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 IOPlac Zlac 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
lac Y-
Absence of induction since lactose will not be taken into the cell
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: The final step in the pathway is the reaction involving the production of tryptophan from indole-glycerol phosphate and serine. This single step is catalyzed by this enzyme tryptophan synthetase, which is a multimer of two proteins that are the product of the B and A genes. Similar to all operons, the trp operon consists of the repressor, promoter, operator and the structural genes. Dissimilarities with lac operon: Unlike the lac operon, the gene for the repressor is not adjacent to the promoter in trp operon, but slightly is located in another part of the E. coli genome. Also, the operator resides entirely within the promoter.
Genetic structure of trp Operon: P/O | L III E | D | C | B | A | _____________________________________________________ Controlling Region III Structural genes
Trp Gene P/O trp L trp E trp D trp C trp B trp A
Operon
Function of gene Promoter; operator sequence is found in the promoter Leader sequence; attenuator (A) sequence is found in the leader Gene encoding the anthranilate synthetase protein subunit Gene encoding the anthranilate synthetase protein subunit Gene encoding the glycerolphosphate synthetase enzyme Gene encoding the tryptophan synthetase protein subunit Gene encoding the tryptophan synthetase protein subunit
Attenuation of the trp Operon: The leader sequence (L) is an important element of the trp operon which is in immediately 5' of the trpE gene. This sequence is about 160 bp is size and controls the expression of the operon through a process called ‘attenuation’. Leader Sequence: This leader sequence has four domains (1-4). Domain 3 (nucleotides 108-121) of the mRNA can base pair with either domain 2 (nucleotides 74-94) or domain 4 (nucleotides 126-134). When tryptophan level is very high in the cell, domain 3 of the leader sequence forms a pair with domain 4. This results in a stem and loop structure on the mRNA and transcription is stopped. When tryptophan level is low in the cell, domain 3 of the leader sequence pairs with domain 2. Hence there is no formation of the stem and loop structure and transcription continues through the operon. Thereby, all of the enzymes required for tryptophan biosynthesis are produced. If domain 4 in the leader sequence is deleted, the stem and loop structure can not form and transcription of the remainder of the operon will occur even in the presence of tryptophan. Since its presence is required to reduce (attenuate) mRNA transcription in the case of high levels of tryptophan, domain 4 is called the attenuator. Domain 1 is also an important constituent of the attenuation process. The section of the leader sequence encodes a 14 amino acid peptide which consists of two tryptophan residues. Transcription in trp Operon under High Tryptophan Levels: When the cellular levels of tryptophan are high, the levels of the tryptophan tRNA are also high. Immediately after transcription process, the mRNA moves quickly through the ribosome complex and the small peptide is translated. This translation is rapid because of the high levels of tryptophan tRNA. Due to this rapid translation, domain 2 becomes associated with the ribosome complex. Then domain 3 binds with domain 4, and transcription is attenuated by the stem and loop formation. Transcription in trp Operon under Low Tryptophan Levels: When the cellular levels of tryptophan are low, the translation of the short peptide on domain 1 is slow. Domain 2 cannot become associated with the ribosome because of the slow translation. Instead, domain 2 associates with domain 3. This structure permits the continued transcription of the operon. Then the trpE-A genes are translated, and the biosynthesis of tryptophan occurs.
Comparison between inducible and repressible systems: In inducible systems, the binding of the effector molecule to the repressor greatly reduces the affinity of the repressor for the operator. The repressor molecule is released and transcription proceeds. The lac operon is an example of such an inducible system. In repressible systems, the binding of effector molecule to the repressor greatly increases the affinity of repressor for the operator. The repressor molecule binds and stops transcription. The trp operon is a repressible system. In trp operon, the addition of tryptophan (the effector molecule) to the E. coli environment shuts off the system since the repressor binds at the operator. The most important difference between repressible and inducible systems is the result that occurs when the effector molecule binds to the repressor. Inducible system: The effector molecule interacts with the repressor protein such that it can not bind to the operator. Repressible system: The effector molecule interacts with the repressor protein such that it can bind to the operator.
