Genetics Chapter 15

Let's suppose a mutation in the glucocorticoid receptor does not prevent the binding of the glucocorticoid hormone to the protein but prevents the ability of the receptor to activate transcription. Make a list of all the possible defects that may explain why transcription cannot be activated.

1. it could be in the DNA binding domain so that the receptor would not recognize GREs 2. it could be in the HSP90 domain so that the HSP90 would not be released when the hormone binds 3. it could be in the dimerization domain, so that the receptor would not dimerize 4. it could be in the nuclear localization domain so that the receptor would not travel into the nucleus 5. it could be in the domain that activates RNA polymerase so that the receptor would not activate transcription, even though it could bind to GREs

A transcription factor is a protein that affects the ability of RNA polymerase to transcribe a gene. A) True B) False

A

Transcription factors usually contain one or more motifs that play key roles in their function. What is the function of the following motifs? A. Helix-turn-helix B. Zinc finger C. Leucine zipper

A. DNA binding. B. DNA binding. C. Protein dimerization.

A CpG island consists of about 2000 copies of the CG dinucleotide. A) True B) False

B

In order for CREB to activate transcription: A) It must act as a heterodimer. B) It must be phosphorylated. C) It must bind to cAMP. D) It must bind to RNA polymerase.

B

Methylation of CpG islands: A) Enhances binding of regulatory transcription factors. B) Prevents activation of enhancers. C) Prevents binding of chromatin remodeling proteins. D) Interferes directly with RNA polymerase binding.

B

Transcription factors, if present, will always bind their appropriate response elements. A) True B) False

B

GREs: A) Are located near promoters of many different genes. B) Are activated in response to increased glucocorticoid levels. C) Are bound by a dimerized regulatory transcription factor. D) All of these. E) None of these.

D

Gene regulation in eukaryotes may be influenced by: A) Environmental conditions. B) Developmental stage. C) Tissue or cell type. D) All of these. E) None of these.

D

In the absence of glucocorticoid: A) No glucocorticoid receptor is present. B) Glucocorticoid receptor is present in an active form. C) Glucocorticoid receptor dimerizes to prevent glucocorticoid binding. D) All of these. E) None of these.

E

How do regulatory transcription factors act on RNA polymerase?

Indirectly, via TFIID or mediator, or through other protein complexes that affect DNA packaging and chromatin structure.

Differentiate between de novo and maintenance methylation.

Maintenance methylation converts hemimethylated sites into fully methylated sites, while de novo methylation adds methyl groups to unmethylated DNA.

Let's suppose that a vertebrate organism carries a mutation that causes some cells that normally differentiate into nerve cells to differentiate into muscle cells. A molecular analysis of this mutation revealed that it was in a gene that encodes a methyltransferase. Explain how an alteration in a methyltransferase could produce this phenotype.

Perhaps the methyltransferase is responsible for methylating and inhibiting a gene that causes a cell to become a muscle cell. The methyltransferase is inactivated by the mutation.

Explain how phosphorylation affects the function of the CREB protein.

Phosphorylation of the CREB protein causes it to act as a transcriptional activator.

Transcription factors such as the glucocorticoid receptor and the CREB protein form homodimers and activate transcription. Other transcription factors form heterodimers. For example, a transcription factor known as myogenic bHLH forms a heterodimer with a protein called the E protein. This heterodimer activates the transcription of genes that promote muscle cell differentiation. However, when myogenic bHLH forms a heterodimer with a protein called the Id protein, transcriptional activation does not occur. (Note: Id stands for Inhibitor of differentiation.) Which of the following possibilities best explains this observation? Only one possibility is correct.

Possibility 2

Discuss the structure and function of regulatory elements. Where are they located relative to the core promoter?

Regulatory elements are relatively short genetic sequences that are recognized by regulatory transcription factors. After the regulatory transcription factor has bound to the regulatory element, it will affect the rate of transcription, either activating it or repressing it, depending on the action of the regulatory protein. Regulatory elements are typically located in the upstream region near the promoter, but they can be located almost anywhere (i.e., upstream and downstream) and even quite far from the promoter.

