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Cell_Biology_Alberts. Quality Control Mechanisms Act to Prevent Translation of Damaged mRNAs In eukaryotes, mRNA production involves both transcription and a series of elaborate RNA processing steps; as we have seen, these take place in the nucleus, segregated from ribosomes, and only when the processing is complete are the mRNAs transported to the cytosol to be translated (see Figure 6–38). However, this scheme is not foolproof, and some incorrectly processed mRNAs are inadvertently sent to the cytosol. In addition, mRNAs that were flawless when they left the nucleus can become broken or otherwise damaged in the cytosol. The danger of


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Cell_Biology_Alberts. Figure 6–75 binding sites for antibiotics on the bacterial ribosome. The small (left) and large (right) subunits of the ribosome are arranged as though the ribosome has been opened like a book. Antibiotic binding sites are marked with colored spheres, and the bound tRNA molecules are shown in purple (see Figure 6–62). Most of the antibiotics shown bind directly to pockets formed by the ribosomal RNA molecules. Hygromycin B induces errors in translation, spectinomycin blocks the translocation of the peptidyl-tRNA from the A site to the P site, and streptogramin B prevents elongation of nascent peptides. Table 6–4 lists the inhibitory mechanisms of the other antibiotics shown in the figure. (Adapted from J. Poehlsgaard and S. Douthwaite, Nat. Rev. Microbiol. 3:870–881, 2005. With permission from Macmillan Publishers Ltd.) translating damaged or incompletely processed mRNAs (which would produce truncated or otherwise aberrant proteins) is apparently so great that the cell has several


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Cell_Biology_Alberts. from Macmillan Publishers Ltd.) translating damaged or incompletely processed mRNAs (which would produce truncated or otherwise aberrant proteins) is apparently so great that the cell has several backup measures to prevent this from happening. To avoid translating broken mRNAs, for example, the 5ʹ cap and the poly-A tail are both recognized by the translation-initiation machinery before translation begins (see Figure 6–70).


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Cell_Biology_Alberts. The most powerful mRNA surveillance system, called nonsense-mediated mRNA decay, eliminates defective mRNAs before they move away from the nucleus. This mechanism is brought into play when the cell determines that an mRNA molecule has a nonsense (stop) codon (UAA, UAG, or UGA) in the “wrong” place. This situation is likely to arise in an mRNA molecule that has been improperly spliced, because aberrant splicing will usually result in the random introduction of a nonsense codon into the reading frame of the mRNA—especially in organisms, such as humans, that have a large average intron size (see Figure 6–31B).


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Cell_Biology_Alberts. The nonsense-mediated mRNA decay mechanism begins as an mRNA molecule is being transported from the nucleus to the cytosol. As its 5ʹ end emerges from a nuclear pore, the mRNA is met by a ribosome, which begins to translate it. As translation proceeds, the exon junction complexes (EJCs) that are bound to the mRNA at each splice site are displaced by the moving ribosome. The normal stop codon will lie within the last exon, so by the time the ribosome reaches it and stalls, no more EJCs will be bound to the mRNA. In this case, the mRNA “passes inspection” and is released to the cytosol where it can be translated in earnest (Figure 6–76). However, if the ribosome reaches a stop codon earlier, when EJCs remain bound, the mRNA molecule is rapidly degraded. In this way, the first round of translation allows the cell to test the fitness of each mRNA molecule as it exits the nucleus.


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Cell_Biology_Alberts. Nonsense-mediated decay may have been especially important in evolution, allowing eukaryotic cells to more easily explore new genes formed by DNA rearrangements, mutations, or alternative patterns of splicing—by selecting only those mRNAs for translation that can produce a full-length protein. Nonsense-mediated decay is also important in cells of the developing immune system, where the extensive DNA rearrangements that occur (see Figure 24–28) often generate mRNA SURVIVES, EFFICIENT TRANSLATION Upf TRIGGERS mRNA DEGRADATION


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Cell_Biology_Alberts. Figure 6–76 Nonsense-mediated mRNA decay. As shown on the right, the failure to correctly splice a pre-mRNA often introduces a premature stop codon into the reading frame for the protein. These abnormal mRNAs are destroyed by the nonsense-mediated decay mechanism. To activate this mechanism, an mRNA molecule, bearing exon junction complexes (EJCs) to mark successfully completed splices, is first met by a ribosome that performs a “test” round of translation. As the mRNA passes through the tight channel of the ribosome, the EJCs are stripped off, and successful mRNAs are released to undergo multiple rounds of translation (left side). However, if an in-frame stop codon is encountered before the final EJC is reached (right side), the mRNA undergoes nonsense-mediated decay, which is triggered by the Upf proteins (green) that bind to each EJC. Note that this mechanism ensures that nonsense-mediated decay is triggered only when the premature stop codon is in the same reading frame as that of


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Cell_Biology_Alberts. the Upf proteins (green) that bind to each EJC. Note that this mechanism ensures that nonsense-mediated decay is triggered only when the premature stop codon is in the same reading frame as that of the normal protein. (Adapted from J. Lykke-Andersen et al., Cell 103:1121–1131, 2000. With permission from Elsevier.) premature termination codons. The surveillance system degrades the mRNAs produced from such rearranged genes, thereby avoiding the potential toxic effects of truncated proteins.


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Cell_Biology_Alberts. The nonsense-mediated surveillance pathway also plays an important role in mitigating the symptoms of many inherited human diseases. As we have seen, inherited diseases are usually caused by mutations that spoil the function of a key protein, such as hemoglobin or one of the blood-clotting factors. Approximately one-third of all genetic disorders in humans result from nonsense mutations or mutations (such as frameshift mutations or splice-site mutations) that place nonsense mutations into the gene’s reading frame. In individuals that carry one mutant and one functional gene, nonsense-mediated decay eliminates the aberrant mRNA and thereby prevents a potentially toxic protein from being made. Without this safeguard, individuals with one functional and one mutant “disease gene” would likely suffer much more severe symptoms. Some Proteins Begin to Fold While Still Being Synthesized


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Cell_Biology_Alberts. Some Proteins Begin to Fold While Still Being Synthesized The process of gene expression is not over when the genetic code has been used to create the sequence of amino acids that constitutes a protein. To be useful to the cell, this new polypeptide chain must fold up into its unique three-dimensional conformation, bind any small-molecule cofactors required for its activity, be appropriately modified by protein kinases or other protein-modifying enzymes, and assemble correctly with the other protein subunits with which it functions (Figure 6–77). The information needed for all of the steps listed above is ultimately contained in the sequence of amino acids that the ribosome produces when it translates an mRNA molecule into a polypeptide chain. As discussed in Chapter 3, when a


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Cell_Biology_Alberts. Figure 6–77 Steps in the creation of a functional protein. As indicated, translation of an mRNA sequence into an amino acid sequence on the ribosome is not the end of the process of forming a protein. To function, the completed polypeptide chain must fold correctly into its three-dimensional conformation, bind any cofactors required, and assemble with its partner protein chains, if any. Noncovalent bond formation drives these changes. As indicated, many proteins also require covalent modifications of selected amino acids. Although the most frequent modifications are protein glycosylation and protein phosphorylation, over 200 different types of covalent modifications are known (see pp. 165–166).


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Cell_Biology_Alberts. protein folds into a compact structure, it buries most of its hydrophobic residues in an interior core. In addition, large numbers of noncovalent interactions form between various parts of the molecule. It is the sum of all of these energetically favorable arrangements that determines the final folding pattern of the polypeptide chain—as the conformation of lowest free energy (see pp. 114–115).


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Cell_Biology_Alberts. Through many millions of years of evolution, the amino acid sequence of each protein has been selected not only for the conformation that it adopts but also for an ability to fold rapidly. For some proteins, this folding begins immediately, as the protein chain emerges from the ribosome, starting from the N-terminal end. In these cases, as each protein domain emerges from the ribosome, within a few seconds it forms a compact structure that contains most of the final secondary features (α helices and β sheets) aligned in roughly the right conformation (Figure 6–78). For some protein domains, this unusually dynamic and flexible state, called a molten globule, is the starting point for a relatively slow process in which many side-chain adjustments occur that eventually form the correct tertiary structure. It takes several minutes to synthesize a protein of average size, and for some proteins much of the folding process is complete by the time the ribosome releases the C-terminal end of a


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Cell_Biology_Alberts. structure. It takes several minutes to synthesize a protein of average size, and for some proteins much of the folding process is complete by the time the ribosome releases the C-terminal end of a protein (Figure 6–79).


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Cell_Biology_Alberts. Molecular Chaperones Help Guide the Folding of Most Proteins Most proteins probably do not fold correctly during their synthesis and require a special class of proteins called molecular chaperones to do so. Molecular chaperones are useful for cells because there are many different folding paths available to an unfolded or partially folded protein. Without chaperones, some of these pathways would not lead to the correctly folded (and most stable) form because the protein would become “kinetically trapped” in structures that are off-pathway. Some of these off-pathway configurations would aggregate and be left as irreversible dead ends of nonfunctional (and potentially dangerous) structures. covalent modifcation by glycosylation, phosphorylation, acetylation etc. binding to other protein subunits


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Cell_Biology_Alberts. covalent modifcation by glycosylation, phosphorylation, acetylation etc. binding to other protein subunits Figure 6–78 The structure of a molten globule. (A) A molten globule form of cytochrome b562 is more open and less highly ordered than the final folded form of the protein, shown in (B). Note that the molten globule contains most of the secondary structure of the final form, although the ends of the α helices are unraveled and one of the helices is only partly formed. (Courtesy of Joshua Wand, from Y. Feng et al., Nat. Struct. Biol. 1:30– 35, 1994. With permission from Macmillan Publishers Ltd.) Figure 6–79 Co-translational protein folding. A growing polypeptide chain is shown acquiring its secondary and tertiary structure as it emerges from a ribosome. The N-terminal domain folds first, while the C-terminal domain is still being synthesized. This protein has not achieved its final conformation at the time it is released from the ribosome. (Modified from A.N. Fedorov and


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Cell_Biology_Alberts. T.O. Baldwin, J. Biol. Chem. 272:32715–32718, 1997.) Molecular chaperones specifically recognize incorrect, off-pathway configurations by their exposure of hydrophobic surfaces, which in correctly folded proteins are typically buried in the interior. The binding of these exposed hydrophobic surfaces to each other is what causes off-pathway conformations to irreversibly aggregate. We saw in Chapter 3 that in some cases of inherited human diseases, aggregates do form and can cause severe symptoms and even death. Chaperones prevent this from happening in normal proteins by binding to the exposed hydrophobic surfaces using hydrophobic surfaces of their own. As we shall see shortly, there are several types of chaperones; once bound to an incorrectly folded protein, they ultimately release it in a way that gives the protein another chance to fold correctly. Cells Utilize Several Types of Chaperones


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Cell_Biology_Alberts. Cells Utilize Several Types of Chaperones Many molecular chaperones are called heat-shock proteins (designated hsp), because they are synthesized in dramatically increased amounts after a brief exposure of cells to an elevated temperature (for example, 42°C for cells that normally live at 37°C). This reflects the operation of a feedback system that responds to an increase in misfolded proteins (such as those produced by elevated temperatures) by boosting the synthesis of the chaperones that help these proteins refold. There are several major families of molecular chaperones, including the hsp60 and hsp70 proteins. Different members of these families function in different organelles. Thus, as discussed in Chapter 12, mitochondria contain their own hsp60 and hsp70 molecules that are distinct from those that function in the cytosol; and a special hsp70 (called BIP) helps to fold proteins in the endoplasmic reticulum.


