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Modern Genetic Analysis Chapter 14. Regulation of Gene Transcription
Regulation of the Lactose System

The salient features of transcriptional regulation in prokaryotes can be seen in the regulation of expression of the enzymes necessary for lactose metabolism in the bacterium Escherichia coli. We should recognize that the existing system is one that, through a long evolutionary process, has been selected to operate in an optimal fashion for the energy efficiency of the bacterial cell. Presumably because of energy-efficiency considerations, two environmental conditions have to be satisfied for the lactose metabolic enzymes to be expressed.

One condition that must be met is that lactose must be present in the environment. This condition makes sense, because it would be inefficient for the cell to produce the lactose metabolic enzymes in circumstances where there is no substrate to metabolize. We shall see that the cell's recognition that lactose is present is accomplished by a repressor protein.

The other condition is that glucose cannot be present in the cell's environment. Because glucose metabolism yields more usable energy to the cell than does lactose metabolism, mechanisms have evolved that prevent the synthesis of the enzymes for lactose metabolism in the presence of glucose. The repression of transcription of the lactose-metabolizing genes in the presence of glucose is an example of catabolite repression. The transcription of proteins necessary for the metabolism of many different sugars is repressed by catabolites. We shall see that catabolite repression works through an activator protein.

A First Look at the lac Regulatory Circuit

Thanks to the pioneering work of François Jacob and Jacques Monod, we have learned a lot about how lactose metabolism is regulated. First, we will deal with the system used to regulate lactose metabolism according to the presence or absence of lactose. Figure 14-3 is a simplified view of the components of this system. The cast of characters for lac regulation includes protein-coding genes and sites on the DNA that are targets for DNA-binding proteins.

The lac Structural Genes

The metabolism of lactose requires two enzymes: a permease to transport lactose into the cell and b -galactosidase to cleave the lactose molecule to yield glucose and galactose (Figure 14-4). Permease and b -galactosidase are encoded by two contiguous genes, Z and Y, respectively. A third gene, the A gene, encodes an additional enzyme, termed transacetylase, but this enzyme is not required for lactose metabolism, and we will not concentrate on it for now. All three genes are transcribed into a single, multigenic messenger RNA (mRNA) molecule. Regulation of the production of this mRNA coordinates the regulation of the synthesis of all three enzymes.

MESSAGE
If the genes encoding proteins of a given pathway are joined into a single transcription unit, the expression of all of these genes will be coordinately regulated.

Regulatory Components of the Lac System
The gene for the Lac repressor.

A fourth gene, the I gene, encodes the Lac repressor protein, so named because it can block the expression of the Z, Y, and A genes. The I gene happens to map fairly near the Z, Y, and A genes, but this proximity does not seem to be important to its function.

The lac promoter site.

The promoter (P) is the site on the DNA to which RNA polymerase binds to initiate transcription.

The lac operator site.

The operator (O) is the site on the DNA to which the Lac repressor binds. It is located between the promoter and the Z gene near the point at which transcription of the multigenic mRNA begins.

The lac Operon: Assaying the Presence or Absence of Lactose through the Lac Repressor

The P, O, Z, Y, and A segments shown in Figure 14-5 constitute an operon, which is a genetic unit of coordinate expression. The interaction between the lac operator site on the DNA and the Lac repressor is crucial to proper regulation of the lac operon. The Lac repressor is a molecule with two recognition sitesa DNA-binding site that can recognize the specific operator DNA sequence for the lac operon and an allosteric site that binds the lactose allosteric effector and similar molecules (analogs of lactose).

The DNA-binding site of the Lac repressor is able to bind with high affinity to only one DNA sequence in the entire E. coli genomethe lac operator. The specificity of high-affinity DNA binding ensures that the repressor will bind only to the site on the DNA near the genes that it is controlling and not to random sites distributed throughout the chromosome. By binding to the operator, the repressor prevents transcription by RNA polymerase that has bound to its lac promoter site.

As already mentioned, the allosteric site of the Lac repressor binds to lactose and structurally similar molecules (lactose analogs). We shall see later in the chapter that some lactose analogs are very useful tools for experimentally inducing lac operon expression. When the repressor binds to lactose or its analogs, the protein undergoes a conformational (allosteric) change; this slight alteration in shape changes the DNA-binding site so that the repressor no longer has high affinity for the operator. Thus, in response to binding lactose, the repressor falls off the DNA, which satisfies one requirement for such a control systemthe ability to recognize conditions under which it is worthwhile to activate expression of the lac genes.

