Chapter 16 Practice Problems (Solutions)

Give four examples of control of bacterial gene expression by RNA.

Attenuation, antisense RNA, riboswitches and ribozymes are all examples of RNA based mechanisms to control gene expression.

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.

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 allolactose binds to the repressor causing an allosteric change in its shape.

What is a ribozyme and how do they control gene expression?

Ribozymes are structures that form in the 5'UTR of bacterial mRNAs. When a metabolite binds to the structure in the 5'UTR it changes shape and becomes catalytically active cleaving the mRNA. Following cleavage, the mRNA is untranslatable and rapidly degraded. Ribozymes differ from riboswitches in that they have catalytic activity and cleave the mRNA, whereas riboswitches adopt a structure that prevents transcription or translation in the presence of the metabolite. Ribozymes are similar to riboswitches in the sense that they must bind a metabolite to become active.

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

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.

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 b. Negative control in an inducible operon

a. inactive repressor b. active repressor

For the Trp operon at which level of gene regulation does attenuation occur?

transcription

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.

The blob operon produces enzymes that convert compound A into compound B. The operon is controlled by a regulatory gene S. Normally, the enzymes are synthesized only in the absence of compound B. If gene S is mutated, the enzymes are synthesized in the presence and in the absence of compound B. Does gene S produce a regulatory protein that exhibits positive or negative control? Is this operon inducible or repressible?

Because the blob operon is transcriptionally inactive in the presence of B, gene S most likely codes for a repressor protein that requires compound B as a corepressor. The data suggest that the blob operon is repressible because it is inactive in the presence of compound B, but active when compound B is absent.

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 attenuation? What is the mechanism by which the attenuator forms when tryptophan levels are high and the antiterminator forms when tryptophan levels are low?

Attenuation is the termination of transcription prior to the structural genes of an operon. It is a result of the formation of a termination hairpin structure or attenuator in the mRNA. Two types of secondary structures can be formed by the mRNA 5′ UTR of the trp operon. If the 5′ UTR forms two hairpin structures from the base pairing of region 1 with region 2 and the pairing of region 3 with region 4, then transcription of the structural genes will not occur. The hairpin structure formed by the pairing of region 3 with region 4 results in a terminator being formed that stops transcription. When region 2 pairs with region 3, the resulting hairpin acts as an antiterminator allowing for transcription to proceed. Region 1 of the 5' UTR also encodes a small protein and has two adjacent tryptophan codons (UGG). Tryptophan levels affect transcription due to the coupling of translation with transcription in bacterial cells. When tryptophan levels are high, the ribosome quickly moves through region 1 and into region 2, thus preventing region 2 from pairing with region 3. Therefore, region 3 is available to form the attenuator hairpin structure with region 4, stopping transcription. When tryptophan levels are low, the ribosome stalls or stutters at the adjacent tryptophan codons in region 1. Region 2 now becomes available to base pair with region 3, forming the antiterminator hairpin. Transcription can now proceed through the 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 Condition 1 Yes Condition 2 No Condition 3 Yes Condition 4 No: Glucose present No Yes Yes 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. Conditions 2 and 4 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.

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.

For E. coli strains with the lac genotypes show below, use a plus sign (+) to indicate the synthesis of β- galactosidase and permease and a minus sign (−) to indicate no synthesis of the proteins.