5. Regulation of Eukaryotic Gene Expression
Gene Regulation in Eukaryotes Recent researches reveal that a human cell (a eukaryotic cell), contains approximately 35,000 genes. Among these 35,000 genes, some of them are expressed in all cells at all times. Such genes are specially termed as ‘housekeeping genes’ which carry out the routine metabolic functions like respiration common to all cells. Some genes are expressed as a cell enters a particular unique pathway. Some genes are expressed all the time in only those cells that have differentiated in a choosy way. For example, a plasma cell expresses continuously the gene for the antibody it synthesizes. Some are expressed only when the conditions around and in the cell change. For example, the arrival of a hormone may turn on (or off) certain genes in that cell. Several methods are used by eukaryotes to have a control over gene expression: Transcription Control: This is the most common type of genetic regulation which involves the turning on and off of mRNA formation.
Post-Transcriptional Control: Regulation of the processing of a pre-mRNA into a mature mRNA Translational Control: This involves the regulation of the rate of Initiation. Post-Translational Control: Regulation of the modification of an undeveloped or inactive protein to form an active protein Transcriptional Control Transcription start site is the place where a molecule of RNA polymerase II (pol II) binds which denotes the location where transcription of the gene into mRNA begins. Pol II is a complex of ten different proteins. Basal promoter: The basal promoter contains a sequence of 7 bases (TATAAAA) called the TATA box which is similar to the Pribnow box found in prokaryotes. It can be bound by Transcription Factor IID which is a complex of ten different proteins including TATA-binding protein. TATA-binding protein (TBP): This peculiar protein recognizes and binds to the basal promoter or TATA box. The basal or core promoter is found in almost all protein-encoding genes. Upstream promoter: The upstream promoter is the one whose structure and associated binding factors differ from gene to gene. Each specific gene will possess its own specific promoter. Binding of transcription factors to each other probably makes the DNA of the promoter into a loop. Many different genes and many different types of cells share the same transcription factors, not only those that bind at the basal promoter but even some of those that bind upstream. The unique combination of promoter sites and the transcription factors that are selected decides what turns on a particular gene in a particular cell.
Binding of proteins to DNA: Protein interactions are important for the function of transcription factors. The modular structure of transcription factors is in such a way that one part of the protein is responsible for DNA binding, another for dimer formation, and another for interacting with the basal transcription machinery. Formation of dimers adds an extra element of complexity and versatility. Mixing and matching of proteins into different heterodimers and homodimers results in the possibility of forming three distinct complexes from two proteins. Even DNA: protein: protein interactions are diverse in nature; various common structures are present as follows: Helix-turn-helix (homeodomain): Three different planes of the helix are established and bind to the grooves of the DNA. Zinc fingers: Cystine and histidine residues bind to a Zn2+ ion, looping the amino acid into a finger-like chain. This finger-like chain will rest in the grooves of DNA. Leucine zipper: Dimers result from leucine residues at every other turn of the a-helix. When a leucine zipper is formed from the a-helical regions, the regions beyond the zipper outline a Y-shaped region that grips the DNA firmly in a scissors-like configuration.
Enhancers: Some of the transcription factors bind to regions of DNA that are located thousands of base pairs away from the gene they control. Binding increases the rate of transcription of the gene. Enhancers can be located upstream, downstream, or even within the gene they control. One of the possibilities is that enhancer-binding proteins, in addition to their DNA-binding site, have sites that bind to transcription factors ("TF") accumulated at the promoter of the gene. This would draw the DNA into a loop like structure. The specific characteristics of enhancers are (i) Enhancers are capable of work even if their normal 5' to 3' orientation is flipped. (ii) Enhancers can be able to function even if they are moved to a new location. Silencers: These are the regulatory sequences with similar characteristics to that of enhancers like located thousands of base pairs away from the gene they control. In working, they perform the opposite effect. When transcription factors bind to them, expression of the gene they control is repressed. Insulators: Enhancers can turn on promoters of genes located thousands of base pairs away from their gene they control. An insulator prevents an enhancer from incorrectly binding to and activating the promoter of some other gene in the same area of the chromosome. Insulators are normally stretches of DNA located between the enhancer(s) and promoter or silencer(s) and promoter of adjacent genes or clusters of adjacent genes. They serve to prevent a gene from being influenced by the activation (or repression) of its neighbor genes. In humans, a phenomenon called as ‘imprinting’ occurs. In this case, only the allele for insulin-like growth factor 2 (Igf2) inherited from one's father is active and that inherited from the mother is inactive. The fact behind this is the mother's allele has an insulator between the Igf2 promoter and enhancer. It is present in the father's allele too, but in his case, the insulator has been methylated. CTCF can no longer bind to the insulator. Hence the enhancer is now free to turn on the father's Igf2 promoter.
