Week 11- Eukaryotic Gene Expression

Review the diagram

Compare and contrast how gene expression is controlled in bacteria and eukaryotes. How does gene expression compare in bacteria and eukaryotes? These two major groups of organisms share a very distant common ancestor, and they often experience different selective pressures. Look at Table 19.2 for a quick comparison of gene expression and its control in these two domains of life.

Asnwer: RNA splicing opens the possibility of splicing one primary transcript in multiple, regulated ways. This can generate different proteins to meet the unique demands of particular cell types. Because splicing is rare in bacteria, these organisms lack the possibilities for regulation offered by alternative splicing in eukaryotes.

Describe how RNA splicing opens possibilities for controlling gene expression that are unavailable to bacteria.

As in bacteria, the promoter in eukaryotes is a site in DNA where RNA polymerase binds to initiate transcription. However, eukaryotic promoters are significantly more complex.

Describe how the initiation of transcription is regulated.

Answer: Acetylation of histones decondenses chromatin and allows transcription to begin, so HATs are involved in positive control. Deacetylation condenses chromatin and inactivates transcription, so HDACs are involved in negative control.

Do HATs and HDACs work in positive control or negative control? Explain your reasoning.

Each nucleosome consists of about 200 base pairs of DNA wrapped almost twice around a core of eight histone proteins. As Figure 19.2b indicates, a histone called H1 "seals" DNA to each set of histone proteins. Between each pair of nucleosomes there is a stretch of linker DNA with no histones.

Explain Figure 19.2b

Promoter-proximal elements are near the core promoter. Enhancers are located farther away, may be upstream or downstream from the promoter, and may even be within introns. Each of the colored areas in the enhancers or the promoter-proximal elements represents a regulatory sequence that is recognized by a protein. This drawing gives you an idea of the arrangement of introns, exons, and regulatory elements, but not their actual sizes, which vary widely.

Explain Figure 19.7

Step 1: Activators bind to DNA and recruit chromatin-remodeling complexes and histone acetyltransferases (HATs). Chromatin decondenses.

Explain Step 1

Step 2: swath of chromatin is exposed that includes the core promoter, promoter-proximal elements (only one is shown in the figure), and enhancers.

Explain Step 2

Step 3: Other activators bind to the exposed enhancers and promoter-proximal elements; DNA loops, allowing DNA-bound activators to bind to the Mediator.

Explain Step 3

Step 4: General transcription factors and RNA polymerase II assemble on the Mediator, then associate with the core promoter. DNA strands are opened, and RNA polymerase II can now begin transcription.

Explain Step 4

Answer: If one species made more extensive use of alternative splicing than another, then more gene products would be produced in the species with more extensive alternative splicing, even with the same number of genes.

Explain how alternative splicing could allow two different species with the same number of genes to produce vastly different numbers of proteins.

Answer: Tumor suppressor genes can be compared crudely to brakes and proto-oncogenes to accelerators. To run out of control, the accelerator must be active when it shouldn't be, and at the same time, the ability to put on the brakes must be lost. This is why tumor suppressor genes—the brakes—must not produce an active product, and proto-oncogenes—the accelerator—must produce an overly active product for cell division to run out of control.

Explain how cancer development requires inactivating mutations in tumor suppressor genes and activating mutations in proto-oncogenes.

Answer: An important role of p53 is to shut down DNA replication if there is DNA damage. With a loss-of-function mutation, p53 would fail to perform this role and cells would continue replicating damaged DNA. Replication of damaged DNA would lead to errors, which in turn would lead to mutations in many genes.

Explain why loss-of-function mutations in p53 often lead to mutations in other genes.

Answer: Regulation at the post-transcriptional level is costly in terms of energy and materials because either an mRNA or a protein already has to have been synthesized for post-transcriptional control to function. The advantage of post-transcriptional control is that it allows a more rapid response than transcriptional control because there are fewer steps between an mRNA or a newly synthesized protein and the final, active gene product (often an active protein). Another advantage of post-transcriptional control is that it can fine-tune transcriptional regulation. For example, if an mRNA is produced in crude cuts of a lot or a little, then miRNA or translational control can work to more closely regulate the precise amount of the molecule.

Explain the costs and benefits to a cell of regulating gene expression at the post-transcriptional level rather than the transcriptional level.

All cancers involve uncontrolled cell division. What allows this unbridled increase in cell number? Each type of cancer is caused by a different set of mutations that lead to cancer when they alter two classes of genes: (1) genes that stop or slow the cell cycle, and (2) genes that trigger cell growth and division. It turns out that many of the genes that are mutated in cancer control gene expression, either directly as transcription factors or indirectly as proteins involved in cell communication. Let's take a closer look at how altered gene regulation can lead to the uncontrolled cell growth that is a hallmark of cancer.

Explain the relationship between cancer and defects in gene regulation.

Answer: The name "RNA interference" is apt because small RNAs, such as miRNAs, interfere with mRNAs by targeting them for destruction or preventing them from being translated.

Explain why RNA interference is aptly named.

Answer: Many different types of mutations can disrupt control of the cell cycle and initiate cancer. These different mutations can, however, result in the same pattern of uncontrolled cell growth.

Explain why cancer has a common pattern of uncontrolled cell growth, but not a common cause.