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Cell_Biology_Alberts. The hsp60 and hsp70 proteins each work with their own small set of associated proteins when they help other proteins to fold. These hsps share an affinity for the exposed hydrophobic patches on incompletely folded proteins, and they hydrolyze ATP, often binding and releasing their protein substrate with each cycle of ATP hydrolysis. In other respects, the two types of hsp proteins function differently. The hsp70 machinery acts early in the life of many proteins (often before the protein leaves the ribosome), with each monomer of hsp70 binding to a string of about four or five hydrophobic amino acids (Figure 6–80). On binding ATP, hsp70 releases the protein into solution allowing it a chance to re-fold. In contrast, hsp60-like proteins form a large barrel-shaped structure that acts after a protein has been fully synthesized. This type of chaperone, sometimes called a chaperonin, forms an “isolation chamber” for the folding process (Figure 6–81).


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Cell_Biology_Alberts. To enter a chamber, a substrate protein is first captured via the hydrophobic entrance to the chamber. The protein is then released into the interior of the chamber, which is lined with hydrophilic surfaces, and the chamber is sealed with a lid, a step requiring ATP. Here, the substrate is allowed to fold into its final conformation in isolation, where there are no other proteins with which to aggregate. When ATP is hydrolyzed, the lid pops off, and the substrate protein, whether folded or not, is released from the chamber.


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Cell_Biology_Alberts. The chaperones shown in Figures 6–80 and 6–81 often need many cycles of ATP hydrolysis to fold a single polypeptide chain correctly. This energy is used to perform mechanical movements of the hsp60 and hsp70 “machines,” converting them from binding forms to releasing forms. Just as we saw for transcription, splicing, and translation, the expenditure of free energy can be used by cells to improve the accuracy of a biological process. In the case of protein folding, ATP hydrolysis allows chaperones to recognize a wide variety of misfolded structures, to halt any further misfolding, and to recommence the folding of a protein in an orderly way.


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Cell_Biology_Alberts. Figure 6–80 The hsp70 family of molecular chaperones. These proteins act early, recognizing a small stretch of hydrophobic amino acids on a protein’s surface. Aided by a set of smaller hsp40 proteins (not shown), ATP-bound hsp70 molecules grasp their target protein and then hydrolyze ATP to ADP, undergoing conformational changes that cause the hsp70 molecules to associate even more tightly with the target. After the hsp40 dissociates, the rapid rebinding of ATP induces the dissociation of the hsp70 protein after ADP release. Repeated cycles of hsp binding and release help the target protein to refold.


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Cell_Biology_Alberts. Figure 6–81 The structure and function of the hsp60 family of molecular chaperones. (A) A misfolded protein is initially captured by hydrophobic interactions with the exposed surface of the opening. The initial binding often helps to unfold a misfolded protein. The subsequent binding of ATP and a cap releases the substrate protein into an enclosed space, where it has a new opportunity to fold. After about 10 seconds, ATP hydrolysis occurs, weakening the binding of the cap. Subsequent binding of additional ATP molecules ejects the cap, and the protein is released. As indicated, only half of the symmetric barrel operates on a client protein at any one time. This type of molecular chaperone is also known as a chaperonin; it is designated as hsp60 in mitochondria, TCP1 in the cytosol of vertebrate cells, and GroEL in bacteria. (B) The structure of GroEL bound to its GroES cap, as determined by x-ray crystallography. On the left is shown the outside of the barrel-like structure, and on the


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Cell_Biology_Alberts. cells, and GroEL in bacteria. (B) The structure of GroEL bound to its GroES cap, as determined by x-ray crystallography. On the left is shown the outside of the barrel-like structure, and on the right a cross section through its center. (B, adapted from B. Bukau and A.L. Horwich, Cell 92:351–366, 1998. With permission from Elsevier.) 10 nm


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Cell_Biology_Alberts. Although our discussion focuses on only two types of chaperones, the cell has a variety of others. The enormous diversity of proteins in cells presumably requires a wide range of chaperones with versatile surveillance and correction capabilities. If radioactive amino acids are added to cells for a brief period, the newly synthesized proteins can be followed as they mature into their final functional forms. This type of experiment demonstrates that the hsp70 proteins act first, beginning when a protein is still being synthesized on a ribosome, and the hsp60-like proteins act only later to help fold completed proteins. We have seen that the cell distinguishes misfolded proteins, which require additional rounds of ATP-catalyzed refolding, from those with correct structures through the recognition of hydrophobic surfaces.


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Cell_Biology_Alberts. Usually, if a protein has a sizable exposed patch of hydrophobic amino acids on its surface, it is abnormal: it has either failed to fold correctly after leaving the ribosome, suffered an accident that partly unfolded it at a later time, or failed to find its normal partner subunit in a larger protein complex. Such a protein is not merely useless to the cell, it can be dangerous. Proteins that rapidly fold correctly on their own do not display such patterns and generally bypass the chaperones. For the others, the chaperones can carry out “protein repair” by giving them additional chances to fold while, at the same time, preventing their aggregation.


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Cell_Biology_Alberts. Figure 6–82 outlines all of the quality-control choices that a cell makes for a difficult-to-fold, newly synthesized protein. As indicated, when attempts to refold a protein fail, an additional mechanism is called into play that completely destroys the protein by proteolysis. This proteolytic pathway begins with the recognition of an abnormal hydrophobic patch on a protein’s surface, and it ends with the delivery of the entire protein to a protein-destruction machine, a complex protease known as the proteasome. As described next, this process depends on an elaborate protein-marking system that also carries out other central functions in the cell by destroying selected normal proteins. The Proteasome Is a Compartmentalized Protease with Sequestered Active Sites


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Cell_Biology_Alberts. The Proteasome Is a Compartmentalized Protease with Sequestered Active Sites The proteolytic machinery and the chaperones compete with one another to recognize a misfolded protein. If a newly synthesized protein folds rapidly, at most only a small fraction of it is degraded. In contrast, a slowly folding protein is vulnerable to the proteolytic machinery for a longer time, and many more of its molecules may be destroyed before the remainder attain the proper folded state. Due to mutations or to errors in transcription, RNA splicing, and translation, some proteins never fold properly, and it is particularly important that the cell destroy these potentially harmful proteins. The apparatus that deliberately destroys aberrant proteins is the proteasome, an abundant ATP-dependent protease that constitutes nearly 1% of cell protein.


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Cell_Biology_Alberts. Figure 6–82 The processes that monitor protein quality following protein synthesis. A newly synthesized protein sometimes folds correctly and assembles on its own with its partner proteins, in which case the quality control mechanisms leave it alone. Incompletely folded proteins are helped to properly fold by molecular chaperones: first by a family of hsp70 proteins, and then, in some cases, by hsp60-like proteins. For both types of chaperones, the substrate proteins are recognized by an abnormally exposed patch of hydrophobic amino acids on their surface. These “protein-rescue” processes compete with another mechanism that, upon recognizing an abnormally exposed hydrophobic patch, marks the protein for destruction by the proteasome. The combined activity of all of these processes is needed to prevent massive protein aggregation in a cell, which can occur when many hydrophobic regions on proteins clump together nonspecifically.


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Cell_Biology_Alberts. Figure 6–83 The proteasome. (A) A cutaway view of the structure of the central 20S cylinder, as determined by x-ray crystallography, with the active sites of the proteases indicated by red dots. (B) The entire proteasome, in which the central cylinder (yellow) is supplemented by a 19S cap (blue) at each end. The complex cap (also called the regulatory particle) selectively binds proteins that have been marked by ubiquitin for destruction; it then uses ATP hydrolysis to unfold their polypeptide chains and feed them through a narrow channel (see Figure 6–85) into the inner chamber of the 20S cylinder for digestion to short peptides. (B, from W. Baumeister et al., Cell 92:367–380, 1998. With permission from Elsevier.)


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Cell_Biology_Alberts. W. Baumeister et al., Cell 92:367–380, 1998. With permission from Elsevier.) Present in many copies dispersed throughout the cytosol and the nucleus, the proteasome also destroys aberrant proteins that have entered the endoplasmic reticulum (ER). In the latter case, an ER-based surveillance system detects proteins that have failed either to fold or to be assembled properly after they enter the ER, and retrotranslocates them back to the cytosol for degradation by the proteasome (discussed in Chapter 12).


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Cell_Biology_Alberts. Each proteasome consists of a central hollow cylinder (the 20S core proteasome) formed from multiple protein subunits that assemble as a stack of four heptameric rings (Figure 6–83). Some of the subunits are proteases whose active sites face the cylinder’s inner chamber, thus preventing them from running rampant through the cell. Each end of the cylinder is normally associated with a large protein complex (the 19S cap) that contains a six-subunit protein ring through which target proteins are threaded into the proteasome core, where they are degraded (Figure 6–84). The threading reaction, driven by ATP hydrolysis, unfolds the target proteins as they move through the cap, exposing them to the proteases lining the proteasome core (Figure 6–85). The proteins that make up the ring structure in the proteasome cap belong to a large class of protein “unfoldases” known as AAA proteins. Many of them function as hexamers, and they share mechanistic features with the ATP-dependent DNA helicases


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Cell_Biology_Alberts. the proteasome cap belong to a large class of protein “unfoldases” known as AAA proteins. Many of them function as hexamers, and they share mechanistic features with the ATP-dependent DNA helicases that unwind DNA (see Figure 5–14).


