5. As you know, the lac promoter is a poor intrinsic promoter: it is transcribed efficiently only when CAP-CAMP is bound nearby. Imagine that a culture of E. coli cells is mutagenized and spread on an agar plate containing IPTG, glucose, and X-Gal. Most of the resulting colonies are white but a few are blue. [IPTG is a lactose analog that binds to and inactivates the lac repressor; unlike lactose, IPTG does not require Lac Permease (LacY) to enter the cell. X-gal is a substrate of B-galactosidase that yields a blue product when cleaved by the enzyme; X-gal does not bind to the lac repressor. For the purpose of this problem, assume that anything less than maximal induction of B-galactosidase results in a white colony. (i) Why was it expected that most of the colonies would be white?

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5. As you know, the lac promoter is a poor intrinsic promoter: it is transcribed efficiently only
when CAP-CAMP is bound nearby.
Imagine that a culture of E. coli cells is mutagenized and spread on an agar plate containing
IPTG, glucose, and X-Gal. Most of the resulting colonies are white but a few are blue.
[IPTG is a lactose analog that binds to and inactivates the lac repressor; unlike lactose, IPTG
does not require Lac Permease (LacY) to enter the cell. X-gal is a substrate of B-galactosidase
that yields a blue product when cleaved by the enzyme; X-gal does not bind to the lac
repressor. For the purpose of this problem, assume that anything less than maximal induction
of B-galactosidase results in a white colony.
(i) Why was it expected that most of the colonies would be white?
(ii) Describe two different mutations mapping within protein-coding DNA that can account for
the blue colonies. The two mutations should map in different genes and need not map to the lac
operon. Name the gene with the mutation and explain how the mutation alters the function of
the protein and thereby yields blue colonies.
(iii) Describe two different mutations that do NOT map within a protein-coding region that
can account for the blue colonies. The two mutations should affect distinct aspects of lac
regulation.
(iv) Predict whether each of your four mutations would be dominant or recessive to wild-type.
Explain briefly.
(v) Which of the two classes of mutations (i.e. those described in parts ii and iii above) influence
"trans-acting" functions and which influence cis-acting functions?
Transcribed Image Text:5. As you know, the lac promoter is a poor intrinsic promoter: it is transcribed efficiently only when CAP-CAMP is bound nearby. Imagine that a culture of E. coli cells is mutagenized and spread on an agar plate containing IPTG, glucose, and X-Gal. Most of the resulting colonies are white but a few are blue. [IPTG is a lactose analog that binds to and inactivates the lac repressor; unlike lactose, IPTG does not require Lac Permease (LacY) to enter the cell. X-gal is a substrate of B-galactosidase that yields a blue product when cleaved by the enzyme; X-gal does not bind to the lac repressor. For the purpose of this problem, assume that anything less than maximal induction of B-galactosidase results in a white colony. (i) Why was it expected that most of the colonies would be white? (ii) Describe two different mutations mapping within protein-coding DNA that can account for the blue colonies. The two mutations should map in different genes and need not map to the lac operon. Name the gene with the mutation and explain how the mutation alters the function of the protein and thereby yields blue colonies. (iii) Describe two different mutations that do NOT map within a protein-coding region that can account for the blue colonies. The two mutations should affect distinct aspects of lac regulation. (iv) Predict whether each of your four mutations would be dominant or recessive to wild-type. Explain briefly. (v) Which of the two classes of mutations (i.e. those described in parts ii and iii above) influence "trans-acting" functions and which influence cis-acting functions?
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