Mod 13

Draw a picture illustrating the general structure of an operon and identify its parts.

(question 3 ch 16)

The lac operon is transcribed at high levels when lactose is the only carbon source. However, transcription of the operon is greatly reduced in the presence of lactose and glucose. Why?

Glucose lowers the levels of cAMP. cAMP binds to CAP and together these proteins help RNA polymerase to bind to the promoter. If cAMP levels are low, CAP and RNA polymerase have difficulty binding to the promoter to initiate transcription.

What is catabolite repression? How does it allow a bacterial cell to use glucose in preference to other sugars?

In catabolite repression, the presence of glucose inhibits or represses the transcription of genes involved in the metabolism of other sugars. Because the gene expression necessary for utilizing other sugars is turned off, only enzymes involved in the metabolism of glucose will be synthesized. Operons that exhibit catabolite repression are under the positive control of catabolic activator protein (CAP). For CAP to be active, it must form a complex with cAMP. Glucose affects the level of cAMP. The levels of glucose and cAMP are inversely proportional—as glucose levels increase, the level of cAMP decreases. Thus, CAP is not activated.

With respect to the lac operon, define the term induction.

Induction is the relief of repression of transcription caused by the repressor protein. This occurs after (allo)lactose binds to the repressor causing an allosteric change in its shape.

Which strand of DNA (upper or lower) in Figure 16.9 is the template strand? Explain your reasoning

Solution: The bottom strand. RNA polymerase binds the promoter and moves downstream, toward the transcription start site, so in this diagram it moves from left to right. The template strand is always read 3′ to 5′, because the newly synthesized RNA is synthesized 5′ to 3′. As the polymerase moves left to right, it is the bottom strand that will be read 3′ to 5′, so it must be the template strand.

A mutation at the operator site prevents the regulator protein from binding. What effect will this mutation have in the following types of operons? a. Regulator protein is a repressor in a repressible operon

Solution: The regulator protein-corepressor complex would normally bind to the operator and inhibit transcription. If a mutation prevented the repressor protein from binding at the operator, then the operon would never be turned off and transcription would occur all the time.

Why does E. coli prefer to use glucose as a carbon source even if lactose is also present?

Lactose must be cleaved to generate glucose and galactose. If free glucose is already available, the cell does not need to make β-galactosidase and permease to utilize lactose. In fact this would be an inefficient use of cellular energy resources.

For the lac operon contrast negative and positive control of transcription

Negative control occurs when the repressor binds to the operator in the absence of lactose. The operon is not transcribed. Positive control occurs in the presence of lactose when CAP-cAMP help RNA polymerase to bind to the promoter and activate transcription.

What is difference between positive and negative control? What is the difference between inducible and repressible operons?

Positive transcriptional control requires an activator protein to stimulate transcription at the operon. In negative control, a repressor protein inhibits or turns off transcription at the operon. An inducible operon normally is not transcribed. It requires an inducer molecule to stimulate transcription either by inactivating a repressor protein in a negative inducible operon or by stimulating the activator protein in a positive inducible operon. Transcription normally occurs in a repressible operon. In a repressible operon, transcription is turned off either by the repressor becoming active in a negative repressible operon or by the activator becoming inactive in a positive repressible operon

How does the term allosteric transition apply to the regulation of the lac operon?

When lactose binds to the repressor, the repressor undergoes a conformational change (allosteric change). The altered repressor is unable to bind to the operator and inhibit transcription by RNA polymerase.

. For each of the following types of transcriptional control, indicate whether the protein produced by the regulator gene will be synthesized initially as an active repressor, inactive repressor, active activator, or inactive activator. b. Positive control in a repressible operon

Solution: Active activator

. For each of the following types of transcriptional control, indicate whether the protein produced by the regulator gene will be synthesized initially as an active repressor, inactive repressor, active activator, or inactive activator. c. Negative control in an inducible operon

Solution: Active repressor

. For each of the following types of transcriptional control, indicate whether the protein produced by the regulator gene will be synthesized initially as an active repressor, inactive repressor, active activator, or inactive activator. d. Positive control in an inducible operon

Solution: Inactive activator

A mutation prevents the catabolite activator protein (CAP) from binding to the promoter in the lac operon. What will the effect of this mutation be on transcription of the operon?

