Let us make an in-depth study of the regulation of gene expression in prokaryotes.
All the activities of an organism are controlled by genes. Most of the genes of an organism express themselves by producing proteins. The genes which produce proteins are called structural genes or cistrons. Every cell of an organism posses all the genes. But all of them are not functional all the time. If all the genes function all the time, enzymatic chaos will prevail and there will not be much cell differentiation.
The products of many genes are needed only occasionally by the cell. Therefore, those proteins are synthesized only when the substrate on which they act is present or when they are needed by the cell. In highly differentiated cells of eukaryotes only a few genes are functional and all other genes are permanently shut off. Even a lowly E. coli bacterium expresses only some of its genes at any given time out of the total of about three thousand genes.
Various mechanisms exist in the cell, which control and regulate the expression of genes. The regulatory system turns the genes “on” when needed and turns “off’ when not needed. This proves that gene activity can be regulated.
There are various stages at which the expression of a gene can be regulated but most common is the initiation of transcription. It is here that bulk of the gene regulation takes place. Other levels of gene regulation are transcriptional elongation, mRNA processing during translation and post translation stage.
Gene Regulation in Prokaryotes:
In bacteria the expression of genes is controlled by extracellular signals often present in the medium in which bacteria are grown. These signals are carried to the genes by regulatory proteins. Regulatory proteins are of two types. They are positive regulators called activators and negative regulators called repressors. These activators and repressors are DNA binding proteins.
Negative Regulators or Repressors:
The repressor or inhibitor protein binds to the target site (operator) on DNA. These block the RNA polymerase enzyme from binding to the promoter, thus preventing the transcription. The repressor binds to the site where it overlaps the polymerase enzyme. Thus, activity of the genes is turned off. It is called negative control mechanism.
An anti-repressor or anti-inhibitor called inducer is needed to inactivate the repressor and thereby activating the genes. Thus, the genes are switched on. This is demonstrated by lactose operon.
Positive Regulators or Activators:
To activate the transcription by the promoter, the activator helps polymerase enzyme to bind to the promoter.
Genes under positive control mechanism are expressed only when an activator or stimulator or active regulator is present.
In bacteria cistrons or structural genes, producing enzymes of a metabolic pathway are organised in a cluster whose functions are related. Polycistronic genes of prokaryotes along with their regulatory genes constitute a system called operon. Operon is a unit of expression and regulation.
1. Lactose Operon or Lac Operon:
This is a negative control mechanism. In 1961 Francois Jacob and Jacques Monod proposed operon model for the regulation of gene expression in E. coli. The synthesis of enzyme (3-galactosidase has been studied in detail. This enzyme causes the breakdown of lactose into glucose and galactose.
In the absence of lactose, β-galactosidase is present in negligible amounts. As soon as lactose is added from outside, the production of β- galactosidase increases thousand times. As soon as the lactose in consumed, the production of the enzyme again drops. The enzymes whose production can be increased by the presence of the substrate on which it acts are called inducible enzymes.
Addition of lactose to the culture medium of E. coli induces the formation of three enzymes (5-galactosidase, permease and transacetylase, which degrade lactose into glucose and galactose. The genes, which code for these enzymes lie in a cluster and are called cistrons or structural genes. They are transcribed simultaneously into a single mRNA chain, which has codons for all the three enzymes. The mRNA transcribed from many genes is called polycistronic. The functioning of structural genes to produce mRNA is controlled by regulatory genes.
There are three structural genes Z, Y and A, which code for enzymes p-galactosidase, lac permease and transacetylase respectively. Regulatory genes consist of Regulator I, Promoter P and a control gene called operator gene O. Regulator I gene produces a protein called repressor or inhibitor. The repressor is active and binds to the operator gene O and switches it “off” and the transcription is stopped.
This happens because RNA polymerase enzyme which binds to the promoter is unable to do so because binding site of RNA polymerase and the binding site of repressor on operator overlap each other. Hence in negative control mechanism, the active genes are turned “off” by the repressor protein.
