Each cell has an ability to best adapt to its environment. This ability to adapt is accomplished by altering the expression of genes. The alteration in the gene expression depending on the environment is called as gene regulation. The gene regulation in prokaryotes is much simpler and involves the genes arranged in sequence or group known as operon.

Now, an operon can be defined as a stretch of DNA consisting of structural genes and a regulatory genes (see fig 1).

Fig 1: The Operon

The structural genes are the ones encoding the functional proteins, like enzymes, which carry out various functions in the cell. These genes are transcribed as a polycistronic mRNA, which are further transcribed and modified to give individual proteins (see fig 2).

Fig 2: The synthesis and translation of polycistronic mRNA

On the other hand, the regulatory genes control the expression of these structural genes as per the requirement for the cell survival. The regulatory genes can further be either cis-acting or trans-acting.

• Cis-acting regulatory genes are region of non-coding DNA, regulating the transcription of neighbouring genes. These are usually DNA regions, where a protein binds and affects the expression of the adjoining genes. The promoter and the operator are examples of cis-acting genes regulatory genes.

– The promoter is the binding site for RNA polymerase, to initiate the transcription while the operator gene is the negative regulatory site located between the promoter and the structual genes (see fig 1).

• The trans-acting regulatory genes are the region of DNA, which usually codes for a diffusible protein, which can bind and regulate the transcription of distant genes. The trans- acting genes include genes encoding proteins like repressor and activator.

– Repressor, a negative regulatory molecule, binds the operator gene and interferes with the expression of genes. Activator, a positive regulatory molecule, enhances the expression of the genes.

– Let’s have a look at an example of the operon, the Lactose Operon, commonly called as lac operon.

Lactose (Lac) Operon:

Lac operon enables the bacteria to metabolize lactose as the source of energy. It is present in E. coli, a bacteria commonly found in the human intestines.

Structure of the lac operon:

As in any operon, the lac operon too, contains the structural and the regulatory genes.

a. Structural genes of lac operon:

The lac operon contains three structural genes: lac Z, lac Y, and lac A (see fig 4). These genes are transcribed from a single promoter as a single polycistronic mRNA. These structural genes encode for proteins that enable the cell to take up and metabolize lactose and other β-galactosides.

The function of the three structural genes are:

i. lac Z codes for the enzyme β-galactosidase, a tetramer of about 500 kD. This enzyme breaks down β-galactoside into its monosaccharide components. For example, lactose is split into glucose and galactose (see fig 3) which can be metabolized further through glycolysis.

Fig 3: Lactose and it’s monosaccharide components, glucose and galactose.

ii. lac Y codes for the β-galactoside permease, a 30 kD membrane-bound protein which transports β-galactosides into the cell.

iii. lac A codes for β-galactoside transacetylase, that transfers an acetyl group from acetyl-CoA to β-galactosides (role in lac operon not clear).

Fig 4: The structure of Lac operon

b. Regulatory genes:

The regulatory genes of lac operon includes promoter gene, operator gene, lac I, and catabolite activator protein (CAP) bindingw site.

i. The promoter is the binding site for RNA polymerase, the enzyme that performs transcription.

ii. The operator is a negative regulatory site where the lac repressor protein binds. It is located between the promoter and the structural genes and overlaps with the promoter (see fig 4).

iii. Lac I (repressor) gene codes for the lac operon repressor, which is a tetramer of identical subunits of 38 kD each. This gene is located adjacent to the promoter of the lac operon, with its own promoter and terminator and is always transcribed, hence the repressor is always synthesized. The repressor is a diffusible product, making Lac I is a trans-acting gene. Repressor binds the operator to repress (turn off) the operon

iv. Catabolite Activator Protein (CAP) binding site is a positive regulatory site located just upstream of the lac operon promoter, where the catabolite activator protein (CAP) binds. The CAP is a dimer protein, which has binding sites for cAMP and DNA. When cAMP binds CAP, its affinity for the DNA increases. When bound to DNA, CAP promotes transcription by aiding RNA polymerase bind to the promoter more efficiently.

Let us see the different environmental conditions and how the different regulatory elements control the lac operon.

