Chapter 19 Book notes
Chapter 19 Book notes BIOL 2311
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Chapter 19 – Control of Gene Expression in Eukaryotes MingHan Lu The cells in a multicellular eukaryote express different genes in response to changes in the internal environment—specifically, to signals from other cells. As a human being develops, cells that are located in different parts of the organism are exposed to different cellcell signals. o As a result, they express different genes. Differential gene expression is responsible for creating different cell types, arranging them into tissues, and coordinating their activity to form the multicellular society we call an individual. 19.1 Gene Regulation in Eukaryotes – An Overview Like bacteria, eukaryotes can control gene expression at levels of transcription, translation, and posttranslation. 3 additional levels of control occur in eukaryotes as genetic information flows from DNA to proteins. 1. The first additional level of control involves the DNAprotein complex at the top of the figure. In eukaryotes, DNA is wrapped around proteins to create a structure called chromatin. Eukaryotic genes have promoters, just as bacterial genes do; but before transcription can begin in eukaryotes, the stretch of DNA containing the promoter must be released from tight interactions with proteins, so that RNA polymerase can make contact with the promoter. a. Chromatin remodeling must occur before transcription. 2. The second level of regulation that is unique to eukaryotes is RNA processing —the steps required to produce a mature, processed mRNA from a primary RNA transcript. Recall that introns have to be spliced out of primary transcripts. In many cases, carefully orchestrated alternative splicing occurs— meaning that different combinations of exons are included in the mRNA. a. If different cells use different splicing patterns, different gene products result. 3. mRNA life span is regulated in eukaryotes: mRNAs that remain in the cell for a long time tend to be translated more than mRNAs that have a short life span. Chapter 19 – Control of Gene Expression in Eukaryotes MingHan Lu 19.2 Chromatin Remodeling For a molecular signal to trigger the transcription of a particular gene, the chromatin around the target gene must be remodeled. What Is Chromatin’s Basic Structure? The most abundant DNAassociated proteins belong to a group called the histones. Chromatin consists of DNA complexed with histones and other proteins. In some preparations for electron microscopy, chromatin looked like beads on a string. The “beads” came to be called nucleosomes. Each nucleosome consists of DNA wrapped almost twice around a core of eight histone proteins. The intimate association between DNA and histones occurs in part because DNA is negatively charged and histones are positively charged. DNA has a negative charge because of its phosphate groups; histones are positively charged because they contain many lysines and arginines, two positively charged amino acids. o There are additional layers of complexity in packaging DNA. H1 histones interact with one another and with histones in other nucleosomes to produce a tightly packed structure. Based on its width, this structure is called the 30nanometer fiber. o Finally, the 30nm fibers are attached at intervals along their length to proteins that form a scaffold or framework inside the nucleus. In this way, the entire chromosome is organized and held in place. When chromosomes condense before mitosis or meiosis the scaffold proteins and 30nm fibers are folded into still larger and more tightly packed structure. A eukaryotic chromosome, then, is made up of chromatin that has several layers of organization: The DNA is wrapped around histones to form nucleosomes, nucleosomes are packed into 30nm fibers, 30nm fibers are attached to scaffold proteins, and the Chapter 19 – Control of Gene Expression in Eukaryotes MingHan Lu entire assembly can be folded into the highly condensed structure observed during cell division. Evidence that Chromatin Structure Is Altered in Active Genes The central idea is that chromatin must be decondensed, to expose the promoter so RNA polymerase can bind to it. DNA in Condensed Chromatin Is Protected from DNase DNases are enzymes that cut DNA. Some DNases cleave DNA at random locations, and these cannot cut efficiently if DNA is tightly with proteins. o DNases function when they are working with a decondensed gene. Histone Mutants The lack of histone proteins prevented the assembly of normal chromatin. If the absence of normal histoneDNA interactions promotes transcription, then the presence of normal histoneDNA interactions must prevent it. Normal, or default, state, eukaryotic genes are turned off. This is a new mechanism of negative control. When DNA is wrapped into a 30nm fiber, the parking brake is on. Summary: So basically, histone proteins interacting with DNA inhibits