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Updated: May 1 2020

Protein Synthesis

  • Ribosome
    • Structure
      • eukaryotes
        • small subunit (40S)
          • synthesized in the nucleus
        • large subunit (60S)
          • synthesized in the nucleolus
        • total = 80S
          • S = sedimentation
      • prokaryotes
        • small subunit (30S) + large subunit (50S) → 70S
    • Catalytic sites
      • A (acceptor) site
        • accepts charged aa-tRNA
      • P (peptidyl) site
        • holds the growing peptide chain and transfers to the aa-tRNA in the A site
      • E (exit) site
        • allows tRNA to exit the ribosome after releasing its amino acid to the aa-peptide chain
    • Forms
      • polysomes
        • single mRNA being translated simultaneously by several ribosomes
        • can be
          • free
            • proteins for nucleus or mitochondria
          • membrane bound
            • found on rough ER
            • proteins for secretion or membrane insertion
  • Steps
    • Initiation
      • initiation complex generated from
        • small ribosomal subunit
        • mRNA
          • 5'-cap (eukaryotes)
          • Shine-Dalgarno sequence (prokaryotes)
        • initiation aa-tRNA
          • met-tRNA (eukaryotes)
          • fmet-tRNA (prokaryotes)
        • initiation factors (IFs)
          • help in assembly
          • released when mRNA joins the ribosomal subunit
      • once assembled, recruits the large ribosomal subunit
        • initiation aa-tRNA occupies the P-site
          • all other aa-tRNAs bind the A-site
        • the A-site is empty
    • Elongation
      • aminoacyl-tRNA binds to A site
        • consumes 1 GTP
      • peptidyl-bond formation
        • peptidyl transferase reaction catalyzed by ribosomal rRNA ("ribozyme")
        • transfers growing polypeptide to amino acid in the A site
      • translocation
        • ribosome moves 3 nucleotides at a time in a 5' to 3' direction
        • moves peptidyl RNA from A site to P site
        • moves empty tRNA from P site to E site for exit
        • A site now empty and ready to accept next aa-tRNA
        • catalyzed by elongation factor-2 (eEF-2)
          • inactivated via ADP-ribosylation by bacterial toxins (Pseudomonas and Diphtheria)
        • consumes 1 GTP
      • cycle is repeated for each amino acid addition to the polypeptide
      • Wobble phenomenon
        • The anticodon of certain tRNA molecules can bind to multiple codons
        • The first two nucleotides of the codon adequately specify which tRNA (and amino acid) binds the codon, thereby permitting multiple nucleotides in the third position to bind the same tRNA.
    • Termination
      • stop codon reached in mRNA
      • release factor binds mRNA
      • peptide bond is hydrolyzed and completed protein is released
      • ribosomal complex dissociates
    • Overall energy requirement
      • 4 high energy bonds
        • ATP to AMP for tRNA charging (2)
        • 2 GTP to GDP for elongation (2)
  • Pharmacologic Inhibition of Prokaryotic Protein Synthesis
    • Bind 30S
      • aminoglycosides
        • inhibit formation of the initiation complex
        • promotes misreading of mRNA
    • Bind 50S
      • chloramphenicol
        • inhibits peptidyl transferase
      • macrolides
        • inhibits translocation
      • clindamycin
        • inhibits translocation
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