I. When the lactose is absent:

In the absense of the lactose, the lactose-metabolizing enzymes are not required. Hence, the lac operon is non-operative in this situation and to saves the cell from spending its resource in making proteins that are not needed.

This shutdown of the lac operon is brought about by the lac repressor. As mentioned earlier, the lac repressor is synthesized always, which binds the operator. Now, the operator partially overlaps with the promoter and interferes with the binding and transcription by the RNA polymerase (which binds the promoter). Hence, RNA polymerase cannot transcribe the structural genes of the lac operon and the lactose-metabolising enzymes are not produced.

Fig 5: Negative regulation of lac operon by the repressor: Repressor binds and occupies operator site. Operator overlaps with promoter, site where RNA polymerase binds to initiate transcription. Hence repressor interferes with binding and transcription by the RNA polymerase.

II. When the lactose is present

When lactose is present (and glucose unavailable), the structural genes should be transcribed to produce lactose-metabolising enzymes. For this, the repressor has to be lifted from the operator, to allow the RNA polymerase bind the promoter and initiate the transcription of the genes. The repressor is lifted up by the action of the molecule called inducer, allolactose (see fig 6) in case of lac operon.

Fig 6: The inducer of lac operon: Allolactose, obtained from lactose using enzyme β-galactosidase.

Hence, the lac operon is considered an inducible operon, which is normally turned off (repressed) and is turned on, in the presence of the inducer..

Fig 7: When lactose is present, few molecules of lactose are converted into allolactose, the inducer of lac operon, which binds the repressor. This brings about conformational changes in the repressor and it can then no longer bind DNA operator site.

The molecule of allolactose is obtained from the lactose (which is now present in the environment), using the enzyme β-galactosidase (low level of which is always present in the cell). Allolactose binds the repressor protein and causes conformational change in the repressor due to which, the repressor protein loses its ability to bind to the operator. As the repressor is lifted from the operator, the RNA polymerase can bind the promoter site and transcribes the genes to further synthesize the lactose-metabolizing proteins (see fig 7).

The effect of availability of glucose:

The E. coli can utilize lactose, however glucose is preferred source of energy for the cell, as the utilization of lactose is energetically more expensive than glucose. Hence, lactose is utilized by the cell, only when the glucose is present in low level or absent. That is, the lac operon is turned on only when glucose is unavailable and it is turned off in presence of the glucose (even if lactose is present).

Let us see, how the lac operon is regulated depending on the availability of glucose.

i. In the absence (or low level) of glucose:

The lac operon is turned on only when the glucose is unavailable. This control, is due to the positive regulatory protein called Catabolite Activator Protein (CAP), which is essential for the expression of lactose metabolising enzymes.

The CAP is a dimer protein with a binding site for cAMP and DNA. When cAMP binds CAP, its affinity for the DNA increases. cAMP bound CAP then binds DNA, just upstream of the lac operon promoter and helps RNA polymerase anchor onto the promoter and the efficiency of transcription is highly enhanced (see fig 8).

Now, CAP is functional only when cAMP is bound to it, and cAMP in the cell is available only in the absence of glucose. Hence CAP allows the transcription of lac operon genes and subsequent metabolism of lactose, only in absence of glucose.

Fig 8: The positive regulatory effect of CAP: CAP helps control the lac Operon depending on the concentration of glucose. When glucose is absent, cAMP binds CAP, which then binds CAP binding site. cAMP bound CAP anchors the RNA polymerase to the promoter and initiated the transcription of lac operon genes.

ii. In the presence of glucose

In case, when glucose is present, the amount of available cAMP decreases. As mentioned before, without cAMP, CAP cannot bind DNA, which in turn causes weak binding of RNA polymerase to the lac operon promoter (fig 9). As a result only few transcripts are produced and lactose utilizing enzymes are not efficiently produced.

Fig 9: When glucose is present, the lac operon is turned off, despite the presence of lactose: With high level of glucose, cAMP for CAP becomes unavailable. Without cAMP, CAP cannot bind and anchor RNA polymerase on the DNA at promoter.

Hence the lac operon is turned on only when ‘glucose‘ that is the preferred source of energy is unavailable and lactose is present. Hence lac operon is great example of how the gene regulation is carried out (in prokaryotes) to best adapt to the environment.

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