It can lead to developmental disorders. For example, Rett syndrome occurs when a mutation leads to the absence of a protein that binds to methylated DNA to induce chromatin condensation. One of the remarkable unexplained features of Rett syndrome is how the lack of a protein that can bind to many methylated DNA sequences found across the entire genome can cause the specific phenotypes of Rett syndrome. Whatever the explanation, the failure to normally condense chromatin leads to this human developmental disorder. Epigenetics is at work again.

Failure to condense chromatin leads to what?

But the stories of their creation and their function are different. siRNAs are generated from much longer double-stranded RNAs than the miRNA precursors—and this sets the stage for differences in how miRNAs and siRNAs function. The double-stranded RNA precursors for siRNAs are invasive molecules that can harm the cell. Often these are RNA molecules that make up viral genomes or that form as part of viral infection cycles. In other cases, the origin of an siRNA is a double-stranded RNA that is generated as another invasive molecule, a transposable element, is in the act of moving within the genome

How are siRNAs different to miRNAs?

At maturity, siRNAs are virtually indistinguishable from miRNAs—both are single-stranded RNAs about 21 nucleotides long that are bound to RISC and that target particular RNAs by complementary base pairing

How are siRNAs similar to miRNAs

Answer: A microRNA can recognize a specific target whenever it can form complementary base pairs with the target. One way that a miRNA could recognize more than one target mRNA would be if different mRNAs shared a common sequence recognized by the miRNA. Another way would be if the miRNA did not pair perfectly with its target sequence. In this case, two different mRNAs could have related but distinct sequences that the same miRNA could bind to.

If you understand RNA interference, you should be able to explain how a microRNA can recognize a specific target mRNA and to propose a way that one miRNA could recognize more than one target mRNA.

Answer: miRNA-mediated control is based on controlling the presence of mRNAs (through cleavage) or the ability to translate mRNAs. This system presupposes that mRNAs are stable enough to bother controlling, whether it's useful to remove them or prevent their use. In the case of mRNAs with a fleeting existence, controlling mRNA stability or translatability takes away most of its advantages.

Knowing that mRNAs in bacteria have very short half-lives (they don't last long), explain why it is reasonable to expect bacteria not to use a system analogous to miRNA-mediated control of gene expression.

Answer: The number of regulatory elements would be predicted to decrease. This is because operons group genes that function in the same pathway into co-transcribed groups, and transcription of the entire set is controlled by the same set of DNA regulatory elements. In this way, a given set of regulatory elements could control many genes. Without this clustering, a separate set of regulatory elements is needed for each gene.

Predict how the number of regulatory elements would change if eukaryotes made extensive use of operons.

Review this

Summary: Elements of Transcriptional Regulation

DNA packaging: The chromatin of eukaryotic DNA must be decondensed for general 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 the condensed chromatin of eukaryotes and have more accessible promoters, is "on." Condensed chromatin prevents transcription and provides a mechanism of negative control that does not exist in bacteria.

The control of gene expression in bacteria and eukaryotes shares many similarities, but also shows significant differences (1/4)

Complexity of transcription: Transcription initiation is much more elaborate in eukaryotes. The sheer number of eukaryotic proteins needed to regulate the start of transcription dwarfs that in bacteria, as does the number of their interactions.

The control of gene expression in bacteria and eukaryotes shares many similarities, but also shows significant differences (2/4)

Coordinated transcription: In bacteria, genes that take part in the same cellular process 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.

The control of gene expression in bacteria and eukaryotes shares many similarities, but also shows significant differences (3/4)

Reliance on post-transcriptional control: Eukaryotes make much greater use of post-transcriptional control, such as alternative splicing and RNA interference. Bacteria use other forms of post-transcriptional control, but use these to a lesser extent.

The control of gene expression in bacteria and eukaryotes shares many similarities, but also shows significant differences (4/4)

On the flip side of the coin are genes that stimulate cell division. These genes are called proto-oncogenes (literally, "first cancer genes"). In normal cells, the proteins produced from proto-oncogenes are active only when conditions are appropriate for division. In cancerous cells, defects in the regulation of proto-oncogenes or their protein products spur cells to divide all the time. In such cases, a mutation has converted the proto-oncogene into an oncogene—a mutant allele that promotes cancer. For cancers to develop, many mutations are required within a single cell, and these alter both tumor suppressor genes and proto-oncogenes.

What are proto-oncogens? What are oncogens?

-Proteins may be activated when a protein kinase adds a phosphate group. One example is activation of cyclin-Cdk complexes by phosphorylation, which triggers entry into M phase of the cell cycle (Ch. 12, Section 12.3). -Proteins may be targeted for destruction. When a protein such as a cyclin needs to be destroyed, enzymes mark it by adding copies of a small polypeptide called ubiquitin. Ubiquitin got its name because it is ubiquitous (widespread and common) in cells. A macromolecular machine called the proteasome (a macromolecular machine that destroys proteins that have been marked by the addition of ubiquitin) recognizes proteins that have a ubiquitin tag and cuts them into short segments

What are some examples of post-translational control of gene expression?

As you learned in the chapter on the cell cycle (Ch. 12, Section 12.3), proteins that stop or slow the cell cycle when conditions are unfavorable for cell division are called tumor suppressors. 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 brake on the cell cycle is eliminated.

What are tumor suppresors?

Like miRNAs and siRNAs, piRNAs work as small single-stranded RNAs that target particular RNAs when the piRNAs are bound to a complex of proteins. For piRNAs, this protein complex is different from RISC. piRNAs were first discovered to prevent the movement of transposable elements in germ-line cells—the cells that produce sperm and eggs. More recent work has shown that piRNAs play a much broader role outside of reproductive cells, where they block the movement of transposable elements and regulate gene expression in every eukaryote that's been examined.

