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Homework
BIO 435/535
NAME ____________________________________

by Scott Cooper (University of WisconsinóLa Crosse, cooper@mail.uwlax.edu)

You are studying the molecular mechanism by which Catabolite Acitvator Protein (CAP) [also called cAMP Receptor Protein (CRP)] binds to the promoter of the lac operon. You mutagenize the DNA in the region of the lac promoter thought to be the CAP binding site, replacing the GC base pair at position 5 with AT. You then assay b -galactosidase activity with and without glucose and galactose, comparing wild-type activity with that of your mutant. You get the following results.
 
 

Glucose
Lactose
Wild type
GC->AT
+
-
0
0
+
+
2
2
-
+
20
2

+ indicates that the carbon source was available in the media

numbers refer to b -galactosidase activity in U/ml media.

Go to the following website and select the model of CRP. Click on the box next to the statement "Close-up view of protein-DNA interactions in one half-site." http://bioweb.uwlax.edu/GenWeb/Molecular/Modeling/Chime_Page/chime_page.htm

Based upon this three dimensional model and your data above answer these questions.

  1. Using the diagram below, label bases G5 and C5 that would be present in the wild type lac promoter. Draw the amino acids in CAP that interact with these bases, indicating the H-bonds that would be formed.
  2. In the mutation you generated you substituted an AT base pair for the GC base pair. Draw a diagram of how the existing amino acids on CAP would interact with this AT base pair. Be sure to label bases, amino acids and add H-bonds.
  3. Briefly explain how your enzymatic data above would be consistent with your model.
  4. Based on your model, what amino acids replacements would you make in the CAP protein to get it to bind to your mutated promoter (i.e. the AT base pair)?
  5.