Isabella Carter
Promoters are functional units of DNA that control the expression of genes. Constitutive promoters are unregulated promoters that are always active and allow for the continual transcription of their associated gene. They are active in a cell under all circumstances and carry out a continuous transcription process. On the other hand, inducible promoters are only active under specific circumstances and can be switched from an 'OFF' to an 'ON' state. They are activated only when they receive a specific stimulus, such as chemical agents, temperature, mechanical injury, or light. Inducible promoters are important tools in cellular and molecular biology as they allow for the depletion, repletion, and overexpression of genes on demand.
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What You'll Learn
Inducible promoters are regulated by chemicals, light, and temperature
Inducible promoters are inactive in their default state, requiring specific stimuli to activate them. They are often regulated by chemicals, light, and temperature, which can induce gene expression by removing repressor proteins from DNA. This allows activator proteins to bind to promoter regions, initiating transcription. Chemically regulated promoters are among the most common inducible promoters, with the Tet-On system being a versatile tool for use in both prokaryotes and eukaryotes. Tetracycline and its derivatives, for instance, serve as inducing agents for promoter activation.
Temperature-sensitive expression systems, such as the heat shock-inducible Hsp70 or Hsp90-derived promoters, are also commonly used. These promoters are typically less leaky than chemically induced promoters and can be activated by heat or cold exposure. For example, a brief heat shock can activate the Hsp70 promoter, releasing heat shock factor 1 (HSF-1), which then binds to heat shock elements in the promoter, activating transcription.
Light-induced promoters are another important tool in synthetic biology, particularly in cyanobacteria, where light-controlled promoters offer an environmentally friendly alternative to chemical induction. Two-component systems, such as the Tabor lab's yellow flame light-regulated system, can use light to regulate transcription. Additionally, the light-oxygen-voltage (LOV) domain from the fungus Neurospora crassa has been used in mammalian cell studies to research essential signaling pathways.
Inducible promoters are valuable tools in biotechnology, allowing for precise control of gene expression and offering a wide range of applications in various organisms, including bacteria, yeast, plants, and cyanobacteria.
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Inducible promoters can be switched from off to on
Inducible promoters are DNA sequences located near the transcription initiation site of genes. They are regulated promoters that can be switched from an inactive "OFF" state to an active "ON" state in response to specific stimuli. In the "OFF" state, the promoter is inactive because a bound repressor protein actively prevents transcription. This can occur when a repressor protein is present and binds to the operator segment of the DNA, preventing transcription.
Once an inducer binds to the repressor protein, the repressor protein is removed from the DNA, turning the promoter "ON" and initiating transcription. Inducers can include chemical compounds, temperature shifts, physical damage, or exposure to light. For example, the negative inducible pLac promoter requires the removal of the lac repressor (lacI protein) for transcription to be activated. In the presence of lactose or lactose analog IPTG, the lac repressor undergoes a conformational change, removing it from the promoter and activating transcription.
Another example is the positive inducible tetracycline ON (Tet-On) system, which is used in both prokaryotes and eukaryotes. In this system, the activator rtTA (reverse tetracycline-controlled transactivator) is normally inactive and cannot bind to the tetracycline response elements (TRE) in a promoter. Tetracycline or its derivatives serve as inducing agents, activating the promoter and allowing transcription to occur.
Inducible promoters are commonly used in research to control gene expression and regulate the production of recombinant proteins. They allow for specific genes to be expressed or repressed on demand, which is particularly useful for functional studies. By using inducers or removing repressors, researchers can switch inducible promoters from an "OFF" to an "ON" state, providing a powerful tool for studying gene expression and protein production.
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Constitutive promoters are unregulated and always active
Promoters are vital DNA sequences located near the transcription initiation site of genes. They facilitate the binding of RNA polymerase and transcription factors to initiate gene transcription. There are different types of promoters, and two of them are inducible promoters and constitutive promoters.
Inducible promoters are regulated promoters that are only active in response to specific stimuli. These stimuli can include chemical compounds, temperature shifts, physical damage, and exposure to light. In the OFF state, the promoter is inactive because a bound repressor protein actively prevents transcription. Once an inducer binds to the repressor protein, it is removed from the DNA, allowing transcription to turn ON.
