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Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002.

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Biochemistry. 5th edition.

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Section 31.3Transcriptional Activation and Repression Are Mediated by Protein-Protein Interactions

We have seen how interactions between DNA-binding proteins such as CAP and RNA polymerase can activate transcription in prokaryotic cells (Section 31.1.6). Such protein-protein interactions play a dominant role in eukaryotic gene regulation. In contrast with those of prokaryotic transcription, few eukaryotic transcription factors have any effect on transcription on their own. Instead, each factor recruits other proteins to build up large complexes that interact with the transcriptional machinery to activate or repress transcription.

A major advantage of this mode of regulation is that a given regulatory protein can have different effects, depending on what other proteins are present in the same cell. This phenomenon, called combinatorial control, is crucial to multicellular organisms that have many different cell types. Even in unicellular eukaryotes such as yeast, combinatorial control allows the generation of distinct cell types.

31.3.1. Steroids and Related Hydrophobic Molecules Pass Through Membranes and Bind to DNA-Binding Receptors

Just as prokaryotes can adjust their patterns of gene expression in response to chemicals in their environment, eukaryotes have many systems for responding to specific molecules with which they come in contact. We first consider a system that detects and responds to estrogens. Synthesized and released by the ovaries, estrogens, such as estrone, are cholesterol-derived, steroid hormones (Section 26.4). They are required for the development of female secondary sex characteristics and, along with progesterone, participate in the ovarian cycle.

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Because they are hydrophobic molecules, estrogens easily diffuse across cell membranes. When inside a cell, estrogens bind to highly specific, soluble receptor proteins. Estrogen receptors are members of a large family of proteins that act as receptors for a wide range of hydrophobic molecules, including other steroid hormones, thyroid hormones, and retinoids.

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These receptors all have a similar mode of action. On binding of the signal molecule (called, generically, a ligand), the ligand-receptor complex modifies the expression of specific genes by binding to control elements in the DNA. The human genome encodes approximately 50 members of this family, often referred to as nuclear hormone receptors. The genomes of other multicellular eukaryotes encode similar numbers of nuclear hormone receptors, although they are absent in yeast. A comparison of the amino acid sequences of members of this family reveals two highly conserved domains: a DNA-binding domain and a ligand-binding domain (Figure 31.22). The DNA-binding domain lies toward the center of the molecule and includes nine conserved cysteine residues. This domain provides these receptors with sequence-specific DNA activity. Eight of the cysteine residues are conserved because of their role in binding zinc ions: the first four cysteine residues bind one zinc ion, and the second four bind a second zinc ion (see Figure 31.22). The zinc ions stabilize the structure of this small domain; without the bound zinc ions, the domains unfold. Such domains are often referred to as zinc finger domains.

Figure 31.22. Structure of Two Nuclear Hormone Receptor Domains.

Figure 31.22

Structure of Two Nuclear Hormone Receptor Domains. Image mouse.jpg Nuclear hormone receptors contain two crucial conserved domains: (1) a DNA-binding domain toward the center of the sequence and (2) a ligand-binding domain toward the carboxyl terminus. The structure (more...)

The structure of the zinc-binding region of a steroid receptor includes an α helix that begins at the end of the first zinc finger domain. This helix lies in the major groove in the specific DNA complexes formed by estrogen receptors and binds with specific DNA sequences, analogously to prokaryotic DNA-binding proteins. Estrogen receptors bind to specific DNA sites (referred to as estrogen response elements or EREs) that contain the consensus sequence 5′-AGGTCANNNTGACCT-3′. As expected from the symmetry of this sequence, an estrogen receptor binds to such sites as a dimer.

The second highly conserved region of the nuclear receptor proteins lies near the carboxyl terminus and is the ligand-binding site. This domain folds into a structure that consists almost entirely of α helices, arranged in three layers. The ligand binds in a hydrophobic pocket that lies in the center of this array of helices (Figure 31.23). The ligand-binding domain also participates in receptor dimerization.

Figure 31.23. Ligand Binding to Nuclear Hormone Receptor.

Figure 31.23

Ligand Binding to Nuclear Hormone Receptor. Image mouse.jpg The ligand lies completely surrounded within a pocket in the ligand-binding domain. The last α helix, helix 12 (shown in purple), folds into a groove on the side of the structure on ligand binding. (more...)

