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In this article we will discuss about:- 1. Introduction to Gene Regulation in Prokaryotes 2. Types of Operon in Gene Regulation 3. Mechanisms.
Introduction to Gene Regulation in Prokaryotes:
Gene regulation refers to the control of the rate or manner in which a gene is expressed. In other words, gene regulation is the process by which the cell determines (through interactions among DNA, RNA, proteins, and other substances) when and where genes will be activated and how much gene product will be produced.
Thus, the gene expression is controlled by a complex of numerous regulatory genes and regulatory proteins. The gene regulation has been studied in both prokaryotes and eukaryotes.In prokaryotes, the operon model of gene regulation is widely accepted.
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This model of gene regulation was proposed by Jacob and Monod in 1961 for which they were awarded Nobel Prize in 1965. The operon refers to a group of closely linked, genes which together code for various enzymes of a particular biochemical pathway.
In other words, operon is a unit of bacterial gene expression and regulation, including structural genes and control elements in-DNA recognized by regulator gene product(s). Thus operon is a model which explains the on-off mechanism of protein synthesis in a systematic manner. The main points of operon model of gene regulation are presented below.
(i) Developed By:
In prokaryotes, the operon model of gene regulation was developed by Jacob and Monod in 1961 for which they were awarded Nobel prize in 1965. Now this model of gene regulation is widely accepted.
(ii) Organism Used:
The operon model was developed working with lactose region [lac region] of human intestine bacteria E. coli. The gene regulation was studied for degradation of the sugar lactose.
(iii) Genes Involved:
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In the operon model of gene regulation, four types of genes viz:
(i) Structural genes,
(ii) Operator gene,
(iii) Promoter gene, and
(iv) Regulator gene are involved.
In addition, repressor, co-repressor, and inducer molecules are also involved.
(iv) Enzymes Involved:
Four types of enzymes are involved in gene regulation of prokaryotes. These are beta-galactosidase, galactosidase permease, transacetylase and RNA polymerase. The beta-galactosidase catalyses the breakdown of lactose into glucose and galactose.
The galactosidase permease permits entry of lactose from the medium into the bacterial cell. The enzyme transacetylase transfers an acetyl group from acetyl co-enzyme A to beta galactosidase. The enzyme mRNA polymerase controls on-off of the transcription.
Types of Operon in Gene Regulation:
In prokaryotes, operons are of two types, viz., inducible and repressible. The example of an inducible operon is the lactose operon, which contains genes that encode enzymes responsible for lactose metabolism. An example of repressible operon is the Trp operon, which encodes enzymes responsible for the synthesis of the amino acid tryptophan (trp for short).
A. Inducible Operon:
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Inducible Enzyme:
An enzyme whose production is enhanced by adding the substrate in the culture medium is called inducible enzyme, and such system is called inducible system. The example of an inducible operon is the lactose operon, which contains genes that encode enzymes responsible for lactose metabolism.
In bacteria, operon refers to a group of closely linked genes which act together and code for the various enzymes of a particular biochemical pathway.
The model of lac operon of E. coli looks like this:
(1) Structural Genes:
There are three structural genes of the lac operon i.e. lac Z, lac Y and lac A. The main function of structural genes is to control of protein synthesis through messenger RNA. Function of these genes is as follows.
(i) lac Z:
It encodes the enzyme beta-galactosidase, which catalyses the breakdown of lactose into glucose and galactose.
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(ii) lac Y:
It encodes the enzyme galactosidase permease, which permits entry of lactose from the medium into the bacterial cell.
(iii) lac A:
It encodes the enzyme transacetylase, which transfers an acetyl group from acetyl co-enzyme A to beta galactosidase.
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(2) Promoter Gene:
The above three structural genes genes are under the control of the promoter gene [designated P]. In the promoter, RNA polymerase binds to the DNA and prepares to initiate transcription. The main function of promoter gene is to initiate mRNS transcription.
(3) Operator Gene:
The other regulatory element in an operon is the operator (designated O). This is the element that determines whether or not the genes of the operon are transcribed. The main function of operator gene is to control function of structural genes.
(4) Regulator Gene:
This is designated as I. It is expressed all the time, or constitutively and plays an important role in operon function. This is the lac I gene, which encodes a protein called the lac repressor. The lac repressor has two functional domains or regions: one that binds to the DNA of the operator region, and one that binds to lactose.
