IGEM:Stanford/2009/Notebook/Marys iGEM Notebook/2009/08/13

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Stuffs we need to find out for the new trp model

If you know anything about these, please put it right after the list, or in "comments". Thanks^^

Let's start with wild type system--

  • kt or ΔG :dissociation constant/Gibbs free energy of WT aporepressor binding with one single L-tryptophan (there are two binding sites in all).
    • Alternative solutions: find out through fitting with equations based on plots in [1], but need to build out the whole system model first.


  • On WT repressor and WT operator binding, I borrowed several assumptions in [2], and need to check out whether it's the same situation for the trp system:
    • (1) The three binding sites are specific, nonoverlapping, DNA sequences; each site binds one repressor at a time.
    • (2) Repressors are bound at the operator sites in their dimeric forms only. Repressor monomers are in equilibrium with dimers.
    • (3) There are no co-operative interactions between WT repressor dimers and any other mutant repressors, including adjacent operator bound mutant repressors. (Is it true???)
    • (4) At an operator with three bound repressor dimers, co-operative interactions only happens between two of them. (Is it true???)
    • (5) What effect does RNA polymerase binding promoter have on WT repressor binding operator? (Need to find out...)
    • (6) Occupancy of operator sites is determined by equilibrium statistical thermodynamic probabilities. (This is the basis of our model, especially on cooperativity.)
  • Thus, it's important to find out the follow values:
    • Gibbs free energy (ΔG) or dissociation constant of every single operator binding site bound with WT repressor.(ΔG_1, ΔG_2, ΔG_3)
    • Coupling free energy between adjacent bound repressors.(ΔG_12, ΔG_23)


As for the mutant system, it's much the same--

  • Data needed for the 5MT binding Leu58 trp-repressor model
    • kt value or ΔG
  • Data needed for the trp-repressor binding trp operator cooperativity model
    • Gibbs free energy (ΔG) or dissociation constant of every single binding site bound with wt-repressor and Lys79 repressor
    • Alternative solutions: estimation based on data in [3], but need to build out the whole system model first.
      • For instance, ΔG of 7C(one of the mutant operator binding sites) bound with Lys79(mutant repressor binding site)
    • Already know the coupling free energy between adjacent Lys79 repressors, which is ~−2 kcal/mol ([4]). Is there cooperativity between adjacent WT and mutant repressor?

References

  1. Dennis N. Arvidson,' Michael Shapiro and Philip Youderian. Mutant Tryptophan Aporepressors With Altered Specificities of Corepressor Recognition.Genetics Society of America,1991
  2. The OR Control System of Bacteriophage Lambda A Physical-Chemical Model for Gene Regulation. Madeline A. Shea and Gary K. Ackersf. J. Mol. Rid. (1985) 181, 211-230
  3. Mutant Trp Repressors with New DNA-Binding Specificities. STEVEN BASS,* VINCENZA SORRELLS,t PHILIP YOUDERLAN. SCIENCE, VOL. 242.
  4. In vivo and in vitro Studies of TrpR-DNA Interactions. Jie Yang, Angelo Gunasekera†, Teresa A. Lavoie, Lihua Jin, Dale E. A. Lewis and Jannette Carey. J. Mol. Biol. (1996) 258, 37–52.

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