Positive control and Catabolite repression (Lac operon regulation)
E.coli and many other bacteria will metabolize glucose preferentially in the presence of lactose and other sugars. They do so because glucose enters glycolysis without further modification and therefore requires less energy to metabolize than do other sugars.
When glucose is available, genes that participate in the metabolism of other sugars are repressed. This phenomenon known as catabolite repression (or the glucose effect).
Catabolite repression is a type of positive control in the lac operon.
(The efficient transcription of the lac operon takes place only if lactose is present and glucose is absent).
How is the expression of the lac operon influenced by glucose? What brings about catabolite repression?
The catabolite repression of the lac operon and several other operons is mediated by:
1) Catabolite Activator Protein (CAP) – Regulatory protein (activator protein)
2) Cyclic Adenosine Monophosphate (cAMP) – small effector molecule
- CAP is a dimeric protein that binds to CAP binding site located within or slightly upstream of the promoter of the lac operon.
- CAP is also called as cyclic AMP receptor protein (CRP) because of its involvement with cAMP. The gene encoding this protein is named crp gene.
- The cAMP Receptor Protein (CRP) is an allosteric protein that is inactive in the free form but is activated by binding to cAMP. A dimer of CRP is activated by a single molecule of cyclic AMP.
- CAP must form a complex with cAMP before binding to the promoter of the lac operon. Only the CAP/cAMP complex binds to the lac promoter. In the absence of cAMP, CAP does not bind.
- RNA polymerase does not bind efficiently to many promoters unless CAP is first bound to the DNA.
- The CAP/cAMP complex must be present at its binding site in the lac promoter in order for the operon to be induced normally.
How does CAP –cAMP complex enhance transcription of genes?
CAP contains a helix-turn-helix DNA-binding motif. When it binds at the CAP site, it causes the DNA helix to bend. The CAP/cAMP and RNA polymerase binding sites are adjacent to one another in the lac promoter. The bent helix enables CAP to interact directly with the RNA polymerase enzyme bound to the promoter and facilitate the initiation of transcription. The CAP/cAMP complex exerts positive control over the transcription of the lac operon by enhancing the affinity of RNA polymerase for the lac promoter. (It has an effect exactly opposite to that of repressor binding to an operator).
- The catabolite activator protein exerts positive control in more than 20 operons of E. coli.
- The response to CAP varies among these promoters; some operons are activated by low levels of CAP, whereas others require high levels.
The promoter consists of two components: (1) the site that binds the CAP/cAMP complex (2) the RNA polymerase binding site.
The catabolite repression in E.coli is achieved through the utilization of phosphotransferase system.
It involves two mechanisms.
1) Inducer exclusion: Glucose prevents the uptake of alternative carbon sources like lactose from the medium.
In case of lac operon, exclusion of lactose results in the absence of inducer allolactose and hence operon cannot be transcribed.
Mechanism of inducer exclusion
- The key molecular component in inducer exclusion is the PTS (phosphoenolpyruvate: glucose phosphotransferase system). It is a complex of proteins in the bacterial membrane, which simultaneously phosphorylates and transports sugars into the cell.
- When the PTS is actively transporting glucose into the cell, one of the proteins of this complex (enzyme IIAGlc) becomes dephosphorylated.
- Dephosphorylated enzyme IIAGlu then binds to the lactose permease located in the membrane.
- This binding will allosterically inhibit lactose permease activity and lactose uptake is blocked.
- Thus, the concentration of lac inducer is very low in the presence of glucose, so the Lac repressor is active and represses transcription of the lac operon genes.
When glucose is absent, enzyme IIAGlu exists in phosphorylated form. So it will not inhibit Lac permease enzyme. As a result lactose will be imported into the cell, allolactose will be formed and the operon is switched on.
2) Controlling the concentration of intracellular cAMP level
- In E. coli, the intracellular cAMP concentration is sensitive to the presence or absence of glucose.
- The concentration of cAMP is inversely proportional to the level of available glucose
- Low concentrations of glucose stimulate high levels of cAMP, resulting in increased cAMP– CAP binding to DNA. This increase enhances the binding of RNA polymerase to the promoter and increases transcription of the lac genes by some 50-fold.
- High concentrations of glucose cause sharp decreases in the intracellular concentration of cAMP. So little cAMP –CAP complex is available to bind to the DNA. Subsequently, RNA polymerase has poor affinity for the lac promoter, and little transcription of the lac operon takes place.
How adenylyl cyclase activity is affected in the presence of glucose?
- Control of adenylate cyclase activity occurs via the phosphotransferase system.
- Glucose is changed to glucose 6-phosphate during its transport through the PTS system.
- As the cell imports glucose, phosphate is continually removed from EIIAGlc. As a result unphosphorylated IIAGlc will be the predominant form of the protein present.
- Unphosphorylated EIIAGlc interacts with and inhibits adenylate cyclase activity (the enzyme that catalyzes the formation of cAMP from ATP).
- The net effect of inhibiting adenylate cyclase is that intracellular cAMP levels fall.
(This happens because cAMP synthesis stops and cAMP is continually degraded by cAMP phosphodiesterase).
How does adenylyl cyclase activity affected in the absence of glucose?
If glucose is not in the medium, it is unavailable for transport. So there is no sugar to receive the phosphate group from phosphorylated EIIA. Phosphorylated form of protein IIAGlc stimulates membrane bound adenylyl cyclase enzyme. As a result cAMP levels rise and cAMP-CRP complex forms, enabling CRP-dependent expression of catabolite-repressible genes.
Refer the link: PEP dependent Phosphotransferase system (PTS) in E.coli
If E. coli grows in a medium containing both glucose and lactose, it uses glucose preferentially until the sugar is exhausted. Then after a short lag, growth resumes with lactose as the carbon source. This biphasic growth pattern or response is called diauxic growth.
Catabolite repression or the glucose effect plays a role in diauxic growth.
Inducer exclusion appears to be the main reason for glucose/lactose diauxic growth.
- At first, the bacterium will metabolize all the glucose and grow at a higher speed.
- Eventually, when all the glucose has been consumed, the bacterium will begin the process of expressing the genes to metabolize the lactose.
Diauxic growth curve have multiple phases:
1) Fast growth phase
The bacterium is consuming exclusively glucose and is capable of rapid growth.
During this phase glucose represses the enzymes for lactose utilization (catabolite repression or the glucose effect).
2) Lag phase (Induction of lac operon)
Cellular machinery used to metabolize sugar lactose is activated and observable cell growth stops.
Once the metabolic enzymes are synthesized, second available sugar is metabolized and growth resumes.
3) Growth phase which is slower than the first fast growth phase
Reason: Sugar lactose is used as the primary energy source instead of glucose.
4) Saturation phase
Growth ceases due to resource exhaustion and/or waste accumulation