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A few of the innovative scientific contributions from ancient Islam A few of the innovative scientific contributions from ancient Islam Polling Question #7 6b. The relationship between glucose and cAMP is: 1. 2. 3. When glucose is high, cAMP is high When glucose is high, cAMP is low Glucose levels have no relationship to cAMP levels Peer Instruction ATP Adenylyl cyclase cAMP Two phosphate groups Glucose allosterically inhibits this enzyme How does glucose influence levels of cAMP? So, high [glucose] = _____ cAMP = ______ binding of RNA pol at the lac operon And low [glucose] = _____ cAMP = ______ binding of RNA pol at the lac operon Polling Question #8 What carbohydrates are present in the scenario as shown? FREQUENT TRANSCRIPTION Glucose LOW Lactose HIGH 1. 2. 3. 4. CAP site Operator High levels of glucose High levels of lactose Low levels of glucose Low levels of lactose lacZ lacY lacA Peer Instruction Describe the positive regulation of lac operon expression. When glucose is high, cAMP is absent: CAP lacZ CAP site lacY RNA polymerase bound loosely to promoter (blue DNA) When glucose is low, cAMP is present: CAP cAMP lacZ CAP site RNA polymerase bound tightly to promoter (blue DNA) lacY Polling Question #9 What will the output of lactose-usage proteins be in the following scenario? INFREQUENT TRANSCRIPTION Glucose HIGH Lactose HIGH CAP site Operator lacZ lacY lacA Inducer-repressor complex 1. Very high levels 2. Low or medium levels 3. It depends on unpredictable outside factors Concept Questions • The consensus promoter sequence of the Tau operon is ATTGCTGATC. Which of the following promoters is more likely to give high expression levels: ATTGGTGATC or AGTGCTTATT? (only one strand is shown for simplicity) • What are the molecular interactions that determine the level of expression in either [high glucose, high lactose] or [low glucose, low lactose]? – Why do these make logical sense for a functional cell? • A regulation scheme in a new cell has genes A, B and C. The products of A and B repress the expression of C, but the product of A increases the binding of the polymerase at the promoter of B. The product of C increases the expression of A. – Is C a positive or negative regulator of B? – Is C a positive or negative regulator of its own expression? – Hint: Draw it out! • Challenge: Imagine that a new strain of E. coli is found that regulates lac operon expression based on levels of mercury in the environment. Design molecular interactions (or new molecules) such that E. coli only produces high levels of lac operon products in low mercury. Tuesday January 31st, 2017 Class 17 Learning Goals Understanding the Cell Cycle • What are the phases of the cell cycle, and how do cells decide when to proceed through each phase? • Why are transitions between phases controlled by checkpoints? • What is the molecular basis of checkpoint control? • How does this relate to prokaryotic gene regulation? Polling Question #1 In the cell cycle shown here, where is Anaphase? 1. In G2 2. In G1 3. In Interphase 4. In M-phase 5. In S-phase 6. Anaphase does not occur in the cell cycle Mitosis M DIVISION G2 G1 S INTERPHASE Peer Instruction 1) Label G1, G2, M, S, and Interphase on this cell cycle. 2) When is DNA replication? 3) When do mitotic spindles need to be correct and ready? G1 S PHASE AND G2 MITOSIS Chromosomes are shown partially condensed to make them visible Parent cell: 4 unreplicated chromosomes Parent cell: 4 replicated chromosomes Sister chromatids Replicated chromosomes condense at the start of mitosis. 1) Explain the reasons for these three checkpoints. Peer Instruction Metaphase Checkpoint checkpoint M-phase checkpoint G2 GCheckpoint 2 Pass this checkpoint if: • all chromosomes are attached to spindle Pass this checkpoint if: • chromosome replication is successfully completed • no DNA damage • activated enzymes present M G2 G1 2) What is G0? Pass this checkpoint if: • nutrients are sufficient • growth factors are present • cell size is adequate • chromosome is undamaged G0 S G1 checkpoint G1 Checkpoint P Inactive Cdk Cyclin Peer Instruction P Cyclin Cdk Cdk P P (Binding) Growth factors Activated Cdk P P Cyclin Rb Cdk S-phase P Rb ATP E2F E2F Expression of the E2F gene E2F ADP Tight Binding Retinoblastoma Protein (constitutively produced) The players involved are: • growth factors sent from nearby cells • CDK (an enzyme that is always present) • Cyclin (an enzyme that is sometimes present) • Rb (a protein with two binding sites) • E2F (a protein with one binding site) Decipher the molecular control of the G1 checkpoint. P Inactive Cdk Cyclin Polling Question #3 P Cyclin Cdk Cdk P P (Binding) Growth factors Activated Cdk P P Cyclin Rb Cdk S-phase P Rb ATP E2F E2F Expression of the E2F gene E2F ADP Tight Binding Retinoblastoma Protein (constitutively produced) Do the social growth factors shown here from nearby cells tend to increase or decrease passage through this checkpoint? 