How Transcription Is Regulated PDF

HOW TRANSCRIPTION IS REGULATED Until 60 years ago, the idea that genes could be switched on and off was revolutionary. This concept was a major advance, and it came originally from studies of how E. coli bacteria adapt to changes in the composition of their growth medium. Many of the same principles...

HOW TRANSCRIPTION IS REGULATED Until 60 years ago, the idea that genes could be switched on and off was revolutionary. This concept was a major advance, and it came originally from studies of how E. coli bacteria adapt to changes in the composition of their growth medium. Many of the same principles apply to eukaryotic cells. However, the enormous complexity of gene regulation in organisms that possess a nucleus, combined with the packaging of their DNA into chromatin, creates special challenges and some novel opportunities for control—as we will discuss. We begin with a look at transcription regulators (often loosely referred to as transcription factors), proteins that bind to specific DNA sequences and control gene transcription. Transcription Regulators Bind to Regulatory DNA Sequences Nearly all genes, whether bacterial or eukaryotic, contain DNA sequences that direct and control their transcription. In Chapter 7, we saw that the promoter region of a gene binds the enzyme RNA polymerase and correctly orients the enzyme to begin its task of making an RNA copy of the gene. The promoters of both bacterial and eukaryotic genes include a transcription initiation site, where RNA synthesis begins, plus nearby sequences that contain recognition sites for proteins that associate with RNA polymerase: sigma factor in bacteria (see Figure 7–9) or the general transcription factors in eukaryotes (see Figure 7–12). In addition to the promoter, the vast majority of genes include https://nerd.wwnorton.com/nerd/231302/r/goto/cfi/128!/4?control=control-toc&lti=true 10/12/23, 8:45 PM Page 1 of 18 regulatory DNA sequences that are used to switch the gene on or off, depending on the needs of the cell. Some regulatory DNA sequences are as short as 10 nucleotide pairs and act as simple switches that respond to a single signal; such simple regulatory switches predominate in bacteria. Other regulatory DNA sequences, especially those in eukaryotes, are very long (sometimes spanning more than 100,000 nucleotide pairs) and act as molecular microprocessors, integrating information from a variety of signals into a command that determines how often transcription of the gene is initiated. Regulatory DNA sequences do not work by themselves. To have any effect, these sequences must be recognized by proteins called transcription regulators. It is the binding of a transcription regulator to a regulatory DNA sequence that acts as the switch to control transcription. The simplest bacterium produces several hundred different transcription regulators, each of which recognizes a different DNA sequence and thereby regulates a distinct set of genes. Humans make many more—2000 or so— indicating the importance and complexity of this form of gene regulation in the development and function of a complex organism. Transcription regulators that recognize a specific nucleotide sequence do so because the surface of the protein fits tightly against the surface features of the DNA double helix in that region. Because these surface features will vary depending on the nucleotide sequence, different DNA-binding proteins will recognize different nucleotide sequences. In most cases, the protein inserts into the major groove of the DNA double helix and makes a series of intimate, noncovalent molecular contacts with the nucleotide pairs within the groove (Figure 8–4, Movie 8.1, and Movie 8.2). https://nerd.wwnorton.com/nerd/231302/r/goto/cfi/128!/4?control=control-toc&lti=true 10/12/23, 8:45 PM Page 2 of 18 Although each individual contact is weak, the 10–20 contacts that typically form at the protein–DNA interface combine to ensure that the interaction is both highly specific and very strong; indeed, protein–DNA interactions are among the tightest and most specific molecular interactions known in biology. Figure 8–4 A transcription regulator binds tightly to the DNA double helix. (A) The regulator shown recognizes a specific DNA sequence via three α helices, drawn as numbered cylinders, which allow the protein to fit into the major groove and form tight associations with the base pairs in a short stretch of DNA. This particular structural motif, called a homeodomain, is found in many eukaryotic DNA-binding proteins (see Movie 8.1). (B) Most of the contacts with the