Gene Expression and Control
Topics: 1. Introduction 2. Operons: Regulatory Units in Prokaryotes 3. Mechanism of lac Operon 4. Mechanism of trp Operon 5. Regulation of Eukaryotic Gene Expression 6. Control of Gene Expression
1. Introduction
Gene expression: Gene expression can be defined as the combination of three processes in which a gene is transcribed into mRNA, the processing of that mRNA, and the course of its translation into protein(in case of protein-encoding genes). In multicellular organisms, all the cells contain the same DNA as genetic material since they all have originated from a diploid zygote. Still, there are a variety of cells like the nerve cell to an epithelial cell that exhibit a prominent difference in the external morphology, also in their functions and other characteristics. In spite of the fact that every cell contains the same DNA, these variations occur as a result of the differences in their respective gene expressions. Control: Gene activity is controlled at the level of transcription where the initial conversion of a gene into an mRNA is started. The majority of the control of gene expression is being done through the interaction between the proteins which bind to the specific DNA sequences and their respective DNA-binding sites. Environmental changes can also induce signals to a cell that can alter its communication which in turn result in changes in the gene expressions.
Regulation: Prokaryotic genomes have their regulatory sites in operons which possess specific DNAbinding proteins. Eukaryotes possess a more complex genome and hence the regulation of gene expression takes place through chromatin interactions rather then specific DNAbinding proteins. Regulation of genes is widely studied in the bacterium E.coli.
2. Operons: Regulatory Units in Prokaryotes
In a living cell of an organism, the genes are expressed to perform a particular function or a collection of functions to achieve an important cellular activity. If we take the bacterium E.coli as an example, it normally uses the simple monosaccharide glucose as its source to derive energy. In case, certain changes in the environment of the cell lead to low availability of glucose and an increase in the availability of lactose, the cell attains the ability to control over its expression of genes. The presence of lactose in the environment induces them to synthesize the enzyme called as beta–galactosidase which is capable of catalyzing the hydrolysis of the disaccharide, lactose into glucose and galactose. Later, this glucose can be used by the bacteria as the source of energy. Along with this beta –galactosidase, two more proteins called as galactoside permease and thiogalactoside transacetylase are also synthesized which assist beta –galactosidase to complete is function effectively. Hence if the environment of the bacteria do not have lactose around it, the synthesis of the enzyme beta –galactosidase is not required. In short, any changes in the metabolism, physiology, or environmental conditions contribute to the control in regulating the expression of genes. Operon: Thus, a collection of enzymes are responsible for the necessary adaptation for the changes happened in the environment of the cell. This synchronized part of gene expression is called as ‘operon’. Activators and Repressors: In prokaryotes, the control of gene expression lies at the initial stage of transcription itself. In a unit of transcription the RNA polymerase recognizes the start sites for initiation of transcription. This activity of RNA polymerase at a given promoter is in turn regulated by its interaction with accessory proteins. These regulatory proteins are called as ‘activators’ if they initiate the process and as ‘repressors’ if they inhibit the process.
Operators: The accessibility of promoter regions of prokaryotic DNA is in many cases regulated by the interaction of proteins with sequences termed operators. The operated region is adjacent to the promoter elements in most operons and in most cases the sequences of the operator binds a repressor protein. Each operon has its specific ‘operator’ and specific ‘repressor’. For example, lac operator is present only in the lac operon and it interacts specifically with lac repressor only.
3. Mechanism of lac Operon
Francois Jacob and Jacques Monod were awarded the Nobel Prize for their proposal of the operon model. Their work described the control of enzymes that are produced as a response to the presence of the sugar lactose in the environment of E. coli cell. Production of the monosaccharides glucose and galactose from the disaccharide sugar molecule of lactose is catalyzed by the enzyme ß-galactosidase.
ß-galactosidase Lactose ⎯⎯ ⎯ ⎯ → Galactose + Glu cos e ⎯
Along with ß-galactosidase, two more proteins play a parallel role in this pathway of lactose metabolism happening in the E. coli cell. These proteins serve effectively as enzymes namely galactoside permease and thiogalactoside transacetylase which makes the work of ß-galactosidase complete. ß-galactosidase - converts lactose into glucose and galactose Galactoside permease - transports lactose across the bacterial cell membrane Galactoside transacetylase – not absolutely required for lactose metabolism. It seems to carry on the detoxification of the compounds which can be transported by the permease. All of the genes involved in controlling this pathway are located next to each other on the E. coli chromosome and together they constitute an operon. Lac operon functions as a cluster of structural genes that are expressed as a group along with their associated promoter and operator regions.
