In the previous, we described the components of genetic switches— regulatory proteins and the specific sequences that these proteins recognize. We shall now discuss how these components operate to turn genes on and off in response to a variety of signals.Only 40 years ago the idea that genes could be switched on and off was revolutionary.
This concept was a major advance, and it came originally from the study of how E. Coli bacteria adapt to changes in the composition of their growth medium. Parallel studies on the lambda bacteriophage led to many of the same conclusions and helped to establish the underlying mechanism. Many of the same principles apply to eucaryotic cells. However, the enormous complexity of regulation in higher organisms, combined with the packaging of their into, creates special challenges and some novel opportunities for control—as we shall see. We begin with the simplest example—an on-off switch in bacteria that responds to a single signal. The Tryptophan Repressor Is a Simple Switch That Turns Genes On and Off in BacteriaThe of the bacterium E.
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Coli, a single-celled organism, consists of a single circular of about 4.6 × 10 6 pairs. This DNA encodes approximately 4300 proteins, although only a fraction of these are made at any one time. The of many of them is regulated according to the available food in the environment. This is illustrated by the five E. Coli genes that code for enzymes that manufacture the tryptophan.
These genes are arranged as a single; that is, they are adjacent to one another on the chromosome and are transcribed from a single as one long molecule. But when tryptophan is present in the growth medium and enters the cell (when the bacterium is in the gut of a mammal that has just eaten a meal of, for example), the cell no longer needs these enzymes and shuts off their production. The clustered genes in E. Coli that code for enzymes that manufacture the amino acid tryptophan. These five genes are transcribed as a single mRNA molecule, a feature that allows their expression to be controlled coordinately.
Clusters of genes transcribedThe molecular basis for this switch is understood in considerable detail. As described in Chapter 6, a is a specific sequence that directs to bind to DNA, to open the DNA, and to begin synthesizing an RNA. Within the promoter that directs transcription of the tryptophan biosynthetic genes lies a regulating element called an (see ).
This is simply a short region of regulatory DNA of defined sequence that is recognized by a, in this case the tryptophan repressor, a member of the helix-turn-helix family (see ). The promoter and are arranged so that when the tryptophan repressor occupies the operator, it blocks access to the promoter by RNA polymerase, thereby preventing of the tryptophan-producing enzymes. Switching the tryptophan genes on and off.
If the level of tryptophan inside the cell is low, RNA polymerase binds to the promoter and transcribes the five genes of the tryptophan (trp) operon. If the level of tryptophan is high, however, the tryptophanThe block to is regulated in an ingenious way: to bind to its, the has to have two molecules of the tryptophan bound to it. As shown in, tryptophan binding tilts the helix-turn-helix of the repressor so that it is presented properly to the DNA major groove; without tryptophan, the motif swings inward and the protein is unable to bind to the operator. Thus the tryptophan repressor and operator form a simple device that switches production of the tryptophan biosynthetic enzymes on and off according to the availability of free tryptophan.
Because the active, DNA-binding form of the protein serves to turn genes off, this mode of gene regulation is called, and the gene regulatory proteins that function in this way are called transcriptional repressors or gene repressor proteins. Transcriptional Activators Turn Genes OnWe saw in Chapter 6 that purified E. Coli (including the σ ) can bind to a and initiate. Some bacterial promoters, however, are only marginally functional on their own, either because they are recognized poorly by RNA polymerase or because the polymerase has difficulty opening the DNA helix and beginning transcription. In either case these poorly functioning promoters can be rescued by regulatory proteins that bind to a nearby site on the DNA and contact the RNA polymerase in a way that dramatically increases the probability that a will be initiated. Because the active, DNA-binding form of such a turns genes on, this mode of gene regulation is called, and the gene regulatory proteins that function in this manner are known as transcriptional activators or gene activator proteins. In some cases, bacterial gene activator proteins aid RNA polymerase in binding to the promoter by providing an additional contact surface for the polymerase.
In other cases, they facilitate the transition from the initial DNA-bound of polymerase to the actively transcribing form, perhaps by stabilizing a.As in by a transcriptional, a transcriptional activator can operate as part of a simple on-off genetic switch. The bacterial activator CAP (catabolite activator protein), for example, activates genes that enable E. Coli to use alternative carbon sources when, its preferred carbon source, is not available. Falling levels of glucose induce an increase in the intracellular signaling cyclic AMP, which binds to the CAP protein, enabling it to bind to its specific sequence near target promoters and thereby turn on the appropriate genes.
