Microbiologists are investigating sugar metabolism in wild-type and mutant strains of Escherichia coli. Both strains are found to grow viable colonies on lactose-containing media. Each strain is then cultured on a new growth medium containing only glucose. Representative colonies of each strain from the new media undergo Western blot processing using a fluorescently labeled probe specific for β-galactosidase. Wild-type bacterial colonies are found to contain only trace quantities of β-galactosidase. However, the mutant colonies express significant amounts of β-galactosidase. Further analysis reveals that the variant strain contains a mutation that inhibits the binding of a certain protein to its regulatory sequence. In which of the following locations did this mutation most likely occur?
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The lac operon consists of a regulatory gene (lac I), a promoter region (lac p), an operator region (lac o), and three structural genes (lac Z, lac Y, and lac A). The lac Z gene codes for β-galactosidase, which is responsible for the hydrolysis of lactose to glucose and galactose. The lac Y gene codes for permease, which allows lactose to enter the bacterium. The lac p region is the binding site for RNA polymerase during the initiation of transcription. The Lac I repressor protein is the product of the lac I gene and is constitutively expressed. Repressor proteins, when bound to the operator region, prevent binding of RNA polymerase to the promoter region, thus decreasing transcription of the lac Z, lac Y, and lac A genes. Culture of E coli in lactose-containing media causes a conformational change in the repressor protein, preventing its attachment to the operator region and increasing transcription of the lac operon structural genes.
Culturing E coli in media containing glucose results in reduced expression of the lac operon, even when the media contains lactose as well. This occurs because the lac operon is positively regulated by the binding of catabolite activator protein (CAP) to a site slightly upstream from the promoter region. This only occurs when cAMP concentrations are high. Since glucose decreases the activity of adenylyl cyclase (reducing intracellular cAMP), the lac operon is repressed in high-glucose conditions. In summary, the lac operon is regulated by 2 distinct mechanisms:
Mutations impairing the binding of the repressor protein to its binding site at the operator region will prevent repression of the genes of the lac operon in the absence of lactose. This results in increased transcription of the genes of the lac operon in lactose-deficient media, although the presence of glucose will prevent maximal transcriptional activity.
(Choices A and D) Mutations that impair the binding of cAMP-CAP to its regulatory site upstream from the promoter will decrease transcription of the lac operon, as cAMP-CAP is a positive regulator.
(Choices C and E) Mutations impairing the binding of RNA polymerase to the promotor region will also reduce transcription of the lac operon.
Educational objective:
The lac operon is regulated by two distinct mechanisms: negatively by binding of the repressor protein to the operator locus and positively by cAMP-CAP binding upstream from the promoter region. Constitutive expression of the structural genes of the lac operon occurs with mutations that impair the binding of the repressor protein (Lac I) to its regulatory sequence in the operator region.