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Protein Translation

Protein Translation. Text Ch 3, 17 Structure Amino acids Folding Synthesis Pre-initiation Initiation Elongation Post-processing. + H O - H. + H 3 N – CR – CO – NH – CR – COO -. Protein structure. Base. Acid. Amino acid Amine Side chain Carboxylic acid Amide backbone

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Protein Translation

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  1. Protein Translation • Text Ch 3, 17 • Structure • Amino acids • Folding • Synthesis • Pre-initiation • Initiation • Elongation • Post-processing

  2. +H O- H +H3N – CR – CO – NH – CR – COO- Protein structure Base Acid • Amino acid • Amine • Side chain • Carboxylic acid • Amide backbone • Side chains • Polarity/charge • Size • Glycine “R” is –H • Tryptophan “R” is C9H8N with two rings H2N – CR – COOH +H3N – CR – COO-

  3. 3-D structure • Solvent interaction – water • Hide the hydrophobes • Charge interaction • Acidic side chains (-) • Alkaline side chains (+) • Polar • Secondary structure • a-helix • b-sheet • Tertiary structure

  4. Protein Translation • Assembly of 5’-cap complex • Annealing of ribosome • t-RNA decoded polypeptide elongation • Trafficking • Co-translational modification • Sugars • Fatty acids • Chaperone mediated folding

  5. 80S Ribosome • Equivalent to RNA PolII or DNA Pold • Two major subunits: 40S & 60S tRNA docking 40S (large) subunit structrue 60S (large) subunit structrue A P E 3 tRNA binding sites Narrow peptide extrusion tunnel (spinnerette) mRNA twisted through 40S Ban et al., 2000

  6. Initiation Transition to elongation Pre-initiation complex Fig 17-9

  7. eEF1 Cycle Elongation Cycle (note: edited from text) Elongation eEF2 cycle Fig 17-10

  8. tRNA Lever • Base complement structure • Codon matching • Structural amplification CCA-amino acid Anticodon

  9. Pre-Initiation complex • 40S ribosomal subunit • eIF1A • 80S dissociation • Pseudo A-site tRNA • eIF3 • 80S dissociation • Initiation complex scaffold • eIF2 • Met-tRNA carrier • GTP dependent

  10. Initiation Complex • 43S Pre-Initiation Complex • mRNA • 7’methylguanosine (7mG) cap • eIF4 • eIF4G scaffold • eIF4E targeting • eIF4A ATP dependent helicase • Scanning • 5’ UTR structure 7mG cap eIF4E specifically binds 7mG cap

  11. Ribosome Assembly • 48S Initiation complex • Scans along mRNA for AUG • eIF5: eIF2 GAP • eIF5B: recruits 60S subunit • GTP hydrolysis displaces eIF5B • 60S subunit • Aminoacyl, peptidyl, exit docking sites • P site initially occupied by t-Met

  12. Elongation • eEF1:tRNA recruitment • eEF2 procession (note: edited from text)

  13. Elongation • eEF1a(bacterial EF-Tu) • GTP dependent • Recruits aa-tRNA to A site • P-protein bound to A-amino acid • Transitional tRNA state • eEF2 (bacterial EF-G) • GTP dependent • Displaces A-tRNA • Ribosomal Release Factor (rRF)

  14. eEF1 Function • eEF1A: codon independent association • Stabilized by codon recognition • Triggers GTP hydrolysis • 60S nuclease center • eEF1 release as eEF1:GDP • Codon hybridization • Peptide binding

  15. Translational accuracy • AA-tRNA synthesis • Codon matching • Structural amplification • 1 Å accuracy CCA-amino acid 2.5 Å H-bonds 70 Å mRNA tRNA Anticodon 9 Å

  16. Ribosome procession • eEF2 • Structurally similar to eEF1+tRNA • Displaces A/P site tRNA to P site • Prime A site • GTP hydrolysis • 60S nuclease center

  17. NH3 NH3 NH3 NH3 Elongation eEF1 mediated tRNA recruitment E P A E P A 3’ 3’ 5’ 5’ reset for next cycle ribosome mediated peptide binding E P A E E P P A A 3’ 3’ 5’ 5’ eEF2 mediated ribosome procession

  18. Termination • eRF1 recruited to stop codon • UAA, UAG, UGA • Another structural analog of tRNA • Breaks P-site peptide bond • GTPase

  19. Termination • eRF3 • eRF1 GAP • Dissociation of eRF1 by activating GTPase • eRF4 • 60S dissociation and recycling • Initiation factors • eIF3 Displaces P-site tRNA • eIF1

  20. Post-translational Processing • Folding • Chaperone proteins • Endoplasmic reticulum • Trafficking • Subcellular localization • Targeting signals

  21. Protein folding • Energy minimization • Hydrophobic domains • Charge balance • Metallic complexes • Ribosome holds ~40 residues denatured • Spontaneous folding • Assisted folding Protein folding may be a stochastic search for the lowest energy configuration

  22. Molecular Chaperones • Heat Shock Proteins (HSP) • HSP70 binds short hydrophobic chains • Delay folding • Prevent aggregation • Chaperonins • Receive HSP complexes • Shield larger molecules during complex folding

  23. Subcellular trafficking • Posttranslational targeting to organelles • Cotranslational targeting to compartments • ER/Golgi • Signal sequence (Start/Stop) • Translocon

  24. Glycosylation • Co-translational addition of oligosaccharides • ER • Extracellular or membrane bound • Negatively charged • Highly hydrated • Glycosaminoglycans (GAG) • Binding/recognition • Synapse • ECM • Growth factor

  25. Acylation – fatty acid transfer • Myristic acid (C14:0) • NH3-Met-Gly- • Co-translational amide bond with Gly • Palmitic acid (C18:0) • N-terminal, near TM domains • Thioester bond with Cysteine • Isoprenoids (C15:3/C20:4) • C-terminal CAAX box • Thioester bond with Cysteine • Cleavage of AAX • Membrane association • Acyl-chain coding of target membrane Saturated fatty acids

  26. Glycophosphoinositol (GPI) Anchor • Complex membrane anchor • Carboxy terminal • Raft Targeting • Extracellular • Acetylcholinesterase • “Self” recognition • Paroxysmal nocturnal haemoglobinuria • Carbonic anhydrase • PLC cleaves PO4

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