Gene Regulation 10 Flashcards
Give reasons for why gene regulation is important
Gene expression is costly: natural selection has shaped genes’ architectures to reduce cost of gene expression - more complex organisms require more regulation
Differential gene expression patterns define cell development into different cell types.
Different environments require different gene sets to be expressed allowing cost efficient adaptation to the environment:
Changing environments require different gene sets to be expressed allowing cells to react quickly to the changes:
Example: infection
What is gene regulation
What can gene regulation be used for to benefit us
Biomedicine: e.g. inhibitors of oncogene expression, antivirals
Production: e.g. induce proteins for commercial applications
What are the 3 main ways of controlling gene regulation
What are the general principles of gene regulation
What are cis and trans elements of gene regulation*
Cis-acting elements: Cis-acting elements are DNA sequences that regulate gene expression by acting locally on the same DNA molecule (i.e., in cis). These elements are typically located near the gene they regulate, often within the promoter region or within the gene itself. Cis-acting elements include:
Promoters: DNA sequences located upstream of a gene that serve as binding sites for transcription factors and RNA polymerase, thereby regulating the initiation of transcription.
Enhancers: DNA sequences that can increase the transcriptional activity of a gene, often by binding transcription factors and promoting the assembly of the transcriptional machinery.
Silencers: DNA sequences that can repress the transcriptional activity of a gene, often by binding transcriptional repressors and preventing the assembly of the transcriptional machinery.
Insulators: DNA sequences that can block the spread of regulatory signals between neighboring genes or between enhancers and promoters.
Cis-acting elements exert their effects on gene expression through direct interactions with proteins or other molecules on the same DNA molecule. Mutations or alterations in cis-acting elements can affect the expression of nearby genes without affecting other genes located elsewhere in the genome.
Trans-acting factors: Trans-acting factors are regulatory proteins or RNA molecules that influence gene expression by acting from a distance (i.e., in trans). Unlike cis-acting elements, trans-acting factors are not physically linked to the DNA sequence they regulate and can act on multiple target genes located throughout the genome. Trans-acting factors include:
Transcription factors: Proteins that bind to specific DNA sequences and regulate the transcription of target genes by promoting or inhibiting the assembly of the transcriptional machinery.
RNA-binding proteins: Proteins that bind to specific RNA sequences and regulate processes such as RNA splicing, stability, and translation.
MicroRNAs (miRNAs): Small RNA molecules that can bind to target mRNAs and regulate their stability and translation.
Trans-acting factors typically recognize specific DNA or RNA sequences through sequence-specific binding domains, allowing them to regulate the expression of target genes regardless of their genomic location
Give an example of a cis element
Give an example of a trans element
What are transcription factors
Are an example of trans elements
What are the main types of transcription factor binding motifs
What is positive regulation of a gene and why isn’t it very effective
What is V1 negative regulation
What is V2 negative regulation
How do you identify cis elements using DNA footprinting*
Preparation of DNA Probe: Begin by generating a DNA fragment containing the region of interest, which may include putative cis-regulatory elements such as transcription factor binding sites. The DNA fragment is typically labeled at one end, either radioactively or fluorescently, to facilitate detection.
Binding of Proteins: Incubate the labeled DNA fragment with nuclear or cytoplasmic protein extract derived from cells or tissues of interest. Allow the proteins to bind to their cognate binding sites within the DNA sequence.
Chemical Cleavage or Nuclease Digestion: Treat the DNA-protein complexes with a chemical cleavage agent, such as DNase I or hydroxyl radicals generated by the Fenton reaction, or with a restriction enzyme that cuts DNA at specific sequences. These cleavage agents will cleave the DNA at sites that are not protected by protein binding.
Denaturing Gel Electrophoresis: After cleavage, separate the DNA fragments by denaturing polyacrylamide gel electrophoresis (PAGE). The gel is run under denaturing conditions to disrupt protein-DNA interactions and to allow the DNA fragments to migrate based on their size.
Visualization of Protected Regions: Visualize the labeled DNA fragments using autoradiography (for radioactive labeling) or fluorescence imaging (for fluorescent labeling). Regions of DNA that are protected from cleavage by protein binding will appear as “footprints” on the gel, where the intensity of the signal is reduced compared to surrounding regions.
Mapping and Characterization: Analyze the footprinting results to map the precise location of the protected regions within the DNA sequence. These protected regions correspond to the binding sites of proteins or other molecules and can be further characterized to identify the specific cis-regulatory elements involved.