transcription. How Is Chromatin Altered? DNA methylation A group of enzymes known as DNA methyltransferases add methyl groups (CH ) to 3 cytosine residues in DNA. Methylated CpG sequences are recognized by proteins that trigger chromatin condensation. Actively transcribed genes (not condensed genes) usually have low levels of methylated CpG near their promoters, and nontranscribed genes (condensed) usually have high levels of methylated CpG. Histone Modification Histone code hypothesis postulates that particular combinations of histone modifications set the state of chromatin condensation for a particular gene. o It plays an important role in regulating transcription. Two different types of enzymes can add or remove acetyl groups from histones. Histone acetyltransferases (HATs) added acetyl groups to the positively charged lysine residues in histones, and histone deacetylases (HDACs) remove them. o Acetylation of histones usually results in decondensed chromatin, a state associated with active transcription. When HATs add acetyl acetyl groups, t he acetyl groups neutralize the positive charge on lysine residues and loosens the close association of nucleosomes with the negatively charged DNA. The addition of acetyl groups also creates a binding site for other proteins that help open the chromatin. Chapter 19 – Control of Gene Expression in Eukaryotes MingHan Lu o In contrast, when HDACs remove acetyl groups from histones, this process usually leads to condensed chromatin, a state associated with no transcription. HATs are an on switch for transcription, and HDACs are an off switch. ChromatinRemodeling Complexes Other major players in chromatin alteration and gene regulation are enzymes that form macromolecular machines called chromatinremodeling complexes. o These machines harness the energy in ATP to reshape chromatin (ATP dependent). Chromatinremodeling complexes cause nucleosomes to slide along the DNA or, in some cases, knock the histones completely off the DNA to open stretches of chromatin and allow gene transcription. DNA methylation, histone modifications, and chromatinremodeling complexes work together to finetune chromatin condensation to specific genes. Chromatin Modifications Can Be Inherited DNA methylation and histone modifications are an example of epigenetic inheritance, the collective term for patterns of inheritance that are due to something other than differences in DNA sequences. The epi of epigenetics comes from the Greek word meaning “upon.” It implies another level of inheritance that adds to standard DNAbased mechanisms to explain how different phenotypes are transmitted. Muscle cells are different from brain cells not because they contain different genes, but largely because they have inherited different patterns of DNA methylation and histone modifications during the course of their development. A regulatory region is a section of DNA that is involved in controlling the activity of a gene. 19.3 Initiating Transcription: Regulatory Sequences and Regulator Proteins The promoter is a site in DNA where RNA polymerase binds to initiate transcription. TATA box Once a promoter that contains a TATA box has been exposed by chromatin remodeling, the first step in initiating transcription is binding of the TATAbinding protein (TBP). PromoterProximal Elements Are Regulatory Sequences Near the Promoter The first regulatory sequences in Eukaryotic DNA were discovered in the late 1970s, when Yasuji Oshima and coworkers set out to understand how yeast cells control the metabolism of sugar galactose. o When galactose is absent, S. cerevisiae cells produce only tiny quantities of the enzymes required to metabolize it. But when galactose is present., transcription of the genes encoding these enzymes increases by a factor of 1000. Chapter 19 – Control of Gene Expression in Eukaryotes MingHan Lu The team’s first major result was the discovery of mutant cells that failed to produce any of the five enzymes required for galactose metabolism, even if galactose was present. o To interpret this observation, they hypothesized that 1. The five genes are regulated together, even though they are not on the same chromosome; 2. Normal cells have an activator protein that exerts positive control over the five genes; 3. The mutant cells have a mutation that completely disables the activator protein. Researchers were able to isolate the regulatory protein and confirm that it binds to a short stretch of DNA located just upstream from the promoter for all five genes required for galactose use. In bacteria, genes that need to be regulated together are often clustered into a single operon and transcribed into a single mRNA. o Eukaryotes coregulated genes are not clustered together, but instead share a regulatory DNA sequence that binds the same regulatory protein. Regulatory sequences like these are located close to the promoter and bind regulatory proteins are termed promoterproximal elements. o