6. Control of Gene Expression
Genetic Control in Prokaryotes Prokaryotes are simple, primitive, single celled organisms, and have simpler systems. Genes are grouped together based on like functions into separate functional units called operons. Operon could be operated by a single on/off switch for the genes’ expressions and repression. Prokaryotes exhibit two levels of metabolic control: By varying the numbers of specific enzymes made. This is accomplished by the regulation of gene expression. It is slow, but can have a remarkable effect on metabolic activity. By regulating enzymatic pathways through feedback inhibition, allosteric control etc. It is rapid and can be adjusted, but if the enzyme system does not have this level of control, then it will be ineffective. Genetic Control in Eukaryotes Genetic Control in case of eukaryotes is much more complicated. In humans, genetic totipotency exists. Every cell except the gametes (sex cells) possesses the same DNA, containing the same information. Usually, every gene has more than one gene regulator and all of which must be on for the gene to function effectively.
Points to Remember:
Gene expression is a combined phenomenon in which three distinct processes ‘transcription of a gene into mRNA’, ‘processing of that mRNA’, and ‘its translation into protein’ happens in a rhythm with one another. Control of gene expression enables the cell to respond and adapt to the changes in the environment. Regulation of prokaryotic genomes happens through their regulatory sites in operons which have specific DNA-binding proteins. Operon is a cluster of genes comprising a single transcription unit which will synthesize the essential mRNA according to the alterations in the surroundings of a cell. Regulatory proteins are called as ‘activators’ if they initiate the process and as ‘repressors’ if they inhibit the process, in case a gene expression.
Operators are the sequences in the prokaryotic DNA where the accessibility of promoter regions is regulated by the interaction of proteins. A molecule which interacts with the repressor and affects the affinity of the repressor for the operator is termed as the ‘effector’ molecule. The lac operon serves as an inducible operon (i.e) is ‘off’ in normal conditions, but when an inducer molecule is present, the operon turns on. The trp operon serves as a repressor operon (i.e) is ‘on’ in normal conditions, but when a repressor molecule is present, the operon turns off. Genetic structure of the lac Operon comprises a promoter, operator, and three structural genes lac Z, lac Y and lac A which encodes for the enzymes beta – galactosidase, galactoside permease and thiogalactoside transacetylase.
Genetic structure of trp Operon comprises a promoter (operator sequence included), a leader sequence for attenuation and five structural genes trp E, trp D, trp C, trp B, and trp A which encodes for the enzymes Anthranilate synthetase, Indole glycerolphosphate synthetase and Tryptophan synthetase.
Basal promoter is a sequence of 7 bases (TATAAAA) called the TATA box can be bound by Transcription Factor IID which is a complex of ten different proteins including TATA-binding protein.
TATA-binding protein (TBP) is the specific protein which recognizes and binds to the basal promoter or TATA box. Upstream promoter is unique for every gene where the sequence and associated binding factors differ from gene to gene. Protein interactions lead to formation of different heterodimers and homodimers. The common structures found in DNA:protein interactions are Helix-turn-helix, Zinc fingers, and Leucine zipper. Enhancers are the regulatory sequences that are located thousands of base pairs away from the gene they control and increase the rate of transcription of the gene when transcription factors bind to them.
Silencers are the regulatory sequences similar to enhancers located thousands of base pairs away from the gene they control and when transcription factors bind to them, expression of the gene they control is repressed.
Insulators prevent an enhancer from incorrectly binding to and activating the promoter of some other gene in the same area of the chromosome.
Prokaryotes exhibit two levels of metabolic control by varying the numbers of specific enzymes made and by regulating enzymatic pathways through feedback inhibition, allosteric control etc.
In eukaryotes, every gene has more than one gene regulator and all of which must be on for the gene to function effectively.