Lac operon- Definition, structure, Inducers, diagram

The lac operon is a set of genes and regulatory elements that control the expression of enzymes involved in lactose metabolism in the bacterium Escherichia coli. The lac operon consists of three structural genes (lacZ, lacY and lacA), an operator site (Olac), a promoter site (Plac) and a regulator gene (lacI). The structural genes encode proteins that are responsible for transporting, hydrolyzing and modifying lactose, while the regulator gene encodes a protein that can bind to the operator site and inhibit the transcription of the structural genes. The operator site is a DNA sequence that lies between the promoter site and the structural genes and acts as a switch for transcription. The promoter site is a DNA sequence that binds RNA polymerase and initiates transcription of the structural genes. The lac operon is an example of an inducible operon, which means that its expression is normally low or absent but can be turned on by the presence of a specific molecule called an inducer. The main inducer of the lac operon is allolactose, which is a derivative of lactose produced by the enzyme β-galactosidase. Allolactose binds to the repressor protein encoded by lacI and prevents it from binding to the operator site, thus allowing transcription of the structural genes. The lac operon is also regulated by another factor called catabolite activator protein (CAP) or cAMP receptor protein (CRP), which enhances transcription in response to low glucose levels. CAP/CRP binds to a site near the promoter site and facilitates the binding of RNA polymerase to Plac. The lac operon is therefore subject to both negative and positive regulation by different molecules depending on the availability of lactose and glucose in the environment.

The lac operon is a cluster of genes that are involved in the transport and metabolism of lactose in E. coli and other bacteria. The lac operon consists of three structural genes and a promoter, an operator, a terminator and a regulator. The three structural genes are:

• lacZ: This gene encodes β-galactosidase, an enzyme that cleaves lactose into glucose and galactose.
• lacY: This gene encodes β-galactoside permease, a membrane protein that transports lactose into the cell.
• lacA: This gene encodes β-galactoside transacetylase, an enzyme that transfers an acetyl group to some β-galactosides.

The promoter (Plac) is a DNA sequence that binds RNA polymerase and initiates transcription of the lac operon. The operator (Olac) is a DNA sequence that overlaps with the promoter and binds the lac repressor protein. The lac repressor protein is encoded by the lacI gene, which has its own promoter (PlacI) and is located upstream of the lac operon. The terminator (Tlac) is a DNA sequence that signals the end of transcription.

The structure of the lac operon can be represented as follows:

PlacI  lacI  Plac  Olac  lacZ  lacY  lacA  Tlac
|------|-----|-----|-----|-----|-----|-----|----|

The lac operon is transcribed as a single polycistronic mRNA that contains the coding sequences of all three structural genes. The polycistronic mRNA is then translated into three separate proteins by the ribosomes.

The expression of the lac operon is regulated by two factors: the presence or absence of lactose and the presence or absence of glucose. These factors affect the binding of two regulatory proteins to the lac promoter: the lac repressor and the catabolite activator protein (CAP).

Lactose is a disaccharide sugar composed of glucose and galactose units. To use lactose as an energy source, E. coli bacteria need to break it down into its constituent monosaccharides, which can then enter the glycolytic pathway. The lac operon contains three genes that encode key enzymes involved in lactose metabolism: lacZ, lacY, and lacA.

• lacZ: encodes an enzyme called β-galactosidase, which digests lactose into glucose and galactose. β-galactosidase also converts some of the lactose into allolactose, which acts as an inducer of the lac operon by binding to the lac repressor and preventing it from inhibiting transcription.
• lacY: encodes a membrane-embedded transporter called galactoside permease (also known as lactose permease). This protein helps to transfer lactose into the cell across the cell membrane, where it can be acted upon by β-galactosidase.
• lacA: encodes an enzyme called thiogalactoside transacetylase, which transfers an acetyl group from acetyl-CoA to certain β-galactosides, such as lactose and IPTG (an artificial inducer of the lac operon). The function of this enzyme is not well understood, but it may help to detoxify some β-galactosides that could otherwise interfere with cell metabolism.

These three enzymes are produced only when lactose is available and glucose is scarce, as regulated by the lac repressor and the catabolite activator protein (CAP). This ensures that the bacteria use the most efficient carbon source available and avoid wasting energy and resources on unnecessary enzyme synthesis.

An inducer is a small molecule that triggers the expression of an operon by binding to and inactivating a repressor protein. In the case of the lac operon, the inducer is allolactose, which is an isomer of lactose produced by the action of β-galactosidase. Allolactose binds to the lac repressor and changes its conformation, preventing it from binding to the operator site and blocking transcription. Thus, allolactose acts as a lactose sensor that allows the lac operon to be expressed only when lactose is available.

