In 1961, F. Jacob and J. Monod proposed the operon model to explain the genetic basis of enzyme induction and repression in prokaryotes.
A few years later (1965), these two investigators were awarded the Nobel Prize for their most incisive work.
Although Jacob and Monod’s original operon model applied specifically to the regulation of the genes for lactose metabolism in E. coli, additional findings by these two scientists as well as the work of many others have revealed the mechanisms by which other operons function.
The components of an operon are illustrated in Figure 11-11a. The operon consists of a series of structural genes (denoted by SGI, SG2, and SG3), a segment of DNA called an operator adjacent to the structural genes, and, next to the operator, an additional DNA segment called a promoter. Elsewhere on the chromosome, there is a segment of DNA called a regulator gene and next to this is a DNA segment that functions as a promoter for the regulator gene.
In the absence of an inducer (e.g., lactose in an E. coli culture) RNA polymerase binds to the promoter of the regulator gene and progresses along the DNA transcribing the regulator gene. The RNA transcript- that is produced is then translated into a repressor protein, which binds to the operator, of the operon.
Binding of the repressor to the operator (see the top half of Fig. 11-11b) prevents RNA polymerase from attaching to this segment of the DNA. As a result, the structural genes cannot be transcribed and no mRNA or enzyme is formed. It is now known that the repressor prevents the binding of the RNA polymerase by steric hindrance.
When Jacob and Monod conducted their studies in the 1960s, the existence of the promoter and the mechanism of repressor function were not known. Today it is recognized that operons also regulate enzyme induction'(Fig. 11-11b) and repression (Fig. 11-11c).
In the case of the operon model for the induction of enzymes (Fig. 11-11b), the inducer (e.g., lactose) combines with the repressor to form a complex that cannot bind to the operator. Therefore, RNA polymerase can associate with the operon promoter and proceeds along the DNA, transcribing it into mRNA, which in turn are translated into polypeptides and enzymes.
In the operon model for enzyme repression (Fig. 11-11c), the repressor cannot bind to the operator unless it first complexes with a corepressor. In the example presented earlier in which histidine-synthesizing enzymes in S. typhimurium are repressed by histidine that is supplied exogenously, histidine serves the role of the corepressor. In the absence of the corepressor, RNA polymerase associates with the operon promoter, and transcription and translation occur.
The mRNA transcripts of multigenic operons (i.e., operons containing two or more structural genes) contain the code for several polypeptides and are said to be polycistronic. Therefore, there is the coordinate expression of all of these genes of the operon.
The Lac Operon:
The progressive unraveling of the molecular organization and function of the lac operon is a classic study in physiology and genetics. Jacob and Monod began their studies of this operon in E. coli in the early 1960s; since then, they and many others have continued the study so that today it is one of the best understood regulatory systems (Fig. 11-12).
In E. coli, the lac operon is inducible, consisting of three structural genes of known lengths, designated z, y, and a, which, respectively, code for β-galactosidase, β-galactoside permease, and β-galactoside iransacetylase. The location and nucleotide sequences are also known for the promoter (P) and operator (O) segments of the operon.
The regulator gene consists of two contiguous portions of known lengths—an i gene and the promoter of the i gene (P (i)) (see Fig. 11-12). The i gene codes for a polypeptide consisting of 360 amino acids; four of these polypeptides combine to form a biologically active tetramer that functions as a repressor of the operator gene. The repressor binds to the operator and, in so doing, prevents RNA polymerase from associating with the promoter of the operon.
The normal inducer of the lac operon is allolactose, which is produced from lactose by β-galactosidase (Fig. 11-13). A few copies of this enzyme are present in E. coli cells even in the uninduced state. When allolactose combines with the repressor, a steric change occurs that causes the repressor to be released from the operator. As a result, transcription of the structural genes by RNA polymerase begins and the three enzymes quickly appear in the cells.
