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 simplified lac operon model. The three genes Z, Y, and A are coordinately expressed. The product of the I gene, the repressor, blocks the expression of the Z, Y, and A genes by interacting with the operator (O). The inducer can inactivate the repressor, thereby preventing interaction with the operator. When this happens, the operon is fully expressed.
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 metabolism of lactose. The enzyme β-galactosidase catalyzes a reaction in which water is added to the β-galactosidase linkage to break lactose into separate molecules of galactose and glucose. The enzyme lactose permease is required to transport lactose into the cell.
).
Permease and β-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.
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.
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 promoter (P) is the site on the DNA to which RNA polymerase binds to initiate transcription.
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.
Regulation of the lac operon. The I gene continually makes repressor. The repressor binds to the O (operator) region, blocking the RNA polymerase bound to P (the promoter region) from transcribing the adjacent structural genes. When lactose is present, it binds to the repressor and changes its shape so that the repressor no longer binds to O. The RNA polymerase is then able to transcribe the Z, Y, and A structural genes, so the three enzymes are produced.
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 sites—a 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 genome—the 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 system—the 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 β-galactosidase, β-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 β-galactosidase, β-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 condition—namely, 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.
An additional control system is superimposed on the repressor–operator 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 β-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.
Catabolite control of the lac operon. The operon is inducible by lactose to the maximal levels when cAMP and CAP form a complex. (a) Under conditions of high glucose, a glucose breakdown product inhibits the enzyme adenylate cyclase, preventing the conversion of ATP into cAMP. (b) Under conditions of low glucose, there is no breakdown product, and therefore adenylate cyclase is active and cAMP is formed. (c) When cAMP is present, it acts as an allosteric effector, complexing with CAP. (d) The cAMP–CAP complex acts as an activator of lac operon transcription by binding to a region within the lac promoter. (CAP = catabolite activator protein; cAMP = cyclic adenosine monophosphate.)
).
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.
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.
), that bind to the allosteric
site of the Lac repressor but are not hydrolyzed by β-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).
| β-GALACTOSIDASE(Z) | PERMEASE(Y) | |||||
|---|---|---|---|---|---|---|
| Strain | Genotype | Noninduced | Induced | Noninduced | Induced | Conclusions |
| 1 | O+Z+Y+ | − | + | − | + | Wild type is inducible |
| 2 | O+Z+Y+/F′O+Z−Y+ | − | + | − | + | Z+ is dominant to Z− |
| 3 | OcZ+Y+ | + | + | + | + | Oc is constitutive |
| 4 | O+Z−Y+/F′OcZ+Y− | + | + | − | + | Operator is cis-acting |
O+ / Oc heterozygotes demonstrate that operators are cis-acting. Because a repressor cannot bind to Oc operators, the lac structural genes linked to an Oc operator are expressed even in the absence of an inducer. However, the lac genes adjacent to an O+ operator are still subject to repression.
).
| β-GALACTOSIDASE(Z) | PERMEASE(Y) | |||||
|---|---|---|---|---|---|---|
| Strain | Genotype | Noninduced | Induced | Noninduced | Induced | Conclusions |
| 1 | I+Z+Y+ | − | + | − | + | I+ is inducible |
| 2 | I−Z+Y+ | + | + | + | + | I− is constitutive |
| 3 | I+Z−Y+/F′I−Z+Y+ | − | + | − | + | I+ is dominant to I− |
| 4 | I−Z−Y+/F′I+Z+Y− | − | + | − | + | I+ is trans-acting |
The recessive nature of I− mutations demonstrates that the repressor is transacting. Although no active repressor is synthesized from the I− gene, the wild-type (I+) gene provides a functional repressor that binds to both operators in a diploid cell and blocks lac operon expression (in the absence of an inducer).
on the next page).
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.
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.
The dominance of Is mutations is due to the inactivation of the allosteric site on the Lac repressor. In an Is / I+ diploid cell, none of the lac structural genes are transcribed, even in the presence of an inducer. In contrast with the wild-type repressor, the Is repressor lacks a functional lactose-binding site (the allosteric site) and thus is not inacti-vated by an inducer. Thus, even in the presence of an inducer, the Is repressor binds irreversibly to all operators in a cell, thereby blocking transcription of the lac operon.