When the inducer (lactose) in supplied from outside, the inducers binds to the repressor. The lactose on entering the bacteria changes into allolactase. Allolactose changes the shape of the repressor (conformational changes) which renders it inactive and unable to bind to the operator. The operator becomes free and is “turned on” and thus transcription starts.
In this way, the presence of the inducer permits the transcription of Lac operon, which is no longer blocked by the repressor protein. The synthesis of enzymes in response to the presence of specific substrate (lactose) is called induction. It is also called de-repression.
The inducible system operates in a catabolic pathway. In the absence of lactose, E. coli cells have an average of only three molecules of P-galactosidase enzyme per cell. Within 2-3 minutes of induction of lactose, 3000 molecules of P-galactosidase are produced in each cell.
2. Tryptophan Operon:
It is also a negative control system but forms a biosynthetic pathway. It is known as repressible system. It works on the principle that when the amino acid tryptophan is present, there is no need to activate the tryptophan operon.
Repressor protein is activated by the co-repressor (tryptophan-the end product) and it binds the operator to switch it “off’. Tryptophan is synthesized in five steps, each step requiring a particular enzyme. The genes for encoding these enzymes lie adjacent to one another, called trp E, trp D, trp C, trp B and trp A.
Tryptophan operon codes for five enzymes that are required for the synthesis of amino acid tryptophan. In repressible system, the regulatory gene produces a repressor protein, which is normally inactive and unable to bind to operator on DNA. The repressor upon joining the co-repressor (which is the end product tryptophan in this case) undergoes conformational changes that activate it and enable it to bind to the operator. This prevents the binding of RNA polymerase enzyme to the promoter. This is opposite to the situation of lac operon in which the repressor is active on its own and loses the affinity for the operator when bound to the inducer.
Here the availability of tryptophan which is the end product regulates the expression of this operon and represses the synthesis of tryptophan. In this way the synthesis of enzymes of a metabolic pathway is stopped by the end product of the metabolic chain. This mechanism enables the bacteria to synthesize enzymes only when they are required. This is known as feed back repression.
In feed back inhibition the end product of a metabolic pathway acts as an allosteric inhibitor of the first enzyme of the metabolic chain.
Induction and repression save valuable energy by preventing the synthesis of unnecessary enzymes.
Positive Control of Transcription:
The system of regulation in lactose and tryptophan operon is essentially a negative control in the sense that the operon is normally “on” but is kept “off’ by the regulator protein. In other words the structural genes are not allowed to express unless required.
Lac operon also shows positive control by catabolic repression. This is an additional control system, which binds the repressor-operator. In E. coli, in the presence of both glucose and lactose, the glucose in first fully utilized and then lactose is taken up for production of energy.
Glucose is richest and more efficient source of energy. Glucose has an inhibitory effect on the expression of lac operon. The mechanism of positive control enables E. coli to adapt more efficiently to the changing environment of its natural habitat, which is the human intestine.
In the presence of glucose, synthesis of β-galactosidase enzyme becomes suppressed. The inhibitory effect of glucose is due to the marked drop in the level of a nucleotide called cyclic AMP (c-AMP), which inhibits the transcription of mRNA.
Lactose operon transcription requires not only cyclic AMP but also another protein called catabolic activator protein (CAP). The cAMP and CAP form a complex called cAMP-CRP complex, which is necessary for the functioning of lactose operon.
A catabolic breakdown product of glucose, called glucose catabolite, prevents the activation of lac operon by lactose. This effect is called catabolic repression. When glucose concentration increases, the cAMP concentration decreases and vice versa. High concentration of cAMP is necessary for the activation of lac operon.
Normally in the presence of glucose, the lactose operon remains inactive.
Glucose catabolite prevents the formation cAMP-CRP complex.
In this way cAMP-CRP system is positive control because expression of lac operon requires the presence of an activating signal which is this case in cAMP-CRP complex.
There are some promoters on DNA at which RNA polymerase cannot initiate transcription without the presence of some additional protein factors such as cAMP-CRP complex. These factors are positive regulators because their presence is necessary to switch on the cistrons. These are called activators or stimulators.