Eleanor Finch
The synthesis of β-galactosidase in Escherichia coli is affected by a number of closely linked mutations. Some mutations cause a loss of the capacity to synthesize the active enzyme, while others allow the enzyme to be synthesized constitutively instead of inducibly. The inducibility of β-galactosidase formation can be measured in cells recovering from various treatments that inhibit protein synthesis. The question of whether β-galactosidase is inducible, constitutive, or non-inducible is a complex one, as it depends on various factors such as the presence of certain substrates or mutations.
| Characteristics | Values |
|---|---|
| Definition | Inducible: the operon is not normally transcribed; however, it can be activated |
| Noninducible: the operon can never be transcribed | |
| Constitutive: the operon is always transcribed | |
| Transcription | Inducible and constitutive β-galactosidase formation was measured in cells recovering from various treatments that inhibit protein synthesis in the cell |
| Undelayed β-galactosidase formation was found in stringent auxotrophs recovering from amino acid starvation, in cells recovering from glycerol or potassium starvation, and in bacteria recovering from puromycin treatment | |
| Delayed β-galactosidase formation was found in relaxed auxotrophs recovering from amino acid starvation and in prototrophs recovering from chloramphenicol or tetracycline treatment | |
| The length of the delay was directly proportional to the duration of the treatment | |
| All cells recovering from the various treatments exhibited a slightly decreased rate of β-galactosidase formation | |
| The lac operon is not transcribed when lactose is absent because the lacI+ gene on the F' plasmid produces lac repressor proteins that bind to both copies of lacO, inhibiting expression | |
| The lac operon is turned on when lactose is present and turned off when it is absent | |
| The lac operon is transcribed at a low level, producing only a few (5-10) copies each of β-galactosidase per cell | |
| The lac operon is transcribed at high levels when an E. coli strain results in constitutive expression |
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What You'll Learn
Inducible, noninducible and constitutive
Gene expression occurs in two steps: transcription and translation. Some genes, known as constitutive genes, are expressed continuously in the cell and synthesize their products. These genes are always "on". The enzymes involved in processes such as glycolysis, the citric acid cycle, transcription, and translation are continuously synthesised, and the constitutive genes code for those enzymes.
In contrast, inducible genes are expressed only under certain conditions when there is a necessity. For example, inducible gene expression occurs when there is an insufficient amount of a particular molecule within the cell. The Lac operon present in bacteria is an example of an inducible gene. In the case of the Lac operon, the operon will be turned on constitutively (the genes will be expressed) when the repressor is inactivated.
Constitutive expression is the expression of a constitutive gene at a constant level. For example, a mutation in the lacZ gene affects only β-galactosidase, not the transacetylase (or other products of the operon), showing that lacZ is a structural gene. A mutation in lacI affects both enzymes, hence lacI is a regulatory gene. Both are expressed in the absence of the inducer, hence the operon is constitutively expressed (the strain shows a constitutive phenotype).
Inducible expression is the expression of an inducible gene under certain conditions only. For example, a mutation in lacIS prevents the binding of the inducer, leading to a non-inducible phenotype. Binding of the inducer to the "core" causes an allosteric shift in the repressor so that the "headpiece" is no longer able to form a high-affinity complex with the DNA, and the repressor can dissociate.
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Mutations in lacZ and lacI
The lacZ gene of Escherichia coli encodes β-galactosidase (β-gal), a lactose metabolism enzyme of the lactose operon. The lacZ gene has played an important role in recombinant DNA technology through colorimetric selection of recombinant clones via the α-complementation phenomenon. β-gal is extensively used in the food industry and in a range of other industries.
The lac operon and the lacZ gene of Escherichia coli have a long history in the field of molecular biology, dating back to the discovery of gene regulation in the pioneering studies of Jacob and Monod that examined the production of β-galactosidase (β-gal) from lacZ in the presence of inducing substrates. Previous chemical modification or site-directed mutagenesis experiments have identified 21 amino acids that are essential for β-gal catalytic activity.
A study examined a total of 6,465 independent mutations in the lacZ gene to establish the importance of different amino acid residues in the function of β-gal. A wide variety of sequenced mutants induced by chemical or radiation exposure, different genetic backgrounds, and spontaneous mutants were compiled into the single dataset used in this study. Missense mutations represented ∼42% of all independent mutations (2,732 out of 6,465) and 33% of all unique mutations (895 unique out of 2,732) detected. Of 492 codons with functional mutations, 384 were identified by NGS and 266 by Sanger sequencing.
The plasmid-based transgenic mouse model, which uses the lacZ gene as the target for mutation, is sensitive to a wide range of in vivo mutations, ranging from point mutations to insertions and deletions extending far into the mouse genome. Sequence analysis confirmed the presence of a point mutation in each lacZ gene of nine different color mutants.
A mutation in lacZ affects only β-galactosidase, not the transacetylase (or other products of the operon), showing that lacZ is a structural gene. A mutation in lacI affects both enzymes, hence lacI is a regulatory gene. Both are expressed in the absence of the inducer, hence the operon is constitutively expressed (the strain shows a constitutive phenotype).
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Role of repressors
The Lac operon is a cluster of coordinately regulated genes that includes structural genes (encoding enzymes) and regulatory genes (encoding proteins that affect gene expression). The Lac operon uses a two-part control mechanism to ensure that the cell only produces the necessary enzymes when lactose is available and no preferable energy source, such as glucose, is present.
The Lac operon consists of three structural genes: lacZ, lacY, and lacA. lacZ encodes β-galactosidase, an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose. lacY encodes β-galactoside permease, a membrane protein that enables the cellular transport of lactose into the cell. lacA encodes β-galactoside transacetylase.