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Cell_Biology_Alberts. (A) (B) ubiquitin target protein with receptor polyubiquitin chain


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Cell_Biology_Alberts. Figure 6–84 Processive protein digestion by the proteasome. (A) The proteasome cap recognizes proteins marked by a polyubiquitin chain (see Figure 3–70), and subsequently translocates them into the proteasome core, where they are digested. At an early stage, the ubiquitin is cleaved from the substrate protein and is recycled. Translocation into the core of the proteasome is mediated by a ring of ATPases that unfold the substrate protein as it is threaded through the ring and into the proteasome core. This unfoldase ring is depicted in Figure 6–85). (B) Detailed structure of the proteasome cap. The cap includes a ubiquitin receptor, which holds a ubiquitylated protein in place while it begins to be pulled into the proteasome core, and a ubiquitin hydrolase, which cleaves ubiquitin from the doomed protein. (A, from S. Prakash and A. Matouschek, Trends Biochem. Sci. 29:593–600, 2004. With permission from Elsevier. B, adapted from G.C. Lander et al., Nature 482:186–191, 2012.)


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Cell_Biology_Alberts. A crucial property of the proteasome, and one reason for the complexity of its design, is the processivity of its mechanism: in contrast to a “simple” protease that cleaves a substrate’s polypeptide chain just once before dissociating, the proteasome keeps the entire substrate bound until all of it is converted into short peptides.


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Cell_Biology_Alberts. One would expect that a machine as efficient as the proteasome would be tightly regulated; in particular, it must be able to distinguish abnormal proteins from those that are properly folded. The 19S cap of the proteasome acts as a gate at the entrance to the inner proteolytic core, and only those proteins marked for destruction are threaded through the cap. The destruction “mark” is the covalent attachment of the small protein ubiquitin. As we saw in Chapter 3, ubiquitin modification of proteins is used for many purposes in the cell. The particular type of ubiquitin linkage that concerns us here is a chain of ubiquitin molecules linked together at lysine 48 (see Figure 3–69); this is the distinguishing feature of the ubiquitin tag that marks a protein for destruction in the proteasome.


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Cell_Biology_Alberts. A special set of E3 molecules (see Figure 3–70B) is responsible for the ubiquitylation of denatured or otherwise misfolded proteins, as well as proteins containing oxidized or other abnormal amino acids. Abnormal proteins tend to display on their surface hydrophobic amino acid sequences or conformational motifs that are recognized as degradation signals by these E3 molecules; these sequences are buried and therefore inaccessible in the normal, properly folded version. However, a proteolytic pathway that recognizes and destroys abnormal proteins must be able to distinguish completed proteins that have “wrong” conformations from the many growing polypeptides on ribosomes (as well as polypeptides just released from ribosomes) that have not yet achieved their normal folded conformation. This is not a trivial problem; in the course of carrying out its main job, the ubiquitin–proteasome system probably destroys many nascent and newly formed protein molecules, not because these proteins are


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Cell_Biology_Alberts. is not a trivial problem; in the course of carrying out its main job, the ubiquitin–proteasome system probably destroys many nascent and newly formed protein molecules, not because these proteins are abnormal as such, but because they have transiently exposed degradation signals that are buried in their mature (folded) state.


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Cell_Biology_Alberts. One function of intracellular proteolytic mechanisms is to recognize and eliminate misfolded or otherwise abnormal proteins, as just described. Indeed, every protein in the cell eventually accumulates damage and is probably degraded by the proteasome. Yet another function of these proteolytic pathways is to confer short lifetimes on specific normal proteins whose concentrations must change promptly with alterations in the state of a cell. Some of these short-lived proteins are degraded rapidly at all times, while many others are conditionally short-lived; that is, they are metabolically stable under some conditions, but become unstable upon a change in the cell’s state. For example, mitotic cyclins are long-lived throughout the cell cycle until their sudden degradation at the end of mitosis, as explained in Chapter 17.


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Cell_Biology_Alberts. Figure 6–85 A hexameric protein unfoldase. (A) The proteasome cap includes proteins (orange) that recognize and hydrolyze ubiquitin and a hexameric ring (blue) through which ubiquitylated proteins are threaded. The hexameric ring is formed from six subunits, each belonging to the AAA family of proteins. (B) Model for the ATP-dependent unfoldase activity of AAA proteins. The ATP-bound form of a hexameric ring of AAA proteins binds a folded substrate protein that is held in place by its ubiquitin tag. A conformational change, driven by ATP hydrolysis, pulls the substrate into the central core and strains the ring structure. At this point, the substrate protein, which is being tugged upon, can partially unfold and enter further into the pore or it can maintain its structure and partially withdraw. Very stable protein substrates may require hundreds of cycles of ATP hydrolysis and dissociation before they are successfully pulled through the AAA protein ring. Once unfolded (and


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Cell_Biology_Alberts. withdraw. Very stable protein substrates may require hundreds of cycles of ATP hydrolysis and dissociation before they are successfully pulled through the AAA protein ring. Once unfolded (and de-ubiquitylated), the substrate protein moves relatively quickly through the pore by successive rounds of ATP hydrolysis. (A, adapted from G.C. Lander et al., Nature 482:186–191, 2012; B, adapted from


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Cell_Biology_Alberts. R.T. Sauer et al., Cell 119:9–18, 2004. With permission from Elsevier.) N phosphorylation unmasking by creation of destabilizing by protein kinase protein dissociation N-terminus How is such a regulated destruction of a protein controlled? Several general mechanisms are illustrated in Figure 6–86. Specific examples of each mechanism are discussed in later chapters. In one general class of mechanism (Figure 6–86A), the activity of a ubiquitin ligase is turned on either by E3 phosphorylation or by an allosteric transition in an E3 protein caused by its binding to a specific small or large molecule. For example, the anaphase-promoting complex (APC) is a multi-subunit ubiquitin ligase that is activated by a cell-cycle-timed subunit addition at mitosis. The activated APC then causes the degradation of mitotic cyclins and several other regulators of the metaphase–anaphase transition (see Figure 17–15A).


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Cell_Biology_Alberts. Alternatively, in response either to intracellular signals or to signals from the environment, a degradation signal can be created in a protein, causing its rapid ubiquitylation and destruction by the proteasome (Figure 6–86B). One common way to create such a signal is to phosphorylate a specific site on a protein that unmasks a normally hidden degradation signal. Another way to unmask such a signal is by the regulated dissociation of a protein subunit. Finally, powerful degradation signals can be created by cleaving a single peptide bond, provided that this cleavage creates a new N-terminus that is recognized by a specific E3 protein as a “destabilizing” N-terminal residue. This E3 protein recognizes only certain amino acids at the N-terminus of a protein; thus not all protein-cleavage events will lead to degradation of the C-terminal fragment produced.


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Cell_Biology_Alberts. In humans, nearly 80% of proteins are acetylated on their N-terminal residue, and we now know that this modification is recognized by a specific E3 enzyme, Figure 6–86 Two general ways of inducing the degradation of a specific protein. (A) Activation of a specific E3 molecule creates a new ubiquitin ligase. Eukaryotic cells have many different E3 molecules, each activated by a different signal. (B) Creation of an exposed degradation signal in the protein to be degraded. This signal binds a ubiquitin ligase, causing the addition of a polyubiquitin chain to a nearby lysine on the target protein. All six pathways shown are known to be used by cells to induce the movement of selected proteins into the proteasome.


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Cell_Biology_Alberts. which directs the ubiquitylation of the protein and sends it to the proteasome for degradation. Thus, the majority of human proteins carry their own signals for destruction. It has been proposed that when a protein is properly folded (and, before that, when it is in contact with a chaperone), this acetylated N-terminus is buried and therefore inaccessible to the E3 enzyme. According to this idea, as a protein ages and becomes damaged (or if it fails to fold correctly from the start), this destruction signal becomes exposed, and the protein is destroyed. There Are Many Steps From DNA to Protein


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Cell_Biology_Alberts. There Are Many Steps From DNA to Protein We have seen so far in this chapter that many different types of chemical reactions are required to produce a properly folded protein from the information contained in a gene (Figure 6–87). The final level of a properly folded protein in a cell therefore depends upon the efficiency with which each of the many steps is performed. We also now know that the cell devotes enormous resources to selectively degrading proteins, particularly those that fail to fold properly or accumulate damage as they age. It is the balance between the rates of synthesis and degradation that determines the final amount of every protein in the cell.


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Cell_Biology_Alberts. In the following chapter, we shall see that cells have the ability to change the levels of their proteins according to their needs. In principle, any or all of the steps in Figure 6–87 could be regulated for each individual protein. As we shall see in Chapter 7, there are examples of regulation at each step from gene to protein. 5˜3˜DNA introns exons INITIATION OF TRANSCRIPTION CLEAVAGE, POLYADENYLATION, AND TERMINATION EXPORT CAPPING, ELONGATION SPLICING AAAA mRNA NUCLEUS CYTOSOL cap poly-A tail Figure 6–87 The production of a protein by a eukaryotic cell. The final level of each protein in a eukaryotic cell depends upon the efficiency of each step depicted.


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Cell_Biology_Alberts. Figure 6–87 The production of a protein by a eukaryotic cell. The final level of each protein in a eukaryotic cell depends upon the efficiency of each step depicted. The translation of the nucleotide sequence of an mRNA molecule into protein takes place in the cytosol on a large ribonucleoprotein assembly called a ribosome. Each amino acid used for protein synthesis is first attached to a tRNA molecule that recognizes, by complementary base-pair interactions, a particular set of three nucleotides (codons) in the mRNA. As an mRNA is threaded through a ribosome, its sequence of nucleotides is then read from one end to the other in sets of three according to the genetic code.


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Cell_Biology_Alberts. To initiate translation, a small ribosomal subunit binds to the mRNA molecule at a start codon (AUG) that is recognized by a unique initiator tRNA molecule. A large ribosomal subunit then binds to complete the ribosome and begin protein synthesis. During this phase, aminoacyl-tRNAs—each bearing a specific amino acid—bind sequentially to the appropriate codons in mRNA through complementary base-pairing between tRNA anticodons and mRNA codons. Each amino acid is added to the C-terminal end of the growing polypeptide in four sequential steps: aminoacyl-tRNA binding, followed by peptide bond formation, followed by two ribosome translocation steps. Elongation factors use GTP hydrolysis both to drive these reactions forward and to improve the accuracy of amino acid selection. The mRNA molecule progresses codon by codon through the ribosome in the 5ʹ-to-3ʹ direction until it reaches one of three stop codons. A release factor then binds to the ribosome, terminating translation and releasing


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Cell_Biology_Alberts. progresses codon by codon through the ribosome in the 5ʹ-to-3ʹ direction until it reaches one of three stop codons. A release factor then binds to the ribosome, terminating translation and releasing the completed polypeptide.