How do you identify cis elements using Reporter Gene Assay: Promoter mapping *
Selection of Reporter Gene: Choose a reporter gene that can be easily assayed and quantified, such as β-galactosidase (lacZ), luciferase (luc), green fluorescent protein (GFP), or chloramphenicol acetyltransferase (CAT). The reporter gene should have minimal endogenous expression in the host cells being used for the assay.
Construction of Reporter Gene Constructs: Design and construct a series of reporter gene constructs containing different lengths of the promoter region of interest. These constructs should consist of the reporter gene fused downstream of the promoter sequence to be tested. Typically, the promoter region is cloned upstream of a minimal promoter element, such as the TATA box, to ensure efficient transcription initiation.
Transient Transfection or Stable Transduction: Introduce the reporter gene constructs into target cells either transiently through transfection or stably through transduction. Ensure that the cells used for the assay are appropriate for the promoter being tested and provide a suitable environment for reporter gene expression.
Reporter Gene Assay: Measure the expression of the reporter gene in the transfected or transduced cells using an appropriate assay method. For example, if using the lacZ reporter gene, you can assay for β-galactosidase activity using a colorimetric substrate such as X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). For luciferase or GFP reporters, you can measure luminescence or fluorescence, respectively.
Promoter Activity Analysis: Compare the expression levels of the reporter gene driven by different promoter constructs to identify regions of the promoter that are essential for transcriptional activity. This analysis allows you to determine the location and function of cis-acting elements, such as transcription factor binding sites, enhancers, and silencers, within the promoter sequence.
How do you identify trans elements using DNA affinity chromatography *
Preparation of DNA Probe: Begin by designing and synthesizing a DNA probe containing the target cis-acting element of interest. The probe should be immobilized onto a solid support, such as agarose beads or magnetic beads, to facilitate chromatographic separation.
Binding of Trans-acting Factors: Incubate the immobilized DNA probe with a nuclear or cytoplasmic protein extract derived from cells or tissues of interest. Allow sufficient time for the trans-acting factors present in the extract to bind to the immobilized DNA probe specifically.
Washing: Wash the DNA-protein complexes with a series of buffers to remove non-specifically bound proteins and contaminants. This step helps increase the specificity of the interaction between the DNA probe and the trans-acting factors.
Elution: Elute the bound proteins from the DNA probe using a high-salt buffer or a buffer containing a competitor DNA sequence that competes with the immobilized DNA probe for binding to the trans-acting factors. This step releases the specifically bound proteins while minimizing non-specific interactions.
Identification of Trans-acting Factors: Analyze the eluted proteins using techniques such as mass spectrometry, Western blotting, or gel electrophoresis coupled with protein staining or immunoblotting. These analyses allow you to identify and characterize the trans-acting factors that specifically interact with the immobilized DNA probe.
Confirmation of Binding Specificity: To confirm the specificity of the interaction between the identified trans-acting factors and the DNA probe, perform control experiments such as competition assays using excess non-labeled competitor DNA or DNA mutations within the binding site.
How do you identify trans elements using Gel shift assay*
Preparation of DNA Probe: Begin by designing and synthesizing a DNA probe containing the target sequence of interest. The probe is typically labeled with a radioactive or fluorescent tag to facilitate detection. The target sequence can be a promoter region, enhancer element, transcription factor binding site, or any other regulatory sequence.
Protein Extraction and Purification: Extract and purify nuclear or cytoplasmic proteins from cells or tissues of interest. These proteins may include transcription factors, RNA-binding proteins, or other trans-acting factors that potentially interact with the target DNA sequence.
Incubation with DNA Probe: Incubate the purified proteins with the labeled DNA probe in a reaction buffer containing appropriate salts, cofactors, and stabilizing agents. This allows the proteins to bind to the DNA probe if they have specific affinity for the target sequence.
Electrophoresis: After incubation, load the reaction mixture onto a polyacrylamide gel and subject it to electrophoresis. The gel electrophoresis separates the protein-DNA complexes from free DNA based on their differential mobility through the gel matrix.