What do piRNAs do?

These observations inspired the hypothesis that p53 is a regulatory transcription factor that works as a master brake on the cell cycle, making it a "guardian of the genome." In this model, shown in Figure 19.12a, p53 activity is induced 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). Expression of these genes allows the cell to halt the cell cycle and repair its DNA, if this is possible, or undergo apoptosis if the DNA damage is too severe.

What does figure 19.12.a demonstrate

In mutant cells with an altered form of p53 unable to bind to enhancers, DNA damage cannot trigger either arrest of the cell cycle or apoptosis, and damaged DNA is replicated (Figure 19.12b). This situation leads to chromosome breaks and mutations that 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.

What does figure 19.12b demonstrate

In bacteria, controlling the activity of proteins after they are made—post-translational regulation—is important in allowing cells to respond rapidly to new conditions (Chapter 18). The same is true for eukaryotes. Instead of waiting for transcription, RNA processing, and translation to occur, the cell can keep an existing but inactive protein handy and then quickly activate it in response to altered conditions. Similarly, an active protein can rapidly be inactivated by removing a modification. This is the essence of post-translational control. There is a trade-off, however: Speed comes at the expense of efficiency. Transcription, RNA processing, and translation use energy and materials.

What is Post-Translational Control?

Researchers began to understand one function of p53 when they exposed noncancerous human cells to UV radiation and noticed that the level of active p53 protein increased markedly. Recall that UV radiation damages DNA (Ch. 15, Section 15.5). Follow-up studies confirmed that there is a close correlation between DNA damage and the activity of p53 in a cell. Analyses of the p53 protein's primary structure suggested that it might contain a DNA-binding region similar to the one shown for the muscle-specific transcription factor in Figure 19.8b.

What is the function of p53 tumor suppresors?

To understand how defects in gene expression can lead to cancer, let's consider research on the gene that is most often defective in human cancers. The gene is called p53 because when it was first discovered, researchers knew only that the protein it codes for has a molecular weight of about 53 kilodaltons. Sequencing studies have revealed that mutant, nonfunctional forms of the p53 gene are found in well over half of all human cancers. This gene codes for a regulatory transcription factor.

What is the p53 tumor suppresor?

In either case, the cell is in trouble and needs to shut down the problem—and this is what siRNAs do. Once bound by the siRNA coupled to RISC, the viral or transposable element RNAs are chopped to bits. The danger has passed.

What so siRNAs do?

Answer: Bacterial regulatory sequences are found close to the promoter; eukaryotic regulatory sequences can be close to the promoter or far from it. Bacterial regulatory proteins interact directly with RNA polymerase to initiate or prevent transcription; eukaryotic regulatory proteins influence transcription by altering chromatin structure or binding to the general transcription complex through Mediator proteins.

Compare and contrast the nature of regulatory sequences and regulatory proteins in bacteria versus eukaryotes.

Answer: A typical eukaryotic gene usually contains introns and many regulatory sequences, including enhancers, and each particular mRNA produced from the gene usually codes for one protein. Bacterial operons usually lack introns and enhancers, and produce an mRNA that codes for two or more distinct proteins.

Compare and contrast the structure of this typical eukaryotic gene and the structure of a bacterial operon.

CONCLUSION: A mother's diet influences chromatin modifications and gene expression patterns throughout her offspring's life.

Could epigenetic inheritance be at work in silencing Hnf4a expression? What was the conclusion?

The central idea is that chromatin must be decondensed to expose the promoter so RNA polymerase can bind to it. If so, then chromatin remodeling would be the first step in the control of eukaryotic gene expression. Studies that examined the accessibility of DNA to DNases have provided strong support for this hypothesis. DNases are enzymes that cut DNA, but they cannot cut efficiently if DNA is tightly wrapped with proteins. As Figure 19.4 shows, DNase works effectively only if DNA is in a decondensed, or open, configuration.

DNase is an enzyme that cuts DNA at random locations. However, it cannot cut DNA in condensed chromatin. Explain.

RNA interference involves more than just miRNAs. Two other types of small RNAs are short interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs). The origins of these two types of small RNAs differ from one another and from microRNAs, but all work to silence gene expression through RISC or RISC-like complexes.

Explain how siRNAs and piRNAs Protect against Viruses and Transposable Elements

Introns are spliced out in the nucleus as the primary RNA is transcribed. Recall that the mRNA that results from splicing consists of sequences encoded by exons, and that it is protected by a cap on the 5′ end and a long poly(A) tail on the 3′ end (Ch. 17, Section 17.2). You may also recall that splicing is accomplished by macromolecular machines called spliceosomes, and that many primary transcripts can be spliced in more than one way. This is a major way of regulating eukaryotic gene expression.

Explain how Primary transcripts are alternatively spliced?

Similar to the situation in eukaryotes, bacterial DNA interacts with proteins, but 30-nm fibers or higher-order arrangements have not been observed in bacterial chromosomes.

Explain how bacterial DNA interacts with proteins

Answer: Addition of acetyl groups to histones or methyl groups to DNA can cause chromatin to decondense or condense, respectively. Different patterns of acetylation or methylation will determine which genes in muscle cells versus liver cells can be transcribed and which genes are not available for transcription.

Explain how certain patterns of histone acetylation or DNA methylation could influence whether a cell became a muscle cell or a liver cell.

