There is a mutation in the lacI gene, called the Id mutation, which results in t

ID: 217987 • Letter: T

Question

There is a mutation in the lacI gene, called the Id mutation, which results in the lac operon being uninducible; that is, no ?-galactosidase is made in the presence or in the absence of lactose (these results are also unaffected by the presence or absence of glucose in the medium). In partial diploids (chromosome + plasmid), lacId acts as a dominant gene. In other words, even a good copy of the lacI gene on a plasmid cannot overcome the defect of the Id mutation on the genomic lacI gene.

a. (4 points) For what protein does the lacI gene code?

b. (4 points) To what region of DNA does the LacI protein bind?

c. (4 points) What is the likely cause of this phenotype?

d. (4 points) You have isolated a different mutant of E. coli (normal lacI gene) that has increased activity of the enzyme phosphodiesterase, which degrades cAMP. Predict the effect of this mutation on the inducibility of the lac operon.

e. (4 points) DNA footprinting using DNaseI revealed much information about how the lac repressor functions. What would DNA footprinting reveal about the DNA binding sites of a normal Lac repressor protein? A normal RNA polymerase?

Explanation / Answer

a. LacI codes for the repressor protein.

b. LacI binds to the operator sequence.

c. LacId mutation renders it to be not bound by lactose (or lactose binding can not induce structural changes in the protein so that it can not dissociate from the operator sequence). Hence, LacId is always bound to the operator sequence leading constitutional repression of the operon.

d. Increased phosphodiesterase activity leads to rapid degradation of cAMP. So, CAP will be inactive. Hence, the affinity of RNA polymerase to the promoter is reduced and the transcription of the operon is suppressed.

e. DNaseI footprinting assay reveals protein binding sites on nucleic acids. RNA polymerase binds to the promoter. SO, promoter sequence will be protected in the DNase footprinting assay with RNA polymerase. Similarly, operator sequence will be protected when the DNA is incubated with the LacI protein.

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There is a mutation in the lacI gene, called the Id mutation, which results in t

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There is a mutation in the lacI gene, called the Id mutation, which results in t

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