UNIT 7 – Gene Regulation in Prokaryotes (Q&A) | MZO-002 | MSCZOO | M.Sc. Zoology | IGNOU
SAQ 1
i) Fill in the blanks:
a) The promoters are .......................... nucleotides long and extend ......................... from the initiation site.
Answer: 50, upstream
b) ........................... DNA sequences are recognized by transcription regulators which bind to DNA.
Answer: Regulatory
c) Genes that specify enzymes that catalyse metabolic processes are ........................ .
Answer: constitutive genes
d) Genes that get induced only under certain conditions are called ........................... .
Answer: inducible genes
e) Binding of the activators or repressors to the ............................. site depends on the presence or absence of molecules (sugars, amino acids) or certain metabolites.
Answer: Regulator Protein Binding Site (RPBS)
ii) State whether these statements are 'True' or 'False'
a) Regulatory DNA sequences found predominantly in bacteria are 10 nucleotide pairs long.
Answer: True
b) Each regulator recognizes a similar DNA sequence and regulates a similar set of genes.
Answer: False
c) Gene expression is turned "on" when the RNA polymerase binds to the promoter region.
Answer: True
d) The binding of the regulator gene product promotes transcription in a positive control system.
Answer: True
e) The binding of the activators or repressors to the Regulator Protein Binding Site (RPBS) is not related to the presence or absence of molecules/metabolites.
Answer: False
SAQ 2
Answer in one word
a) A regulatory system found in bacteria in which genes coding for functionally related proteins are clustered along the DNA.
Answer: Operon
b) A CoA-dependent acetyltransferase enzyme that transfers an acetyl group from acetyl-CoA to galactosides, lactosides and glucosides.
Answer: β-galactoside transacetylase
c) Inducer of the lac operon which is derived from lactose via β-galactosidase activity.
Answer: Allolactose
d) binding of this complex to the lac promoter is essential for the induction of the lac operon.
Answer: CAP/cAMP complex
e) This process ensures that the cell primarily metabolises glucose in preference to other, less efficient, energy sources.
Answer: Catabolite repression
SAQ 3
Fill in the blanks
a) Each gene of the trp operon encodes for different enzymes required for the conversion of ............................. to tryptophan (Fig. 3.8).
Answer: chorismic acid
b) The tryptophan repressor is an ......................... protein.
Answer: allosteric
c) The repressor ............................. production of a set of biosynthetic enzymes according to the availability of the end product of the pathway that the enzymes catalyze.
Answer: regulate
d) The presence of ............................. in the growing medium of E. coli cells, results in the premature termination of transcription at a site near the end of the mRNA leader sequence encoded by trpL.
Answer: tryptophan
e) The formation of the ............................ inhibits progress of transcription resulting in attenuation of gene expression.
Answer: transcription-termination hairpin
TERMINAL QUESTIONS
1. Differentiate between constitutive, inducible, and repressible gene expression.
Gene expression is the process by which information from a gene is used to produce a functional product like a protein. In living organisms, all genes are not expressed in the same way or at the same time. Some genes are always active, while others are turned on or off depending on the cell's needs or external signals. Based on how the expression is controlled, gene expression is broadly classified into three types: constitutive, inducible and repressible. These types differ in terms of regulation, activity pattern and response to cellular or environmental conditions. The following points explain the major differences among these three types are:
1. Based on Expression Pattern
Constitutive genes are expressed continuously. They remain active all the time because their products are always needed for basic functions like energy production or protein synthesis. For example, genes for ribosomal proteins are constantly required and thus are always expressed.
Inducible genes are normally inactive and get activated only when a specific molecule is present. For example, in bacteria, the lac operon stays off unless lactose is present in the environment, which acts as an inducer and switches the gene on.
Repressible genes are generally active but can be switched off when a certain product accumulates. In the trp operon, the genes are expressed to produce tryptophan but stop functioning when tryptophan becomes abundant in the cell.
2. Based on Control Mechanism
Constitutive gene expression does not depend on external signals or cellular feedback. It is unregulated and always ON.
Inducible gene expression depends on an inducer that interacts with a repressor protein to start transcription. In the absence of the inducer, the gene stays silent. This helps in saving energy and producing proteins only when needed, as seen when the lac operon turns on only in the presence of lactose.
