Ch.16 Control of Gene Expression in Bacteria

Name six different levels at which gene expression might be controlled.

(1) Alteration or modification of the gene structure at the DNA level (2) Transcriptional regulation (3) Regulation at the level of mRNA processing (4) Regulation of mRNA stability (5) Regulation of translation (6) Regulation by posttranslational modification of the synthesized protein

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?

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.

Why is gene regulation important for bacterial cells?

Gene regulation allows for biochemical and internal flexibility while maintaining energy efficiency by the bacterial cells.

Which strand of DNA (upper or lower) in Figure 16.8 is the template strand? Explain your reasoning.

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.

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 or an inactive repressor.

a. Negative control in a repressible operon --> Inactive repressor b. Negative control in an inducible operon --> Active repressor

Examine Figure 16.7. What would be the effect of a drug that altered the structure of allolactose so that it was unable to bind to the regulator protein?

Allolactose is produced when lactose is present; allolactose normally binds to the repressor protein and makes it inactive, allowing transcription to occur when lactose is present. If a drug altered the structure of allolactose, it would not bind to the repressor and the repressor would continue to bind to the operator, keeping transcription off. The result would be that transcription was repressed even in the presence of lactose; thus, no -galactosidase or permease would be produced.

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.

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.

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.

What is antisense RNA? How does it control gene expression?

Antisense RNA molecules are small RNA molecules that are complementary to other DNA or RNA sequences and that form RNA-protein complexes. In bacterial cells, antisense RNA molecules can bind to a complementary region in the 5' UTR of a mRNA molecule, blocking the attachment of the ribosome to the mRNA and stopping translation or they pair with specific regions of the mRNA and cleave the mRNA stopping translation.

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.

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.

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. --> The regulator protein-corepressor complex would normally bind to the operator and inhibit transcription. If a mutation prevented the repressor protein from 340 Chapter Sixteen: Control of Gene Expression in Prokaryotes binding at the operator, then the operon would never be turned off and transcription would occur all the time. b. Regulator protein is a repressor in an inducible operon. --> 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.