The promoter of the lac operon contains two active components that regulate transcription. One is the RNA polymerase binding site and the second is the catabolite activator protein (CAP) binding site. This CAP site functions to prevent transcription of the lac operon when sufficient glucose is present. Today, a number of operons are known in addition to the lac operon. Some of these are listed in Table 11-3.
Catabolic Repression:
Catabolic repression is a specific type of repression of enzyme production in which a metabolite such as glucose acts to repress the formation of enzymes that would allow the catabolism of other, related metabolites. For example, when E. coli cells are cultured in a medium that is rich in glucose, the glucose represses the formation of β-galactosidase even if lactose (an inducer of this enzyme) is added to the medium. Glucose is even known to repress the production of enzymes that formerly were thought to be constitutive.
A common phenomenon in bacteria is the suppression of aerobic respiration and electron transport by high glucose concentrations, even in the presence of ample oxygen. Under this condition, the cells utilize the glycolytic and fermentative pathways. The manner in which the catabolite brings about the effect is only partially understood. In the case of catabolite repression by glucose in bacteria, it appears that glucose affects the amount of cyclic AMP (cAMP) present in the cells.
When the concentration of glucose is high, the concentration of cAMP is low; and low levels of glucose are accompanied by high concentrations of cAMP. It is possible that glucose affects the synthesis of cAMP, which is formed from ATP by adenylcyclase (Fig. 11-14).
cAMP is necessary for the CAP to bind to the promoter site. cAMP binds to the CAP, and once the cAMP-CAP complex binds to the promoter (Fig. 11-15), the RNA polymerase can attach to the promoter and begin transcription. For example, in the case of the lac operon, when the glucose level is high (even if lactose is also present), the cAMP is low, and therefore the cAMP—CAP complex is not available to bind to the promoter and allow transcription to start.
However, in the absence of glucose and in the presence of lactose (which forms a complex with the repressor), cAMP is plentiful and is available to combine with the CAP so that transcription proceeds. The rate of lac operon transcription in the absence of glucose is 50 times as great as in the presence of glucose.
Translational Control:
Regulatory mechanisms usually function at the beginning of a sequence of events rather than at the end. For example, allosteric feedback control is usually achieved by regulating the enzyme that catalyzes the first reaction in a sequence of reactions. Likewise, control of the number of enzyme molecules produced in a cell is most commonly brought about at the transcriptional level rather than at the level of translation.
Regulatory mechanisms conserve cell energy and prevent the buildup of materials that will not be used. Therefore, translational control mechanisms would be expected to be rare and at the present time there is little evidence for very much control being exercised at this level.
Indirect evidence suggests that translational control of enzyme synthesis may occur for enzymes that are part of the same operon. For example, because the three structural genes in the lac operon are controlled by the same operator, one would expect that equal quantities of the three enzymes would be produced.
However, induced cells contain more copies of one enzyme over another. This could be explained if the ribosome detaches from mRNA before completing the translation of the entire polycistronic message.
The economic impact of the use of control mechanisms is reflected also in the synthesis of ribosomal RNA (rRNA). When E. coli cells are cultured in a medium that is deficient in amino acids, they are unable to synthesize proteins. As a consequence, the cells also stop producing rRNA—a mechanism called stringent control.
Mutants of E. coli do exist, however, that continue to synthesize rRNA under these conditions and are called relaxed mutants. Paper chromatographic analysis of nucleotide extracts of the stringent cells and relaxed mutants reveals that there are two spots in the chromatogram of stringent cells that are absent in the chromatogram of the relaxed mutants.
Initially these spots were called “magic spot I” and “magic spot II,” but they have now been identified as guanosine-5′-diphosphate-2′- (or 3′) diphosphate (ppGpp) and guanosine-5′-triphos- phate-2′- (or 3′) diphosphate (pppGpp), respectively. The studies of Cashel and Gallant and their colleagues indicate that these nucleotides trigger the events that turn off rRNA synthesis when the amino acids needed by the cells for protein synthesis are lacking.