A comparison of the structures of the ligand-binding domains with and without bound ligand reveals that ligand binding leads to substantial structural rearrangement. In particular, the last α helix (referred to as helix 12), which has hydrophobic residues lining one face but extends out from the receptor in the ligand-free form, folds into a shallow groove on the side of the receptor on ligand binding (see Figure 31.23). How does ligand binding lead to changes in gene expression? The simplest model would have the binding of ligand alter the DNA-binding properties of the receptor, analogously to the lac repressor in prokaryotes. However, the results of experiments with purified nuclear hormone receptors revealed that ligand binding does not significantly alter DNA-binding affinity and specificity. Another mechanism must be operative.

31.3.2. Nuclear Hormone Receptors Regulate Transcription by Recruiting Coactivators and Corepressors to the Transcription Complex

Because ligand binding does not alter the ability of nuclear hormone receptors to bind DNA, investigators sought to determine whether specific proteins might bind to the nuclear hormone receptors only in the presence of ligand. Such searches led to the identification of several related proteins called coactivators, such as SRC-1 (steroid receptor coactivator-1), GRIP-1 (glucocorticoid receptor interacting protein-1), and NcoA-1 (nuclear hormone receptor coactivator-1). These coactivators, referred to as the p160 family because of their size, have a common modular structure (Figure 31.24). Each coactivator protein contains three sequences of the form Leu-X-X-Leu-Leu within a central region of 200 amino acids. These sequences form short α helices that bind to a hydrophobic patch on the surface of the ligand-binding domains of a nuclear hormone receptor (Figure 31.25). The binding site for the coactivator is fully formed only when ligand is bound, inasmuch as it is adjacent to helix 12. It is likely that a coactivator molecule binds to the ligand-binding domains of a receptor dimer through two of its three Leu-X-X-Leu-Leu sequences. Thus, the binding of ligand to the receptor induces a conformational change that allows the recruitment of a coactivator (Figure 31.26).

Figure 31.24. Coactivator Structure.

Figure 31.24

Coactivator Structure. Image mouse.jpg The p160 family of coactivators includes a number of domains that can be recognized at the amino acid sequence level, including a basic helix-loop-helix domain that takes part in DNA binding, a PAS domain that participates in protein-protein (more...)

Figure 31.25. Coactivator-Nuclear Hormone Receptor Interactions.

Figure 31.25

Coactivator-Nuclear Hormone Receptor Interactions. Image mouse.jpg The structure of a complex between the ligand-binding domain of the estrogen receptor with estradiol bound and a peptide from a coactivator reveals that the Leu-X-X-Leu-Leu (LXXLL) sequence forms a helix (more...)

Figure 31.26. Coactivator Recruitment.

Figure 31.26

Coactivator Recruitment. The binding of ligand to a nuclear hormone receptor induces a conformational change in the ligand-binding domain. This change in conformation generates favorable sites for the binding of a coactivator.

Some members of the nuclear hormone receptor family, such as the receptors for thyroid hormone and retinoic acid, repress transcription in the absence of ligand. This repression also is mediated by the ligand-binding domain. In their unbound forms, the ligand-binding domains of these receptors bind to corepressor proteins. Members of this family of proteins include SMRT (Silencing mediator for retinoid and thyroid hormone receptors) and N-Cor (nuclear hormone receptor corepressor). Such a corepressor binds to a site in the ligand-binding domain that overlaps the coactivator binding site; ligand binding triggers the release of the corepressor and frees the ligand-binding domain for binding to a coactivator.

31.3.3. Steroid-Hormone Receptors Are Targets for Drugs

Image caduceus.jpg Molecules such as estradiol that bind to a receptor and trigger signaling pathways are called agonists. Athletes sometimes take natural and synthetic agonists of the androgen receptor, a member of the nuclear hormone receptor family, because their binding to the androgen receptor stimulates the expression of genes that enhance the development of lean muscle mass.

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Referred to as anabolic steroids, such compounds used in excess are not without side effects. In men, excessive use leads to a decrease in the secretion of testosterone, to testicular atrophy, and sometimes to breast enlargement (gynecomastia) if some of the excess androgen is converted into estrogen. In women, excess testosterone causes a decrease in ovulation and estrogen secretion; it also causes breast regression and growth of facial hair.

Other molecules bind to nuclear hormone receptors but do not effectively trigger signaling pathways. Such compounds are called antagonists and are, in many ways, like competitive inhibitors of enzymes. Some important drugs are antagonists that target the estrogen receptor. For example, tamoxifen and raloxifene are used in the treatment and prevention of breast cancer, because some breast tumors rely on estrogen-mediated pathways for growth. These compounds are sometimes called selective estrogen receptor modulators (SERMs).

