CFM 1 - Protein Synthesis

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  1. Prokaryote ribosome subunits
    30S + 50S = 70S
  2. Eukaryote ribosome subunits
    40S +60S = 80S
  3. Shine-Dalgarno sequence: what does it do and how does it do it?
    • Controls initiation of translation in prokaryotes
    • 1. Spacing of AUG from S/D (5-10 nts)
    • 2. Complementarity of mRNA to S/D sequence
  4. Translation initiation in prokaryotes
    • Small subunit binds mRNA
    • Met tRNA + small subunit + IF2 + GTP (ternary complex)
    • Large subunit joins; GTP hydrolyzed to GDP
  5. Translation initiation in eukaryotes
    • eIF4E binds 5'G cap
    • Recruits eIF4G (scaffolding protein)
    • eIF4G binds to PABP (polyA binding protein), eIF3 (recruits 40S subunit), eIF4A (ATP dependent helicase)
  6. eIF4E
    Translation initiation factor that binds to 5' 7mG cap
  7. eIF4G
    Adapter molecule that binds eIF4E, PABP, eIF3
  8. eIF3
    Translation initiation factor that recruits 40S ribosome to mRNA
  9. eIF4A
    ATP dependent RNA helicase
  10. eIF2 ternary complex and difference between prokaryotes and eukaryotes
    • eIF2, GTP, Met tRNA, eIF3
    • Prokaryotes: small subunit can bind to mRNA first
    • Eukaryotes: ternary complex must be bound to 40S rRNA for rRNA to bind to mRNA
  11. AUG context
    A/G at -3; G at +4
  12. Leaky scanning
    Ribosome bypasses start codon not in the right context
  13. eIF2B
    Ras GTPase that converts inactive eIF2-GDP to active eIF2-GTP
  14. eIF2 kinases that block GTP regeneration on eIF2
    • PKR
    • PERK
    • GCN2
    • HRI
  15. GADD34
    Phosphatase that removes phosphate from eIF2 to allow regeneration of GTP
  16. There is much (more / less) eIF2B compared to eIF2
  17. Small amount of eIF2 needs to be phosphorylated to shut down translation. Why?
    Phosphorylation of eIF2 increases the affinity of eIF2B 100-fold, so all the eIF2B will bind to the small amount of phosphorylated eIF2 (eIF2B << eIF2) and will not be able to be separated. Thus no more eIF2B to regenerate GTP to make more active eIF2.
  18. PKR
    • eIF2 alpha kinase
    • Activated by binding dsRNA of virus; dimerizes and autophosphorylates; phosphorylates eIF2, sequestering eIF2B, blocking translation
  19. Examples of viruses preventing PKR activation
    • Flu NS1: dsRNA sequestering protein; no dsRNA, PKR won't activate, translation can't be stopped
    • Adenovirus: sRNA that looks like dsRNA; competitively inhibits dsRNA binding site on PKR
    • EBV and HIV: same as adenovirus
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CFM 1 - Protein Synthesis
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