Unlike the promoter itself, promoterproximal elements have sequences that are unique to specific sets of genes. In this way They furnish a mechanism for eukaryotic cells to express certain genes but not others. The discovery of promoterproximal elements and a mechanisms of positive control suggested a satisfying parallel between gene regulation in bacteria and in eukaryotes. Enhancers Are Regulatory Sequences Far from the Promoter Regulatory sequences that are far from the promoter and activate transcription are termed enhancers. Enhancers are regulatory DNA sequences unique to eukaryotes. When regulatory proteins called transcriptional activators bind to enhancers, transcription begins. o Thus, enhancers and activators are like a gas pedal—an element in positive control. o Eukaryotes also possess regulatory sequences that are similar in structure and share key characteristics with enhancers but work to inhibit transcription. These DNA sequences are called silencers. When regulatory proteins called repressors bind to silencers, transcription is shut down. Silencers and repressors are like a brake an element in negative control. The Role of Transcription Factors in Differential Gene Expression Enhancers and silencers are binding sites for activators and repressors that regulate transcription. These proteins are termed regulatory transcription factors, or often transcription factors. Different types of cells express different genes because they have different transcription factors. In multicellular species, the transcription factors, in turn, are produced in response to signals that arrive from other cells early in embryonic development. Chapter 19 – Control of Gene Expression in Eukaryotes MingHan Lu For example, if a signal that says “become a muscle cell in the early embryo, it triggers a signal transduction cascade that leas to the production of transcription factors specific to muscle cells. o Because different transcription factors bind to specific regulatory sequences, they turn on the production of musclespecific proteins. Differential gene expression is a result of the production or activation of specific transcription factors. Eukaryotic genes are turned on when transcription factors bind to enhancers and promoterproximal elements; the genes are turned off when transcription factors bind to silencers or when chromatin is condensed. How Do Transcription Factors Recognize Specific DNA Sequences? Each transcription factor must be able to recognize and bind to a specific DNA sequences. o Recall that DNA bases are partially exposed in the major and minor grooves of the DNA double helix. The edges of an AT base pair and a GC base pair that project into the grooves of the DNA helix contain different sets of atoms and have different surface shapes. These differences in composition and shape can be recognized by transcription factors. Just as base pairs come together by complementary molecular interactions, so too can proteins and specific DNA sequences. A transcription factor that is essential for the development of muscle cells inserts amino acid side chains into two major grooves of DNA. This particular transcription factor binds to a specific enhancer sequence because of complementary interactions between base pairs and its amino acids. Without such specific interactions between transcription factors and DNA, the development of muscle cells – or any other specialized cell type—would not be possible. A Model for Transcription Initiation The transcription factors must interact with regulatory sequences to initiate transcription. Basal transcription factors – these proteins interact with the promoter and are not restricted to particular genes or cell types. o The term basal implies that these proteins are necessary for transcription to occur, but they do not provide much in the way of regulation. The promoterrecognized TATAbinding protein is an example of a basal transcription factor that is common to many genes. Chapter 19 – Control of Gene Expression in Eukaryotes MingHan Lu In addition to transcription factors, a large complex of proteins called Mediator acts as a bridge between regulatory transcription factors, basal transcription factors, and RNA polymerase II. Activators work not only to stimulate transcription but also to bring chromatin remodeling proteins to the right place the right time. None of the proteins that remodel chromatin can recognize specific DNA sequences. It is the transcriptional activators that bind to regulatory sequences at particular genes to recruit the proteins needed to remodel chromatin. Getting RNA polymerase to initiate transcription requires interactions between many proteins, including transcriptional activators that are bound to enhancers and promoter proximal elements, Mediator, basal transcription factors, and RNA polymerase itself. The result is a large, macromolecular machine that is positioned at a gene’s start site and capable of starting transcription. 