Question

Lab 6 Beta-Galactosidase Activity (Gene Expression): Preparation

1. When lactose is present and glucose is absent, explain what will occur with the lac operon; specifically to the repressor protein, CAP-cAMP complex, RNA polymerase and the production levels of the -galactosidase enzyme

2. When lactose is present and glucose is present, explain what will occur with the lac operon; specifically to the repressor protein, CAP-cAMP complex, RNA polymerase and the production levels of the b-galactosidase enzyme.

3. When lactose is absent and glucose is present, explain what will occur with the lac operon; specifically to the repressor protein, CAP-cAMP complex, RNA polymerase and the production levels of the b-galactosidase enzyme.

4. When lactose is absent and glucose is absent, explain what will occur with the lac operon; specifically to the repressor protein, CAP-cAMP complex, RNA polymerase and the production levels of the b-galactosidase enzyme.

Answer 1

1. When lactose is present and glucose is absent, explain what will occur with the lac operon; specifically to the repressor protein, CAP-cAMP complex, RNA polymerase and the production levels of the galactosidase enzyme

Answer:

· When glucose is absent, adenylate cyclase is not inhibited, the level of intracellular cAMP rises and binds to CRP.

· Therefore, when glucose is absent but lactose is present, the CRP–cAMP complex stimulates transcription of the lac operon and allows the lactose to be used as an alternative carbon source.

· When there is a shortage of glucose catabolites and CAP-cAMP forms, the enzymes taking part in lactose transport and metabolism are produced only if lactose is present.

2. When lactose is present and glucose is present, explain what will occur with the lac operon; specifically to the repressor protein, CAP-cAMP complex, RNA polymerase and the production levels of the b-galactosidase enzyme.

Answer:

· If both glucose and lactose are both present, lactose binds to the repressor and prevents it from binding to the operator region.

· In the presence of glucose, the catabolite activator protein (CAP), required for production of the enzymes, remains inactive, and  shuts down lactose permease to prevent transport of lactose into the cell

· If both lactose and glucose are present, synthesis of β-galactosidase is not induced until all the glucose has been utilized. Thus, the cell conserves its metabolic machinery (that, for example, induces the lac enzymes) by utilizing any existing glucose before going through the steps of creating new machinery to metabolize the lactose.

· In this case the RNA polymerase can sit on the promoter site, but it is unstable and it keeps falling off.

3. When lactose is absent and glucose is present, explain what will occur with the lac operon; specifically to the repressor protein, CAP-cAMP complex, RNA polymerase and the production levels of the b-galactosidase enzyme.

Answer:

· If lactose is absent and glucose is present, the lac repressor binds to the operator region

· When glucose is present in high concentrations, the cAMP concentration is low; as the glucose concentration decreases, the concentration of cAMP increases correspondingly. The high concentration of cAMP is necessary for activation of the lac operon.

4. When lactose is absent and glucose is absent, explain what will occur with the lac operon; specifically to the repressor protein, CAP-cAMP complex, RNA polymerase and the production levels of the b-galactosidase enzyme.

Answer:

In the absence of lactose, the lac repressor, lacI, halts production of the enzymes encoded by the lac operon. When lactose is not available, the lac repressor binds tightly to the operator, preventing transcription by RNA polymerase.

The activator protein only woks when glucose is absent. In this way coli only makes enzymes to metabolise other sugars in the absence of glucose promoter site, transcription activator protein steadies the RNA polymerase.