The relief of repression for systems such as lac is termed induction; lactose and its analogs that allosterically inactivate the repressor and lead to expression of the lac genes are termed inducers. We now know that the Lac repressor is a protein consisting of four identical subunits, each with a molecular weight of approximately 38,000. Each tetrameric Lac repressor molecule contains four inducer-binding allosteric sites. (A more detailed description of the repressor is given later in the chapter.)

Let's examine the implications of the last few paragraphs. In the absence of an inducer (lactose or an analog), the Lac repressor binds to the lac operator site and prevents transcription of the lac operon. Most of the effect of Lac repressor's binding to the operator is to block the progression of RNA polymerase transcription. In this sense, the Lac repressor acts as a roadblock on the DNA. Consequently, all of the structural genes of the lac operon (the Z, Y, and A genes) are repressed, and there is no b -galactosidase, b -galactoside permease, or transacetylase in the cell. In contrast, when an inducer is present, it binds to the allosteric site of the Lac repressor, thereby inactivating the operator DNA-binding site of the Lac repressor protein. This inactivation permits the induction of transcription of the structural genes of the lac operon and, through the translation of the multigenic mRNA, the enzymes b -galactosidase, b -galactoside permease, and transacetylase now appear in the cell in a coordinated fashion.

Before going any further, however, we should note that there is more to the regulation of lac operon transcription. Recall that the induction of transcription of the lac operon also requires a second environmental conditionnamely, that glucose is not present in the environment of the cell. We shall consider the reasons for this condition and the mechanisms governing glucose repression next.

Catabolite Repression of the lac Operon: Choosing the Best Sugar to Metabolize

An additional control system is superimposed on the repressoroperator system. This control system is thought to have evolved because the cell can capture more energy from the breakdown of glucose than it can from the breakdown of other sugars. If both lactose and glucose are present, the synthesis of b -galactosidase is not induced until all the glucose has been utilized. Thus, the cell conserves its energy pool used, for example, to synthesize the Lac enzymes by utilizing any existing glucose before going through the energy-expensive process of creating new machinery to metabolize lactose.

Studies indicate that a breakdown product of glucose (the identity of this catabolite is as yet unknown) prevents activation of the lac operon by lactosethe catabolite repression referred to earlier. The glucose catabolite modulates the level of an important cellular constituentcyclic adenosine monophosphate (cAMP). When glucose is present in high concentrations, the cell's cAMP concentration is low; as the glucose concentration decreases, the cellular concentration of cAMP increases correspondingly. The high concentration of cAMP is necessary for activation of the lac operon. Mutants that cannot convert ATP into cAMP cannot be induced to produce b -galactosidase, because the concentration of cAMP is not great enough to activate the lac operon. In addition, other mutants that do make cAMP cannot activate the Lac enzymes, because they lack yet another protein, called CAP (catabolite activator protein), made by the crp gene. By itself, CAP cannot bind to the CAP site of the lac operon. However, by binding to its allosteric effector, cAMP, CAP is able to bind to the CAP site. The DNA-bound CAP is then able to interact physically with RNA polymerase and essentially increase the affinity of RNA polymerase for the lac promoter. In this way, the catabolite repression system contributes to the selective activation of the lac operon (Figure 14-6).

MESSAGE
The lac operon has an added level of control so that the operon remains inactive in the presence of glucose even if lactose also is present. High concentrations of glucose catabolites produce low concentrations of an allosteric effector, cyclic adenosine monophosphate (cAMP), which binds to the activator, CAP, to permit the induction of the lac operon.

Genetic Aspects of the lac Operon Model

One way to be certain that we understand how the lac operon works is by considering the genetic consequences of mutations in the various components of the lac operon. The properties of mutations in the structural genes and the regulatory elements of the lac operon are quite different. Indeed, the phenotypes of these mutations in homozygotes and hemizygotes, as well as their complementation behavior, were important clues for Jacob and Monod in unraveling the mechanisms of gene regulation in bacteria. Here, we shall examine the kinds of mutations that occur and how they can be explained by the lac operon model.