In this way the of a target is switched on or off, depending on whether cyclic AMP levels in the cell are high or low, respectively. Summarizes the different ways that positive and negative control can be used to regulate genes. Summary of the mechanisms by which specific gene regulatory proteins control gene transcription in procaryotes. (A) Negative regulation; (B) positive regulation. Note that the addition of an “inducing” ligand can turn on a gene eitherIn many respects transcriptional activators and transcriptional repressors are similar in design. The tryptophan and the transcriptional activator CAP, for example, both use a helix-turn-helix (see ) and both require a small in order to bind. In fact, some bacterial proteins (including CAP and the bacteriophage lambda repressor) can act as either activators or repressors, depending on the exact placement of the DNA sequence they recognize in relation to the: if the for the overlaps the promoter, the polymerase cannot bind and the protein acts as a repressor.
A Transcriptional Activator and a Transcriptional Repressor Control the lac OperonMore complicated types of genetic switches combine positive and negative controls. The lac in E.
Coli, for example, unlike the trp operon, is under both negative and positive transcriptional controls by the lac and CAP, respectively. The lac operon codes for proteins required to transport the lactose into the cell and to break it down. CAP, as we have seen, enables bacteria to use alternative carbon sources such as lactose in the absence of.
It would be wasteful, however, for CAP to induce of the lac operon if lactose is not present, and the lac repressor ensures that the lac operon is shut off in the absence of lactose. This arrangement enables the control region of lac operon to respond to and integrate two different signals, so that the operon is highly expressed only when two conditions are met: lactose must be present and glucose must be absent. Any of the other three possible signal combinations maintain the cluster of genes in the off state. Dual control of the lacoperon.
Glucose and lactose levels control the initiation of transcription of the lac operon through their effects on the lac repressor protein and CAP. Lactose addition increases the concentration of allolactose, which binds toThe simple logic of this genetic switch first attracted the attention of biologists over 50 years ago. As explained above, the molecular basis of the switch was uncovered by a combination of genetics and biochemistry, providing the first insight into how is controlled. Although the same strategies are used to control gene expression in higher organisms, the genetic switches that are used are usually much more. Regulation of Transcription in Eucaryotic Cells Is ComplexThe two-signal switching mechanism that regulates the lac is elegant and simple. However, it is difficult to imagine how it could grow in complexity to allow dozens of signals to regulate transcription from the operon: there is not enough room in the neighborhood of the to pack in a sufficient number of regulatory sequences.
How then have eucaryotes overcome such limitations to create their more genetic switches?The regulation of transcription in eucaryotes differs in three important ways from that typically found in bacteria. Second, as we saw in the last chapter, eucaryotic II, which transcribes all -coding genes, cannot initiate transcription on its own. It requires a set of proteins called general transcription factors, which must be assembled at the before transcription can begin. (The term “general” refers to the fact that these proteins assemble on all promoters transcribed by RNA polymerase II; in this they differ from regulatory proteins, which act only at particular genes.) This assembly process provides, in principle, multiple steps at which the rate of transcription initiation can be speeded up or slowed down in response to regulatory signals, and many eucaryotic gene regulatory proteins influence these steps. Eucaryotic Gene Regulatory Proteins Control Gene Expression from a DistanceLike bacteria, eucaryotes use regulatory proteins (activators and repressors) to regulate the of their genes but in a somewhat different way. The sites to which the eucaryotic gene activators bound were originally termed, since their presence “enhanced,” or increased, the rate of transcription dramatically.
It came as a surprise when, in 1979, it was discovered that these activator proteins could be bound thousands of pairs away from the. Moreover, eucaryotic activators could influence transcription of a gene when bound either upstream or downstream from it. How do sequences and the proteins bound to them function over these long distances? How do they communicate with the promoter?Many models for “action at a distance” have been proposed, but the simplest of these seems to apply in most cases. The between the and the loops out to allow the activator proteins bound to the enhancer to come into contact with proteins (, one of the general transcription factors, or other proteins) bound to the promoter (see ). The DNA thus acts as a tether, helping a bound to an enhancer even thousands of pairs away to interact with the of proteins bound to the promoter. This phenomenon also occurs in bacteria, although less commonly and over much shorter lengths of DNA.
A Eucaryotic Gene Control Region Consists of a Promoter Plus Regulatory DNA SequencesBecause eucaryotic regulatory proteins can control transcription when bound to far away from the, the DNA sequences that control the of a gene are often spread over long stretches of DNA. We shall use the term to refer to the whole expanse of DNA involved in regulating transcription of a gene, including the, where the general transcription factors and the polymerase assemble, and all of the to which gene regulatory proteins bind to control the rate of the assembly processes at the promoter.