Visualization and Analysis: Visualize the DNA bands on the gel using autoradiography (for radioactive probes) or fluorescence imaging (for fluorescent probes). The gel shift assay reveals one or more shifted bands corresponding to protein-DNA complexes, as well as unshifted bands corresponding to free DNA probes
How do you identify trans elements nowadays
What are the differences between gene regulation in prokaryotes and eukaryotes
What is an operon
What components make up an operon
How is an operon structured
Where may operons have come from
What is the role of sigma factor 70 *
Sigma factor 70, also known as σ70, is a key component of bacterial RNA polymerase and plays a crucial role in the initiation of transcription. In bacteria such as Escherichia coli (E. coli), σ70 is the primary sigma factor responsible for recognizing and binding to specific promoter sequences on DNA, thereby initiating the transcription of genes involved in housekeeping functions and growth-related processes.
Here are the key roles of sigma factor 70:
Promoter Recognition: Sigma factor 70 recognizes specific DNA sequences known as promoter regions, which are located upstream of genes. These promoter sequences typically contain conserved elements, such as the -10 and -35 regions, which are recognized by σ70. The binding of σ70 to the promoter region helps to position RNA polymerase correctly for the initiation of transcription.
Initiation of Transcription: Once bound to the promoter region, σ70 facilitates the assembly of the RNA polymerase holoenzyme complex, which consists of core RNA polymerase (composed of multiple subunits) and the σ70 subunit. This holoenzyme complex is responsible for initiating transcription by synthesizing an RNA molecule complementary to the DNA template strand.
Housekeeping Genes: Sigma factor 70 is primarily associated with the transcription of housekeeping genes, which are genes that are required for basic cellular functions and are constitutively expressed under normal growth conditions. These genes encode essential proteins involved in processes such as metabolism, DNA replication, and cell division.
Growth-Related Processes: In addition to housekeeping genes, σ70 is also involved in regulating the expression of genes required for bacterial growth and adaptation to environmental changes. This includes genes involved in nutrient uptake, stress response, and virulence factors in pathogenic bacteria.
Alternative Sigma Factors: While σ70 is the primary sigma factor in many bacteria, some bacteria possess multiple sigma factors with specialized functions. These alternative sigma factors can recognize distinct promoter sequences and regulate the transcription of specific sets of genes in response to environmental cues or developmental signals. In E. coli, for example, other sigma factors such as σS (sigma factor 38) and σE (sigma factor 24) play roles in stress response and envelope stress, respectively.
What is transcription and translation coupling and what does it do *
Transcription and translation coupling refers to the spatial and temporal coordination between the processes of transcription and translation in prokaryotic cells. In prokaryotes, such as bacteria like Escherichia coli (E. coli), transcription and translation can occur simultaneously on the same mRNA molecule, which remains associated with ribosomes as it is being transcribed. This coupling ensures efficient and rapid gene expression and is a characteristic feature of prokaryotic gene regulation.
Here’s how transcription and translation coupling works and what it does:
Simultaneous Processes:
In prokaryotic cells, mRNA synthesis (transcription) and protein synthesis (translation) are closely linked processes that occur simultaneously.
As RNA polymerase synthesizes the mRNA molecule, ribosomes bind to the mRNA near the transcription start site and begin translating the mRNA into protein even before transcription is complete.
The mRNA molecule emerges from the RNA polymerase complex and is rapidly captured by ribosomes, which then move along the mRNA transcript, synthesizing protein as they go.
Efficiency and Speed:
Coupling transcription and translation allows for the rapid production of proteins from newly transcribed mRNAs.
Because translation begins while transcription is still ongoing, there is minimal delay between mRNA synthesis and protein production, enabling cells to respond quickly to environmental changes or metabolic demands.
Coupling Factors:
The coupling of transcription and translation is facilitated by various factors, including the physical proximity of ribosomes to the RNA polymerase complex and the absence of a nuclear membrane in prokaryotic cells.
The close association between ribosomes and RNA polymerase prevents premature degradation of the mRNA molecule and allows ribosomes to immediately access the nascent mRNA for translation.
Regulation and Efficiency:
Transcription and translation coupling is an efficient mechanism for gene expression in prokaryotes, but it can also have regulatory implications.
Regulatory elements within the mRNA transcript, such as riboswitches or RNA secondary structures, can modulate the rate of transcription and translation to ensure proper gene expression levels.
Additionally, coupling transcription and translation allows for the co-translational folding of nascent polypeptides, which can influence protein folding, stability, and function.
Give 3 examples of sigma 70 regulated operons
What is the lactose operon
Why does bacteria stop growing at 3 hours when cultures with lactose and glucose
What is catabolite repression of the lactose operon *
Catabolite repression is a regulatory mechanism observed in bacteria that allows them to preferentially utilize certain carbon sources over others based on their availability.
