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The study of human anatomy and physiology is one of the crucial Biology Topics for medical professionals and researchers.
Regulation of Gene Expression: Operons, Epigenetics, and Transcription Factors
Any living organism contains a large number of genes to maintain its life on earth and it may transmit all its genetic properties to its progeny which ensures the stability of species and continuity of life on the earth. The genetic structure of the living being not only determines the organization of body structure, but it also controls the physiological functioning and behaviour of an organism in the living world. Though an organism possesses a large number of genes, to maintain life all its genes need not function at any particular time. Keeping aside the unicellular forms and some lower eukaryotes, the life of higher organisms is initiated from the zygotic stage which is formed by the union of male and female gametes. Gradually the process of differentiation leads to the establishment of the body in total in the shape of a miniature form of the adult organism. The organism being formed starts functioning independently in its own way.
Ultimately an organism (especially higher forms) grows old, senescence comes when death becomes its final fate. From the very beginning of life, i.e., even when the organism is at the zygotic stage, it becomes complete by its genetic makeup. Normally no further addition to this genetic pool may occur in the life period of the organism, though by default some alteration in this makeup may be possible by the environmental influence. It is also true that the genes that show activity in the initial stage of life, get shut down at the later stage of life and new type of genes may start functioning then. It has been found that in bacteria (for e.g., Escherichia coli) about 1000 of about 4000 genes function at a time. When conditions are altered, some genes are turned off and others are switched on. Simply a change in growth condition such as a shift in temperature may lead to altered expression of 50 to 100 genes.
However, the functioning of all the genes together with their expression is not necessary for an organism and it is consistent with the economic use of resources. Nature in its own way, has arranged the system of regulation of gene expression, so as to promote expression of a gene only in necessity. Single-celled organisms regulate their genes in response to their environment as well as to the internal state of the cell. In multicellular organisms, the system of cell communication and developmental progression is also important for the regulation of gene expression. There are a number of stages at which gene expression may be regulated and these steps are
- Transcription: At this stage, a gene produces an RNA as the primary product of the gene in the way of gene expression.
- Post-Transcriptional Processing: At this stage primary transcript undergoes some modification to form functional RNA.
- Stability of RNA: This is true for mRNA molecules produced in the cell. Short-lasting mRNA molecules can not give a protein unless it is stable.
- Translation: When mRNA gives a polypeptide chain, the regulatory mechanism ensures the formation of a polypeptide chain in the way of the production of protein.
- Post Translational Processing: when a polypeptide chain after its formation undergoes modification so that the processed polypeptide chain may be used for the formation of a protein.
- Control of the activity of an enzyme needed for gene expression.
- Degradation of Protein: If the gene expression is mediated through the production of a protein, then the protein after its production is degraded and it cannot help in the expression of the gene. Therefore, protein degradation may be one way in regulating gene expression.
Though all the above mechanisms may be involved in the regulation of gene expression, this regulation is most frequently involved at the level of transcription rather than at the later stages or at the level of translation. Nature has developed strategies to optimize regulation so that it may be less wasteful. This regulation may again be +ve or -ve. In +ve regulation, a gene is incapable of expression unless it receives a +ve signal for functioning. Whereas in -ve regulation a gene is inherently active but is prevented from expressing itself unless some inhibitory influence is removed.
However, some genes in the living cell never comes under designed regulation and the genes are constitutive by nature which means that these genes are always active. These genes are rather autoregulated. These are known as housekeeping genes. Ribosomal RNA-producing genes and the genes-producing proteins involved in protein synthesis are the housekeeping genes and they express themselves constitutively. A generalized model indicating regulation at different levels for expression may be shown in the following manner.
Regulation of Gene Expression in Prokaryotes
In the living world, prokaryotes represent the simpler form of organisms containing single-celled bodies with naked nuclei with circular DNA molecules as their genome. Within this circular DNA there remain thousands of genes. Having a single-celled body, the prokaryotic forms are easily affected by changes in environmental conditions. Therefore, they need to shift from one functional state to another in order to survive in the fluctuating environmental scenario. Hence, unless there is a well-coordinated regulation of gene function, the bacterial organism must have to face jeopardy in adapting to the changed environmental condition.