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Cell_Biology_Alberts. Eukaryotic and bacterial ribosomes are closely related, despite differences in the number and size of their rRNA and protein components. The rRNA has the dominant role in translation, determining the overall structure of the ribosome, forming the binding sites for the tRNAs, matching the tRNAs to codons in the mRNA, and creating the active site of the peptidyl transferase enzyme that links amino acids together during translation.


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Cell_Biology_Alberts. In the final steps of protein synthesis, two distinct types of molecular chaperones guide the folding of polypeptide chains. These chaperones, known as hsp60 and hsp70, recognize exposed hydrophobic patches on proteins and serve to prevent the protein aggregation that would otherwise compete with the folding of newly synthesized proteins into their correct three-dimensional conformations. This pro-tein-folding process must also compete with an elaborate quality control mechanism that destroys proteins with abnormally exposed hydrophobic patches. In this case, ubiquitin is covalently added to a misfolded protein by a ubiquitin ligase, and the resulting polyubiquitin chain is recognized by the cap on a proteasome that unfolds the protein and threads it into the interior of the proteasome for proteolytic degradation. A closely related proteolytic mechanism, based on special degradation signals recognized by ubiquitin ligases, is used to determine the lifetimes of many normally folded


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Cell_Biology_Alberts. for proteolytic degradation. A closely related proteolytic mechanism, based on special degradation signals recognized by ubiquitin ligases, is used to determine the lifetimes of many normally folded proteins as well as to remove selected proteins from the cell in response to specific signals.


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Cell_Biology_Alberts. We have seen that the expression of hereditary information requires extraordinarily complex machinery and proceeds from DNA to protein through an RNA intermediate. This machinery presents a central paradox: if nucleic acids are required to synthesize proteins and proteins are required, in turn, to synthesize nucleic acids, how did such a system of interdependent components ever arise? One view is that an RNA world existed on Earth before modern cells arose (Figure 6–88). According to this hypothesis, RNA both stored genetic information and catalyzed the chemical reactions in primitive cells. Only later in evolutionary time did DNA take over as the genetic material and proteins become the major catalysts and structural components of cells. If this idea is correct, then the transition out of the RNA world was never complete; as we have seen in this chapter, RNA still catalyzes several fundamental reactions in modern-day cells, which can be viewed as molecular fossils from an earlier


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Cell_Biology_Alberts. of the RNA world was never complete; as we have seen in this chapter, RNA still catalyzes several fundamental reactions in modern-day cells, which can be viewed as molecular fossils from an earlier world.


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Cell_Biology_Alberts. formed with DNA Figure 6–88 Time line for the universe, suggesting the early existence of an RNA world of living systems. The RNA world hypothesis relies on the fact that, among present-day biological molecules, RNA is unique in being able to act as both a carrier of genetic information and as a ribozyme to catalyze chemical reactions. In this section, we discuss these properties of RNA and how they may have been especially important in early cells.


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Cell_Biology_Alberts. We have seen in this chapter that RNA can carry genetic information in mRNAs, and we saw in Chapter 5 that the genomes of some viruses are composed solely of RNA. We have also seen that complementary base-pairing and other types of hydrogen bonds can occur between nucleotides in the same chain of RNA, causing an RNA molecule to fold up in a unique way determined by its nucleotide sequence (see, for example, Figures 6–50 and 6–67). Comparisons of many RNA structures have revealed conserved motifs, short structural elements that are used over and over again as parts of larger structures (Figure 6–89).


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Cell_Biology_Alberts. Protein catalysts require a surface with unique contours and chemical properties on which a given set of substrates can react (discussed in Chapter 3). In exactly the same way, an RNA molecule with an appropriately folded shape can serve as a catalyst (Figure 6–90). Like some proteins, many of these ribozymes work by positioning metal ions at their active sites. This feature gives them a wider range of catalytic activities than provided by the limited chemical groups of a polynucleotide chain. Much of our inference about the RNA world has come from experiments in which large pools of RNA molecules of random nucleotide sequences are generated in the laboratory. Those rare RNA molecules with a property specified by the experimenter are then selected out and studied (Figure 6–91). Such experiments have created RNAs that can catalyze a wide variety of biochemical reactions (Table 6–5), with reaction rate enhancements only a few orders of magnitude lower than


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Cell_Biology_Alberts. Figure 6–89 Some common elements of RNA structure. Conventional, complementary base-pairing interactions are indicated by red “rungs” in double-helical portions of the RNA. Figure 6–90 A ribozyme. This simple RNA molecule catalyzes the cleavage of a second RNA at a specific site. This ribozyme is found embedded in larger RNA genomes—called viroids—which infect plants. The cleavage, which occurs in nature at a distant location on the same RNA molecule that contains the ribozyme, is a step in the replication of the viroid genome. Although not shown in the figure, the reaction requires a magnesium ion positioned at the active site. (Adapted from T.R. Cech and O.C. Uhlenbeck, Nature 372:39–40, 1994. With permission from Macmillan Publishers Ltd.)


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Cell_Biology_Alberts. Figure 6–91 In vitro selection of a synthetic ribozyme. Beginning with a large pool of nucleic acid molecules synthesized in the laboratory, those rare RNA molecules that possess a specified catalytic activity can be isolated and studied. Although a specific example (that of an autophosphorylating ribozyme) is shown, variations of this procedure have been used to generate many of the ribozymes listed in Table 6–5. During the autophosphorylation step, the RNA molecules are kept sufficiently dilute to prevent the “cross”phosphorylation of additional RNA molecules. In reality, several repetitions of this procedure are necessary to select the very rare RNA molecules with this catalytic activity. Thus, the material initially eluted from the column is converted back into DNA, amplified many fold (using reverse transcriptase and PCR, as explained in Chapter 8), transcribed back into RNA, and subjected to repeated rounds of selection. (Adapted from J.R. Lorsch and


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Cell_Biology_Alberts. J.W. Szostak, Nature 371:31–36, 1994. With permission from Macmillan Publishers Ltd.) those of the “fastest” protein enzymes. Given these findings, it is not clear why protein catalysts greatly outnumber ribozymes in modern cells. Experiments have shown, however, that RNA molecules may have more difficulty than proteins in binding to flexible, hydrophobic substrates. In any case, the availability of 20 types of amino acids presumably provides proteins with a greater number of catalytic strategies. RNA molecules have one property that contrasts with those of polypeptides: they can directly guide the formation of copies of their own sequence. This capacity depends on complementary base-pairing of their nucleotide subunits, which enables one RNA to act as a template for the formation of another. As we have seen in this and the preceding chapter, these complementary templating mechanisms lie at the heart of DNA replication and transcription in modern-day cells.


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Cell_Biology_Alberts. large pool of double-stranded DNA molecules, each with a different, randomly generated nucleotide sequence large pool of single-stranded RNA molecules, each with a different, randomly generated nucleotide sequence only the rare RNA molecules able to phosphorylate themselves incorporate sulfur molecules that fail to bind to the rare RNA molecules that can catalyze their own phosphorylation using ATP as a substrate But the efficient synthesis of RNA by such complementary templating mechanisms requires catalysts to promote the polymerization reaction: without catalysts, polymer formation is slow, error-prone, and inefficient.


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Cell_Biology_Alberts. Because RNA has all the properties required of a molecule that could catalyze a variety of chemical reactions, including those that lead to its own synthesis (Figure 6–92), it has been proposed that RNAs served long ago as the catalysts for template-dependent RNA synthesis. Although self-replicating systems of RNA molecules have not been found in nature, scientists have made significant progress toward constructing them in the laboratory. While such demonstrations would not prove that self-replicating RNA molecules were central to the origin of life on Earth, they would establish that such a scenario is plausible. How Did Protein Synthesis Evolve?


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Cell_Biology_Alberts. How Did Protein Synthesis Evolve? The molecular processes underlying protein synthesis in present-day cells seem inextricably complex. Although we understand most of them, they do not make conceptual sense in the way that DNA transcription, DNA repair, and DNA replication do. It is especially difficult to imagine how protein synthesis evolved because it is now performed by a complex interlocking system of protein and RNA molecules; obviously the proteins could not have existed until an early version of the translation apparatus was already in place. As attractive as the RNA world idea is for envisioning early life, it does not explain how the modern-day system of protein synthesis arose. Although we can only speculate on the origins of the genetic code, several experimental observations have provided plausible scenarios.


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Cell_Biology_Alberts. In modern cells, some short peptides (such as antibiotics) are synthesized without the ribosome; peptide synthetase enzymes assemble these peptides, with their proper sequence of amino acids, without mRNAs to guide their synthesis. It is plausible that this noncoded, primitive version of protein synthesis first developed in the RNA world, where it would have been catalyzed by RNA molecules. This idea presents no conceptual difficulties because, as we have seen, rRNA catalyzes peptide bond formation in present-day cells. However, it leaves unexplained how the genetic code—which lies at the core of protein synthesis in today’s cells— might have arisen. We know that ribozymes created in the laboratory can perform specific aminoacylation reactions; that is, they can match specific amino acids to specific tRNAs. It is therefore possible that tRNA-like adaptors, each matched to a specific amino acid, could have arisen in the RNA world, marking the beginnings of a genetic code.


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Cell_Biology_Alberts. Once coded protein synthesis evolved, the transition to a protein-dominated world could proceed, with proteins eventually taking over the majority of catalytic and structural tasks because of their greater versatility, with 20 rather than 4 different subunits. Although these ideas are highly speculative, they are consistent with the known properties of RNA and protein molecules. All Present-Day Cells Use DNA as Their Hereditary Material If the evolutionary speculations embodied in the RNA world hypothesis are correct, early cells would have differed fundamentally from the cells we know today in having their hereditary information stored in RNA rather than in DNA (Figure 6–93). Evidence that RNA arose before DNA in evolution can be found


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Cell_Biology_Alberts. Figure 6–92 An RNA molecule that can catalyze its own synthesis. This hypothetical process would require catalysis both of the production of a second RNA strand of complementary nucleotide sequence (not shown) and the use of this second RNA molecule as a template to form many molecules of RNA with the original sequence. The red rays represent the active site of this hypothetical RNA enzyme. Figure 6–93 The hypothesis that RNA preceded DNA and proteins in evolution. In the earliest cells, RNA molecules (or their close analogs) would have had combined genetic, structural, and catalytic functions. In present-day cells, DNA is the repository of genetic information, and proteins perform the vast majority of catalytic functions in cells. RNA primarily functions today as a go-between in protein synthesis, although it remains a catalyst for a small number of crucial reactions.


