Lecture 11: Tryptophan operon, tryptophan attenuator,

1. Distinguish between induction and repression in a manner that makes it clear that you know what each is and how they differ. (Be careful, this is a tricky question that needs to be approached at multiple levels).

2. Describe two distinctly different ways in which the trp operon is controlled by the overall availability of tryptophan.

3. How does the interaction between the repressor protein and the operator site differ between the lac operon and the trp operon? What role does the ligand that binds to the repressor protein play in each case?

4. Describe the mechanism responsible for shutdown of the trp operon when a plentiful supply of free tryptophan is available.

5. Describe the mechanism by which the leader-attenuator region fine tunes the extent of transcription of the structural genes in the trp operon when the availability of tryptophan is marginal.

6. The leader-attenuation system utilizes a conditional transcription termination signal. Explain how that signal is activated and deactivated.

7. Leader-attenuator mechanisms have been found to control operons coding for enzymes involved in the synthesis of a number of different amino acids in various bacterial species, sometimes as the only regulatory mechanism. What do you consider to be the most likely reason for failure to find similar regulatory mechanisms controlling rates of synthesis of other types of biologically important molecules, such as vitamins and nucleic acid bases? (This requires analysis beyond the level of information that has been presented in class, but will be very obvious once you realize what the correct answer is.)

8. Would it be possible to reverse positions, placing the leader/attenuator sequence upstream from the promoter/operator sequence of the trp operon? Explain the reasoning behind your answer.

9. Some operons are controlled only by a leader/attenuator mechanism. Does this indicate that the trp operon in E. coli could function normally with the promoter/operator sequence completely removed? Explain the reasoning behind your answer and think carefully about what you are saying..

10. What effects on control of the trp operon would you expect from a frameshift mutation resulting from addition of one base in each of the following locations? In each case, would you expect the mutant strain to be able to multiply in a medium that did not contain tryptophan, and would you expect the operon to be turned off in the presence of an adequate amount of tryptophan?

a. Near the 5'-end of the coding sequence for the trp repressor protein.

b. Near the 5'-end of the coding sequence for the trp attenuator peptide. The codon for tryptophan is UGG, and the complete coding sequence for the attenuator peptide, including the UGA stop codon is

5'-AUGAAAGCAAUUUUCGUACUGAAAGGUUGGUGGCGCACUUCCUGA-3' (Consider this one carefully -- it could get complicated!).

c. Near the 3'-end of the coding sequence for the trp attenuator peptide (beyond the trp codons). (This one could also get complicated).

d. Near the 5'-end of the trp E gene (the first structural gene in the operon).

e. How would the answer to part b differ if the mutation were a missense mutation in the second codon of the attenuator peptide?

11. A strain of bacteria is auxotrophic because of a missence mutation in the trpE gene (the first one in the operon). Explain the reasoning behind your answers to each of the following questions.

a. What effect would this have on the the ability of the strain to grow in a medium containing tryptophan?

b. What effect would this have on the the ability of the strain to grow in a medium lacking tryptophan?

c. Would you expect the trpA gene to be transcribed in this strain when tryptophan is absent from the culture medium?

d. Would you expect the trpA gene to be trainscribed in this strain when the culture medium contains tryptophan?

e. Would you expect the trpE gene product to be translated in a medium lacking tryptophan? (Be careful, this one is tricky).

12. As illustrated in figure 8.14, The trpA and trpB gene products are needed only for the final step in tryptophan synthesis in E. coli, namely conversion of indole-3-glycerol phosphate (InGP) to tryptophan. The other three genes in the trp operon are involved in steps leading to the synthesis of InGP. Explain the reasoning behind your answers to each of the following quesitons.

a. In a wild-type (non-mutant) strain, what effect would you expect the presence of InGP in a culture medium that contained no added tryptophan to have on transcription of the trp operon

b. Would you expect a strain with a loss-of-function mutation in trpE to be able to grow in a medium that contains InGP but no tryptophan?

c. What would be the transcriptional state of the trp operon in question b?

d. Would you expect a strain with a loss-of-function mutation in trpA to be able to grow in a medium that contains InGP but no tryptophan?

e. What would be the transcriptional state of the trp operon in question d?

13. The gene coding for the tryptophan repressor protein is at a remote location relative to the tryptophan operon. What effect would complete deletion of the repressor gene have on the response of the operon to varying levels of tryptophan in the environment?

14. What effect would an operator constituitive mutation (unable to bind the repressor protein) have on the response of the tryptophan operon to varying levels of tryptophan in the environment?

15. What effect would you expect complete deletion of the leader-attenuator sequence from the tryptophan operon to have on its response to varying levels of tryptophan?

The following questions related to bacteriophage lambda are presented only for your entertainment. Aspects of bacteriophage lambda that are not included in other lectures or in assigned parts of the textbook other than section 8.4 will not be included on any examinations.

Lambda-1. What is antitermination, and how is it used to control gene expression? What objective is usually achieved through the use of an antitermination mechanism.

Lambda-2. Describe the major steps that are required for initiation of the lysogenic state in bacteriophage lambda.

Lambda-3. How is production of the lambda repressor maintained once it has been initiated?

Lambda-4. What mechanism is responsible for reversion from the lysogenic state to an active lytic infection in bacteriophage lambda?

Lambda-5. What causes the plaques produced by an infection with wild-type bacteriophage lambda to be somewhat cloudy?

Lambda-6. Summarize the molecular competition that ultimately determines whether bacteriophage lambda becomes lysogenic or lytic.

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