Bacteria such as E. coli usually rely on glucose as their source of carbon and energy. However, when glucose is scarce, E. coli can use lactose as their carbon source even though this disaccharide does not lie on any major metabolic pathways. An essential enzyme in the metabolism of lactose is β-galactosidase, which hydrolyzes lactose into galactose and glucose (Figure-1)
Figure-1- Action of Beta galactosidase on lactose
An E. coli cell growing on a carbon source such as glucose or glycerol contains fewer than 10 molecules of β -galactosidase. In contrast, the same cell contains several thousand molecules of the enzyme when grown on lactose. The presence of lactose in the culture medium induces a large increase in the amount of β -galactosidase by eliciting the synthesis of new enzyme molecules rather than by activating a preexisting but inactive precursor. A crucial clue to the mechanism of gene regulation is that two other proteins are synthesized in concert with β -galactosidase namely, galactoside permease and thiogalactoside transacetylase. The permease is required for the transport of lactose across the bacterial cell membrane. The transacetylase is not essential for lactose metabolism but appears to play a role in the detoxification of compounds that also may be transported by the permease. Thus, the expression levels of a set of enzymes that all contribute to the adaptation to a given change in the environment change together. Such a coordinated unit of gene expression is called an operon.
The parallel regulation of β -galactosidase, the permease, and the transacetylase suggested that the expression of genes encoding these enzymes is controlled by a common mechanism. Francois Jacob and Jacques Monod proposed the operon model (Figure-2) to account for this parallel regulation as well as the results of other genetic experiments .The genetic elements of the model are a regulator gene, a regulatory DNA sequence called an operator site, and a set of structural genes.
The regulator gene encodes a repressor protein that binds to the operator site. The binding of the repressor to the operator prevents transcription of the structural genes. The operator and its associated structural genes constitute the operon. For the lactose (lac) operon, the i gene encodes the repressor, o is the operator site, and the z, y, and a genes are the structural genes for β -galactosidase, the permease, and the transacetylase, respectively (Figure-2). The operon also contains a promoter site (denoted by p), which directs the RNA polymerase to the correct transcription initiation site. The z, y, and a genes are transcribed to give a single mRNA molecule that encodes all three proteins. A mRNA molecule encoding more than one protein is known as a polygenic or polycistronic transcript.
Figure-2- (A) The general structure of an operon as conceived by Jacob and Monod. (B) The structure of the lactose operon. In addition to the promoter (p) in the Operon, a second promoter is present in front of the regulator gene (i) to drive the synthesis of the regulator.
Regulation of lac operon
(a) Negative Control- Repression
How does the lac repressor inhibit the expression of the lac operon?
The lac repressor can exist as a dimer of 37-kd subunits, and two dimers often come together to form a tetramer. In the absence of lactose, the repressor binds very tightly and rapidly to the operator. When the lac repressor is bound to DNA, it prevents bound RNA polymerase from locally unwinding the DNA to expose the bases that will act as the template for the synthesis of the RNA strand.(Figure-3).Thus, very little β-galactosidase, permease, or transacetylase are produced.
Figure-3- In the absence of lactose lac Operon is off
(b) Double negative control- Derepression
How does the presence of lactose trigger expression from the lac operon?
Interestingly, lactose itself does not have this effect; rather, allolactose, a combination of galactose and glucose with an α-1,6 rather than an α -1,4 linkage, does. Allolactose is thus referred to as the inducer of the lac operon. Allolactose is a side product of the β-galactosidase reaction produced at low levels by the few molecules of β-galactosidase that are present before induction.
Figure- 4- structure of Allolactose(a) and Isopropyl thiogalactoside (IPTG)
A lactose analog that is capable of inducing the lac operon while not itself serving as a substrate for β-galactosidase is an example of a gratuitous inducer. An example is isopropylthiogalactoside (IPTG) -Figure-4-(b).
IPTG is useful in the laboratory as a tool for inducing gene expression. The addition of lactose or of 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.
When the lac repressor is bound to the inducer, the repressor’s affinity for operator DNA is greatly reduced. This binding leads to local conformational changes so that it cannot easily contact DNA simultaneously, leading to a dramatic reduction in DNA-binding affinity and the release of DNA by the lac repressor. With the operator site unoccupied, RNA polymerase can then transcribe the other lac genes and the bacterium produces the proteins necessary for the efficient utilization of lactose (Figure-5)
Figure-5- Presence of Lactose (substrate is actually allolactose) changes conformation of the lac repressor. Inactive form unable to bind to operator ->GENE TURNED ON
In such a manner, an inducer derepresses the lac operon and allows transcription of the structural genes for -β -galactosidase, galactoside permease, and thiogalactoside transacetylase.