Setting aside the housekeeping genes, genes of the other category belong to regulatory mechanisms for their expression in the organisms. In the most studied prokaryotic species, Escherichia coli, there are two principal mechanisms in the regulation of gene expression, namely the inducible system and the repressible system. Francis Jacob and Jacques Monod in 1961 first noticed an inducible system of gene expression in E. coli. In this system in the presence of the metabolite, the enzymes for its utilization may be synthesized. They found that E. coli normally utilizes glucose as an energy source in the environment, but in the absence of glucose, if lactose is present in the environment, the bacteria utilizes lactose with the help of two enzymes, β-galactosidase and lac permease from two functional units of their genome.
They noted that lactose induced the synthesis of these two enzymes together from the genome and when lactose was withdrawn from the medium, synthesis of the enzymes was blocked. They advocated the presence of some regulatory segments on the genome for switching on and off the modes of activity of the genes and they proposed an operon model to explain the process of gene regulation in E. coli. For lactose utilization, the system of regulation of gene expression has been designated as an inducible system. The genes whose function is regulated in this manner has been categorized as inducible genes and the enzymes produced by them are known as inducible enzymes. Subsequently, similar other functional units have also been discovered in E. coli. Gal operon and Arabinose operon are two other examples of inducible systems.
Following this discovery an opposite mode of gene functioning was also discovered in a prokaryotic organism, E. coli in which the system is associated with metabolite synthesis, and in a converse fashion the presence of metabolite stops the synthesis of enzymes from the genes, required for the synthesis of the metabolite. Unlike the inducible system, the metabolite acts as a repressor. Hence, this system of regulation of gene expression is called a repressible system. Tryptophan operon and histidine operon of E. coli belong to this system of regulation of gene expression.
Operon Model
As proposed by Jacob and Monod gene expression and its regulation may be explained by the operon model. According to this model, gene functions and expresses as a unit. A group of genes act as a unit and expression of a character occurs under the conjoint action of these group of genes. In this group, there are operator, promoter, and structural genes. The structural genes include the genetic elements that come into expression and the promoter and operator act as controlling elements of the structural genes. In prokaryotic organisms, gene expression occurs in a well-coordinated fashion with the involvement of operator, promoter, and structural genes. They are present in the genome in a contiguous fashion. Hence, an operon comprises a contiguous functional unit of a genome consisting of operator, promoter, and structural genes.
The modes of functioning of different operons may vary and normally one operon remains under the control of a regulator gene. On the basis of the methods of functioning, the operon may be categorized into two types namely inducible operon and repressible operon. The inducible operon denotes a system where the operon is switched on under the influence of an inducer which is normally the metabolite. On the other hand, the repressible operon denotes a system where the operon remains switched off in the presence of the metabolite. Besides this system is related to an anabolic process, while the inducible system is concerned with a catabolic process. Sometimes an operon may be constitutive by nature when it does not have any regulation to stop the functioning of the operon.
Lactose Operon of E. coli
The genetic unit that is concerned with lactose utilization in E. coli is known as the lac operon. Lactose is a disaccharide and during its utilization, it is broken down into glucose and galactose by enzymatic action. The enzyme involved in this process is β-galactosidase which is produced by a gene of this operon. Besides β-galactosidase there are also other two enzymes, namely lac-permease and trans acetylase which are also synthesized along with β-galactosidase, by the genes of the same operon.
However, only Lac-permease is needed for lactose utilization and this promotes entry of lactose molecules into the bacterial cell from the surrounding medium. It is to be mentioned that Lac-permease is a membrane protein of the bacterial cell. The expression of the genes in the operon is regulated in a unique fashion with the involvement of the controlling elements and a regulator gene and synthesis of the enzymes for lactose utilization occurs when lactose remains present in the medium. In the absence of lactose, the operon does not function and enzymes for lactose utilization cannot be synthesized. Lactose being present in the medium induces the functioning of the operon, it is called the inducer, and as already mentioned lac operon is an example of the inducible operon.
According to the definition, an operon is a functional unit comprising two controlling elements a promoter and operator, and several structural genes. A look at the organization of lac-operon indicates the component parts of this element and their orientation in the genome. The structural organization of this operon indicates the following features.
- Position: In the E. coli genome this operon is located between pro B and tsx loci at the 9′ and 11′ positions of the genome.
- Dimension: The DNA segment comprising lac-operon contains about 6000 bp.