19.4 PostTranscriptional Control Once a gene is transcribed, a series of events has to occur before a final product appears. Each of these events offers an opportunity to regulate gene expression, and each is used in some cells at least some of the time. Chapter 19 – Control of Gene Expression in Eukaryotes MingHan Lu o These control mechanisms include (1) splicing RNAs in various ways, (2) modifying the life span of mRNAs, (3) altering the rate at which translation is initiated, and (4) activating or inactivating proteins after translation has occurred. Alternative Splicing of mRNAs Introns are spliced out in the nucleus as the primary RNA is transcribed. Splicing is accomplished by macromolecular machines called spliceosomes, and that many primary transcripts can be spliced in more than none way. o This turns out to be a major way of regulating eukaryotic gene expression. During splicing, gene expression is regulated when selected exons are removed from the primary transcript along with the introns. As a result, the same primary RNA transcript can yield more than one kind of mature, processed mRNA, consisting of different combinations of exons. Since these mature mRNAs contain differences in their sequences, the polypeptides translated from them will likewise differ. Splicing the same primary RNA transcript in different ways to produce different mature mRNAs and thus different proteins is referred to as alternative splicing. o Alternative splicing is controlled by proteins that bind to RNAs in the nucleus and interact with spliceosomes to influence which sequences are used for splicing. o When cells that are destined to become skeletal muscle or smooth muscle are developing, they receive signals leading to the production of specific proteins that are active in the regulation of splicing. Given the extent of alternative splicing, the definition of proteincoding genes has been changed to the coding and regulatory sequences that direct the production of one or more related mRNAs and polypeptides. Alternative splicing is a major mechanism in the control of gene expression in multicellular eukaryotes. mRNA Stability and RNA Interference One splicing is complete and processed mRNAs are exported to the cytoplasm, new regulatory mechanisms come into play. RNA interference occurs when a tiny, singlestranded RNA held by a protein complex binds to a complementary sequence in an mRNA. This event unleashes either the destruction of the mRNA or a block to the mRNA’s translation. STEP 1 RNA interference begins when RNA polymerase transcribes genes that code for RNAs that double back on themselves to form a hairpin. Hairpin formation occurs because pairs of bases within the RNA transcription are complementary. STEP 2 Some of the RNA is trimmed by enzymes in the nucleus; then the doublestranded hairpin that remains is exported to the cytoplasm STEP 3 In the cytoplasm, another enzyme cuts out the hairpin loop to form doublestranded RNA molecules that are only about 22 nucleotides. Chapter 19 – Control of Gene Expression in Eukaryotes MingHan Lu STEP 4 One of the strands from this short RNA is taken up by a group of proteins called the RNAinduced silencing complex, or RISC. The RNA strand held by the RISC is a microRNA (mRNA). STEP 5 Once it is part of a RISC, the miRNA binds to its complementary sequences in a target mRNA. STEP 6 If the match between a miRNA and an mRNA is perfect, an enzyme in the RISC destroys the mRNA by cutting it in two. In effect, tight binding by a miRNA is a “kiss of death” for the mRNA. If the match isn’t perfect, however, the mRNA is not destroyed. Instead, its translation is inhibited. Either way, miRNAs “interfere” with gene expression. Current data suggest that a typical animal or plant species has at least 500 genes that code for miRNAs and that each miRNA regulates more than one mRNA. Because of this evidence, it is estimated that a large percentage of all animals and plant genes are controlled by these tiny molecules. miRNAs are critical for normal development, and mutations in miRNA genes are associated with many diseases. How is Translation Controlled? Chapter 19 – Control of Gene Expression in Eukaryotes MingHan Lu Cells may slow or stop translation in response to a sudden increase in temperature or infection by a virus. The slowdown occurs because regulatory proteins that are activated by the temperature spike or viral invasion add a phosphate group to a protein that is part of the ribosome. High temperatures disrupt protein folding, so shutting down translation prevents the production of improperly folded polypeptides. If the problem is an invading virus, the cell stops the infection because it avoids manufacturing viral proteins. PostTranslational Control Instead of waiting for transcription, RNA processing, and translation to occur, the cell can keep an existing but inactive protein waiting in the wings and then quickly activate it in response to altered conditions. This is the essence of posttranslational control. There is a tradeoff, however: Speed is gained at the expense of efficiency. Another key mechanism of posttranslational control—the targeted destruction of proteins—was first introduced by describing the short life span of cyclin proteins. When a protein such as cyclin needs to be destroyed, enzymes mark it by adding many copies of a small polypeptide called ubiquitin. Ubiquitin got its name because it is ubiquitous in cells. A macromolecular machine called the proteasome recognizes proteins that have a ubiquitin tag and cuts them into short segments. 