 REGULATION OF GENE EXPRESSION  10 1. Constitutive genes are those genes which are constantly expressing themselves in a cell because their products are required for the normal cellular activities, e.g., genes for glycolysis, ATPase. 2. Non-constitutive genes are not always expressing themselves in a cell. These are called Luxury genes. They are switched on or off according to the requirement of cellular activities, e.g., gene for nitrate reductase in plants, lactose system in Escherichia coli.          (1) (2) In eukaryotes, the control of gene expression involves several mechanisms at different levels. The control of gene expression is just a fancy way of saying that the cell controls whether or not it makes a particular protein. In any case, it all starts with a single gene. DNA unpacking in the nucleus- if the DNA of a chromosome remains coiled up tightly, there is no way that the gene can be transcribed. Without transcription, there is no mRNA to translate Transcription in the nucleus- chemical signals can be used to stop RNA polymerase from transcribing a gene, even if that section of the chromosome is uncoiled mRNA processing in the nucleus- the exons can be spliced together in a different order to make a slightly different protein. Translation in the cytoplasm- the cell can stop translation of a particular mRNA if it "decides" that it no longer needs that particular protein. Protein activity in the cytoplasm- if the cell does not need the protein after it has made it, it can simply break it down (destroy that protein) This provides maximum functional efficiency to the cell. Such regulated genes, therefore are required to be switched 'on' and 'off' when a particular function is to begin or stop. This regulation can be achieved by anyone of the following two processes: Induction. This is a process of gene regulation where addition of a substrate stimulates or induces synthesis of enzymes needed for its own breakdown. Repression. In this process of gene regulation, addition of end product stops the synthesis of enzymes needed for its formation. This phenomenon is also known as feedback repression.  Francois Jacob and Jacques Monod (1961) proposed a model of gene regulation, known as Operon model. Fig: In prokaryotes, structural genes of related function are often organized together on the genome and transcribed together under the control of a single promoter. The operon’s regulatory region includes both the promoter and the operator. If a repressor binds to the operator, then the structural genes will not be transcribed. Alternatively, activators may bind to the regulatory region, enhancing transcription.  Operon is a co-ordinated group of genes such as structural genes, operator gene, promoter gene, regulator gene which function or transcribe together and regulate a metabolic pathway as a unit. Inducible operon (lac operon)    In Escherichia coli, breakdown of lactose requires three enzymes. These enzymes are synthesized together in a co-ordinated manner and the unit is known as lac operon. Since the addition of lactose itself stimulates the production of required enzymes, it is also known as inducible system. Fig: The three structural genes that are needed to degrade lactose in E. coli are located next to each other in the lac operon. When lactose is absent, the repressor protein binds to the operator, physically blocking the RNA polymerase from transcribing the lac structural genes. When lactose is available, a lactose molecule binds the repressor protein, preventing the repressor from binding to the operator sequence, and the genes are transcribed. Lac operon  lac operon consists of following genes : (1) Structural genes. These genes code for the proteins needed by the cell which include enzymes or other proteins having structural functions  In lac-operon, there are following three structural genes: (i) lac a -gene coding for enzyme transacetylase (ii) lac y -gene coding for enzyme permease (iii) lac z -gene coding for enzyme -galactosidase (2) Operator gene (o). It interacts with a protein molecule (the regulator molecule), which promotes (induce) or prevents (repress) the transcription of structural genes. (3) Promoter gene (p). This gene is the recognition point where RNA polymerase remains associated. (4) Regulator gene (i). This is generally known as inhibitory gene (i). This gene codes for a protein called the repressor protein. It is an allosteric protein which can exist in two forms. In one form, it is paratactic to an operator and binds with it and in another form it is paratactic to inducer (like lactose). The operon is switched 'off' and 'on'.  The transcription or function of lac genes or structural genes depends upon operator gene.  