- Component Elements: There are two component parts of the operon namely structural genes and the controlling elements.
(i) Structural Genes: There are three structural genes in this operon and they are designated as lac z, lac y, and lac a. The arrangement of the structural genes from left to right is z-y-a, indicating that lac z is the initial genetic element and lac remains at the terminal end of the operon. Three structural genes produce three enzymes namely β- galactosidase, lac permease, and transacetylase. The structural gene lac z is related to the production of b-galactosidase, lac y is related to the production of lac-permease, and lac a is responsible for the synthesis of transacetylase. These three genetic elements have their individual dimensions. Lac z comprises 3510 bp region in the genome, lac y is composed of 780 bp region, and lac a is formed of 825 bp region in the genome. All these genes are arranged in a contiguous fashion in the genome.
(ii) Controlling Elements: The operator and promoter are the two controlling elements of this operon. Location-wise operator site is present before the structural gene lac z. The operator site is preceded by the promoter site. They may be designated as lac o and lac p. Dimension-wise lac o and lac p are constituted of 35 bp and 80 bp respectively.
The Operator (Lac o)
Jacob and Monod identified one operator site in the lac operon, but subsequently, two other operator sites were discovered. They are designated as O1, O2, and O3. The operator O1 is located between lac z and lac p and lac O2 is located downstream from lac O1 within the structural gene lac z. Whereas, lac O3 is present upstream of the lac promoter. The repressor protein i.e., a product from the regulator gene called lac I in this case, binds with the operators for repressing synthesis from the structural genes. Each active repressor is a tetramer and each remains in two dimeric units. The function of the operator is related to the repression of the operon. The repressor protein produced from the regulator gene may bind with the double-stranded DNA at the operator site when RNA polymerase cannot carry out transcription from the structural genes.
The Promoter (Lac p)
The promoter region is the most important controlling element of the operon. Recognition of RNA polymerase and its binding with this region only may promote transcription of the required genetic segments of the operon. The promoter is present upstream of the structural genes and is present before the operator locus. However, the operator and promoter are so arranged that they have a small segment of overlapping zone.
Lac promoter is extended from +1 to -80 sequence in the operon segment of the genome. The region contains two separate components namely the RNA polymerase binding site and CAP (catabolite activator protein) binding site. The CAP binding site is extended from -55 to -80 in the upstream region. Within this, a palindromic sequence is present extending from -60 to -70. This region is very important in relation to transcription from the genes of the operon. The CAP binds with a cyclic AMP and then it may bind with the CAP site of the promoter. The binding of this moiety with the CAP site promotes proper RNA polymerase binding with the promoter.
If the CAP site remains unoccupied by the CAP —cAMP complex, then promoter binding with RNA polymerase will be imperfect, and synthesis from the structural genes will be prevented. The second site is for RNA polymerase binding and this binding is needed for transcription of the structural genes. This region contains two sequences that are important for RNA polymerase interaction. One of these sequences is the -10 sequence or Pribnow box which is found to be present in almost all prokaryotic promoters with consensus base sequence. The other sequence is known as the -35 sequence which is also common in all prokaryotic promoters containing consensus sequence.
The Regulator Gene (Lac I)
The lac operon remains under the regulation of a regulator gene designated as lac I which encodes a protein called repressor measuring 360 amino acids long. The repressor in active form contains 4 polypeptides, the product of the same gene I. In the absence of an inducer (lactose), the repressor binds to the lac operator thereby preventing RNA polymerase from catalyzing transcription of the three structural genes. Therefore, the regulator gene exerts a negative control of the operon functioning by inhibiting synthesis from the structural genes.
The regulator gene is about 1040 bp long and this genic region is present before the promoter of this operon. The regulator gene has its own promoter. The product of the regulator gene is diffusible and functions as an active repressor. In the presence of lactose, the active repressor is inactivated. The repressor protein has two binding sites, one for binding with the DNA at the operator region and the other for binding with lactose molecules. However, lactose molecules cannot bind directly with the repressor, but one isomeric form of lactose called allolactose binds with the specific site of the repressor.
It is to be mentioned here that the repressor protein shows allosteric activity. In the presence of lactose when the protein binds with allolactose at the specific site, its DNA binding site becomes altered conformationally. At this stage, the repressor cannot bind with the operator and therefore, in the presence of lactose, the operon cannot be repressed. It is also noteworthy that allolactose molecules may be produced from lactose molecules in the presence of the enzyme β-galactosidase.