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Cell_Biology_Alberts. 366 Chapter 6: How Cells Read the Genome: From DNA to Protein in the chemical differences between them. Ribose, like glucose and other sim-WHAT WE DON’T KNOW ple carbohydrates, can be formed from formaldehyde (HCHO), a simple chemical which is readily produced in laboratory experiments that attempt to simulate • How did the present relationships conditions on the primitive Earth. The sugar deoxyribose is harder to make, and in present-day cells it is produced from ribose in a reaction catalyzed by a pro- evolve? How did the genetic code tein enzyme, suggesting that ribose pre-dates deoxyribose in cells. Presumably, originate?


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Cell_Biology_Alberts. DNA appeared on the scene later, but then proved more suitable than RNA as a permanent repository of genetic information. In particular, the deoxyribose in • The information carried in genomes its sugar-phosphate backbone makes chains of DNA chemically more stable than specifies the sequences of all proteins chains of RNA, so that much greater lengths of DNA can be maintained without and RNA molecules in the cell, breakage. and it determines when and where these molecules are synthesized. The other differences between RNA and DNA—the double-helical structure of DNA and the use of thymine rather than uracil—further enhance DNA stability by making the many unavoidable accidents that occur to the molecule much easier discovered? to repair, as discussed in detail in Chapter 5 (pp. 271–273). • Cells go to great length to correct mistakes in the processes of DNA replication, transcription, splicing, and translation. Are there analogous


Cell_Biology_Alberts

Cell_Biology_Alberts. • Cells go to great length to correct mistakes in the processes of DNA replication, transcription, splicing, and translation. Are there analogous From our knowledge of present-day organisms and the molecules they contain, it strategies to correct mistakes in the seems likely that the development of the distinctive autocatalytic mechanisms fun- selection of which genes are to be damental to living systems began with the evolution of families of RNA molecules expressed in a given cell type? Could that could catalyze their own replication. DNA is likely to have been a late addition: the great complexity of transcription as the accumulation of protein catalysts allowed more efficient and complex cells to initiation in animals and plants reflect evolve, the DNA double helix replaced RNA as a more stable molecule for storing the such a strategy? increased amounts of genetic information required by such cells. Which statements are true? explain why or why not.


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Cell_Biology_Alberts. Which statements are true? explain why or why not. 6–1 The consequences of errors in transcription are less severe than those of errors in DNA replication. 6–2 Since introns are largely genetic “junk,” they do not Figure Q6–1 Supercoils around a moving RNA polymerase (Problem have to be removed precisely from the primary transcript 6–6). during RNA splicing. 6–3 Wobble pairing occurs between the first position 6–7 You have attached an RNA polymerase molecule in the codon and the third position in the anticodon. to a glass slide and have allowed it to initiate transcription on a template DNA that is tethered to a magnetic bead as 6–4 During protein synthesis, the thermodynamics of shown in Figure Q6–2. If the DNA with its attached mag-base-pairing between tRNAs and mRNAs sets the upper netic bead moves relative to the RNA polymerase as indilimit for the accuracy with which protein molecules are cated in the figure, in which direction will the bead rotate? made.


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Cell_Biology_Alberts. made. 6–5 Protein enzymes are thought to greatly outnum-Figure Q6–2 System for measuring the rotation of DNA ber ribozymes in modern cells because they can catalyze magnet a much greater variety of reactions and all of them have (Problem 6–7). The magnet faster rates than any ribozyme. holds the bead upright (but doesn’t interfere with its Discuss the following problems. bead rotation), and the attached tiny fluorescent beads allow 6–6 In which direction along the template must the the direction of motion to DNA be visualized under the RNA polymerase in Figure Q6–1 be moving to have gen- microscope. RNA polymerase is erated the supercoiled structures that are shown? Would you expect supercoils to be generated if the RNA poly-RNA the glass slide. merase were free to rotate about the axis of the DNA as it progressed along the template? glass slide


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Cell_Biology_Alberts. merase were free to rotate about the axis of the DNA as it progressed along the template? glass slide Figure Q6–3 Alternatively spliced mRNAs from the human α-tropomyosin gene (Problem 6–8). (A) Exons in the human α-tropomyosin gene. The locations and relative sizes of exons are shown by the blue and red rectangles, with alternative exons in red. (B) Splicing patterns for four α-tropomyosin mRNAs. Splicing is indicated by lines connecting the exons that are included in the mRNA.


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Cell_Biology_Alberts. (B) Splicing patterns for four α-tropomyosin mRNAs. Splicing is indicated by lines connecting the exons that are included in the mRNA. 6–8 The human α-tropomyosin gene is alternatively spliced to produce different forms of α-tropomyosin mRNA in different cell types (Figure Q6–3). For all forms of the mRNA, the protein sequences encoded by exon 1 are the same, as are the protein sequences encoded by exon 10. Exons 2 and 3 are alternative exons used in different mRNAs, as are exons 7 and 8. Which of the following statements about exons 2 and 3 is the most accurate? Is that statement also the most accurate one for exons 7 and 8? Explain your answers. A. Exons 2 and 3 must have the same number of nucleotides. b. Exons 2 and 3 must each contain an integral number of codons (that is, the number of nucleotides divided by 3 must be an integer). C. Exons 2 and 3 must each contain a number of nucleotides that when divided by 3 leaves the same remainder (that is, 0, 1, or 2).


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Cell_Biology_Alberts. C. Exons 2 and 3 must each contain a number of nucleotides that when divided by 3 leaves the same remainder (that is, 0, 1, or 2). 6–9 After treating cells with a chemical mutagen, you isolate two mutants. One carries alanine and the other carries methionine at a site in the protein that normally contains valine (Figure Q6–4). After treating these two mutants again with the mutagen, you isolate mutants from each that now carry threonine at the site of the original valine (Figure Q6–4). Assuming that all mutations involve single-nucleotide changes, deduce the codons that are used for valine, methionine, threonine, and alanine at the affected site. Would you expect to be able to isolate valineto-threonine mutants in one step? 6–10 Which of the following mutational changes would you predict to be the most deleterious to gene function? Explain your answers.


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Cell_Biology_Alberts. 6–10 Which of the following mutational changes would you predict to be the most deleterious to gene function? Explain your answers. frst Ala second Figure Q6–4 Two rounds of treatment treatment mutagenesis and the altered amino acids at a single position in Val Thr a protein (Problem 6–9). 1. Insertion of a single nucleotide near the end of the coding sequence. 2. Removal of a single nucleotide near the beginning of the coding sequence. 3. Deletion of three consecutive nucleotides in the middle of the coding sequence. 4. Substitution of one nucleotide for another in the middle of the coding sequence. 6–11 Prokaryotes and eukaryotes both protect against the dangers of translating broken mRNAs. What dangers do partial mRNAs pose for the cell?


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Cell_Biology_Alberts. 6–11 Prokaryotes and eukaryotes both protect against the dangers of translating broken mRNAs. What dangers do partial mRNAs pose for the cell? 6–12 Both hsp60-like and hsp70 molecular chaperones share an affinity for exposed hydrophobic patches on proteins, using them as indicators of incomplete folding. Why do you suppose hydrophobic patches serve as critical signals for the folding status of a protein? 6–13 Most proteins require molecular chaperones to assist in their correct folding. How do you suppose the chaperones themselves manage to fold correctly? 6–14 What is so special about RNA that it is hypothesized to be an evolutionary precursor to DNA and protein? What is it about DNA that makes it a better material than RNA for storage of genetic information?


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Cell_Biology_Alberts. 6–15 If an RNA molecule could form a hairpin with a symmetric internal loop, as shown in Figure Q6–5, could the complement of this RNA form a similar structure? If so, would there be any regions of the two structures that are identical? Which ones? Figure Q6–5 An RNA hairpin with 5’-G-C-A C-C-G a symmetric internal loop (Problem U 6–15). 6–16 Imagine a warm pond on the primordial Earth. Chance processes have just assembled a single copy of an RNA molecule with a catalytic site that can carry out RNA replication. This RNA molecule folds into a structure that is capable of linking nucleotides according to instructions in an RNA template. Given an adequate supply of nucleotides, will this single RNA molecule be able to use itself as a template to catalyze its own replication? Why or why not? Atkins JF, Gesteland RF & Cech TR (eds) (2011) The RNA Worlds: From Life’s Origins to Diversity in Gene Regulation. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.


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Cell_Biology_Alberts. Atkins JF, Gesteland RF & Cech TR (eds) (2011) The RNA Worlds: From Life’s Origins to Diversity in Gene Regulation. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Berg JM, Tymoczko JL & Stryer L (2012) Biochemistry, 7th ed. New York: WH Freeman. Brown TA (2007) Genomes 3. New York: Garland Science. Darnell J (2011) RNA: Life’s Indispensable Molecule. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Hartwell L, Hood L, Goldberg ML et al. (2011) Genetics: from Genes to Genomes, 4th ed. Boston: McGraw Hill. Judson HF (1996) The Eighth Day of Creation, 25th anniversary ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Lodish H, Berk A, Kaiser C et al. (2012) Molecular Cell Biology, 7th ed. New York: WH Freeman. Stent GS (1971) Molecular Genetics: An Introductory Narrative. San Francisco: WH Freeman. The Genetic Code (1966) Cold Spring Harb. Symp. Quant. Biol. 31. The Ribosome (2001) Cold Spring Harb. Symp. Quant. Biol. 66.


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Cell_Biology_Alberts. The Genetic Code (1966) Cold Spring Harb. Symp. Quant. Biol. 31. The Ribosome (2001) Cold Spring Harb. Symp. Quant. Biol. 66. Watson JD, Baker TA, Bell SP et al. (2013) Molecular Biology of the Gene, 7th ed. Menlo Park, CA: Benjamin Cummings. From DNA to RNA Berget SM, Moore C & Sharp PA (1977) Spliced segments at the 5ʹ terminus of adenovirus 2 late mRNA. Proc. Natl. Acad. Sci. USA 74, 3171–3175. Brenner S, Jacob F & Meselson M (1961) An unstable intermediate carrying information from genes to ribosomes for protein synthesis. Nature 190, 576–581. Chow LT, Gelinas RE, Broker TR et al. (1977) An amazing sequence arrangement at the 5ʹ ends of adenovirus 2 messenger RNA. Cell 12, 1–8. Conaway CC & Conaway JW (2011) Function and regulation of the Mediator complex. Curr. Opin. Genet. Dev. 21, 225–230. Cooper TA, Wan L & Dreyfuss G (2009) RNA and disease. Cell 136, 777–793.


