Question

1. For the lac operon, explain what would happen and why regarding expression of the operon in each of the following scenarios. Be sure to include both positive and negative regulation in your explanation. a.) lactose is present, glucose is absent b.) lactose is absent, glucose is present c.) both lactose and glucose are present d.) lactose is present, glucose is absent, but there is a mutation to the sequence in the CAP site

Answer 1

1. The lac operon is present in E.coli and is required for metabolism of lactose. This operon is turned on and off in response to lactose. There is catabolite activator protein (CAP) binding site, a promoter site, an operator site, followed by the three genes lacy, lac Z and lac A. There is also a lacI regulatory gene, which synthesizes the repressor protein. The CAP binding site is the positive regulatory site while the operator is the negative regulatory site. CAP binding site overlaps with the RNA polymerase binding site in promoter. Binding of cAMP-CAP complex to CAP binding site will allow the RNA polymerase to bind the promoter. As a result, there will be optimal transcription of lac genes. When repressor binds to the operator, it will prevent RNA polymerase from transcribing the lac genes. However, allolactose can bind to repressor and prevent it from binding the operator. This will induce transcription of lac genes.

a) Lactose is present, glucose is absent: When glucose is absent, the levels of cAMP in the cell will increase. Thus, cAMP will bind to CAP. The CAP-cAMP complex will bind to the CAP binding site in the operon. Allolactose that diffuses in the cell will bind to the repressor, thereby preventing it from binding to the operator. RNA polymerase will bind to the promoter and will transcribe lac ZYA genes. Thus, lactose will be taken in by the cell by permease (product of lac Y) and then metabolized by beta galactosidase (product of lac Z). Thus, lac operon will be induced.

b) Lactose is absent, glucose is present: When glucose is present, it is utilized as a carbon source and is metabolized. As a result, cAMP levels decline in the cell. Hence, cAMP cannot bind to CAP. Thus, CAP binding site is empty, preventing RNA polymerase from binding the promoter. Simultaneously, due to absence of allolactose, repressor will bind to the operator. This will also prevent transcription of lac genes. As RNA polymerase is not bound to promoter and there is repressor attached to operator, lac operon is not operational. Only glucose is used as a carbon source and is metabolized.

c) Both glucose and lactose is present: Glucose will be metabolized first and lac operon is not operational during glucose metabolism. Synthesis of beta galactosidase will not occur until all glucose is utilized. When glucose is metabolized, cAMP levels decline. As a result, CAP cannot bind to CAP binding site preventing the binding of RNA polymerase to promoter. Repressor is also bound to lac operator when glucose is being metabolized. A breakdown product of glucose will also inhibit formation of cAMP-CAP complex.

When glucose gets completely metabolized, cAMP levels will start increasing. As a result, cAMP binds to the CAP, and this complex will bind to the CAP binding site. As a result, RNA polymerase can bind to the promoter. The repressor will now bind to the allolactose that diffuses in the cell. Hence, the operator site is not empty, allowing RNA polymerase to initiate transcription of lac genes and produce beta galactosidase that is required to break down lactose. Thus, the cell will conserve energy and use lactose only when there is no glucose is left for metabolism.

d) Lactose is present, glucose is absent, but there is a mutation to the sequence in the CAP site: As glucose is absent, the cell will allow a small amount of allolactose to enter the cell. The levels of cAMP in the cell will be high, as there is no glucose metabolism. cAMP will bind to CAP to form a cAMP-CAP complex. However, as there is a mutation in CAP binding site, the cAMP-CAP complex will not bind the CAP binding site. Hence, RNA polymerase cannot bind the promoter.

Although, allolactose will bind the repressor protein, resulting in a free operator, there is minimal transcription and synthesis of beta galactosidase. This is because RNA polymerase is prevented from binding to promoter to initiate transcription of lac genes. Thus, even though lactose is present, the lac operon is not functional due to mutation in CAP site. There is prevention of high levels of expression of lac operon. Only minimal transcription of lac genes will occur.