Lac Operon Activity: The functional activity of the lac operon comes through the transcription of the structural genes and ultimately by the production of three enzymes, β-galactosidase, lac permease, and transacetylase as the products from three structural genes lac z, lac y, and lac a. It is noteworthy that three structural genes are transcribed together producing a single mRNA molecule. Such an mRNA is called polycistronic mRNA which then may produce three enzymes separately.
Normally the operon remains in inactive condition when no enzyme from the structural genes may be synthesized. Such a mode of function may be possible due to the activity of the repressor, the protein from the regulator gene I. The repressor protein is active and it may bind to the operator site. This binding blocks RNA polymerase to move towards the structural genes for their transcription. Therefore, the operon is repressed without producing any lactose utilizing enzyme. This is to be kept in mind that such repressing activity is observed in the absence of lactose in the culture medium.
On the other hand, if lactose remains present in the medium, operon activity may be induced. In that case, the repressor protein produced from the regulator gene gets inactivated due to the binding of allolactose with the active repressor. The binding of allolactose with the repressor results in a conformational change of the repressor when its DNA binding site is altered in conformation and it can no longer bind with the operator site. In such a situation no block may be developed at the O site, permitting the RNA polymerase to move along the structural genes. Therefore, in this scenario, the structural genes may be transcribed together forming the polycistronic mRNA. This ultimately leads to the synthesis of the enzymes namely β-galactosidase, lac permease, and transacetylase.
Trp Operon of E. coli
The Tryptophan operon or trp operon of E. coli is related to the expression of genes concerned with tryptophan biosynthesis in the bacterial cell. Unlike lac operon, this operon is related to a metabolic pathway (anabolism) in the cell, when the essential amino acid tryptophan is produced. When the operon is active five essential protein components in the form of enzymes for tryptophan biosynthesis from a precursor called chorismic acid. When sufficient tryptophan molecules are present in the cell, operon activity is repressed inhibiting further synthesis of tryptophan biosynthetic enzymes. Thus operon represents a repressible system of regulation of gene expression in the prokaryotic organism.
That tryptophan operon is concerned with tryptophan biosynthesis in E. coli cells was first discovered by Monod and his colleagues in 1953 and it was described as a repressible system. Subsequently, Monod pointed out that the presence of tryptophan in adequate amounts in the cell may stop the biosynthesis of enzymes for the production of tryptophan in the cell. The operon represents a repressible system of regulation of gene expression in prokaryotic organisms. This exercise by the bacterial cell is energetically advantageous for the prokaryotic cell.
However, a detailed study on the genes involved in the synthesis of tryptophan biosynthetic enzymes and the adjacent regulatory sequence of the trp operon was carried out by Yanofsky and his colleagues during the subsequent period. It has also been revealed that trp operon gene expression is regulated by both repressions of transcription of the structural genes of the operon and attenuation when transcription of the genes may be stopped prematurely. In the presence of sufficient tryptophan in the medium both the process of regulation of gene expression may be operative. The transcription of the operon genes may only occur when the tryptophan level in the medium as well as in the intracellular pool is low.
Structure of trp operon:
Similar to the structure of the operon of E. coli, the trp operon is also composed of several structural genes namely trp E, trp D, trp C, trp B, and trp A. The controlling elements as trp P and trp O are present in this case, but additionally, there is another controlling element known as trp L present prior to the trp E gene. The regulatory trp L region contains an attenuator sequence for control of transcription from the structural genes.
Organization of trp operon:
The trp operon of E. coli is located between the lac operon and histidine operon within its circular chromosome. The location and different component parts of the operon may be shown in the following manner.
Besides the genetic elements as mentioned in the table, there are two other controlling elements t containing 36 base pairs and t’ containing about 250 bp are present after the trp A. trp operon also contains a regulator gene designated as trp R which is present in the genome somewhat away from the pi site of the operon. This gene may produce a protein known to be an inactive repressor. This inactive repressor is also allosteric in nature having two binding sites, one for the double-stranded DNA and the other for binding with metabolite, tryptophan. With the binding of tryptophan molecule to the specific site of the inactive repressor a conformational change occurs in the repressor when it becomes active. The active repressor then can bind to the DNA at the operator site of the trp operon. Because of this tryptophan is called a corepressor and in a repressible operon, the metabolite acts as a corepressor.