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Cell_Biology_Alberts. Conaway CC & Conaway JW (2011) Function and regulation of the Mediator complex. Curr. Opin. Genet. Dev. 21, 225–230. Cooper TA, Wan L & Dreyfuss G (2009) RNA and disease. Cell 136, 777–793. Cramer P, Armache KJ, Baumli S et al. (2008) Structure of eukaryotic RNA polymerases. Annu. Rev. Biophys. 37, 337–352. Fica SM, Tuttle N, Novak T et al. (2013) RNA catalyses nuclear premRNA splicing. Nature 503, 229–234. Grunberg S & Hahn S (2013) Structural insights into transcription initiation by RNA polymerases II. Trends Biochem. Sci. 38, 603–611. Grunwald D, Singer RH & Rout M (2011) Nuclear export dynamics of TNA-protein complexes. Nature 475, 333–341. Kornblihtt AR, Schor IE, Allo M et al. (2013) Alternative splicing: a pivotal step between eukaryotic transcription and translation. Nature 14, 153–165. Liu X, Bushnell DA & Kornberg RD (2012) RNA polymerase II transcription: Structure and mechanism. Biochim. Biophys. Acta 1829, 2–8.


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Cell_Biology_Alberts. Liu X, Bushnell DA & Kornberg RD (2012) RNA polymerase II transcription: Structure and mechanism. Biochim. Biophys. Acta 1829, 2–8. Makino DL, Halibach F & Conti E (2013) The RNA exosome and proteasome: common principles of degradation control. Nature 14, 654–660. Malik S & Roeder RC (2010) The metazoan mediator co-activator complex as an integrative hub for transcriptional regulation. Nat. Rev. Genet. 11, 761–772. Mao YS, Zhang B & Spector DL (2011) Biogenesis and function of nuclear bodies. Trends Genet. 27, 295–306. Matera AG & Wang Z (2014) A day in the life of the spliceosome. Nature 15, 108–121. Matsui T, Segall J, Weil PA & Roeder RG (1980) Multiple factors required for accurate initiation of transcription by purified RNA polymerase II. J. Biol. Chem. 255, 11992–11996. Opalka N, Brown J, Lane WJ et al. (2010) Complete structural model of Escherichia coli RNA polymerase from a hybrid approach. PLoS Biol. 9, 1–16.


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Cell_Biology_Alberts. Opalka N, Brown J, Lane WJ et al. (2010) Complete structural model of Escherichia coli RNA polymerase from a hybrid approach. PLoS Biol. 9, 1–16. Ruskin B, Krainer AR, Maniatis T et al. (1984) Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro. Cell 38, 317–331. Schneider C & Tollervey D (2013) Threading the barrel of the RNA exosome. Trends Biochem. Sci. 38, 485–493. Semlow DR & Staley JP (2012) Staying on message: ensuring fidelity in pre-mRNA splicing. Trends Biochem. Sci. 37, 263–273. From RNA to Protein Anfinsen CB (1973) Principles that govern the folding of protein chains. Science 181, 223–230. Crick FHC (1966) The genetic code: III. Sci. Am. 215, 55–62. Forster F, Unverdorben P, Sledz P et al. (2013) Unveiling the long-held secrets of the 26S proteasome. Structure 21, 1551–1562. Hershko A, Ciechanover A & Varshavsky A (2000) The ubiquitin system. Nat. Med. 6, 1073–1081.


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Cell_Biology_Alberts. Hershko A, Ciechanover A & Varshavsky A (2000) The ubiquitin system. Nat. Med. 6, 1073–1081. Horwich AL, Fenton WA, Chapman E et al. (2007) Two families of chaperonin: physiology and mechanism. Annu. Rev. Cell Dev. Biol. 23, 115–145. Ling J, Reynolds N & Ibba M (2009) Aminoacyl-tRNA synthesis and translational quality control. Annu Rev. Microbiol. 63, 61–78. Moore PB (2012) How should we think about the ribosome? Annu. Rev. Biophys. 41, 1–19. Noller HF (2005) RNA structure: reading the ribosome. Science 309, 1508–1514. Popp MW & Maquat LE (2013) Organizing principles of mammalian nonsense-mediated mRNA decay. Annu. Rev. Genet. 47, 139–165. Saibil H (2013) Chaperone machines for protein folding, unfolding and disaggregation. Nature 14, 630–642. Schmidt M & Finley D (2013) Regulation of proteasome activity in health and disease. Biochim. Biophys. Acta 1843, 13–25. Steitz TA (2008) A structural understanding of the dynamic ribosome machine. Nature 9, 242–253.


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Cell_Biology_Alberts. Steitz TA (2008) A structural understanding of the dynamic ribosome machine. Nature 9, 242–253. Varshavsky A (2012) The ubiquitin system, an immense realm. Annu. Rev. Biochem. 81, 167–176. Voorhees RM & Ramakrishnan V (2013) Structural basis of the translational elongation cycle. Annu. Rev. Biochem. 82, 203–236. Wilson DN (2014) Ribosome-targeting antibiotics and mechanisms of bacterial resistance. Nat. Rev. Microbiol. 12, 35–48. Zaher HS & Green R (2009) Fidelity at the molecular level: Lessons from protein synthesis. Cell 136, 746–762. The RNA World and the Origins of life Blain JC & Szostak JW (2014) Progress Towards Synthetic Cells. Annu. Rev. Biochem. 83, 615–640. Cech TR (2009) Crawling out of the RNA world. Cell 136, 599–602. Kruger K, Grabowski P, Zaug P et al. (1982) Self-splicing RNA: Autoexcision and autocyclization of the ribosomal RNA intervening seuence of Tetrahymena. Cell 31, 147–157.


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Cell_Biology_Alberts. Kruger K, Grabowski P, Zaug P et al. (1982) Self-splicing RNA: Autoexcision and autocyclization of the ribosomal RNA intervening seuence of Tetrahymena. Cell 31, 147–157. Orgel L (2000) Origin of life. A simpler nucleic acid. Science 290, 1306–1307. Robertson MP& Joyce GF (2012) The origins of the RNA world. Cold Spring Harb. Perspect. Biol. 4, a003608. Control of Gene expression 7 An organism’s DNA encodes all of the RNA and protein molecules required to construct its cells. Yet a complete description of the DNA sequence of an organism—be it the few million nucleotides of a bacterium or the few billion nucleotides of a human—no more enables us to reconstruct the organism than a list of English words enables us to reconstruct a play by Shakespeare. In both cases, the problem is to know how the elements in the DNA sequence or the words on the list are used. Under what conditions is each gene product made, and, once made, what does it do?


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Cell_Biology_Alberts. In this chapter, we focus on the first half of this problem—the rules and mechanisms that enable a subset of genes to be selectively expressed in each cell. These mechanisms operate at many levels, and we shall discuss each level in turn. But first we present some of the basic principles involved. The different cell types in a multicellular organism differ dramatically in both structure and function. If we compare a mammalian neuron with a liver cell, for example, the differences are so extreme that it is difficult to imagine that the two cells contain the same genome (Figure 7–1). For this reason, and because cell differentiation often seemed irreversible, biologists originally suspected that genes might be selectively lost when a cell differentiates. We now know, however, that cell differentiation generally occurs without changes in the nucleotide sequence of a cell’s genome. the Different Cell types of a Multicellular Organism Contain the Same DnA


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Cell_Biology_Alberts. the Different Cell types of a Multicellular Organism Contain the Same DnA The cell types in a multicellular organism become different from one another because they synthesize and accumulate different sets of RNA and protein molecules. The initial evidence that they do this without altering the sequence of their DNA came from a classic set of experiments in frogs. When the nucleus of a fully differentiated frog cell is injected into a frog egg whose nucleus has been removed, the injected donor nucleus is capable of directing the recipient egg to


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Cell_Biology_Alberts. Figure 7–1 A neuron and a liver cell share the same genome. the long branches of this neuron from the retina enable it to receive electrical signals from many other neurons and convey them to neighboring neurons. the liver cell, which is drawn to the same scale, is involved in many metabolic processes, including digestion and the detoxification of alcohol and other drugs. Both of these mammalian cells contain the same genome, but they express different sets of rnAs and proteins. (neuron adapted from S. ramón y Cajal, histologie du Systeme nerveux de l’homme et de vertebres, 1909–1911. paris: Maloine; reprinted, Madrid: C.S.i.C, 1972.) 370 Chapter 7: Control of Gene expression produce a normal tadpole (Figure 7–2A). The tadpole contains a full range of differentiated cells that derived their DNA sequences from the nucleus of the original donor cell. Thus, the differentiated donor cell cannot have lost any important DNA sequences. A similar conclusion came from experiments performed


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Cell_Biology_Alberts. their DNA sequences from the nucleus of the original donor cell. Thus, the differentiated donor cell cannot have lost any important DNA sequences. A similar conclusion came from experiments performed with plants. When differentiated pieces of plant tissue are placed in culture and then dissociated into single cells, often one of these individual cells can regenerate an entire adult plant (Figure 7–2B). And the same principle has been more recently demonstrated in mammals that include sheep, cattle, pigs, goats, dogs, and mice (Figure 7–2C).