19.5 How Does Gene Expression Compare in Bacteria and Eukaryotes 1. DNA Packaging – The chromatin of eukaryotic DNA must be decondensed for basal and regulatory transcription factors to gain access to genes and for RNA polymerase to initiate transcription. The tight packaging of eukaryotic DNA means that the default state of transcription in eukaryotes is “off.” In contrast, the default state of transcription in bacteria, which lack histone proteins and have freely accessible promoters, is “on.” Chromatin structure provides a mechanism of negative control that does not exist in bacteria. 2. Complexity of transcription – Transcriptional control is much more complex in eukaryotes than in bacteria. The sheer number of eukaryotic proteins involved in regulating transcriptions dwarfs that in bacteria, as does the complexity of their interactions. 3. Coordinated transcription – In bacteria, genes that take part in the same cellular response are often organized into operons and transcribed together from a single promoter. In contrast, operons are rare in eukaryotes. Instead, for coordinated gene expression, eukaryotes rely on the strategy used in bacterial regulons—physically scattered genes are expressed together when the same regulatory transcription factors trigger the transcription of genes with the same DNA regulatory sequences. 4. Greater reliance on posttranscriptional control – Eukaryotes make greater use of posttranscriptional control such as alternative splicing. Alternative splicing allows eukaryotes to production of many proteins from each gene. Alternative splicing, Chapter 19 – Control of Gene Expression in Eukaryotes MingHan Lu microRNAs, and regulation of RNA stability are seldom in bacteria, but these constitute major elements of control in eukaryotes. Normal regulation of gene expression results in the orderly development of an embryo and appropriate responses to environmental change in adults. 19.6 Linking Cancer with Defects in Gene Regulation Each type of cancer is caused by a different set of mutations that lead to cancer when they affect one of two classes of genes: (1) genes that stop or slow the cell cycle, and (2) genes that trigger cell growth and division by initiating specific phases in the cell cycle. Many of the genes that are mutated in cancer influence gene regulation. The Genetic Basis of Uncontrolled Cell Growth proteins that stop or slow the cell cycle when conditions are unfavorable for cell division are called tumor suppressors. Logically enough, the genes that code for these proteins are called tumor suppressor genes. If the function of a tumor suppressor gene is lost because of mutation, then a key brake on the cell cycle is eliminated. Genes that stimulate cell division are called protooncogenes (literally, “first cancer genes”). In normal cells, the proteins produced from protooncogenes are active only when conditions are appropriate for growth. In cancerous cells, defects in the regulation of protooncogenes causes these genes to stimulate growth at all times. In such cases, a mutation has converted the protooncogene into an oncogene—an allele that promotes cancer development. Chapter 19 – Control of Gene Expression in Eukaryotes MingHan Lu The p53 Tumor Suppressor: A Case Study The gene is called p53 because the protein it codes for has a molecular weight of approximately 53 kilodaltons. Close correlation between DNA damage and the amount of p53 in a cell. These observations inspired the hypothesis that p53 is a regulatory transcription factor that serves as a master brake on the cell cycle. P53 is activated by DNA damage. Activated p53 binds to the enhancers of genes that arrest the cell cycle, repair DNA damage, and when all else fails trigger apoptosis (cell death). In mutant cells that lack a form of p53 that can bind to enhancers, DNA damage cannot arrest the cell cycle, the cell cannot kill itself, and damaged DNA is replicated. This situation leads to mutations that can move the cell farther down the road to cancer. The p53 protein is like a quality control officer. If it is missing, errors are made and things go downhill. The role of p53 in preventing cancer is so important that biologists call this gene “the guardian of the genome.”
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