Repressible and Inducible enzymes are both an example of negative control of a pathway. Activating the repressor proteins shuts off the pathway. Positive control requires that an activator molecule switch on transcription.
(c) Positive control
There are also DNA-binding proteins that stimulate transcription. One particularly well-studied example is the catabolite activator protein (CAP), which is also known as the cAMP response protein (CRP). When bound to cAMP, CAP, which also is a sequence-specific DNA-binding protein, stimulates the transcription of lactose catabolizing genes. Within the lac operon, CAP binds to an inverted repeat that is centered near position -61 relative to the start site for transcription (Figure-6).
The CAP-cAMP complex stimulates the initiation of transcription by approximately a factor of 50. A major factor in this stimulation is the recruitment of RNA polymerase to promoters to which CAP is bound. Studies have been undertaken to localize the surfaces on CAP and on the α subunit of RNA polymerase that participate in these interactions.
These energetically favorable protein-protein contacts increase the likelihood that transcription will be initiated at sites to which the CAP-cAMP complex is bound. Thus, in regard to the lac operon, gene expression is maximal when the binding of allolactose relieves the inhibition by the lac repressor, and the CAP-cAMP complex stimulates the binding of RNA polymerase. The E. coli genome contains many CAP-binding sites in positions appropriate for interactions with RNA polymerase.
Thus, an increase in the cAMP level inside an E. coli bacterium results in the formation of CAP-cAMP complexes that bind to many promoters and stimulate the transcription of genes encoding a variety of catabolic enzymes.
When grown on glucose, E. coli have a very low-level of catabolic enzymes such as β-galactosidase. Clearly, it would be wasteful to synthesize these enzymes when glucose is abundant. The inhibitory effect of glucose, called catabolite repression, is due to the ability of glucose to lower the intracellular concentration of cyclic AMP. By an independent mechanism, the bacterium accumulates cAMP only when it is starved for a source of carbon. In the presence of glucose—or of glycerol in concentrations sufficient for growth—the bacteria will lack sufficient cAMP to bind to CAP because the glucose inhibits adenylyl cyclase, the enzyme that converts ATP to cAMP. Thus, in the presence of glucose or glycerol, cAMP-saturated CAP is lacking, so that the DNA-dependent RNA polymerase cannot initiate transcription of the lac operon (Figure-7).
Thus, the CAP-cAMP regulator is acting as a positive regulator because its presence is required for gene expression. The lac operon is therefore controlled by two distinct DNA binding factors; one that acts positively (cAMP-CRP complex) and one that acts negatively (LacI repressor). Maximal activity of the lac operon occurs when glucose levels are low (high cAMP with CAP activation) and lactose is present (LacI is prevented from binding to the operator) (Figure-7).
Figure-6- The CAP binding site on DNA is adjacent to the position at which RNA polymerase binds.
Figure-7- Catabolite repression and the role of CRP-c AMP complex
Constitutive Expression and continuous repression
When the lacI gene has been mutated so that its product, LacI, is not capable of binding to operator DNA, the organism will exhibit constitutive expression of the lac operon. In a contrary manner, an organism with a lacI gene mutation that produces a LacI protein which prevents the binding of an inducer to the repressor will remain repressed even in the presence of the inducer molecule, because the inducer cannot bind to the repressor on the operator locus in order to derepress the operon. Similarly, bacteria harboring mutations in their lac operator locus such that the operator sequence will not bind a normal repressor molecule constitutively express the lac operon genes.
Thus the repression/derepression and induction of lac operon can be summarized as follows-
1) In the absence of lactose- Lac operon remains repressed due to the presence of lac repressor at the operator site- (Negative control).
2) In the presence of only Lactose- Lac operon is derepressed, the structural genes are transcribed and the lactose metabolizing enzymes are synthesized (Double negative control).
3) In the presence of both glucose and lactose- CAP -cAMP complex is not formed, RNA polymerase can not initiate the transcription of structural genes( absence of positive regulation), the operator site is though still vacant due to the binding of lactose/allolactose with lac repressor (Double negative regulation) but Lac operon remains in the repressed state. Thus glucose is consumed first and as it is exhausted , c AMP level rises, CAP-cAMP complex is formed and lac operon is expressed, lactose metabolizing enzymes are synthesized.
4) In the presence of only glucose, cAMP level is low, CAP-cAMP complex is not formed, no transcription of lactose metabolizing enzyme (Lack of positive control. Besides that lac repressor is bound to the operator site, RNA polymerase cannot transcribe the genes. (Negative control). Thus lac operon remains in the repressed state.
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