Bi1011 Biochemistry and Molecular Biology

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Bi1011 Biochemistry and Molecular Biology
2013-05-17 13:56:53

18th May, 9am, Talybont Sport
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  1. What is Chargaff's Rule?
    • %A = %T
    • %C = %G
  2. Give the difference between a nucleoside and nucleotide.
    • Nucleoside is sugar and base e.g deoxyadenosine.
    • Nucleotide is sugar, base and phosphate e.g. deoxyadenosine monophosphate.
  3. Give two proposed structures for the 30nm fibre.
    • Solenoid Model: a helix with 6 nucleosomes per turn.
    • Zig-Zag Model: Nucleosomes arrange in a zig-zag fashion.
  4. Describe the process of DNA compacting in eukaryotes, beginning with a DNA molecule and ending with a chromosome.
    • 1. DNA wraps around a histone in a 1 and 3/4 turn. This is the 10nm fibre.
    • 2. Histones arrange into the 30nm fibre.
    • 3. 30nm fibre attaches to a non-histone protein scaffold in loops of 80 kbp. 
    • 4. Condenses and chromosome forms.
  5. Give an overview of eukaryotic DNA replication, mentioning enzymes.
    • 1. DNA helicase unwinds a section of DNA. DNA gyrase acts as a topoisomerase to relieve supercoiling.
    • 2. Primase synthesises RNA primers.
    • 3. dNTP's and DNA Poly III begin continual DNA synthesis down the leading strand, running 5' to 3'. 
    • 4. Lagging strand runs 3' to 5' and so is replicated by laying down of Okazaki fragments between primers. 
    • 5. DNA poly 1 removes RNA primers via nick translation. 
    • 6. Gaps left by primers filled by DNA ligase.
  6. Which enzyme acts as both a 5'-3' exonuclease and a 3'-5' exonuclease?
    • DNA poly I.
    • DNA poly III only acts as a 3'-5' exonuclease.
  7. What is meant by nick translation and proof reading?
    • Nick translation - acting as a 5' - 3' exonuclease, to overwrite sections of DNA during replication.
    • Proof reading - acting as a 3' - 5' exonuclease to go back and remove faulty bases during replication.
  8. What is hyperchromic shift?
    As DNA denatures, UV absorption rises as bases are exposed.
  9. Give 3 functions of RNA and the eukaryotic RNA polymerases that transcribe them.
    • Catalytic: rRNA: RNA poly I
    • Information: mRNA: RNA poly II
    • Transport: tRNA: RNA poly III
  10. What is present at the ori?
    Large amounts of A-T base pairs because of their lower hydrogen bond count.
  11. Give the components of a histone.
    2 molecules of the following: H2A, H2B, H3 and H4, with 1 molecule of H1.
  12. Give the promoter regions used in transcription in prokaryotes.
    • -35 hexamer: TTGACA
    • Pribnow box: TATAAT
  13. Give an overview of the stages of transcription.
    • 1. Initiation: RNA poly binds to the promoter. DNA strands partially unwind, RNA synthesis begins.
    • 2. Elongation: RNA moves along the DNA, transcribing mRNA as it goes. 12-15 bp of DNA are exposed at any one time. 
    • 3. Termination: RNA poly dissociates from the DNA, mRNA released.
  14. Describe the two forms of RNA polymerase used by E.coli.
    • Holoenzyme: α2ββ’σ. Carries out initiation but not elongation.
    • Core enzyme: α2ββ’. Carries out elongation. σ dissociates and rebinds following termination.
  15. Give 3 eukaryotic post-translational processes.
    • Intron splicing.
    • 3' polyadenylation.
    • 5' methylated cap.
  16. Regarding transcription, what 3 features apply to eukaryotes?
    • A single core promoter.
    • Additional upstream promoter elements. 
    • Transcription factors (form pre-intiation complex when bound to UPE's)
  17. In translation, what is the start codon and which amino acid does it associate with?
    • AUG.
    • In eukaryotes: methionine.
    • In prokaryotes: N-formylmethionine.
  18. What is the functional difference between methionine and N-formylmethionine?
    N-formylmethionine has an aldehyde group added to it's amino group to allow binding to the N-terminus.
  19. Give 5 properties of tRNA's.
    • 1. Conserved CCA for amino acid binding.
    • 2. High internal base pairing.
