CFM 2: Protein Synthesis

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CFM 2: Protein Synthesis
2015-09-10 18:50:52
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  1. Eukaryotic Translation initiation machinery
    • eIF4E: binds to cap
    • eIF4G: adapter protein
    • PABP: Poly-A binding protein
    • eIF3: recruits 40S ribosome
    • Ternary complex: eIF2, GTP, met tRNA, 40S ribosome
    • eIF4A: RNA helicase
  2. eIF2B
    Converts GDP to GTP on gamma subunit of eIF2
  3. eIF2 regulation
    • eIF2B converts GDP to GTP on gamma subunit of eIF2
    • eIF2α kinases phosphorylate eIF2 on alpha subunit; this binds to eIF2B and sequesters it
    • GADD34 dephosphorylates eIF2 alpha subunit, allowing translation to continue
  4. 4 examples of eIF2α kinases
    • HCR: heme controlled repressor (hemoglobin sensor)
    • PKR: dsRNA activated
    • PERK: responds to ER stress
    • GCN2: histidine regulated kinase
  5. Examples of viral mechanisms on PKR
    • Flu NS1: masks its dsRNA appearance, so PKR does not detect
    • Adenovirus and EBV: ssRNA looks like dsRNA, competitively inhibits dsRNA binding site on PKR
    • VV and HCV: something that looks like eIF2, plugs PKR
  6. mTOR effect on translation in normal cell and in hypoxic cell
    • Normal: mTOR, responding in a signaling pathway signaled by growth factors, inactivates 4E binding protein, allowing eIF4E to bind to cap and initiate translation
    • Hypoxic: mTOR inactivated, 4EBP active, sequesters eIF4E, no translation
  7. Features of picornavirus RNA
    • No cap
    • Long 5'UTR
    • Multiple possible translation start sites
    • Uses cellular translation machinery to synthesize one long polyprotein, which is cleaved into many proteins by proteases
  8. Poliovirus translational control
    • Cleaves eIF4G
    • Uses IRES to translate its own stuff
    • Human IRESs still need eIF4G for translation, since human IRESs bring eIF4G directly without eIF4E
  9. What kinds of cellular mRNAs would use IRESs?
    Ones that need to be translated under stress conditions; e.g. cell cycle proteins, growth factors, transcription factors, apoptosis proteins, etc.
  10. VEGFA is a gene encoding a growth factor that has an IRES region. Why is inhibiting the IRES region of VEGFA theoretically a good cancer treatment?
    IRESs are only used under hypoxia, which is the state of tumor cells but not normal cells. So, translation of VEGFA inhibited in tumor cells but not normal cells.
  11. What happens to mTOR in cancer cells?
    • Cancer cells are hypoxic, so mTOR should be inactivated to inhibit translation
    • However, in cancer cells, mTOR regulation is uncoupled from cell stress
    • So, mTOR is still active and actually upregulates protein synthesis in spite of the stress conditions
  12. Most stop codons are located where on the mRNA?
    Last exon
  13. How does the cell know if there is a premature stop codon?
    • EJCs
    • Most stop codons are located on the last exon of the mRNA, so there is no EJC downstream
    • EJCs are removed by ribosomes during translation
    • If there is a premature stop codon, EJC will still remain, signaling nonsense-mediated decay
  14. Why is translation termination slower if there is a premature stop codon? How can drugs take advantage of this?
    • EJCs bind to elongation termination factors
    • EJCs still present on mRNA if PTC, but not normally (because stop codon should be on last exon)
    • Termination factors bound to downstream EJC stalls ribosome
    • Drugs can take advantage of this by inserting a cognate tRNA (e.g. tryptophan) while the ribosome is stalled so that the ribosome will read through the PTC
    • Example: Duchenne Muscular Dystrophy
  15. Where do most miRNAs come from?
    Intronic sequences
  16. miRNA formation
    • Transcribed in nucleus
    • Drosha trims it
    • Exportin exports it to cytoplasm
    • Dicer trims it to its final form
  17. _______ pair miRNA with complementary regions in mRNA
    AGO proteins
  18. miRNAs typically complementary bind to mRNAs in what region of the mRNA?
    3' NCR
  19. What happens when miRNAs bind to mRNAs?
    • Perfectly base paired: degrades mRNA
    • Imperfectly base paired: represses transcription
  20. How are miRNAs good for diagnostics?
    • They are excellent biomarkers
    • The expression profiles of miRNAs correlate well with certain diseases
  21. How can miRNAs be useful for treatment?
    You can specifically target certain mRNAs to cause their degradation