Repressible gene expression is controlled by a feedback system where the end product acts as a corepressor. When the concentration of the product becomes high, it binds to a repressor protein and halts gene expression, as in the case of tryptophan regulating its own synthesis.
3. Based on Functional Purpose
Constitutive genes are responsible for essential cellular activities and are needed at all times. These include enzymes involved in central metabolism and DNA repair.
Inducible genes allow the cell to respond quickly to environmental changes. They help in adapting to new conditions like the sudden availability of a sugar which was previously absent.
Repressible genes help in maintaining metabolic balance. They stop unnecessary production of molecules that are already available in sufficient amount, preventing waste of cellular resources.
4. Based on Energy Utilization
Constitutive genes consume more energy because they keep producing proteins all the time, even when those proteins are not currently needed.
Inducible genes help save energy because they make proteins only when the required molecule or condition is present.
Repressible genes are also energy-efficient as they stop making proteins when the end product is already available in enough quantity.
2. What are the roles of an activator and a repressor in regulating gene expression in eukaryotes?
In eukaryotic cells, gene expression is a highly regulated process that ensures genes are turned on or off at the right time and in the right cells. This precise control is necessary for development, differentiation and response to environmental signals. Two major types of regulatory proteins that control gene expression are activators and repressors. Both of these proteins bind to specific DNA sequences and influence the transcription of genes but in opposite ways.
Role of Activators:
Activators increase gene expression by binding to DNA regions called enhancers or promoter-proximal elements. Their binding helps attract and stabilize the transcription machinery, including RNA polymerase II and general transcription factors, at the gene's promoter. Activators also recruit proteins that modify chromatin structure, making DNA more open and accessible for transcription. This action results in increased transcription and higher production of mRNA. Activators are important in processes where rapid or high levels of gene expression are needed, such as during cell differentiation or in response to external signals like hormones.
Role of Repressors:
Repressors decrease or block gene expression by binding to DNA sequences called silencers or operator regions near the target gene. They prevent transcription by blocking the binding of activators or the transcription machinery. Additionally, repressors can recruit proteins that modify chromatin into a compact form, which makes the gene less accessible for transcription. This reduces or completely stops the production of mRNA. Repressors are essential for turning off genes that are not required in a particular cell type or under specific conditions, thus helping maintain cell identity and proper functioning.
3. Distinguish between positive and negative control systems of gene expression.
Gene expression in both prokaryotes and eukaryotes is controlled by regulatory mechanisms that decide whether a gene will be transcribed or not. These mechanisms mainly work through two types of control systems: Positive Control System and Negative Control System. These systems regulate gene expression based on the presence or absence of specific regulatory proteins, which either help or block the process of transcription.
1. Positive Control System
In positive control, a regulatory protein called an activator is required to start or increase transcription. This activator protein binds to the DNA at a specific site and helps RNA polymerase to attach to the promoter and begin transcription. If the activator is not present or is inactive, transcription does not occur or occurs at a very low level.
This system stimulates gene expression by making the environment favorable for RNA polymerase to function.
Example:
In the lac operon of E. coli, when glucose levels are low, cyclic AMP (cAMP) levels rise. The cAMP binds to the catabolite activator protein (CAP) and this complex then binds to the promoter region of the lac operon. This binding enhances the binding of RNA polymerase and activates transcription of the genes needed for lactose metabolism.
2. Negative Control System
In negative control, a regulatory protein called a repressor binds to a specific region on the DNA (usually an operator) and blocks transcription. This prevents RNA polymerase from attaching to the promoter or moving forward. When the repressor is removed or inactivated (usually by a signal molecule), transcription can proceed.
This system inhibits gene expression until the proper signal turns it off or removes the block.
Example:
In the lac operon again, when lactose is absent, a repressor protein binds to the operator site and prevents transcription. When lactose is present, it binds to the repressor, changes its shape and causes it to detach from the DNA. This allows transcription to happen.
4. What is an operon? Describe the organisation of a typical operon.
An operon is a functional unit of DNA found mainly in prokaryotes like bacteria. It consists of a group of related genes that are transcribed together under the control of a single promoter. These genes usually have related functions and are regulated together, allowing the cell to efficiently respond to environmental changes.
The concept of the operon was first described by François Jacob and Jacques Monod in 1961 based on their studies of the lac operon in E. coli.