    • 3. Function as adapter molecules.
    • 4. Possess an anti-codon.
    • 5. Incorporates bases other than GCAT.
  20. What is a base wobble?
    Not dubstep. A base wobble is when a tRNA anti-codon may recognise more than one mRNA codon due to their incorporation of alternate bases.
  21. Give the 3 tRNA binding sites in ribosomes.
    • E = Exit.
    • P = Peptidyl.
    • A = Aminoacyl.
  22. How are ribosomal subunits measured?
    Sedimentation rate during centrifugation. Unit is Svedbergs.
  23. What are the functions of the large and small subunits of a ribosome?
    • Large - catalyse peptide bond formation.
    • Small - bind mRNA.
  24. What are elongation factors?
    • Unassociated proteins that deliver appropriate tRNA's to the ribosome. 
    • EF-Tu: delivers amino acyl tRNA.
    • EF-G: assists translocation of the ribosome.
  25. Regarding the lac operon in E.coli, give the events arising from these 3 scenarios:
    1. glucose is present, lactose is absent.
    2. glucose absent, lactose present.
    3. both glucose and lactose are present.
    • 1. Lac i is translated into the repressor protein. RNA poly binds the promoter but is blocked by the repressor binding the operator. No operon expression.
    • 2. Lac i is translated into the repressor protein, which is inhibited by allolactose and so does not bind to the operator. RNA
    • poly initiates transcription. cAMP is abundant because glucose concentration is
    • low. CAP-cAMP complex binds to CAP site. Transcription greatly enhanced. 
    • 3. Lac i is translated into the repressor protein, which is inhibited by allolactose and so does not bind to the operator. cAMP is not abundant, so not CAP-cAMP complex binds the CAP site. Weak transcription results.
  26. What is the entropic effect?
    The energetically favourable clustering of hydrophobic amino acid residues into the center of the protein so as to prevent water caging which defies entropy.
  27. How is L / D configuration of an amino acid determined?
    • CORN rule: COOH, R group, N group.
    • Clockwise: D
    • Anticlockwise: L.
  28. What is β-branching?
    More than 1 non-hydrogen substituent on the β-carbon.
  29. Give properties of the α-helix.
    Right handed helix, 3.6 residues per turn, supported by the n+4 hydrogen bonding rule.
  30. Which amino acids break α-helix structure?
    Tyrosine and Proline, due to rigid ring structures and lack of hydrogen bonding potentiality.
  31. Give differences between the anti-parallel and parallel forms of β-pleated sheet.
    • Anti-parallel: opposite running order, hydrogen bonding groups line up. Stronger bond results. High tensile strength.
    • Parallel: complementary running order, hydrogen bonding groups staggered. Weaker bond results.
  32. What is a β-turn?
    A hairpin turn that allows a polypeptide chain to change direction. Uses a n+3 hydrogen bonding rule. Responsible for globular nature of proteins.
  33. What is super-secondary structure?
    Clusters of secondary structure. Often conforms to motifs e.g. βαβ is an α-helix between two β-sheets.
  34. What are domains?
    Globular, compact regions of protein with specific function encoded by exons. Often 100 - 400 residues long.
  35. What are 5 forces that determine tertiary structure?
    • 1. Entropic effect.
    • 2. Van der waals.
    • 3. Covalent interactions e.g. S-S bridges.
    • 4. Hydrogen bonding
    • 5. Electrostatic interactions
  36. Give the Michaelis-Menten Equation.
    V = Vmax x [S] / (Km+[S])

    • v = velocity
    • Vmax = optimal velocity
    • [S] = substrate concentration
    • Km = Michaelis constant. [S] that gives 1/2 Vmax.
  37. Define the following: K1, K2, Kcat, specificty constant.
    • K1: Binding constant.
    • K2: Dissociation constant.
    • Kcat: catalysis constant. Represents turnover number.
    • Specificity constant: Kcat / Km. Represents affinity. How fast enzyme goes / how dependent on substrate enzyme is.
  38. By deriving the Michaelis-Menten equation into y = mx + c, which plot may we produce?
    Lineweaver Burke plot / double reciprocal plot.
  39. Give the 3 types of inhibition and their effects on enzyme kinetics.
    • 1. Competitive: reversible, competes with substrate for active site. Vmax remains same, Km increased. All enzymes active, but not all binding substrate.
