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Week 10 to 13 Lecture Review Booklet.docx

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University of British Columbia
BIOL 112
Karen Smith

Week 10 to 13 Lecture Review Booklet Week 10 Learning Objectives  • Compare and contrast regulation of the Lac and Mal operons in bacteria to regulation of galactose  catabolism genes in yeasts. • Apply the concepts of positive and negative regulation to galactose catabolism genes under inducing (+  galactose) and non­inducing conditions • Regulation of gene expression in eukaryotes using galactose catabolism in yeast.  • Distinguish b/t anabolic and catabolic reactions.  • Describe redox reaction in organic compounds and identify reduced and oxidized states. • Explain why cells need nutrients and the roles of different nutrients and what source it serves as. • Explain the role of the enthalpic(∆ H) and entropic( ∆ S) factors in the Free Energy (∆ G) equation and how they determine whether a chemical reaction is spontaneous. • Compare the metabolic diversity b/t photolithotrophs, chemolithotrophs, chemo‐organotrophs with respect to their primary source of energy. Lecture 23 Bacteria gene regulation malPQ operons Genes encoding proteins for metabolizing sugar maltose(carbon and energy source). Genes: 3 to 4 letter abbreviation) no caps written in italics or underlined. Ex. malP, malQ, malT Proteins (gene product) : 3 to 4 letters with caps. Ex. MalP, MalQ, Mal T mal P and malQ encodes enzymes that break down maltose malT – encodes a regulator (regulator protein) of mal PQ. When maltose is not present, MalT can’t bind to DNAat the operator region. RNApol doesn’t bind frequently and there is little transcription. The promoter for Mal PQ operons is weak. When maltose is present, maltose bind to Mal T and changes its shape. The MalT-maltose complex has an increased affinity for the operator and RNApol binds more and leads to transcription of the mal PQ operons. As maltose gets used up, the concentration decreases. Due to the binding equilibrium, maltose dissociates from Mal T, Mal T changes shape and cannot binds to the operator. This turns down the malPQ gene expression. Mal T is a positive regulator of the malPQ operon. Clicker: In the absence of the regulatory protein, the promoter is likely to be weak (less transcription) for a positively regulated gene, and to be strong(more transcription) for a negatively regulated gene. lac operons Genes encoding proteins for metabolizing sugar lactose. To use lactose, the cell needs a permease (LacY) to transport it into cell and an enzyme to break it down, called β-galactosidase (LacZ) to break it into glucose and galactose. Lac I is made all the time and will bind to the operator region. It’s not part of the lac operons, it has its own promoter and terminator. The lacI gene is expressed at very low levels (weak promoter) so not many copies of LacI are made. Lac I is a negative regulator of the lac operons. When lactose is not present, Lac I bind tightly to operators. Since the promoter and operator are close together, binding of Lac I to the operator blocks RNA pol binding to the promoter. RNA pol rarely access the promoter and only small amounts of the LacZ, Y,Aproteins are made. When lactose is present, lactose binds to LacI and cause it to change shape and the LacI-lactose complex only bins loosely to the operator due to the binding equilibrium. Then RNA pol can access the promoter more often, Thus large amounts of the LacZ, Y, andAproteins are made. As lactose gets used up, the concentration decreases. Due to the binding equilibrium, lactose dissociates from lacI and Lac I changes shape and binds tightly to the operator. This turns down the lac gene expression. Comparing the lac and mal PQ gene expression regulatory system Similarity: Both gene expression regulatory systems function to metabolize the sugars. The protein for metabolizing lactose and maltose are only made at high levels when they are present. Difference: lac system exhibits negative regulation. Mal system exhibits positive regulation. 3 components of the system that regulate gene expression based on the environment 1. The DNAregulatory sequence: a sequence near to, or partially overlapping with the promoter. Ex. Lac and malPQ operators. 