Different Genic Elements of Trp Operon and their Functions:
The structural genes in the trp operon are five in number as mentioned before. They are arranged in sequence from left to right as EDCBA. The structural genes are sandwiched between L (the leader sequence) on the left and t (of 36 base pairs) on the right. These five structural genes produce single polycistronic mRNA from which five polypeptides of different lengths are produced by the activity of RNA polymerase. As mentioned in the table the product of the structural genes is polypeptide ε, polypeptide δ, polypeptide Indole phosphate synthetase, polypeptide β, and polypeptide a respectively.
Of the five polypeptides first two contribute to the formation of the enzyme anthranilic acid synthetase. This enzyme converts chorismic acid into anthranilic acid and the second enzyme also converts anthranilic acid into PRA (Phosphorybosyl anthranilate). The third enzyme, indole glycerol phosphate synthesis catalyzes the conversion of PRA into CDRP and then CDRP in InGR The fourth and fifth gene produce, polypeptide β and polypeptide α to form tryptophan synthetase that catalyzes the production of tryptophan from InGP (Indole glycerol phosphate). The production of five polypeptides mediated through the formation of a polycistronic mRNA is regulated in two ways as mentioned before, i.e., repression of transcription attenuation by leader attenuator sequence (La).
Regulation of Gene Expression in Trp Operon
Regulation of expression of trp operon genes though is affected in two ways as mentioned before, but in both the mechanisms transcription of genes is prevented. In case if first mechanism transcription cannot be started at all and in the other method a process of premature termination of transcription occurs. This is known as attenuation control.
(a) Repression of Transcription:
This is the principal method of regulation of gene expression in the trp operon when no synthesis of mRNA from the structural gene is possible. This is effected through the production of repressor protein from the regulator gene of trp operon, trp R, and the metabolic tryptophan. The protein that comes from the regulator gene appears inactive and by binding with the metabolite it becomes an active repressor. The tryptophan molecules thus act as corepressors. The active repressor binds with the operator site of the operon when RNA polymerase binding with the first promoter (p1) may be prevented and thereby this enzyme will be unable to initiate transcription from the structural genes. But in the absence of tryptophan (corepressor), the repressor cannot bind with tryptophan for its activation and therefore, the repressor remains in its inactive form. In such a condition, the inactive repressor fails to bind with the operator region in the operon. As a result, the RNA polymerase binds with the promoter site (p1) and initiates transcription from the structural genes. Thus on the basis of the presence or absence of tryptophan in the culture medium or in the cell, the activity of the operon to produce biosynthetic enzymes may be determined.
(b) Control of Trp Operon Gene Expression by Attenuation:
Regulation of the expression of structural genes may also occur by leader attenuator (La) sequence by a phenomenon called attenuation. Before a discussion on this aspect, the organization and location of the leader attenuator region should be known. The leader attenuator region represents a site of the E. coli genome in between15 trp E gene and the trp O region. This region is marked as trp L and is having a dimension of 162 bp DNA. When transcription occurs the leader (trp L) region is also transcribed forming leader mRNA which remains prior to the transcript from the trp E gene. The 162 nucleotide long leader mRNA sequence contains several (4) complementary sequences that may be marked as 1, 2, 3, and 4. Because of their complementarity region 1 may pair with region 2 and similarly, region 3 may pair with region 4. When such base pairing occurs hairpin loops may be formed.
The hairpin loop formed by Region 3 and Region 4 is known attenuator or terminator. This loop is structurally identical with the terminator loop of prokaryotic gene termination and the terminator loop is also followed by some U residues as found in the natural condition. The loop formed by region 3 and region 4 may terminate transcription of the trp operon genes and therefore, the terminator loop is called attenuator. But when a hair pin loop is formed between Region 2 and Region 3, the 3-4 loop cannot be formed to terminate transcription of the trp operon genes. Hence, 2-3 loop is called gene terminators.
