Principles of Gene Regulation:- The lac Operon Is Subject to Negative Regulation
The lactose (lac) operon (Fig. 1a) includes the genes for-galactosidase (Z), galactoside permease (Y), and thiogalactoside transacetylase (A). The last of these enzymes appears to modify toxic galactosides to facilitate their removal from the cell. Each of the three genes is preceded by a ribosome binding site (not shown in Fig. 1) that independently directs the translation of that gene . Regulation of the lac operon by the lac repressor protein (Lac) follows the pattern outlined in Figure 28–4a. The study of lac operon mutants has revealed some details of the workings of the operon’s regulatory system. In the absence of lactose, the lac operon genes are repressed. Mutations in the operator or in another gene, the I gene, result in constitutive synthesis of the gene products. When the I gene is defective, repression can be restored by introducing a functional I gene into the cell on another DNA molecule, demonstrating that the I gene encodes a diffusible molecule that causes gene repression. This molecule proved to be a protein, now
called the Lac repressor, a tetramer of identical monomers. The operator to which it binds most tightly (O1) abuts the transcription start site (Fig. 1a). The I gene is transcribed from its own promoter (PI) independent of the lac operon genes. The lac operon has two secondary binding sites for the Lac repressor. One (O2) is centered near position +410, within the gene encoding -galactosidase (Z); the other (O3) is near position 90, within the I gene. To repress the operon, the Lac repressor appears to bind to both the main opera tor and one of the two secondary sites, with the intervening DNA looped out (Fig. 1b, c). Either binding arrangement blocks transcription initiation.
Despite this elaborate binding complex, repression is not absolute. Binding of the Lac repressor reduces the rate of transcription initiation by a factor of 103. If the O2 and O3 sites are eliminated by deletion or mutation, the binding of repressor to O1 alone reduces tran scription by a factor of about 102. Even in the repressed state, each cell has a few molecules of β-galactosidase and galactoside permease, presumably synthesized on the rare occasions when the repressor transiently dissociates from the operators. This basal level of tran scription is essential to operon regulation.
When cells are provided with lactose, the lac operon is induced. An inducer (signal) molecule binds to a specific site on the Lac repressor, causing a conformational change (Fig. 1d) that results in dissociation of the repressor from the operator. The inducer in the lac operon system is not lactose itself but allolactose, an isomer of lactose . After entry into the E. coli cell (via the few existing molecules of permease), lactose is converted to allolactose by one of the few existing β-galactosidase molecules. Release of the opera tor by Lac repressor, triggered as the repressor binds to allolactose, allows expression of the lac operon genes and leads to a 103-fold increase in the concentration of β-galactosidase.
Several β-galactosides structurally related to allolactose are inducers of the lac operon but are not substrates for β-galactosidase; others are substrates but not inducers. One particularly effective and nonmetabolizable inducer of the lac operon that is often used experimentally is isopropylthiogalactoside (IPTG):
An inducer that cannot be metabolized allows researchers to explore the physiological function of lactose as a car bon source for growth, separate from its function in the regulation of gene expression. In addition to the multitude of operons now known in bacteria, a few polycistronic operons have been found in the cells of lower eukaryotes. In the cells of higher eukaryotes, however, almost all protein-encoding genes are transcribed separately. The mechanisms by which operons are regulated can vary significantly from the simple model presented in Figure 1. Even the lac operon is more complex than indicated here, with an activator also contributing to the overall scheme, as we shall see in Section 28.2. Before any further discussion of the layers of regulation of gene expression, however, we examine the critical molecular interactions between DNA-binding proteins (such as repressors and activators) and the DNA sequences to which they bind.
FIGURE 1 The lac operon. (a) The lac operon in the repressed state. The I gene encodes the Lac repressor. The lac Z, Y, and A genes encode -galactosidase, galactoside permease, and thiogalactoside transacetylase, respectively. P is the promoter for the lac genes, and PI is the promoter for the I gene. O1 is the main operator for the lac operon; O2 and O3 are secondary operator sites of lesser affinity for the Lac repressor. (b) The Lac repressor binds to the main operator and O2 or O3, apparently forming a loop in the DNA that might wrap around the repressor as shown. (c) Lac repressor bound to DNA (de rived from PDB ID 1LBG). This shows the protein (gray) bound to short, discontinuous segments of DNA (blue). (d) Conformational change in the Lac repressor caused by binding of the artificial inducer isopropylthiogalactoside, IPTG (derived from PDB ID 1LBH and 1LBG). The structure of the tetrameric repressor is shown without IPTG bound (transparent image) and with IPTG bound (overlaid solid image; IPTG not shown). The DNA bound when IPTG is absent (transparent structure) is not shown. When IPTG is bound and DNA is not bound, the repressor’s DNA-binding domains are too disordered to be defined in the crystal structure.
