You are doing a genetic engineering experiment. You use restrictions enzymes to cut the regulatorysequences from the lac operon and replace them with the regulatory sequences of the trp operon.Specifically, you will eliminate everything upstream of the beginning of lacZ and replace them withthe trp sequences upstream of the beginning of trpE, including trpR. Now, describe the regulation ofyour ñew constructed gene. What will you do to get expression of the three lac genes?The lac Operon and its Control ElementslaclCAP PlaczlacYlacAgenes5DNAbindingsite3AUGAUGAUGmessenger RNARNA polymerase blocked fromtranscribing trp operonRegulatory genetrp operonTPRPtrpDNAtrpRtrpEtrpDtrpCtrpBtrpARepressor boundto operatorPromoter5'trpR-MRNAR(a) Tryptophan present, repressor bound to operator, operon repressed.When complexed with tryptophan, the repressor protein produced by thetrpR gene binds tightly to the trp operator, thereby preventing RNApolymerase from transcribing the operon genes.Inactive formActive formTof repressorof repressorTryptophan

introduction pGLO is a genetically engineered plasmid which incorporates parts of the arabinose operon to drive the expression of the green fluorescent protein (GFP). The plasmid also has an antibiotic resistance gene ampR. The bacterium can survive in the LB medium and show growth. When ampicillin is added to the medium, the antibiotic kills the bacterium and there is no growth. When ampicillin is added to the medium and the bacterium contains pGLO, the bacterium is provided resistance and survives. When the plate contains arabinose and ampicillin, the bacteria without pGLO will die due to inability to use arabinose and antibiotic ampicillin will kill the bacteria. When the medium contains arabinose and ampicillin, the bacteria with pGLO will survive. It will use arabinose as sugar source because the arabinose operon will produce enzymes to digest the arabinose. The antibiotic resistance gene with the GFP will provide resistance to the bacterium. How Genetic Switches Work In the previous section, we described the basic components of genetic switches—gene regulatory proteins and the specific DNA 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 gene regulation in higher organisms, combined with the packaging of their DNA into chromatin, 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 Bacteria The chromosome of the bacterium E. coli, a single-celled organism, consists of a single circular DNA molecule of about 4.6 × 106 nucleotide pairs. This DNA encodes approximately 4300 proteins, although only a fraction of these are made at any one time. The expression 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 amino acid tryptophan. These genes are arranged as a single operon; that is, they are adjacent to one another on the chromosome and are transcribed from a single promoter as one long mRNA molecule (Figure 7-33). 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 protein, for example), the cell no longer needs these enzymes and shuts off their production. Figure 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 transcribed (more...) The molecular basis for this switch is understood in considerable detail. As described in Chapter 6, a promoter is a specific DNA sequence that directs RNA polymerase to bind to DNA, to open the DNA double helix, and to begin synthesizing an RNA molecule. Within the promoter that directs transcription of the tryptophan biosynthetic genes lies a regulating element called an operator (see Figure 7-33). This is simply a short region of regulatory DNA of defined nucleotide sequence that is recognized by a repressor protein, in this case the tryptophan repressor, a member of the helix-turn-helix family (see Figure 7-14). The promoter and operator are arranged so that when the tryptophan repressor occupies the operator, it blocks access to the promoter by RNA polymerase, thereby preventing expression of the tryptophan-producing enzymes (Figure). Figure 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 tryptophan (more...) The block to gene expression is regulated in an ingenious way: to bind to its operator DNA, the repressor protein has to have two molecules of the amino acid tryptophan bound to it. As shown in , tryptophan binding tilts the helix-turn-helix motif 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 negative control, and the gene regulatory proteins that function in this way are called transcriptional repressors or gene repressor proteins. Figure The binding of tryptophan to the tryptophan repressor protein changes the conformation of the repressor. The conformational change enables this gene regulatory protein to bind tightly to a specific DNA sequence (the operator), thereby blocking transcription (more...) Transcriptional Activators Turn Genes On We saw in Chapter 6 that purified E. coli RNA polymerase (including the σ subunit) can bind to a promoter and initiate DNA transcription. 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 gene 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 transcript will be initiated. Because the active, DNA-binding form of such a protein turns genes on, this mode of gene regulation is called positive control, 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 conformation of polymerase to the actively transcribing form, perhaps by stabilizing a transition state. As in negative control by a transcriptional repressor, a transcriptional activator can operate as part of a simple on-off genetic switch. The bacterial activator protein CAP (catabolite activator protein), for example, activates genes that enable E. coli to use alternative carbon sources when glucose, its preferred carbon source, is not available. Falling levels of glucose induce an increase in the intracellular signaling molecule cyclic AMP, which binds to the CAP protein, enabling it to bind to its specific DNA sequence near target promoters and thereby turn on the appropriate genes. In this way the expression of a target gene is switched on or off, depending on whether cyclic AMP levels in the cell are high or low, respectively. Figure summarizes the different ways that positive and negative control can be used to regulate genes. Figure 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 either (more...) In many respects transcriptional activators and transcriptional repressors are similar in design. The tryptophan repressor and the transcriptional activator CAP, for example, both use a helix-turn-helix motif (see Figure and both require a small cofactor in order to bind DNA. 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 promoter: if the binding site for the protein overlaps the promoter, the polymerase cannot bind and the protein acts as a repressor (Figure 7-37). Figure Some bacterial gene regulatory proteins can act as both a transcriptional activator and a repressor, depending on the precise placement of its binding sites in DNA. An example is the bacteriophage lambda repressor. For some genes, the protein acts as (more...) A Transcriptional Activator and a Transcriptional Repressor Control the lac Operon More complicated types of genetic switches combine positive and negative controls. The lac operon in E. coli, for example, unlike the trp operon, is under both negative and positive transcriptional controls by the lac repressor protein and CAP, respectively. The lac operon codes for proteins required to transport the disaccharide 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 glucose. It would be wasteful, however, for CAP to induce expression 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. Figure Dual control of the lac operon. 