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In this article, we will discuss about the regulation of gene expression in prokaryotes.
The Operon Model:
Francois Jacob and Jacques Monod (1961), two French geneticists, discovered while studying bacteria, that the enzymes synthesized by them can be placed in two categories:
(i) Those that are synthesized all the time and occur in relatively constant concentrations, such as enzymes of glycolysis, and
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(ii) Those that are synthesized only after a specific stimulation. The first type was named constitutively synthesized and the latter the inducible enzymes.
Analyzing a variety of E. coli that were defective for the induction of the lactose utilizing enzymes, Jacob and Monod hit upon the possible molecular mechanism that controls the repression and de-repression of a set of genes. The E. coli requires a set of three genes to be able to metabolize lactose. When a little lactose is added to a glucose-free growth medium, it is seen that these three lactose utilizing genes (lac genes) named lac z, lac y and lac a are synthesized simultaneously.
The product of lac z is the enzyme β-gaIactosidase that catalyzes the conversion of lactose into galactose and glucose. These genes are note expressed in the absence of lactose. Jacob and Monod (1961) proposed the operon model to explain the genetic basis of induction and repression of lac genes in prokaryotes. They were awarded Nobel Prize for this work in 1965.
The Operon:
1. Operons are segments of genetic material (DNA) that function as regulated unit that can be switched on or off.
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2. An operon consists of minimum four types of genes: regulator, operator, promoter and structural (Fig. 8.4.A).
3. Regulator gene is a gene which forms a biochemical for suppressing the activity of operator gene.
4. Operator gene is a gene which receives the product of regulator gene. It allows the functioning of the operon when it is not covered by the biochemical produced by regulator gene.
5. The functioning of operon is stopped when operator gene is covered.
6. Promoter gene is the gene which provides point of attachment to RNA polymerase required for transcription of structural genes.
7. Structural genes are genes which transcribe mRNA for polypeptide synthesis.
8. An operon may have one or more structural genes, e.g., 3 in lac operon, 5 in tryptophan operon, 9 in histidine operon.
9. The polypeptides may become component of structural proteins, enzymes, transport proteins, hormones, antibodies, etc. Some structural genes also form non-coding RNAs.
10. The mechanism of regulation of protein synthesis utilizing operon model can be illustrated using two examples (lac & tryptophan) in bacteria.”
Inducible Operon System (Induction of Operon):
1. Inducible operon system is (a) regulated operon system in which the structural genes remain switched off unless and until an inducer is present in the medium. (Fig. 8.4B)
2. It occurs in catabolic pathways.
3. Lac operon of Escherichia coli is an inducible operon system which was discovered by Jacob and Monod (1961).
4. Lac operon of Escherichia coli has three structural genes, z, y, and a.
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5. In the induced operon the structural genes transcribe a polycistronic mRNA which produces three enzymes. These are β-galactosidase, galactoside permease and galactoside acetylase.
6. β-galactoside brings about hydrolysis of lactose or galactoside to form glucose and galactose.
7. Galactoside permease is required for entry of lactose or galactoside into the bacterium.
8. Galactoside acetylase is a transacetylase which can transfer acetyle group to β-galactoside.
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9. The initiation codon of structural gene z is TAG (corresponding to AUG of mRNA) and is located 10 base pairs away from the end of the operator gene.
10. The substance whose addition induces the synthesis of enzyme is called inducer.
11. Inducer is a chemical which attaches to repressor and changes the shape of operator binding site so that repressor no more remains attached to operator.
12. In the lac operon allolactose is the actual inducer while lactose is the apparent (visible) inducer.
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13. Inducers which induce enzyme synthesis without getting metabolized are called gratuitous inducers, e.g. IPTG (Isopropyl thiogalactoside).
14. Regulator gene (gene) produces mRNA that synthesises a biochemical repressor.
15. Repressor is a small protein formed by regulator gene which binds to operator gene and blocks structural enzyme thus checking mRNA synthesis.
16. The represseor of lac operon is a tetrameric protein having a molecular weight of 1, 60,000. It is made up of 4 subunits each having molecular weight of 40,000.
17. The repressor protein has two sites, a head for attaching to operator gene and a groove for attachment of inducer.
18. Promoter gene functions as a recognition point for RNA polymerase. RNA polymerase initially binds to this gene. It becomes functional only when it is able to pass over the operator gene and reach structural genes.
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19. Operator gene controls the expressibility of the operon. It is normally switched off due to binding of repressor over it.
20. However, if the repressor is withdrawn by the inducer, the gene allows RNA polymerase to pass from promoter gene to structural gene.
