Biochem Test 3 Review Replication Terms to know: Semiconservative: each new DNA molecule contains one original strand and one new strand Leading Strand: continuous, replication proceeds towards replication fork (still 5?-3?) Lagging Strand: Discontinuous (okazaki fragments), proceeds away from fork DNA ligase: joins lagging strand fragments DNA Gyrase: (prokaryotic) undergoes supercoiling in order to help separate strands, re-supercoils DNA after replication DNA helicase: ?unzips? DNA, separates strands DNA Pol III: (prokaryotic) large poylmerizing enzyme, exonuclease activity only 3?-5? DNA Pol (delta): eukaryotic version of Pol III DNA Pol I: (prokaryotic) removes RNA primer and replicates with correct complementary nucleotides (Exonuclease in both directions) Primase: Synthesizes RNA primer Concepts: DNA is always synthesized 5?-3? The new nucleotide is added to the 3? OH of the growing strand. This 3? OH serves as the nucleophile for the generation of the Phosphodiester bond between nucleotides The Exonuclease function of polymerases allows for repair/proofreading and maintains high fidelity DNA must be separates before it can be replicated Require Single Strand Binding Proteins to support sDNA Eukaryotes have topoisomerases instead of DNA gyrase (same function) Possible Exam Questions What is meant by semi-conservative replication? There?s one parent strand and one newly formed strand What is the function of single strand binding proteins? Keeps single strands from coming back together. They are necessary because if they were not present the body would degrade them. What are Okazaki fragments and why are they necessary? What is the Role of DNA Pol 1 in bacteria? Exonuclease activity?Alpha in humans Explain how a recombinant DNA molecule can be produced in a laboratory setting? Cut the DNA with the same endonuclease as another bacterial DNA. Which cuts it in the middle, and because you used the same Enzyme, you have the same Sticky ends. Then these can be joined. What enzyme would you use to join strands? DNA ligase. You need 2 enzymes: Endonuclease and DNA ligase Transcription Terms: RNA polymerase: adds ribonucelotides to the Growing RNA Strand Promotor: upstream of the structural gene, very A-T rich, *Signals start of transcription* Constitutive genes: a gene that is always transcribed ( always turned on) ex: mitochondrial genes Pribnow Box: 10 bases upstream, first promoter element, AT rich (TATA box in eukaryotes) Spliceosome: made up of pre-mRNA, and snRPS Removes introns Only in Eukaryotes Enhancer: upstream of promoter, can bind transcription factors **Operon: (prokaryotes) genes are controlled as a group, can be turned on/off by repressors or inducers Initiation: RNA Polymerase binds to promoter and forms closed complex ( Starts transcription Elongation:: Addition of ribonucleotides in a 5?-3? direction by RNA Polymerase Termination: end of transcription either by: 1. Inverted repeats (termination sites) 2. Rho factor Both create hairpin loops which stops transcription Post-Transcriptional modification: (of eukaryotic mRNA) 5? cap, 3? Polymerase A tail, and splicing out introns Concepts: The Lac Operon (Group of lac Genes) ?-Galactosidase breaks down lactose, and is encoded by the lacZ structural gene (made up of other lac genes) The production of this enzyme only occurs in the presence of lactose and absence of glucose How it works: In the presence of Glucose ( the repressor is bound to the operator (no transcription of lac genes) In absence of glucose ( inducer binds repressor making it inactive (transcription of lac genes proceeds) The bacteria recognizes the presence of glucose based on the binding of CAP to the promoter region Without glucose ( cAMP is formed and binds to CAP, this complex then binds to the promoter This allows RNA Polymerase to bind and transcription to take place With Glucose ( low cAMP, no complex made, no CAP at promoter, no transcription **Suggested to read this part in the book. Guarantee there will be a question on this Possible Test Questions: Describe the function of the spliceosome & what is it made up of? Removes introns?snRPS & pre-mRNA Draw a Line Diagram of an operon. Glycolysis Reactions to know: Investment Phase: Glucose ( Glucose-6-phosphate by Hexokinase/Glucokinase Fructose-6-Phosphate ( Fructose 1,6 biphosphate by PFK-1 The commitment Step! (know Why: Because once you make it you have to go through glycolysis; you can?t shuttle it out to another process. Every Exergonic! You also commit ATP to this process. Lots of Gibbs Free