Weaver eoc answers 09

Weaver eoc answers 09 - Answers to Weaver end of chapter...

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Answers to Weaver end of chapter questions Chapter 9 DNA-Protein Interactions in Prokaryotes 1. See Figure 9.2: 2. See Figures 9.3 and 9.4. Amino acid swapping experiments can be used to show which amino acids are important in specifying binding of repressors to their operators. In these experiments, amino acid residues that are predicted by structural studies to play a role in binding a particular operator are altered by site-directed mutagenesis. To use the example given in the text of the λ-like phages 434 and P22, we would predict that if we were to swap the amino acids we hypothesize as being important for operator-binding in the P22 repressor for those hypothesized to perform the same function in phage 434, the engineered P22 phage repressor would now bind 434 operators. In other words, the key amino acids in operator binding from one repressor can confer the same operator binding specificity on a repressor from a different phage. We can visualize the change in DNA binding specificities by DNase footprinting experiments. We would expect to observe binding of the altered 434 repressor to P22 operators and vice versa. Functional studies can also be used to determine the binding specificity repressors to their operators. E. coli lysogenic for phage 434 (actually, lysogenic for a lambda phage bearing the 434 immunity region), for example, will be immune to superinfection by another lambda phage bearing the 434 immunity region because the repressor in the lysogen will inactivate the genes in the incoming phage. If however, we were to engineer a lambda phage with a 434 immunity region such that it now had the amino acids in its repressor binding domain that are key to binding the P22 operators, a lysogen for this recombinant phage would no longer be immune to superinfection by the lambda phage bearing the 434 immunity region, but would be immune to superinfection by lambda phage bearing the P22 immunity region. 3. The λ repressor and Cro bind with different affinities to different regions of the same operators. Specifically, repressor binds with the highest affinity to O R 1 and the lowest affinity to O R 3 while Cro binds with its highest affinity to O R 3 and its lowest affinity to O R 1. These specificities are mediated by specific amino acid-base pair interactions 1
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between the recognition helix and the major grove of the DNA. The critical amino acids and base pairs are different for Cro and for the λ repressor. 4.
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Weaver eoc answers 09 - Answers to Weaver end of chapter...

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