Q5/ Chapter 12 practice problems

The histone 3 methylase H3K4me3 adds 3 methyl groups to the lysine 4 in the tail of histone 3. These modifications typically occur near the transcription start site of genes. If four H3K4me3 modifications have been identified, it suggests that at least four genes are present within this region of DNA.

A geneticist is trying to determine how many genes are found in a 300,000-bp region of DNA. Analysis shows that four different areas within the 300,000-bp region have H3K4me3 modifications. What might their presence suggest about the number of genes located there?

negative repressible

An operon is controlled by a repressor. When the repressor binds to a small molecule, it binds to DNA near the operon. The operon is constitutively expressed if a mutation prevents the repressor from binding to the small molecule. The type of control is

5' regulator protein gene | Promoter, Structural gene 3'

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

Allolactose binds to the lacI regulator protein, which does not bind to the operator, and RNA polymerase transcribes the structural genes.

For lac operon, which of the following occurs if lactose is present?

Acetyltransferase enzymes add acetyl groups to histone proteins preventing the proteins from forming the 30-nm chromatin fiber. Essentially, the chromatin structure is destabilized, which allows for transcription to occur. If the cells are raised to 40°C, then the acetyltransferase enzyme would not function and acetyl groups would not be added to the histone proteins that are the target of this enzyme. The result would be that the nucleosomes and the chromatin would remain stabilized and block transcriptional activation.

In a line of human cells grown in culture, a geneticist isolates a temperature-sensitive mutation at a locus that encodes an acetyltransferase enzyme; at temperatures above 38°C, the mutant cells produce a nonfunctional form of the enzyme. What would be the most likely effect of this mutation when the cells are grown at 40°C?

Regulator gene: B When sequence B is mutated, gene expression is not repressed by the presence of mmm. Promoter: D When sequence D is mutated, no gene expression occurs either in the presence or absence of mmm. Structural gene for enzyme 1: A When sequence A is mutated, enzyme 1 is not produced. Structural gene for enzyme 2: C When sequence C is mutated, enzyme 2 is not produced.

Indicate which sequence (A, B, C, or D) is part of the following components of the operon:

repressible

Is the mmm operon inducible or repressible?

(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 post-translational modification of the synthesized protein

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

The C gene is a regulator gene. The C gene is trans acting, thus it affects the expression of the A gene located on a different DNA molecule, which is typical of a gene encoding a regulatory protein. If the C gene was an operator, it would be cis acting and only able to regulate the expression of the A gene found on the same DNA molecule, which is not the case as demonstrated from genotype 3.

On the basis of the results of these experiments, is the C gene an operator or a regulator gene? Explain your reasoning.

Lactose present/Glucose present Condition 1 Yes No Condition 2 No Yes Condition 3 Yes Yes Condition 4 No No

Under which of the following conditions would a lac operon produce the greatest amount of β-galactosidase? The least? Explain your reasoning.

Epigenetic effects are differences in the expression of genes that are passed on to other cells and sometimes to other generations. They are due to alterations in DNA and chromatin structure but without changes of the DNA nucleotide sequence.

What are epigenetic effects? How do they differ from other genetic traits?

A non-coding, regulatory DNA sequence that is bound by a repressor protein.

Which of the following is the most appropriate definition of an operator?

lacI+ lacP+ lacOc lacZ+ lacY+ lacA+

A mutant E. coli strain, grown under conditions that normally induce the lac operon, produces particularly high amounts of ß-galactosidase (coded by lacZ). What is a possible genotype of the cells?

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.

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

a. Regulator protein is a repressor in a repressible operon. The operon would never be turned off, and transcription will take place all the time. b. Regulator protein is a repressor in an inducible operon. The result will be constitutive expression, and the transcription will take place all the time.

A mutation at the operator prevents the regulator protein from binding. What effect will this mutation have in the following types of operons?

RNA polymerase will bind to the lac promoter poorly, significantly decreasing the transcription of the lac structural genes.

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 the transcription of the operon?

