Operons
Control of Gene Activity in Prokaryotic Cells

I. The activity of genes is controlled by the cell and the environment. A. Inducible genes are inactive unless circumstances cause them to be activated (“turned on”). B. Repressible genes are active unless circumstances cause them to be inactivated (“turned off”). C. Constitutive gene functions are active continually, with no control exerted. This is generally an abnormal situation.

II. In prokaryotic cells (and viruses) the control of gene activity is often in the form of operons. A. Operons are a form of transcriptional control. B. An operon consists of the structural gene (or genes) which actually code for specific proteins and the controlling elements associated with the control of those genes. An operon typically contains several genes, all under the same control mechanism. C. Though rather similar controlling systems have been found for some eukaryotic genes, control mechanisms in eukaryotes are generally more diverse and more complex, and except for a few examples in simple eukaryotic organisms like yeasts, multiple genes are not found to function under a single control mechanism. In other words, eukaryotic cells do not have operons.

III. The first operon investigated was the lac operon in E. coli. This work came from Jacob and Monod (1959 Journal of Molecular Biology V. 1). A. The primary carbohydrate source for the cell is the sugar glucose, but there are a large number of sugars which can be used if there isn’t enough glucose available to support the energy needs of the cell. Sugars like lactose are “backup” carbohydrates. This means that the cell only metabolizes lactose if glucose is low and lactose is plentiful. B. The cell uses a negative control system (the lac operon) to respond to the availability of lactose in the environment. Negative control means that it’s a system in which the active substance acts to turn off function. It is very wasteful to manufacture the enzymes needed to metabolize lactose if there is no lactose to be used, so under normal conditions the genes for producing the enzymes for metabolizing lactose need to be inactive. They should be activated only when lactose is plentiful. So the purpose of the operon is to keep these genes turned off if there’s no lactose around, and to turn them on if lactose is plentiful. 1. The lac operon contains three genes which code for enzymes necessary for the metabolism of the sugar lactose. These three genes are side by side on the DNA molecule, and they are transcribed as a single, polycistronic message. The operon consists of a promoter, an operator, the structural genes, a termination sequence, and a repressor gene. a. The lac z gene codes for beta-galactosidase. b. The lac y gene codes for permease c. The lac a gene codes for transacetylase 2. There is a single promoter region which precedes the lac z gene (the first gene in the sequence). The promoter region is where the RNA polymerase binds for transcription. 3. Between the Promoter and the lac z gene is a region called the Operator. This is the primary control site for the operon. 4. The repressor gene (lac I) is not contiguous with the other portions of the operon. The repressor codes for the production of a diffusible repressor protein. So our entire operon could be diagrammed like this: [pic] lac I is the name for the repressor gene; P is the promoter, O the operator, and T the termination sequence. Note that the prokaryotic genome is a circular DNA molecule, and we are viewing just one segment of that circle. Also note that the normal condition for a prokaryotic cell is to have a single copy of its genome in the cell. 5. When glucose is plentiful and/or lactose is low, the desired situation is for these genes to be repressed (inactive). 6. The repressor gene codes for the production of a diffusible repressor protein, which is present in low numbers in the cell at all times. The repressor has an affinity for the operator of the lac operon, and unless something happens to intervene, the repressor molecule will bind to the O site and block the movement of an RNA polymerase from the promoter to the structural genes, thus preventing transcription of those genes. [pic] 7. When lactose is plentiful (and glucose is not) the repression needs to be removed so the genes can be activated. The trigger for this activation needs to be lactose. Besides being attracted to the operator sequence of the operon, the repressor protein also has an affinity for a slightly unusual form of lactose called allolactose, which will be present as a small percentage of the lactose available whenever there’s lactose around. When the repressor binds to allolactose, the configuration (three-dimensional shape) of the repressor is altered, and it is no longer able to bind to the operator. It falls off, and there is nothing to prevent RNA polymerase from reading through the operator to the structural genes, and the three enzymes will thus be made. [pic] 8. As the cell metabolizes the available lactose, eventually the concentration of lactose will fall far enough that there won’t be enough around to maintain the complexes with the available repressor proteins, the repressor will return to its former configuration, and will once again bind to the operator and repress the structural genes. C. There are actually two conditions which must be met if the cell needs these enzymes to be produced. Not only must lactose be plentiful, but glucose must be low. The cell responds to glucose availability through a positive control mechanism involving cyclic AMP. Positive control means that it’s a system in which the active substance acts to turn on function. 1. When abundant glucose is available, it isn’t efficient for the cell to metabolize lactose, even if it is plentiful, because it takes more energy to metabolize lactose than to metabolize glucose. So the lac operon has a second control system which keeps the operon turned off when glucose is abundant. 2. This repression is called catabolite repression, and involves a protein called catabolite-activating protein (CAP). 3. CAP has an affinity for the promoter region of the lac operon, and unless CAP is bound to that region, RNA polymerase will not bind to the promoter, and transcription will not occur. (Contrast this to the situation with the lac repressor. The repressor prevents transcription when bound to the Operator site; CAP allows transcription when bound to the operator site. This is why catabolite repression is a positive control mechanism and the repressor system is a negative control mechanism.) 4. In order to bind to the promoter, CAP must first be combined with a molecule called cyclic AMP (cAMP), which is produced from ATP through the action of the enzyme adenyl cyclase. 5. The presence of high levels of glucose in the cell inhibits the activity of adenyl cyclase, thus reducing the production of cAMP. By reducing the level of cAMP in the cell, glucose thus reduces the level of CAP. With the reduction of CAP, and its unavailability for binding to the Promotor of the lac operon, activity of that operon is repressed. D. So these three lactose-metabolysis genes are under dual control. 1. The CAP system prevents lac operon activity when glucose is plentiful because high glucose levels lead to reduced CAP availability, and CAP is necessary for this operon to function. 2. The repressor system prevents lac operon activity when lactose is not available because the repressor protein binds to the Operator site and prevents transcription. The repression can only be removed when lactose is high because allolactose is necessary to inactivate the repressor. 3. The combination of these two control mechanisms ensures that these enzymes will be produced only under conditions in which glucose is low and lactose is high. E. In low glucose conditions (when the only control mechanism functioning is the repressor system), mutations in the various components of the operon have been studied. 1. Some mutations in the I gene (which codes for the repressor) produced an altered protein which is unable to bind to the Operator. These mutations would result in constitutive gene activity. 2. There are also I gene mutations which produce a repressor which is unable to bind to allolactose. These mutations produce cells which are unable to activate the lac operon genes, and which can therefore not metabolize lactose, even though the structural genes themselves may be completely fine. 3. There are also mutations which alter the sequence of the Operator region such that the repressor molecule is no longer able to bind. These would be constitutive for all of the genes in the operon.