Answer: Promoter-proximal elements are binding sites for regulatory transcription factors. If the DNA sequence in a promoter-proximal element changed, this would be likely to alter how tightly, or if at all, the transcription factor associated with this element could bind to DNA. In turn, this change in binding would increase or decrease the amount of time the transcription factor was associated with DNA, therefore changing how efficiently transcription initiation was promoted by this transcription factor.

Explain how changes in a promoter-proximal element DNA sequence could lead to changes in the rate of transcription initiation.

studies of many different genes in various cell types led to similar conclusions—chromatin is decondensed in genes that are being transcribed. These results suggest that in their normal, or default, state, eukaryotic genes are turned off because DNA is normally wrapped tightly in chromatin. Gene expression then depends on opening up chromatin in the promoter region.

Explain how chromatin is decomposed in genes that are being transcribed

These findings support one of the most important statements researchers can make about gene regulation in eukaryotes: Different types of cells express different genes primarily because they have different transcription factors. In multicellular species, the genes encoding transcription factors, in turn, are expressed largely in response to signals that arrive from other cells, especially during embryonic development.

Explain how different types of cells express different genes

Recent studies have found that each chromosome lies in its own distinct region, or territory, within the interphase nucleus. This arrangement is shown in Figure 19.3. Portions of these segregated chromosomes can fold out to reach sites within the nucleus where genes are actively transcribed and then fold back when transcription is finished. As in real estate, location matters. This image represents a cross section through the chromosomes (numbered) in the nucleus of one human cell during interphase.

Explain how each chromosome lies in its own distinct region

Answer: The existence of chromatin means that genes are often inaccessible for transcription, but the fact that mechanisms must be in place to open chromatin to access genes offers the possibility of regulating whether genes are ready for, or resistant to, transcription.

Explain how eukaryotic chromatin poses both challenges and opportunities for regulating gene expression.

Answer: Genetic inheritance is based on differences in DNA sequences, essentially different alleles that lead to differences in phenotype. Epigenetic inheritance is inheritance that is due to anything else. In reality, however, the mechanism of epigenetic inheritance appears to be the inheritance of different patterns of chromatin condensation.

Explain how genetic inheritance and epigenetic inheritance differ.

Even after an mRNA is produced and exported to the cytoplasm, other crucial levels of gene regulation come into play (see Figure 19.1). Let's begin with a look at how the translation of particular mRNAs can be regulated by a set of small RNAs that have a long reach in controlling gene expression.

Explain how mRNA Stability and Translation Are Important Mechanisms of Post-Transcriptional Control

Individual nucleosomes are linked to each other in increasingly complex structures to form chromatin. First, H1 histones interact with one another and with histones in other nucleosomes to produce a tightly packed structure like that shown in Figure 19.2b. Based on its width, this structure is called the 30-nanometer fiber. (Recall that a nanometer is one-billionth of a meter and is abbreviated nm.)

Explain how nucleosomes are linked to each other to form chromatin

With epigenetic inheritance, when a cell receives a "become a skin cell" signal early in development, the chromatin is modified in distinctive ways, and those modifications are passed on to its descendants. Skin cells are different from liver cells, for example, not because they contain different genes, but largely because they have inherited different patterns of DNA methylation and histone modifications during their development. The shared set of genes in these cells are differentially accessible for expression because of distinct and stable patterns of chromatin condensation.

Explain how, for example, skin cells are different from liver cells

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.

Explain the association between DNA and histones

In bacteria, genes that need to be regulated together are often clustered into a single operon and transcribed into a single mRNA. In contrast, eukaryotes use the strategy uncovered by Oshima for galactose-metabolizing genes in yeast—instead of being clustered together, each co-regulated gene has the same regulatory DNA sequence that binds the same type of regulatory protein. Regulatory DNA sequences exist in all eukaryotic genes.

Explain the differences in regulation between bacteria and eukaryotes

The elaborate structure of eukaryotic chromatin does more than just package DNA so that it fits into the nucleus. Chromatin structure also has profound implications for the control of gene expression.

Explain the elaborate structure of eukaryotic chromatin

The first additional level of control involves the DNA-protein complex at the top of the figure. In eukaryotes, DNA is wrapped around proteins to create a structure called chromatin

Explain the first additional level of control

Moving up the organizational ladder, 30-nm 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. Finally, when chromosomes condense before mitosis or meiosis, the scaffold proteins and 30-nm fibers are folded into even more tightly packed structures that ultimately lead to the chromosomes that are visible during cell division.

Explain the scaffold framework inside the nucleus

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. To capture this idea, biologists say that chromatin remodeling must occur before transcription, transitioning from a condensed or "closed" state to a decondensed or "open" state.

Explain the similarities between promotors in eukaryotic genes and bacterial genes

The pattern of chromatin modifications varies from one cell type to another. For example, suppose you analyzed the same gene in a skin cell and a liver cell from one individual. This gene and others are likely to have different patterns of DNA methylation and histone modifications in the two cell types, as well as different patterns of chromatin condensation.

Explain the variety in chromatin modifications

(a) The edges of different base pairs projecting into the major and minor grooves of DNA present different shapes and chemical groups. Atoms of these chemical groups that participate in hydrogen bonding with amino acids of transcription factors are indicated by green arrows. The methyl group on thymine (T), indicated with a blue arrow, is also important in recognition. (b) A transcription factor (green) binding to a regulatory sequence in DNA. This transcription factor recognizes edges of base pairs that project into the major groove. The bases within the regulatory sequence recognized by the protein are highlighted in red.

