The Bacterial Operon In bacteria, the genes that encode the enzymes of a metabolic pathway are usually clustered

together on the chromosome in a functional complex called an operon. All of the genes of an operon are coordinately controlled by a mechanism first described in 1961 by Francois Jacob and Jacques Monod of the Pasteur Institute in Paris. A typical bacterial operon consists of structural genes, a promoter region, an operator region, and a regulatory gene (Figure 12.28).

Structural genes code for the enzymes themselves. The structural genes of an operon usually lie adjacent to oneanother, and the RNA polymerase moves from one structuralto the next, transcribing all of the genes into a single mRNA. This extended mRNA is then translated into the various individual enzymes of the metabolic pathway. Consequently, turning on one gene turns on all the enzyme-producing genes of an operon. The promoter is the site where the RNA polymerase binds to the DNA prior to beginning transcription. The operator, which typically resides adjacent to or overlaps with the promoter (see Figure 12.30), serves asthe binding site for a protein, called the repressor. The repressor is an example of a gene regulatory proteina protein that recognizes a specific sequence of base pairs within the DNA and binds to that sequence with high affinity. As will be evident in the remaining sections of this chapter, DNA-binding proteins, such as bacterial repressors, play a predominant role in determining whether or not a particular segment of the genome is transcribed. The regulatory gene encodes the repressor protein. The key to operon expression lies in the repressor.When the repressor binds the operator (Figure 12.29), the promoter is shielded from the polymerase, and transcription of the structural genes is prevented. The capability of the repressor to bind the operator and inhibit transcription depends on the conformation of the repressor, which is regulated allosterically by a key compound in the metabolic pathway, such as lactose or tryptophan, as described shortly. It is the concentration of this key metabolic substance that determines whether the operon is active or inactive at any given time. The lac Operon The interplay among these various elements is illustrated by the lac operon the cluster of genes that regulates production of the enzymes needed to degrade lactose in bacterial cells. The lac operon is an example of an inducible operon, that is, one in which the presence of a key metabolic substance (in this case, lactose) induces transcription of the structural genes (Figure 12.29a). The lac operon contains three tandem structural genes: the z gene, which encodes _-galactosidase; the y gene, which encodes galactoside permease, a protein that promotes the entry of lactose into the cell; and the a gene, which encodes thiogalactoside transacetylase, an enzyme whose physiologic role is unclear. If lactose is present in the medium, the disaccharide enters the cell where it binds to the lac repressor, changing the conformation of the repressor and making it unable to attach to the DNA of the operator. In this state, the structural genes are transcribed, the enzymes are synthesized, and the lactose molecules are

catabolized. Thus, in an inducible operon, such as the lac operon, the repressor protein is able to bind to the DNA only in the absence of lactose, which functions as the inducer. As the concentration of lactose in the medium decreases, the disaccharide dissociates from its binding site on the repressor molecule. Release of lactose enables the repressor to bind to the operator, which physically blocks the polymerase from reaching the structural genes, turning off transcription of the operon. Positive control by Cyclic AMP. Repressors, such as those of the lac and trp operons, exert their influence by negative control, as the interaction of the DNA with this protein inhibits gene expression. The lac operon is also under positive control, as was discovered during an early investigation of a phenomenon called the glucose effect. If bacterial cells are supplied with glucose as well as a variety of other substrates, such as lactose or galactose, the cells catabolize the glucose and ignore the other compounds. The glucose in the medium acts to suppress the production of various catabolic enzymes, such as _-galactosidase, needed to degrade these other substrates. In 1965, a surprising finding was made: cyclic AMP (cAMP), previously thought to be involved only in eukaryotic metabolism, was detected in cells of E. coli. It was found that the concentration of cAMP in the cells was related to the presence of glucose in the medium; the higher the glucose concentration, the lower the cAMP concentration. Furthermore, when cAMP was added to the medium in the presence of glucose, the catabolic enzymes that were normally absent were suddenly synthesized by the cells. Although the exact means by which glucose lowers the concentration of cAMP has still not been elucidated, the mechanism by which cAMP overcomes the effect of glucose is well understood. As might be expected, a small molecule such as cAMP (see Figure 15.11) could not serve on its own to stimulate the expression of a specific battery of genes. As in eukaryotic cells, cAMP acts in prokaryotic cells by binding to a protein, in this case the cAMP receptor protein (CRP). By itself, CRP is unable to bind to DNA. However, the cAMPCRP complex recognizes and binds to a specific site in the lac control region (Figure 12.30). The presence of the bound CRP causes a change in the conformation of the DNA, which makes it possible for RNA polymerase to transcribe the lac operon. Thus the presence of the bound cAMPCRP complex is necessary for transcription of the operon, even when lactose is present and the repressor is inactivated. As long as glucose is abundant, cAMP concentrations remain below that required to promote transcription of the operon. The trp Operon In a repressible operon, such as the tryptophan (or trp) operon, the repressor is unable to bind to the operator DNA by itself. Instead, the repressor is active as a DNA-binding protein only when complexed with a specific factor, such as tryptophan (Figure 12.29b), which functions as a corepressor. In the absence of tryptophan, the operator site is open to binding by RNA polymerase, which transcribes the structural genes of the trp operon and leads to the production of the enzymes that synthesize tryptophan. Once tryptophan becomes available, the enzymes of the tryptophan synthetic pathway are no longer required. Under these conditions, the increased concentration of tryptophan leads to the formation of the tryptophanrepressor complex, which blocks transcription.