In higher eucaryotes it is not unusual to find the regulatory sequences of a gene dotted over distances as great as 50,000 pairs. Although much of this DNA serves as “spacer” sequence and is not recognized by gene regulatory proteins, this spacer DNA may facilitate transcription by providing the flexibility needed for communication between DNA-bound proteins.
It is also important to keep in mind that, like other regions of eucaryotic chromosomes, much of the DNA in gene control regions is packaged into nucleosomes and higher-order forms of, thereby compacting its length. The gene control region of a typical eucaryotic gene. The promoter is the DNA sequence where the general transcription factors and the polymerase assemble (see Figure 6-16). The regulatory sequences serve as binding sites for gene regulatory proteins,In this chapter we generally use the term to refer only to a segment of that is transcribed into (see ).
However, the classical view of a would include the as well. The different definitions arise from the different ways in which genes were historically identified.
The discovery of has further complicated the definition of a gene—a point we discussed briefly in Chapter 6 and will return to later in this chapter.Although many regulatory proteins bind to sequences and activate gene transcription, many others function as negative regulators, as we see below. In contrast to the small number of general transcription factors, which are abundant proteins that assemble on the promoters of all genes transcribed by II, there are thousands of different gene regulatory proteins. For example, of the roughly 30,000 human genes, an estimated 5–10% encode gene regulatory proteins. These regulatory proteins vary from one to the next, and each is usually present in very small amounts in a cell, often less than 0.01% of the total. Most of them recognize their specific sequences using one of the DNA-binding motifs discussed previously, although as we discuss below, some do not recognize DNA directly but instead assemble on other DNA-bound proteins.The regulatory proteins allow the individual genes of an organism to be turned on or off specifically. Different selections of gene regulatory proteins are present in different cell types and thereby direct the patterns of gene that give each cell type its unique characteristics. Each gene in a eucaryotic cell is regulated differently from nearly every other gene.
Given the number of genes in eucaryotes and the complexity of their regulation, it has been difficult to formulate simple rules for gene regulation that apply in every case. We can, however, make some generalizations about how gene regulatory proteins, once bound to a on, influence the rate of transcription initiation, as we now explain.
Eucaryotic Gene Activator Proteins Promote the Assembly of RNA Polymerase and the General Transcription Factors at the Startpoint of TranscriptionMost regulatory proteins that activate gene transcription—that is, most —have a modular design consisting of at least two distinct domains. One usually contains one of the structural motifs discussed previously that recognizes a specific regulatory sequence. In the simplest cases, a second domain—sometimes called an activation domain—accelerates the rate of transcription initiation. This type of modular design was first revealed by experiments in which genetic engineering techniques were used to create a hybrid containing the activation domain of one protein fused to the DNA-binding domain of a different protein. The modular structure of a gene activator protein. Outline of an experiment that reveals the presence of independent DNA-binding and transcription-activating domains in the yeast gene activator protein Gal4. A functional activator can be reconstitutedOnce bound to, how do eucaryotic activator proteins increase the rate of transcription initiation?
As we will see shortly, there are several mechanisms by which this can occur, and, in many cases, these different mechanisms work in concert at a single. But, regardless of the precise biochemical pathway, the main function of activators is to attract, position, and modify the general transcription factors and II at the promoter so that transcription can begin. They do this both by acting directly on the transcription machinery itself and by changing the structure around the promoter.We consider first the ways in which activators directly influence the positioning of the general transcription factors and at promoters and help kick them into action. Although the general transcription factors and RNA polymerase II assemble in a stepwise, prescribed order (see ), there are cases in living cells where some of them are brought to the as a large pre-assembled that is sometimes called the. In addition to some of the general transcription factors and RNA polymerase, the holoenzyme typically contains a 20- complex called the mediator, which was first identified biochemically as being required for activators to stimulate transcription initiation.Many activator proteins interact with the holoenzyme and thereby make it more energetically favorable for it to assemble on a that is linked through to the site where the activator is bound.
In this sense, eucaryotic activators resemble those of bacteria in helping to attract and position on specific sites on DNA (see ). One type of experiment that supports the idea that activators attract the holoenzyme complex to promoters creates an “activator bypass”. Here, a sequence-specific DNA-binding is experimentally fused directly to a component of the mediator; this hybrid protein, which lacks an activation domain, strongly stimulates transcription initiation when the DNA sequence to which it binds is placed in proximity to a promoter. Activation of transcription initiation in eucaryotes by recruitment of the eucaryotic RNA polymerase II holoenzyme complex. (A) An activator protein bound in proximity to a promoter attracts the holoenzyme complex to the promoter. According to this model,Although recruitment of the holoenzyme to promoters provides a conceptually simple mechanism for envisioning activation, the effect of activators on the holoenzyme complex is probably more complicated.