Explain this figure

For example, if a signal that says "become a muscle cell" reaches a cell in the early embryo, it triggers a signal transduction cascade (Chapter 11) that leads to the production of transcription factors specific to muscle cells. Because different transcription factors bind to specific regulatory sequences, they turn on the production of muscle-specific proteins. But if a become-a-muscle-cell signal isn't present, then no active muscle-specific transcription factors are produced and no muscle-specific gene expression takes place in that embryonic cell.

Explain transcription factors in relation to gene expression

Each transcription factor must be able to recognize and bind to a specific DNA sequence. How can it do this? Recall that DNA bases are partially exposed in the grooves of the DNA double helix, and that one of these grooves, the major groove, is wide, and the other groove, the minor groove, is narrow. The edges of an A-T base pair and a C-G base pair that project into the major and minor grooves contain different sets of atoms and have different surface shapes (Figure 19.8a). These differences in chemical composition and shape can be recognized by transcription factors.

How Do Transcription Factors Recognize Specific DNA Sequences?

There appear to be two answers. Some transcription factors can bind to DNA that is associated with histones. It also turns out that most chromatin is dynamic. DNA occasionally dissociates from the histone proteins in nucleosomes, particularly DNA sequences near the linker DNA that connects nucleosomes, exposing regulatory sequences to activators.

How can an activator bind to DNA in the first place if chromatin is condensed?

Just as base pairs come together by complementary molecular interactions, so too can proteins and specific DNA sequences. An example is shown in Figure 19.8b. In this case, 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 regulatory sequence because of complementary interactions between its amino acids and a particular sequence of base pairs in DNA. Without such specific interactions between transcription factors and DNA, the development of muscle cells—or any other cell—would not be possible.

How can proteins and specific DNA sequences come together?

DNA methylation, histone modifications, and chromatin-remodeling complexes work together to fine-tune chromatin condensation at specific genes. The take-home message is that the condensation state of chromatin is critical in determining whether transcription can occur.

How do DNA methylation, histone modification and chromatin remodeling complexes work together?

To fit inside the nucleus, the DNA must be packed tightly—so tightly that RNA polymerase can't access it. How is DNA packaged? And how can it be unpacked at particular genes so RNA polymerase can transcribe it?

How does DNA fit inside the nucleus

RNA interference (RNAi) occurs when a tiny, single-stranded RNA held by a protein complex binds to a complementary sequence in another RNA. In the case of mRNAs, depending on how well the small RNA matches its mRNA target, this binding of complementary RNAs either leads to the destruction of the mRNA or blocks the mRNA's translation. How does it work? RNA Degradation- Degradation of an mRNA molecule or inhibition of its translation following its binding by a short RNA whose sequence is complementary to a portion of the mRNA

How does the RNA interface control expression of RNAs

Answer: The general transcription factors found in muscle and nerve cells are similar or identical; the sets of regulatory transcription factors found in the two cell types are different.

If you understand this concept, you should be able to compare and contrast the regulatory and general transcription factors expected to be found in muscle cells versus nerve cells.

Answer: DNA forms loops when distant regulatory regions, such as silencers and enhancers, are brought close to the core promoter through binding of regulatory transcription factors to Mediator.

If you understand this model, you should be able to explain why DNA forms loops near the core promoter in order for transcription to begin.

Investigators examined what happened to rats born to mothers fed low-protein diets during pregnancy and while nursing. Even when provided a normal diet after these early deprivations, these animals have a greatly increased risk of developing disorders in later life that are similar to type 2 diabetes. Type 2 diabetes is a serious and increasingly common disease that alters the cellular uptake of glucose (Chapter 41). Both genetic factors and environmental factors, such as diet, play important roles in diabetes development.

Long-Lasting Epigenetic Marks Imposed during Development. Explain: One example of how events in early development can make lifelong differences comes from a study of rats.

Answer: (There are many possibilities because alternative splicing allows any exon or intron to be skipped or retained in the final mRNA.)

MODEL If you understand alternative splicing, you should be able to draw a model showing some possible mRNAs that could be spliced from a primary transcript containing two exons that surround an intron.

Provide rat mothers with a normal or a low-protein diet during pregnancy and while nursing. After weaning, feed rat pups a normal diet and raise to old age. Determine types of histone modifications for a regulatory gene involved in diabetes and measure transcription.

One significant gene associated with diabetes is Hnf4a. The Hnf4a gene codes for a regulator of genes involved in glucose uptake. The diabetic rats born to protein-deprived mothers express the Hnf4a gene at lower levels than normal rats. What was the experimental setup?

Answer: Many more genes than normal are predicted to be expressed because the inability to methylate DNA would lead to more decondensed chromatin.

Predict how gene expression will be affected if a cell is grown with compounds that prevent DNA methylation.

For a molecular signal to trigger the transcription of a specific gene, the chromatin around the target gene must be remodeled. To appreciate why, consider that a typical cell in your body contains about 6 billion base pairs of DNA. Lined up end to end, these nucleotide pairs would form a double helix about 2 m (6.5 feet) long. But the nucleus that holds this DNA is only about 5 µm in diameter—far less than the thickness of a piece of paper

Suppose a cell detects a signal that tells it to produce a specific protein. What happens next?

Answer: Regulatory sequences are predicted to be the same because different cells of an individual contain the same DNA sequence. In contrast, transcription factors are predicted to differ because it is largely differences in transcription factors that allow gene expression patterns to differ between cell types.

