Gene Regulation (Lac Operon) PDF

Gene Regulation Gene Regulatio n in Prokaryot es  TERMINOLOGIES Induction: The ability of bacteria to synthesis certain enzymes only when their substrates are present. It refers to switching on transcription as a result of interaction of the inducer with the regulator...

Gene Regulation Gene Regulatio n in Prokaryot es  TERMINOLOGIES Induction: The ability of bacteria to synthesis certain enzymes only when their substrates are present. It refers to switching on transcription as a result of interaction of the inducer with the regulator protein. Repression: The ability of bacteria to prevent synthesis of certain enzymes when their products are present. It refers to inhibition of transcription by binding of repressor protein to a specific site on DNA (Negative regulation).  Classification of gene with respect to their Expression Constitutive ( house keeping) genes: 1- Are expressed at a fixed rate, irrespective to the cell condition. 2- Their structure is simpler Controllable genes: 1- Are expressed only as needed. Their amount may increase or decrease with respect to their basal level in different condition. 2- Their structure is relatively complicated with some response elements  Positive regulation : Transcription factor is required to bind at the promoter in order to enable RNA polymerase to initiate transcription. Inducer: Small molecule that triggers gene transcription by binding to a regulator protein. Gratuitous inducers: Molecule that induce enzyme synthesis but not metabolized. Co- repressor: Small molecule that triggers repression of transcription by binding to a regulator protein. Constituitive gene expression: The continued expression of a gene that does not respond  With in any cell, not all its genes are active at the same time. Some gene products need to be continously synthesized, where others are necessary only during certain phases of the life cycle or perhaps only when particular environments are encountered. Even when genes are “ Turned on” the quantity of proteins they specify may need to be controlled. Some proteins need to be synthesized in large amounts and others only in small amounts. Therefore, the activity of virtually all genes The control of gene expression can take place at the levels of Transcription, Translation and Protein functioning. The most efficient region to control gene expression is at the level of transcription. E.coli messenger RNAs are short lived in vivo. They degrade enzymatically with in about two minutes. The complete turn over in the cells messenger RNA occurs rapidly and continually, and this rapid turnover is a prerequisite for transcriptional control.  Types of gene regulation  Negative inducible control (Lac operon)  Negative repressible control ( Tryptophan operon)  Positive inducible control (Arabinose operon)  Global regulation or Multiple controls (Lac operon)  Post translational control  Distinguished between two types of sequences in DNA  Sequences that code for trans acting products and cis acting products.  Gene activity is regulated by the specific interactions of the trans acting products (usually proteins) with the cis acting sequences (usually sites in DNA)  The sequences that mark the beginning and end of the transcription unit, the promoter and terminator, are examples of cis acting sites (genes physically connected to it on the same stretch of DNA).  Additional cis acting regulatory sites are often juxtaposed to, or interspersed with, the promoter.  Operon The entire system, including structural genes and the control elements that control their expression, forms a common unit of regulation is called Operon.  OPERON MODEL If metabolite is not present , enzymes for its breakdown are not useful, and synthesizing these enzymes is wasteful. Cell produces enzymes only when the carbon source is present in the environment, the enzyme system is known as an inducible system. Fig. 18.21 egative regulation: ubstrate induction Fig. 18.22a Positive regulation of the lac operon Fig. 18.22b Positive regulation of the lac operon OPERON MODEL  LACTOSE CATABOLISM Lactose: milk sugar; a disaccharide; is a P –galactoside. Wild type E. coli in lactose free medium. After its transfer to medium containing lactose.  STRUCTURAL GENES o Codes for any RNA or protein product other than a regulator. o Structural genes represent an enormous variety of protein structures and functions including, structural proteins, enzymes with catalytic activities, proteins responsible for transporting the small substrate into the cell and regulatory proteins. o Bacterial structural genes are often organised in to cluster.  ACTIVITY OF STRUCTURAL GENES lac Z codes for the enzyme P- galactosidase ( 500 KD). The enzyme breaks a P- galactoside into its component sugar. For example, lactose is cleaved into glucose and galactose. Its secondary function is to convert the 1-4 linkage of glucose and galactose to a 1-5 linkage in allolactose. lac Y codes for the P-galactoside permease, a 30KD membrane bound protein constituents of the transport system. This transports P- galactosides into cell. lac A codes for P-galactoside transacetylase, an enzyme that transfers an acetyl group from acetyl CoA to P- galactosides. Lac operon Phenomenon: 1. On glucose- Bacteria grow fine 2. On lactose- bacteria don’t grow, then grow because they induce an enzyme that breaks lactose down into glucose b-galactosidase 3. ON lactose AND glucose- No b-galactosidase !  By acetylating galactosides the transferase prevents P- galactosidase from cleaving them. The transferase is believed to protect the cell from the build up of toxic products created by P- galactosidases acting on other galactosides.  Four situations are possible 1. When glucose is present and lactose is absent the E. coli does not produce β-galactosidase. 2. When glucose is present and lactose is present the E. coli does not produce β- galactosidase. 3. When glucose is absent and lactose is absent the E. coli does not produce β-galactosidase. 4. When glucose is absent and lactose is present the E. coli does produce β-galactosidase © 2007 Paul Billiet ODWS  Regulator gene  It is a totally independent transcriptional entity.  The regulator specifies a protein called a repressor.  Repressor proteins interferes with the transcription of the genes involved in lactose metabolism.  The structure of the repressor and its interaction with the operator sites was worked recently with X ray crystallography.  The functional repressor is a homo tetramer (formed from four identical copies of the repressor protein).  Each operator site has two fold symmetry. The lac repressor tetramer binds two operators Fig. 7.12  Repressor tetramer binds to Three operator sequences dimer p O1: the original operator CAP O3 O2 O2: 410 bp downstream i z in lacZ O1 O:3 83 bp upstream in lacI * This structure enhanced RNA polrmerase binding (100x)/store at promoter  PROMOTER  The DNA region that RNA polymerase associates with immediately before beginning transcription.  The promoter is the important part of the gene expression in both prokaryotes and eukaryotes.  Promoters contain the information for transcription initiation.  Operator  Site on DNA at which a repressor binds to prevent transcription from initiating at the adjacent promoter.  When the repressor is bound to the operator, it interferes with RNA polymerase binding.  The repressor is released when it combines with an inducer , a derivative of lactose called allolactose.  Catabolite Repression  Lac and Ara operons are repressed in the presence of glucose.  Glucose is catabolised in preference to other sugars.  Catabolite repression involves cAMP.  Cyclic AMP in eukaryotes- second messenger- an intracellular messenger reg. by extracellular hormones.  Cyclic AMP works in conjunction with another regulatory protein CAP to control the transcription of certain operons.  Absence of glucose: c AMP + CAP complex – binds to a distal part of the promoter(CAP sites) – enhances the affinity of RNApolymerase for the promoter.  Presence of glucose: uptake- loss of cAMP ( inhibits the  Glucose × Adenyl cyclase ATP cAMP CAP cAMP-CAP complex Gene turn The binding of CAP- cAMP to the CAP site causes the DNA to bend more than 90 degrees. This bending , by itself , may enhance transcription, making the DNA more available to RNA polymerase. Catabolite repression is an example of positive regulation. Binding of the CAP- cAMP complex at the CAP site enhances the transcription rate of that transcriptional unit. Thus, the lac operon is both positively and negatively regulated; the repressor exerts negative control, and the CAP+cAMP complex exerts positive control of transcription. CAP cause DNA to bend