Biology 2581B Lecture Notes - Lecture 2: Dna-Binding Domain, Lac Operon, Lac Repressor
15 Mar 2015
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Small proportion of lactose entering cell becomes allolactose. Repressor protein has to physically bind wild-type sequence but not lac oc mutant. Repressor protein must have two distinct domains one binds dna & the other. Take labeled fragments and mix them with dna-binding protein (repressor) If protein does have dna binding activity prevents it from being cut by enzyme (dnase cuts randomly) Experiment (w/ protein) run on gel segments missing showing that protein binds to domain. Dna fragment containing binding sequence labeled radioactively at 5" with phosphates. Control (no protein) run on gel. Take dna that contains wildtype copy of lac operator. Dna & protein will sediment to the point where density is equal. Dna & protein co-localize part of a complex. Take dna that contains mutant copy of lac operator. Mix that with radioactively labeled repressor protein. Dna & protein should sediment to the same spot but don"t no complex.