Discuss the common points of control in eukaryotic gene regulation.

See figure 15.1

An enhancer, located upstream from a gene, has the following sequence: 5ʹ-GTAG-3ʹ 3ʹ-CATC-5ʹ This enhancer is orientation-independent. Which of the following sequences also works as an enhancer? A. 5ʹ-CTAC-3ʹ 3ʹ-GATG-5ʹ B. 5ʹ-GATG-3ʹ 3ʹ-CTAC-5ʹ C. 5ʹ-CATC-3ʹ 3ʹ-GTAG-5ʹ

The enhancer found in A would work, but the ones found in B and C would not. The sequence that is recognized by the transcriptional activator is 5ʹ-GTAG-3ʹ in one strand and 3ʹ-CATC-5ʹ in the opposite strand. This is the same arrangement found in A. In B and C, however, the arrangement is 5ʹ-GATG-3ʹ and 3ʹ-CATC-5ʹ. In the arrangement found in B and C, the two middle bases (i.e., A and T) are not in the correct order.

List four main levels of gene expression that can be regulated.

Transcription, RNA processing, translation, posttranslational modifications.

The gene that encodes the enzyme called tyrosine hydroxylase is known to be up regulated by the CREB protein. Tyrosine hydroxylase is expressed in nerve cells and is involved in the synthesis of catecholamine, a neurotransmitter. The exposure of cells to adrenaline normally up regulates the transcription of the tyrosine hydroxylase gene. A mutant cell line was identified in which the tyrosine hydroxylase gene was not up regulated when exposed to adrenaline. List all the possible mutations that could explain this defect. How would you explain the defect if only the tyrosine hydroxylase gene was not up regulated by the CREB protein, whereas other genes having CREs were properly up regulated in response to adrenaline in this cell line?

adrenaline receptor g protein adenylyl cyclase protein kinase a CREB protein CREs of the tyrosine hydroxylase gene

What is DNA methylation? When we say that DNA methylation is heritable, what do we mean? How is it passed from a mother to a daughter cell?

the attachment of a methyl group to a base within the DNA. In many eukaryotic species, this occurs in cytosine at a CG sequence. After de novo methylation has occurred it is passed from mother to daughter cell. Because DNA replication is semiconservative the newly made DNA contains one strand that is methylated and one that is not. DNA methyl transferase recognized the hemimethylated DNA and methylates the cytosine in the unmethylated DNA strand; this event is called maintenance methylation

The glucocorticoid receptor and the CREB protein are two examples of transcriptional activators. These proteins bind to response elements and activate transcription. (Note: The answers to this question are not directly described in this chapter. You have to rely on your understanding of the functioning of other proteins that are modulated by the binding of effector molecules, such as the lac repressor.) A. How would the function of the glucocorticoid receptor be shut off? B. What type of enzyme would be needed to shut off the activation of transcription by the CREB protein?

A. Eventually, the glucocorticoid hormone will be degraded by the cell. The glucocorticoid receptor binds the hormone with a certain affinity. The binding is a reversible process. Once the concentration of the hormone falls below the affinity of the hormone for the receptor, the receptor will no longer have the glucocorticoid hormone bound to it. When the hormone is released, the glucocorticoid receptor will change its conformation, and it will no longer bind to the DNA. B. An enzyme known as a phosphatase will eventually cleave the phosphate groups from the CREB protein. When the phosphates are removed, the CREB protein will stop activating transcription. C14. The enhancer found in A would work, but the ones found in B and

What do GREs and CREs have in common?

Both bind their respective proteins as a result of a signaling molecule; both are DNA response elements; other answers are possible.

A homodimer: A) is a protein with a helix-loop-helix DNA binding motif. B) is a protein made of two unique subunits. C) is a pair of identical proteins. D) typically prevents DNA binding.