For example, a stepwise assembly of the general transcription factors (see ) may occur on some promoters. On others, their rearrangement, once brought to as part of the holoenzyme, may be required.
In addition, most forms of the holoenzyme complex lacks some of the general transcription factors (notably TFIID and TFIIA), and these must be assembled on the separately (see ). In principle, any of these assembly processes could be a slow step on the pathway to transcription initiation, and activators could facilitate their completion. In fact, many activators have been shown to interact with one or more of the general transcription factors, and several have been shown to directly accelerate their assembly at the promoter. Eucaryotic Gene Activator Proteins Modify Local Chromatin StructureIn addition to their direct actions in assembling the holoenzyme and the general transcription factors on, activator proteins also promote transcription initiation by changing the structure of the regulatory sequences and promoters of genes. As we saw in Chapter 4, the two most important ways of locally altering chromatin structure are through covalent modifications and remodeling (see and ). Many gene activator proteins make use of both these mechanisms by binding to and thereby recruiting histone transferases (HATs), commonly known as histone acetylases, and ATP-dependent chromatin remodeling complexes to work on nearby chromatin.
In general terms, the local alterations in chromatin structure that ensue allow greater accessibility to the underlying DNA. This accessibility facilitates the assembly of the general transcription factors and the RNA polymerase holoenzyme at the, and it also allows the binding of additional gene regulatory proteins to the control region of the gene. Two specific ways that local histone acetylation can stimulate transcription initiation.
(A) Some gene activator proteins can bind directly to DNA that is packaged in unmodified chromatin. By attracting histone acetylases (and nucleosome remodeling complexes),The general transcription factors seem unable to assemble onto a that is packaged in a conventional. In fact, such packaging may have evolved in part to ensure that leaky, or, transcription initiation (initiation at a promoter in the absence of bound upstream of it) does not occur. As well as making the more generally accessible, local acetylation has a more specialized role in promoting transcription initiation. As discussed in Chapter 4 (see ), certain patterns of histone acetylation are associated with transcriptionally active, and gene activator proteins, by recruiting histone acetylases, produce these patterns.
One such pattern is directly recognized by one of the subunits of the TFIID, and this recognition apparently helps the factor assemble DNA that is packaged in chromatin. Thus gene activator proteins, through the action of histone acetylases, can indirectly aid in the assembly of the general transcription factors at a promoter and thereby stimulate transcription initiation.
Gene Activator Proteins Work SynergisticallyWe have seen that eucaryotic activator proteins can influence several different steps in transcription initiation, and this property has important consequences when different activator proteins work together. In general, where several factors work together to enhance a rate, the joint effect is generally not merely the sum of the enhancements caused by each factor alone, but the product. If, for example, factor A lowers the free-energy barrier for a reaction by a certain amount and thereby speeds up the reaction 100-fold, and factor B, by acting on another aspect of the reaction, does likewise, then A and B acting in parallel will lower the barrier by a double amount and speed up the reaction 10,000-fold. Similar multiplicative effects occur if A and B speed the reaction by each helping to recruit necessary proteins to the reaction site. Thus, gene activator proteins often exhibit what is called transcriptional synergy, where the transcription rate produced by several activator proteins working together is much higher than that produced by any of the activators working alone.
Transcriptional synergy is observed both between different gene activator proteins bound upstream of a gene and between multiple -bound molecules of the same activator. It is therefore not difficult to see how multiple gene regulatory proteins, each binding to a different regulatory DNA sequence, could control the final rate of transcription of a eucaryotic gene. Transcriptional synergy.
In this experiment, the rate of transcription produced by three experimentally constructed regulatory regions is compared in a eucaryotic cell. Transcriptional synergy, the greater than additive effect of the activators, is observedSince activator proteins can influence many different steps on the pathway to transcriptional activation, it is worth considering whether these steps always occur in a prescribed order. For example does remodeling necessarily precede acetylation or vice versa? When does recruitment of the holoenzyme occur relative to the chromatin modifying steps?
The answers to these questions appears to be different for different genes—and even for the same gene under different conditions. Whatever the precise mechanisms and the order in which they are carried out, a must be bound to either directly or indirectly to influence transcription of its target, and the rate of transcription of a gene ultimately depends upon the spectrum of regulatory proteins bound upstream and downstream of its transcription start site. Eucaryotic Gene Repressor Proteins Can Inhibit Transcription in Various WaysLike bacteria, eucaryotes use in addition to activator proteins to regulate transcription of their genes. However, because of differences in the way transcription is initiated in eucaryotes and bacteria, eucaryotic repressors have many more possible mechanisms of action. For example, we saw in Chapter 4 that whole regions of eucaryotic chromosomes can be packaged into, a form of that is normally resistant to transcription. We will return to this feature of eucaryotic chromosomes later in this chapter.