Another feature of the leader mRNA is that Region 2 also carries a complementary sequence with part of Region 3. Therefore, the hairpin loop may also be formed between region 2 and region 3, the 3-4 loop cannot be formed to terminate the transcription of the trp operon genes. Hence, the 2-3 loop is termed an antitermination. Region 1, 2, 3, and 4 span the sequence 60-68, 75-83, 110-121, and 126-134 nucleotides in the leader mRNA. Now the question may arise at what condition the attenuator loop may be formed to promote transcription and at what condition the attenuator is formed. It has been observed that only at low tryptophan concentration in the bacterial cell the terminator or attenuator loop cannot be formed; instead, the anti-terminator is formed to promote transcription from the structural genes.
In the leader mRNA, there is a ribosome binding site at its 5′ end which is followed by one initiation codon and after that, there are thirteen other codons including two UGG codons for tryptophan at the 10th and 11th position. At the end of the leader mRNA, there is a termination codon UGA. Because of this from a leader mRNA a short polypeptide called ‘leader polypeptide’ of 14 amino acids may be formed. It is to be pointed out that the UGG codons in the leader mRNA are so positioned that they are present within Region 1. It is a well-established fact that in the prokaryotic cell transcription and translation are coupled processes and therefore, before the completion of transcription translation may be initiated. Under this condition the leader mRNA part from the trp operon when is released in the cytosol. It may bind with a ribosome and starts synthesizing leader polypeptide.
When tryptophan concentration is low in the cell, during translation of leader polypeptide charged tRNA with tryptophan becomes unavailable to supply tryptophan to the growing polypeptide and therefore, the ribosome gets stalled at region 1 of the leader mRNA. However, by this time transcription along region 3 becomes complete. In this condition region 2 gets a chance to form a hairpin loop with region 3 when there remains no scope for the formation of a 3-4 attenuator loop. Transcription of the region past region 4 may also be continued in the situation and the whole of the structural gene region is transcribed into single polycistronic mRNA.
Conversely, when tryptophan concentration is high in the cellular medium as well as in the intercellular pool, the charged tRNA with tryptophan may be available to supply tryptophan to the growing leader polypeptide from leader mRNA from trp operon. In this situation when leader polypeptide is complete in its formation, the ribosome partly covers region 3 of the leader mRNA when region 3 and region 4 after their transcription by RNA polymerase pair with their complementary sequence forming the attenuator loop. If an attenuator or terminator loop is formed, the RNA polymerase cannot move further and transcription is terminated at this point. The combined effect of repression and attenuation may give about 700-fold efficiency in regulating transcription from the trp operon.
Mutation in Trp Operon
Similar to lac operon the trp operon genes also undergo mutation changing the properties of the operon. The structural genes of this operon are designated as trp E, trp D, trp C, trp B, and trp A. Mutations at these loci are loss of function mutations and these may be designated as E-, D-, C-, B, and A-, trp O gene shows a mutation known as Oc. This mutation converts one operon into a constitutive one when synthesis from the structural genes cannot be regulated. The other controlling element of this operon is trp P, but there are two promoters as P1 and P2. The wild-type form of this gene may be designated as P+. A mutant form of the locus is known as p-. But this mutation is confined to P1 and such a mutation prevents RNA polymerase binding with the promoter and because of this mutation transcription from the structural genes of this operon cannot be promoted. Whereas P2 is a weak promoter that is concerned with the rate of synthesis from the loci C, B, and A. The promoter enhances the rate of synthesis.
Therefore, a mutation at this site promotes a reduced rate of synthesis of indole glycerol phosphate synthetase, tryptophan synthetase B, and tryptophan synthetase A. The regulator gene of the trp operon is known as trp R. When wild type gene is designated as trp R+ and a mutation at this locus is trp R”. The protein produced from this operon mutation cannot bind with corepressor, tryptophan. Hence, in the case of the mutation trp R’, synthesis from the structural genes cannot be regulated by a product from the regulator gene. Another mutation in this locus called trpSR is concerned with the production of a super-repressor and this repressor does not require metabolite, tryptophan for binding with the inactive repressor to make it active. Super-repressor is always active and may bind with the operator site of the trp operon for blocking synthesis from the structural genes.
Comparison between Lac Operon and Trp Operon
The lac operon and trp operon of E. coli are comparable because are opposite in their mode. of functioning. A comparative account on the operons may be shown by the following table.
