A-T,A,A,D,M,T-A,Minor,A-T,T-A,A,A,A,A,A,A,D,H,G-C,C-G,G-C,C-G,Major,H,D,A,A,A,A,D,A,A,D,Minor,The 4 Major Groove Patterns Are Distinct,2,A,A,A,A,D,D,H,1,Hyrdophobic Groups on Bases,3,Helix-Turn-Helix Motif,34 A,3,4,1,2,5,N-term,3,4,6,Gln33 Networks with Gln44,Ser, G = 4.5 kcal,Leu, G = 6.2 kcal,7,Conclusion,DNA binding specificity from major groove interactions Mutation in one AA affects neighbors Protein-DNA interfaces contain an intricate Network of hydrogen bonds,B DNA,434 Complex,9,4 bases dont contact protein But very Important!,Filter Binding,vacuum,% Bound,Protein nM,0 20 40 60 80 100,100,50,Kd = 20 nM,10,Immunoprecipitaiton,% Bound,Protein nM,0 20 40 60 80 100,100,50,Kd = 20 nM,10,A,Sepharose,Gel-Shift,% Shifted,Protein nM,0 20 40 60 80 100,100,50,Kd = 20 nM,Protein,11,DNAse I Footprinting,% Protected,Protein nM,0 20 40 60 80 100,100,50,Kd = 20 nM,Protein,+DNAse I,12,Cooperative DNA binding By the repressor,13,Interference Footprinting,DNA + MMS,+ Saturating Protein,Gel Shift,14,Electron Microscopy,15,YFP,Tilt,+Roll,Tilt,-Roll,19,Tilt,Roll,Interaction of TBP (TATA Binding Protein) With DNA,Major Groove,Minor Groove,17,Protein-Induced DNA Bending From Positive Roll,18,5,3,Pyrimidine-Purine Steps Have Little Base Stacking,19A (Lecture 1),For advanced reading: Nucleic Acids: Structures, Properties, and Functio