When repressor protein produced by inhibitory gene (i) or regulatory gene binds to operator (o) gene, RNA polymerase gets blocked. There would be no transcription and      the operon model remains in 'switched off' position. On the other hand, when inducer like lactose is added, the repressor protein (produced by gene gets bound to it. The repressor, therefore, gets released from the operator. RNA polymerase is now permitted to act. Hence, the transcription of lac genes now takes place and the operon model is in 'switched on' position. All the three genes are transcribed by RNA polymerase to form a single mRNA strand. Each gene segment of mRNA is called cistron and, therefore, mRNA strand transcribed by more than one gene is known as polycistronic. Fig: When ATP levels decrease due to depletion of glucose, some remaining ATP is converted to cAMP by adenylyl cyclase. Thus, increased cAMP levels signal glucose depletion. Fig: (a) In the presence of cAMP, CAP binds to the promoters of operons, like the lac operon, that encode genes for enzymes for the use of alternate substrates. (b) For the lac operon to be expressed, there must be activation by cAMP-CAP as well as removal of the lac repressor from the operator. Tryptophan Operon -Repressible Operon System  Operon model can also be explained using feed-back repression.  In tryptophan (trp) operon, three enzymes are necessary for the synthesis of amino acid tryptophan. These enzymes are synthesized by the activity of five different genes in a co-ordinated manner. The addition of tryptophan, however, stops the production of these enzymes. Thus, the system is known as repressible system.  In this system, there are five structural genes, trp A, trp B, trp C, trp D and trp E, coding for three enzyme needed for the synthesis of tryptophan, an amino acid. Regulatory gene (R) produces repressor protein which is known as apo-repressor because it does not get bound to the operator directly. Hence, the operator gene remains in 'switched on' position.  Fig: The five structural genes needed to synthesize tryptophan in E. coli are located next to each other in the trp operon. When tryptophan is absent, the repressor protein does not bind to the operator, and the genes are transcribed. When tryptophan is plentiful, tryptophan binds the repressor protein at the operator sequence. This physically blocks the RNA polymerase from transcribing the tryptophan biosynthesis genes.   Tryptophan when added, binds to the apo-repressor and is called co-repressor This apo-repressor and co-repressor complex (activated repressor) now binds to operator gene and blocks the function of RNA polymerase.  Thus, transcription would not occur and tryptophan operon would be in 'switched off' position.  Feed-back repression is functional when there is no further need of the end product and, hence, there is no requirement of continuation of this anabolic pathway. This  operon stops the process. In the prokaryotes, control of the rate of transcriptional initiation is the predominant site for control of gene expression. Regulation of Gene Expression in Eukaryotes  In eukaryotes, the regulation of gene expression could be exerted at four levels: (i) (ii) (iii) (iv) Transcriptional level (formation of primary transcript) Processing level (regulation of splicing) Transport of mRNA from nucleus to the cytoplasm Translational level  In eukaryotes, functionally related genes do not represent an operon but are present on different sites in chromosomes.  Here, structural gene is called split gene which is a mosaic of exons and introns, i.e., base triplet -amino acid matching is not continuous.   Britten -Davidson gene battery model is most popular for eukaryotic genes. It proposes the occurrence of 5-types of genes-producer, receptor, integrator, sensor and enhancer-silencer. Exons are coding part of cistron that forms RNA.     Introns are non-translated part of DNA called IVS (intervening sequences) or spacer DNA. An intron begins with GU and ends up with an AG. However, the entire split gene is transcribed to form a continuous strip of hn-RNA. This removal of non-coding intronic part and fusion of exonic coding parts of RNA is called RNA splicing. About 50-90% of primary transcribed RNA is discarded during processing Fig: In eukaryotes, an enhancer is a DNA sequence that promotes transcription. Each enhancer is made up of short DNA sequences called distal control elements. Activators bound to the distal control elements interact with mediator proteins and transcription factors. Two different genes may have the same promoter but different distal control elements, enabling differential gene expression.