In chapter 8, we surveyed the metabolic reactions in cells and the enzymes involved in those reactions. There we learned that some enzymes are regulated and that one form of regulation occurs at the genetic level. This control mechanism ensures that genes are active only when their products are required. In this way, enzymes will be produced as they are needed and the waste of energy and materials in dead-end synthesis is prevented. A major form of gene regulation in prokaryotes is through systems called operons. An operon is a stretch of DNA containing several structural genes along with a regulatory region that controls the transcription of the genes. This arrangement allows all the genes for a particular metabolic pathway to be stimulated or inhibited simultaneously by the same regulatory element, much as a single switch may control many lights in a room.
Operons are described as either inducible or repressible. The category of an operon is determined by how it is affected by the environment within the cell. Many catabolic operons are inducible, meaning that the operon is turned on (induced) by the substrate of the enzyme for which the structural genes code. In this way, the enzymes needed to metabolize a nutrient (lactose, for example) are produced only when that nutrient is present. Repressible operons often contain genes coding for anabolic enzymes, such as those used to synthesize amino acids. In the case of these operons, several genes in series are turned off (repressed) by the product synthesized by the enzyme.
The Lactose Operon: A Model for Inducible Gene Regulation in Bacteria The best understood cell system for explaining control through genetic induction is the lactose (lac) operon. The system, first described in 1961 by François Jacob and Jacques Monod, accounts for the regulation of lactose metabolism in Escherichia coli. Many other operons with similar modes of action have since been identified, and together they furnish convincing evidence that a cell’s metabolic actions can have great impact on gene expression.
The lactose operon has three important features (process figure 1):
1. the regulator, a gene that codes for a protein capable of repressing the operon (a repressor);
2. the control locus, composed of two areas, the promoter recognized by RNA polymerase and the operator, a sequence that acts as an on/off switch for transcription; and
3. the structural locus, made up of three genes, each coding for a different enzyme needed to catabolize lactose. One of the enzymes, β-galactosidase (LacZ), hydrolyzes the lactose into its monosaccharides; another, permease (LacY), brings lactose across the cell membrane.
Process Fig1. The lactose operon in bacteria: How inducible genes are controlled by substrate.
The promoter, operator, and structural components lie adjacent to one another, but the regulator can be at a distant site.
e another, but the regulator can be at a distant site. In inducible systems such as the lac operon, the operon is normally in an off mode, and enzymes are not produced when the substrate is absent (process figure 1, step 1). How is the operon maintained in this mode? The key is in the repressor protein that is encoded by the regulatory gene. This relatively large molecule is allosteric, meaning that its activity can be altered, depending on which active site and substrate are in play. A substrate binding to one site can distort a different site and prevent it from accepting its substrate (see figure 8.10, right). In the absence of lactose, the re pressor protein interacts with the operator and causes the operator to distort into a temporary loop configuration. This loop blocks access of the RNA polymerase to the DNA of the operator and pre vents transcription. Think of the repressor as a lock on the operator, and if the operator is locked, the structural genes cannot be transcribed. Importantly, the regulator gene is located in a separate site from the operator region and is not affected by this block on the operon.
If lactose is added to the cell’s environment, it triggers several events that turn the operon on. First, the binding of lactose to the repressor protein creates a shape change in the repressor that dislodges it from the operator segment (process figure1, step 2). This opens up the operator to allow RNA polymerase to bind to it and begin transcription. The structural genes are transcribed in a single unbroken transcript coding for all three enzymes. During translation, however, each protein is synthesized separately. Because lactose is ultimately responsible for stimulating the chain of events leading to protein synthesis, it is considered an inducer.
As lactose is depleted, further enzyme synthesis is not necessary, so the order of events reverses. At this point, there is no longer sufficient lactose to inhibit the repressor; hence, the repressor re gains its active shape for attachment to the operator. The operator is locked, and transcription of the structural genes and enzyme syn thesis related to lactose are arrested.
A fine but important point about the lac operon is that it functions only in the absence of glucose or if the cell’s energy needs are not being met by the available glucose. Glucose is the preferred carbon source because it can be used immediately in growth and does not require induction of an operon. When a supply of glucose is present, a second regulatory system ensures that the lac operon is inactive, regardless of lactose levels in the environment.
A Repressible Operon
Bacterial systems for synthesis of amino acids, nucleotides, and other processes work on a principle that is the reverse of the lac operon—that of repression. Similar factors such as repressor proteins, operators, and a series of structural genes are part of a repressible operon, but with some important differences. Unlike the lac operon, this operon is normally in the on mode and will be turned off only when the product of the pathway is no longer required. Excess product plays a role as a corepressor that slows the transcription of the operon.
A metabolically active cell that is consuming large amounts of the amino acid arginine (arg) will serve to illustrate the operation of a repressible operon. Under these conditions, the arg operon is set to on, and arginine is being actively synthesized through the action of the operon’s enzymatic products (process figure 2, step 1). In such an active cell, the arginine will be used immediately, and the repressor will remain inactive (unable to bind the operator) because there is too little free arginine to activate it. As the cell’s metabolism begins to slow down, however, the synthesized arginine is no longer used up and accumulates. The free arginine is then available to act as a corepressor by attaching to the repressor. This reaction changes the shape of the repressor, making it capable of binding to the operator and stopping transcription. Arginine will cease to be synthesized until the cell once again requires it in metabolism ( process figure 2, step 2).
Fig2. Repressible operon: Genetic control through excess product.
RNA and Gene Expression
For decades, RNA was considered little more than a stopping point on the way to proteins, but this view has changed dramatically as more has been learned about the dynamic nature of RNA, especially its ability to regulate the expression of other genes. Some mRNA molecules contain a riboswitch, a short segment of nucleotides that regulates translation of the same mRNA of which they are a part. The lysine riboswitch helps to control the production of the amino acid lysine, for example. If lysine is abundant in the cell, the riboswitch will bind the amino acid, changing the shape of the mRNA, preventing translation, and slowing the production of lysine (figure 3).
Fig3. Riboswitches regulate translation at the ribosome. When the riboswitch is on, the leader sequence of mRNA forms a loop with itself that exposes the ribosome binding sequence of the mRNA. This allows the ribosome to bind the mRNA and begin protein synthesis. The riboswitch is turned off when a ligand binds the mRNA, changing its shape, and repositioning the ribosome binding sequence. This makes the sequence on the mRNA inaccessible, blocking translation. Barry Chess/McGraw Hil
Other regulatory RNAs, rather than binding metabolic end products like lysine, work by binding mRNA itself. Described as RNA interference, these noncoding RNA molecules—including micro RNA (miRNA), antisense RNA (asRNA), and small interfering RNA (siRNA)—all work in a similar fashion. A noncoding RNA complementary to the gene to be regulated (or at least a portion of it) is transcribed and allowed to bind to its complementary mRNA, creating a double-stranded RNA molecule that cannot be translated and is rapidly degraded. RNA interference is seen primarily in eukaryotic organisms. Gene regulation in eukaryotes is more complex, including not only the response to environmental stimuli such as nutrients, toxin levels, or temperature, but also growth and development. It is only through the regulation of thousands of genes that the hundreds of different tissues seen in a multicellular organism can be produced.