DNA bases are made by helix 3 (red), which is shown here end-on. (C) An asparagine residue from helix 3 forms two hydrogen bonds with the adenine in an A-T base pair. The view faces down the center of the DNA double helix, and the protein contacts the base pair from the major-groove side. The interactions between the protein and DNA take place along the edges of the nucleotide base and do not disrupt the hydrogen bonds that hold the base pairs together. For simplicity, only one amino acid–base contact is shown; in reality, transcription regulators form hydrogen bonds (as shown here), ionic bonds, and hydrophobic interactions with multiple bases. Most of these contacts occur in the major groove, but some proteins also interact with bases in the minor groove, as shown in (B). Typically, a protein–DNA interface consists of 10–20 such contacts, each involving a different amino acid and each contributing to the overall strength of the protein–DNA interaction. A typical transcription regulator will recognize about six to eight nucleotide pairs of DNA. However, many transcription regulators bind to the DNA helix as dimers. Such dimerization roughly doubles the area of contact with the DNA, thereby greatly increasing the https://nerd.wwnorton.com/nerd/231302/r/goto/cfi/128!/4?control=control-toc&lti=true 10/12/23, 8:45 PM Page 3 of 18 potential specificity and strength of the protein–DNA interaction (Figure 8–5 and Movie 8.3). Figure 8–5 Transcription regulators bind to specific DNA sequences. (A) Nanog, a homeodomain family member, is a key regulator in embryonic stem cells. Like all sequence-specific DNA-binding proteins, it recognizes a collection of closely related DNA sequences. In this diagram, called a “logo,” the height of each letter is proportional to the frequency with which this base is found in the collection of DNA sequences recognized by Nanog in the cell. In the first position, for example, T is found more often than C, while A is the only nucleotide found in the second and third positions of the sequence. Although the regulatory sequences in the cell are double-stranded, a logo typically shows the sequence of only one DNA strand; the other strand is simply the complementary sequence. Logos are useful because they reveal at a glance the range of DNA sequences recognized by a given transcription regulator—and the relative preference with which the regulator will bind to each. (B) Here, and throughout the book, regulatory sequences are represented by colored bars (in this example, light green); each bar represents a double-helical segment of DNA, like the ones shown in Figure 8–4A and B. (C) Many transcription regulators bind to DNA as dimers, rather than monomers. In this case, the regulatory sequences to which it binds are repeated, increasing the strength and specificity of the overall protein-DNA interaction. Transcription Switches Allow Cells to Respond to Changes in Their Environment The simplest and best-understood examples of gene regulation occur in bacteria. The genome of the bacterium E. coli consists of a single, circular DNA molecule of about 4.6 × 106 nucleotide pairs. This DNA encodes approximately 4300 proteins, although only a fraction of these are made at any one time. Bacteria regulate the https://nerd.wwnorton.com/nerd/231302/r/goto/cfi/128!/4?control=control-toc&lti=true 10/12/23, 8:45 PM Page 4 of 18 expression of many of their genes according to the food sources that are available in the environment. In E. coli, for example, five genes code for enzymes that manufacture tryptophan when this amino acid is scarce. These genes are arranged in a cluster on the chromosome and are transcribed from a single promoter as one long mRNA molecule; such coordinately transcribed clusters are called operons (Figure 8–6). Although operons are common in bacteria (see Figure 7–42), they are rare in eukaryotes, where genes are usually transcribed and regulated individually. Figure 8–6 A cluster of bacterial genes can be transcribed from a single promoter. Each of these five genes encodes a different enzyme; all of the enzymes are needed to synthesize the amino acid tryptophan from simpler molecular building blocks. The genes are transcribed as a single mRNA molecule, a feature that allows their expression to be coordinated. Such clusters of genes, called operons, are common in bacteria. In this case, the entire operon is controlled by a single regulatory DNA sequence, called the Trp operator (green), situated within the promoter. The yellow blocks in the promoter represent DNA sequences that bind RNA polymerase (see Figure 7–10A). When tryptophan concentrations are low, the operon is transcribed; the resulting mRNA is translated to produce a full set of biosynthetic enzymes, which work in tandem to synthesize the amino acid from simpler precursor molecules. When tryptophan is abundant, however—for example, when the bacterium is in the gut https://nerd.wwnorton.com/nerd/231302/r/goto/cfi/128!