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The determination of the structures of complexes between the estrogen receptor and these drugs revealed the basis for their antagonist effect (Figure 31.27). Tamoxifen binds to the same site as estradiol. However, tamoxifen (and other antagonists) have groups that extend out of the normal ligand-binding pocket. These groups prevent helix 12 from binding in its usual position; instead, this helix binds to the site normally occupied by the coactivator. Tamoxifen blocks the binding of coactivators and thus inhibits the activation of gene expression.

Figure 31.27. Estrogen Receptor-Tamoxifen Complex.

Figure 31.27

Estrogen Receptor-Tamoxifen Complex. Image mouse.jpg Tamoxifen binds in the pocket normally occupied by estrogen. However, part of the tamoxifen structure extends from this pocket, and so helix 12 cannot pack in its usual position. Instead, this helix blocks the coactivator-binding (more...)

31.3.4. Chromatin Structure Is Modulated Through Covalent Modifications of Histone Tails

We have seen that nuclear receptors respond to signal molecules by recruiting coactivators and corepressors to the chromatin. Now we can ask, How do coactivators and corepressors modulate transcriptional activity? Much of their effectiveness appears to result from their ability to covalently modify the amino-terminal tails of histones and perhaps other proteins. Some of the p160 coactivators and, in addition, the proteins that they recruit catalyze the transfer of acetyl groups from acetyl CoA to specific lysine residues in the amino-terminal tails of histones.

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Enzymes that catalyze such reactions are called histone acetyltransferases (HATs). The histone tails are readily extended; so they can fit into the HAT active site and become acetylated (Figure 31.28).

Figure 31.28. Structure of Histone Acetyltransferase.

Figure 31.28

Structure of Histone Acetyltransferase. Image mouse.jpg The amino-terminal tail of histone H3 extends into a pocket in which a lysine side chain can accept an acetyl group from acetyl CoA bound in an adjacent site.

What are the consequences of histone acetylation? Lysine bears a positively charged ammonium group at neutral pH. The addition of an acetyl group generates an uncharged amide group. This change dramatically reduces the affinity of the tail for DNA and modestly decreases the affinity of the entire histone complex for DNA. The loosening of the histone complex from the DNA exposes additional DNA regions to the transcription machinery. In addition, the acetylated lysine residues interact with a specific acetyllysine-binding domain that is present in many proteins that regulate eukaryotic transcription. This domain, termed a bromodomain, comprises approximately 110 amino acids that form a four-helix bundle containing a peptide-binding site at one end (Figure 31.29).

Figure 31.29. Structure of a Bromodomain.

Figure 31.29

Structure of a Bromodomain. Image mouse.jpg This four-helix-bundle domain binds peptides containing acetyllysine. An acetylated peptide of histone H4 is bound in the structure shown.

Bromodomain-containing proteins are components of two large complexes essential for transcription. One is a complex of more than 10 polypeptides that binds to the TATA-box-binding protein. Recall that the TATA-box-binding protein is an essential transcription factor for many genes (Section 28.2.4). Proteins that bind to the TATA-box-binding protein are called TAFs (for TATA-box-binding protein associated factors). In particular, TAFII250 (named for its participation in RNA polymerase II transcription and its apparent molecular weight of 250 kd) contains a pair of bromodomains near its carboxyl terminus. The two domains are oriented so that each can bind one of two acetyllysine residues at positions 5 and 12 in the histone H4 tail. Thus, acetylation of the histone tails provides a mechanism for recruiting other components of the transcriptional machinery.

Bromodomains are also present in some components of large complexes known as chromatin-remodeling engines. These complexes, which also contain domains homologous to those of helicases (Section 27.2.5), utilize the free energy of ATP hydrolysis to shift the positions of nucleosomes along the DNA and to induce other conformational changes in chromatin (Figure 31.30). Histone acetylation can lead to reorganization of the chromatin structure, potentially exposing binding sites for other factors. Thus, histone acetylation can activate transcription through a combination of three mechanisms: by reducing the affinity of the histones for DNA, by recruiting other components of the transcriptional machinery, and by initiating the active remodeling of the chromatin structure.

Figure 31.30. Chromatin Remodeling.

