Regulation of Gene Expression in Prokaryotes:- The lac Operon Undergoes Positive Regulation
The operator-repressor-inducer interactions described earlier for the lac operon provide an intuitively satisfying model for an on/off switch in the regulation of gene expression. In truth, operon regulation is rarely so simple. A bacterium’s environment is too complex for its genes to be controlled by one signal. Other factors besides lactose affect the expression of the lac genes, such as the availability of glucose. Glucose, metabolized directly by glycolysis, is E. coli’s pre ferred energy source. Other sugars can serve as the main or sole nutrient, but extra steps are required to prepare them for entry into glycolysis, necessitating the syn thesis of additional enzymes. Clearly, expressing the genes for proteins that metabolize sugars such as lactose or arabinose is wasteful when glucose is abundant. What happens to the expression of the lac operon when both glucose and lactose are present? A regulatory mechanism known as catabolite repression restricts expression of the genes required for catabolism of lactose, arabinose, and other sugars in the presence of glucose, even when these secondary sugars are also present. The effect of glucose is mediated by cAMP, as a coactivator, and an activator protein known as cAMP receptor protein, or CRP (the protein is sometimes called CAP, for catabolite gene activator protein). CRP is a homodimer (subunit Mr 22,000) with binding sites for DNA and cAMP. Binding is mediated by a helix-turn helix motif within the protein’s DNA-binding domain (Fig. 1). When glucose is absent, CRP-cAMP binds to a site near the lac promoter (Fig. 2a) and stimulates RNA transcription 50-fold. CRP-cAMP is therefore a positive regulatory element responsive to glucose levels, whereas the Lac repressor is a negative regulatory element responsive to lactose. The two act in concert. CRP-cAMP has little effect on the lac operon when the Lac repressor is blocking transcription, and dissociation of the repressor from the lac operator has little effect on transcription of the lac operon unless CRP cAMP is present to facilitate transcription; when CRP is not bound, the wild-type lac promoter is a relatively weak promoter (Fig. 2b). The open complex of RNA polymerase and the promoter does not form readily unless CRP-cAMP is present. CRP inter acts directly with RNA polymerase (at the region shown in Fig.1) through the polymerase’s α subunit.
FIGURE 1 CRP homodimer. (PDB ID 1RUN) Bound molecules of cAMP are shown in red. Note the bending of the DNA around the protein. The region that interacts with RNA polymerase is shaded yellow.
FIGURE 2 Activation of transcription of the lac operon by CRP. (a) The binding site for CRP-cAMP is near the promoter. As in the case of the lac operator, the CRP site has twofold symmetry (bases shaded beige) about the axis indicated by the dashed line. (b) Sequence of the lac promoter compared with the promoter consensus sequence. The differences mean that RNA polymerase binds relatively weakly to the lac promoter until the polymerase is activated by CRP-cAMP.
The effect of glucose on CRP is mediated by the cAMP interaction (Fig. 3). CRP binds to DNA most avidly when cAMP concentrations are high. In the presence of glucose, the synthesis of cAMP is inhibited and efflux of cAMP from the cell is stimulated. As [cAMP] declines, CRP binding to DNA declines, thereby de creasing the expression of the lac operon. Strong in duction of the lac operon therefore requires both lactose (to inactivate the lac repressor) and a lowered concentration of glucose (to trigger an increase in [cAMP] and increased binding of cAMP to CRP). CRP and cAMP are involved in the coordinated regulation of many operons, primarily those that encode enzymes for the metabolism of secondary sugars such as lactose and arabinose. A network of operons with a common regulator is called a regulon. This arrange ment, which allows for coordinated shifts in cellular functions that can require the action of hundreds of genes, is a major theme in the regulated expression of dispersed networks of genes in eukaryotes. Other bacterial regulons include the heat-shock gene system that responds to changes in temperature (p. 1083) and the genes induced in E. coli as part of the SOS response to DNA damage, described later.
FIGURE 3 Combined effects of glucose and lactose on expression of the lac operon. (a) High levels of transcription take place only when glucose concentrations are low (so cAMP levels are high and CRP-cAMP is bound) and lactose concentrations are high (so the Lac repressor is not bound). (b) Without bound activator (CRP-cAMP), the lac promoter is poorly transcribed even when lactose concentrations are high and the Lac repressor is not bound.