When the repressor binds to the operator, it prevents RNA polymerase advancing along the operon, and transcription does not occur. The regulation of the operon depends on regulating whether or not the repressor binds to the operator. The function of regulator gene is to direct synthesis of repressor, a protein molecule. Its function differs in the presence and absence of lactose as discussed below.
When Lactose is absent:
When the lactose is absent in the environment, events take place in this way. The lac I gene is transcribed [constitutively i.e. continuously] and the mRNA is translated, producing the lac repressor. The repressor binds to the operator, and blocks RNA polymerase.
When RNA polymerase is blocked, there is no transcription. Thus the enzymes for lactose metabolism are not synthesized, because there is no lactose to metabolize. Thus when lactose is absent, lactose-metabolizing enzymes are not produced.
When Lactose is Present:
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When the lactose is present in the environment, the events occur in a different way. A small amount of the lactose enters into the cell and affects regulation of the operon. The lac repressor is still synthesized. The repressor can bind to lactose.
After binding to lactose, the repressor undergoes a conformational change (change of shape). Molecules that change shape when they bind to another molecule are called allosteric molecules. With this change, the lac repressor is unable to bind to the operator region. Hence RNA polymerase is not blocked, and is able to transcribe the genes of the operon.
The enzymes encoded by those genes are produced. The lac permease transports more lactose into the cell and beta-galactosidase cleaves the lactose into glucose and galactose. This can be further metabolized by other enzymes, producing energy for the cell.
Lactose, therefore, is able to induce the synthesis of the enzymes necessary for its metabolism (by preventing the action of the repressor). As such, lactose is the inducer of the lac operon. Thus when lactose is absent, lactose-metabolizing enzymes are not produced, and when lactose is present, those enzymes are produced.
Mutations of the Lac Operon:
Mutations can affect the regulation of the lac operon in different ways as given below:
(i) Mutation of the lac I gene in such a way that the repressor encoded no longer binds to lactose. In this case, the repressor would bind to the operator regardless of the presence or absence of lactose, and the operon would never be transcribed at high levels.
(ii) Mutation of the lac I gene in such a way that the repressor no longer binds to the operator. In this case, the operon would never be repressed, and transcription would be carried out continuously. This is known as constitutive transcription.
(iii) Mutation in the operator region in such a way that the wild-type repressor does not recognize it (the repressor recognizes the specific DNA sequence of the operator legion): In this case, there will be no binding of the repressor to the operator, and transcription will go on continuously.
Catabolite Repression:
Expression of the lac operon can also be regulated in another way. Glucose is preferable to lactose as an energy source. Hence if glucose is present in the environment, the transcription is reduced or lac operon is down-regulated.
Transcription of the lac operon requires another protein, called catabolite activator protein (CAP for short). This CAP protein binds to the lac promoter and enhances transcription. But it occurs only after CAP binds to a small molecule called cyclic AMP (cAMP).
Without cAMP, CAP will not bind to the promoter, and no transcription will occur. In the previous examples involving the lac operon, we can assume that cAMP was present, and the CAP-cAMP complex was bound to the promoter.
The cAMP is produced by an enzyme called adenyl-cyclase. In the presence of glucose in the environment, adenyl-cyclase is inhibited, and cAMP production drops. Thus there is no cAMP to bind to CAP. In this situation, the CAP will not bind to the lac promoter, and no lac transcription takes place.
In this way, the bacterium does not produce enzymes for lactose metabolism when they are not necessary because of the presence of glucose. Beta-galactosidase breaks lactose in to glucose and galactose. When enough lactose has been metabolized, glucose (one of the products) accumulates and causes repression of the lac operon.
Merits of Operon Model in Gene Regulation:
1. It is a very simple yet informative model of gene regulation in prokaryotes.
2. It is a very well understood model of gene regulation in prokaryotes.
3. This model is based on empirical results and has been studied on different prokaryotes.
4. This model is of two types, viz:
(i) Inducible operon and
(ii) Repressible.
B. Repressible Operon:
A protein molecule which prevents transcription is called repressor and the process of inhibition of transcription is called repression. Repressible operons are regulated by the end product of the metabolic pathway and not by a reactant in the metabolic pathway (such as lactose in lac operon).
