GENE REGULATION.pdf

GENE REGULATION  Gene regulation is the process of controlling which genes in a cell's DNA are expressed (used to make a functional product such as a protein). Different cells in a multicellular organism may express very different sets of genes, even though they contain the same DNA. T...

GENE REGULATION  Gene regulation is the process of controlling which genes in a cell's DNA are expressed (used to make a functional product such as a protein). Different cells in a multicellular organism may express very different sets of genes, even though they contain the same DNA. The set of genes expressed in a cell determines the set of proteins and functional RNAs it contains, giving it its unique properties. In eukaryotes like humans, gene expression involves many steps, and gene regulation can occur at any of these steps. However, many genes are regulated primarily at the level of transcription.  Some genes are expressed continuously, as they produce proteins involved in basic metabolic functions; some genes are expressed as part of the process of cell differentiation; and some genes are expressed as a result of cell differentiation. Mechanisms of gene regulation include: Regulating the rate of transcription. This is the most economical method of regulation. Regulating the processing of RNA molecules, including alternative splicing to produce more than one protein product from a single gene. Regulating the stability of mRNA molecules. Regulating the rate of translation.  Gene Regulation Makes Cells Different Gene regulation is how a cell controls which genes, out of the many genes in its genome, are "turned on" (expressed). Due to gene regulation each cell type in your body has a different set of active genes – despite the fact that almost all the cells of your body contain the exact same DNA. These different patterns of gene expression cause your various cell types to have different sets of proteins, making each cell type uniquely specialized to do its job.  For example, one of the jobs of the liver is to remove toxic substances like alcohol from the bloodstream. To do this, liver cells express genes encoding subunits (pieces) of an enzyme called alcohol dehydrogenase. This enzyme breaks alcohol down into a non-toxic molecule. The neurons in a person's brain don’t remove toxins from the body, so they keep these genes unexpressed, or “turned off.” Similarly, the cells of the liver don’t send signals using neurotransmitters, so they keep neurotransmitter genes turned off.  Structural Genes Structural genes are genes that code for proteins in the body needed for structure or function. Proteins are the building blocks of our cells. They create physical structures inside cells like the cytoskeleton, which gives our cells shape and support. Proteins also do important jobs inside the cell. Enzymes speed up chemical reactions, allowing our cells to grow, divide, and perform their functions for the body. Proteins also act as messengers, sending messages between cells and around the body. Structures are physical creations you can see. So, you can remember that structural genes code for physical things inside cells. Let's look at a few examples.  Lac Operon In bacteria, genes are arranged in functional groups called operons. These contain all the structural genes needed to carry out a task, as well as other genes to regulate their function. One specific operon helps bacteria break down lactose, a sugar in milk, called the lac operon. Lactose is made of two sugars called galactose and glucose. Cells need to break down lactose into these parts to be able to use them for energy. When lactose is in the environment, the cell starts making the structural genes needed to use lactose as an energy source. Sugars like lactose aren't very useful to cells if they stay in the environment, so the first step is to bring it into the cell. A structural gene, lac Y codes for beta-galactoside permease, which brings lactose into the cell. The next structural gene, lac Z codes for beta- galactosidase, which breaks lactose into its component sugars, glucose and galactose. The cell can then use these sugars to make energy.  Structure of the lac operon The lac operon contains three genes: lacZ, lacY, and lacA. These genes are transcribed as a single mRNA, under control of one promoter. Genes in the lac operon specify proteins that help the cell utilize lactose. lacZ encodes an enzyme that splits lactose into monosaccharides (single- unit sugars) that can be fed into glycolysis. Similarly, lacY encodes a membrane-embedded transporter that helps bring lactose into the cell.  The lacZ gene encodes an enzyme called β- galactosidase, which is responsible for splitting lactose (a disaccharide) into readily usable glucose and galactose (monosaccharides). The lacY gene encodes a membrane protein called lactose permease, which is a transmembrane "pump" that allows the cell to import lactose. The lacA gene encodes an enzyme known as a transacetylase that attaches a particular chemical group to target molecules. It's not clear if this enzyme actually plays any role in lactose breakdown.  The lac operon also contains a number of regulatory DNA sequences. These are regions of DNA to which particular regulatory proteins can bind, controlling transcription of the operon.  The promoter is the binding site for RNA polymerase, the enzyme that performs transcription. The operator is a negative regulatory site bound by the lac repressor protein. The operator overlaps with the promoter, and when the lac repressor is bound, RNA polymerase cannot bind to the promoter and start transcription. The CAP binding site is a positive regulatory site that is bound by catabolite activator protein (CAP). When CAP is bound to this site, it promotes transcription by helping RNA polymerase bind to the promoter.  Actin In eukaryotic cells, like our own, genes are a bit more complicated. They are no longer arranged in neat operons based on function and have more complex regulation. However, eukaryotic cells still have structural genes that code for proteins needed for cell function. One important structural gene family codes for the protein actin. Actin is a cytoskeletal protein that gives cells structure and support, allows for movement, and cell division. Differential expression of each of the different sub-types of actin allows for many unique functions in the cell.  