In addition to molecules that shut down large regions of chromatin, eucaryotic cells also contain regulatory proteins that act only locally to repress transcription of nearby genes. Unlike bacterial repressors, most do not directly compete with the for access to the; rather they work by a variety of other mechanisms, some of which are illustrated in. Like gene activator proteins, many eucaryotic proteins act through more than one mechanism, thereby ensuring robust and efficient repression.
Eucaryotic Gene Regulatory Proteins Often Assemble into Complexes on DNASo far we have been discussing eucaryotic regulatory proteins as though they work as individual polypeptides. In reality, most act as parts of complexes composed of several (and sometimes many) polypeptides, each with a distinct function. These complexes often assemble only in the presence of the appropriate sequence.
In some well-studied cases, for example, two gene regulatory proteins with a weak affinity for each other cooperate to bind to a DNA sequence, neither having a sufficient affinity for DNA to efficiently bind to the DNA site on its own. Once bound to DNA, the protein dimer creates a distinct surface that is recognized by a third protein that carries an activator that stimulates transcription.
This example illustrates an important general point: protein-protein interactions that are too weak to cause proteins to assemble in solution can cause the proteins to assemble on DNA; in this way the DNA sequence acts as a “crystallization” site or seed for the assembly of a protein. Eucaryotic gene regulatory proteins often assemble into complexes on DNA. Seven gene regulatory proteins are shown in (A). The nature and function of the complex they form depends on the specific DNA sequence that seeds their assembly.
In (B), some assembledAn individual can often participate in more than one type of regulatory. A protein might function, for example, in one case as part of a complex that activates transcription and in another case as part of a complex that represses transcription (see ).
Thus individual eucaryotic gene regulatory proteins are not necessarily dedicated activators or repressors; instead, they function as regulatory units that are used to generate complexes whose function depends on the final assembly of all of the individual components. This final assembly, in turn, depends both on the arrangement of control region sequences and on which gene regulatory proteins are present in the cell.Gene regulatory proteins that do not themselves bind but assemble on DNA-bound regulatory proteins are often termed coactivators or corepressors, depending on their effect on transcription initiation. As shown in, the same coactivator or corepressor can assemble on different DNA binding proteins.
Coactivators and corepressors typically carry out multiple functions: they can interact with remodeling complexes, modifying enzymes, the holoenzyme, and several of the general transcription factors.In some cases, the precise sequence to which a regulatory directly binds can affect the of this protein and thereby influence its subsequent transcriptional activity. When bound to one type of DNA sequence, for example, a interacts with a corepressor and ultimately turns off transcription. When bound to a slightly different DNA sequence, it assumes a different conformation and interacts with a coactivator, thereby stimulating transcription.Typically, the assembly of a group of regulatory proteins on is guided by a few relatively short stretches of sequence (see ). However, in some cases, a more elaborate -DNA structure, termed an enhancesome, is formed. A hallmark of enhancesomes is the participation of architectural proteins that bend the DNA by a defined angle and thereby promote the assembly of the other enhancesome proteins. Since formation of the enhancesome requires the presence of many regulatory proteins, it provides a simple way to ensure that a gene is expressed only when the correct combination of these proteins is present in the cell. We saw earlier how the formation of gene regulatory heterodimers in solution provides a mechanism for the of gene.
The assembly of larger complexes of gene regulatory proteins on DNA provides a second important mechanism for combinatorial control, offering far richer opportunities. The nonuniform distribution of four gene regulatory proteins in an early Drosophila embryo. At this stage the embryo is a syncytium, with multiple nuclei in a common cytoplasm. Although not illustrated in these drawings, all of these proteins are concentratedThe regulatory sequences of the eve are designed to read the concentrations of gene regulatory proteins at each position along the length of the embryo and to interpret this information in such a way that the eve gene is expressed in seven stripes, each initially five to six nuclei wide and positioned precisely along the - axis of the embryo. How is this remarkable feat of information processing carried out?