Techniques for Mutational Analysis

For studying the Lac repressor and the lac operator, we shall consider enzymatic assays for b -galactosidase and the permease under two sets of environmental conditions. In both conditions, no glucose will be present, so we do not have to consider the effects of catabolite repression. Genotypes will be assayed in the uninduced state, in which no inducer molecule is present. Tests of uninduced bacteria permit us to assess whether the genetic circuitry necessary for repression is present. Genotypes will also be assayed in the induced state; that is, in the presence of inducers in the culture medium. Natural inducers, such as lactose, are not optimal for these experiments, because they are hydrolyzed by b -galactosidase; the inducer concentration decreases during the experiment, and so the measurements of enzyme induction become quite complicated. Rather, for such experiments, we use synthetic inducers, such as isopropyl- b -d-thiogalactoside (IPTG; Figure 14-7), that bind to the allosteric site of the Lac repressor but are not hydrolyzed by b -galactosidase. By assaying wild-type and mutant genotypes in the induced state, we determine whether the genetic circuitry necessary to overcome repression is functioning normally.

We shall see that several different classes of mutations can alter the expression of the structural genes of the lac operon. Genetic complementation tests, in which we construct bacterial genotypes heterozygous for various lac operon mutations, are essential for distinguishing between these different mutant classes. F factors make this possible. Ordinarily, bacteria are haploid, making such complementation analysis difficult. However, by using F factors (see Chapter 9) carrying the lac region of the genome, we can produce bacteria that are diploid and heterozygous for the desired lac mutations. We shall see that complementation tests allow us to distinguish mutations in the lac operator from mutations in the I gene (encoding the Lac repressor).

Results of Genetic Analysis

Let's begin to look at the effects of various combinations of mutations, in the induced and noninduced state, on the production of b -galactosidase and permease. The first thing that we learn from examining mutations that inactivate the structural genes for b -galactosidase and permease (designated Z and Y, respectively) is that Z and Y are recessive to their respective wild-type alleles (Z+ and Y+). For example, strain 2 in Table 14-1 is inducible for b -galactosidase (like the wild-type haploid strain 1 in this table), even though it is heterozygous for mutant and wild-type Z alleles. This demonstrates that the Z+ allele is dominant to its Z counterpart.

Mutations in the repressor and operator cause global misregulation of the lac operon structural genes. Let's first consider operator mutations (Table 14-1), Oc mutations that make the operator nonfunctionalthat is, that make the operator incapable of binding to repressor. Such Oc mutations cause the lac operon structural genes to be constitutive in expression (Table 14-1, strain 3). That is, regardless of whether inducer is present, these mutations lead to the transcription and translation of the lac operon genes. Furthermore, the constitutive effects of Oc mutations are restricted solely to those lac structural genes on the same chromosome; for this reason, we say that operators are cis-acting. (More generally, we use the term cis-acting to refer to genetic interactions that are restricted to elements on the same DNA molecule.) This is demonstrated by the phenotype of strain 4 in Table 14-1. Here, because the wild-type permease (Y+) gene is in cis to the wild-type operator, permease activity is inducible. In contrast, the wild-type b -galactosidase (Z+) gene is in cis to the Oc mutant operator, and hence b -galactosidase expression is constitutive. How can we understand the cis-acting nature of the operator? The explanation is that the operator acts solely as a protein-binding siteit makes no gene product. The operator binding site thus regulates only the expression of the structural genes linked to it (Figure 14-8).

Now let's consider the effects of constitutive I mutations (Table 14-2). In I mutations, the DNA-binding site of the repressor has been mutated, so no functional operator-binding repressor protein is made. Thus, unlike the inducible phenotype of the wild-type I+ (strain 1), I mutations are constitutive (strain 2). In addition, we see that the inducible phenotype of I+ is dominant to the constitutive phenotype of I (strain 3). This tells us that the amount of wild-type repressor encoded by one copy of the gene is sufficient to regulate both copies of the operator in a diploid cell. Finally, we see that the I+ gene product is trans-acting (strain 4), which means that the gene product regulates all structural lac operon genes, both those in cis and those in trans (residing on different DNA molecules). How do we explain the trans-action of the I+ gene product? The I gene is a standard protein-coding gene. The protein product of the I gene is able to diffuse and act on all operators in the cell (Figure 14-9 on the next page).

MESSAGE
Mutations in a target DNA site reveal that such a site is cis-acting; that is, the target site regulates the expression of an adjacent transcription unit on the same DNA molecule. In contrast, mutations in the gene encoding an activator or repressor protein reveal that this protein is trans-acting; that is, it can act on any copy of the target DNA site in the cell.

