HW 3

Homework Set #2 – Chapters 16 and 17 Most of these questions are taken from the end of each chapter in Brooker: Concepts of Genetics 2nd ed. For each question, write out your answer on separate piece of paper OR type your answer in a Word document. Chapter 16 What is the difference between a constitutive gene and a regulated gene? A constitutive gene is unregulated, which means that its expression level is relatively constant. In contrast, the expression of a regulated gene varies under different conditions. In bacteria, the regulation of genes often occurs at the level of transcription by combinations of regulatory proteins and small effector molecules. In addition, gene expression can be regulated at the level of translation or the function of a protein can be regulated after translation is completed. If a gene is repressible and under positive control, describe what kind of effector molecule and regulatory protein are involved. Explain how the binding of the effector molecule affects the regulatory protein. In this case, an inhibitor molecule and an activator protein are involved. The binding of the inhibitor molecule to the activator protein would prevent it from binding to the DNA and thereby inhibit its ability to activate transcription. Transcriptional regulation often involves a regulatory protein that binds to a segment of DNA and a small effector molecule that binds to the regulatory protein. Do the following terms apply to a regulatory protein, a segment of DNA, or a small effector molecule? Repressor Regulatory protein Inducer Effector molecule Operator site DNA segment Corepressor Effector molecule Activator Regulatory protein Attenuator DNA segment Inhibitor Effector molecule An operon is repressible—a small effector molecule turns off transcription. Which combinations of small effector molecules and regulatory proteins could be involved? In both B and C, the presence of the small effector molecule will turn off transcription. In contrast, the presence of an inducer turns on transcription. An inducer plus a repressor No A corepressor plus a repressor Yes An inhibitor plus an activator Yes An inducer plus an activator No Some mutations have a cis-effect on gene expression, whereas others have a trans-effect. Explain the molecular differences between cis- and trans-mutations. A mutation that has a cis-effect is within a genetic regulatory sequence, such as an operator site, that affects the binding of a genetic regulatory protein. A cis-effect mutation affects only the adjacent genes that the genetic regulatory sequence controls. A mutation having a trans-effect is usually in a gene that encodes a genetic regulatory protein. In the lac operon, how would gene expression be affected if one of the following segments was missing? lac operon promoter No transcription would take place. The lac operon could not be expressed. Operator site No regulation would take place. The operon would be continuously turned on. lacA gene The rest of the operon would function normally but none of the transacetylase would be made. If an abnormal repressor protein could still bind allolactose, but the binding of allolactose did not alter the conformation of the repressor protein, how would this affect the expression of the lac operon? It would be impossible to turn the lac operon on even in the presence of lactose because the repressor protein would remain bound to the operator site. Mutations may have an effect on the expression of the lac operon and the trp operon. Would the following mutations have a cis- or trans-effect on the expression of the protein-encoding genes in the operon? A mutation in the operator site that prevents the lac repressor from binding to it Cis-effect. It would affect only the genes that are in the adjacent operon A mutation in the lacI gene that prevents the lac repressor from binding to DNA Trans-effect. This is a mutation that affects a protein that can move throughout the cell. A mutation in trpL that prevents attenuation Cis-effect. It would affect only the genes that are in the adjacent operon. Would a mutation that inactivated the lac repressor and prevented it from binding to the lac operator site result in the constitutive expression of the lac operon under all conditions? Explain. What is the disadvantage to the bacterium of having a constitutive lac operon? A mutation that prevented the lac repressor from binding to the operator would make the lac operon constitutive only in the absence of glucose. However, this mutation would not be entirely constitutive because transcription would be inhibited in the presence of glucose. The disadvantage of constitutive expression of the lac operon is that the bacterial cell would waste a lot of energy transcribing the genes and translating the mRNA when lactose was not present. What is meant by the term attenuation? Is it an example of gene regulation at the level of transcription or translation? Explain your answer. Attenuation means that transcription is ended before it has reached the end of an operon. Because it causes an end to transcription, it is a form of transcriptional regulation even though the translation of the trpL region plays a key role in the attenuation mechanism. As described in Figure 16.12, four regions within the trpL mRNA can form stem-loops. Let's suppose that mutations have been previously identified that prevent the ability of a particular region to form a stem-loop with a complementary region. For example, a region 1 mutant cannot form a 1–2 stem-loop, but it can still form a 2–3 or 3–4 stem-loop. Likewise, a region 4 mutant can form a 1–2 or 2–3 stem-loop but not a 3–4 stem-loop. Under the following conditions, would attenuation occur? Region 1 is mutant, tryptophan is high, and translation is not occurring. Attenuation will not occur because loop 2–3 will form. Region 2 is mutant, tryptophan is low, and translation is occurring. Attenuation will occur because 2–3 cannot form, so 3–4 will form. Region 3 is mutant, tryptophan is high, and translation is not occurring. Attenuation will not occur because 3–4 cannot form Region 4 is mutant, tryptophan is low, and translation