UNIT 8 – Gene Regulation in Eukaryotes (Q&A) | MZO-002 | MSCZOO | M.Sc. Zoology | IGNOU
SAQ 1
Fill in the blanks with appropriate words:
a) In prokaryotes, genes are organized into ........................ .
Answer: operons
b) ........................... elements recognize, bind transcription factors and increase the rate of transcription of a gene.
Answer: Enhancer
c) The enzyme ............................. is required to bind a promoter to initiate transcription.
Answer: RNA polymerase
d) The transcription factors bind to specific DNA sequences called .......................... located at the proximal (near) end of the promoter promoter of of a gene or distal end (far) of the gene.
Answer: cis regulatory elements
e) ........................... is the factor that causes the DNA in the promoter region to bend like the CAP protein in bacteria and helps in the attachment of transcription factor to the promoter.
Answer: TFIID or TATA binding protein
SAQ 2
Answer in one word
a) The factors that bind to specific sequences of DNA upstream of coding genes and promote transcription of genes.
Answer: Transcription factors (TFs)
b) In this family of transcription factors, zinc forms an important component of the DNA-binding region.
Answer: Zinc finger motif
c) It binds to DNA as dimers which are comprised of two a-helices held together.
Answer: Leucine zipper
d) The sequences that repress transcription of the gene(s) by inhibiting the transcription-promoting activity of the associated promoter of a given gene.
Answer: Repressor
e) The other name for repressor.
Answer: Silencer
SAQ 3
Match the following:
Answer: (a) → (v); (b) → (iv); (c) → (i); (d) → (ii); (e) → (iii)
TERMINAL QUESTIONS
1. Describe various factors involved in the regulation of gene expression.
Gene expression in eukaryotes and prokaryotes is controlled at multiple levels. Each level involves different molecular factors that can increase or decrease the transcription, translation, or stability of gene products. These factors ensure that the right genes are expressed at the right time, in the right cell type and in response to environmental or developmental signals.
There are the following six major categories of factors involved in the regulation of gene expression:
1. Transcriptional Factors
These factors control whether a gene is transcribed into mRNA or not. The most important components include:
- Transcription factors: These are DNA-binding proteins that bind to promoter or enhancer regions. They can act as activators or repressors. For example, TATA-binding protein (TBP) binds to the TATA box in eukaryotic promoters.
- RNA polymerase: This enzyme synthesizes mRNA from the DNA template. In eukaryotes, RNA polymerase II transcribes protein-coding genes.
- Promoters and enhancers: Promoters are sequences where transcription begins. Enhancers are regulatory regions that increase transcription efficiency, often located far from the gene.
2. Epigenetic Factors
These are heritable changes that affect gene expression without altering the DNA sequence. The main mechanisms are:
- DNA methylation: Methyl groups are added to cytosine bases, usually at CpG sites, which repress transcription by blocking transcription factor binding.
- Histone modification: Enzymes like HATs (Histone Acetyltransferases) and HDACs (Histone Deacetylases) modify histones to either loosen or tighten DNA-histone interaction. Acetylation generally activates transcription, while deacetylation represses it.
- Chromatin remodeling complexes: These proteins rearrange chromatin structure to allow or prevent access to DNA.
3. Post-transcriptional Factors
After mRNA is synthesized, various factors influence its stability, processing and translation:
- Alternative splicing: Different combinations of exons are joined to produce different proteins from the same gene.
- RNA editing: Chemical modifications alter nucleotide sequences in mRNA.
- mRNA stability: The length of the poly-A tail and specific RNA-binding proteins influence how long an mRNA lasts in the cytoplasm.
4. Translational Factors
Translation of mRNA into protein can be regulated through:
- Initiation factors: Proteins like eIFs (eukaryotic initiation factors) help the ribosome bind to mRNA.
- Ribosome availability: The number of ribosomes can influence translation efficiency.
- miRNAs: MicroRNAs bind to mRNA and block translation or cause degradation.
5. Post-translational Factors
After a protein is made, its activity or lifespan can still be controlled by:
- Protein modifications: Such as phosphorylation, methylation, ubiquitination.
- Protein degradation: Proteins tagged with ubiquitin are destroyed by the proteasome.
- Transport and folding: Proteins must be properly folded and may need to be transported to their target location to function.
6. Environmental and Cellular Signals
External and internal signals often initiate changes in gene expression. These include:
- Hormones: Like steroid hormones that bind to intracellular receptors and directly affect transcription.