21. In lac operon the operator gene is small, 27 base pairs long. The gene is made of palindromic or self-complementary sequences.
22. If lactose is added, the repressor is rendered inactive so that it cannot attach on operator gene and synthesis of mRNA takes place.
23. Transcription is under negative control when lac repressor is inactivated by inducer.
24. Transcription in lac operon is under positive control through cyclic AMP receptor protein (CAP).
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25. The catabolite gene activator protein (Cga protein) or cyclic AMP receptor protein (CAP) binds to the Cga site.
26. When CAP is attached to the binding site the promoter becomes a stronger one.
27. CAP only attaches to the binding site when bound with cAMP.
28. When glucose level is high cAMP does not occur and so CAP does not bind and hence RNA polymerase do not bind, resulting in low transcription.
29. Lac operon will not however remain operative indefinitely despite presence of lactose in the external environment.
30. It will stop its activity with the accumulation of glucose & galactose in the cell beyond the capacity of the bacterium for their metabolism.
Repressible Operon System (Repression of Operon e.g. Tryptophan Operon of E.coli):
1. A repressible operon system is a regulated segment of genetic material which normally remains operational but can be switched off when its product is either not required or crosses a threshold value.
2. This system is commonly found in anabolic pathways.
3. Tryptophan operon of Escherichia coli is one such repressible operon system. (Fig. 8.5).
4. Tryptophan operon has 5 structural gene – E, D, C, and B A.
5. The gene E and D encodes for enzyme anthranilate synthetase, gene C for glycerol phosphate synthetase, gene B for β subunit of tryptophan synthetize and A for α subunit of tryptophan synthetize.
6. Regulator gene (trp-R) produces a biochemical, generally a proteinaceous substance, called aporepressor.
7. Aporepressor alone is unable to block the operator gene because of the absence of the binding head. Therefore, the operon system remains switched on.
8. A complete repressor is formed only when a non-proteinaceous corepressor joins the aporepressor,
9. Corepressor is a non-proteinaceous component or repressor which is also an end product of reaction catalysed by enzymes produced through the activity of structural genes.
10. It (corepressor) combines with aporepressor and forms repressor which then blocks the operator gene to switch off the operon.
11. The structural genes stop transcription and the phenomenon is known as feed-back repression.
12. Corepressor of tryptophan operon is amino acid tryptophan.
13. In tryptophan the repressor gene is not adjacent to promoter but located in another part of E. coli genome.
16. Promoter gene (trp-P) is the recognition as well as initiation point for RNA polymerase. RNA polymerase attaches to promoter gene. It can pass to structural genes provided the operator gene is in the functional state.
17. Operator gene (trp-O) lies in the passage-way between promoter and structural genes. Normally it remains switched on so that RNA polymerase can pass over from promoter gene to structural gene and bring about transcription.
18. The operator gene can be switched off when both aporepressor and corepressor join together to form repressor. The repressor binds to operator gene to interrupt movement of RNA polymerase.
19. In absence of tryptophan, the RNA polymerase binds to the operator site and thus structural genes are transcribed.
20. The transcription of structural gene leads to the production of enzyme (tryptophan synthetize) that synthesizes tryptophan.
21. When tryptophan becomes available, the enzymes for synthesizing tryptophan are not needed, co-repressor (tryptophan) – repressor complex blocks transcription.
22. One element of tryptophan operon is the leader sequence ‘L’ that is immediately 5′ end of trp. E gene.
23. This ‘L’ sequence controls expression of the operon through a process called attenuation.
24. Attenuation is the termination of the transcription prematurity at the leader region.
25. The tryptophan operon is a negative control.
The two operon models described above can be summarized as given below:
(i) Inducible System:
Active Repressor + Operator → System OFF
Active Repressor + Inducer = Inactive Repressor → System ON
(ii) Repressible System:
Apo-repressor and co-repressor complex = Active repressor → System OFF
Apo-repressor = Inactive Repressor → System ON
Importance of Gene Regulation:
1. There are two types of gene action – constitutive and regulated.
2. The constitutive gene action occurs in those systems which operate all the time and the cell cannot live without them, e.g., glycolysis. It does not require repression. Therefore, regulator and operator genes are not associated with it.
3. In regulated gene action all the genes required for a multistep reaction can be switched on or off simultaneously.
4. The genes are switched on or off in response to particular chemicals whether required for metabolism or are formed at the end of a metabolic pathway.
5. Gene regulation is required for growth, division and differentiation of cells. It brings about morphogenesis.
