Lecture 10: Prokaryotic gene expression: lac operon
1. Draw a diagram showing the arrangement of the genetic sites that are involved in the lac operon and briefly summarize the role of each site.
2. Starting with lactose in the extracellular environment, describe all of the steps that must occur to induce enhanced expression of the genes in the lac operon.
3. What effect will each of the following have on induction of the lac operon by lactose in a medium that lacks glucose?
a. A mutation that inactivates beta-galactosidase.
b. A mutation that inactivates galactoside permease.
c. A mutation that makes the lac repressor protein incapable of binding to the lac operator site.
d. A mutation that makes the lac operator site incapable of binding the lac repressor protein.
e. A mutation that makes the lac repressor protein incapable of binding allolactose.
4. What advantage is provided to E. coli by the presence of two separate regulatory mechanisms for the lac operon, with one responsive to glucose and the other responsive to lactose?
5. What is meant by a polycistronic transcript, and how is it related to the lac operon?
6. What is the normal function of the operator site and what is the effect of loss of that function?
7. What is the catabolite activator protein (CAP) and how does it affect gene expression at the lac operon?
8. What are the minimum set of components that must be present to have an operon?
9. What is a merozygote and how is it used to analyze the control mechanisms of an operon?
10. What is meant by cis and trans when these terms are used to describe regulatory mechanisms within an operon. Give an example of a mutation that is cis -dominant and one that has dominant effects both cis and trans.
11. Describe two different roles of beta-galactosidase and explain the importance of each in the overall function of the lac operon.
12. Explain how the presence of glucose in the external environment represses transcription of genes in the lac operon.
13. What is the advantage of using an artificial inducer, such as IPTG, to study the regulation of the lac operon?
14. An engineered F' plasmid carrys a lac operon that is fully functional and intact except that each of its 3 enzymes have been mutated in ways that allow them to be distinguished from host enzymes, but do not affect their enzymatic activity. What will be the effect of adding that plasmid to each of the following mutant strains of E. coli ? Include in your answers a description of the properties of the mutant strain without the plasmid and a description of the ways in which those properties are changed by addition of the plasmid.
a. An operator constituitive (Oc) strain whose operator is incapable of binding the repressor protein.
b. A strain with a non-functional repressor protein (I-) that is incapable of binding the operator.
c. A strain with a mutation that renders beta-galactosidease nonfunctional (lac Z-).
d. A strain with a mutation that renders the lactose permease nonfunctional (lac Y-)
e. A strain with a mutation in the repressor protein that renders it incapable of binding allolactose or IPTG, without altering any of its other funcitons.
15. Is the Lac I gene that codes for the lactose repressor protein considered to be part of the lactose operon? Explain your answer.
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