Although the molecular details are not yet all understood, several general principles have emerged from studies of eve and other Drosophila genes that are similarly regulated. The seven stripes of the protein encoded by the even-skipped (eve) gene in a developing Drosophila embryo. Two and one-half hours after fertilization, the egg was fixed and stained with antibodies that recognize the Eve protein (green) and antibodiesThe regulatory region of the eve is very large (approximately 20,000 pairs). It is formed from a series of relatively simple regulatory modules, each of which contains multiple regulatory sequences and is responsible for specifying a particular stripe of eve along the embryo.
This modular organization of the eve is revealed by experiments in which a particular regulatory (say, that specifying stripe 2) is removed from its normal setting upstream of the eve gene, placed in front of a reporter gene (see ), and reintroduced into the Drosophila. When developing embryos derived from flies carrying this genetic construct are examined, the reporter gene is found to be expressed in precisely the position of stripe 2 (see ).
Similar experiments reveal the existence of other regulatory modules, each of which specifies one of the other six stripes or some part of the expression pattern that the gene displays at later stages of. Distribution of the gene regulatory proteins responsible for ensuring that eve is expressed in stripe 2. The distributions of these proteins were visualized by staining a developing Drosophila embryo with antibodies directed against each of the four proteinsWe have already discussed two mechanisms of of —heterodimerization of gene regulatory proteins in solution (see ) and the assembly of combinations of gene regulatory proteins into small complexes on (see ). It is likely that both mechanisms participate in the regulation of eve expression.
In addition, the regulation of stripe 2 just described illustrates a third type of combinatorial control. Because the individual regulatory sequences in the eve stripe 2 are strung out along the DNA, many sets of gene regulatory proteins can be bound simultaneously and influence the of a gene. The promoter integrates the transcriptional cues provided by all of the bound proteins. Integration at a promoter. Multiple sets of gene regulatory proteins can work together to influence transcription initiation at a promoter, as they do in the eve stripe 2 module illustrated previously in Figure 7-55.
It is not yet understood in detailThe regulation of eve is an impressive example of. Seven combinations of regulatory proteins—one combination for each stripe—activate eve expression, while many other combinations (all those found in the interstripe regions of the embryo) keep the stripe elements silent. The other stripe regulatory modules are thought to be constructed along lines similar to those described for stripe 2, being designed to read provided by other combinations of gene regulatory proteins. The entire, strung out over 20,000 pairs of, binds more than 20 different proteins. A large and control region is thereby built from a series of smaller modules, each of which consists of a unique arrangement of short DNA sequences recognized by specific gene regulatory proteins. Although the details are not yet understood, these gene regulatory proteins are thought to employ a number of the mechanisms previously described for activators and repressors.
In this way, a single gene can respond to an enormous number of combinatorial inputs. Complex Mammalian Gene Control Regions Are Also Constructed from Simple Regulatory ModulesIt has been estimated that 5–10% of the coding capacity of a mammalian is devoted to the synthesis of proteins that serve as regulators of transcription.
This large number of genes reflects the exceedingly network of controls governing of mammalian genes. Each gene is regulated by a set of gene regulatory proteins; each of those proteins is the product of a gene that is in turn regulated by a whole set of other proteins, and so on.
Moreover, the regulatory molecules are themselves influenced by signals from outside the cell, which can make them active or inactive in a whole variety of ways. Thus, pattern of gene expression in a cell can be viewed as the result of a complicated molecular computation that the intracellular gene control network performs in response to information from the cell's surroundings. We shall discuss this further in Chapter 21, dealing with multicellular, but the complexity is remarkable even at the level of the individual genetic switch, regulating activity of a single gene.
It is not unusual, for example, to find a mammalian gene with a control region that is 50,000 pairs in length, in which many modules, each containing a number of regulatory sequences that bind gene regulatory proteins, are interspersed with long stretches of spacer. Some ways in which the activity of gene regulatory proteins is regulated in eucaryotic cells. (A) The protein is synthesized only when needed and is rapidly degraded by proteolysis so that it does not accumulate. (B) Activation by ligand binding.
(C)One of the best-understood examples of a mammalian regulatory region is found in the human β-globin, which is expressed exclusively in red blood cells and at a specific time in their. A complex array of gene regulatory proteins controls the of the gene, some acting as activators and others as repressors. The concentrations (or activities) of many of these gene regulatory proteins are thought to change during development, and only a particular combination of all the proteins triggers transcription of the gene. The human β-globin gene is part of a cluster of globin genes. The five genes of the cluster are transcribed exclusively in erythroid cells, that is, cells of the lineage. Godzilla monster of monsters neca.
