Bio 99 Final Lec 19

  1. Elongation Step 1 – incoming tRNA
    • EF-Tu escorts new aminoacyl-tRNA into A site of ribosome
    • EF-Tu is bound by GTP
    • EF-Tu protects aminoacyl-tRNA from hydrolysis
  2. Elongation Step 1b
    • Once the tRNA is in the A site, EF-Tu GTP is hydrolyzed to GDP and EF-Tu leaves
    • EF-Tu-GDP is recycled and GTP regenerated by EF-Ts
  3. Elongation Step 1c
    • Accommodation – correct tRNA basepairing brings its amino acid in close proximity to polypeptide
    • Proofreading occurs here by ribosome to ensure correct tRNA is in A site (reject or accommodate)
  4. Elongation Step 2 – Peptidyl Transferase Reaction
    • 1. Peptide Bond forms
    • 2. aminoacyltRNA bond severed
    • Peptidyl transferase reaction creates a hybrid state
  5. Elongation Step 3 – Translocation
    In order to resolve weird hybrid state, the ribosome must shift over one codon to move the A site’s tRNA into the P site, and the P site’s tRNA into the E site
  6. Elongation Step 3a
    EF-G (GTP) enters A site
  7. Elongation Step 3b
    • GTP on EF-G is hydrolyzed, pulling ribosome over one codon
    • This fixes hybrid state
  8. Elongation Step 3c
    EF-G (GDP) is released, and the ribosome is ready for the next aminoacyltRNA
  9. A mutation if EF-G will halt elongation at which step?

    D. )
  10. GTP hydrolysis powers accomodation and translocation, what provides the
    energy for the peptidyl-transferase reaction?

    B. )
  11. Why is the hybrid state called a hybrid state?
    A. Because the peptide bond is unstable
    B. Because the anticodon arm of the incoming tRNA is in the A site, but
    the amino acid arm is in the P site
    C. Because EF-Tu is still in the A site but linked to GDP, which is
    D. Because the growing polypeptide is attached to the A site tRNA
    outside of the exit tunnel
  12. Translation Termination
    • what happens when ribosome encounters a stop codon
    • There is no tRNA that recognizes a stop codon, but rather a protein factor called RF (RF-1 or RF-2)
  13. Translation Termination Step 1 – entry of RF (release factor)
    • Recognizes stop codons
    • RF-1 – UAG, UAA
    • RF-2 – UGA, UAA
    • Protein that mimics shape of tRNA
    • Drives last peptidyl transferase reaction (to water molecule)
    • Releases polypeptide
  14. Translation Termination Step 2 – removal of RF
    • RF-3 binds to RF to kick it out of ribosome
    • Uses weird GDP/GTP flipping (not
    • conventional GTP hydrolysis)
    • RF-3-GDP binds to RF
    • After kicking out RF, RF-3-GDP is replaced by RF-3-GTP
    • GTP is hydrolyzed to kick out RF-3
  15. Ribosome Recycling
    • Ribosome Release Factor (RRF)
    • • Protein mimic of tRNA
  16. Ribosome Recycling Step 1
    RRF inserts into A site, base pairs with stop codon
  17. Ribosome Recycling Step 2
    • EF-G (GTP) enters A site
    • Pulls ribosome over one codon
    • RRF is now in P site
  18. Ribosome Recycling Step 3
    • GTP hydrolysis separates small and large subunits
    • IF-3 binds to small subunit to block large subunit rejoining
  19. Proteins that mimic tRNA
    • RF-1
    • RRF
    • EF-Tu:tRNA
    • RF-G
  20. The following non-tRNAs can enter the A site. Which one can get all the
    way into the P site?

    C. )
  21. Which factor does not bind to GTP or GDP?

    C. )
  22. Why is it important that IF-3 is involved with ribosome recycling?

    C. )
  23. How much energy does it take to make
    a protein?
    • for 10 AA protein, total of 20 ATP and 20 GTP
    • (2 ATP, 2 GTP per amino acid of protein)
  24. Puromycin
    • mimics 3’ end of tRNA
    • Enters A site and forms peptide bond with growing polypeptide
    • Cannot attach new amino acids to it, so it halts translation
    • Powerful antibiotic
  25. How to remove defective mRNAs in Eukaryotes
    Case 1 – No stop codon Ribosome just keeps on going, even through the poly A tail
    • Non-stop mRNA decay
    • Ribosome translates AAA to produce long string of Lysines (poly-lysine)
    • Ski7 recognizes poly-lysine
    • Dissociates ribosome
    • Recruits exosome to eat mRNA
    • Recruits protease (that recognizes poly-lysine) to eat protein
  26. How to remove defective mRNAs in Eukaryotes
    Case 2 – premature stop codon (nonsense
    • Nonsense-mediated mRNA decay
    • Normally, exon junctions are marked by a protein complex (EJC)
    • As translation proceeds, ribosome displaces all EJCs
    • In an mRNA with a premature stop codon, some EJCs remain bound to the mRNA even after the ribosome dissociates
    • Upf1 and Upf2 recognize EJCs and recruit decapping enzyme to remove 5’ cap of defective mRNA
    • Without 5’ cap, mRNA is rapidly degraded by endonucleases
  27. Puromycin can act as a substrate for the peptidyl-transferase reaction.
    What other non-aminoacyl-tRNA can do this?

    D. )
  28. What does the non-stop mRNA degradation machinery actually recognize?

    C. )
  29. How does Upf1 and Upf2 cause degradation of mRNAs with premature
    stop codons?

    C. )
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Bio 99 Final Lec 19