    • 2. Uncompetitve: reversible, binds to intermediate. Km and Vmax lowered. 
    • 3. Non-competitive: irreversible, binds to active site. Vmax is lowered, Km remains same.
  40. What is the equation for Km Apparent and why do we use it?
    KmApp = Km [1 + [I]/Ki]

    • KmApp = Apparent value of Km.
    • [I] = Inhibitor concentration
    • Ki = Inhibitor constant. 

    We use this equation because KmApp is the Km in presence of an inhibitor, not the true Km.
  41. Give 4 protein purification techniques.
    • 1. Solubility / Salting out: separating proteins from mixtures based on their solubility, which is a function of surface polarity. 
    • 2. Size Exclusion Chromatography: separating based on size through polydextran gel filtration. Small proteins take longest to sink through the gel, larger proteins sink quickest. Time series produced. 
    • 3. Ion exchange chromatography: separation based on ionic charge. Interior of column lined with charged polymer. Charged proteins delayed or not depending on interaction. 
    • 4. Affinity chromatography: separation based on selective affinity. Proteins may be delayed or not delayed depending on the substance used. Adding affinity groups to proteins pre-transcription artificially inserts affinity.
  42. What factors influence enzyme turnover number?
    • 1. Stability of mRNA signal.
    • 2. Alteration of transcription factors externally.
    • 3. Translation rate. 
    • 4. Protein degradation rate
  43. In what ways is ATP stable or unstable.
    Thermodynamically unstable, kinetically stable.
  44. Define these two in terms of ΔG: exergonic, endergonic.
    • Exergonic: ΔG < 0. Loss of energy, reaction is spontaneous.
    • Endergonic: ΔG > 0. No loss of energy, reaction is not spontaneous.
  45. Define catabolism and anabolism.
    • Anabolism: Synthetic pathway, requires ATP, endergonic.
    • Catabolism: Degradative pathway, produces ATP, exergonic.
  46. Why does oxidation of molecules produce energy?
    Oxidation is loss of electrons. As electrons flow through intermediates towards oxygen down a redox gradient, a current is produced.
  47. What is the difference between SubPhos and OxPhos?
    • SubPhos: substrate level phosphorylation. Transfer of phosphoryl to ADP, producing ATP.
    • OxPhos: oxidative phosphorylation. ETC produces ATP using redox energy.
  48. Give the 3 big players in hormonal metabolism and their effects.
    • 1. Insulin: removes glucose from blood, promotes storage and uptake. Anabolic effect. 
    • 2. Glucagon: releases glucose from glycogen. Promotes mobilisation of fuel and catabolism, inhibits storage and uptake. 
    • 3. Adrenaline: acts similarly to glucagon with respect to glucose.
  49. Which cell-surface receptor do glucagon and adrenaline utilise? Describe it's functionality.
    The G-coupled protein receptor. Consists of 7-transmembrane subunits. When a receptor is activated, it induces conformational change in the Gα subunit, which dissociates GDP and binds GTP, becoming active. Gα dissociates from other subunits and activates effector molecule. GTP is hydrolysed to GDP, Gα reassociates with subunits.
  50. Give the two adrenaline receptors.
    α-adrenogenic and β-adrenogenic receptors.
  51. What are the regulatory points in Krebs cycle?
    Steps 1, 3 and 4. Step 3 is a critical step.
  52. Outline the progression of chemicals in Krebs cycle.
    • 1. Citrate.
    • 2. Isocitrate.
    • 3. α-ketoguterate.
    • 4. Succinyl Co-A
    • 5. Succinate
    • 6. Fumarate
    • 7. Malate
    • 8. Oxaloacetate.
  53. Give the enzymes in Krebs cycle and the processes they catalyse, in order.
    • 1. Citrate synthase.
    • 2. Aconitase.
    • 3. Isocitrate dehydrogenase.
    • 4. α-ketogluterate dehydrogenase.
    • 5. Succinyl CoA synthetase
    • 6. Succinate dehydrogenase
    • 7. Fumarase.
    • 8. Malate dehydrogenase.
  54. What is the ATP yield of one Krebs cycle?
    10 ATP.
  55. How may Krebs cycle be regulated?
    • 1. Key enzymes are inhibited by their products.
    • 2. Intra-cycle regulation at steps 1, 3 and 4.