).
Specific DNA sequences are important for efficient transcription of E. coli genes by RNA polymerase. The boxed sequences at approximately 35 and 10 nucleotides before the transcription start site are highly conserved in all E. coli promoters, an indication of their role as contact sites on the DNA for RNA polymerase binding. Mutations in these regions have mild (gold) and severe (brown) effects on transcription. The mutations may be changes of single nucleotides or pairs of nucleotides, or a deletion (Δ) may occur. (From J. D. Watson, M. Gilman, J. Witkowski, and M. Zoller, Recombinant DNA, 2d ed. Copyright © 1992 by James D. Watson, Michael Gilman, Jan Witkowski, and Mark Zoller.)
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 DNA base sequences of (a) the lac operator, to which the Lac repressor binds, and (b) the CAP-binding site, to which the CAP–cAMP complex binds. Sequences exhibiting twofold rotational symmetry are indicated by the colored boxes and by a dot at the center point of symmetry. (Part a from W. Gilbert, A. Maxam, and A. Mirzabekov, in N. O. Kjeldgaard and O. Malløe, eds., Control of Ribosome Synthesis. Academic Press, 1976. Used by permission of Munksgaard International Publishers, Ltd., Copenhagen.)
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
protein–DNA 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.
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.
The base sequence and the genetic boundaries of the control region of the lac operon, with partial sequences for the structural genes. (After R. C. Dickson, J. Abelson, W. M. Barnes, and W. S. Reznikoff, “Genetic Regulation: The Lac Control Region,” Science 187, 1975, 27. Copyright © 1975 by the American Association for the Advancement of Science.)
Negative and positive control of the lac operon by the Lac repressor and catabolite activator protein (CAP), respec-tively. (a) In the absence of lactose to serve as an inducer, the Lac repressor is able to bind the operator; regardless of the levels of cAMP and the presence of CAP, mRNA production is repressed. (b) With lactose present to bind the repressor, the repressor is unable to bind the operator; however, only small amounts of mRNA are produced because the presence of glucose keeps the levels of cAMP low, and thus the cAMP–CAP complex does not form and bind the promoter. (c) With the repressor inactivated by lactose and with high levels of cAMP present (owing to the absence of glucose), cAMP binds CAP. The cAMP–CAP complex is then able to bind the promoter; the lac operon is thus activated, and large amounts of mRNA are produced. (d) When CAP binds the promoter, it creates a bend greater than 90° in the DNA. Apparently, RNA polymerase binds more effectively when the promoter is in this bent configuration. (e) CAP bound to its DNA recognition site. This part is derived from the structural analysis of the CAP–DNA complex. [Parts a–d redrawn from B. Gartenberg and D. M. Crothers, Nature 333, 1988, 824. (See H. N. Lie-Johnson et al., Cell 47, 1986, 995.) Adapted from H. Lodish, D. Baltimore, A. Berk, S. L. Zipursky, P. Matsudaira, and J. Darnell, Molecular Cell Biology, 3d ed. Copyright © 1995 by Scientific American Books. Part e from L. Schultz and T. A. Steitz.]
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 protein–protein contacts with RNA polymerase, through the RNA
polymerase α 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 CAP–cAMP complex required for the RNA polymerase to attach at the lac promoter site. Even when there is a shortage of glucose catabolites and CAP–cAMP 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.
Comparison of repression and activation. (a) In repression, an active repressor (encoded by the R gene in the example shown here) blocks gene expression of the A,B,C operon by binding to an operator site (O). An inactive repressor allows gene expression. The repressor can be inactivated either by an inducer or by mutation. (b) In activation, a functional activator is required for gene expres-sion, as shown for the X,Y,Z operon here. Small molecules can convert a nonfunctional activator into a functional one, as in the case of cyclic AMP and the CAP protein. A nonfunctional activator results in no gene expression. The activator binds to the control region of the operon, termed I in this case. (The positions of both O and I with respect to the promoter, P, in the two examples are arbitrarily drawn.)
outlines these two basic
types of control systems.
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.
