Regulation of Lac operon
The regulation of lac operon is very important within E.coli cells when it comes to energy production, utilization, and conservation. There are two key stimuli that regulate the amount of Lac operon gene expression – glucose and lactose. E.coli cells prefer to use glucose as their energy source because it is able to provide more energy than lactose when broken down. If glucose is present, the lac operon will be negatively regulated in order to save energy. When glucose becomes scarce within the E.coli cell, the lac operon is positively regulated in order to take advantage of the lactose present. Although lactose doesn’t produce as much energy as glucose when broken down, it’s better than not creating any energy at all.
A) Negative control – Repression
Repression: A mechanism often used to decrease or inhibit the expression of a gene.
Negative control: The regulatory protein is a repressor.
(It binds to the operon and inhibits transcription. Like a switch, the regulatory protein turns gene expression off).
What is lac repressor?
The lac repressor (produced by regulatory gene lacI) is a DNA-binding protein which inhibits the expression of genes regulating lactose metabolism in bacteria. These genes are repressed when lactose is not available to the cell.
Advantage of repression: Saving energy
- Bacterium invests energy in the production of machinery necessary for uptake and utilization of lactose only when lactose is present.
- The lac repressor protein is a homo-tetramer, which contains two DNA-binding surfaces that recognize a DNA sequence named “operator.” Each dimer binds to the operator sequence via a helix-turn-helix motif.
How does the lac repressor inhibit the expression of the lac operon?
- The lac repressor acts as a lactose sensor.
- In the absence of lactose, the repressor binds very tightly to the operator. When lac repressor is bound to DNA, it prevents bound RNA polymerase from locally unwinding the DNA to expose the base that will act as the template for the synthesis of mRNA.
- That means repressor prevents transcription of structural genes in lac operon.
B) Double negative control – Derepression
Derepression: Removal of repression
- An inducer derepresses the lac operon (The process can be called as induction).
- The lac operon is an example of inducible operon.
Permease is required to transport lactose into the cell. If the lac operon is repressed and no permease is being produced, how does lactose get into the cell to inactivate the repressor and turn on transcription?
- The answer is that repression never completely shuts down transcription of the lac operon. Even with active repressor bound to the operator, there is a low level of transcription and a few molecules of β-galactosidase, permease, and transacetylase are synthesized.
- When lactose appears in the medium (glucose is absent), the permease that is present transports a small amount of lactose into the cell. There, the few molecules of β-galactosidase that are present convert some of the lactose into allolactose. The allolactose then attaches to the repressor and alters its shape so that the repressor no longer binds to the operator.
- When the operator site is clear, RNA polymerase can bind and transcribe the structural genes of the lac operon and the lac enzymes are produced.
- (If lactose is added to the growth medium, the rate of synthesis of all three enzymes simultaneously increases about a thousand fold within 2 to 3 minutes).
Coordinate induction: The simultaneous synthesis of several enzymes, stimulated by a specific molecule called as the inducer.
What is allolactose? How it is formed?
- Allolactose is referred to as the inducer of the lac operon because it turns on or induces the expression of the lac genes.
- Allolactose is a disaccharide of galactose and glucose with a β-1, 6 linkage rather than β-1, 4 linkages.
- Allolactose is an isomer of lactose and also side product of the β-galactosidase reaction.
- Conversion of lactose to allolactose is by transglycosylation reaction.
How does the allolactose induce lac operon or how does the presence of the inducer modulate gene expression?
Allolactose binds to an allosteric site on the repressor protein causing a conformational change. As a result of this change, the repressor’s affinity for operator DNA is greatly reduced. Hence the repressor can no longer bind to the operator region and falls off. RNA polymerase can then binds to the promoter and transcribe the lac genes.
Artificial inducer of lac operon
These are compounds related to allolactose that can bind to the lac repressor and induce transcription of the lac operon.
Example: Isopropylthiogalactoside (IPTG)
- Although IPTG inactivates the repressor and allows the transcription of lacZ, lacY, and lacA, IPTG is not metabolized by β-galactosidase. They are not substrates of the enzyme.
- IPTG is useful in the laboratory as a tool for inducing gene expression independent of metabolism.
- The addition of lactose or a gratuitous inducer such as IPTG to bacteria growing on a poorly utilized carbon source (such as succinate) results in prompt induction of the lac operon enzymes.
Gratuitous inducer: A gratuitous inducer is a molecule which is structurally similar to another molecule that induces transcription for a specific product but they are not a substrate for the induced enzymes.
Feedback Control of the lac Operon
- When the enzymes encoded by the lac operon are produced, they break down lactose and allolactose, eventually releasing the repressor to stop additional synthesis of lac mRNA.
- The lac mRNA is extremely unstable, and decays with a half-life of only ~3 minutes. This feature allows induction to be reversed rapidly.
- Transcription ceases as soon as the inducer is removed and in a very short time all the lac mRNA has been destroyed. Finally In the absence of inducer, the operon is transcribed at a very low basal level.
C) Positive regulation of lac operon