IV. The tryptophan (trp) operon in E. coli is a negative control repressible system. It’s negative control because the system produces a repressor which functions to turn off the operon. It’s repressible because, unlike the lac operon, repression occurs when a critical substance is abundant in the cell. (For the trp operon, that critical substance is the amino acid tryptophan; for the lac operon the critical substance is lactose, and its presence removes repression.) A. Tryptophan is an important amino acid which most E. coli can acquire in two ways. They can extract it from the materials the cell consumes, or they can manufacture it themselves. The genes controlled by the trp operon produce enzymes which are necessary for the cell to produce its own tryptophan. Again, contrast to the lac operon. The lac operon enzymes function in the digestion (catabolism) of a “food” molecule (lactose); the trp operon enzymes function in the manufacture (anabolism) of a necessary amino acid for the construction of the cell’s own proteins. The lac operon needs to be active when there’s lots of lactose around; the trp operon needs to be active when tryptophan levels in the cell are low. B. The components of the trp operon are basically the same as those for the lac operon. 1. This operon controls five structural genes which code for enzymes needed for the production of tryptophan, including the key enzyme tryptophan synthetase. These genes are called trp E, trp D, trp C, trp B and trp A. 2. In the trp operon, the repressor gene is named trp R. [pic] C. The repressor gene (trp R) codes for a repressor protein. Unlike the lac repressor, this one is inactive as it is produced by the cell. It requires a co-repressor in order to bind to the Operator. [pic] D. The co-repressor is the amino acid tryptophan. Thus, when tryptophan is abundant, the repressor is active and will bind to the Operator, preventing transcription of these genes. But if tryptophan levels fall, the repressor will lose its trp co-repressor and will fall off the Operator, and the genes will be transcribed and the enzymes constructed. This leads to exactly the control needed—if there’s a lot of tryptophan around, the cell doesn’t want to make more, and it would be a waste of energy and materials to have this set of genes active. It’s when tryptophan levels fall that the cell needs to manufacture more, and needs this operon to be active. E. This is an example of feedback control, a very common control mechanism in living systems. Feedback control happens when the end product of a process (in this cases, tryptophan) functions to inactivate the process (in this case, to repress the operon). This allows a pretty consistent steady-state control over the level of the key substance in the system.

V. Since these early discoveries, many additional operon systems have been studied in a variety of prokaryotic organisms. Operons are also of significance among viruses. For example, the lambda phage (a temperate virus which parasitizes E. coli) had two competing operon systems which begin to function as soon as the virus attacks a cell. The “winner” of this competition determines whether the virus will follow a lysogenic or a lytic pathway.

VI. Operons as such are not known in eukaryotic cells (other than some possible candidates in yeast). Some of the control mechanisms known for eukaryotic genes bear a resemblance to the operon control system, but strings of contiguous genes, all under the control of a single promoter/operator region, are not found in eukaryotic cells.