Genetic Evidence for Allostery

Another class of repressor mutations reveals the importance of allostery. Recall that the Lac repressor has to inhibit transcription of the lac operon in the absence of an inducer but must permit transcription when the inducer is present. This is accomplished through a second site on the repressor protein, the allosteric site, which binds to the inducer. When bound to the inducer, the repressor is changed in overall structure such that the DNA-binding site can no longer function.

Is mutations (super-repressors) cause repression even in the presence of an inducer (compare strain 2 in Table 14-3 with the inducible wild-type strain 1). Unlike the case for I, Is mutations are dominant to I+ (Table 14-3, strain 3). Is mutations alter the stereospecific allosteric site such that it can no longer bind to an inducer. In the absence of an ability to bind an inducer, Is-encoded repressor protein continually binds to the operatorpreventing transcription of the lac operon even when the inducer is present in the cell. On this basis, we can see why Is is dominant to I+. Mutant Is protein will bind to all operators in the cell, even in the presence of an inducer and regardless of the fact that I+ protein may be present in the same cell (Figure 14-10).

Genetic Analysis of the lac Promoter

Genetic experiments demonstrated that an element essential for lac transcription is located between I and O. This element, termed the promoter (P), serves as the initiation site for transcription. There are two binding regions for RNA polymerase in a typical prokaryotic promoter, shown in Figure 14-11 as the two highly conserved regions at 35 and 10. Promoter mutations affect the transcription of all structural genes in the operon in a similar manner. The dominance of promoter mutations is cis-acting, because promoters, like operators, are sites on the DNA molecule that are bound by proteins.

The Structures of Target DNA Sites

The DNA sequences to which the lac repressor and the CAPcAMP complex bind are now known. These sequences (Figure 14-12 on the following page) are very different from one another, and these differences underlie the specificity of binding by these very different DNA-binding proteins. One property that they do share, and which is common to many other DNA-binding sites, is rotational twofold symmetry. In other words, if we rotate the DNA sequence 180° within the plane of the page, the sequence of the highlighted bases of the binding sites will be identical. The highlighted bases are thought to constitute the important contact sites for proteinDNA interactions. This rotational symmetry is thought to be reflective of the fact that many DNA-binding proteins are homodimers or homotetramers, and that the symmetries of the target-site DNA sequences reflect symmetries within the multimeric DNA-binding proteins. We shall consider the structures of some DNA-binding proteins later in the chapter.

MESSAGE
Generalizing from the lac operon story, we can envision the chromosome as heavily decorated by regulatory proteins binding to the operator sites that they control. The exact pattern of decorations will depend on which genes are turned on or off and whether particular operons are regulated by activators or repressors.

A Summary of the lac Operon

We can now fit the known repressor, CAPcAMP, and RNA polymerase binding sites into the detailed model of the lac operon, as shown in Figures 14-13 and 14-14. In Figure 14-14d, the DNA is shown as being bent when CAP binds. This may aid RNA polymerase binding to the promoter. There is also evidence to suggest that CAP makes direct proteinprotein contacts with RNA polymerase, through the RNA polymerase a subunit, that are important for the CAP activation effect (Figure 14-14e).

Glucose control is accomplished because a glucose-breakdown product inhibits maintenance of the high cAMP levels necessary for formation of the CAPcAMP complex required for the RNA polymerase to attach at the lac promoter site. Even when there is a shortage of glucose catabolites and CAPcAMP forms, the mechanism for lactose metabolism will be implemented only if lactose is present. This level of control is accomplished because lactose must bind to the repressor protein to remove it from the operator site and permit transcription of the lac operon. Thus, the cell conserves its energy and resources by producing the lactose-metabolizing enzymes only when they are both needed and useful.

Inducerrepressor control of the lac operon is an example of repression, or negative control, in which expression is normally blocked. In contrast, the CAPcAMP system is an example of activation, or positive control, because its expression requires the presence of an activating signalin this case, the interaction of the CAPcAMP complex with the CAP site. Figure 14-15 outlines these two basic types of control systems.

MESSAGE
The lac operon is a cluster of structural genes that specify enzymes taking part in lactose metabolism. These genes are controlled by the coordinated actions of cis-acting promoter and operator regions. The activity of these regions is, in turn, determined by a repressor molecule specified by a separate regulator gene.

© 1999 by W. H. Freeman and Company. All rights reserved.