Moreover, each gene is turned on at a different stage of development (see ) and in different organs: the ε-globin gene is expressed in the embryonic sac, γ in the yolk sac and the fetal liver, and δ and β primarily in the adult bone marrow. Each of the globin genes has its own set of regulatory proteins that are necessary to turn the gene on at the appropriate time and tissue. In addition to the individual regulation of each of the globin genes, the entire cluster appears to be subject to a shared control region called a control region (LCR). The LCR lies far upstream from the gene cluster (see ), and we shall discuss its function next. The cluster of β-like globin genes in humans. (A) The large chromosomal region shown spans 100,000 nucleotide pairs and contains the five globin genes and a locus control region (LCR).
(B) Changes in the expression of the β-like globinIn cells where the globin genes are not expressed (such as brain or skin cells), the whole cluster appears tightly packaged into. In erythroid cells, by contrast, the entire gene cluster is still folded into nucleosomes, but the higher-order packing of the chromatin has become decondensed This change occurs even before the individual globin genes are transcribed, suggesting that there are two steps of regulation. In the first, the chromatin of the entire globin becomes decondensed, which is presumed to allow additional gene regulatory proteins access to the. In the second step, the remaining gene regulatory proteins assemble on the DNA and direct the of individual genes.The LCR appears to act by controlling condensation, and its importance can be seen in patients with a certain type of thalassemia, a severe inherited form of anemia. In these patients, the β-globin is found to have undergone deletions that remove all or part of the LCR, and although the β-globin and its nearby regulatory regions are intact, the gene remains transcriptionally silent even in erythroid cells. Deus ex machina examples.
Moreover, the β-globin gene in the erythroid cells fails to undergo the normal chromatin decondensation step that occurs during erythroid cell.Many LCRs (that is, regulatory sequences that regulate the accessibility and of distant genes or clusters) are present in the human, and they regulate a wide variety of cell-type specific genes. The way in which they function is not understood in detail, but several models have been proposed. The simplest is based on principles we have already discussed in this chapter: the gene regulatory proteins that bind to the LCR interact through DNA looping with proteins bound to the control regions of the genes they regulate. In this way, the proteins bound at the LCR could attract remodeling complexes and modifying enzymes that could alter the chromatin structure of the before transcription begins. Other models for LCRs propose a mechanism by which proteins initially bound at the LCR attract other proteins that assemble cooperatively and therefore spread along the DNA toward the genes they control, modifying the chromatin as they proceed. Insulators Are DNA Sequences That Prevent Eucaryotic Gene Regulatory Proteins from Influencing Distant GenesAll genes have control regions, which dictate at which times, under what conditions, and in what tissues the will be expressed. We also have seen that eucaryotic gene regulatory proteins can act across very long stretches of.
How then are control regions of different genes kept from interfering with one another? In other words, what keeps a bound on the control region of one gene from inappropriately influencing transcription of adjacent genes?Several mechanisms have been proposed to account for this regulatory compartmentalization, but the best understood rely on, also called boundary elements.
Insulator elements (insulators, for short) are sequences that bind specialized proteins and have two specific properties. First, they buffer genes from the repressing effects of.
When a (from a fly or a mouse, for example) and its normal control region is inserted into different positions in the, it is often expressed at levels that vary depending on its site of insertion in the genome and are especially low when it is inserted amid heterochromatin. We saw an example of this in Chapter 4, where genes inserted into heterochromatin are transcriptionally silenced (see ). When insulator elements that flank the gene and its control region are included, however, the gene is usually expressed normally, irrespective of its new position in the genome. The second property of insulators is in some sense the converse of this: they can block the action of enhancers (see ). For this to occur, the insulator must be located between the and the of the target gene. Schematic diagram summarizing the properties of insulators. Insulators both prevent the spread of heterochromatin (right-hand side of diagram) and directionally block the action of enhancers (left-hand side).
Thus gene B is properly regulated and geneThus insulators can define domains of, both buffering the gene from outside effects and preventing the control region of the gene (or cluster of genes) from acting outside the. For example, the globin LCR (discussed above) is associated with a neighboring insulator which allows the LCR to influence only the cluster of globin genes. Presumably, another insulator is located on the distal side of the globin cluster, serving to define the other end of the domain.The distribution of insulators in a is therefore thought to divide it into independent domains of regulation and structure. Consistent with this idea, the distribution of insulators across a genome is roughly correlated with variations in chromatin structure. For example, an insulator-binding from flies is localized preferentially to interbands (and also to the edges of puffs) in polytene chromosomes. Localization of a Drosophila insulator-binding protein on polytene chromosomes. A polytene chromosome (see pp.
218–220) was stained with propidium iodide (red) to show its banding patterns—with bands appearing bright red and interbandsThe mechanisms by which insulators work are not currently understood, and different insulators may function in different ways. At least some pairs of insulators may define the basis of a looped chromosomal (see ).
