1. To understand the concept of the gene function control. To understand the concept of the gene function control. To describe the operon model of prokaryotic gene regulation. To describe the operon model of prokaryotic gene regulation. To know the genetic sequence involved in the regulation To know the genetic sequence involved in the regulation To discuss the evident role of genetic induction & repression. To discuss the evident role of genetic induction & repression. To identify the level of regulation control in eukaryotic cells. To identify the level of regulation control in eukaryotic cells. Objectives: Control of Gene Expression Control of Gene Expression = Gene Regulation in = Gene Regulation in Prokaryotic cell Prokaryotic cell

2. Gene regulation is Economic: Gene regulation is Economic: - E. Coli Contains constitutive genes encode enzymes that are - E. Coli Contains constitutive genes encode enzymes that are needed (e.g. enzymes of glycolysis). needed (e.g. enzymes of glycolysis). - Activation other genes occurs only under special condition - Activation other genes occurs only under special condition (e.g. absence of glucose & presence of lactose in the media). (e.g. absence of glucose & presence of lactose in the media). General levels of Gene Expression Control: General levels of Gene Expression Control: 1) Transcriptional level; - Positive control ( activation) - Positive control ( activation) - Negative control ( repression) - Negative control ( repression) 2) Translation level; - Increases or decreases the rate of translation - Increases or decreases the rate of translation ( rate of ribosomal function). ( rate of ribosomal function). 3) Post-translation level; - activation or inhibition the function of - activation or inhibition the function of the enzymes. the enzymes. ( feedback inhibition mechanism). ( feedback inhibition mechanism). Gene Regulation in Prokaryotes:

3. Inducible and Repressible Operons: Two Types of Negative Gene Regulation An inducible operon is one that is usually off; a molecule called an inducer inactivates the repressor and turns on transcription An inducible operon is one that is usually off; a molecule called an inducer inactivates the repressor and turns on transcription The classic example of an inducible operon is the lac operon, which contains genes coding for enzymes in hydrolysis and metabolism of lactose The classic example of an inducible operon is the lac operon, which contains genes coding for enzymes in hydrolysis and metabolism of lactose A repressible operon is one that is usually on; binding of a repressor to the operator shuts off transcription A repressible operon is one that is usually on; binding of a repressor to the operator shuts off transcription The trp operon is a repressible operon The trp operon is a repressible operon

4. LAC OPERON Composition: 1) Structural genes: - Group of linked genes with related function - Group of linked genes with related function ( Lactose operon contains 3 linked genes). ( Lactose operon contains 3 linked genes). - Form unit on bacterial DNA. - Form unit on bacterial DNA. - Coding for group of enzymes with related functions. - Coding for group of enzymes with related functions. - Their transcription results in single mRNA. - Their transcription results in single mRNA. - Translation results in separate 3 enzymes, because: - Translation results in separate 3 enzymes, because: Each enzyme is marked by initiation and termination codons on Each enzyme is marked by initiation and termination codons on mRNA. 1) Permease mRNA. 1) Permease 2) ß- galactosidase 2) ß- galactosidase 3) Galactoside transacetylase 3) Galactoside transacetylase Transcriptional levelTranscriptional level 1. LACTOSE OPERON = System of Gene Complex

5. 2) Operator: - DNA –sequence - DNA –sequence - Switch the transcription on or off - Switch the transcription on or off - Overlapping the promoter - Overlapping the promoter 3) Promoter: - Binding site of RNA-polymerase - Binding site of RNA-polymerase 123 O PR Carries binding site of repressor protein Composition of Operon repressor gene promoter operator structural linked genes DNA strand

6. Repressor protein - Encoded by a repressor gene Constitutive gene (its always on), so it produces continuously small amount of repressor -protein. Located upstream from the promoter site. -Repressor protein binds to operator, so it switches the transcription off. - Inactivated by inducer that switches the transcription (operator) on. How works ? The LAC OPERON Lac operon is inducible system of genes (= An inducible gene is not transcribed unless a specific inducer inactivates its repressor). (= An inducible gene is not transcribed unless a specific inducer inactivates its repressor). - becomes active under certain conditions such as absence of glucose & presence of lactose. It works to: Transform lactose into glucose to be used as a source of energy in absence of glucose. Transform lactose into glucose to be used as a source of energy in absence of glucose. -Catabolism is the metabolic pathway.