C

Histones are thought to be displaced as RNA polymerase is transcribing a gene. What would be the potentially harmful consequences if histones were not put back into a gene after RNA polymerase had passed?

That transcription may be initiated at multiple points within a gene, thereby producing many nonfunctional transcripts. This would be a waste of energy.

Response elements may be located within the gene itself. A) True B) False

A

What is a histone variant?

A histone with an amino acid sequence that is slightly different from a core histone. Histone variants play specialized roles with regard to chromatin structure and function

A particular drug inhibits the protein kinase that is responsible for phosphorylating the CREB protein. How would this drug affect the following events? A. The ability of the CREB protein to bind to CREs B. The ability of extracellular hormones to enhance cAMP levels C. The ability of the CREB protein to stimulate transcription D. The ability of the CREB protein to dimerize

A. no effect B. no effect C. it would be inhibited D. no effect

ATP dependent chromatin remodeling can activate a gene by:

Moving a histone off the core promoter (TATA box); may be a first step followed by histone acetyltransferase activity; others may be possible.

The DNA-binding domain of each CREB protein subunit recognizes the sequence 5ʹ-TGACGTCA-3ʹ. As a matter of random chance, how often would you expect this sequence to occur in the human genome, which contains approximately 3 billion base pairs? Actually, only a few dozen genes are activated by the CREB protein. Does the value of a few dozen agree with the number of random sites found in the human genome? If the number of random sites in the human genome is much higher than a few dozen, provide at least one explanation why the CREB protein is not activating more than a few dozen genes.

1. to create a functional CREB there needs to be two of these sequences close together because the CREB protein functions as a homodimer 2. CREs might not be near a gene 3. the conformation of chromatin containing a CRE might not be accessible to binding by the CREB protein

DNA in an open configuration is more likely to be transcribed than DNA in a closed configuration. A) True B) False

A

Housekeeping genes: A) Are constitutively expressed. B) Have highly methylated CpG islands. C) Encode proteins required only in specific tissues. D) All of these. E) None of these.

A

Methylation of DNA is a heritable trait. A) True B) False

A

What is a CpG island? Where would you expect one to be located? How does the methylation of CpG islands affect gene expression?

A CpG island is a stretch of 1000 to 2000 base pairs in length that contains a high number of CpG site. CoG islands are often located near promoters. When the island is methylated, this inhibits transcription. This inhibition may be the result of the inability of the transcriptional activators to recognize the methylated promoter and/or the effects of methyl-CpG-binding proteins which may promote a closed chromatin conformation.

What is a nucleosome-free region? Where are they typically found in a genome? How are nucleosome-free regions thought to be functionally important?

A nucleosome-free region (NFR) is a location in the genome where nucleosomes are missing. They are typically found at the beginning and ends of genes. An NFR at the beginning of a gene is thought to be important so that genes can be activated. The NFR at the end of a gene may be important for its proper termination.

Are the following statements true or false? A. An enhancer is a type of regulatory element. B. A core promoter is a type of regulatory element. C. Regulatory transcription factors bind to regulatory elements. D. An enhancer may cause the down regulation of transcription.

A. true B. false C. true D. false it causes up regulation

What is an insulator? Describe two different ways that insulators may exert their effects.

An insulator is a segment of DNA that functions as a boundary between two adjacent genes. An insulator may act as a barrier to changes in chromatin structure or block the effects of a neighboring enhancer.

DNA sites bound by regulatory transcription factors: A) Are trans-acting factors. B) Are cis-acting elements. C) Are promoters. D) Have similar sequences in all cells. E) None of the above.

B

In comparing effects of regulatory transcription factors that affect TFIID and those that affect mediator: A) TFIID binding prevents transcription while mediator binding allows transcription. B) Mediator allows binding of RNA polymerase but transcription can be prevented by transcription factors binding to silencers. C) Transcription factors which bind to enhancers stimulate binding of both proteins. D) Transcription factors which bind to silencers prevent binding of both proteins.

