1. 1 9/21/2010 Gene regulatory mechanism: [1] [2] [3] Negative control: lac operon gal operon trp operon Positive control: ara operon mal operon tol operon Global Regulatory Mechanism: Catabolic repression ntr operon, general stress response, etc Positive Regulation The ara Operon

2. 9/21/2010 2 Model of ara operon Bind to Model of ara operon AraC protein synthesized by araC exist in two states: P1 and P2 In the absence of L-arabinosa (inducer), AraC is in the P1 state -------  inactive If arabinosa is present, it binds to AraC and changes the protein conformation to P2 state. P2 state binds to the DNA (araI) in the promoter region activate transcription of araBAD Responsible for the utilization of the five-carbon sugar

3. 9/21/2010 3 Model of ara operon AraC is not just an activator: Ara C is too high will repress transcription by binding of AraC to O1. The Maltose Operon Positive Regulation

4. 9/21/2010 Model of Maltose Operon Inducer of mal operon: Maltotriose (3 mol glucose) can be synthesized from maltose or degradation of polymer of maltose. The enzyme that degrade maltose polymer-----  play an indirect role in the regulating the operons. Genes in three clusters are regulated by activator encoded by MalT (MalT activator protein) MalT binds the inducer “maltotriose” MalK is repressor of mal operon activate transcription. may bind to MalT and hold it in an inactive state if maltose is not present. Model of Maltose operon Maltodextrin-  Maltose-  Glucose Maltose transport Degrade Maltose Break down Maltose 4

5. 9/21/2010 5 The Maltose Transport System Protein product of the mal operon are involved in transporting Maltodextrin and maltose Product of lamB in the outer membrane-------  it can bind Maltodextrin in the media Lamb forms a large channel in the outer membrane------  Maltodextrin can pass MalS protein in the periplasm degrade maltodextrin into smaller Polymers. Small polymers of maltose binds to MalE in periplasm. MalF, G, K ----transport maltodextrin through inner membrane MalP, Q operon break the maltodextrin----  maltose-----  Glucose-6-P-----  metabolism/physiology Global Regulatory Mechanisms

6. 9/21/2010 6 Changes in environment require regulatory system Simultaneously regulate numerous operons Global Regulatory Mechanisms

7. Log Cell No Polycistronic mRNA produced Lactose 9/21/2010 Structure of the lac operon — However, in the presence of lactose, the operon is switched on by inactivation of the lacI repressor protein (an example of Negative Control) LacI repressor I ZYA DNA p o L L L L RNA polymerase ( ) can now bind to the promoter and begin transcription After a "lag period", the cells start to grow on lactose Cells stop growing when all glucose is used up Time 7 Lag period However— can this be the whole story? Surely not! Recall E.coli growing on glucose and lactose:

8. 8 9/21/2010 In the presence of glucose Ratio of IIA GLC to IIA GLC~P high IIA GLC inhibits lactose permease In the absence of glucose The IIA GLC~P is high IIA GLC~P activates adenylate cyclase lactose permease: lactose transport is permitted. ATP Adenylate cyclase Glucose cAMP CAP cAMP-CAP complex Gene turn on Out of the cell

9. CAP RNA polymerase 9/21/2010 Why is glucose used first from a glucose / lactose mixture? The lac operon is not activated by a mixture of glucose and lactose until all of the glucose has been used up. Why not? Energetically, it makes sense to use the monosaccharide first. RNA polymerase binds poorly to the lac promoter. It requires a co-factor protein, CAP, in order to bind well. AZYp CAP is Catabolite Activator Protein, and exists in two different states: 1) Inactive (normal state) - does not bind to DNA AZYp inactive CAP 9

10. AZYp 9/21/2010 CAP is Catabolite Activator Protein, and exists in two different states: 1) Inactive (normal state) - does not bind to DNA 2) Active (protein is bound to cyclic AMP— cAMP) — CAP-cAMP complex binds to a specific DNA sequence just upstream of the lac promoter AZYp CAP cAMP CAP is Catabolite Activator Protein, and exists in two different states: 1) Inactive (normal state) - does not bind to DNA 2) Active (protein is bound to cyclic AMP— cAMP) — CAP-cAMP complex binds to a specific DNA sequence just upstream of the lac promoter CAP is inactive when glucose is present (cAMP low) CAP is active when glucose is absent (cAMP high) 10

11. AZYp 9/21/2010 CAP is Catabolite Activator Protein, and exists in two different states: 1) Inactive (normal state) - does not bind to DNA 2) Active (protein is bound to cyclic AMP— cAMP) — CAP-cAMP complex binds to a specific DNA sequence just upstream of the lac promoter mRNA 3) Binding of CAP-cAMP complex to its DNA site promotes binding of RNA polymerase to the lac promoter — only when all the glucose has GONE and the cAMP level has increased The CAP-cAMP complex system is an example of POSITIVE control — the lac operon is switched on by activation of CAP So, to summarize: lacI repressor = Negative Control system CAP-cAMP complex = Positive Control system inactivation of the lacI repressor activates the lac operon in the presence of lactose activation of CAP co-activates the lac operon ONLY when glucose is absent BOTH systems are required to activate the operon 11

12. 9/21/2010 12 Positive control of gene expression promoter

13. 9/21/2010 13 The CAP dimer binding site with a conserved TGTGA pentamer CAP biding to different region relative to promoter

14. 14 Enduracidin ? Bacitracin MM bcrC (~30 genes) RsbSTUVW cascade BB General Stress Response bceAByvcRS YvqE BceSYvcQ BceR YvcP YvqC ? ? yvqH ? 9/21/2010 The network of B. subtilis responses to cell wall inhibitor antibiotics Vancomycin (~60 genes) ? YhdLK YbbM ww   X Sigma factors (~150 genes) Two-component regulatory system