Genes VII

Bacteria need to respond swiftly to changes in their environment. Fluctuations in the supply of nutrients can occur at any time; survival depends on the ability to switch from metabolizing one substrate to another. Yet economy also is important, since a bacterium that indulges in energetically expensive ways to meet the demands of the environment is likely to be at a disadvantage. So a bacterium avoids synthesizing the enzymes of a pathway in the absence of the substrate; but is ready to produce the enzymes if the substrate should appear.

The synthesis of enzymes in response to the appearance of a specific substrate is called induction. This type of regulation is widespread in bacteria, and occurs also in unicellular eukaryotes (such as yeasts). The lactose system of E. coli provides the paradigm for this sort of control mechanism.

When cells of E. coli are grown in the absence of a β-galactoside, there is no need for β-galactosidase, and they contain very few molecules of the enzyme Vsay, <5. When a suitable substrate is added, the enzyme activity appears very rapidly in the bacteria. Within 2-3 minutes some enzyme is present, and soon there are ~5000 molecules of enzyme per bacterium. (Under suitable conditions, β-galactosidase can account for 5 V10% of the total soluble protein of the bacterium.) If the substrate is removed from the medium, the synthesis of enzyme stops as rapidly as it had originally started.

Figure 10.6 summarizes the essential features of induction. Control of transcription of the lac genes responds very rapidly to the inducer, as shown in the upper part of the figure. In the absence of inducer, the operon is transcribed at a very low basal level (see below). Transcription is stimulated as soon as inducer is added; the amount of lac mRNA increases rapidly to an induced level that reflects a balance between synthesis and degradation of the mRNA.

The lac mRNA is extremely unstable, and decays with a half-life of only ~3 minutes. This feature allows induction to be reversed rapidly. Transcription ceases as soon as the inducer is removed; and in a very short time all the lac mRNA has been destroyed, and the cellular content has returned to the basal level.

The production of protein is followed in the lower part of the figure. Translation of the lac mRNA produces β-galactosidase (and the products of the other lac genes). There is a short lag between the appearance of lac mRNA and appearance of the first completed enzyme molecules (it is ~2 min after rise of mRNA from basal level before protein begins to increase). There is a similar lag between reaching maximal induced levels of mRNA and protein. When inducer is removed, synthesis of enzyme ceases almost immediately (as the mRNA is degraded), but the β-galactosidase in the cell is more stable than the mRNA, so the enzyme activity remains at the induced level for longer.

This type of rapid response to changes in nutrient supply not only provides the ability to metabolize new substrates, but also is used to shut off endogenous synthesis of compounds that suddenly appear in the medium. For example, E. coli synthesizes the amino acid tryptophan through the action of the enzyme tryptophan synthetase. But if tryptophan is provided in the medium on which the bacteria are growing, the production of the enzyme is immediately halted. This effect is called repression. It allows the bacterium to avoid devoting its resources to unnecessary synthetic activities.

Induction and repression represent the same phenomenon. In one case the bacterium adjusts its ability to use a given substrate for growth; in the other it adjusts its ability to synthesize a particular metabolic intermediate. The trigger for either type of adjustment is the small molecule that is the substrate for the enzyme, or the product of the enzyme activity, respectively. Small molecules that cause the production of enzymes able to metabolize them are called inducers. Those that prevent the production of enzymes able to synthesize them are called corepressors.

The ability to act as inducer or corepressor is highly specific. Only the substrate/product or a closely related molecule can serve. But the activity of the small molecule does not depend on its interaction with the target enzyme. Some inducers resemble the natural inducers for β-galactosidase, but cannot be metabolized by the enzyme. The example par excellence is isopropylthiogalactoside (IPTG), one of several thiogalactosides with this property. Although it is not recognized by β-galactosidase, IPTG is a very efficient inducer of the lac genes.

Molecules that induce enzyme synthesis but are not metabolized are called gratuitous inducers. They are extremely useful because they remain in the cell in their original form. (A real inducer would be metabolized, interfering with study of the system.) The existence of gratuitous inducers reveals an important point. The system must possess some component, distinct from the target enzyme, that recognizes the appropriate substrate; and its ability to recognize related potential substrates is different from that of the enzyme.

The component that responds to the inducer is the repressor protein coded by lacI. Its role in controlling transcription of the lacZYA structural genes in response to the environment is summarized in Figure 10.7. The structural genes are transcribed into a single mRNA from a promoter just upstream of lacZ. The state of the repressor determines whether this promoter is turned off or on:

• In the absence of an inducer, the genes are not transcribed, because repressor protein is in an active form that is bound to the operator.
• When an inducer is added, the repressor is converted into an inactive form that leaves the operator. Then transcription starts at the promoter and proceeds through the genes to a terminator located somewhere beyond lacA.

The crucial features of the control circuit reside in the dual properties of the repressor: it can prevent transcription; and it can recognize the small-molecule inducer. The repressor has two binding sites, one for the operator and one for the inducer. When the inducer binds at its site, it changes the conformation of the protein in such a way as to influence the activity of the operator-binding site. The ability of one site in the protein to control the activity of another is called allosteric control. (For an introduction see the supplement on Allostery).

Induction accomplishes a coordinate regulation: all the genes are expressed (or not expressed) in unison. The mRNA is translated sequentially from its 5′ end, which explains why induction always causes the appearance of β-galactosidase, β-galactoside permease, and β-galactoside transacetylase, in that order. Translation of a common mRNA explains why the relative amounts of the three enzymes always remain the same under varying conditions of induction.

Induction throws a switch that causes the genes to be expressed. Inducers vary in their effectiveness, and other factors influence the absolute level of transcription or translation, but the relationship between the three genes is predetermined by their organization.

We notice a potential paradox in the constitution of the operon. The lactose operon contains the structural gene (lacZ) coding for the β-galactosidase activity needed to metabolize the sugar; it also includes the gene (lacY) that codes for the protein needed to transport the substrate into the cell. But if the operon is in a repressed state, how does the inducer enter the cell to start the process of induction?

Two features ensure that there is always a minimal amount of the protein present in the cell, enough to start the process off. There is a basal level of expression of the operon: even when it is not induced, it is expressed at a residual level (0.1% of the induced level). And some inducer enters anyway via another uptake system.