THINK CAREFULLY Predict whether regulatory sequences or transcription factors—or both—would be the same in muscle cells and brain cells within an individual.

As Figure 19.10a shows, the rat α-tropomyosin gene contains 13 exons. However, in each cell, a different subset of the 13 exons present in the primary transcript is spliced together to produce distinctly different mRNAs (Figure 19.10b). Each of these mRNAs is referred to as an isoform. As a result of alternative splicing, the same tropomyosin gene is expressed to produce six distinct proteins. Tropomyosin is important in muscle function, and one reason skeletal muscle and smooth muscle are different is that they contain different types of tropomyosin protein. (There are six isoforms (types) of tropomyosin mRNA, including two different types in skeletal muscle cells. Figure 19.10)

To see how alternative splicing works, consider the protein tropomyosin. The tropomyosin gene is expressed in at least five different types of cells, including two kinds of muscle cells that make up skeletal muscle responsible for voluntary movement and smooth muscle that lines the gut and certain blood vessels.

Another major player in chromatin alteration and gene regulation are proteins that form macromolecular machines called chromatin-remodeling complexes. These complexes harness the energy in ATP to reshape chromatin. Chromatin-remodeling complexes either cause nucleosomes to slide along the DNA or to knock the histones completely off the DNA to open up stretches of chromatin for transcription.

What are chromatin-remodeling complexes?

a set of regulatory sequences in eukaryotic DNA that may be located upstream or downstream of the start site of transcription, in nontranscribed regions, or in introns. Enhancers are often far from the gene they control. Binding of a specific regulatory transcription factor proteins to an enhancer promotes the transcription of genes.

What are enhancers?

These proteins interact with the core promoter and are not restricted to particular genes or cell types. The term "general" implies that these proteins are necessary for transcription to occur, but they do not provide much in the way of regulation. The TATA-binding protein (TBP) that you learned about is part of a large general transcription factor that is used for many genes.

What are general transcription factors?

Later work documented that a group of proteins called histones are the most abundant DNA-associated proteins. Chromatin consists of DNA complexed with histones and other proteins. Histones: a member of a class of positively charged (basic) proteins associated with DNA in the chromatin of eukaryotic cells

What are histones?

Chromatin remodeling and transcription are just the opening to the story of gene regulation. Once a gene is transcribed, a series of events has to occur before a final product appears (see Figure 19.1). Each of these events offers an opportunity to regulate gene expression. Any regulation that occurs after transcription is a form of post-transcriptional control. These regulatory mechanisms include (1) different ways of splicing the same primary transcript, (2) altering the ability to translate particular mRNAs, or destroying them, and (3) altering the activity of proteins after translation has occurred. Let's consider each mechanism in turn.

What are post-translational modifications?

Follow-up work supported the hypothesis that enhancers and silencers are binding sites for activators and repressors that regulate transcription. Collectively, these proteins are termed regulatory transcription factors, or often transcription factors for short. There are hundreds of transcription factors that bind to enhancers, silencers, and promoter-proximal elements.

What are regulatory transcription factors? What are transcription factors?

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 of negative control.

What are silencers? What are repressors?

1) RNA polymerase transcribes genes coding for miRNAs; the newly transcribed RNAs double back on themselves to form hairpins (only one hairpin is shown in the figure). 2) Hairpins form because sets of bases within the RNA are complementary. Step 2Hairpin-containing precursor miRNA is bound to proteins in the nucleus that form an RNA-processing complex. The single-stranded 5′ and 3′ ends are trimmed off. 3) The partially processed miRNA is exported to the cytoplasm and bound by another RNA-processing complex called Dicer. Dicer trims off the loop, leaving a small (~21 base pair), double-stranded RNA with short single-stranded overhangs at each end.

What are steps 1-3 in figure 19.11

4) The double-stranded miRNA fragments are bound by the RNA-induced silencing complex (RISC) proteins. 5) The RNA strands are unwound. One strand of RNA is expelled and ultimately degraded, and the other strand, called the guide RNA, is retained by RISC. The guide RNA is the mature miRNA. 6) As part of RISC, the miRNA binds to its complementary sequences in a target mRNA. 7) If the match between an miRNA and an mRNA is perfect, an enzyme in RISC cuts the mRNA in two. If the match is imperfect, the guide RNA bound to the mRNA with RISC inhibits translation without destroying the mRNA. Either way, miRNAs interfere with gene expression.

What are steps 3-7 in figure 19.11

Research on chromatin remodeling has advanced at a furious pace, and biologists have succeeded in identifying key players that work to change the state of chromatin condensation. There are three major ways to remodel chromatin: DNA methylation, histone modification, and the use of chromatin-remodeling complexes.

What are the 3 main ways to remodel chromatin

-Enhancers can be vast distances (sometimes more than 100,000 base pairs) away from the promoter. They can be located in introns or in nontranscribed sequences and can be upstream, downstream, or even within a gene (Figure 19.7). -Like promoter-proximal elements, there are many types of enhancers. -Most genes have more than one enhancer. -An enhancer is composed of many short regulatory sequences that each bind a different specific regulatory protein. -Enhancers can work even if they are flipped from their normal 5′→3′5′→3′ orientation or moved to new locations near the gene.

What are the key characteristics of enchancers?

Besides the core promoter, other DNA sequences—called regulatory sequences—allow the binding of proteins that control the initiation of transcription. Any segment of DNA or RNA that is involved in controlling the expression of a specific gene of process such as RNA splicing by binding a regulatory transcription factor protein or other protein

What are the regulatory sequences?