When glucose is present, E. coli preferentially metabolizes it because it is a more efficient carbon source compared to lactose. As a result, the lac operon, which is responsible for lactose metabolism, is subject to repression by glucose through a mechanism involving catabolite repression.
Catabolite repression of the lactose operon involves the global regulatory protein known as catabolite activator protein (CAP), also called the cAMP receptor protein (CRP). CAP is a transcriptional activator that requires cyclic AMP (cAMP) as a cofactor for its activity. When glucose levels are high, intracellular cAMP levels decrease due to its repression by glucose metabolism. Consequently, low levels of cAMP result in reduced CAP activity.
CAP normally binds to specific DNA sequences, known as cAMP-responsive elements (CREs), located near promoters of target genes. In the case of the lac operon, CAP binds to a CRE located upstream of the lac promoter region, called the lac promoter (Plac). When bound to CAP, CAP-cAMP complex enhances RNA polymerase binding to the lac promoter, thereby stimulating transcription of the lac operon.
However, in the presence of glucose, low levels of cAMP lead to reduced binding of CAP to CREs, including the one upstream of the lac promoter. As a result, RNA polymerase has reduced access to the lac promoter, leading to decreased transcription of the lac operon genes. This repression mechanism ensures that E. coli preferentially utilizes glucose as a carbon source when available, while lactose metabolism is suppressed.
What are The interactions formed between the CAP and CAP-binding site in catabolite repression
How is the lactose operon governed by a single molecule trigger*
The lac operon is governed by a single molecule trigger, which is the inducer molecule, specifically allolactose. Here’s how it works:
In the Absence of Lactose (Allolactose):
By default, the lac operon is repressed. This repression is mediated by a regulatory protein called the lac repressor (encoded by the lacI gene).
The lac repressor binds to a specific DNA sequence called the operator, located between the promoter and the structural genes of the lac operon.
When bound to the operator, the lac repressor physically blocks the binding of RNA polymerase to the promoter region, preventing transcription of the structural genes.
In the absence of lactose, allolactose is not present, and the lac repressor remains bound to the operator, maintaining repression of the operon.
In the Presence of Lactose (Allolactose):
When lactose is present in the environment, it can enter the bacterial cell and be converted by the enzyme β-galactosidase, encoded by the lacZ gene, into allolactose.
Allolactose acts as an inducer molecule by binding to the lac repressor, causing a conformational change in the protein.
This conformational change weakens the lac repressor’s affinity for the operator, causing it to dissociate from the DNA.
With the lac repressor no longer bound to the operator, RNA polymerase can access the promoter region and initiate transcription of the lacZ, lacY, and lacA genes.
The lacZ gene encodes β-galactosidase, which further hydrolyzes lactose into glucose and galactose, facilitating lactose metabolism.
The lacY gene encodes lactose permease, a membrane protein that transports lactose into the cell.
The lacA gene encodes a transacetylase enzyme, which is less involved in lactose metabolism and has a regulatory role in acetylation of other sugars.
What are the effects of [glucose] and [lactose] on the lactose operon
What is the arabinose operon *
It is responsible for the metabolism of the sugar arabinose. The arabinose operon consists of a cluster of genes that encode enzymes involved in the catabolism of arabinose, allowing the bacteria to utilize arabinose as a carbon source when it is available.
cis elements: araO1, araO2 and araI (araI1 & araI2) and cAMP-CAP binding site
trans elements: AraC and cAMP-CAP
How can the arabinose operon be regulated
What is negative auto regulation with regards to the arabinose operon*
Negative auto-regulation, also known as autogenous regulation, is a regulatory mechanism in which a transcription factor controls its own expression by binding to regulatory sites within its own promoter region. This mechanism allows cells to maintain tight control over the expression levels of specific genes or operons.
How does the conc. of arabinose and glucose affect the arabinose operon
What is repression with respect to the tryptophan operon*
Repression refers to the inhibition of gene expression. In the case of the tryptophan operon, repression occurs when there is an abundance of tryptophan in the cell. The tryptophan repressor protein, encoded by the trpR gene, binds to the operator sequence within the tryptophan operon. This binding prevents RNA polymerase from transcribing the genes required for tryptophan synthesis. As a result, the operon is effectively “turned off” or repressed in the presence of excess tryptophan.