Genetic structure of the lac Operon: I P O III Z | Y | A | _________________________________________________________ Controlling III Region lac Operon Gene I P O lac Z lac Y lac A Structural genes
Function of Gene Gene for repressor protein Promoter Operator Gene for ß-galactosidase Gene for ß-galactoside permease Gene for ß-galactoside transacetylase
• • •
When lactose is absent in the cell environment, the repressor protein binds to the operator and prevents the read through of RNA polymerase into the three structural genes. With lactose present in the cell, lactose binds to the repressor. This binding causes a structural change in the repressor and it loses its affinity for the operator. RNA polymerase can then binds to the promoter and transcribe the structural genes. Lactose acts as an effector molecule in this system.
Effector molecule: A molecule which interacts with the repressor and affects the affinity of the repressor for the operator is termed as the ‘effector’ molecule. Various mutations will have an effect on lac operon gene expression as follows: Mutant gene IO
-
lac
Mutant Phenotype constitutive expression since the operator is never closed constitutive expression due to the inability of the repressor protein to bind RNA polymerase cannot bind which results in non expression of the operon Glucose or galactose cannot be produced from lactose Absence of induction since lactose will not be taken into the cell
Plac Zlac Y
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Catabolite Repression: Glucose is the prime source of carbohydrate for E. coli while lactose is not the preferred one. Hence if both lactose and glucose are present, the cell will use all of the glucose before the lac operon is turned on. Such a type of control is termed as ‘catabolite repression’. A second level for control of gene expression exists in order to prevent lactose metabolism. The promoter of the lac operon has two binding sites. First site is the place where RNA polymerase binds. Second location is the binding site for a complex between the catabolite activator protein (CAP) and cyclic AMP (cAMP). For transcription of the lac operon to happen, the binding of the CAP-cAMP complex to the promoter site is essential.
Importance of CAP-cAMP complex: The presence of this complex is closely related with the presence of glucose in the cell. When the concentration of glucose is high, the amount of cAMP will start going low. As the cAMP decreases, obviously the amount of complex decreases. Decrease in the complex inactivates the promoter, as there is no binding on the promoter site. As a result, the lac operon is turned off. Since the CAP-cAMP complex is needed for transcription, it exerts a positive control over the expression of the lac operon. Lac operon in absence of lactose: • If lactose is absent, the repressor gene produces repressor, which binds to the operator. This binding blocks the action of RNA polymerase, and hence transcription does not take place.
Lac operon in presence of lactose: • • • If lactose is present, the repressor gene produces repressor, which has a site for binding with allolactose. The allolactose/repressor compound is incompetent to bind with the operator. So, the RNA polymerase is uninhibited and transcription proceeds. Once the concentration of lactose decreases, the repressor-allolactose complex decreases and transcription is again inhibited.
Inducible operon: The lac operon is ‘off’ in normal conditions, but when a molecule called an inducer is present, the operon turns on. Hence lac operon serves as an example of an inducible operon.
Repressible operon: The trp operon is ‘on’ in normal conditions, but when a molecule called a repressor is present, the operon turns off. Hence trp operon serves as an example of a repressor operon.
4. Mechanism of trp Operon
Biosynthesis of tryptophan in the cell from its initial precursor chorismic acid is controlled by the trp operon of E. coli. Trp operon contains genes which encode for five proteins which are essential to produce three enzymes.
Anthranilate synthetase: The products of the E and D genes present in trp operon combine together to form a multimeric protein. This protein is comprised of two copies of each protein to form the enzyme anthranilate synthetase. This enzyme catalyzes the first two reactions in the tryptophan pathway.
Indole glycerolphosphate synthetase: This enzyme is responsible for catalyzing the next two steps in the tryptophan pathway. Indole glycerolphosphate synthetase is the product of the C gene.
Tryptophan synthetase