The lac Operon in E. coli

The lac operon in Escherichia coli (E. coli) is a well-known example of an inducible operon. Operons are functional units of DNA that consist of a promoter region, an operator, and one or more genes. The lac operon controls the expression of genes involved in the metabolism of lactose, a sugar found in milk.

The operon model was first proposed in 1961 by Jacob and Monod and remains pivotal in our understanding for gene regulation. It is seen as a paradigm in thinking but for many years was one of a number of models put forward with many of them radically different to each other.

The lac operon is composed of three structural genes: lacZ, lacY, and lacA, which are responsible for the breakdown and utilization of lactose. These genes are transcribed together as a single mRNA molecule and share a common regulatory region.

This next section describes the function of the various components of the operon.

The Structural Genes

lacZ Gene

The lacZ gene encodes an enzyme called β-galactosidase, which plays a crucial role in lactose metabolism. β-galactosidase is responsible for the conversion of lactose into glucose and galactose, which can be further utilized by the bacterium as an energy source.

lacY Gene

The lacY gene encodes a lactose permease, a membrane protein that facilitates the transport of lactose molecules across the bacterial cell membrane. Lactose permease allows lactose to enter the cell, making it available for β-galactosidase to break down.

lacA Gene

The lacA gene encodes a transacetylase, which is involved in acetylation reactions that modify certain molecules.

The Regulatory Sections Of The Operon

Regulatory Region

The regulatory region consists of the promoter, operator, and the lacI gene. It controls the expression of the lac operon by interacting with regulatory proteins and molecules.

Promoter (P)

The promoter is a DNA sequence located upstream of the lac operon. It serves as the binding site for RNA polymerase, which initiates transcription. In the absence of lactose, RNA polymerase binds weakly to the promoter, leading to low levels of transcription.

Operator (O)

The operator is another DNA sequence located between the promoter and the structural genes. It acts as a switch, controlling the access of RNA polymerase to the promoter. When the operator is unoccupied by a repressor protein, RNA polymerase can bind to the promoter and initiate transcription.

Repressor Protein (lacI)

The lac repressor protein, encoded by the lacI gene located elsewhere in the genome, is a key regulator of the lac operon. In the absence of lactose, the lac repressor binds to the operator, preventing RNA polymerase from binding to the promoter and thereby blocking transcription. This state is known as repression.

Regulation Of The Lac Operon

The regulation of the lac operon depends on the presence or absence of lactose and glucose in the environment.

The Mechanism of Negative Control

The negative control of the transcription of the lac operon in Escherichia coli is primarily governed by the lac repressor protein (LacI). This mechanism ensures that the lac operon is not transcribed when lactose is absent, thereby conserving cellular resources. It also means that when lactose is absent and glucose is present, the lac operon is repressed because the lac repressor protein binds to the operator, preventing transcription.

1. Lac Repressor Binding: In the absence of lactose, the lac repressor (LacI) binds to the operator region (lacO) of the lac operon, which overlaps the promoter (lacP). This binding physically blocks RNA polymerase from accessing the promoter, thereby preventing transcription of the downstream genes (lacZ, lacY, and lacA).
2. Induction by Lactose: When lactose is present in the cell, it is converted into allolactose, which acts as an inducer by binding to the lac repressor. This binding causes a conformational change in LacI, reducing its affinity for the operator and leading to its release from the DNA. This derepression allows RNA polymerase to access the promoter and initiate transcription of the lac operon genes.

Experimental Evidence

1. Mutational Analysis

• Operator Mutations (lacO): Mutations in the operator region that prevent LacI binding result in constitutive expression of the lac operon, meaning the operon is transcribed regardless of the presence or absence of lactose. This demonstrates the necessity of the operator sequence for repressor binding and operon repression.
• Repressor Mutations (lacI): Mutations in the lacI gene that produce a non-functional repressor also lead to constitutive expression of the lac operon. Classic experiments by Jacob and Monod identified lacI^- mutants that lacked repressor activity, highlighting the importance of LacI in negative control.

2. Allolactose as an Inducer

• Inducer Binding and Repression Release: Experiments showing that the addition of lactose (or allolactose) to E. coli cultures results in the derepression of the lac operon provide direct evidence of the inducer mechanism. Inducer binding to LacI reduces its affinity for the operator, leading to increased transcription of the lac operon.