What is attenuation with respect to the tryptophan operon*
Attenuation is a regulatory mechanism that controls gene expression in prokaryotic cells by modulating the premature termination of transcription. It is particularly well-known in the context of the tryptophan (trp) operon in bacteria such as Escherichia coli.
In the tryptophan operon, attenuation regulates the expression of the structural genes involved in tryptophan biosynthesis in response to the availability of tryptophan. The operon contains regions known as leader sequences, located upstream of the structural genes, which harbor specific elements that participate in the attenuation process.
Here’s how attenuation works in the tryptophan operon:
Leader Sequence: The leader sequence contains two regions called the leader peptide coding region and the attenuator region. The leader peptide coding region encodes a short peptide called the leader peptide, which includes a sequence of consecutive tryptophan residues.
Regulatory Elements: The attenuator region contains specific regulatory elements, including a pair of tandem tryptophan codons followed by a series of RNA secondary structures called attenuator loops (stem-loop structures).
Ribosome Stalling: During transcription initiation, ribosomes begin translating the leader peptide coding region. As ribosomes translate the leader peptide mRNA, they pause at the tandem tryptophan codons due to the scarcity of charged tRNAs for tryptophan, causing ribosome stalling.
Antitermination vs. Termination: The ribosome stalling at the tandem tryptophan codons determines the fate of transcription elongation. If intracellular tryptophan levels are low (resulting in a shortage of charged tRNAs for tryptophan), ribosome stalling at the tryptophan codons is prolonged. In this case, the attenuator region adopts a conformation that favors the formation of an antiterminator hairpin structure, which allows RNA polymerase to continue transcribing the structural genes of the trp operon. However, if intracellular tryptophan levels are high (resulting in an abundance of charged tRNAs for tryptophan), ribosome stalling at the tryptophan codons is reduced. In this scenario, the attenuator region adopts a conformation that favors the formation of a terminator hairpin structure, leading to premature transcription termination before the structural genes.
Regulation: Thus, attenuation in the tryptophan operon allows the cell to finely regulate tryptophan biosynthesis in response to intracellular tryptophan levels. When tryptophan is abundant, attenuation causes premature termination of transcription, resulting in reduced expression of the trp operon. Conversely, when tryptophan is scarce, attenuation allows for continued transcription of the operon, enabling increased tryptophan synthesis.
What is the effect of [tryptophan] on the tryptophan operon
What is a riboswitch*
A riboswitch is a regulatory segment of a messenger RNA (mRNA) molecule that can directly bind to a specific small molecule ligand, typically a metabolite or ion, and alter the gene expression or RNA processing in response to the ligand binding. Riboswitches are found in the untranslated regions (UTRs) of mRNA, often in the 5’ untranslated region (5’ UTR), and sometimes in the 3’ UTR.
What is transcriptional noise
What are the differences between mRNA production in pro and eukaryotes
What is epigenetics
Give an overview of mRNA production
What is histone modification and how does it affect gene expression
Christian condensed = transcription repressed and vice versa
What is DNA methylation and how does it affect gene expression
What does methylation of RNA do*
Overall, RNA methylation plays crucial roles in the regulation of gene expression, RNA metabolism, regulation of RNA stability, regulating splicing and cellular homeostasis. Dysregulation of RNA methylation has been associated with various human diseases, including cancer, neurological disorders, and metabolic disorders, highlighting the importance of this epitranscriptomic modification in health and disease.
What do the different RNApol do
What is the RNApol II initiation complex*
The RNA polymerase II (RNAP II) initiation complex, also known as the preinitiation complex (PIC), is a multiprotein complex that assembles at the promoter region of genes to initiate transcription in eukaryotic cells. It is responsible for recruiting RNAP II and other transcription factors to the promoter, unwinding the DNA, and initiating RNA synthesis. The assembly of the RNAP II initiation complex is a highly regulated and coordinated process involving multiple steps and protein-protein interactions. Here’s a simplified overview of the components and steps involved:
Basal transcription factors: These are a group of proteins that initially bind to the core promoter region of genes. Basal transcription factors include:
TATA-binding protein (TBP): Binds to the TATA box element in the promoter.
Transcription factor IID (TFIID): A multi-subunit complex containing TBP and TBP-associated factors (TAFs). TFIID recognizes and binds to the TATA box, facilitating the recruitment of other transcription factors.
Transcription factor IIB (TFIIB): Interacts with TBP and helps position RNAP II at the transcription start site.
