MMG /BIOC 352

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MMG /BIOC 352. Protein-DNA Interactions: Kinetics and Thermodynamics Example: the Bacteriophage l System. Spring 2006. Scott W. Morrical with special thanks to Margaret A. Daugherty. Contact Information. Scott W. Morrical Given B407 656-8260 Scott.Morrical@uvm.edu. - PowerPoint PPT Presentation

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  • MMG /BIOC 352Spring 2006Protein-DNA Interactions: Kinetics and ThermodynamicsExample: the Bacteriophage l SystemScott W. Morricalwith special thanks toMargaret A. Daugherty

  • Contact InformationScott W. MorricalGiven B407656-8260Scott.Morrical@uvm.edu

  • Lecture outline:Introduction to the system Bacteriophage lambda Lysogeny vs. lysisThe molecular switch PR, PRM, cI repressor, cro

    Specific vs. Non-specific Interactions What makes a good DNA binding protein?

    Thermodynamic Primer DG = DH - TDS: importance Intrinsic Free Energy Cooperativity

    Techniques Quantitative DNAse Footprinting

    cI repressor protein Structure Dimerization Data

    Cro protein Structure Dimerization & DNA Binding An example of induced fit Data

    Kinetic Aspects of cI and cro binding Facilitated diffusion cro-DNA interactions

    Structure Analysis? cro-DNA vs. cI-DNA interactions

  • Reference list for this topic:Ref 1: Ptashne, M. (1992) A Genetic Switch, 2nd ed., Cell Press & Blackwell Scientific Publications, Cambridge, MA. **This an excellent general review of bacteriophage l with simple descriptions of thermodynamics and regulation.

    Ref 2: Johnson, A.D., Poteete, A.R., Lauer, G., Sauer, R.T., Ackers, G.K. & Ptashne, M. (1981) l Repressor and cro - components of an efficient molecular switch. Nature 294: 217-223. Review article of bacteriophage l, outdated, but ok for understanding the system in general.

    Ref 3: Chattophadhyay, R. & Ghosh, K. (2003) A comparative three-dimensional model of the carboxy-terminal domain of the lambda repressor and its use to build intactrepressor tetramer models bound to adjacent operator sites. J. Struct. Biol. 141:103-114.

    Ref 4: Oda, M. & Nakamura, H. (2000) Thermodynamic and kinetic analyses for understanding sequence-specific DNA recognition. Genes to Cells 5: 319-326.Just one of many reviews on thermo & kinetic aspects of DNA binding.

    Ref 5: Brenowitz, M., Senear, D.F., Shea, M.A. & Ackers, G.K. (1986) Footprint titrations yield valid thermodynamic isotherms. P.N.A.S. USA 83: 8462-8466

  • Reference list - continuedRef 6: Koblan, K.S. & Ackers, G.K. (1992) Site-Specific Regulation of DNA Transcription at Bacteriophage l OR, Biochemistry 31: 57-67.

    Ref 7: Darling, P.J., Holt, J.M. & Ackers, G.K. (2000) Coupled Energetics of l cro Repressor Self-assembly and Site-specific DNA Operator Binding II: Cooperative Interactions of cro Dimers. J. Mol. Biol. 302: 625-638.

    Ref 8: Albright, R.A. & Matthews, B.W. (1998) Crystal structure of l-cro bound to a Consensus Operator at 3.0 Resolution, J. Mol. Biol. 280: 137-151.

    Ref 9: Spolar, R.S. and Record, M.T. (1994) Coupling of Local Folding to Site-Specific Binding of Proteins to DNA. Science 263: 777 - 784 a classic must know paper!Ref 10: von Hippel (1994) Protein - DNA Recognition : New Perspectives and Underlying Themes. Science 263: 769-770. (a review of Spolar & Record)

    Ref 11: Frankel, A.D. & Kim, P.S. (1991) Modular Structure of Transcription Factors: Implications for Gene Regulation. Cell 65: 717-719 (quick reading - introduces notion of induced fit)

    Ref 12: Takeda, Y., Ross.P.D. & Mudd,C.P. (1992) Thermodynamics of Cro-protein DNAinteractions. Proc. Natl. Acad. Sci. USA 89: 8180-8184.

  • Reference list - continuedRef 13: von Hippel, P.H. & Berg, O.G. (1989) Facilitated Target Location in Biological Systems. J. Biol. Chem. 264: 675-678. Nice mini-review.

    Ref 14:Kim, J.G., Takeda, Y., Matthews, B.W. & Anderson, W.F. (1987) Kinetic Studies of Cro-Repressor Operator DNA Interaction, J. Mol. Biol. 196: 149-158

    Ref 15: Albright, R.A. and Matthews, B.W. (1998) How Cro and l-repressor distinguish between operators: The structural basis underlying a genetic switch. Proc. Natl.Acad. Sci. USA 95: 3431-3436.

  • Bacteriophage l: an obligate parasite100,000x8,000xRef 1: Ptashne (1992) A Genetic Switch, 2nd ed., Cell Press & Blackwell Scientific Publications, Cambridge, MA.