Regulatory Genes Regulatory genes code for protein products that control other genes, instead of making structures of their own. Regulatory genes code for proteins that act like switches, turning other genes on or off. These genes are essential to controlling cell function, and without them, cells can grow out of control, causing diseases, like cancer, in the body.  Lac Repressor The lac repressor is a protein that represses (inhibits) transcription of the lac operon. It does this by binding to the operator, which partially overlaps with the promoter. When bound, the lac repressor gets in RNA polymerase's way and keeps it from transcribing the operon. When lactose is not available, the lac repressor binds tightly to the operator, preventing transcription by RNA polymerase. However, when lactose is present, the lac repressor loses its ability to bind DNA. It floats off the operator, clearing the way for RNA polymerase to transcribe the operon.  Control Of Gene Expression In Prokaryotes Gene expression in prokaryotes is controlled in two ways: positive control and negative control. The positive inducible system (positive control) is also known as ‘switch on’ of the lac- operon. The positive control of gene expression in prokaryotes occurs in the presence of lactose, which is the inducer.  Steps in Positive Control 1.The regulatory gene is expressed by the repressor. 2.After expression of a regulatory gene, the repressor produces repressor proteins. 3.Repressor protein has binding sites for the operator and the inducer i.e. lactose. When lactose is present (inducer) it binds with the repressor protein and forms “R+I complex”.  After the binding of the inducer to the repressor, it blocks the binding of the repressor to the operator. As the repressor protein does not block the operator, the RNA polymerase binds to the promotor and moves further to transcribe mRNA  Negative Control The negative control of the lac-operon is also called the ‘switch-off’ mechanism. It occurs in the absence of the inducer (lactose). Steps in negative control -First, the regulatory gene is expressed by the repressor. -After expression of a regulatory gene, the repressor produces a repressor protein.  In the absence of inducer or lactose the, repressor protein directly binds to an operator. This blocks the movement of RNA polymerase and its attachment to the promoter. At last, inhibits the mRNA transcription. Inducers and Induction of Lac-operon The inducer suppresses the activity and binding of the repressor protein to the operator and makes it “inactive repressor” from the active repressor. In the Lac-operon, lactose or allolactose acts as an inducer. Another inducer of the lac operon is isopropylthiogalactoside (IPTG).  Allolactose is formed by the enzyme betagalactosidase as a result of isomerization of lactose (i.e. galactose links to the Carbon 6 instead of Carbon 4). In the absence of an inducer such as allolactose or IPTG, the Lac I gene is transcribed.  The resulting repressor protein binds to the operator site of the lac operon, Lac O, and prevents transcription of the lac-Z, lac-Y and lac-A genes. In the presence of inducers, the inducer binds to the repressor. This causes a change in the conformation of the repressor that greatly reduces its affinity for the lac operator site.  The lac repressor now dissociates from the operator site and allows the RNA polymerase (already in place on the adjacent promoter site) to begin transcribing the lac-Z, lac-Y and lac-A genes. These genes are transcribed to yield a single polycistronic mRNA that is then translated to produce all three enzymes in large amounts.  The existence of a polycistronic mRNA ensures that the amounts of all three gene products are regulated co-ordinately. If the inducer is removed, the lac repressor rapidly binds to the lac operator site and transcription is inhibited almost immediately.  High-level transcription of the lac operon requires the presence of a specific activator protein called catabolite activator protein (CAP), also called cAMP receptor protein (CRP). This protein, which is a dimer, cannot bind to DNA unless it is complexed with 3’5′ cyclic AMP (cAMP)  The CRP–cAMP complex binds to the lac promoter just upstream from the binding site for RNA polymerase. It increases the binding of RNA polymerase and so stimulates transcription of the lac operon. Whether or not the CRP protein is able to bind to the lac promoter depends on the carbon source available to the bacterium.  When glucose is present, E. coli does not need to use lactose as a carbon source and so the lac operon does not need to be active. The system has evolved to be responsive to glucose. Glucose inhibits adenylate cyclase, the enzyme that synthesizes cAMP from ATP.  Thus, in the presence of glucose the intracellular level of cAMP falls, so CRP cannot bind to the lac promoter, and the lac operon is only weakly active (even in the presence of lactose). When glucose is absent, adenylate cyclase is not inhibited, the level of intracellular cAMP rises and binds to CRP.  Therefore, when glucose is absent but lactose is present, the CRP–cAMP complex stimulates transcription of the lac operon and allows the lactose to be used as an alternative carbon source. In the absence of lactose, the lac repressor, of course, ensures that the lac operon remains inactive.  These combined controls ensure that the lacZ, lac-Y and lac-A genes are transcribed strongly only if glucose is absent and lactose is present. The regulation that the lac operon undergoes is termed negative inducible because gene is turned off by the regulator factor (lac repressor). The regulator gene produces a repressor molecule which in the absence of lactose, inhibits the structural genes directly but acts through the operator gene.  In eukaryotes gene regulation may occur when DNA is uncoiled & loosened from the nucleosomes (structural unit of a eukaryotic chromosome consisting of a length of DNA coiled around a core of histones) to bind to transcriptional factors (epigenetic level), when the RNA is transcribed (transcriptional level).  When RNA is processed and exported to the cytoplasm after it is transcribed (posttranscriptional level), when RNA is translated into protein (translational level), or after the protein has been made (post- translational level). In prokaryotes, RNA transcription and protein translation occur simultaneously. Gene expression in prokaryotes is regulated at the transcriptional level.