2. The regulator proteins: a protein that binds to the regulatory sequence that affects whether RNA pol can initiate transcription or not. The gene for the protein is expressed at a low level. Ex. LacI and MalT. 3. Asignal chemical: an environmental chemical that binds to a regulatory protein and affects the DNA binding properties of the protein. Ex. Lactose and maltose. Gene Regulation: Negative regulation The regulator binds to the DNAand blocks transcription. To activate the gene, the regulatory protein must be modified so it doesn’t bind DNA. Regulatory protein called a “repressor” protein. Ex. Lac I Gene Regulation: Positive regulation The regulator binds to the DNAand activate transcription. When the regulator doesn’t bind DNA, there is no transcription. Regulatory protein called an “active” protein. Ex. MalT Induction versus repression Induction: binding of an effector (signal molecule) to a regulatory protein “turns up” gene expression. Ex. Binding of lactose to Lac I (- reg) and binding of maltose to MalT(+ reg) Effectors (lactose and maltose) are called: inducers Repression: binding of an effector to a regulatory protein “turns down” gene expression. Effectors are called: co-repressors The presence of effectors in the cytoplasm is a reflection of its presence in the environment. It’s necessary to always make a small amount of membrane transporters so the cell can sense the environment. Lecture 24 Eukaryotic gene regulations Cis acting elements : DNAsequence, can influence expression of adjeacnet genes on same DNAe.g. promoter- proximal elements, enhancers and silences. Trans acting elements : transcription factors, proteins, can diffuse through cytoplasm. e.g activators, co- activators. Galactose metabolism in yeasts Yeast genes: italicized CAPS Proteins are written with CAPS no italics 5 enzymes are required to metabolize galactose to glucose in yeast so that it can be used as an energy and carbon source. 3 (GAL 1, 7, 10) are coordinately inducible to a 1000 fold when galactose is present and glucose is not present. They are on the same chromosome. But they are transcribed separately and have their own promoter and terminator sequence. Regulatory protein (GAL 4) is on a separate chromosome. It’s a positive regulator. When galactose is present, GAL 4 binds to the promoter proximal element of GAL 1, 7, 10 which activates the basal transcription complex associated with each gene’s promoter and transcription of GAL 1, 7, 10. GAL 80 is another regulatory protein, GAL 80 is a negative regulator. It binds to GAL 4 and prevents its interaction with the basal transcription complex when there is no galactose as shown below. Lecture 25 Metabolism All of the enzyme catalyzed reaction that occur in a cell = anabolism and catabolism (The building and breaking down of carbon sources to harness or release energy) . CoA(enters the citric acid cycle) and glycerol. Glycerol enters the glycolytic pathway once it has been oxidized and phosphorylated. Proteins can be broken down to produceATP. Anabolic: reaction that synthesize large molecules from small components. It needs energy. Precursor molecules required for synthesizing.Acetyl CoAis the starting point for anabolic pathways from the synthesis of fatty acids. Catabolic: reaction that breaks down molecules and produces ATP. Enzymes break down fats to acetyl Redox Reactions Oxidation: Loss of election Reduction: gain of electron Electron donor: loses electron becoming oxidized. Electron acceptor: gains electron becoming reduced Atoms gaining bond to O and losing bonds with H are oxidized.Atoms gaining bonds to H and losing bonds with O are reduced. Metabolism begins with nutrients Nutrients are sued as source of Major bio elements such as C,N,P,S,O,H. • Required to build organic cellular molecules. Eg. Protein, DNA, RNA, lipids, polysaccharides • Nutrient sources differ widely. • Autotrophs: uses inorganic-C (from CO2) • Heterotrophs: uses organic-C (from sugars, fats etc) Minor bio elements: Fe, Ca, Mg, Zn, Cu, others • Required for enzyme functions: most form part of the structure of enzyme active sties. • Nutrient sources are the same for all organisms: found in soil and rocks and water and everywhere. Vitamins (Composed of the major bioelements ) • Small organic molecules required for enzyme function by all life. • Most vitamins synthesized by plants (plants don’t need them)  Some microbes (like plant) can synthesize vitamins from other nutrients and don’t require them.  