/4?control=control-toc&lti=true 10/12/23, 8:45 PM Page 5 of 18 of a mammal that has just eaten a protein-rich meal—the amino acid is imported into the cell and shuts down production of the biosynthetic enzymes, which are no longer needed. We understand in considerable detail how this repression of the tryptophan operon comes about. Within the operon’s promoter is a short DNA sequence, called the operator (see Figure 8–6), which is recognized by a transcription regulator. When this regulator binds to the operator, it blocks access of RNA polymerase to the promoter, thus preventing transcription of the operon and, ultimately, the production of the tryptophan-synthesizing enzymes. The transcription regulator is known as the tryptophan repressor, and it is controlled in an ingenious way: the repressor can bind to DNA only if it is also bound to tryptophan (Figure 8–7). Figure 8–7 Genes can be switched off by repressor proteins. If the concentration of tryptophan inside a bacterium is low (left), RNA polymerase (blue) binds to the promoter and transcribes the five genes of the tryptophan operon. However, if the concentration of tryptophan is high (right), the repressor protein (dark green) becomes active and binds to the operator (light green), where it blocks the binding of RNA polymerase to the promoter. When the concentration of intracellular tryptophan drops, the repressor falls off the DNA, allowing the polymerase to again transcribe the operon. The promoter contains two key blocks of DNA sequence information, the –35 and –10 https://nerd.wwnorton.com/nerd/231302/r/goto/cfi/128!/4?control=control-toc&lti=true 10/12/23, 8:45 PM Page 6 of 18 regions, highlighted in yellow, which are recognized by RNA polymerase (see Figure 7–10). The complete operon is shown in Figure 8–6. The tryptophan repressor is an allosteric protein (see Figure 4–42): the binding of tryptophan causes a subtle change in its threedimensional structure so that the protein can now bind tightly to the operator sequence. When the concentration of free tryptophan in the bacterium drops, the repressor loses its tryptophan and falls off the DNA, allowing the tryptophan operon to be transcribed. The repressor is thus a simple device that switches production of a set of biosynthetic enzymes on and off according to the availability of tryptophan—a form of feedback inhibition (see Figure 4–40). The tryptophan repressor protein itself is always present in the cell. The gene that encodes it is continuously transcribed at a low level, so that a small amount of the repressor protein is always being made. Thus the bacterium can respond nimbly to changes in tryptophan concentration. Repressors Turn Genes Off and Activators Turn Them On The tryptophan repressor, as its name suggests, is a transcriptional repressor protein: in its active form, it switches genes off, or represses them. Some bacterial transcription regulators do the opposite: they switch genes on, or activate them. These transcriptional activator proteins work on promoters that—in contrast to the promoter for the tryptophan operon—are only marginally able to bind and position RNA polymerase on their own. These inefficient promoters can be made fully functional by activator proteins that bind to a nearby regulatory sequence and make contact with the RNA polymerase, helping it to initiate https://nerd.wwnorton.com/nerd/231302/r/goto/cfi/128!/4?control=control-toc&lti=true 10/12/23, 8:45 PM Page 7 of 18 transcription (Figure 8–8). Figure 8–8 Genes can be switched on by activator proteins. An activator protein binds to a regulatory sequence on the DNA and then interacts with the RNA polymerase to help it initiate transcription. Without the activator, the promoter fails to initiate transcription efficiently. In bacteria, the binding of the activator to DNA is often controlled by the interaction of a metabolite or other small molecule (red circle) with the activator protein. (Dynamic Figure) Interaction with a small molecule (red) allows an activator protein (dark blue) to bind to DNA; there it attracts an RNA polymerase (light blue) that can then transcribe the gene into mRNA. https://nerd.wwnorton.com/nerd/231302/r/goto/cfi/128!