Transcription factor IIF (TFIIF): Stabilizes the binding of RNAP II to the promoter and helps recruit other factors.
Transcription factor IIE (TFIIE) and transcription factor IIH (TFIIH): Involved in promoter melting and DNA unwinding.
RNA polymerase II: Once the basal transcription factors are assembled at the promoter, RNAP II is recruited to form the preinitiation complex. RNAP II is the enzyme responsible for synthesizing RNA transcripts from DNA templates.
Coactivators and regulatory factors: In addition to the basal transcription machinery, various coactivators and regulatory factors can interact with the initiation complex to modulate transcriptional activity. These factors may include chromatin remodelers, histone modifiers, and specific transcriptional activators or repressors.
Promoter DNA melting and transcription initiation: The assembled initiation complex facilitates the melting of the DNA duplex at the transcription start site, allowing RNAP II to initiate RNA synthesis. The initiation of transcription involves the synthesis of short RNA transcripts (abortive transcripts) before RNAP II escapes from the promoter and transitions into productive elongation.
Overall, the RNA polymerase II initiation complex is essential for the accurate initiation of transcription in eukaryotic cells. It coordinates the assembly of transcriptional machinery at gene promoters, ensuring the proper regulation of gene expression.
What is GAL regulation in yeast*
In yeast, the GAL (galactose utilization) regulatory system controls the expression of genes involved in the metabolism of galactose. It primarily operates in the budding yeast Saccharomyces cerevisiae and has been extensively studied as a model for gene regulation.
The GAL regulatory system consists of several components:
Galactose metabolism genes: These are the genes responsible for the utilization of galactose as a carbon source. They include GAL1, GAL2, and GAL7, which encode enzymes involved in galactose metabolism.
GAL regulatory proteins: The main regulatory proteins of the GAL system include:
Gal4p: A transcriptional activator that binds to specific DNA sequences called upstream activating sequences (UAS_GAL or GAL4 binding sites) located upstream of the GAL genes. Gal4p is normally inactive in the absence of galactose.
Gal80p: Acts as a repressor of Gal4p activity. It binds to Gal4p and inhibits its ability to activate transcription of GAL genes. Gal80p is sensitive to galactose levels.
Gal3p: Functions as a sensor of intracellular galactose levels. It binds galactose and interacts with Gal80p, preventing Gal80p from inhibiting Gal4p activity in the presence of galactose.
The regulation of the GAL system can be summarized as follows:
In the absence of galactose: Gal4p is bound by Gal80p, rendering it inactive. As a result, GAL genes are not expressed, and the yeast cells utilize other carbon sources, such as glucose, for growth.
In the presence of galactose: Galactose enters the cell and binds to Gal3p. This binding causes a conformational change in Gal3p, leading to its interaction with Gal80p. This interaction prevents Gal80p from inhibiting Gal4p. As a result, Gal4p becomes active and stimulates transcription of the GAL genes, allowing the yeast cells to metabolize galactose.
The GAL regulatory system in yeast is a classic example of transcriptional regulation, where the presence or absence of a specific molecule (galactose) modulates the activity of transcription factors (Gal4p) to control the expression of target genes involved in a specific metabolic pathway.
What is the GAL4-UAS system in drosophila
The GAL4-UAS system is a powerful genetic tool used primarily in Drosophila melanogaster (fruit fly) research for targeted gene expression manipulation. It consists of two main components: the GAL4 transcription factor and the upstream activating sequence (UAS).
GAL4: GAL4 is a yeast transcriptional activator protein. In the GAL4-UAS system, GAL4 is expressed under the control of a tissue-specific or inducible promoter. This promoter dictates where GAL4 will be active within the organism.
UAS: The upstream activating sequence (UAS) is a DNA sequence containing GAL4 binding sites. It is typically placed upstream of a gene of interest. When GAL4 binds to the UAS sequence, it activates transcription of the downstream gene.
Here’s how the system works:
GAL4 is expressed in specific tissues or developmental stages of the fruit fly, depending on the promoter used to drive GAL4 expression. This creates spatial and temporal control over where and when the GAL4 protein is active.
Transgenic flies carrying a UAS-regulated gene of interest are generated. This gene can be one that promotes the expression of a protein, such as a fluorescent marker or a mutant protein, or one that silences gene expression, such as a RNA interference (RNAi) construct.