Another inducer of the lac operon is isopropylthiogalactoside (IPTG). Unlike allolactose, this inducer is not metabolized by E. coli and so, is useful for experimental studies of induction only. IPTG also binds to the lac repressor and induces a conformational change that releases it from the operator site.

The process of induction of the lac operon can be summarized as follows:

1. In the absence of an inducer, the lac repressor binds to the operator site and inhibits transcription of the lacZ, lacY and lacA genes.
2. In the presence of an inducer, such as allolactose or IPTG, the inducer binds to the lac repressor and alters its shape, reducing its affinity for the operator site.
3. The lac repressor dissociates from the operator site and allows RNA polymerase to bind to the promoter site and initiate transcription of the lacZ, lacY and lacA genes.
4. The resulting polycistronic mRNA is translated into β-galactosidase, permease and transacetylase proteins that enable lactose metabolism.
5. If the inducer is removed, the lac repressor resumes its original shape and binds to the operator site again, shutting off transcription of the lac operon.

Figure 1: Induction of Lac operon by allolactose or IPTG

Source: Microbe Notes

In the absence of an inducer, such as allolactose or IPTG, the lac operon is turned off by the action of the lac repressor protein. The lac repressor is encoded by the lacI gene, which has its own promoter and is constitutively expressed. The lac repressor binds to the operator site of the lac operon, Olac, which overlaps with the promoter site, Plac. By binding to the operator, the lac repressor blocks the access of RNA polymerase to the promoter and prevents transcription of the structural genes lacZ, lacY and lacA. This ensures that the cell does not waste energy and resources to produce enzymes that are not needed when lactose is absent. The lac repressor has a high affinity for the operator site and can bind to it even in very low concentrations. However, when an inducer is present, it can bind to the lac repressor and change its conformation, making it unable to bind to the operator. This allows transcription of the lac operon to proceed.

When lactose is present in the medium, some of it enters the cell through the action of the permease and is converted to allolactose by the β-galactosidase. Allolactose acts as an inducer of the lac operon by binding to the lac repressor and changing its shape. This reduces the affinity of the repressor for the operator site and allows it to detach from the DNA. As a result, the RNA polymerase can access the promoter and initiate transcription of the lacZ, lacY and lacA genes. These genes are transcribed as a single polycistronic mRNA that encodes for the three enzymes involved in lactose metabolism: β-galactosidase, permease and transacetylase. The production of these enzymes enables the cell to utilize lactose as a carbon and energy source.

Another inducer of the lac operon is IPTG (isopropylthiogalactoside), which is a synthetic analog of allolactose. IPTG can bind to the repressor and induce the lac operon in a similar way as allolactose, but it is not metabolized by the cell. Therefore, IPTG can be used experimentally to induce the lac operon without affecting the lactose levels.

The induction of the lac operon by allolactose or IPTG is an example of allosteric regulation, which is a type of regulation where a small molecule binds to a protein and alters its activity or conformation. In this case, the binding of the inducer to the repressor changes its conformation and prevents it from binding to the operator site.

The induction of the lac operon is also influenced by glucose levels in the medium. When glucose is present, the cAMP levels in the cell are low and the CRP-cAMP complex cannot bind to the lac promoter. This reduces the transcriptional activity of the lac operon even if lactose is present. When glucose is absent, the cAMP levels are high and the CRP-cAMP complex binds to the lac promoter and enhances transcription. Therefore, glucose exerts a catabolite repression on the lac operon, which means that it inhibits the expression of genes involved in catabolism of alternative substrates.

The induction of the lac operon by lactose and glucose is an example of dual control, which means that two different signals regulate gene expression in opposite ways. In this case, lactose acts as a positive signal that activates transcription, while glucose acts as a negative signal that represses transcription.

The induction of the lac operon allows E. coli to adapt to different environmental conditions and use lactose efficiently when glucose is scarce. The regulation of gene expression by inducers and repressors ensures that only the necessary enzymes are produced at the right time and amount.

High-level transcription of the lac operon requires the presence of a specific activator protein called catabolite activator protein (CAP), also called cAMP receptor protein (CRP). This protein, which is a dimer, cannot bind to DNA unless it is complexed with 3’5′ cyclic AMP (cAMP). The CRP–cAMP complex binds to the lac promoter just upstream from the binding site for RNA polymerase. It increases the binding of RNA polymerase and so stimulates transcription of the lac operon.

Whether or not the CRP protein is able to bind to the lac promoter depends on the carbon source available to the bacterium. When glucose is present, E. coli does not need to use lactose as a carbon source and so the lac operon does not need to be active. Thus the system has evolved to be responsive to glucose. Glucose inhibits adenylate cyclase, the enzyme that synthesizes cAMP from ATP. Thus, in the presence of glucose the intracellular level of cAMP falls, so CRP cannot bind to the lac promoter, and the lac operon is only weakly active (even in the presence of lactose).