Once the nucleosome-based structure of chromatin was established, scientists realized that the close physical interaction between DNA and histones must be altered for RNA polymerase to make contact with DNA. They hypothesized that a gene could not be transcribed until the condensed chromatin near its promoter was remodeled.

What did scientists hypothesize about transcription?

An important point in this model of transcription initiation is the dual role of transcriptional activators. Activators work not only to stimulate transcription but also to bring chromatin-remodeling proteins to the right place at the right time. None of the proteins that remodel chromatin can recognize specific DNA sequences. Transcriptional activators bind to regulatory sequences of particular genes to recruit the proteins needed to alter chromatin structure.

What do activators work to do?

Like bacteria, eukaryotes can control gene expression at the levels of transcription, translation, and post-translation. But as Figure 19.1 shows, additional levels of control occur in eukaryotes as genetic information flows from DNA to proteins.

What does Figure 19.1 show?

Differential gene expression results largely from the production or activation of specific transcription factors. Eukaryotic genes are turned on when transcription factors bind to enhancers and promoter-proximal elements; the genes are turned off when transcription factors bind to silencers, when a particular transcription factor is not present, or when chromatin is condensed. Distinctive sets of transcription factors are what make a muscle cell a muscle cell and a liver cell a liver cell.

What does different gene expression result from?

There are several types of RNA interference. As Figure 19.11 shows, one form of RNA interference works through a small RNA called a microRNA (miRNA) that is derived from transcription of cellular genes.

What does figure 19.11 demonstrate

Histone acetylation usually promotes decondensed chromatin, a state associated with active transcription. The addition of acetyl groups also creates a binding site for other proteins that help open the chromatin.

What does histone acetylation promote?

Modifying histones with these and other chemical groups alters the association of DNA with histone proteins and promotes condensed or decondensed chromatin, depending on the specific set of modifications made to particular histones.

What does modifying histones do?

Adding an acetyl group to lysines in histones neutralizes the positive charge on this amino acid. Addition of an acetyl group reduces the electrostatic interactions between the negatively charged phosphates in the DNA backbone with histones, and it interferes with the assembly of nucleosomes into condensed chromatin.

What does the addition of an acetyl group do?

Think of what alternative splicing of tropomyosin means—one gene, one pre-mRNA, six different mRNAs, six different polypeptides. Across the entire set of protein-coding genes, this principle of "from one, many" made possible by alternative splicing amplifies the number of proteins that can be specified by a much smaller set of genes.

What does the alternative splicing of tropomyosin means?

As the graph on the left in the "Results" section of Figure 19.6 shows, they found that histone modifications of the Hnf4a gene that led to condensed chromatin were elevated in rats born to malnourished mothers as compared to control offspring.Conversely, histone modifications associated with decondensed chromatin were significantly reduced in the treatment group.

What does the graph on the left demonstrate?

The graph on the right confirms that transcription of Hnf4a was much lower in the treatment group than the control group. Together these results demonstrate correlations between maternal diet, altered histone modifications, and decreased levels of Hnf4a gene expression in adult offspring

What does the graph on the right demonstrate?

Once a core promoter that contains a TATA box has been exposed by chromatin remodeling, the first step in initiating transcription is binding of the TATA-binding protein (TBP). The TBP works in the context of a large, multi-subunit protein complex. There are also proteins, related in function to the TBP, that work on promoters with other conserved sequences. But the binding of TBP or any of its relatives does not guarantee that a gene will be transcribed. A wide array of other DNA sequences and proteins must work together with RNA polymerase to allow transcription to begin.

What happens once the core promoter is exposed?

A group of enzymes known as DNA methyltransferases add methyl groups (—CH3)(—CH3) to cytosine residues in DNA, by a process called DNA methylation. In mammals, the sequence recognized by these enzymes is a C next to a G in one strand of the DNA. This sequence is abbreviated CpG and is shown here in its methylated form:

What is DNA methylation?

Another 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. If different cells use different splicing patterns, different gene products result.

What is RNA processing?

In addition to transcription factors, a large complex of proteins called the Mediator acts as a bridge between regulatory transcription factors, general transcription factors, and RNA polymerase II. The Mediator plays a critical role in integrating the input of many regulatory transcription factors and delivers a signal to RNA polymerase to initiate transcription. Figure 19.9 summarizes a model for how transcription is initiated in eukaryotes.

What is a mediator?

Enhancers are regulatory DNA sequences primarily found in eukaryotes. When regulatory proteins called transcriptional activators, or activators for short, bind to enhancers, transcription begins. Thus, enhancers and activators are like a gas pedal—an element of positive control

What is a transcriptional activator? What an is activator?

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. Embryonic cells that develop into skeletal muscle or smooth muscle receive signals leading to the production or activation of proteins that regulate splicing.

What is alternative splicing controlled by?

During splicing, gene expression is regulated by differential processing of introns and exons—introns can be retained and exons can be skipped. As a result, the same primary RNA transcript can be spliced together in different ways, yielding more than one kind of mature, processed mRNA. Because 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 is alternative splicing. In other words, in eukaryotes, the splicing of primary RNA transcripts from a single gene in different ways to produce different mature mRNAs and thus polypeptides

What is alternative splicing?