/4?control=control-toc&lti=true 10/12/23, 8:45 PM Page 8 of 18 Like the tryptophan repressor, activator proteins often have to interact with a second molecule to be able to bind DNA. For example, the bacterial catabolite activator protein (CAP) has to bind cyclic AMP (cAMP) before it can bind to DNA (see Figure 4– 20). Genes activated by CAP are switched on in response to an increase in intracellular cAMP concentration, which occurs when glucose, the bacterium’s preferred carbon source, is no longer available; as a result, CAP drives the production of enzymes that https://nerd.wwnorton.com/nerd/231302/r/goto/cfi/128!/4?control=control-toc&lti=true 10/12/23, 8:45 PM Page 9 of 18 allow the bacterium to digest other sugars. The Lac Operon Is Controlled by an Activator and a Repressor In many instances, the activity of a QUESTION 8–1 single promoter is controlled by two Bacterial cells can take up the different transcription regulators. The amino acid tryptophan (Trp) from their surroundings or, if there is an Lac operon in E. coli, for example, is insufficient external supply, they can synthesize tryptophan from controlled by both the Lac repressor other small molecules. The Trp and the CAP activator that we just repressor is a transcription discussed. The Lac operon encodes regulator that shuts off the proteins required to import and digest transcription of genes that code for the enzymes required for the the disaccharide lactose. In the absence synthesis of tryptophan (see of glucose, the bacterium makes cAMP, Figure 8–7). which activates CAP to switch on genes 1. What would happen to the regulation of the tryptophan that allow the cell to utilize alternative operon in cells that express a mutant form of the sources of carbon—including lactose. It tryptophan repressor that would be wasteful, however, for CAP to (1) cannot bind to DNA, (2) induce expression of the Lac operon if cannot bind tryptophan, or (3) binds to DNA even in the lactose itself were not present. Thus the absence of tryptophan? Lac repressor shuts off the operon in 2. What would happen in the absence of lactose. This scenarios (1), (2), and (3) if the cells, in addition, arrangement enables the control region produced normal tryptophan of the Lac operon to integrate two repressor protein from a different signals, so that the operon is second, normal gene? highly expressed only when two conditions are met: glucose must be absent and lactose must be present (Figure 8–9). This circuit thus behaves much like a switch that carries out a logic operation in a computer. When lactose is present AND glucose is absent, the cell executes the appropriate https://nerd.wwnorton.com/nerd/231302/r/goto/cfi/128!/4?control=control-toc&lti=true 10/12/23, 8:45 PM Page 10 of 18 program—in this case, transcription of the genes that permit the uptake and utilization of lactose. None of the other combinations of conditions produce this result. Figure 8–9 The Lac operon is controlled by two transcription regulators, the Lac repressor and CAP. When lactose is absent, the Lac repressor binds to the Lac operator and shuts off expression of the operon. Addition of lactose increases the intracellular concentration of a related compound, allolactose; allolactose binds to the Lac repressor, causing it to undergo a conformational change that releases its grip on the operator DNA (not shown). When glucose is absent, cyclic AMP (red circle) is produced by the cell, and CAP binds to DNA. For the operon to be transcribed, glucose must be absent (allowing the CAP activator to bind) and lactose must be present (releasing the Lac repressor). LacZ, the first gene of the operon, encodes the enzyme β-galactosidase, which breaks https://nerd.wwnorton.com/nerd/231302/r/goto/cfi/128!/4?control=control-toc&lti=true 10/12/23, 8:45 PM Page 11 of 18 down lactose into galactose and glucose (Movie 8.4). The elegant logic of the Lac operon first attracted the attention of biologists more than 60 years ago. The molecular basis of the switch in E. coli was uncovered by a combination of genetics and biochemistry, providing the first insight into how transcription is controlled. In a eukaryotic cell, similar transcription regulatory devices are combined to generate increasingly complex circuits— including those that enable a fertilized egg to form the tissues and organs of a multicellular organism, as we discuss later in the chapter. Eukaryotic Transcription Regulators Control Gene Expression from a Distance Eukaryotes, too, use transcription QUESTION 8–2 regulators—both activators and Explain how DNA-binding proteins repressors—to regulate the expression can make sequence-specific contacts