3. DNA Footprinting

• Repressor-Operator Interaction: DNA footprinting experiments have been used to map the exact binding site of LacI on the lac operator. These studies show protected regions on the DNA where LacI binds, preventing DNase I digestion, thereby confirming the direct interaction between LacI and the operator.

4. Gel Mobility Shift Assays (EMSA)

• Repressor Binding Visualization: EMSAs have demonstrated the binding of LacI to the operator DNA. In these assays, the addition of LacI to a DNA fragment containing the operator results in a slower migrating complex, indicative of protein-DNA binding. The addition of allolactose can be shown to disrupt this complex, further supporting the role of the inducer in releasing the repressor.

However, when lactose is present, it acts as an inducer. Lactose molecules bind to the lac repressor protein, causing a conformational change that prevents it from binding to the operator. As a result, RNA polymerase can bind to the promoter, initiating transcription of the structural genes lacZ, lacY, and lacA genes. The transcript is a single piece of mRNA which is processed further. The enzymes produced by these genes enable E. coli to metabolize lactose for energy production.

Additionally, the lac operon exhibits catabolite repression. It is an example of positive control of transcription and as well as negative control is a model system of gene regulation. That model system involves regulation of the operon by the catabolite activator protein (CAP) which is also known as the also known as the cyclic AMP receptor protein (CRP). The control mechanism acts in such a way that the lac operon is transcribed only when glucose levels are low. This bacterium can preferentially use glucose over other sugars such as lactose.

The Mechanisms of Positive Control

1. cAMP-CAP Complex Formation: When glucose levels are low in the cell, the concentration of cyclic AMP (cAMP) increases. cAMP binds to CAP, causing a conformational change in CAP that allows it to bind to a specific site in the promoter region of the lac operon.

2. Binding to DNA: The cAMP-CAP complex binds to a site upstream of the lac promoter (lacP), which is located at approximately -60 base pairs from the transcription start site. This binding enhances the affinity of RNA polymerase for the lac promoter, facilitating the initiation of transcription.

3. Recruitment of RNA Polymerase: The bound cAMP-CAP complex interacts with RNA polymerase, increasing the rate of transcription initiation of the lac operon. This leads to the transcription of the structural genes lacZ, lacY, and lacA, which are necessary for the uptake and metabolism of lactose.

When glucose is present at high concentrations, even if lactose is available, the lac operon is less likely to be induced. In the presence of glucose, cAMP levels are low, so the capability of binding to CAP is markedly reduced. This reduces the activity of CAP which further decreases lac operon expression.

The Experimental Evidence For Positive Control

1. Mutational Analysis

• CAP Binding Site Mutations: Experiments involving mutations in the CAP binding site demonstrate its critical role. Mutations that prevent CAP binding result in a significant decrease in lac operon transcription, even in the presence of lactose and low glucose levels, indicating the necessity of CAP for positive control.

2. cAMP Levels and Transcription

• cAMP Concentration Manipulation: Studies manipulating intracellular cAMP levels show a direct correlation with lac operon activity. For instance, addition of exogenous cAMP to E. coli cultures grown in high glucose conditions can induce lac operon transcription, mimicking the low-glucose condition.

3. Electrophoretic Mobility Shift Assays (EMSAs)

• CAP-DNA Binding: EMSAs have been used to demonstrate that CAP binds to the lac promoter region only in the presence of cAMP. The formation of the cAMP-CAP-DNA complex can be visualized as a shift in the mobility of DNA in a gel, providing direct evidence of the CAP binding site’s role in the positive regulation of the lac operon.

4. Reporter Gene Assays

• lacZ Reporter Fusions: The use of reporter gene fusions, such as lacZ (which encodes β-galactosidase) fused to the lac promoter, allows for quantitative measurement of transcriptional activity. These experiments show increased β-galactosidase activity in conditions where cAMP-CAP complex formation is promoted, thus supporting the role of CAP in enhancing transcription.

 

Overall, the lac operon provides E. coli with a regulatory mechanism that allows it to effectively utilize lactose as an energy source only when it is available and glucose is scarce. The positive control of the lac operon in E. coli by the cAMP-CAP complex is a well-documented regulatory mechanism. The CAP protein, in the presence of cAMP, binds to the promoter region of the lac operon, facilitating the binding of RNA polymerase and enhancing transcription. This process is critical for the efficient utilization of lactose when glucose is scarce.