On the other hand, constitutive promoters are unregulated promoters that are always active in all circumstances within a cell. They facilitate a continuous transcription process within the target gene. Unlike inducible promoters, constitutive promoters are not influenced or affected by transcriptional factors. Some examples of constitutive promoters include plant ubiquitin promoter, rice actin 1 promoter, maize alcohol dehydrogenase promoter, opine promoter, and CaMV 35S promoter.
Constitutive promoters have a wide range of applications, such as expressing selective marker genes and consistent overexpression of proteins. They are routinely used to drive ectopic gene expression in mammalian systems, with examples including the simian virus 40 early promoter (SV40), cytomegalovirus immediate-early promoter (CMV), and human Ubiquitin C promoter (UBC).
In summary, constitutive promoters are unregulated and always active, facilitating continuous transcription in the desired gene regardless of the prevailing circumstances in the cell.
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Constitutive promoters are used for expressing selective marker genes
Constitutive promoters are DNA sequences that are always active and are not influenced by transcriptional factors. They are used for expressing selective marker genes and consistent overexpression of proteins.
Constitutive promoters have a wide range of applications, including expressing selective marker genes. For example, in a study, a strong constitutive promoter was used to express the pyrE2 gene in vectors as a selective transformation marker. This promoter, PfdX, was used to control the expression of the msfGFP gene, which is a fluorescent protein. The msfGFP gene was inserted under the control of the PfdX promoter, and the resulting construct was used as a benchmark to compare the activity of inducible promoters.
In another study, a set of overexpression vectors were developed using constitutive promoters. The Pcap promoter sequence from the pBUS1_Pcap_HC_cat plasmid was replaced with a new DNA fragment containing an individual selected promoter sequence and a ribosomal-binding site (RBS). This generated a set of vectors, each carrying a different constitutive promoter. These vectors were then used for gene expression and the production of tagged fusion proteins.
Constitutive promoters are also used for expressing selective marker genes in crop transgenic breeding. For example, the Arabidopsis serine carboxypeptidase-like gene AtSCPL30 has been characterized as a strong and constitutive promoter for crop transgenic breeding. The full-length promoter, PD1, and eight 5' deleted fragments (PD2-PD9) were fused with GUS to produce promoter::GUS plasmids, which were then translocated into Nicotiana benthamiana. These plasmids conferred strong and constitutive expression of transgenes in almost all tissues and development stages in the transgenic plants.
Constitutive promoters differ from inducible promoters, which are only active under specific circumstances. Inducible promoters can be regulated by chemicals, light, temperature, or mechanical injury. They are essential tools for molecular and cell biology studies as they allow control over the timing and strength of gene expression.
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Inducible promoters are used in mammalian cell studies
Inducible promoters are essential tools for molecular and cell biology studies, allowing researchers to control the expression of genes of interest in terms of both timing and strength of expression. They are particularly useful in mammalian cell studies for several reasons.
Firstly, inducible promoters allow for the fine-tuning of gene expression, which is necessary when studying lethality-causing genes or essential genes that are impossible to overexpress or knock out. By being able to switch gene expression on and off or modulate the level of expression, researchers can reveal the direct consequences of specific genetic changes and exclude side and off-target effects. This level of control is especially beneficial when working with intricate genetic networks in mammalian cells.
Secondly, inducible promoters are reversible and thus more flexible than stable expression systems. This reversibility also contributes to their higher efficiency and fewer side effects, such as reduced cell death and more regular growth and development.
Thirdly, inducible promoters enable researchers to conduct dynamic studies of gene expression in vivo, perform timed expression of tagged protein fusions, and carry out depletion assays.
Finally, the choice of inducible promoter depends on the specific experimental requirements. For instance, the metallothionein promoter, which is regulated by heavy metals, is unsuitable for studies involving hypoxia, oxidative stress, and hormones. In such cases, other inducible systems, such as the bacterial operons, can be used.
In summary, inducible promoters are valuable tools in mammalian cell studies due to their ability to provide precise control over gene expression, their flexibility and efficiency, their range of applications, and their compatibility with specific experimental needs.
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Frequently asked questions
A constitutive promoter is an unregulated promoter that is always active in a cell, regardless of the circumstances. It facilitates a continuous transcription process within the target gene.
An inducible promoter is a regulated promoter that is only active in response to specific stimuli, such as light, temperature, or chemical agents. It can be switched from an OFF to an ON state.
Some examples of inducible promoters include chemically inducible promoters, temperature-inducible promoters, and light-inducible promoters. Tetracycline and its derivatives, for example, can serve as inducing agents for promoter activation.