A comparison between the lac operon and trp operon of E. coli:
| Point of Difference | Lac Operon | Trp Operon |
| Metabolic Relation | Functional activity is related to catabolism | Functional activity is related to anabolism |
| Role of Metabolie | The presence of metabolite induces synthesis from the operon genes | The presence of metabolite represses synthesis from the operon genes |
| Regulator Gene Product | The regulator gene produces an active repressor. | The regulator gene produces an inactive repressor. |
| Catabolic Repression | Catabolite repression is present. | Catabolite repression is absent. |
Regulation of Gene Expression Post-transcriptionally at the level of Translation
The regulation of gene expression in prokaryotes occurs mainly at the level of transcription. However, there are instances when gene expression is regulated at the post-transcriptional level during the process of translation. Since mostly gene expression denotes the formation of some protein, then inhibition of protein formation comes under the regulation of gene expression. In this consideration, different mechanisms associated with such regulation of gene expression could be revealed. Several such mechanisms may be presented in the following manner.
(a) Regulation mediated by unequal efficiency of translation initiation of altered efficiency or ribosome movement or different rate of mRNA degradation:
In prokaryotes usually, transcription, translation, and mRNA degradation are coupled processes, and more than one gene may be transcribed as polycistronic mRNA. Further, though several genes are transcribed together, but gene products vary in amount in the cell. For example, under the regulation of lac operon three genes, namely lac z, lac y, and lac a are transcribed together into one polycistronic mRNA. Translation of mRNA occurs together forming three enzymes P galactosidase, galactoside permease, and transacetylase. It is worth mentioning that these proteins translated in the E. coli cell vary in amount as 3000 molecules of p galactosidase, 1500 molecules of galactoside permease, and 600 molecules of transacetylase. This synthesis of differential amounts of protein enzymes may be possible by post-translational regulation or altered efficiency of ribosome movement along the inter-genic region or by differential rates of mRNA degradation.
(b) Regulation Mediated by Repressor Protein or by Antisense RNA:
Translational regulation though is less important for prokaryotic cells, yet interference with translation initiation by some repressor proteins cannot be ruled out. The repressor protein if binds to the region adjacent to the Shine Dalgarno sequence of mRNA or near the initiation codon, ribosome may fail to initiate the process of polypeptide synthesis. Control of gene expression via some antisense RNA could also be observed in several cases. In E. coli a membrane protein encoded by the omp F gene is important for osmoregulation. When osmolarity is low omp F is produced more compared to the state of high osmolarity. However, this inhibition has been found to be mediated by the product of another gene of the bacteria named mic F. This gene produces RNA (mic RNA) that is complementary to the omp F mRNA.
When mic F RNA binds to the omp F RNA due to their complementarity, the omp F mRNA cannot be translated into a protein, thus inhibiting the expression of omp F gene. In bacterial cells, most of the time omp F gene is transcribed forming omp F RNA which in turn produces membrane protein that functions as a diffusion pore. But in the condition when the osmolarity of the medium is increased the regulatory gene called mic F is activated producing mic RNA which is an mRNA interfering complementary RNA having the capacity to bind with omp F mRNA inhibiting its translation.
(c) Regulation mediated by Riboswitch:
Riboswitch represents a regulatory sequence of mRNA that when bound with some small molecules may induce the formation of secondary structures in the mRNA. The formation of such a secondary structure may affect the ribosome binding site of mRNA and prevent translation initiation. Riboswitches were discovered in 2002 and are found to regulate about 4% of all bacterial genes.
In bacteria, the genes associated with the formation of en-zymes for the purpose of vitamin B12 synthesis were found to be regulated by riboswitch. When co-enzyme B12 (an activated form of vitamin B12) is present in the cell, it binds with the riboswitch when mRNA folds into a secondary structure that prevents ribosome binding causing cessation of translation of mRNA. On the other hand in the absence of co-enzyme B12, the mRNA takes a different configuration allowing the synthesis of enzymes for vitamin B12 formation.
Sometimes riboswitch carries catalytic function as ribozyme and results degradation of mRNA. This is known as RNA-mediated repression which is found in olms glms gene expression of Bacillus subtilis. This gene encodes an enzyme called glutamine fructose 6 phosphate (GlcN6P). In the glm S mRNA, a part of the 75 nucleotide-long regions at the 5’ end acts as a riboswitch. It has been found that when there is enough HlcN6P in the cell, they bind with the ribozyme part of glm S mRNA and promote degradation of the mRNA.