The Lac operon also includes a regulatory gene, lacI, which encodes the lactose repressor. The repressor plays a crucial role in the control mechanism of the Lac operon. In the absence of lactose, the lac repressor binds to the operator region of the DNA, preventing the transcription of the structural genes and halting the production of the corresponding enzymes. This ensures that the cell does not waste energy producing enzymes when no lactose is available.
When lactose is present, a lactose metabolite called allolactose binds to the repressor, causing an allosteric shift. This conformational change prevents the repressor from binding to the operator, allowing RNA polymerase to transcribe the structural genes and leading to increased levels of the encoded proteins. As a result, the cell can produce the necessary enzymes to utilize lactose as an energy source.
Additionally, the Lac operon exhibits a response to glucose levels. In the presence of glucose, the catabolite activator protein (CAP) remains inactive, and EIIAGlc shuts down lactose permease, preventing the transport of lactose into the cell. This dual control mechanism ensures the sequential utilization of glucose and lactose in two distinct growth phases, known as diauxie.
In summary, the repressor encoded by the lacI gene plays a critical role in regulating the expression of the Lac operon. By binding to the operator region of the DNA in the absence of lactose, the repressor prevents the transcription of the structural genes. When lactose is present, the repressor undergoes a conformational change, allowing transcription to occur and enabling the cell to utilize lactose as an energy source.
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Role of inducers
The role of inducers is crucial in the regulation of gene expression, specifically in the context of the lac operon and the production of β-galactosidase. Inducers are molecules that bind to repressor proteins, causing a conformational shift that prevents the repressor from binding to DNA and blocking gene transcription. This process is essential for the expression of genes involved in the breakdown and utilisation of lactose by bacteria, such as E. coli.
In the lac operon, the lacZ gene encodes for β-galactosidase, an enzyme necessary for the initial breakdown of lactose into galactose and glucose. The lac operon is regulated by a negative inducible mechanism, where the presence of a regulatory factor, the lac repressor, turns off the gene unless an inducer molecule is present. The inducer molecule for the lac operon is typically lactose or its isomer, allolactose. When lactose is present, it binds to the lac repressor, inactivating it and allowing the gene to be expressed. This enables the bacterium to hydrolyse lactose and utilise it as a carbon and energy source.
The concentration of the inducer molecule also plays a role in the expression of the lac operon. At low concentrations, inducers such as isopropyl-β-D-thiogalactopyranoside (IPTG) and thiomethyl galactoside (TMG) can enter the cell through lactose permease, while at high concentrations they can enter the cell independently. IPTG is a commonly used synthetic analogue of lactose that acts as an inducer by binding to the repressor and inactivating it. However, IPTG is not a substrate for β-galactosidase, which makes it useful for laboratory studies as its concentration remains constant.
Gratuitous inducers, such as IPTG, can maintain induction for a longer period as they are not metabolised by the encoded enzymes. This is particularly useful in laboratory settings for studying gene expression. Additionally, the presence of glucose can impact the expression of the lac operon through a process called catabolite repression. Glucose is the preferred source of carbon for E. coli, and its presence represses the expression of the lac operon. In the absence of glucose, the concentration of cyclic adenosine monophosphate (cAMP) increases, which in turn increases the production of β-galactosidase.
In summary, inducers play a critical role in the regulation of gene expression by binding to repressor proteins and preventing them from blocking gene transcription. This is particularly important for the expression of genes involved in lactose utilisation, such as β-galactosidase. The concentration and type of inducer molecule, as well as the presence of other substrates like glucose, all influence the expression of the lac operon and the production of β-galactosidase.
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Operon response to different sugars
The operon model was developed based on lactose metabolism in Escherichia coli. Operons are polycistronic transcripts that can produce multiple proteins from a single mRNA transcript. They are made up of structural genes, regulatory genes, and regulatory sites. Structural genes encode enzymes, regulatory genes encode activators or repressors, and regulatory sites are where regulatory substances act to change gene expression.
The lac operon is a well-studied example of an operon that responds to different sugars. It consists of three genes: lacZ, lacY, and lacA. lacZ encodes β-galactosidase, an enzyme that cleaves lactose into glucose and galactose. lacY encodes β-galactoside permease, a membrane protein that facilitates the transport of lactose into the cell. lacA encodes β-galactoside transacetylase.
In the presence of lactose, the lac operon is turned on, and the genes are transcribed and translated. The operon is regulated by a negative inducible mechanism, meaning that the gene is turned off by a regulatory factor (lac repressor) unless lactose is present. When lactose is present, it binds to the lac repressor, causing an allosteric shift that prevents the repressor from binding to the operator region of DNA. This allows RNAP to transcribe the lac genes, leading to higher levels of the encoded proteins.
In the absence of lactose, the repressor binds to the operator region, preventing transcription of the lac genes. Additionally, the presence of glucose can also repress the expression of the lac operon. Glucose is the preferred source of carbon for E. coli, and it will be consumed before lactose. The presence of glucose leads to a decrease in [cAMP] inside the cell, which represses the expression of the lac operon.
The response of the lac operon to different sugars is a classic example of how genetic mechanisms can be altered in response to changing environmental stimuli to regulate bacterial metabolic activities.
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Frequently asked questions
β-galactosidase is an enzyme that breaks down lactose.
Inducible genes are not normally transcribed but can be activated, non-inducible genes can never be transcribed, and constitutive genes are always transcribed.
β-galactosidase is inducible. It is only expressed in the presence of lactose and the absence of glucose.
The lac operon is a set of genes in E. coli that are involved in the metabolism of lactose. It consists of structural genes (Z, Y, A) and regulatory elements (promoter P, operator O, and the repressor gene I).
The lac operon is transcribed at a low level, producing only a few (5-10) copies of β-galactosidase per cell. The presence of lactose inactivates the repressor, allowing transcription to begin.
