To understand the concept of the gene function control.

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

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

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 • 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 • The trp operon is a repressible operon

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

5. 2) Operator: - DNA –sequence - Switch the transcription on or off - Overlapping the promoter 3) Promoter: - Binding site of RNA-polymerase Composition of Operon R P 1 2 3 O repressor gene promoter operator structural linked genes DNA strand Carries binding site of repressor protein

6. 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). • - 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. • Catabolism is the metabolic pathway. • • 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.

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

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

10. Inducer + repressor a) High lactose, high glucose, low cAMP(inactive operator) due to low affinity of promoter to RNA-polymerase inactive repressor R P 1 2 3 O No transcription CAP is irrelevant RNA- polymerase b) High lactose, low glucose, high cAMP.Activation of promoter by CAP-cAMP complex. R P 1 2 3 O Transcription of Lac operon On CAP-cAMP- RNA-complex polymerase In presence of Lactose and absence or low of glucose concentration repressor helps RNA-polymerase to bind promoter so it activates gene expression allolactose 1 2 3 Single mRNA Inactive repressor

11. 1) Negative Control. •- 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. 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 formCAP- cAMP- complex. - cAMP is regulated by glucose (it is inversely proportional to glucose concentration). glucose cAMP Types of transcriptional control of Lac Operon

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

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

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