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Cell_Biology_Alberts. Most recently, detailed DNA sequencing has confirmed the conclusion that the changes in gene expression that underlie the development of multicellular organisms do not generally involve changes in the DNA sequence of the genome. Different Cell types Synthesize Different Sets of rnAs and proteins As a first step in understanding cell differentiation, we would like to know how many differences there are between any one cell type and another. Although we


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Cell_Biology_Alberts. Figure 7–2 Differentiated cells contain all the genetic instructions necessary to direct the formation of a complete organism. (A) the nucleus of a skin cell from an adult frog transplanted into an enucleated egg can give rise to an entire tadpole. the broken arrow indicates that, to give the transplanted genome time to adjust to an embryonic environment, a further transfer step is required in which one of the nuclei is taken from an early embryo that begins to develop and is put back into a second enucleated egg. (B) in many types of plants, differentiated cells retain the ability to “de-differentiate,” so that a single cell can form a clone of progeny cells that later give rise to an entire plant. (C) A nucleus removed from a differentiated cell from an adult cow and introduced into an enucleated egg from a different cow can give rise to a calf. Different calves produced from the same differentiated cell donor are all clones of the donor and are therefore genetically identical. (A,


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Cell_Biology_Alberts. enucleated egg from a different cow can give rise to a calf. Different calves produced from the same differentiated cell donor are all clones of the donor and are therefore genetically identical. (A, modified from


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Cell_Biology_Alberts. J.B. Gurdon, Sci. Am. 219:24–35, 1968.) unfertilized MEIOTIC SPINDLE egg cell AND ASSOCIATED CHROMOSOMES REMOVED no. of reads no. of reads (B) start of transcription exons introns CELL LINE still do not have an exact answer to this fundamental question, we can make several general statements. 1. Many processes are common to all cells, and any two cells in a single organism therefore have many gene products in common. These include the structural proteins of chromosomes, RNA and DNA polymerases, DNA repair enzymes, ribosomal proteins and RNAs, the enzymes that catalyze the central reactions of metabolism, and many of the proteins that form the cytoskeleton such as actin (Figure 7–3A). 2.


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Cell_Biology_Alberts. 2. Some RNAs and proteins are abundant in the specialized cells in which they function and cannot be detected elsewhere, even by sensitive tests. Hemoglobin, for example, is expressed specifically in red blood cells, where it carries oxygen, and the enzyme tyrosine aminotransferase (which breaks down tyrosine in food) is expressed in liver but not in most other tissues (Figure 7–3B). 3.


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Cell_Biology_Alberts. 3. Studies of the number of different RNAs suggest that, at any one time, a typical human cell expresses 30–60% of its approximately 30,000 genes at some level. There are about 21,000 protein-coding genes and a roughly estimated 9000 noncoding RNA genes in humans. When the patterns of RNA expression in different human cell lines are compared, the level of expression of almost every gene is found to vary from one cell type to another. A few of these differences are striking, like those of hemoglobin and tyrosine aminotransferase noted above, but most are much more subtle. But even those genes that are expressed in all cell types usually vary in their level of expression from one cell type to the next. 4.


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Cell_Biology_Alberts. 4. Although there are striking differences in coding RNAs (mRNAs) in specialized cell types, they underestimate the full range of differences in the final pattern of protein production. As we discuss in this chapter, there are many steps after RNA production at which gene expression can be regulated. And, as we saw in Chapter 3, proteins are often covalently modified after they are synthesized. The radical differences in gene expression between cell types are therefore most fully revealed through methods that directly display the levels of proteins along with their post-translational modifications (Figure 7–4).


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Cell_Biology_Alberts. Figure 7–3 Differences in RNA levels for two human genes in seven different tissues. to obtain the rnA data by the technique known as RNA-seq (see p. 447), rnA was collected from human cell lines grown in culture, derived from each of the seven indicated tissues. Millions of “sequence reads” were obtained and mapped across the human genome by matching rnA sequences to the DnA sequence of the genome. At each position along the genome, the height of the colored trace is proportional to the number of sequence reads that match the genome sequence at that point. As seen in the figure, the exon sequences in transcribed genes are present at high levels, reflecting their presence in mature mrnAs. intron sequences are present at much lower levels and reflect pre-mrnA molecules that have not yet been spliced plus intron sequences that have been spliced out but not yet degraded. (A) the gene coding for “all-purpose” actin, a major component of the cytoskeleton. note that the left-hand end of the


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Cell_Biology_Alberts. spliced plus intron sequences that have been spliced out but not yet degraded. (A) the gene coding for “all-purpose” actin, a major component of the cytoskeleton. note that the left-hand end of the mature β-actin mrnA is not translated into protein. As explained later in this chapter, many mrnAs have 5ʹ untranslated regions that regulate their translation into protein. (B) the same type of data displayed for the enzyme tyrosine aminotransferase, which is highly expressed in liver cells but not in the other cell types tested. (information for both panels from the University of California, Santa Cruz, Genome Browser (http://genome.ucsc.edu), which provides this type of information for every human gene. See also S. Djebali et al., Nature 489:101–108, 2012.) external Signals Can Cause a Cell to Change the expression of its Genes


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Cell_Biology_Alberts. Although the specialized cells in a multicellular organism have characteristic patterns of gene expression, each cell is capable of altering its pattern of gene expression in response to extracellular cues. If a liver cell is exposed to a glucocorticoid hormone, for example, the production of a set of proteins is dramatically increased. Released in the body during periods of starvation or intense exercise, glucocorticoids signal the liver to increase the production of energy from amino acids and other small molecules; the set of proteins whose production is induced includes the enzyme tyrosine aminotransferase, mentioned above. When the hormone is no longer present, the production of these proteins drops to its normal, unstimulated level in liver cells.


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Cell_Biology_Alberts. Other cell types respond to glucocorticoids differently. Fat cells, for example, reduce the production of tyrosine aminotransferase, while some other cell types do not respond to glucocorticoids at all. These examples illustrate a general feature of cell specialization: different cell types often respond very differently to the same extracellular signal. Other features of the gene expression pattern do not change and give each cell type its permanently distinctive character. Gene expression Can Be regulated at Many of the Steps in the pathway from DnA to rnA to protein


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Cell_Biology_Alberts. If differences among the various cell types of an organism depend on the particular genes that the cells express, at what level is the control of gene expression exercised? As we saw in the previous chapter, there are many steps in the pathway leading from DNA to protein. We now know that all of them can in principle be regulated. Thus a cell can control the proteins it makes by (1) controlling when and how often a given gene is transcribed (transcriptional control), (2) controlling the splicing and processing of RNA transcripts (RNA processing control), (3) selecting which completed mRNAs are exported from the nucleus to the cytosol and determining where in the cytosol they are localized (RNA transport and localization control), (4) selecting which mRNAs in the cytoplasm are translated by ribosomes (translational control), (5) selectively destabilizing certain mRNA molecules in the cytoplasm (mRNA degradation control), or (6) selectively activating, inactivating, degrading, or


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Cell_Biology_Alberts. by ribosomes (translational control), (5) selectively destabilizing certain mRNA molecules in the cytoplasm (mRNA degradation control), or (6) selectively activating, inactivating, degrading, or localizing specific protein molecules after they have been made (protein activity control) (Figure 7–5).


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Cell_Biology_Alberts. For most genes, transcriptional controls are paramount. This makes sense because, of all the possible control points illustrated in Figure 7–5, only transcriptional control ensures that the cell will not synthesize superfluous intermediates. In the following sections, we discuss the DNA and protein components that perform this function by regulating the initiation of gene transcription. We shall then return to the additional ways of regulating gene expression.


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Cell_Biology_Alberts. Figure 7–4 Differences in the proteins expressed by two human tissues, (A) brain and (B) liver. in each panel, the proteins are displayed using two-dimensional polyacrylamide-gel electrophoresis (see pp. 452–454). the proteins have been separated by molecular weight (top to bottom) and isoelectric point, the ph at which the protein has no net charge (right to left). the protein spots artificially colored red are common to both samples; those in blue are specific to that tissue. the differences between the two tissue samples vastly outweigh their similarities: even for proteins that are shared between the two tissues, their relative abundances are usually different. note that this technique separates proteins by both size and charge; therefore a protein that has several different phosphorylation states will appear as a series of horizontal spots (see upper right-hand portion of right panel). Only a small portion of the complete protein spectrum is shown for each sample.


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Cell_Biology_Alberts. Methods based on mass spectrometry (see pp. 455–457) provide much more detailed information, including the identity of each protein, the position of each modification, and the nature of the modification. (Courtesy of tim Myers and leigh Anderson, large Scale Biology Corporation.) The genome of a cell contains in its DNA sequence the information to make many thousands of different protein and RNA molecules. A cell typically expresses only a fraction of its genes, and the different types of cells in multicellular organisms arise because different sets of genes are expressed. Moreover, cells can change the pattern of genes they express in response to changes in their environment, such as signals from other cells. Although all of the steps involved in expressing a gene can in principle be regulated, for most genes the initiation of RNA transcription provides the most important point of control.


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Cell_Biology_Alberts. How does a cell determine which of its thousands of genes to transcribe? Perhaps the most important concept, one that applies to all species on Earth, is based on a group of proteins known as transcription regulators. These proteins recognize specific sequences of DNA (typically 5–10 nucleotide pairs in length) that are often called cis-regulatory sequences, because they must be on the same chromosome (that is, in cis) to the genes they control. Transcription regulators bind to these sequences, which are dispersed throughout genomes, and this binding puts into motion a series of reactions that ultimately specify which genes are to be transcribed and at what rate. Approximately 10% of the protein-coding genes of most organisms are devoted to transcription regulators, making them one of the largest classes of proteins in the cell. In most cases, a given transcription regulator recognizes its own cis-regulatory sequence, which is different from those recognized by all the other


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Cell_Biology_Alberts. of the largest classes of proteins in the cell. In most cases, a given transcription regulator recognizes its own cis-regulatory sequence, which is different from those recognized by all the other regulators in the cell.


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Cell_Biology_Alberts. Transcription of each gene is, in turn, controlled by its own collection of cis-regulatory sequences. These typically lie near the gene, often in the intergenic region directly upstream from the transcription start point of the gene. Although a few genes are controlled by a single cis-regulatory sequence that is recognized by a single transcription regulator, the majority have complex arrangements of cis-regulatory sequences, each of which is recognized by a different transcription regulator. It is therefore the positions, identity, and arrangement of cis-regulatory sequences—which are an important part of the information embedded in the genome—that ultimately determine the time and place that each gene is transcribed. We begin our discussion by describing how transcription regulators recognize cis-regulatory sequences. the Sequence of nucleotides in the DnA Double helix Can Be read by proteins


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Cell_Biology_Alberts. We begin our discussion by describing how transcription regulators recognize cis-regulatory sequences. the Sequence of nucleotides in the DnA Double helix Can Be read by proteins As discussed in Chapter 4, the DNA in a chromosome consists of a very long double helix that has both a major and a minor groove (Figure 7–6). Transcription regulators must recognize short, specific cis-regulatory sequences within Figure 7–5 Six steps at which eukaryotic gene expression can be controlled. Controls that operate at steps 1 through 5 are discussed in this chapter. Step 6, the regulation of protein activity, occurs largely through covalent post-translational modifications including phosphorylation, acetylation, and ubiquitylation (see table 3–3, p. 165). Step 6 was introduced in Chapter 3 and is subsequently discussed in many chapters throughout the book.