A typical operon has the following components:
There are five major components found in the organisation of a typical operon:
1. Regulator Gene
This gene is located outside the operon. It codes for a repressor protein or activator protein. These proteins control the activity of the operon by either preventing or helping transcription. For example, in the lac operon, the lacI gene is a regulator gene that produces a repressor.
2. Promoter (P)
The promoter is a specific DNA sequence where RNA polymerase binds to initiate transcription. It lies upstream of the structural genes. The efficiency of promoter binding directly affects how often transcription happens.
3. Operator (O)
The operator is a short DNA region located near or overlapping the promoter. It acts as a regulatory switch. A repressor protein can bind to this operator and block the path of RNA polymerase, preventing transcription. In inducible operons, a small molecule (inducer) can bind to the repressor and inactivate it.
4. Structural Genes
These are the main genes in the operon that are transcribed together as a single mRNA. They usually encode enzymes or proteins involved in a particular metabolic pathway. For example, in the lac operon, the structural genes are lacZ, lacY and lacA, which are involved in lactose metabolism.
5. Terminator
It is a sequence downstream of the structural genes that signals the end of transcription. Once RNA polymerase reaches this region, transcription stops and the mRNA is released.
5. How does the presence/absence of allolactose regulate the expression of structural genes in the lac operon?
The lac operon is a well-known example of gene regulation in Escherichia coli. It is responsible for the metabolism of lactose, a disaccharide sugar. The operon includes three structural genes: lacZ, lacY and lacA, which code for β-galactosidase, permease and transacetylase respectively. These enzymes help in the uptake and breakdown of lactose into simpler sugars.
The regulation of these structural genes depends on the presence or absence of a molecule called allolactose, which acts as an inducer. Allolactose is a modified form of lactose, produced when a small amount of lactose is converted by basal levels of β-galactosidase.
Regulation in the Absence of Allolactose (Lactose Absent)
When lactose is absent in the environment, there is no allolactose available in the cell. In this condition:
- A protein called lac repressor, produced by the lacI gene, is active.
- This active repressor binds to the operator region of the lac operon.
- By binding to the operator, the repressor physically blocks RNA polymerase from transcribing the structural genes.
- As a result, the transcription of lacZ, lacY and lacA does not occur.
Thus, in the absence of allolactose, the lac operon remains off or repressed to avoid wasting energy on producing enzymes that are not needed.
Regulation in the Presence of Allolactose (Lactose Present)
When lactose becomes available, a small amount enters the cell through permease and some of it is converted into allolactose. In this condition:
- Allolactose binds to the lac repressor protein.
- This binding causes a conformational change in the repressor, making it unable to bind to the operator.
- As the operator is now free, RNA polymerase can bind to the promoter and proceed with the transcription of the structural genes.
- The enzymes β-galactosidase, permease and transacetylase are produced, which help in breaking down lactose into glucose and galactose.
Thus, in the presence of allolactose, the lac operon is induced and becomes active.
6. What is the role of CAP/cAMP in the catabolite repression of the lac operon?
Catabolite repression is a type of positive control where the presence of glucose stops the use of other sugars like lactose. In E. coli, this repression is controlled by the CAP/cAMP complex. The full form of CAP is Catabolite Activator Protein and cAMP stands for cyclic Adenosine Monophosphate.
There are the following three key roles that CAP/cAMP plays in the regulation of the lac operon:
1. Acts as a Positive Regulator of Transcription
CAP cannot bind to DNA by itself. When glucose is low, cAMP levels increase. cAMP binds with CAP and forms the CAP–cAMP complex. This complex attaches to a specific DNA site just upstream of the lac promoter. Its role is to help RNA polymerase bind strongly to the promoter so that transcription of the lac operon can happen efficiently. So, CAP/cAMP acts like a helper that increases the transcription of lac genes when glucose is not available.
2. Links Gene Expression to Glucose Availability
The CAP/cAMP complex allows E. coli to give preference to glucose. When glucose is present, cAMP levels drop. Without cAMP, CAP stays inactive and cannot help in transcription. Even if lactose is available, lac operon expression remains low. So the role of CAP/cAMP is to connect gene expression with the cell's energy condition. It ensures that lactose is used only when glucose is absent.
3. Controls the Timing of Lac Operon Activation
CAP/cAMP does not control whether the operon is ON or OFF. That is done by the lac repressor and allolactose. But it controls how much transcription happens. It prepares the promoter to accept RNA polymerase. So the role of CAP/cAMP is not about repression directly, but about activating transcription only under right conditions.