    • 3. Regulation of pyruvate dehydrogenase complex, which feeds Link reaction and, by extension, Krebs.
    • 4. External hormonal control.
  56. Give the four complexes in Electron Transport Chain.
    • Complex I: NADH dehydrogenase. 
    • Complex II: Succinate Q reductase.
    • Complex III: Q-cytochrome c reductase
    • Complex IV: Cytochrome c oxidase. 
    • Acronym: NADQd-SucQr-Qcr-Cox
  57. What is the acronym for the ETC complexes?
  58. What is the acronym for the ETC complexes? (repeat question)
  59. In which GPCR's is cAMP used as a secondary messenger?
    Glucagonic and β-adrenogenic receptors.
  60. Which steps of Krebs produce NADH?
    3, 4, 8.
  61. Which step in Krebs gives GTP?
    Step 5.
  62. Which step in Krebs gives FADH2?
    Step 6.
  63. Describe the structure and function of ATP synthase, giving detail to both F0 and F1 sections, as well as the Binding Exchange Mechanism. OR draw it.
    • F0 is the transmembrane section. Consists of a C-ring and A-subunit containing proton channel.
    • F1 protrudes into the matrix. γ-ε stalk comes up from C-ring and into the α-β hexameric ring. Arm contacting α-β hexameric ring is made from a B-δ arm. 
    • The Binding Exchange Mechanism is that only the β-subunits in the hexameric ring catalyse ATP synthesis. Rotation of the stalk causes the β-subunits to alternate between 3 forms:
    • 1. Open; binds ADP and Pi
    • 2. Tight; ATP synthesis
    • 3. Loose; ATP is released. 
    • BONUS: Since the stalk only rotates when protons flow, it is not ATP synthesis that the Proton Motive Force drives, but its release.
  64. Give the progress of chemicals in glycolysis.
    • 1. Glucose
    • 2. Glucose-6-phosphate
    • 3. Fructose-6-phosphate
    • 4. Fructose-1,6-bisphosphate
    • 5. G3P (via DHAP)
    • 6. 1,3-BPG
    • 7. 3-PG
    • 8. 2-PG
    • 9. PeG
    • 10. Pyruvate
  65. Give the enzymes in glycolysis.
    • 1. Hexokinase
    • 2. Phosphoglucose isomerase
    • 3. Phosphofructose kinase
    • 4. Aldolase 
    • 5. TIM (triose phosphate isomerase)
    • 6. G3P dehydrogenase.
    • 7. Phosphoglycerate kinase
    • 8. Phosphoglycero-mutase
    • 9. Enolase.
    • 10. Pyruvate kinase.
  66. In gluconeogenesis, why are 2 isozymes of PEP carboxykinase used?
    • Depends on precursor used: pyruvate or lactate.
    • Lactate uses cytosolic PEP carboxykinase to give NADH. 
    • Pyruvate uses Mitochondrial PEP carboxykinase.
  67. Give the two systems of numbering carbons in fatty acids.
    • Systematic: Numbering from the carbon in COOH group, which is 1. 
    • Alternative: Numbering backwards from the Ω-carbon, the last carbon in the chain.
  68. Give the basic structure of a steroid. OR draw it.
    3 cyclohexane groups bonded to a cyclopentane group in a staggered arrangement.
  69. What is meant by the term epimers?
    A group of carbohydrates that only differ from one another in that the orientation of ONE of their groups (not the anomeric) is reversed e.g. glucose and galactose.
  70. What is the difference between α-glucose and β-glucose?
    On β-glucose, the anomeric-OH group is orientated upwards. (Beta-up = beat-up)
  71. What is the difference between a white adipocyte and a brown adipocyte?
    Brown adipocytes have lots of mitochondria and are more metabolically active.
  72. Give an overview of β-fatty acid oxidation.
    • 1. Activation by Acyl CoA synthase joining CoA to the fatty acid.
    • 2. Transportation into the mitochondrial matrix. 
    • 3. Oxidation by FAD.
    • 4. Hydration.
    • 5. Oxidation by NAD.
    • 6. Thiolysis. Cleaves Acetyl CoA to leave an acyl CoA which is fed back in.
  73. Describe how you name fatty acids.
    • Single fatty acid: Trifattyacidoylglycerol
    • Mixed fatty acids: Numbered according to position on glycerol, follow above pattern.