is not occurring. Attenuation will not occur because 3–4 cannot form. As described in Chapter 15, enzymes known as aminoacyl-tRNA synthetases are responsible for attaching amino acids to tRNAs. Let's suppose that tryptophanyl-tRNA synthetase was partially defective at attaching tryptophan to tRNA; its activity was only 10% of that found in a normal bacterium. How would that affect attenuation of the trp operon? Would it be more or less likely to be attenuated? Explain your answer. A defective tryptophanyl-tRNA synthetase would make attenuation less likely. This is because the bacterial cell would have a lower amount of charged tRNATrp. Therefore, it would be more likely for the ribosome to stall at the tryptophan codons found within the trpL gene, even if the concentration of tryptophan amino acids in the cell was high. When the ribosome stalls at these tryptophan codons, this prevents attenuation. With regard to cellular efficiency, why do you think the cell has several mechanisms to regulate gene expression at the level of translation? It takes a lot of cellular energy to translate mRNA into a protein. A cell wastes less energy if it prevents the initiation of translation rather than a later stage such as elongation or termination. C19. C20. What is antisense RNA? How does it affect the translation of a complementary mRNA? Antisense RNA is RNA that is complementary to a functional RNA such as mRNA. The binding of antisense RNA to mRNA inhibits translation. A species of bacteria can synthesize the amino acid histidine so it does not require histidine in its growth medium. A key enzyme, which we will call histidine synthetase, is necessary for histidine biosynthesis. When these bacteria are given histidine in their growth medium, they stop synthesizing histidine intracellularly. Based on this observation alone, propose three different regulatory mechanisms to explain why histidine biosynthesis ceases when histidine is in the growth medium. To explore this phenomenon further, you measure the amount of intracellular histidine synthetase protein when cells are grown in the presence and absence of histidine. In both conditions, the amount of this protein is identical. Which mechanism of regulation would be consistent with this observation? One mechanism is that histidine could act as corepressor that shuts down the transcription of the histidine synthetase gene. A second mechanism would be that histidine could act as an inhibitor via feedback inhibition. A third possibility is that histidine inhibits the ability of the mRNA encoding histidine synthetase to be translated. Perhaps it induces a gene that encodes an antisense RNA. If the amount of histidine synthetase protein was identical in the presence and absence of extracellular histidine, a feedback inhibition mechanism is favored, because this affects only the activity of the histidine synthetase enzyme, not the amount of the enzyme. The other two mechanisms would diminish the amount of this protein In what ways are the actions of the lac repressor and trp repressor similar and how are they different with regard to their binding to operator sites, their effects on transcription, and the influences of small effector molecules? The two proteins are similar in that both bind to a segment of DNA and repress transcription. They are different in three ways. (1) They recognize different effector molecules (i.e., the lac repressor recognizes allolactose, and the trp repressor recognizes tryptophan. (2) Allolactose causes the lac repressor to release from the operator, whereas tryptophan causes the trp repressor to bind to its operator. (3) The sequences of the operator sites that these two proteins recognize are different from each other. Otherwise, the lac repressor could bind to the trp operator, and the trp repressor could bind to the lac operator. Transcriptional repressor proteins (e.g., lac repressor), antisense RNA, and feedback inhibition are three different mechanisms that turn off the expression of genes and gene products. Which of these three mechanisms would be most effective in each of the following situations? Shutting down the synthesis of a polypeptide Antisense RNA or a translational repressor would shut down protein synthesis the fastest. A transcriptional repressor would also shut down the synthesis of mRNA, so it would eventually shut down protein synthesis once all of the preexisting mRNA had been degraded. Feedback inhibition would have no effect on protein synthesis. Shutting down the synthesis of mRNA Only a transcriptional repressor protein would shut down the synthesis of mRNA. Shutting off the function of a protein Feedback inhibition is the fastest way to shut down the function of a protein. Antisense RNA and transcriptional repressors eventually prevent protein function once all of the pre-existing mRNA and proteins have been degraded. Chapter 17 What are the common points of control in eukaryotic gene regulation. 1. Transcription. This includes regulatory transcription factors; DNA methylation (the attachment of methyl groups, which usually inhibits transcription); and changes in the arrangements and composition of histones and nucleosomes. 2. RNA level. This includes RNA processing, regulation of alternative splicing via SR proteins; RNA stability, regulation of RNA half-life and RNA translation via RNA binding proteins; regulation via miRNA and siRNA. 3. Protein level. This includes feedback inhibition, small molecules that modulate enzyme activity; and posttranslational modification-covalent changes to protein structure that affect protein activity. 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 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 regulatory 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 may interact with TFIID and/or mediator to inhibit RNA polymerase. Are the following statements true or false? An enhancer is a type of regulatory element. True A core promoter is a type of regulatory element. False Regulatory transcription factors bind to regulatory elements. True An enhancer may cause the down regulation of transcription. False, it causes up regulation. 