In this article we will discuss about the function of regulator gene.
The genetic basis for induction and repression was studied for several years by Jacob and Monod at the Pasteur Institute in Paris. They investigated regulation of the activities of genes which control fermentation of lactose through synthesis of the enzyme β-galactosidase in E. coli. They were awarded Nobel Prize in 1965.
If wild type E. coli cells are grown on a medium containing glucose, the cells are not able to utilise lactose and contain very small quantities of the enzyme β-galactosidase. But if wild type E. coli are grown on a medium devoid of glucose, but containing lactose as the only carbon source, within two minutes they start synthesizing β-galactosidase.
The synthesis of enzyme continues until very large amounts (about 3000 molecules per cell) have been produced. It was found that along with β-galactosidase, lactose induces the synthesis of two other enzymes viz. β-galactoside permease, which facilitates entry of lactose into the cells and β-galactoside transacetylase, whose function is obscure.
The three collectively are known as lac enzymes. Jacob and Monod studied gene regulation by isolating lactose mutants of E. coli which had one defect or the other in this regulation.
The mutants revealed following different types of genes performing different functions in regulation:
(a) There are mutants which on growing on lactose medium, do not have one of the three enzymes synthesised on induction. Mapping techniques have shown that they have defects in three adjacent genes, each of which directs the synthesis of one of the enzymes. These are called structural genes and were shown by Lederberg and his colleagues to be arranged continuously on the chromosome in the order β-galactosidase (denoted z gene), permease (y) and transacetylase (a).
(b) Constitutive Mutants: Enzymes may be constitutive or induced. Constitutive enzymes are those made in constant amounts in a cell, without regard to the metabolic state of the cell. Induced enzymes are made when required in response to the presence of their substrates in a cell.
Constitutive mutants of E. coli studied by Jacob and Monod are those that synthesise the three enzymes regardless of the presence or absence of the inducer. The gene showing this defect was called the regulator gene (denoted by i) and was found by mapping techniques to lie before the z gene.
(c) E. coli cells which were diploid as they had one complete chromosome and a second chromosome fragment homologous with a portion of the first chromosome. Such a bacterial cell is partially diploid for some genes and is called meroploid. Mutants with two chromosomes in a cell were analysed as follows: one chromosome had an active i but a defective z gene (i+ z–); the other chromosome having active z but defective i gene (i– z+).
Such mutants produce β-galactosidase only in presence of inducer. It means that the active regulator gene (i+) on one chromosome can regulate the active structural gene (z+) on the other chromosome. Obviously, the regulator gene must be controlling the synthesis of an intermediary molecule which diffuses through the cytoplasm.
Some other experiments showed that the regulator gene codes for the amino acid sequence of a specific protein called repressor. The repressor molecule diffuses from the ribosomes where it is formed and becomes physically bound to a specific site on DNA near the structural gene.
(d) Further understanding of the repressor molecule came from mutants which were constitutive even though they had an active regulator gene. Such mutants failed to respond to the repressor because of a defect in a small specific region of the chromosome to which the repressor becomes bound. This was called the operator (denoted O) situated near the beginning of the β-galactoside structural gene (z).
The existence of operators was first revealed by genetic analysis. A mutation in the operator can make it inactive, preventing the binding of the repressor. When this happens, then constitutive enzyme synthesis occurs on the z, y and a genes. These mutants are therefore called operator constitutive Oc mutants.
The operator constitutive mutants can be distinguished from mutations in repressor genes by measuring enzyme synthesis in partially diploid cells for certain chromosomal regions.
If such a partially diploid cell contains one mutant and one functional repressor gene, repression occurs because repressor molecules produced by one functional locus can bind to both operators. But if there is one non-functional operator locus, the cells would always be constitutive.
From genetic studies in mutants combined with biochemical evidence, Jacob and Monod derived the following conclusions: the lac operon regulates the metabolism of lactose. When E. coli cells are grown on a medium containing lactose, the lac operon becomes functional and synthesizes enzymes required for the transport and breakdown of lactose.
The lac operon does not function when glucose is present or when lactose is absent from the medium. The lac operon contains a promoter (p), an operator (o), and three structural genes (z, y and a). It also has a transcription terminator gene (t) which gives the chain termination signal during mRNA synthesis.
The regulator gene directs the formation of a repressor protein. This protein has affinity for the sequence of nucleotides of the operator and can bind to the operator. When the repressor is bound to the operator it prevents movement of RNA polymerase towards the three structural genes; no mRNA is synthesised, and therefore the three proteins are not formed.
When inducer (lactose) is present, its molecules can bind to another active site of the repressor protein. This binding changes the three dimensional conformation of the protein, so that it loses its affinity for the operator. The operator is made free, mRNA is transcribed by the structural genes and all the three enzymes are synthesised (Fig. 16.1).
Jacob and Monad also postulated repression of enzyme synthesis. For example, if histidine is added to the culture medium in which E. coli cells are growing, the enzymes leading to the formation of histidine become repressed, and histidine is not synthesised. The process is called feedback inhibition or end-product repression.
By itself the repressor molecule is inactive. But when a co-repressor binds with it, the repressor-co-repressor complex binds with the operator gene that is specific for the structural genes of this operon, and prevents transcription. Thus there are two types of repressor molecules, one which binds with the inducer and promotes synthesis of enzymes; the other binds the co-repressor resulting in end-product repression.