An example of repressible operon is the Trp operon. This encodes enzymes which are responsible for the synthesis of the amino acid tryptophan (trp for short). The trp operon is regulated by trp, which is the product of the metabolic pathway.
In trp operon, the trp repressor only binds to the operator when trp is present, (opposite to the lac repressor). The repressor binds to trp, and undergoes a conformational change [change of shape]. This change in shape allows it to bind to the operator, blocking transcription. Because trp is needed for repression, it is referred to as a co-repressor in this system (as opposed to lactose being an inducer).
When trp is absent, the repressor will not bind to the operator, and transcription occurs. Thus, if there is plenty of trp around [and no more is needed], the transcription is blocked. If there is no trp around [it needs to be synthesized], transcription occurs. In other words, it allows production of the enzymes for trp synthesis.
Repressible operons are organized in much the same way as inducible operons: there are structural genes under the control of a promoter and operator, and there is a gene encoding a repressor.
The mutation will affect the gene regulation as follows:
(i) mutation in the repressor gene in such a way that it no longer binds trp; When repressor does not bind trp, there will be no change in its structure and it will not bind with operator and transcription will occur.
(ii) mutation in the repressor gene in such a way that it no longer binds the repressor: In such situation transcription will take place.
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(iii) mutation in the operator in such a way that it no longer binds to the repressor: In such situation also transcription will occur.
Mechanism of Gene Regulation:
The mechanism of gene regulation is of two types, viz:
(1) Negative regulation, and
(2) Positive regulation.
The mechanism of gene regulation in E. coli operon and tryptophan operon are discussed below:
1. Negative Control:
The first switch in the lac operon of E. coli, is the repressor protein. In negative control, the transcription is controlled by repressor protein, which is an allosteric protein. The repressor protein binds to operator region and prevents transcription. It prevents transcription by blocking RNA polymerase. Thus, when repressor is bound to operator, the transcription is switched off.
Thus the on-off switch of protein synthesis is governed by free or occupied position of the operator gene. When the operator is free, transcription will take place and when the operator gene is blocked, the transcription is prevented. If an isomer of lactose [allolaptose] is present, it will bind to repressor protein and change its shape. The changed repressor does not bind to operator and thus allows transcription.
2. Positive Control:
The second switch in the lac operon of E. coli, is the catabolite activator protein [CAP].The CAP is an allosteric protein. The CAP binds to DNA and small molecule called cyclic adenosine mono phosphate [cAMP], The CAP only binds to promoter region and stimulates transcription when cAMP binds to allosteric site.
The concentration of cAMP is controlled by ATP concentrations. The low ATP leads to high cAMP and high ATP leads to low cAMP. If E. coli is growing on glucose, there will be high [ATP] & low [cAMP], If no glucose is present, there will be a low [ATP] & high [cAMP]
In the absence of glucose, [cAMP] is high, binds to CAP which binds to promoter region and stimulates transcription. If glucose is present, [cAMP] is low. doesn’t bind to CAP which cannot bind to promoter and doesn’t allow transcription.
Tryptophan Operon:
The tryptophan operon [in short trp operon] is regulated by trp, which is the product of .the metabolic pathway. The trp operon contains genes that make 5 enzymes in the biosynthetic pathway for the production of amino acid tryptophan.
In trp operon, the negative control is associated with a repressor protein. However, the repressor protein only binds with operator gene when an allosteric effector is bound to it. The tryptophan is an allosteric effector, which is called a co-repressor in trp operon also, the transcription is controlled by the free or occupied position of repressor.
If the repressor protein doesn’t bind with operator gene, transcription will take place. If tryptophan is present, there is no need to synthesize enzymes. In such situation tryptophan binds to repressor protein and both these [trp and repressor] bind to operator gene preventing transcription. When trp is absent, the repressor will not bind to the operator, and transcription will take place.
In the negative control, repressor protein binds DNA and stops transcription. In positive control, activator protein binds DNA and stimulates transcription. In the inducible system, allosteric effector binds and releases repressor protein from DNA resulting in transcription. In the repressible system, allosteric effector binds and causes repressor protein to bind to DNA preventing transcription.