7. 123 O PR NO Transcription of Lac operon genes 123 O PR Transcription of Lac operon genes repressor Inactive repressor Can’t link with operator, so the Lac operon genes expression switches on In presence of Lactose and absence of glucose RNA- polymerase 12 3 Single mRNA 12 3 Translation to 3 separate enzymes - Permease - ß- galactosidase - Galactoside transacetylase Glucose Lactose Converts into blocked Operator unblocked Inducer ( allolactose) Small molecule formed from lactose 1)Binding of repressor to the operator switches operator off. (negative Control) 2) In presence of lactose, few molecules enter the cell and act as inducer. (positive Control) repressor

8. lactose Permease Transport Lac across the Pl.m. - ß- galactosidase - Galactoside transacetylase - Galactose - Glucose E. Coli plasma membrane Lactose allolactose (Inducer) Few molecules enter the cell & form allolactose Lac operon of E. Coli : Catabolizes the disaccharide lactose into glucose (in presence of lactose & absence of glucose).

9. Binding of inducer to repressor Inactivate the repressor by conformational change, It becomes unable to recognize and bind the operator. RNA –polymerase binds to the unblocked promoter Switch the transcription on

10. 123 O PR repressor Transcription of Lac operon On In presence of Lactose and absence or low of glucose concentration CAP-cAMP- RNA- complex polymerase 12 3 helps RNA- polymerase to bind promoter so it activates gene expression 123 O PR allolactose Inactive repressor inactive repressor No transcription a) High lactose, high glucose, low cAMP (inactive operator) due to low affinity of promoter to RNA-polymerase CAP is irrelevant RNA- polymerase b) High lactose, low glucose, high cAMP. Activation of promoter by CAP- cAMP complex. Single mRNA Inducer + repressor

11. 1) Negative Control. - Inhibit the activity of Lac operon as economical process in presence of glucose. - Inhibit the activity of Lac operon as economical process in presence of glucose. - The controlling -element is the repressor protein that switches the transcription off. - in presence of glucose: bacteria produces repressor binds to operator inactive operator turn transcription off. bacteria produces repressor binds to operator inactive operator turn transcription off. 2) Positive Control. - activation of lactose catabolism. - Pomoter of Lac operon has low affinity for RNA-polymerase, although the repressor protein is inactive by allolactose. - Activation of Lac operon; - Takes place by CAP (catabolic activator protein). - CAP is inactive, becomes active as it combines with cAMP (co-activator) to form CAP- cAMP- complex. - cAMP is regulated by glucose (it is inversely proportional to glucose concentration). glucose cAMP Types of transcriptional control of Lac Operon

12. Promoter DNA trpR Regulatory gene RNA polymerase mRNA 3 5 Protein Inactive repressor Tryptophan absent, repressor inactive, operon on mRNA 5 trpE trpD trpC trpBtrpA Operator Start codon Stop codon trp operon Genes of operon E Polypeptides that make up enzymes for tryptophan synthesis DCBA Repressible system Anabolic pathway (synthesis of amino acid tryptophan)

13. DNA Protein Tryptophan (corepressor) Tryptophan present, repressor active, operon off mRNA Active repressor No RNA made

14. Synthesis of enzymes Switch transcription on Turn operator on Inactive repressor co-repressor - rep. complex Switch operator off Turn transcription off Stop enzyme synthesis Tryptophan level ( tryptophan act as co-repressor ) Decrease Increase

15. Types of Transcriptional Control in Prokaryotes NEGATIVE CONTROL 1) Inducible genes - Represser protein alone - Represser protein alone lactose repressor alone lactose repressor alone - Represser protein + inducer - Represser protein + inducer lactose repressor + allolactose lactose repressor + allolactose 2) Repressible genes - Repressor protein alone - Repressor protein alone Tryptophan repressor alone Tryptophan repressor alone - Repressor + corepressor - Repressor + corepressor Tryptophan repr. + tryptophan Tryptophan repr. + tryptophan Active represser "turns off" regulated gene. Lactose operon not transcribed. Inactive repressor/inducer complex fails to "turn off" regulated gene(s). Lactose operon transcribed. Inactive represser fails to "turn off" regulated gene(s). Typrtophan operon transcribed. Active repressor-corepressor complex "turns off" regulated gene(s). Tryptophan operon not transcribed. POSITIVE CONTROL - Activator protein alone - Activator protein alone CAP alone CAP alone - Activator protein + coactivator - Activator protein + coactivator CAP + cAMP CAP + cAMP Activator alone cannot stimulate transcription of regulated gene(s). Transcription of lactose operon not stimulated Functional activator-coactivator complex stimulates transcription of regulated gene(s). Transcription of lactose operon stimulated.