An important feature of miRNA-based control of gene expression is that a single type of miRNA can regulate many different mRNAs. Conversely, one mRNA can be targeted by many different miRNAs. Considering the large number of miRNAs found in most species—for example, there are at least 1100 different human miRNAs—this suggests a huge network of gene regulation. Many, if not most, genes of multicellular eukaryotes are controlled by miRNAs. miRNAs are critical for development, and mutations in miRNA genes are associated with many diseases. These tiny RNAs are big elements of gene regulation.

What is an important feature of miRNA-based control of gene expression?

If the skin or liver cells divide, the patterns of DNA methylation and histone modifications such as acetylation can be passed on to their daughter cells. This provides a way for each daughter cell to inherit patterns of gene expression by epigenetic inheritance—the collective term for any mechanism of inheritance that is due to something other than differences in DNA sequences

What is epigenetic inheritence?

Figure 19.5 shows two different types of enzymes that add or remove acetyl groups from histones. Shown to the right, histone acetyltransferases (HATs) add acetyl groups to the positively charged lysine residues in histones, and to the left, histone deacetylases (HDACs) remove them. Histone acetyltransferases (HATs) cause chromatin to decondense; histone deacetylases (HDACs) cause it to condense. Figure 19.5 Full Alternative Text Description

What is histone acetyltransferases (HATs)? What are histone deacetylases (HDACs)?

A large set of enzymes adds a variety of chemical groups to specific amino acids of histone proteins. These include acetyl groups (—COCH3), methyl groups, phosphate groups, and short polypeptide chains.

What is histone modification?

A protein that binds to the TAT box in eukaryotic promoters and is a component of the transcription initiation complex

What is the TAT Binding protein (TBP)?

A short DNA sequence in many eukaryotic promoters that is important for assembling general transcription factors and RNA polymerase at the core promoter; located about 30 base pairs upstream from the transcription start site

What is the TATA Box?

In eukaryotes the term core promoter is often used to indicate the specific sequence where RNA polymerase binds, as opposed to the other sequences needed for regulation of transcription. When you hear the word "promoter" in the context of a eukaryote, it is important to think about whether this refers narrowly to the core promoter or if it is meant more broadly to include other DNA sequences involved in controlling the initiation of transcription. The most intensively studied core promoter sequence is a short stretch of DNA known as the TATA box.

What is the core promoter in eukaryotes?

A gene is now viewed as a nucleotide sequence that allows the production of one or more related RNAs or polypeptides. Over 90 percent of human genes undergo alternative splicing. Alternative splicing is a major mechanism in the control of gene expression in multicellular eukaryotes.

What is the definition of a gene?

Researchers have proposed that particular combinations of histone modifications on specific amino acids of histone proteins set the state of chromatin condensation for a particular gene. This idea is the histone code hypothesis. Let's take a look at one way histone modifications can control chromatin structure and the accessibility of DNA sequences needed to initiate transcription.

What is the histone code?

A group of eight histone proteins with about 200 nucleotides of DNA wrapped twice around it; the fundamental unit of chromatin. Later work documented that a group of proteins called histones are the most abundant DNA-associated proteins. Chromatin consists of DNA complexed with histones and other proteins.

What is the nucleosome?

In eukaryotic DNA, a regulatory sequence that is close to a promoter and can bind regulatory transcription factors Regulatory sequences such as the ones discovered in yeast that are close to the promoter are termed promoter-proximal elements. Unlike the core promoter, different types of promoter-proximal elements are associated with each gene. In this way, they allow eukaryotic cells to express certain genes but not others.

What is the promoter-proximal element?

There are a lot of different types, and only a few of these genes are primarily devoted to producing miRNAs. In most cases, the double-stranded RNA precursors for miRNAs are coded for in both protein-coding and noncoding exons of mRNAs, in the introns of pre-mRNAs, and in the introns of non-protein-coding RNAs. In short, almost any region of almost any type of RNA transcript can encode an miRNA.

What kind of genes code for miRNAs?

The gene studied by Tonegawa's group was broken into many introns and exons. Recall that introns are transcribed sequences that are spliced out of the primary transcript; exons are transcribed regions that are included in the mature RNA once splicing is complete (Ch. 17, Section 17.2). The researchers found a regulatory sequence required for enhanced transcription within one of the introns.

What was discovered while exploring how human cells regulate gene expression?

In contrast, when HDACs remove acetyl groups from histones, this reverses the events promoted by histone acetylation, reverting chromatin to a condensed state associated with no transcription. HATs are an on switch for transcription, while HDACs are an off switch.

When HDACs remove acetyle groups from histones, what happens?

The first eukaryotic regulatory sequences were discovered in the late 1970s, when Yasuji Oshima and co-workers set out to understand how yeast cells control the metabolism of the sugar galactose. 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.

When galactose is present, what happens?

Methylated CpG sequences are recognized by proteins that trigger chromatin condensation. Actively transcribed genes usually have relatively few methylated CpG sequences near their promoters, while non-transcribed genes usually have many methylated CpG sequences.

Why is DNA methylation important?

This finding was remarkable for two reasons: (1) The regulatory sequence was thousands of bases away from the promoter, and (2) it was downstream of the promoter. Regulatory sequences that are far from the promoter and activate transcription are termed enhancers. Later work showed that enhancers occur in all eukaryotes.

Why were the findings significant?

In addition, mRNA stability is regulated in eukaryotes. Those mRNAs that remain in the cell for a long time tend to be translated more than mRNAs that have a shorter life span. In this chapter you'll explore all the control points shown in Figure 19.1. Let's begin with the series of events that occur as a cell responds to an external signal.

how is mRNA stability regulated?