B

Extracellular signaling molecules: A) Are usually able to directly affect transcription. B) Are required to enter the cytoplasm and bind a receptor molecule. C) Usually bind plasma membrane receptors which then pass along the signal. D) Are unable to affect transcription.

C

The best explanation for interference of DNA methylation with transcription factor binding is: A) Methyl-CpG binding protein competes with the transcription factor for binding to methylated DNA. B) Methylation of the CpG island modifies the shape of the transcription factor. C) Methylation of the CpG island modifies the shape of the response element to which the transcription factor normally binds. D) Methylation prevents dimerization of DNA binding proteins.

C

Changes in function of which of the following proteins by posttranslational modification (such as phosphorylation or acetylation) would be likely to, in turn, affect the expression of specific genes? A) TFIID B) eIF4F C) Histones D) IRP E) None of these.

D

Receptor tyrosine kinases are transmembrane proteins that are activated by binding of an extracellular signal and subsequent phosphorylation of the receptor. Control of the function of receptor tyrosine kinases primarily occurs at what level? A) Transcription. B) RNA processing. C) Translation. D) Posttranslational modification. E) None of these.

D

Describe the steps that occur for the glucocorticoid receptor to bind to a GRE.

For the glucocorticoid receptor to bind to a GRE, a steroid hormone must first enter the cell. The hormone then binds to the glucocorticoid receptor, which releases HSP90. The release of HSP90 exposes a nuclear localization signal (NLS) within the receptor, which enables it to dimerize and then enter the nucleus. Once inside the nucleus, the dimer binds to a GRE, which activates transcription of the adjacent genes.

Name at least two motifs common to DNA binding proteins.

Helix-turn-helix; helix-loop-helix; zinc finger; leucine zipper.

The binding of an effector molecule, protein-protein interactions, and covalent modifications are three common ways to modulate the activities of transcription factors. Which of these three mechanisms are used by steroid receptors and the CREB protein?

Steroid receptors: binding of an effector molecule and protein-protein interactions CREB protein: covalent modification and protein-protein interactions

Explain how the acetylation of core histones may loosen chromatin packing.

The attraction between DNA and histones occurs because the histones are positively charged and the DNA is negatively charged. The covalent attachment of acetyl groups decreases the amount of positive charge on the histone proteins and thereby may decrease the binding of the DNA. In addition, histone acetylation may attract proteins to the region that loosen chromatin compaction.

Briefly describe three ways that ATP-dependent chromatin- remodeling complexes may change chromatin structure.

The enhancer found in A would work, but the ones found in B and C would not. The sequence that is recognized by the transcriptional activator is 5ʹ-GTAG-3ʹ in one strand and 3ʹ-CATC-5ʹ in the opposite strand. This is the same arrangement found in A. In B and C, however, the arrangement is 5ʹ-GATG-3ʹ and 3ʹ-CATC-5ʹ. In the arrangement found in B and C, the two middle bases (i.e., A and T) are not in the correct order.

What is meant by the term histone code? With regard to gene

The histone code hypothesis is the idea that the patterns of covalent modifications of histones are recognized by proteins that affect chromatin structure and function. In this way, the modification of histones plays a role in gene regulation.

What is meant by the term transcription factor modulation? List three general ways that this can occur.

Transcription factor modulation refers to different ways that the function of transcription factors can be regulated. The three general ways are the binding of an effector molecule, protein-protein interactions, and covalent modifications.

What are the functions of transcriptional activator proteins and repressor proteins? Explain how they work at the molecular level.

Transcriptional activation occurs when a regulatory transcription factor binds to a response element and activates transcription. Such proteins, called activators, may interact with TFIID and/or mediator to promote the assembly of RNA polymerase and general transcription factors at the promoter region. They also could alter the structure of chromatin so that RNA polymerase and transcription factors are able to gain access to the promoter. Transcriptional inhibition occurs when a regulatory transcription factor inhibits transcription. Such repressors also may interact with TFIID and/or mediator to inhibit RNA polymerase.