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Cell_Biology_Alberts. Figure 7–6 Double-helical structure of DNA. A space-filling model of DnA showing the major and minor grooves on the outside of the double helix (see Movie 4.1). the atoms are colored as follows: carbon, dark blue; nitrogen, light blue; hydrogen, white; oxygen, red; phosphorus, yellow.


Cell_Biology_Alberts

Cell_Biology_Alberts. this structure. When first discovered in the 1960s, it was thought that these proteins might require direct access to the interior of the double helix to distinguish between one DNA sequence and another. It is now clear, however, that the outside of the double helix is studded with DNA sequence information that transcription regulators recognize: the edge of each base pair presents a distinctive pattern of hydrogen-bond donors, hydrogen-bond acceptors, and hydrophobic patches in both the major and minor grooves (Figure 7–7). Because the major groove is wider and displays more molecular features than does the minor groove, nearly all transcription regulators make the majority of their contacts with the major groove—as we shall see.


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Cell_Biology_Alberts. Molecular recognition in biology generally relies on an exact fit between the surfaces of two molecules, and the study of transcription regulators has provided some of the clearest examples of this principle. A transcription regulator recognizes a specific cis-regulatory sequence because the surface of the protein is extensively complementary to the special surface features of the double helix that displays that sequence. Each transcription regulator makes a series of contacts with the DNA, involving hydrogen bonds, ionic bonds, and hydrophobic interactions. Although each individual contact is weak, the 20 or so contacts that are typically formed at the protein–DNA interface add together to ensure that the interaction is both highly specific and very strong (Figure 7–8). In fact, DNA–protein


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Cell_Biology_Alberts. Figure 7–7 How the different base pairs in DNA can be recognized from their edges without the need to open the double helix. the four possible configurations of base pairs are shown, with potential hydrogen-bond donors indicated in blue, potential hydrogen-bond acceptors in red, and hydrogen bonds of the base pairs themselves as a series of short, parallel red lines. Methyl groups, which form hydrophobic protuberances, are shown in yellow, and hydrogen atoms that are attached to carbons, and are therefore unavailable for hydrogen-bonding, are white. from the major groove, each of the four base-pair configurations projects a unique pattern of features. (from C. Branden and J. tooze, introduction to protein Structure, 2nd ed. new York: Garland publishing, 1999.) outer limit of sugar-phosphate of DNAbackbone on outside of double helix interactions include some of the tightest and most specific molecular interactions known in biology.


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Cell_Biology_Alberts. Although each example of protein–DNA recognition is unique in detail, x-ray crystallographic and nuclear magnetic resonance (NMR) spectroscopic studies of hundreds of transcription regulators have revealed that many of them contain one or another of a small set of DNA-binding structural motifs (Panel 7–1). These motifs generally use either α helices or β sheets to bind to the major groove of DNA. The amino acid side chains that extend from these protein motifs make the specific contacts with the DNA. Thus, a given structural motif can be used to recognize many different cis-regulatory sequences depending on the specific side chains present. Dimerization of transcription regulators increases their Affinity and Specificity for DnA


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Cell_Biology_Alberts. Dimerization of transcription regulators increases their Affinity and Specificity for DnA A monomer of a typical transcription regulator recognizes about 6–8 nucleotide pairs of DNA. However, sequence-specific DNA-binding proteins do not bind tightly to a single DNA sequence and reject all others; rather, they recognize a range of closely related sequences, with the affinity of the protein for the DNA varying according to how closely the DNA matches the optimal sequence. Hence, cis-regulatory sequences are often depicted as “logos” which display the range of sequences recognized by a particular transcription regulator (Figure 7–9A and B). In Chapter 6, we saw this same representation at work for the binding of RNA polymerase to promoters (see Figure 6–12).


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Cell_Biology_Alberts. The DNA sequence recognized by a monomer does not contain sufficient information to be picked out from the background of such sequences that would occur at random all over the genome. For example, an exact six-nucleotide DNA sequence would be expected to occur by chance approximately once every 4096 nucleotides (46), and the range of six-nucleotide sequences described by a typical logo would be expected to occur by chance much more often, perhaps every 1000 nucleotides. Clearly, for a bacterial genome of 4.6 × 106 nucleotide pairs, not to mention a mammalian genome of 3 × 109 nucleotide pairs, this is insufficient information to accurately control the transcription of individual genes. Additional contributions to DNA-binding specificity must therefore be present. Many transcription regulators form dimers, with both monomers making nearly identical contacts with DNA (Figure 7–9C). This arrangement doubles the length of the cis-regulatory sequence recognized and greatly increases both


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Cell_Biology_Alberts. form dimers, with both monomers making nearly identical contacts with DNA (Figure 7–9C). This arrangement doubles the length of the cis-regulatory sequence recognized and greatly increases both the affinity and the specificity of transcription regulator binding. Because the DNA sequence


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Cell_Biology_Alberts. Figure 7–8 The binding of a transcription regulator to a specific DNA sequence. On the left, a single contact is shown between a transcription regulator and DnA; such contacts allow the protein to “read” the DnA sequence. On the right, the complete set of contacts between a transcription regulator (a member of the homeodomain family—see panel 7–1) and its cis-regulatory sequence is shown. the DnA-binding portion of the protein is 60 amino acids long. Although the interactions in the major groove are the most important, the protein is also seen to contact both the minor groove and phosphates in the sugar–phosphate DnA backbone. (See C. wolberger et al., Cell 67:517–528, 1991.) tryptophan repressor lambda Cro lambda repressor fragment CAP fragment DNA DNA 3.4 nm (A) Arg C 3 1 2 N 2 Ser Arg Asn 3 1 (B) Originally identifed in bacterial transcription regulators, this motif has since been found in many hundreds of DNA-binding proteins from both eukaryotes and prokaryotes. It is constructed


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Cell_Biology_Alberts. 3 1 (B) Originally identifed in bacterial transcription regulators, this motif has since been found in many hundreds of DNA-binding proteins from both eukaryotes and prokaryotes. It is constructed from two ˜ helices (blue and red) connected by a short extended chain of amino acids, which constitutes the “turn.” The two helices are held at a fxed angle, primarily through interactions between the two helices. The more C-terminal helix (in red) is called the recognition helix because it fts into the major groove of DNA; its amino acid side chains, which differ from protein to protein, play an important part in recognizing the specifc DNA sequence to which the protein binds. All of the proteins shown here bind DNA as dimers in which the two copies of the recognition helix (in red) are separated by exactly one turn of the DNA helix (3.4 nm); thus both recognition helices of the dimer can ft into the major groove of DNA. Not long after the frst transcription regulators were discovered in


Cell_Biology_Alberts

Cell_Biology_Alberts. by exactly one turn of the DNA helix (3.4 nm); thus both recognition helices of the dimer can ft into the major groove of DNA. Not long after the frst transcription regulators were discovered in bacteria, genetic analyses of the fruit fy Drosophila led to the characterization of an important class of genes, the homeotic selector genes, that play a critical part in orchestrating fy development (discussed in Chapter 21). It was later shown that these genes coded for transcription regulators that bound DNA through a structural motif named the homeodomain. Two different views of the same structure are shown. (A) The homeodomain is folded into three ˜ helices, which are packed tightly together by hydrophobic interactions. The part containing helices 2 and 3 closely resembles the helix–turn–helix motif. (B) The recognition helix (helix 3, red) forms important contacts with the major groove of DNA. The asparagine (Asn) of helix 3, for example, contacts an adenine, as shown in Figure 7–8. A


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Cell_Biology_Alberts. motif. (B) The recognition helix (helix 3, red) forms important contacts with the major groove of DNA. The asparagine (Asn) of helix 3, for example, contacts an adenine, as shown in Figure 7–8. A fexible arm attached to helix 1 forms contacts with nucleotide pairs in the minor groove. The leucine zipper motif is named because of the way the two ˜helices, one from each monomer, are joined together to form a short coiled-coil. These proteins bind DNA as dimers where the two long ˜helices are held together by interactions between hydrophobic amino acid side chains (often on leucines) that extend from one side of each helix. Just beyond the dimerization interface, the two ˜helices separate from each other to form a Y-shaped structure, which allows their side chains to contact the major groove of DNA. The dimer thus grips the double helix like a clothespin on a clothesline. recognition helix recognition helix DNA dimerization interface HELIX–TURN–HELIX PROTEINS HOMEODOMAIN PROTEINS LEUCINE


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Cell_Biology_Alberts. of DNA. The dimer thus grips the double helix like a clothespin on a clothesline. recognition helix recognition helix DNA dimerization interface HELIX–TURN–HELIX PROTEINS HOMEODOMAIN PROTEINS LEUCINE ZIPPER PROTEINS 376 PANEL 7–1: Common Structural Motifs in Transcription Regulators


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Cell_Biology_Alberts. In the other DNA-binding motifs displayed in this panel, ˜ helices are the primary mechanism used to recognize specifc DNA sequences. In one large group of transcription regulators, however, a two-stranded ° sheet, with amino acid side chains extending from the sheet toward the DNA, reads the information on the surface of the major groove. As in the case of a recognition ˜ helix, this °-sheet motif can be used to recognize many different DNA sequences; the exact DNA sequence recognized depends on the sequence of amino acids that make up the ° sheet. Shown is a transcription regulator that binds two molecules of S-adenosyl methionine (red). On the left is a dimer of the protein; on the right is a simplifed diagram showing just the two-stranded ° sheet bound to the major groove of DNA. This group of DNA-binding motifs includes one or more zinc atoms as structural components. All such zinc-coordinated DNA-binding motifs are called zinc fngers, referring to their appearance in early


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Cell_Biology_Alberts. This group of DNA-binding motifs includes one or more zinc atoms as structural components. All such zinc-coordinated DNA-binding motifs are called zinc fngers, referring to their appearance in early schematic drawings (left). They fall into several distinct structural groups, only one of which we consider here. It has a simple structure, in which the zinc atom holds an ˜ helix and a ° sheet together (middle). This type of zinc fnger is often found in clusters with the ˜ helix of each fnger contacting the major groove of the DNA, forming a nearly continuous stretch of ˜ helices along that groove. In this way, a strong and specifc DNA–protein interaction is built up through a repeating basic structural unit. Three such fngers are shown on the right.N C C N C His Cys Zn His N Cys CCGLEHHZnKYKNQRVRRSFKEVLSASCOOHNH2COOHNH2ZnZnZnDNADNA° SHEET DNA RECOGNITION PROTEINS ZINC FINGER PROTEINS 377