to a double-stranded of their genes. The DNA sites to which DNA molecule without breaking the hydrogen bonds that hold the eukaryotic gene activators bind are bases together. Indicate how, often called enhancers, because their through such contacts, a protein presence dramatically enhances the can distinguish a T-A from a C-G pair. Indicate the parts of the rate of transcription. However, unlike nucleotide base pairs that could their bacterial counterparts, eukaryotic form noncovalent interactions— hydrogen bonds, electrostatic activator proteins can enhance attractions, or hydrophobic transcription even when they are bound interactions (see Panel 2–3, pp. thousands of nucleotide pairs upstream 74–75)—with a DNA-binding —or even downstream—of the gene’s protein. The structures of all the base pairs in DNA are given in promoter. These observations raised Figure 5–4. several questions. How do enhancer sequences and the proteins bound to them function over such long https://nerd.wwnorton.com/nerd/231302/r/goto/cfi/128!/4?control=control-toc&lti=true 10/12/23, 8:45 PM Page 12 of 18 distances? How do they communicate with a gene’s promoter? Many models for this “action at a distance” have been proposed, but the simplest of these seems to apply in most cases. The DNA between the enhancer and the promoter loops out, bringing the activator protein into close proximity with the promoter (Figure 8– 10). The DNA thus acts as a tether, allowing a protein that is bound to an enhancer—even one that is thousands of nucleotide pairs away—to interact with the proteins in the vicinity of the promoter (see Figure 7–12). Often, additional proteins serve as adaptors to close the loop; the most important of these is a large complex of proteins known as Mediator. Together, all of these proteins ultimately attract and position the general transcription factors and RNA polymerase at the promoter, forming a transcription initiation complex (see Figure 8–10). Eukaryotic repressor proteins do the opposite: they decrease transcription by blocking the assembly of this complex—or by keeping the formed complex locked in place, preventing RNA polymerase from moving forward. https://nerd.wwnorton.com/nerd/231302/r/goto/cfi/128!/4?control=control-toc&lti=true 10/12/23, 8:45 PM Page 13 of 18 Figure 8–10 In eukaryotes, gene activation can occur at a distance. An activator protein bound to a distant enhancer attracts RNA polymerase and the general transcription factors to the promoter. Looping of the intervening DNA permits contact between the activator and the transcription initiation complex bound to the promoter. In the case shown here, a large protein complex called Mediator serves as a go-between. The broken stretch of DNA signifies that the segment of DNA between the enhancer and the start of transcription varies in length, sometimes reaching tens of thousands of nucleotide pairs. The TATA box is a DNA recognition sequence for the first general transcription factor that binds to the promoter (see Figure 7–12). Although many eukaryotic activator proteins bind to DNA as dimers, some bind DNA as monomers, as shown. Eukaryotic Transcription Regulators Help Initiate Transcription by Recruiting Chromatin-modifying Proteins In a eukaryotic cell, the proteins that guide the formation of the https://nerd.wwnorton.com/nerd/231302/r/goto/cfi/128!/4?control=control-toc&lti=true 10/12/23, 8:45 PM Page 14 of 18 transcription initiation complex must also deal with the problem of DNA packaging. As discussed in Chapter 5, eukaryotic DNA is wound around clusters of histone proteins to form nucleosomes, which, in turn, are folded into higher-order structures. How do transcription regulators, general transcription factors, and RNA polymerase gain access to the underlying DNA? Although some of these proteins can bind efficiently to DNA that is wrapped up in nucleosomes, others are thwarted by these compact structures. More critically, nucleosomes that are positioned over a promoter can inhibit the initiation of transcription by physically blocking the assembly of the general transcription factors and RNA polymerase on the promoter. Such packaging may have evolved in part to prevent leaky gene expression by blocking the initiation of transcription in the absence of the proper activator proteins. In eukaryotic cells, activator and QUESTION 8–3 repressor proteins can exploit the Some transcription regulators mechanisms used to package DNA to bind to DNA and cause the double helix to bend at a sharp angle. help turn genes on and off. As we saw in Such “bending proteins” can Chapter 5, chromatin