In determining if expression of the β-galactosidase and the permease gene will occur, you should consider several factors. The presence of lacZ+ and lacY+ on the same DNA molecule as a functional promoter (lacP+) is required because the promoter is a cis-acting regulatory element. However, the lacI+ gene product or lac repressor is trans-acting and does not have to be located on the same DNA molecule as β-galactosidase and permease genes to inhibit expression. For the repressor to function, it does require that the cis-acting lac operator be on the same DNA molecule as the functional β-galactosidase and permease genes. Finally, the dominant lacIs gene product is also trans-acting and can inhibit transcription at any functional lac operator region. Genotype of strain Lactose absent -galactosidase Permease Lactose present -galactosidase Permease lacI+ lacP+ lacO+ lacZ+ lacY+ − lacI- lacP+ lacO+ lacZ+ lacY+ + lacI+ lacP+ lacOc lacZ+ lacY+ + lacI- lacP+ lacO+ lacZ+ lacY- + lacI- lacP- lacO+ lacZ+ lacY+ − lacI+ lacP+ lacO+ lacZ- lacY+/ − lacI- lacP+ lacO+ lacZ+ lacY- lacI- lacP+ lacOc lacZ+ lacY+/ + lacI+ lacP+ lacO+ lacZ- lacY- lacI- lacP+ lacO+ lacZ+ lacY-/ − lacI+ lacP- lacO+ lacZ- lacY+ lacI+ lacP- lacOc lacZ- lacY+/ − lacI- lacP+ lacO+ lacZ+ lacY- − + + + + + + + + − + − − − − − + + + + + − + − − + − Genotype of strain lacI+ lacP+ lacO+ lacZ+ lacY+/ lacI+ lacP+ lacO+ lacZ+ lacY+ Lactose absent -galactosidase Permease − − Lactose present -galactosidase Permease + + lacIs lacP+ lacO+ lacZ+ lacY-/ −−−− lacI+ lacP+ lacO+ lacZ- lacY+ lacIs lacP- lacO+ lacZ- lacY+/ −−−− lacI+ lacP+ lacO+ lacZ+ lacY+

Give all the possible genotypes of a lac operon that produces -galactosidase and permease under the following conditions. Do not give partial diploid genotypes.

Lactose absent β-Galactosidase Permease Lactose present β-Galactosidase Permease Genotype lacI+ lacP+ lacO+ lacZ+ lacY+ lacI+ lacP+ lacO+ lacZ- lacY+ lacI+ lacP+ lacO+ lacZ+ lacY- lacI- lacP+ lacO+ lacZ+ lacY+ or lacI+ lacP+ lacOc lacZ+ lacY+ lacIs lacP+ lacO+ lacZ+ lacY+ or lacI+ lacP- lacO+ lacZ+ lacY+ or lacI+ lacP+ lacO+ lacZ- lacY- lacI- lacP+ lacO+ lacZ+ lacY- or lacI+ lacP+ lacOc lacZ+ lacY- lacI- lacP+ lacO+ lacZ- lacY+ or lacI+ lacP+ lacOc lacZ- lacY+ a. − − + b. − − − c. − − + d. + + + e. − − − f. + − + g. − + − + + − + − − +

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 B-galactosidase and permease to utilize lactose. This would be an inefficient use of cellular energy resources.

For the last 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 o lactose when CAP-cAMP help RNA polymerase to bind to the promoter and activate transcription.

With respect to control of gene expression 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 stimulation 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.

What are riboswitches? How do they control gene expression? How do riboswitches differ from RNA- mediated repression?

Riboswitches are regulatory sequences in RNA molecules. Most can fold into compact secondary structures consisting of a base stem and several branching hairpins. At riboswitches, regulatory molecules bind and influence gene expression by affecting the formation of secondary structures within the mRNA molecule. The binding of the regulatory molecule to a riboswitch sequence may result in repression or induction. Some regulatory molecules bind the riboswitch sequence and stabilize a terminator structure in the mRNA, which results in premature termination of the mRNA molecule. Other regulatory molecules bind riboswitch sequences resulting in the formation of secondary structures that block the ribosome binding sites of the mRNA molecules, thus preventing translation initiation. In induction, the regulatory molecule acts as an inducer, stimulating the formation of a secondary structure in the mRNA that allows for transcription or translation to occur. RNA- mediated repression occurs through the action of a ribozyme. In RNA-mediated repression, an RNA sequence within the 5' untranslated region can act as a ribozyme that when stimulated by the presence of a regulatory molecule can induce self-cleavage of the mRNA molecule, which prevents translation of the molecule. When bound by a regulatory molecule, RNA mediated repression results in the self-cleavage of the mRNA molecule. When bound by a regulatory molecule, riboswitch sequences stimulate changes in the secondary structure of the mRNA molecule that affect gene expression.

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.

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 changed (allosteric transition). The altered repressor is unable to bind to the operator and inhibit transcription by RNA polymerase.

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. b. Regulator protein is a repressor in an inducible operon.

a. 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. b. 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.