- Nutrient availability: Presence of glucose or amino acids can switch operons on or off.
- Stress signals: Such as heat shock, which induces specific stress-response genes.
2. What is transcriptional regulation of gene expression?
Transcriptional regulation of gene expression is the process by which a cell controls when, where and how much of a gene's product is made, by regulating the transcription step of gene expression. In simple terms, it means controlling the making of mRNA from DNA, which is the first and most important step in determining whether a gene will produce its final product or not.
In all cells, not all genes are active all the time. The cell needs to decide which genes to express depending on its type, developmental stage and external environment. This decision is mainly made at the transcriptional level.
This regulation ensures that:
- Only the necessary genes are turned on in a particular cell type.
- Genes are activated only at the right time (such as during development, cell division, or in response to a signal).
- The amount of mRNA made from a gene is adjusted based on the cell's need.
- Unwanted or harmful gene expression is turned off or repressed.
This regulation occurs mostly in the nucleus, before the process of translation begins. It involves several internal and external signals that influence whether RNA polymerase can bind to the gene's promoter and begin transcription or not.
For example, in prokaryotes, transcriptional regulation helps bacteria switch on genes only when a certain nutrient is present. In eukaryotes, it allows cells in the same body to behave differently, like a liver cell expressing liver-specific genes while a muscle cell expresses muscle-specific genes, even though both have the same DNA.
So, transcriptional regulation is a central control point that helps cells use their genetic information efficiently and safely, without wasting energy and resources.
3. Citing relevant examples explain how genomic imprinting via methylation regulates gene expression in eukaryotes.
In eukaryotes, genomic imprinting is a process where the expression of a gene depends on whether it is inherited from the mother or the father. This selective expression happens due to methylation, which is a type of epigenetic modification. It does not change the DNA sequence but affects how genes are turned on or off. In this process, one allele (either maternal or paternal) becomes methylated and silenced, and the other allele remains active. This kind of regulation ensures that only one copy of a gene is expressed while the other remains inactive.
Methylation usually takes place at cytosine bases within CpG dinucleotides in DNA, often in regulatory regions like imprinting control regions (ICRs). These methylation marks are added during gamete formation (spermatogenesis or oogenesis). Once established, they are maintained through cell divisions by DNA methyltransferase enzymes (mainly DNMT1).
This parent-specific gene silencing plays an important role during embryonic development, especially in genes related to growth, metabolism and brain function. If this imprinting is disturbed, it can lead to serious genetic disorders.
Examples
1. Igf2 and H19 Genes
This is the best-studied example of genomic imprinting in mammals.
- Igf2 (Insulin-like Growth Factor 2) is only expressed from the paternal allele
- H19 is expressed from the maternal allele.
The region between these two genes contains an imprinting control region (ICR).
- On the paternal chromosome, the ICR is methylated, which prevents binding of the CTCF protein. This allows an enhancer to activate Igf2, so Igf2 is expressed. H19 is silenced due to methylation.
- On the maternal chromosome, the ICR is not methylated, allowing CTCF to bind. This blocks the enhancer from activating Igf2. So, H19 is expressed and Igf2 is silenced.
This example shows how methylation at ICR decides which gene is active.
2. Prader-Willi Syndrome and Angelman Syndrome
Both conditions involve the same region on chromosome 15, but the gene expression depends on the parent of origin.
- In Prader-Willi Syndrome, the paternal genes are deleted or silenced by methylation. The maternal copy is naturally imprinted (inactive), so those genes are not expressed at all.
- In Angelman Syndrome, the maternal genes (like UBE3A) are deleted or methylated. The paternal copy is imprinted (inactive), so again, the required gene expression is missing.
Both disorders clearly show that if methylation-based imprinting is incorrect, gene expression is disturbed, leading to disease.
3. Beckwith-Wiedemann Syndrome
In this overgrowth disorder, abnormal methylation at the Igf2-H19 locus results in both alleles of Igf2 being expressed instead of just the paternal one. This leads to excessive growth and higher risk of cancer. This disorder shows how loss of proper imprinting affects gene dosage and health.
4. Describe alternative splicing as a mechanism for regulation of gene expression in eukaryotes.
Alternative splicing is a process that takes place after transcription. In this process, the same pre-mRNA transcript can be spliced in different ways to produce more than one type of mature mRNA. This leads to the production of different protein variants from a single gene. This mechanism helps in increasing protein diversity and is commonly seen in eukaryotic cells.