We would also expect that the adults would show increased fear and heightened hormonal response to stress. We would expect to see differences in DNA methylation and histone acetylation that altered expression of genes involved in response to stress

A scientist does an experiment in which she removes the offspring of rats from their mother at birth and has her genetics students feed and rear the offspring. Assuming that the students do not lick and groom the baby rats like their mothers normally do, what longterm behavioral and epigenetic effects would you expect to see in the rats when they grow up?

Translation from the APETALA2 is inhibited by a miRNA that binds within the coding region of the mRNA. Thus, deleting much of the 3′ untranslated region of the APETALA2 mRNA will likely not affect the translation regulation by the miRNA molecule. However, the 3′ untranslated region could potentially be needed for mRNA stability and binding of the ribosome to the mRNA molecule, so the deletion could result in a decrease in expression of the APETALA2 gene.

A strain of Arabidopsis thaliana possesses a mutation in the APETALA2 gene, in which much of the 3′ untranslated region of mRNA transcribed from the gene is deleted. What is the most likely effect of this mutation on the expression of the APETALA2 gene?

negative inducible.

An operon is controlled by a repressor. When the repressor binds to a small molecule, it is released from binding to DNA in the operon. The operon is never expressed if a mutation prevents the repressor from binding to the small molecule. The type of control is

The lac operon consists of three structural genes—the lacZ gene, the lacY gene, and the lacA gene, which encode β-galactosidase, permease, and thiogalactoside transacetylase, respectively. All three genes share a promoter and operator region. Upstream from the lactose operon is the lacI gene that encodes the lac operon repressor, which 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, which binds to the lac repressor, altering its shape and reducing the repressor's affinity for the operator. Because the allolactose-bound repressor does not bind to the operator, RNA polymerase can initiate transcription of the lac structural genes from the lac promoter.

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

Transcriptional activator proteins stimulate transcription by binding DNA at specific base sequences such as an enhancer or regulatory promoter and attracting or stabilizing the basal transcriptional factor apparatus. Repressor proteins bind to silencer sequences or promoter regulator sequences. These proteins may inhibit transcription by blocking access to the enhancer sequence by the activator protein, preventing the activator from interacting with the basal transcription apparatus, or preventing the basal transcription factor from being assembled.

Briefly explain how transcriptional activator and repressor proteins affect the level of transcription of eukaryotic genes.

Through Slicer activity, which cleaves mRNA sequences; through the binding of miRNAs to complementary regions in mRNA, which prevents translation; and through transcriptional silencing, in which siRNAs play a role in altering chromatin structure.

Briefly list some of the ways in which siRNAs and miRNAs regulate genes.

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 on a different molecule of DNA. The lacO gene encodes the operator. It affects the binding of RNA polymerase to the DNA and therefore affects the expression of genes only on the same molecule of DNA.

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

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.

For E. coli strains with the lac genotypes given 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.

Negative control in a repressible operon: Inactive repressor b. Positive control in a repressible operon: Active activator c. Negative control in an inducible operon: Active repressor d. Positive control in an inducible operon: Inactive 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.

Methyltransferase enzymes recognize the hemimethylated state of CpG dinucleotides following replication and add methyl groups to the unmethylated cytosines, resulting in two new DNA molecules that are fully methylated.

How are patterns of DNA methylation maintained across cell divisions?

The phenotypic differences in traditional genetic traits such as the color and shape of peas that Mendel studied are due to differences in the DNA base sequences within the alleles. In epigenetics, the phenotypic differences are not due to changes in allele DNA base sequences, but are differences in the expression of genes that are passed on to other cells and sometimes to other generations.

How do epigenetic traits differ from traditional genetic traits, such as the differences in color and shape of peas that Mendel studied?

Much of gene regulation in bacteria occurs at the level of transcription, whereas gene regulation in eukaryotes often takes place at multiple levels. Modification of chromatin structure plays an important role in regulating eukaryotic transcription; chromatin structure is absent in bacteria. Bacterial genes are often organized in operons and coordinately expressed. In contrast, most eukaryotic genes have their own promoters. Eukaryotic transcription is controlled by a machinery more complex than that of bacteria; this complex machinery includes numerous transcription factors and transcriptional activators. RNA processing plays a larger role in eukaryotic gene regulation. Small RNA molecules (siRNAs and miRNAs) play an important role in eukaryotic gene regulation but are absent from most bacteria.