When GAL4 is active in a particular tissue or developmental stage, it binds to the UAS sequence upstream of the gene of interest. This activates transcription of the downstream gene, resulting in the expression of the protein encoded by that gene or the silencing of gene expression if the construct is designed for RNAi.
By crossing GAL4-expressing flies with flies carrying UAS-regulated genes, researchers can precisely control where and when the gene of interest is expressed or suppressed in the offspring. This allows for the investigation of gene function, developmental processes, and disease mechanisms in a highly targeted manner.
The GAL4-UAS system offers remarkable versatility and specificity, making it an indispensable tool for studying gene function and regulatory networks in Drosophila and other model organisms.
How does regulation by miRNA work*
Regulation by microRNAs (miRNAs) involves a post-transcriptional mechanism that fine-tunes gene expression in various biological processes. Here’s how it generally works:
Biogenesis of miRNAs: miRNAs are initially transcribed from genomic DNA by RNA polymerase II or RNA polymerase III into long primary miRNA transcripts (pri-miRNAs). These pri-miRNAs are processed in the nucleus by the microprocessor complex, which includes the enzyme Drosha and its cofactor DGCR8, to generate precursor miRNAs (pre-miRNAs). Pre-miRNAs are typically hairpin structures around 70 nucleotides in length.
Export to cytoplasm: After processing in the nucleus, pre-miRNAs are exported to the cytoplasm by exportin-5, where they are further processed into mature miRNAs.
Mature miRNA formation: In the cytoplasm, pre-miRNAs are cleaved by the enzyme Dicer into double-stranded miRNA duplexes, typically around 22 nucleotides in length. One strand of the miRNA duplex, known as the guide strand, is then loaded into the RNA-induced silencing complex (RISC), while the other strand, known as the passenger strand, is typically degraded.
Target recognition: The guide strand of the mature miRNA within the RISC complex binds to specific target mRNAs through complementary base pairing. Usually, this binding occurs in the 3’ untranslated region (UTR) of the target mRNA, although it can also occur in the coding region or 5’ UTR in some cases.
Gene expression regulation: The binding of the miRNA to its target mRNA can lead to gene expression regulation through two main mechanisms:
mRNA degradation: The binding of the miRNA to the target mRNA can induce mRNA degradation by recruiting enzymes that cleave the mRNA, leading to reduced protein expression.
Translation repression: Alternatively, miRNA binding can inhibit translation initiation or elongation, preventing the ribosome from translating the mRNA into protein without necessarily causing mRNA degradation.
Effects on protein expression: Ultimately, the regulation by miRNAs leads to decreased protein levels of the target gene, thereby influencing various cellular processes, including development, differentiation, metabolism, and response to stress or environmental cues.
How is miRNA needed for C elegans development*
In the nematode Caenorhabditis elegans, microRNAs (miRNAs) play crucial roles in various developmental processes, including the transition from larval stages L1 to L4. One of the most well-known examples of miRNA involvement in C. elegans development is the lin-4 miRNA.
lin-4 miRNA: This was one of the first discovered miRNAs, and it regulates the timing of larval development in C. elegans. lin-4 miRNA acts by repressing the translation of its target messenger RNA (mRNA) lin-14 during the transition from the first larval stage (L1) to the second larval stage (L2). The LIN-14 protein, when expressed, inhibits the transition from L1 to L2. By repressing LIN-14, lin-4 promotes this transition. Thus, lin-4 acts as a temporal regulator of developmental timing in C. elegans.
L1 to L4 Transition: During the developmental progression of C. elegans, there are four larval stages (L1 to L4) before adulthood. Each larval stage involves specific morphological and physiological changes. The transition from L1 to L2 is one of the critical checkpoints regulated by lin-4, as mentioned above. Additionally, other miRNAs and regulatory factors are involved in subsequent transitions.
Role of miRNAs in L1 to L4 Transition: While lin-4 specifically regulates the L1 to L2 transition, other miRNAs likely play roles in the subsequent transitions (L2 to L3, L3 to L4). These miRNAs may target different mRNAs encoding proteins involved in larval development, metabolism, and other processes necessary for the transition between stages. Through their regulatory activity, miRNAs help ensure the proper timing and progression of developmental events in C. elegans.
In summary, miRNAs such as lin-4 play crucial roles in C. elegans development by regulating the expression of target genes involved in larval transitions. While lin-4 specifically controls the L1 to L2 transition by targeting lin-14, other miRNAs likely contribute to subsequent larval transitions, ensuring the orderly progression of development in this model organism.