It plays an important role in regulating gene expression because it decides which protein will be formed, in what form and in which condition or tissue.
Mechanism of Alternative Splicing
There are the following steps in the mechanism of alternative splicing:
1. Transcription of Pre-mRNA
The gene is first transcribed in the nucleus by RNA polymerase II. This forms a primary transcript known as pre-mRNA which includes both exons (coding sequences) and introns (non-coding sequences).
2. Spliceosome Recognition and Assembly
A large molecular complex called the spliceosome is formed. This complex is made of snRNPs (small nuclear ribonucleoproteins) and other splicing proteins. It identifies three key regions of the pre-mRNA:
- 5’ splice site
- Branch point sequence
- 3’ splice site
These are necessary to decide where cutting and joining will happen.
3. Regulation of Splice Site Selection
The selection of which splice site to use is not random. It is regulated by special proteins called:
- SR proteins (splicing enhancers) – promote usage of nearby splice sites
- hnRNPs (splicing silencers) – inhibit usage of nearby splice sites
These proteins bind to specific sequences on the pre-mRNA called ESE (exonic splicing enhancers) or ESS (exonic splicing silencers).
The balance between enhancers and silencers decides which exons are kept and which are removed.
4. Splicing Patterns
Based on splice site selection, the following patterns may occur:
- Exon skipping – certain exons are skipped
- Mutually exclusive exons – only one exon out of two is kept
- Alternative 5' or 3' splice site – different cutting points at ends of exons
- Intron retention – sometimes an intron is not removed and is retained
Each of these patterns results in different mRNAs and hence different protein products.
5. Outcome on Gene Expression
The final spliced mRNA is exported to the cytoplasm. Depending on the way splicing was done:
- A functional protein can be produced
- A non-functional or altered protein can be formed
- The mRNA may be degraded before translation
Thus, gene expression is regulated not just by whether a gene is transcribed or not, but also by how that transcript is processed through splicing.
5. Describe how gene expression is regulated by RNAi.
RNA interference (RNAi) is a natural mechanism in eukaryotic cells which controls gene expression after transcription. This mechanism prevents the synthesis of certain proteins by targeting messenger RNA (mRNA) molecules and either degrading them or blocking their translation.
RNAi works mainly through two types of small RNA molecules:
- Small interfering RNA (siRNA)
- MicroRNA (miRNA)
Both these molecules are non-coding RNAs that do not code for proteins but regulate gene expression by interacting with mRNAs.
There are four main steps involved in the RNAi pathway which regulates gene expression. These steps are similar for both siRNA and miRNA, with some minor differences in their origin and pairing behavior.
1. Initiation: Formation of Small RNAs
The RNAi process starts when double-stranded RNA (dsRNA) or primary microRNA (pri-miRNA) is present inside the cell. These may come from viral infections, transposons, or endogenous gene regulation.
An enzyme called Dicer recognizes and cuts the long dsRNA into small fragments, each about 21 to 23 nucleotides in length. These fragments are known as:
- siRNA, which is derived from perfect dsRNA
- pre-miRNA, which is processed from pri-miRNA
Dicer activity is crucial in initiating RNA interference by generating the key regulatory molecules.
2. Assembly of RISC Complex
After processing by Dicer, the small RNA molecules bind to a protein complex called RISC (RNA-induced silencing complex). Within this complex:
- One strand of the RNA, which is called the guide strand is retained
- The other strand, which is called the passenger strand is degraded
The guide strand helps the RISC to recognize and bind to the complementary sequence on the target mRNA.
3. Target Recognition and Binding
RISC uses the guide strand to find the complementary sequence in the mRNA:
- If the guide RNA is from siRNA, the binding is usually perfect to its target mRNA
- If it is from miRNA, the binding is usually partial, often with mismatches
This base-pairing is essential for the next step, which is the actual silencing of gene expression.
4. Gene Silencing
Once the RISC is bound to its target mRNA, it can regulate gene expression in two main ways:
- mRNA Cleavage: In the case of siRNA, the binding is exact, so the RISC cuts the mRNA strand. This cleavage makes the mRNA unstable and it gets degraded quickly. No protein is formed.
- Translational Repression: In the case of miRNA, the RISC complex usually does not cut the mRNA but binds to it and blocks the ribosome from translating it into protein.
In both situations, the result is reduced gene expression of the target gene.
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