Despite their simplicity of form, bacteria have in common with larger and more complex organisms the need to regulate expression of their genes. One of the main reasons is that they are nutritional opportunists. Consider how bacteria obtain the many important compounds, such as sugars, amino acids, and nucleotides, needed for metabolism. Bacteria swim in a sea of potential nutrients. They can either acquire the compounds that they need from the environment or synthesize them by enzymatic pathways. But synthesizing these compounds also requires expending energy and cellular resources to produce the necessary enzymes for these pathways. Thus, given the choice, bacteria will take compounds from the environment instead. Natural selection favors efficiency and selects against the waste of resources and energy. To be economical, bacteria will synthesize the enzymes necessary to produce compounds only when there is no other option—in other words, when compounds are unavailable in their local environment.

Bacteria have evolved regulatory systems that couple the expression of gene products to sensor systems that detect the relevant compound in a bacterium’s local environment. The regulation of enzymes taking part in sugar metabolism provides an example. Sugar molecules can be broken down to provide energy or they can be used as building blocks for a great range of organic compounds. However, there are many different types of sugar that bacteria could use, including lactose, glucose, galactose, and xylose. A different import protein is required to allow each of these sugars to enter the cell. Further, a different set of enzymes is required to process each of the sugars. If a cell were to simultaneously synthesize all the enzymes that it might possibly need, the cell would expend much more energy and materials to produce the enzymes than it could ever derive from breaking down prospective carbon sources. The cell has devised mechanisms to shut down (repress) the transcription of all genes encoding enzymes that are not needed at a given time and to turn on (activate) those genes encoding enzymes that are needed. For example, if only lactose is in the environment, the cell will shut down the transcription of the genes encoding enzymes needed for the import and metabolism of glucose, galactose, xylose, and other sugars. Conversely, E. coli will initiate the transcription of the genes encoding enzymes needed for the import and metabolism of lactose. In sum, cells need mechanisms that fulfill two criteria:

400

Let’s preview the current model for prokaryotic transcriptional regulation and then use a well-understood example—the regulation of the genes in the metabolism of the sugar lactose—to examine it in detail. In particular, we will focus on how this regulatory system was dissected with the use of the tools of classical genetics and molecular biology.

The regulation of transcription depends mainly on two types of protein–DNA interactions. Both take place near the site at which gene transcription begins.

One of these DNA–protein interactions determines where transcription begins. The DNA that participates in this interaction is a DNA segment called the promoter (Chapter 8, Section 8.2), and the protein that binds to this site is RNA polymerase. When RNA polymerase binds to the promoter DNA, transcription can start a few bases away from the promoter site. Every gene must have a promoter or it cannot be transcribed.

The other type of DNA–protein interaction determines whether promoter–driven transcription takes place. DNA segments near the promoter serve as binding sites for sequence-specific regulatory proteins called activators and repressors. In bacteria, most binding sites for repressors are termed operators. For some genes, an activator protein must bind to its target DNA site as a necessary prerequisite for transcription to begin. Such instances are sometimes referred to as positive regulation because the presence of the bound protein is required for transcription (Figure 11-2). For other genes, a repressor protein must be prevented from binding to its target site as a necessary prerequisite for transcription to begin. Such cases are sometimes termed negative regulation because the absence of the bound repressor allows transcription to begin.

How do activators and repressors regulate transcription? Often, a DNA-bound activator protein physically helps tether RNA polymerase to its nearby promoter so that polymerase may begin transcribing. A DNA-bound repressor protein typically acts either by physically interfering with the binding of RNA polymerase to its promoter (blocking transcription initiation) or by impeding the movement of RNA polymerase along the DNA chain (blocking transcription). Together, these regulatory proteins and their binding sites constitute genetic switches that control the efficient changes in gene expression that occur in response to environmental conditions.

401

Both activator and repressor proteins must be able to recognize when environmental conditions are appropriate for their actions and act accordingly. Thus, for activator or repressor proteins to do their job, each must be able to exist in two states: one that can bind its DNA targets and another that cannot. The binding state must be appropriate to the set of physiological conditions present in the cell and its environment. For many regulatory proteins, DNA binding is effected through the interaction of two different sites in the three-dimensional structure of the protein. One site is the DNA-binding domain. The other site, the allosteric site, acts as a sensor that sets the DNA-binding domain in one of two modes: functional or nonfunctional. The allosteric site interacts with small molecules called allosteric effectors.