When glucose is absent, adenylate cyclase is not inhibited, the level of intracellular cAMP rises and binds to CRP. Therefore, when glucose is absent but lactose is present, the CRP–cAMP complex stimulates transcription of the lac operon and allows the lactose to be used as an alternative carbon source. In the absence of lactose, the lac repressor, of course, ensures that the lac operon remains inactive. These combined controls ensure that the lacZ, lacY and lacA genes are transcribed strongly only if glucose is absent and lactose is present.

The CAP/CRP involved in regulating the lac operon is a good example of an activator. Positive control or regulation of gene expression is when the regulatory protein binds to DNA and increases the rate of transcription. In this case, the regulatory protein is called an activator. Thus the lac operon is subject to both negative and positive control.

The lac operon is not only regulated by the presence or absence of lactose, but also by the availability of glucose. Glucose is the preferred carbon source for E. coli, and when it is present, the bacterium does not need to use lactose as an alternative energy source. Therefore, the lac operon is repressed when glucose is abundant, even if lactose is also available. This phenomenon is called catabolite repression.

The mechanism of catabolite repression involves a molecule called cyclic adenosine monophosphate (cAMP), which is derived from ATP by the enzyme adenylate cyclase. cAMP acts as a signal of low glucose levels in the cell, and it binds to a protein called catabolite activator protein (CAP), also known as cAMP receptor protein (CRP). The CAP-cAMP complex is an activator of the lac operon, meaning that it enhances the transcription of the lac genes by binding to a specific site on the lac promoter, near the RNA polymerase binding site. The CAP-cAMP complex facilitates the binding of RNA polymerase to the promoter and increases the rate of transcription initiation.

However, when glucose is present, adenylate cyclase is inhibited by a glucose transport protein called PtsG. This reduces the level of cAMP in the cell, and prevents the formation of the CAP-cAMP complex. Without CAP-cAMP, the lac promoter is less efficient in binding RNA polymerase, and the transcription of the lac operon is greatly reduced. This ensures that E. coli does not waste energy and resources by producing enzymes that are not needed.

Therefore, the lac operon is subject to dual control by both lactose and glucose. In order for the lac operon to be fully active, both lactose and cAMP must be present. Lactose acts as an inducer that relieves the negative control by the lac repressor, while cAMP acts as a co-activator that enhances the positive control by CAP. Conversely, in order for the lac operon to be fully repressed, both glucose and the absence of lactose are required. Glucose acts as a repressor that inhibits the positive control by CAP, while the absence of lactose allows the negative control by the lac repressor.

The following table summarizes the different states of the lac operon depending on the presence or absence of glucose and lactose:

The lac operon is a good example of how gene expression can be regulated by both positive and negative mechanisms. Positive regulation means that the binding of a regulatory protein to DNA enhances the transcription of a gene or a set of genes. Negative regulation means that the binding of a regulatory protein to DNA inhibits the transcription of a gene or a set of genes.

In the case of the lac operon, the negative regulator is the lac repressor protein, which is encoded by the lacI gene. The lac repressor binds to the operator site (Olac) of the lac operon and blocks the access of RNA polymerase to the promoter site (Plac), thus preventing the transcription of the structural genes (lacZ, lacY and lacA). The lac repressor can be inactivated by an inducer, such as allolactose or IPTG, which binds to the repressor and changes its conformation, reducing its affinity for the operator site. This allows RNA polymerase to bind to the promoter site and initiate transcription.

The positive regulator is the catabolite activator protein (CAP), also known as cAMP receptor protein (CRP). This protein is a dimer that can bind to DNA only when it is complexed with cyclic AMP (cAMP). The CAP-cAMP complex binds to a specific site on the lac promoter, just upstream of the RNA polymerase binding site. The binding of CAP-cAMP enhances the affinity of RNA polymerase for the promoter site and increases the rate of transcription. The level of cAMP in the cell depends on the availability of glucose, which is the preferred carbon source for E. coli. When glucose is present, it inhibits adenylate cyclase, the enzyme that synthesizes cAMP from ATP. Thus, in the presence of glucose, the intracellular level of cAMP is low and CAP cannot bind to the lac promoter. When glucose is absent, adenylate cyclase is not inhibited and cAMP is produced at a high level. Thus, in the absence of glucose, CAP can bind to the lac promoter and stimulate transcription.

These combined controls ensure that the lac operon is transcribed strongly only when glucose is absent and lactose is present. In other words, E. coli uses lactose as an alternative carbon source only when glucose is not available. This allows E. coli to optimize its energy metabolism and adapt to different environmental conditions.

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