Regulation of Gene Expression at Post Translational Level
The phenomenon of post-translational regulation of gene expression is affected through feedback inhibition or end-product inhibition. It has been found in many living organisms that in the biosynthetic pathway of various substances in the body when the amount of the metabolite becomes sufficient in the cell, it may bind to the first enzyme causing its inactivation so that no further production of the component becomes possible in the cell. In support of this phenomenon, the biosynthetic pathway of tryptophan in E. coli may be cited. Tryptophan is synthesized in the bacterial cell from a precursor called chorismic acid with the involvement of several enzymes produced from the structural genes of the trp operon.
The first enzyme in this pathway of tryptophan biosynthesis is anthranylate synthetase. When tryptophan is synthesized sufficiently in the cell, it may bind with the enzyme, anthralate synthetase, arresting the catalytic activity of the enzyme completely. It has been found that the enzyme has a site for binding with the end product in addition to its substrate binding site. When the end product binds with the enzyme to its specific site, it exhibits a conformational change of the enzyme when its substrate binding site is altered. As a result, the enzyme fails to bind with the substrate for initiation of the catalytic process. The proteins that may show such conformational change are called allosteric proteins. The inhibition of enzyme action due to such conformational change is sometimes called allosteric inhibition.
Regulation of Gene Expression in Eukaryotes
Similar to the prokaryotic organisms the eukaryotes also exhibit regulation of expression of their genes. However, the mechanisms involved in the process are more complicated than those as found in the prokaryotes. Such complicacy might have arisen due to some characteristic features of the eukaryotes, for example
- The genetic materials are membrane-bound in eukaryotes while in prokaryotes they are exposed in the cytosol.
- The extranuclear region is highly compartmentalized in eukaryotes and protein formation solely occurs in cytoplasm.
- Transcription and translation are not coupled in eukaryotes and eukaryotic genes are transcribed monocistronic ally.
- Transcription in eukaryotes occurs primarily in the nucleus and transcripts in most cases are processed within the nucleus.
- Multicellularity with the division of labour is another advanced feature in most of the eukaryotes.
Because of all these special features, eukaryotes have developed some unique complexities in regulating their gene expression. Besides their mode of leading life on the earth, such as early embryonic life, young and charting growth phase, and finally, period of senescence, tissue grade organization with external and internal body environment, the response of the body through a well-coordinated system of communication system impart additional complexity to the mechanism of regulation of gene expression. DNA is the genetic material in the body and they are confined to chromatin being complexed with chromatin at a highly compact orientation and the genomic size is also enormously large compared to that in any prokaryotic organism.
In this respect, the regulatory mechanism also has to face many hurdles to express the genes of eukaryotes organized in a complex fashion. In spite of all these phenomena, the genes are expressed by way of two processes namely transcription when RNA is formed and translation when protein is synthesized, as a prokaryote also exhibits in its gene expression. Associated with these two main aspects of gene expression, regulation may be observed at the level of transcription, at the level of post-transcription in RNA processing, at the level of translation during polypeptide formation, and also at the level of post-translational phase during protein formation. The steps of gene regulation and associated mechanisms have been discussed before, in this chapter.
Levels of Control of Gene Expression
In the living body, there are some special categories of genes and they are called housekeeping genes. These genes are present in all the body cells in active condition and genes fulfill the daily need of the organisms. These special categories of genes are expressed in the cells based on the need on a timely basis and control in the expression of these genes does not tally with the conventional regulatory phenomenon following certain principles. The other genes that are also present in all the body cells face regulation of expression by different methods. Further, these genes may not be equally expressed in all the body cells. For example, insulin is one protein hormone that is only synthesized in the cells of Islets of Langerhans in the pancreas of vertebrates, not in the cells of the other organs. Similarly, ptyalin is an enzyme synthesized in the cells of salivary glands of our mouth, not in other organs though all the cells of different organs contain a similar set of enzymes. Based on these facts gene regulation in the body cells of higher organisms as well as eukaryotes may come under short-term regulation (as for housekeeping genes) and long-term regulation (as for other cellular genes). The genes involved in the development and differentiation of the organisms come under long-term regulation. Therefore, eukaryotic genes may be regulated at six levels as-
- Transcription
- RNA processing
- mRNA transport
- mRNA translation
- mRNA degradation
- Protein degradation