How does bacterial gene regulation differ from eukaryotic gene regulation? How are they similar?

The presence of AU-rich elements is associated with rapid degradation of the mRNA molecules that contain them through a RNA silencing mechanism. If the AU-element was deleted, then the miRNA would not be able to bind to the consensus sequence of the AU-rich element and the RISC degradation would not be initiated. It is likely that this mRNA molecule would be more stable resulting in increased gene expression of the protein coded for by the mRNA.

Some eukaryotic mRNAs have an AU-rich element in the 3′ untranslated region. What would be the effect on gene expression if this element were mutated or deleted?

Because the blob operon is transcriptionally inactive in the presence of B, gene S most likely codes for a regulatory protein that exhibits negative control. 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 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?

Changes in chromatin structure can result in repression or stimulation of gene expression. The acetylation of histone proteins increases transcription. The reverse reaction by deacetylases restores repression. Chromatin-remodeling complexes bind directly to the DNA, altering chromatin structure without acetylating histone proteins and allowing transcription to be initiated by making the promoters accessible to transcriptional factors. The methylation of DNA sequences represses transcription. The demethylation of DNA sequences often increases transcription.

What changes take place in chromatin structure and what role do these changes play in eukaryotic gene regulation?

An enhancer is a DNA sequence that, when bound to transcriptional activator proteins, can affect the transcription of a distant gene. Transcription at a distant gene is affected when the DNA sequence between the gene's promoter and the enhancer loops out, bringing the promoter and the enhancer close together and allowing the transcriptional activator proteins to directly interact with the basal transcription apparatus at the promoter, which stimulates transcription.

What is an enhancer? How does it affect transcription of distant genes?

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.

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

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.

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

The histone code refers to modifications to the tails of histone proteins. These modifications include the addition or removal of phosphate groups, acetyl groups, or methyl groups to the tails. Information imparted by these modifications affects how genes are expressed.

What is the histone code?

The total amount of protein synthesized depends on the amount of mRNA available for translation. The amount of available mRNA depends on the rates of mRNA synthesis and degradation. Less-stable mRNAs degrade faster than stable mRNAs, and so fewer copies of the mRNA are available as templates for translation. The 5′ cap, 3′ poly(A) tail, the 5′ UTR, 3′ UTR, and the coding region in the mRNA molecule affect its stability. Poly(A)-binding proteins bind at the 3′ poly(A) tail. These proteins contribute to the stability of the tail and protect the 5′ cap through direct interaction. When a critical number of adenine nucleotides have been removed from the tail, the protection is lost and the 5′ cap is removed. The removal of the 5′ cap enables 5′-to-3′ nucleases to degrade the mRNA.

What role does mRNA stability play in gene regulation? What controls mRNA stability in eukaryotic cells?

Changes in chromatin structure, brought about by DNA methylation, the alteration of histone proteins, and the reposition of nucleosomes.

What types of changes are thought to be responsible for epigenetic traits?

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 βgalactosidase or permease would be produced.

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?

The transcription will be turned off, as the transcription will require an activator protein

When a structural gene is under positive inducible control, what would be the result of a mutation that eliminates the activator protein?

The operator is the binding site for the lacI regulator protein.

Which of the following is true of the lac operon?

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

Why is gene regulation important for bacterial cells?

Cancer cells are typically rapidly dividing cells. DNA methylation particularly in regions with many CpG sequences (CpG islands) is associated with transcriptional repression. If the X31b molecules can be uptaken by the rapidly dividing cancers cells and then stimulate methylation of DNA sequences in the cancer cells, transcriptional repression of genes in the cancer cells would be expected. The repression of transcription could affect the growth of the cancer cells and potentially cause a loss of viability of these cells.

X31b is an experimental compound that is taken up by rapidly dividing cells. Research has shown that X31b stimulates the methylation of DNA. Some cancer researchers are interested in testing X31b as a possible drug for treating prostate cancer. Offer a possible explanation for why X31b might be an effective anticancer drug.