In lactose metabolism, it is actually an isomer of the sugar lactose (called allolactose) that is an allosteric effector: the sugar binds to a regulatory protein that inhibits the expression of genes needed for lactose metabolism. In general, an allosteric effector binds to the allosteric site of the regulatory protein in such a way as to change its activity. In this case, allolactose changes the shape and structure of the DNA-binding domain of a regulatory protein. Some activator or repressor proteins must bind to their allosteric effectors before they can bind DNA. Others can bind DNA only in the absence of their allosteric effectors. Two of these situations are shown in Figure 11-3.

The pioneering work of François Jacob and Jacques Monod in the 1950s showed how lactose metabolism is genetically regulated. Let’s examine the system under two conditions: the presence and the absence of lactose. Figure 11-4 is a simplified view of the components of this system. The cast of characters for lac operon 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: (1) a permease to transport lactose into the cell and (2) β-galactosidase to modify lactose into allolactose and to cleave the lactose molecule to yield glucose and galactose (Figure 11-5). The structures of the β-galactosidase and permease proteins are encoded by two adjacent sequences, Z and Y, respectively. A third contiguous sequence encodes an additional enzyme, termed transacetylase, which is not required for lactose metabolism. We will call Z, Y, and A structural genes—in other words, segments encoding proteins—while reserving judgment on this categorization until later. We will focus mainly on the Z and Y genes. All three genes are transcribed into a single messenger RNA molecule. Regulation of the production of this mRNA coordinates the synthesis of all three enzymes. That is, either all or none of the three enzymes are synthesized. Genes whose transcription is controlled by a common means are said to be coordinately controlled.

402

Regulatory components of the lac system Key regulatory components of the lactose metabolic system include a gene encoding a transcription regulatory protein and two binding sites on DNA: one site for the regulatory protein and another site for RNA polymerase.

The induction of the lac system The P, O, Z, Y, and A segments (shown in Figure 11-6) together constitute an operon defined as a segment of DNA that encodes a multigenic mRNA as well as an adjacent common promoter and regulatory region. The lacI gene, encoding the Lac repressor, is not considered part of the lac operon itself, but the interaction between the Lac repressor and the lac operator site is crucial to proper regulation of the lac operon. The Lac repressor has a DNA-binding site that can recognize the operator DNA sequence and an allosteric site that binds allolactose or analogs of lactose that are useful experimentally. The repressor will bind tightly only to the O site on the DNA near the genes that it is controlling and not to other sequences distributed throughout the chromosome. By binding to the operator, the repressor prevents transcription by RNA polymerase that has bound to the adjacent promoter site; the lac operon is switched “off.”

403

When allolactose or its analogs bind to the repressor protein, the protein undergoes an allosteric transition, a change in shape. This slight alteration in shape in turn alters the DNA-binding site so that the repressor no longer has high affinity for the operator. Thus, in response to binding allolactose, the repressor falls off the DNA, allowing RNA polymerase to proceed (transcribe the gene): the lac operon is switched “on.” The repressor’s response to allolactose satisfies one requirement for such a control system—that the presence of lactose stimulates the synthesis of genes needed for its processing. The relief of repression for systems such as lac is termed induction. Allolactose and its analogs that allosterically inactivate the repressor, leading to the expression of the lac genes, are termed inducers.

404

Let’s summarize how the lac switch works. In the absence of an inducer (allolactose or an analog), the Lac repressor binds to the lac operator site and prevents transcription of the lac operon by blocking the movement of RNA polymerase. In this sense, the Lac repressor acts as a roadblock on the DNA. Consequently, all the structural genes of the lac operon (the Z, Y, and A genes) are repressed, and there are very few molecules of β-galactosidase, permease, or transacetylase in the cell. In contrast, when an inducer is present, it binds to the allosteric site of each Lac repressor subunit, thereby inactivating the site that binds to the operator. The Lac repressor falls off the DNA, allowing the transcription of the structural genes of the lac operon to begin. The enzymes β-galactosidase, permease, and transacetylase now appear in the cell in a coordinated fashion. So, when lactose is present in the environment of a bacterial cell, the cell produces the enzymes needed to metabolize it. But when no lactose is present, resources are not wasted.