Some microbes (like animal) require vitamins as nutrients because they cannot synthesize them. Electrons (e-) • Organisms need e- to reduce major bioelements C, N, and S during anabolism. • All organisms require a nutrient as a source of e- Energy or components of reactions that yield energy (terminal electron acceptors TEA) • Energy is needed to do work. • Mechanical work: movement of organisms • Electrical work: separating oppositely charged chemicals across membrane against the force of electrical attractions to create a gradient. • Concentration work: transporting uncharged chemicals across membranes to create concentration differences across membrane. • Synthetic work: building large molecules from smaller ones. Change in G between 2 states of a chemical system is given by the equation ΔG = ΔH + (-TΔS) ΔG = difference in stability b/t 2 states of chemical system (expressed in energy units J) ΔG > 0 non-spontaneousΔG< 0 spontaneous ΔH = difference in the amound of E needed to break all bonds in the initial and final stages. It’s usually the key factor in theΔG value of metabolic reactions. Bacteria and archaea produceATP in three ways 1. Phtotroph(light feeders) uses light energy to promote electrons to the top of ETC.ATC is produced by photophosphorylation. 2. Chemoorganotrophs oxidize organic molecules with high Ep such as sugar(e-donor).ATP is produced by cellular respiration or fermentation pathways. Humans are chemoorganoheterotrophs. 3. Chemolithotrophs (rock feeders) oxidize inorganic molecules with high Ep such as ammonia or methane.ATP is produced by cellular respiration with inorganic compounds as the electron donor. Week 11 Learning Objectives • Discuss how energy is used to achieve different types of activities(work) in cells. • Explain the energetic role played byATP in polymer biosynthesis (anabolism) • Know the High Energy Intermediates involved in Glycolysis and cellular respiration. • Locate the compartment in which the various components of the complete pathway of cellular respiration occur in eukaryotes, compare that to in bacteria. • Identify the inputs and the outputs of cellular respiration. • Locate glycolysis in bacterial and eukaryotic cell. • Identify inputs and the out puts of the glycolytic path. • Describe the process of substrate level phosphorylation in generatingATP in Glycolysis. • Describe the role of NADH produced or predict the fate of NADH produced. • Discuss the role of fermentation and how end product such as lactate and ethanol play a role in NAD+ regeneration. Lecture 26 Metabolism part 2 I.C. ΔH < 0 and TΔS>0 contribute to the availability of free energy to do work. Energy Organisms need it to do synthetic work: building large molecules from smaller ones. (anabolism) I.C. Biosynthetic reactions requireATP, have positiveΔG(cannot happen spontaneously), are enzyme-catalyzed reactions and it’s small molecules joined to form large molecules. H2O is not a product, but the product components are equal. Anabolism Ex. Protein synthesis(translation), RNAsynthesis(transpiration) and DNAsynthesis(replication) are all polymerization reactions and require energy in the form ofATP. Step 1:ΔG > 0, it cannot happen spontaneously. In order for enzymes to catalyze polymerization, the monomers must first be activated (monomers have been enzymatically reacted with a phosphate-containg group or NTP e.g ATP, UTP, CTP, GTP and the types of activated monomers differ) Step 2Activated monomers are combined with an inactivated monomers High-energy phosphate groups from NTPs are transferred to intermediates, increasing the free energy of the intermediates (lower stability). Removal of the high-energy phosphate groups from the activated intermediates in coupled reactions result in a decrease in free energy (higher stability). ATP hydrolysis yields so much free energy because a transition from a weakly bonded state (reactants)ATP to a more strongly bonded state (product) ADP + Pi contributes to an increase in system stability (TΔS) . Increases stability Decreases stability Overall stability ΔG<0 free energy ΔG>0 free energy Enthalpic stability ΔH<0 product more stable ΔH>0 reactant more stable Entropic stability TΔS > 0 TΔS < 0 Three mechanism ofATP synthesis Light-independent synthesis: substrate-level phosphorylation (SLP) and oxidative phosphorylation (oxphos). Light-dependent synthesis: photophosphorylation (photophos) 1. Substrate-level phosphorylation (cytosol or mitochondrial matrix if cell have mitochondria) Enzyme catalyzes transfer of phosphate from a phosphorylated molecules toADP to formATP. ChemiosmoticATP synthesis 2. Oxidative phosphorylation (oxphos) (Inner mitochondrial membrane(E) or cytoplasmic membrane(P)) High energy intermediates: NADH, FADH2, NADPH,ATP e-donor: NADH/FADH2 come from food   3.Photophosphorlation ( photophos) e-donor: H2O L27 Metabolism Part 3: Glycolysis Glycolysis Phase I: energy investment phase 2ATP used to give 6C sugar 2 negativ
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