Protein Synthesis - Biochemistry

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

References