Regulation of transcription initiation: Repression and activation


Transcriptional Repressors

You have four strains of E. coli:
a. lacP- (class I)
b. lacI- lacP- (class I)
c. lacI(Ts)
d. lacI(Ts) lacP-
(The lacP- mutation is identical in a, b, and d)

All other markers in the strains are wild type. How could you determine which strain is which? [Describe an experiment which identifies each strain (i.e. not simply by a process of elimination). You should be able to do this without DNA sequencing.]


You have isolated 3 regulatory mutants for the lac operon. P1 transduction analyses have shown that all three are linked to the lac operon structural genes. In haploid cells, all of the mutations cause high levels of operon expression in the absence of IPTG. Below are the complementation results you have obtained.

Give a complete description of each type in terms of the Jacob-Monod model.


A mutant of E. coli cannot be induced to synthesize large amounts of B-galactosidase or B-galactoside permease when lactose is added to the medium. However, a partial diploid formed from this mutant and an episome bearing the genes lacI- P+ Oc Z- Y+ synthesizes large amounts of lactose permease in the presence or absence of lactose. The partial diploid synthesizes large amounts of B-galactosidase only in the presence of lactose. What is the genotype of the haploid mutant?


A mutant of E. coli synthesizes large amounts of B-galactosidase in the presence or absence of lactose. It does not synthesize B-galactoside permease. A partial diploid formed from this mutant and an episome bearing the genes R+ P+ O+ Z- Y+ synthesizes large amounts of B-galactosidase in the presence or absence of lactose. The partial diploid synthesizes B-galactoside permease in the presence of lactose but not its absence. When an episome bearing the genes R- P+ O+Z-Y+ is introduced into this mutant, the resultant partial diploid strain synthesizes large amounts of B-galactoside and B-galactoside permease in the presence or absence of lactose. What is the genotype of the haploid mutant?


A mutant of E. coli synthesizes large amounts of the first two enzymes involved in histidine degradation whether or not histidine is present. However, this mutant does not synthesize the last two enzymes in the presence or absence of histidine. When an episome bearing the genes hutP+ O+ U- H- R+ P+ O+ I+ G+ is introduced into this mutant, the resultant partial diploid strain behaves in the same manner as the haploid mutant. What might the genotype of this haploid mutant be?


You have a strain of E. coli which contains a class I lacP mutation and is lacI. You isolate mutants which increase lac expression. Some of the mutants are true revertants which restore the class I mutation to its wild type sequence. A second class of mutants contain mutations which do not map in the site where class I mutations map. Where are these mutations likely to map? Explain at the molecular level how they would increase lac expression.


Explain the phenotypes of the following partial diploids (i.e. the cis/trans dominance of the regulatory mutants):

GenotypeRegulation of
lacZ expression
Rationale
a. lacO+ lacZ+ / lacOc lacZ+Constitutive?
b. lacOc lacZ+ / lacOc lacZ+Constitutive?
c. lacI+ lacZ+ / lacI- lacZ+Inducible?
d. lacI- lacZ+ / lacI+ lacZ-Inducible?
e. lacI- lacZ+ / lacOc lacZ+Constitutive?
f. lacO+ lacZ+ / lacOc lacZ-Inducible?
g. lacO+ lacZ- / lacOc lacZ+Constitutive?


How does growth in medium with glucose as a carbon source affect induction of the lac operon?


If you isolated a mutation that disrupted a positive regulatory gene for an operon, what would the regulatory phenotype be?


Compare and contrast the positive regulation of expression of the lac operon by cAMP CRP complex with the negative regulation of expression of the lac operon by the LacI protein.


Your lab received the following four strains of E. coli in the mail:

(a) lacP-
(b) lacI- lacP-
(c) lacI(TS)
(d) lacI(TS) lacP-

The lacP- mutation is identical in each of the strains. This is a "Class I" promoter mutation that inactivates the promoter. All other markers in the strains are wild type. When the strains were recieved they were not labeled. How could you determine which strain is which? Propose an experiment which directly identifies each strain (i.e. not simply by the process of elimination). DNA sequencing is not required!


Starting with a strain of E. coli which contains a "Class I" lacP mutation and is lacI-, you isolate mutants which increase lac expression. Some of the mutants are true revertants which restore the lacP mutation to its wild-type sequence. A second class of mutants contain mutations still retain the original lacP mutation. Where are these secondary mutations likely to map? Explain at the molecular level how they would increase lac expression.


You have a strain of E. coli that contains a deletion of the crp gene which makes it Lac-and Gal-. By plating a culture of this strain on Lactose minimal medium, you isolate mutants that are able to grow on lactose as a sole carbon source. You find that these mutants fall into two classes with respect to their gal phenotype. Class 1 is Gal+ and class 2 is Gal-. Suggest a plausible explanation for each class of mutation.


A mutant of E. coli synthesizes large amounts of the first two enzymes involved in histidine degradation whether or not histidine is present. However, this mutant does not synthesize the last two enzymes in the presence or absence of histidine. When an episome bearing the genes hisP+ O+ U- H- R+ P+ O+ I+ G+ is introduced into this mutant, the resultant partial diploid strain behaves in the same manner as the haploid mutant. What might the genotype of this haploid mutant be?


Transcriptional Activators

A mutant of E. coli grows well on glucose but is unable to use a large number of other carbon sources. Genetic studies indicate that the mutation is in a single site on the chromosome. What two types of mutants would give this phenotype and how could you distinguish these two types of mutants?


A strain of E. coli that contains a deletion of the crp gene is Lac- and Gal-. It is possible to isolate revertants that are able to grow on lactose as a sole carbon source. These mutants fall into two classes: Class 1 is Gal+ and Class 2 is Gal-. Suggest a plausible molecular explanation for each class of mutation.


A unique class of mutations in the crp gene are designated crp*. The properties of the crp* and crp+ mutations in either a cya or cya+ background are shown in the table below. The cells were grown in minimal medium with glycerol (as the carbon source) and IPTG.

StrainB-galactosidase
activity
crp+ cya+1000
crp+ cya-20
crp* cya+1000
crp* cya-1000

  1. What is the probable molecular mechanism for the phenotype of the cap* mutants. Briefly explain your answer.

  2. How would the crp* mutation affect expression of other catabolic operons such as gal or ara?

  3. How would the crp* mutation affect lac expression in a strain which contains a class I lac promoter mutation?

  4. How about a class II lac promoter mutation?

  5. If you grew a crp* strain in Min + glucose + IPTG, what level of B-galalactosidase would you predict?

  6. If you grew a crp* strain in minimal medium + glycerol without IPTG what level of B-galactosidase would you predict?

  7. If you constructed a partial diploid [F' crp*/crp+] which mutation would be dominant and why? If you added a column to the table with this mutant, what would the predicted B-galactosidase levels be?


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Last modified October 30, 2003