3. Researchers studying Lambda Repressor binding to three operator sites produce

Question

3. Researchers studying Lambda Repressor binding to three operator sites produced the data in the table below. In the experiment they performed DNasel footprinting assay for a range of repressor concentrations. The data provided a relative concentration of repressor dimers required to occupy a specific binding site. Relative Repressor Concentration for Each Site DNA Wild-type Mutant X Mutant Y OR2 2 R3 25 25 2 A. Based on the data for wild-type DNA, explain why the relative concentrations for OR and OR2 are almost the same despite the fact that OR1 has a 10-fold higher affinity for CI repressor than OR2 does? B. Based on the data for wild-type DNA, explain why the relative concentration of repressor needed to bind OR3 is much higher than for OR1 and OR2. C. Explain why the relative repressor concentration required to bindOR3 goes down to 5 for Mutant X relative to wild-type DNA in terms of the mechanism for 1 repressor binding DNA.

Explanation / Answer

A) Binding is dependent on the affinity to each other as well as their effective concentration. At lower concentrations, CI can only bind OR1 and OR2 cooperatively. That is, each lambda repressor dimer binds to another, forming a tetramer, with one half binding OR1 and the other binding OR2. Binding both operators (OR1 and OR2 are located in the PR promoter) as tetramer allows for a stronger interaction

b) The repressor is made up of helixes. The role of the helixes are to make contact with DNA, each helix has an affinity for a a specific sequence.The repressor is able to make more contacts with OR1 making it more stable. Or3 does not have this protein interaction, therefore their is weak affinity for binding. At higher concentrations, CI can bind OR3, located in the PRM promoter.

C) If the affinity of OR1 for repressor is reduced by mutation, cooperative interactions take place between repressor molecules bound at OR2 and OR3.

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