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 a GRE. 2. It could be in the HSP90 domain, so that 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. The glucocorticoid receptor is an example of a transcriptional activator that binds to response elements and activates transcription. (Note: The answer to this question is 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.) How would the function of the glucocorticoid receptor be shut off? 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. Briefly describe three ways that ATP-dependent chromatin- remodeling complexes may change chromatin structure. ATP-dependent chromatin remodeling complexes may change the positions of nucleosomes, evict histones, and/or replace histones with histone variants. 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. 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. What is an insulator? 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 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? DNA methylation is the attachment of a methyl group to a base within the DNA. In many eukaryotic species, this occurs on 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 methyltransferase recognizes this hemimethylated DNA and methylates the cytosine in the unmethylated DNA strand; this event is called maintenance methylation. 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 1,000 to 2,000 base pairs in length that contains a high number of CpG sites. CpG 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. Define epigenetics. Are all epigenetic changes passed from parent to offspring? Explain. One way to define epigenetics is the study of mechanisms that lead to changes in gene expression that can be passed from cell to cell and are reversible, but do not involve a change in the sequence of DNA. Not all epigenetic changes are passed from parent to offspring. For example, those that occur in somatic cells, such as lung cells, would not be passed to offspring. How can environmental agents that do not cause gene mutations contribute to cancer? Would these epigenetic changes be passed to offspring? Though they don’t change the DNA sequence, epigenetic modifications can affect gene expression. Such changes could increase gene expression and thereby result in oncogenes or they could inhibit the expression of tumor suppressor genes. Either type of change could contribute to cancer. For example, DNA methylation of a tumor suppressor gene could promote cancer. What is the function of a splicing factor? Explain how splicing factors can regulate the tissue-specific splicing of mRNAs. The function of splicing factors is to influence the selection of splice sites in RNA. In certain cell types, the concentration of particular splicing factors is higher than in other tissues. The high concentration of particular splicing factors, and the regulation of their activities, may promote the selection of particular splice sites and thereby lead to tissue-specific splicing. What are the advantages and disadvantages of mRNAs with a short half-life compared with mRNAs with a long half-life? A disadvantage of mRNAs with a short half-life is that the cells probably waste a lot of energy making them. If a cell needs the protein encoded by a short-lived mRNA, the cell has to keep transcribing the gene that encodes the mRNA because the mRNAs are quickly degraded. An advantage of short-lived mRNAs is that the cell can rapidly turn off protein synthesis. If a cell no longer needs the polypeptide encoded by a short-lived mRNA, it can stop transcribing the gene, and the mRNA will be quickly degraded. This will shut off the synthesis of more proteins rather quickly. With most long-lived mRNAs, it will take much longer to shut off protein synthesis after transcription has been terminated. What is the relationship between mRNA stability and mRNA concentration? What factors affect mRNA stability? If mRNA stability is low, this means that it is degraded more rapidly. Therefore, low stability results in a low mRNA concentration. The length of the polyA tail is one factor that affects stability. A longer tail makes mRNA more stable. Certain mRNAs have sequences that affect their half-lives. For example, AU-rich elements (AREs) are found in many short-lived mRNAs. The AREs are recognized by cellular proteins that cause the mRNAs to be rapidly degraded. In response to potentially toxic substances (e.g., high levels of iron), eukaryotic cells often use translational or posttranslational regulatory mechanisms to prevent cell death, rather than using transcriptional regulatory mechanisms. Explain why. A cell may need to respond rather quickly to a toxic substance in order to avoid cell death. Translational and posttranslational mechanisms are much faster than transcriptional activation, in which it is necessary to up regulate the gene, synthesize the mRNA, and then translate the mRNA to make a functional protein. A research study indicated that an agent in cigarette smoke caused the silencing of a tumor suppressor gene called p53. However, upon sequencing, no mutation was found in the DNA sequence for this gene. Give two possible explanations for these results. The agent in cigarette smoke may have caused the methylation of CpG islands near the promoter of the p53 gene, thereby inhibiting transcription. Another possibility is that it may affect covalent histone modifications or chromatin remodeling in a way that causes the gene to be in a closed conformation. Let's suppose you were interested in developing drugs to prevent epigenetic changes that may contribute to cancer. What cellular proteins would be the target of your drugs? What possible side effects might your drugs cause? You would want to target proteins that carry out epigenetic changes, such as DNA methyltransferase, enzymes that covalently modify histone proteins, and chromatin remodeling complexes. The possible side effects are somewhat difficult to predict but the general problem is that they may cause genes in normal cells to be turned on or turned off when they should not be. Such effects may result in many different types of cellular dysfunctions.