energy. This is the input of energy that you need for the process) Payout Phase PEP ( Pyruvate by Pyruvate Kinase For each reaction know: Where it occurs in the cell (for Glycolysis= cytosol) How the structure changes (# of Carbons) Which part of the reaction is endergonic and exergonic Hydrolizing ATP: exergonic Use/release ATP? Reduction of NAD+? The Regulation (if it?s regulated). Memorize inhibitors and activators Possible Test Questions: The mechanism of the pyruvate dehydrogenase step is a complex-catalyzed reaction involving 3 enzymes and 5 vitamin containing coenzymes?Name these 5 vitamins: Thiamin Lipoic Acid Pantothenic acid (part of CoA) Nyosine (NAD) Riboflavin (FAD) The final step in glycolysis is catalyzed by +the enzyme PYRUVATE KINASE. A positive allosteric effector of both the enzyme, and PFK-1 is AMP, another being the ?feed forward? positive allosteric effector FRUCTOSE-1,6-BIP, produced in the reaction catalyzed by PFK-1. A negative allosteric effector of this enzyme and PFK-1 is ATP. Two other negative allosteric Effectors of this enzyme are ACETYL COA, and ALANINE TCA Cycle: Reactions to Know: Pyruvate + CoA ( Acetyl CoA by Pyruvate dehydrogenase Redox Reaction Lose CO2. This step is Exergonic. Add electrons and a Proton to NAD: this is endergonic Inhibitor of enzyme Pyruvate Dehydrogenase: ATP, NADH, Acetyl CoA Acetyl CoA ( Citrate by citrate synthase Synthesizing citrate Enzyme Citrate Synthase: Inhibitors: ATP, NADH, Succinyl CoA, Citrate Adding to 6-C oxaloacetate to give 4-C citrate We break CoA off by water Isocitrate ( alpha-ketoglutarate by isocitrate dehydrogenase Redox (Oxidative decarboxylation) Loss of CO2 & Making of NADH Making of NADH is endergonic Inhibited by ATP and NADH Activated by ADP and NAD+ Alpha-keotglutarate ( succinyl-CoA by Alpha-keotglutaratedehydrogenase Redox Reaction is Exergonic Lose CO2 : Decarboxylation Inhibited by ATP, NADH, & Succinyl CoA (it?s Product) Endergonic: Making NADH Know: Cytology (mitochondria) Endergonic/exergonic parts of the step Keep up with # of carbons & Regulation Possible Test Questions: Vertebrate animals are unable to achieve a net synthesis of oxaloacetate from acetyl-CoA using only the enzymes of the TCA cycle. Briefly describe how this is overcome in plants, invertebrates, and microorganisms: These organisms do this through a glyoxalate cycle (figure 88). Glyoxalate adds to succinate to form malate. This creates an extra 4-C compound and you don?t lose the carbons as CO2. Use this cycle to add carbon to the system without losing it as CO2. Briefly describe the TCa cycle ETS and Oxidative Phosphorylation Electron Transport: Electrons are transferred from NADH and FADH2 to complexes in the mitochondrial membrane This releases Gibbs Free Energy (exergonic) Electron Transport drives the pumping of protons from the mitochondrial matrix to the intermembrane space This creates a chemiosmotic gradient (this is an uphill process which stores energy) Terminal electron acceptor: Oxygen?? Oxidative Phosphorylation Protons are allowed to move down their gradient back into the matrix This releases Gibbs Free Energy This energy allows the ATP synthase enzyme to change shape, synthesize and release ATP 2.5 ATP per NADH, and 1.5 per FADH2 30-32 ATP total Questions: What are the 2 different shuttle mechanisms for transporting the NADH made in glycolysis into the mitochondrial membrane? How do they differ in regards to energy production of the cell? Malate-Aspartate shuttle: still get NADH & 2.5 ATP Glycerol-Phosphate Shuttle: Regenerate FADH2, so you get 1.5 ATP. Advantage is that there is a NADH gradient. Mitochondria has high concentration of NADH. So going from the cytosol to Mitochondria makes the cycle not have to go through the gradient. So this can go against the gradient without energy being input into the system. Describe the structure and function of ATP-Synthase Synthesis of ATP. But only one part of the protein actually synthesizes. Two parts: F0 & F1. F0: Goes through the membrane. Integral protein. Alpha helices are what rotates in F0. Protons go through F0 domain F1: Inside the matrix of the mitochondria. This synthesizes ATP. Glycogen metabolism Glycogen is made up of glucose subunits joined by alpha-1,4 glycosideic linkages Every 12 residues, there is a branch point with alpha-1,6 linkages Breakdown: Glycogen phosphorylase Phosphoglucomutase Debranching enzymes Synthesis: Glycogen metabolism produces glucose-6-phosphate to use in Glycolysis (Save 1 ATP)
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