  • Lambda, lysogeny and lysisRef 1lysogenylysisprophageinfectinject

  • An overview of l growth: Patterns of gene expression23101012l chromosomepattern of gene expressionRef 1NcrocI repressorint

  • The molecular switch: Lysogeny to LysisPolymerase can bind to PRM or PRORi = right operator sites where i = 1,2 3PR = right promoter; polymerase transcribes cro proteinPRM = promoter of repressor maintenance; polymerase boundhere transcribes cI repressor protein.cI repressor protein = maintains bacteria in lysogenic statecro protein = control of repressor and other genes; causes switch to lytic lifestyle

  • l repressor vs. croKey points: same operator sites; reverse affinities; cooperativity; bind as dimersRef 1

  • cI repressor: keeps cro turned off!X

  • cro: turns off production of cI!X

  • The switch: completing the storyRef 1

  • Designing an efficient DNA binding proteinPurpose: To understand the factors that influence how efficiently a repressor protein occupies its operator in the cell.Given: the fraction of time that an operator is bound by repressor is determined by two factorsi). Affinity of repressor for operatorii). Concentration of free repressor Problem: Non-specific binding!Goal: Understand how we can increase efficiencyleads us to idea of cooperativity

  • Designing an efficient DNA binding proteinEquations on boardThe rationale for the arguments are taken fromAppendix One in reference 1

  • Designing an efficient DNA binding proteinHow can we increase specificity?1). Increase protein concentration

    2). Improve specificity directly*play with the KD/KOP ratiohold KD constant; improve KOP*increase number of contacts by increasing repressortwice the contacts, twice the energy!KOP = 10-20 M; KD = 10-8 M good idea, but. Affinities become a problem, which give rise to kinetic problems!

  • Increased efficiency with cooperativity

  • cI binding to PRRef 1Cooperativity>99% occupiedNocooperativity10% 90%

  • cI binding to PR: OR1-OR2 species predominatescI binds strongly to OR1 and OR2; weakly to OR3; cooperative interactions enhance interactions at OR2Ref 1

  • fast switch for gene expressionBiological advantage of cooperativityRef 1

  • cI repressor structure: low resolutionKd ~ 6 nMRef 1Dimerization & regulation

  • cI repressor structure: low resolutionN-terminus: major groove interactionsLinker region: flexibleC-terminus: protein-protein interactions that give rise to cooperativityRef 1

  • cI repressor structure: high resolution(pdb1j5g) J. Struct. Biol. 141, 103-114; 2003

  • REVIEW OF TUESDAYS LECTURElysislysogenyDesigning an efficient DNA-binding proteinnon-specific interactions mess up specific binding!KD/KOP and [R]T/[D]T determine binding efficienciesBest way to improve binding - COOPERATIVITY!Cooperativity gives rise to faster biological responses

  • Cooperativity and Free EnergyHow do we determine that there is cooperativity?OR+DG3DG1DG2

  • Thermodynamic Primer: Gibbs Free EnergyP + DNA PDNAKeqDGo = -RTln KeqRemember: more negative, more favorable reaction!

  • vant Hoff equation: temperature dependence of KeqA B Keq = [B]/[A]Measure K as a function of TDH = +533 kJ/molLinear: no DCp changeCurvature: DCp changeDG = DH - TDS62C48C

  • Thermodynamics and biological reactionsDG = DH - TDSDG = criteria for spontaneitynegative - reaction is favorable

    DH = direction of energy flownegative - exothermicinformation about chemical interactions

    DS = tells us about system organizationpositive - increase disorder can reflect conformational entropy or H20 entropy

    DCp = proportional to a change in hydrophobic surface area-- see Spolar reference - very important reference!e.g., negative - organization of protein structure upon DNA binding

  • Constraint: cooperativity can only occur between adjacent operatorsMicroscopic binding configurations} Intrinsic binding DG

  • Brenowitz et al., (1986) P.N.A.S. 83: 8462-8466Autoradiogram of a footprint: false color imagestandardOR3OR1OR2standard

  • Individual site binding isothermsFractional saturationOR3Langmuir isotherm-- single site interactions:Y = K1[X] / (1 + K1[X])K1 = 1/[X] at Y = 0.5

    For 2-site cooperative interaction:Y1 = (K1[X] + K1K2K12[X]2) / BY2 = (K2[X] + K1K2K12[X]2) / Bwhere B = (1 + (K1 + K2)[X] + K1K2K12[X]2)

    K1 and K2 are intrinsic binding constants for sites 1 & 2, and K12 is the interaction (or cooperativity) constant.K12 defines the extra free energy of binding 2 sites simultaneously compared to sum of individual free energies, i.e. DG12 = DGtotal - (DG1 + DG2) where DG = RTlnK.

  • Individual site binding isotherms for cI - OR interactionsKoblan, K. and Ackers, G.K., (1992) Biochemistry 31: 57-65.12313

  • Temperature dependence of DG values for cI-OR Koblan, K. and Ackers, G.K., (1992) Biochemistry 31: 57-65.OR1OR3OR2vant Hoff plots forcI-OR single-siteinteractions

  • Koblan, K. and Ackers, G.K., (1992) Biochemistry 31: 57-65.cI-OR Interactions are Enthalpically Driven

  • Cro repressor structure & induced fitAlbright & Matthews, J. Mol. Biol. (1998) 280, 137-151Cro: helix-turn-helix (like cI and CAP)

    Dimer subunits rotate 53o wrt each other upon binding to consensus OR

    Creation of extensive H-bond networkplus van der Waals contacts along protein-DNA interface.DNA is bent 40o through 19 bp.

    Recognition helices of Cro dimer makeextensive contacts with bp edges in