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 to (more...) The 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 gene expression is controlled. Although the same basic strategies are used to control gene expression in higher organisms, the genetic switches that are used are usually much more complex.Regulation of Transcription in Eucaryotic Cells Is Complex The two-signal switching mechanism that regulates the lac operon 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 promoter to pack in a sufficient number of regulatory DNA sequences. How then have eucaryotes overcome such limitations to create their more complex genetic switches? The regulation of transcription in eucaryotes differs in three important ways from that typically found in bacteria. First, eucaryotes make use of gene regulatory proteins that can act even when they are bound to DNA thousands of nucleotide pairs away from the promoter that they influence, which means that a single promoter can be controlled by an almost unlimited number of regulatory sequences scattered along the DNA. Second, as we saw in the last chapter, eucaryotic RNA polymerase II, which transcribes all protein-coding genes, cannot initiate transcription on its own. It requires a set of proteins called general transcription factors, which must be assembled at the promoter 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 gene 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. Third, the packaging of eucaryotic DNA into chromatin provides opportunities for regulation not available to bacteria. Having discussed the general transcription factors for RNA polymerase II in Chapter 6 (see pp. 309–312), we focus here on the first and third of these features and how they are used to control eucaryotic gene expression selectively. Go to: Eucaryotic Gene Regulatory Proteins Control Gene Expression from a Distance Like bacteria, eucaryotes use gene regulatory proteins (activators and repressors) to regulate the expression of their genes but in a somewhat different way. The DNA sites to which the eucaryotic gene activators bound were originally termed enhancers, 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 nucleotide pairs away from the promoter. Moreover, eucaryotic activators could influence transcription of a gene when bound either upstream or downstream from it. How do enhancer 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 DNA between the enhancer and the promoter loops out to allow the activator proteins bound to the enhancer to come into contact with proteins (RNA polymerase, one of the general transcription factors, or other proteins) bound to the promoter (see Figure ). The DNA thus acts as a tether, helping a protein bound to an enhancer even thousands of nucleotide pairs away to interact with the complex of proteins bound to the promoter (Figure . This phenomenon also occurs in bacteria, although less commonly and over much shorter lengths of DNA . Figure Binding of two proteins to separate sites on the DNA double helix can greatly increase their probability of interacting. (A) The tethering of one protein to the other via an intervening DNA loop of 500 nucleotide pairs increases their frequency of collision. (more...) Figure Gene activation at a distance. (A) NtrC is a bacterial gene regulatory protein that activates transcription by facilitating the transition between the initial binding of RNA polymerase to the promoter and the formation of an initiating complex (discussed (more...)A Eucaryotic Gene Control Region Consists of a Promoter Plus Regulatory DNA Sequences Because eucaryotic gene regulatory proteins can control transcription when bound to DNA far away from the promoter, the DNA sequences that control the expression of a gene are often spread over long stretches of DNA. We shall use the term gene control region to refer to the whole expanse of DNA involved in regulating transcription of a gene, including the promoter, where the general transcription factors and the polymerase assemble, and all of the regulatory sequences to which gene regulatory proteins bind to control the rate of the assembly processes at the promoter (Figure ). In higher eucaryotes it is not unusual to find the regulatory sequences of a gene dotted over distances as great as 50,000 nucleotide 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 chromatin, thereby compacting its length. Figure 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 ). The regulatory sequences serve as binding sites for gene regulatory proteins, (more...) In this chapter we generally use the term gene to refer only to a segment of DNA that is transcribed into RNA (see Figure ). However, the classical view of a gene would include the gene control region as well. The different definitions arise from the different ways in which genes were historically identified. The discovery of alternative RNA splicing 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 gene regulatory proteins bind to enhancer 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 RNA polymerase 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 gene control region to the next, and each is usually present in very small amounts in a cell, often less than 0.01% of the total protein. Most of them recognize their specific DNA 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 gene 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 expression 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 gene control region on DNA, influence the rate of transcription initiation, as we now explain.CONCLUSION Summary The transcription of individual genes is switched on and off in cells by gene regulatory proteins. In procaryotes these proteins usually bind to specific DNA sequences close to the RNA polymerase start site and, depending on the nature of the regulatory protein and the precise location of its binding site 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 promoter 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 nucleotide pairs from the promoter generally control gene expression. Eucaryotic activators and repressors act by a wide variety of mechanisms—generally causing the local modification of chromatin structure, the assembly of the general transcription factors at the promoter, and the recruitment of RNA polymerase. Whereas the transcription of a typical procaryotic gene is controlled by only one or two gene regulatory proteins, the regulation of higher eucaryotic genes is much more complex, commensurate with the larger genome size and the large variety of cell types that are formed. The control region of the Drosophila eve gene, for example, encompasses 20,000 nucleotide pairs of DNA 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 expression. Eucaryotic genes and their control regions are often surrounded by insulators, DNA sequences recognized by proteins that prevent cross-talk between independently regulated genes.