Solution: Catabolite activator protein binds the CAP site of the lac operon and stimulates RNA polymerase to bind the lac promoter, thus resulting in increased levels of transcription from the lac operon. If a mutation prevents CAP from binding to the site, then RNA polymerase will bind the lac promoter poorly. This will result in significantly lower levels of transcription of the lac structural genes.

Under which of the following conditions would a lac operon produce the greatest amount of β-galactosidase? The least? Explain your reasoning Lactose present Glucose present Condition 1 Yes No Condition 2 No Yes Condition 3 Yes Yes Condition 4 No No

Condition 1 will result in the production of the maximum amount of β-galactosidase. For maximum transcription, the presence of lactose and the absence of glucose are required. Lactose (or allolactose) binds to the lac repressor reducing the affinity of the lac repressor to the operator. This decreased affinity results in the promoter being accessible to RNA polymerase. The lack of glucose allows for increased synthesis of cAMP, which can complex with CAP. The formation of CAP-cAMP complexes improves the efficiency of RNA polymerase binding to the promoter, which results in higher levels of transcription from the lac operon. Condition 2 will result in the production of the least amount of β-galactosidase. With no lactose present, the lac repressor is active and binds to the operator, inhibiting transcription. The presence of glucose results in a decrease of cAMP levels. A CAP-cAMP complex does not form, and RNA polymerase will not be stimulated to transcribe the lac operon.

A mutation at the operator site prevents the regulator protein from binding. What effect will this mutation have in the following types of operons? b. Regulator protein is a repressor in an inducible operon.

Solution: In an inducible operon, a mutation at the operator site that blocks binding of the repressor would result in constitutive expression and transcription would occur all the time.

. For each of the following types of transcriptional control, indicate whether the protein produced by the regulator gene will be synthesized initially as an active repressor, inactive repressor, active activator, or inactive activator. a. Negative control in a repressible operon

Solution: Inactive repressor

A mutant strain of E. coli produces β-galactosidase in the presence and the absence of lactose. Where in the operon might the mutation in this strain occur?

Solution: Within the operon, the operator region is the most probable location of the mutation. If the mutation prevents the lac repressor protein from binding to the operator, then transcription of the lac structural genes will not be inhibited. Expression will be constitutive. Outside of the operon, a mutation in the lacI gene that inactivates the repressor or keeps it from binding to the operator could also lead to constitutive expression of the structural genes.

Briefly describe the lac operon and how it controls the metabolism of lactose.

The lac operon consists of three structural genes involved in lactose metabolism, the lacZ gene, the lacY gene, and the lacA gene. Each of these three genes has a different role in the metabolism of lactose. The lacZ gene codes for the enzyme β-galactosidase, which breaks the disaccharide lactose into galactose and glucose, and converts lactose into allolactose. The lacY gene, located downstream of the lacZ gene, codes for lactose permease. Permease is necessary for the passage of lactose through the E. coli cell membrane. The lacA gene, located downstream of lacY, encodes the enzyme thiogalactoside transacetylase whose function in lactose metabolism has not yet been determined. All of these genes share a common overlapping promoter and operator region. Upstream from the lactose operon is the lacI gene that encodes the lac operon repressor. The repressor binds at the operator region and inhibits transcription of the lac operon by preventing RNA polymerase from successfully initiating transcription. When lactose is present in the cell, the enzyme β-galactosidase converts some of it into allolactose. Allolactose binds to the lac repressor, altering its shape and reducing the repressor's affinity for the operator. Since this allolactose-bound repressor does not occupy the operator, RNA polymerase can initiate transcription of the lac structural genes from the lac promoter.

Explain why mutations in the lacI gene are trans in their effects, but mutations in the lacO gene are cis in their effects.

The lacI gene encodes the lac repressor protein, which can diffuse within the cell and attach to any operator. It can therefore affect the expression of genes on the same or different molecules of DNA. The lacO gene encodes the operator. The binding of the lac repressor to the operator affects the binding of RNA polymerase to the DNA, and therefore affects only the expression of genes on the same molecule of DNA.