1. Increase 2. Decrease 3. It is not possible to predict P Inactive Cdk Cyclin Polling Question #3 P Cyclin Cdk Cdk P P (Binding) Growth factors Activated Cdk P P Cyclin Rb Cdk S-phase P Rb ATP E2F E2F Expression of the E2F gene E2F ADP Tight Binding Retinoblastoma Protein (constitutively produced) What happens when Cyclin and CDK bind? 1. CDK loses a phosphate group 2. CDK gains a phosphate group 3. CDK probably changes shape 4. Rb is can be phosphorylated by Cyclin/CDK 5. Rb is can be dephosphorylated by Cyclin/CDK P Inactive Cdk Cyclin Polling Question #4 P Cyclin Cdk Cdk P P (Binding) Growth factors Activated Cdk P P Cyclin Rb Cdk S-phase P Rb ATP E2F E2F Expression of the E2F gene E2F ADP Tight Binding Retinoblastoma Protein (constitutively produced) E2F is able to start S-phase: 1. 2. 3. 4. When bound to RB. When unbound by RB. Always. Whenever any cyclin is present. P Inactive Cdk Cyclin Polling Question #5 P Cyclin Cdk Cdk P P (Binding) Growth factors Activated Cdk P P Cyclin Rb Cdk S-phase P Rb ATP E2F E2F Expression of the E2F gene E2F ADP Tight Binding Retinoblastoma Protein (constitutively produced) Would you classify Rb as a positive or negative controller of passage through the G1 checkpoint? 1. Neither positive nor negative 2. Positive 3. Negative P Inactive Cdk Cyclin Peer Instruction P Cyclin Cdk Cdk P P (Binding) Growth factors Activated Cdk P P Cyclin Rb Cdk S-phase P Rb ATP E2F E2F Expression of the E2F gene E2F ADP Tight Binding Retinoblastoma Protein (constitutively produced) How do you think E2F initiates S-phase? Polling Question #6 P Inactive Cdk Cyclin P Cyclin Cdk Cdk P P (Binding) Growth factors Activated Cdk P P Cyclin Rb Cdk S-phase P Rb ATP E2F E2F Expression of the E2F gene E2F ADP Tight Binding Retinoblastoma Protein (constitutively produced) How do you think E2F initiates S-phase? 1. 2. 3. 4. Directly catalyzing needed reactions Increasing expression of S-phase-related genes Destroying G2-phase-related proteins Binding the promoter region of S-phase-related genes and helping to recruit RNA polymerase P Inactive Cdk Cyclin Peer Instruction P Cyclin Cdk Cdk P P (Binding) Growth factors Activated Cdk P P Cyclin Rb Cdk S-phase P Rb ATP E2F E2F Expression of the E2F gene E2F ADP Tight Binding Retinoblastoma Protein (constitutively produced) Draw E2F, and include binding sites for other molecules. -Where are they in relation to each other? Does E2F binds to Rb or promoters more tightly? Why is E2F called a ‘transcription factor’? Peer Instruction 1) Explain the reasons for these three checkpoints. Metaphase Checkpoint checkpoint M-phase checkpoint G2 GCheckpoint 2 P P M Cycli n Cdk P Cdk P P (Binding) G2 E2F Expression of the E2F gene E 2 F E 2 F F G1 checkpoint G1 Checkpoint R E 2b S G0 Cycli n Rb E2 F P Cdk Rb Sphase P ATP AD P E2 F Concept Questions • • • • • • The consensus promoter sequence of the Tau operon is ATTGCTGATC. Which of the following promoters is more likely to give high expression levels: ATTGGTGATC or AGTGCTTATT? (only one strand is shown) What are the molecular interactions that determine the level of expression in either [high glucose, high lactose] or [low glucose, low lactose]? – Why do these make logical sense for a functional cell? A regulation scheme in a new cell has genes A, B and C. The products of A and B repress the expression of C, but the product of A increases the binding of the polymerase at the promoter of B. The product of C increases the expression of A. Hint: Draw it out! – Is C a positive or negative regulator of B? – Is C a positive or negative regulator of its own expression? Challenge: Imagine that a new strain of E. coli is found that regulates lac operon expression based on levels of mercury in the environment. Design molecular interactions (or new molecules) such that E. coli only produces high levels of lac operon products in low mercury. Why does it make sense that a checkpoint would stop the cell cycle when it senses DNA damage? What about low nutrients? What is the effect on G1 checkpoint passage of: – A loss of E2F proteins? A loss of Rb proteins? – An increase of the binding strength between Rb and E2F proteins?