structure can be stimulate the initiation of transcription without contacting altered by ATP-dependent chromatin- either the RNA polymerase, any of remodeling complexes and by enzymes the general transcription factors, or any other transcription that covalently modify the histone regulators. Can you devise a proteins that form the core of the plausible explanation for how nucleosome (see Figures 5–27 and 5– these proteins might work to modulate transcription? Draw a 28). Many gene activators take diagram that illustrates your advantage of these mechanisms by explanation. attracting such chromatin-modifying proteins to promoters. For example, the recruitment of histone acetyltransferases promotes the attachment of acetyl groups to https://nerd.wwnorton.com/nerd/231302/r/goto/cfi/128!/4?control=control-toc&lti=true 10/12/23, 8:45 PM Page 15 of 18 selected lysines in the tail of histone proteins; these acetyl groups themselves attract proteins that promote transcription, including some of the general transcription factors (Figure 8–11). And the recruitment of ATP-dependent chromatin-remodeling complexes makes nearby DNA more accessible. These actions enhance the efficiency of transcription initiation. Figure 8–11 Eukaryotic transcriptional activators can recruit chromatin-modifying proteins to help initiate gene transcription. On the left, the recruitment of histone-modifying enzymes such as histone acetyltransferases adds acetyl groups to specific histones, which can then serve as binding sites for proteins that stimulate transcription initiation (not shown). On the right, ATPdependent chromatin-remodeling complexes render the DNA packaged in nucleosomes more accessible to other proteins in the cell, including those required for transcription initiation; notice, for https://nerd.wwnorton.com/nerd/231302/r/goto/cfi/128!/4?control=control-toc&lti=true 10/12/23, 8:45 PM Page 16 of 18 example, the increased exposure of the TATA box. In a similar way, gene repressor proteins can modify chromatin in ways that reduce the efficiency of transcription initiation. For example, many repressors attract histone deacetylases—enzymes that remove the acetyl groups from histone tails, thereby reversing the positive effects that acetylation has on transcription initiation. Although some eukaryotic repressor proteins work on a gene-bygene basis, others can orchestrate the formation of large swathes of transcriptionally inactive chromatin. As discussed in Chapter 5, these transcription-resistant regions of DNA include the heterochromatin found in interphase chromosomes and the inactive X chromosome in the cells of female mammals. The Arrangement of Chromosomes into Looped Domains Keeps Enhancers in Check We have seen that all genes have regulatory regions, which dictate at which times, under what conditions, and in what tissues the gene will be expressed. We have also seen that eukaryotic transcription regulators can act across very long stretches of DNA, with the intervening DNA looped out. What, then, prevents a transcription regulator—bound to the control region of one gene— from looping in the wrong direction and inappropriately influencing the transcription of a neighboring gene? To avoid such unwanted cross-talk, the chromosomal DNA of plants and animals is arranged in a series of loops that hold individual genes and their regulatory regions in rough proximity. This localization restricts the action of enhancers, preventing them https://nerd.wwnorton.com/nerd/231302/r/goto/cfi/128!/4?control=control-toc&lti=true 10/12/23, 8:45 PM Page 17 of 18 from straying toward the wrong genes. The chromosomal loops are formed by specialized proteins that bind to sequences that are then drawn together to form the base of the loop (Figure 8–12). Figure 8–12 Animal and plant chromosomes are arranged in DNA loops. In this schematic diagram, specialized proteins (dark purple) hold chromosomal DNA in loops, thereby favoring the association of each gene with its proper enhancer. The loops, sometimes called topological associated domains (TADs), range in size between thousands and millions of nucleotide pairs and are typically much larger than the loops that form between regulatory sequences and promoters (see Figure 8–10). The importance of these loops is highlighted by the effects of mutations that prevent the loops from properly forming. Such mutations, which lead to genes being expressed at the wrong time and place, are found in numerous cancers and inherited diseases. https://nerd.wwnorton.com/nerd/231302/r/goto/cfi/128!/4?control=control-toc&lti=true 10/12/23, 8:45 PM Page 18 of 18