蛋白质和DNA的关系.pptVIP

Protein-DNA Interactions Equilibria and free energies Estimate binding site occupancy Forces driving protein-DNA interactions Lambda repressor Methods to study protein-DNA interactions Conclusion DNA binding specificity from major groove interactions Mutation in one AA affects neighbors --Protein-DNA interfaces contain an intricate Network of hydrogen bonds For advanced reading:Nucleic Acids:Structures, Properties, and FunctionsV.A. BloomfieldD.M. CrothersI. Tinoco 免疫学信息网 * A A A A D D A A A A D M 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 A A A A D M H 1 Hyrdophobic Groups on Bases 3 Helix-Turn-Helix Motif 34 Ao 3 4 1 2 5 N-term 3’ 4 Specificity Determinants? Generic Interactions? 5 6 104 103 Gln33 Networks with Gln44 Ser ? ?G = 4.5 kcal Leu ? ?G = 6.2 kcal 7 B DNA 434 Complex 9 4 bases don’t 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 5’ 3’ 3’ 5’ C G A T 19A (Lecture 1) 免疫学信息网 *