It has been proposed that chromosomes of all eucaryotes are divided by insulators into independent looped domains, each regulated separately from all the others. Bacteria Use Interchangeable RNA Polymerase Subunits to Help Regulate Gene TranscriptionWe have seen the importance of regulatory proteins that bind to regulatory sequences in and signal to the transcription apparatus whether or not to start the synthesis of an chain. Although this is the main way of controlling transcriptional initiation in both eucaryotes and procaryotes, some bacteria and their viruses use an additional strategy based on interchangeable subunits of. As described in Chapter 6, a sigma (σ) is required for the bacterial RNA polymerase to recognize a.
Many bacteria make several different sigma subunits, each of which can interact with the RNA polymerase core and direct it to a different set of promoters. This scheme permits one large set of genes to be turned off and a new set to be turned on simply by replacing one sigma subunit with another; the strategy is efficient because it bypasses the need to deal with the genes one by one.
It is often used subversively by bacterial viruses to take over the host polymerase and activate several sets of viral genes rapidly and sequentially. Interchangeable RNA polymerase subunits as a strategy to control gene expression in a bacterial virus. The bacterial virus SPO1, which infects the bacterium B. Subtilis, uses the bacterial polymerase to transcribe its early genes immediately after theIn a sense, eucaryotes employ an analogous strategy through the use of three distinct polymerases (I, II, and III) that share some of their subunits.
Procaryotes, in contrast, use only one type of core, but they modify it with different sigma subunits. Gene Switches Have Gradually EvolvedWe have seen that the control regions of eucaryotic genes are often spread out over long stretches of, whereas those of procaryotic genes are typically closely packed around the start point of transcription. Several bacterial regulatory proteins, however, recognize DNA sequences that are located many pairs away from the, as we saw in. This case provided one of the first examples of DNA looping in gene regulation and greatly influenced later studies of eucaryotic gene regulatory proteins.It seems likely that the close-packed arrangement of bacterial genetic switches developed from more extended forms of switches in response to the evolutionary pressure on bacteria to maintain a small size.
This compression comes at a price, however, as it restricts the complexity and adaptability of the control device. The extended form of eucaryotic control regions, in contrast, with discrete regulatory modules separated by long stretches of spacer, would be expected to facilitate a reshuffling of the regulatory modules during evolution, both to create new regulatory circuits and to modify old ones. Unraveling the history of how control regions evolved presents a fascinating challenge, and many clues can be found in present-day DNA sequences. We shall take up this issue again at the end of this chapter.
SummaryThe transcription of individual genes is switched on and off in cells by regulatory proteins. In procaryotes these proteins usually bind to specific sequences close to the start site and, depending on the nature of the regulatory and the precise location of its relative to the start site, either activate or repress transcription of the gene. The flexibility of the DNA helix, however, also allows proteins bound at distant sites to affect the RNA polymerase at the by the looping out of the intervening DNA. Such action at a distance is extremely common in eucaryotic cells, where gene regulatory proteins bound to sequences thousands of pairs from the promoter generally control gene. Eucaryotic activators and repressors act by a wide variety of mechanisms—generally causing the local modification of structure, the assembly of the general transcription factors at the promoter, and the recruitment of RNA polymerase.Whereas the transcription of a typical procaryotic is controlled by only one or two gene regulatory proteins, the regulation of higher eucaryotic genes is much more, commensurate with the larger size and the large variety of cell types that are formed. The control region of the Drosophila eve gene, for example, encompasses 20,000 pairs of and has binding sites for over 20 gene regulatory proteins.
Some of these proteins are transcriptional activators, whereas others are transcriptional repressors. These proteins bind to regulatory sequences organized in a series of regulatory modules strung together along the DNA, and together they cause the correct spatial and temporal pattern of gene. Eucaryotic genes and their control regions are often surrounded by insulators, DNA sequences recognized by proteins that prevent cross-talk between independently regulated genes.
How it WorksThe first part of the bind command tells the game what keyboard key to bind the toggle hand command to: i.e. 'bind l' tells the game 'bind what comes next to the L key'. The command 'bind m' would bind what command comes next to the M key, etc.The next part of this command, 'toggle clrighthand 0 1' is the part that toggles the 'right hand setting'. When the 'clrighthand' setting is set to 0, your gun appears on the left.
When the 'clrighthand' setting is set to 1, your gun appears on the right. The 'toggle' command will toggle a setting between two different values. So, after toggle we specify 'clrighthand' (the setting we want the toggle the value of) and then the two settings we want to toggle it between: 0 and 1.