Biology 265
2
Problem-based Learning cAMP Regulation of the Lactose Operon Received for publication, March 17, 2004 Jozsef Szeberenyi‡ From the Department of Medical Biology, School of Medicine, University of Pe ´ cs, H-7624 Pe ´ cs, Hungary Terms to be familiar with before you start to solve the test: lactose operon, adenylate cyclase, cAMP, catabolite activator protein (CAP), expression plasmid, lac operator, lac repressor, lactose, glucose, promoter, cis- and trans-acting factors. THE EXPERIMENT Two mutant E. coli strains were compared in this study. In one strain the gene coding for adenylate cyclase, while in the other one the gene of catabolite activator protein (CAP), 1 were altered so that the respective proteins be- came inactivated. Considering the role of these proteins in cell metabolism, solve the following multiple-choice questions! FOUR-CHOICE ASSOCIATION In this type of question a set of lettered headings is followed by a list of numbered words, phrases or state- ments. Select the response A if the word, phrase or statement is associated with a only; B if the word, phrase or statement is associated with b only; C if the word, phrase or statement is associated with a andb; D if the word, phrase or statement is associated with neither a nor b. a Adenylate cyclase mutant cells b Catabolite activator protein mutant cells c Both of them d Neither of them 1. These cells would become normal if transformed by an expression plasmid containing the adenylate cy- clase gene. 2. Adding lactose to their medium would increase their cAMP content. 3. These cells would become normal if transformed by a plasmid containing the lac operator. 4. Withdrawing glucose from their medium would in- crease their cAMP content. 5. Lac repressor is unable to bind to the operator under any conditions in these cells. 6. They can use only lactose as carbon source. 7. Lac repressor is constitutively bound to the oper- ator in these cells. 8. In a medium containing glucose, CAP binds to their lactose operon promoter. 9. They do not survive if their only source of carbon is lactose. 10. They do not survive if their only source of carbon is glucose. EXPLANATIONS To solve this test based on a fictitious experiment knowledge of the structure and understanding the regula- tion of the lactose operon is required (Fig. 1). The operon consists of the promoter that serves as the RNA polymer- ase binding site, the operator, attachment site for lac re- pressor, and the structural genes coding for the three enzymes required for the breakdown of lactose. In the absence of lactose, the operator is occupied by the lac repressor, preventing the binding and functioning of RNA polymerase. The other “branch” of regulation is controlled by glucose in the medium. In the presence of glucose adenylate cy- clase is inactive, cAMP is not produced, and CAP is not able to bind upstream of the promoter (this is required for stable RNA polymerase binding). In the absence of glu- cose, the adenylate cyclase/cAMP/CAP axis is activated. This dual regulation ensures that the lactose operon is expressed at a high level only in the presence of lactose ‡ To whom correspondence should be addressed: Department of Medical Biology, School of Medicine, University of Pe ´ cs, H-7624 Pe ´ cs, Hungary. E-mail: [email protected]. 1 The abbreviations used are: CAP, catabolite activator protein; MCQ, multiple-choice question. FIG. 1. The structure and regulation of lactose operon. G, Glucose; L, lactose. © 2004 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Printed in U.S.A. Vol. 32, No. 3, pp. 198–199, 2004 This paper is available on line at http://www.bambed.org 198
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Molecular genetics
Transcript of Biology 265
Problem-based Learning
cAMP Regulation of the Lactose Operon
Received for publication, March 17, 2004
Jozsef Szeberenyi‡
From the Department of Medical Biology, School of Medicine, University of Pecs, H-7624 Pecs, Hungary
Terms to be familiar with before you start to solve the test: lactose operon, adenylate cyclase, cAMP,catabolite activator protein (CAP), expression plasmid, lac operator, lac repressor, lactose, glucose,promoter, cis- and trans-acting factors.
THE EXPERIMENT
Two mutant E. coli strains were compared in this study.In one strain the gene coding for adenylate cyclase, whilein the other one the gene of catabolite activator protein(CAP),1 were altered so that the respective proteins be-came inactivated. Considering the role of these proteins incell metabolism, solve the following multiple-choicequestions!
FOUR-CHOICE ASSOCIATION
In this type of question a set of lettered headings isfollowed by a list of numbered words, phrases or state-ments. Select the response
A if the word, phrase or statement is associated with aonly;
B if the word, phrase or statement is associated with bonly;
C if the word, phrase or statement is associated with aandb;
D if the word, phrase or statement is associated withneither a nor b.
a Adenylate cyclase mutant cellsb Catabolite activator protein mutant cellsc Both of themd Neither of them
1. These cells would become normal if transformed byan expression plasmid containing the adenylate cy-clase gene.
2. Adding lactose to their medium would increase theircAMP content.
3. These cells would become normal if transformed bya plasmid containing the lac operator.
4. Withdrawing glucose from their medium would in-crease their cAMP content.
5. Lac repressor is unable to bind to the operatorunder any conditions in these cells.
6. They can use only lactose as carbon source.7. Lac repressor is constitutively bound to the oper-
ator in these cells.8. In a medium containing glucose, CAP binds to their
lactose operon promoter.9. They do not survive if their only source of carbon is
lactose.10. They do not survive if their only source of carbon is
glucose.
EXPLANATIONS
To solve this test based on a fictitious experimentknowledge of the structure and understanding the regula-tion of the lactose operon is required (Fig. 1). The operonconsists of the promoter that serves as the RNA polymer-ase binding site, the operator, attachment site for lac re-pressor, and the structural genes coding for the threeenzymes required for the breakdown of lactose. In theabsence of lactose, the operator is occupied by the lacrepressor, preventing the binding and functioning of RNApolymerase.
The other “branch” of regulation is controlled by glucosein the medium. In the presence of glucose adenylate cy-clase is inactive, cAMP is not produced, and CAP is notable to bind upstream of the promoter (this is required forstable RNA polymerase binding). In the absence of glu-cose, the adenylate cyclase/cAMP/CAP axis is activated.This dual regulation ensures that the lactose operon isexpressed at a high level only in the presence of lactose
‡ To whom correspondence should be addressed: Departmentof Medical Biology, School of Medicine, University of Pecs,H-7624 Pecs, Hungary. E-mail: [email protected].
1 The abbreviations used are: CAP, catabolite activator protein;MCQ, multiple-choice question.
FIG. 1. The structure and regulation of lactose operon. G,Glucose; L, lactose.
© 2004 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATIONPrinted in U.S.A. Vol. 32, No. 3, pp. 198–199, 2004
This paper is available on line at http://www.bambed.org198
and in the absence of glucose (see Fig. 1).In both mutants described in this test, the cAMP path-
way is knocked out (MCQ 4: B; MCQ 6: D; MCQ 8: D;MCQ 9: C; MCQ 10: D), while the lac repressor mecha-nism is intact (MCQ 3: D; MCQ 5: D; MCQ 7: D). Lactosedoes not have an impact on the cAMP mechanism (MCQ2: D). The genes for adenylate cyclase and CAP act astrans-regulatory elements in this system: expressing exog-enous wild-type genes coding for these proteins in the
respective mutants would most likely “cure” lactose me-tabolism in the “sick” cells (MCQ 1: A).
CORRECT ANSWERS
1. A 6. D2. D 7. D3. D 8. D4. B 9. C5. D 10. D
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