Figure 31.30

Chromatin Remodeling. Eukaryotic gene regulation begins with an activated transcription factor bound to a specific site on DNA. One scheme for the initiation of transcription by RNA polymerase II requires five steps: (1) recruitment of a coactivator, (more...)

31.3.5. Histone Deacetylases Contribute to Transcriptional Repression

Just as in prokaryotes, some changes in a cell's environment lead to the repression of genes that had been active. The modification of histone tails again plays an important role. However, in repression, a key reaction appears to be the deacetylation of acetylated lysine, catalyzed by specific histone deacetylase enzymes.

In many ways, the acetylation and deacetylation of lysine residues in histone tails (and, likely, in other proteins) is analogous to the phosphorylation and dephosphorylation of serine, threonine, and tyrosine residues in other stages of signaling processes. Like the addition of phosphoryl groups, the addition of acetyl groups can induce conformational changes and generate novel binding sites. Without a means of removing these groups, however, these signaling switches will become stuck in one position and lose their effectiveness. Like phosphatases, deacetylases help reset the switches.

31.3.6. Ligand Binding to Membrane Receptors Can Regulate Transcription Through Phosphorylation Cascades

In Chapter 15, we examined several signaling pathways that begin with the binding of molecules to receptors in the cell membrane. Some of these pathways lead to the regulation of gene expression. Let us review the pathway initiated by epinephrine. The binding of epinephrine to a 7TM receptor results in the activation of a G protein. The activated G protein, in turn, binds to and activates adenylate cyclase, increasing the intracellular concentration of cAMP. This cAMP binds to the regulatory subunit of protein kinase A (PKA), activating the enzyme. We have previously examined the role of phosphorylation by PKA of a variety of enzymes—for example, those controlling glycogen metabolism. PKA also phosphorylates the cyclic AMP-response element binding protein (CREB), a transcription factor that binds specific DNA sequences as a dimer. Each monomer contributes a long α helix; together, the two helices grab the DNA in the manner of a pair of chopsticks (Figure 31.31).

Figure 31.31. Cyclic AMP-Response Element Binding Protein (CREB).

Figure 31.31

Cyclic AMP-Response Element Binding Protein (CREB). Image mouse.jpg Each of two CREB subunits contributes a long α helix. The two helices coil around each other to form a dimeric DNA-binding unit. CREB is phosphorylated on a specific serine residue by protein (more...)

How does the phosphorylation of CREB affect its ability to activate transcription? Phosphorylation does not appear to alter the DNA-binding properties of this protein. Instead, phosphorylated CREB binds a coactivator protein termed CBP, for CREB-binding protein. CBP possesses a highly revealing domain structure (Figure 31.32).

Figure 31.32. Domain Structure of CREB-Binding Protein (CBP).

Figure 31.32

Domain Structure of CREB-Binding Protein (CBP). Image mouse.jpg The CREB-binding protein includes at least three types of protein-protein interaction domains in addition to a histone acetyltransferase domain that lies near the carboxyl terminus. The kinase-inducible (more...)

Its domains include a KIX domain (for kinase-inducible interaction) that binds to the phosphorylated region of CREB (Figure 31.33); a bromodomain that binds acetylated histone tails, and two TAZ domains, zinc-binding domains that facilitate the binding of CBP to a variety of proteins through a remarkable triangular structure (see Figure 31.32). Thus, the pathway initiated by epinephrine binding induces the phosphorylation of a transcription factor, the recruitment of a coactivator, and the assembly of complexes that participate in chromatin remodeling and transcription initiation.

Figure 31.33. Interaction between CBP and CREB.

Figure 31.33

Interaction between CBP and CREB. Image mouse.jpg The KIX domain of CBP binds a region of CREB in its phosphorylated form.

31.3.7. Chromatin Structure Effectively Decreases the Size of the Genome

The transcriptional regulatory mechanisms utilized by prokaryotes and eukaryotes have some significant differences, many of which are related to the significant difference in genome sizes between these classes of organisms. However, much of the DNA in a eukaryotic cell is stably assembled into chromatin. The packaging of DNA with chromatin renders many potential binding sites for transcription factors inaccessible—in effect, reducing the size of the genome. Thus, rather than scanning through the entire genome, a eukaryotic DNA-binding protein scans a set of accessible binding sites that is close in size to the genome of a prokaryote. The cell type is determined by the genes that are accessible.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2002, W. H. Freeman and Company.
Bookshelf ID: NBK22496