7. How is gene expression in the trp operon regulated by the amino acid tryptophan?
The trp operon in Escherichia coli controls the synthesis of the amino acid tryptophan. This operon is a classic example of negative feedback regulation, where the end product (tryptophan) controls its own synthesis. The operon contains five structural genes (trpE, trpD, trpC, trpB and trpA) that together code for enzymes involved in tryptophan biosynthesis.
There are the following two main mechanisms by which tryptophan regulates the expression of genes in the trp operon:
1. Repression by the Trp Repressor Protein (Negative Control)
When tryptophan levels in the cell are high, the tryptophan molecule binds to a regulatory protein called the Trp repressor, which is coded by the trpR gene (located outside the operon). In its inactive form, the repressor cannot bind to the operator. But when tryptophan binds to it, the repressor becomes active and undergoes a conformational change. The tryptophan–repressor complex now binds to the operator region of the trp operon, which lies between the promoter and structural genes.
By binding to the operator, the complex blocks the movement of RNA polymerase, preventing the transcription of downstream structural genes. This means that no mRNA is produced, and the enzymes required to synthesize more tryptophan are not made. This is a negative feedback loop because the presence of the end product (tryptophan) turns off its own synthesis.
2. Attenuation (Transcriptional Regulation at the Leader Sequence)
In addition to repression, the trp operon also uses a second method of regulation called attenuation, which fine-tunes gene expression when tryptophan is present in moderate amounts. This regulation depends on a short region called the leader sequence (trpL), located just before the first structural gene.
The leader sequence is transcribed into a short mRNA that includes a region that can fold into alternative secondary structures (stem-loop structures). This mRNA also includes two tryptophan codons that act as sensors.
- When tryptophan is abundant, ribosomes quickly translate these codons and a terminator loop is formed in the mRNA. This loop causes premature termination of transcription and the structural genes are not transcribed.
- When tryptophan is scarce, the ribosome stalls at the tryptophan codons due to lack of available tRNA^Trp. This allows the formation of an anti-terminator loop and transcription continues through the structural genes.
So, attenuation acts like a fine control that depends on the actual levels of tryptophan during transcription.
8. Describe the role of the trpL leader sequence in regulating the expression of the trp operon.
The trpL leader sequence is a short coding region located upstream of the structural genes in the trp operon of E. coli. It plays a crucial role in regulating gene expression through a process called attenuation. This mechanism allows the operon to respond precisely to intracellular levels of the amino acid tryptophan.
There are the following four main roles of the trpL leader sequence in regulating the trp operon:
1. Sensing Tryptophan Availability through Leader Peptide Translation
The trpL region contains a leader peptide-coding sequence with two adjacent tryptophan codons. These codons act as a sensor for the availability of charged tRNA^Trp. When tryptophan levels are high, ribosomes can quickly translate the leader peptide. When tryptophan is low, ribosomes stall at these codons. This stalling influences the downstream RNA structure and determines whether transcription continues or terminates.
2. Controlling Transcription through Attenuation Mechanism
The RNA sequence of trpL contains four complementary regions (1, 2, 3 and 4) that can form different secondary structures in the mRNA:
- If tryptophan is abundant, ribosomes do not stall. This allows formation of a 3–4 terminator loop, which causes premature termination of transcription before the structural genes are transcribed.
- If tryptophan is scarce, ribosomes stall at the Trp codons. This allows the formation of a 2–3 anti-terminator loop, which prevents the formation of the terminator structure, allowing transcription of the downstream genes to continue.
This attenuation mechanism allows the cell to fine-tune gene expression based on current tryptophan levels.
3. Providing Graded or Conditional Regulation
Unlike the trp repressor, which works as an on-off switch, the trpL sequence allows for conditional regulation. It does not completely block or fully allow gene expression. Instead, it adjusts the level of transcription depending on how much tryptophan is present, making the regulation more dynamic and energy-efficient.
4. Coordinating Transcription and Translation
The function of trpL also depends on the simultaneous coupling of transcription and translation, which is a feature of prokaryotic cells. The behavior of the ribosome during translation directly affects the folding of the mRNA as it is being transcribed. This coordination ensures that gene expression is rapidly adjusted based on cellular conditions.
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