New version page

UVM MMG 352 - Protein-DNA Interactions

Upgrade to remove ads
Upgrade to remove ads
Unformatted text preview:

PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43Slide 44Slide 45Slide 46Slide 47Slide 48Slide 49Slide 50Slide 51Slide 52Slide 53Slide 54Slide 55Slide 56Slide 57Slide 58Slide 59Slide 60Slide 61Slide 62Slide 63Slide 64Slide 65Slide 66Slide 67Slide 68MMG /BIOC 352Spring 2006Protein-DNA Interactions: Kinetics and ThermodynamicsExample: the Bacteriophage  SystemScott W. Morricalwith special thanks toMargaret A. DaughertyContact InformationScott W. MorricalGiven [email protected] outline:Introduction to the system Bacteriophage lambda Lysogeny vs. lysisThe molecular switch PR, PRM, cI repressor, croSpecific vs. Non-specific Interactions What makes a good DNA binding protein?Thermodynamic “Primer” G = H - TS: importance Intrinsic Free Energy CooperativityTechniques Quantitative DNAse FootprintingcI repressor protein Structure Dimerization DataCro protein Structure Dimerization & DNA Binding An example of induced fit DataKinetic Aspects of cI and cro binding Facilitated diffusion cro-DNA interactionsStructure Analysis? cro-DNA vs. cI-DNA interactionsReference 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 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) Repressor and cro - components of an efficient molecular switch. Nature 294: 217-223. Review article of bacteriophage , outdated, but ok for understanding the system in general.Ref 3: Chattophadhyay, R. & Ghosh, K. (2003) A comparative three-dimensional model of the carboxy-terminal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: 8462-8466Reference list - continuedRef 6: Koblan, K.S. & Ackers, G.K. (1992) Site-Specific Regulation of DNA Transcription at Bacteriophage  OR, Biochemistry 31: 57-67.Ref 7: Darling, P.J., Holt, J.M. & Ackers, G.K. (2000) Coupled Energetics of 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 -cro bound to a Consensus Operator at 3.0 Å 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: 149-158Ref 15: Albright, R.A. and Matthews, B.W. (1998) How Cro and -repressor distinguish between operators: The structural basis underlying a genetic switch. Proc. Natl.Acad. Sci. USA 95: 3431-3436.Bacteriophage : an obligate parasite100,000x8,000xRef 1: Ptashne (1992) A Genetic Switch, 2nd ed., Cell Press & Blackwell Scientific Publications, Cambridge, MA.Lambda, lysogeny and lysisRRRRRRef 1lysogenylysisprophageRRRRRinfectinjectAn overview of  growth: Patterns of gene expression23101012PRPL chromosomepattern of gene expressionRef 1NcrocI repressorintThe 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 repressor vs. croKey points: same operator sites; “reverse” affinities; cooperativity; bind as dimersRef 1cI repressor: keeps cro turned off!Xcro: turns off production of cI!XUV irradiation activates RecA cleavage of cI monomers.P-DNA complex is reversible. cI dimerization is reversible. When cI dimers fall off, they attempt to reestablish equilibrium; monomers get cleaved. Decrease in [cI dimers], hence DNA opens up for cro binding!The switch: completing the storyRef 1Designing 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 cooperativityDesigning an efficient DNA binding proteinEquations on boardThe rationale for the arguments are taken fromAppendix One in reference 1Designing an efficient DNA binding proteinHow can we increase specificity?1). Increase protein concentration2). Improve specificity directly*play with the KD/KOP ratiohold KD constant; improve KOP*increase number of contacts by increasing repressortwice the contacts, twice the


View Full Document
Download Protein-DNA Interactions
Our administrator received your request to download this document. We will send you the file to your email shortly.
Loading Unlocking...
Login

Join to view Protein-DNA Interactions and access 3M+ class-specific study document.

or
We will never post anything without your permission.
Don't have an account?
Sign Up

Join to view Protein-DNA Interactions 2 2 and access 3M+ class-specific study document.

or

By creating an account you agree to our Privacy Policy and Terms Of Use

Already a member?