Lecture 21

• AUG/Met


  Initiation codon
• UAA, UGA, UAG


  Termination codons
• Ribosome


  A complex supramolecular machine

  
• Eukaryotic ribosomes


  • Larger (~80S) and more complex than bacterial ribosomes
  • Have two subunits (60S and 40S on average)
  • Very similar to bacterial ribosomes

  
• Amino acid arm


  Carries a specific amino acid esterified by its carboxyl group to the 2'-OH or 3'-OH group of the A residue at the 3' end of the tRNA

  
• Anticodon arm


  Contains the anticodon
• Stage 1: Aminoacyl-tRNA synthetases attach the correct amino acids to their tRNAs


  1. Occurs in cytosol

  2. Activates the carboxyl group of each amino acid

  3. Establishes a link between each new amino acid and the information encoding it in the mRNA

  
• Aminoacyl-tRNA synthetases


  Esterify the 20 amino acids to their corresponding tRNAs

  
• Stage 2: A specific amino acid initiates protein synthesis


  All organisms have two tRNAs for methionine: one for when (5')-most AUG is the initiation codon, one for when a Met residue in an internal position in a polypeptide

  
• Met-tRNAMet


  Inserts methionine in interior positions in eukaryotic cells

  
• Polypeptides synthesized by mitochondrial and chloroplast ribosomes begin with N-formylmethionine

  
• Initiation in bacteria


  Requires the 30S ribosomal subunit, mRNA, the initiating fMet-tRNAfMet, three initiation factors (IF1, IF2, and IF3), GTP, the 50S ribosomal subunit, Mg2+

  
• Shine-Dalgarno sequence


  Region of mRNA that guides the initiating AUG to its correct position
• Formation of the initiation complex in bacteria


  1. Step 1:

  2. Step 2:

  3. Step 3: Initiation complex = a functional 70S ribosome containing mRNA and the initiating fMet-tRNAfMet

  
• Elongation


  1. Requires the initiation complex, aminoacyl-tRNAs, elongation factors (EF-Tu, EF-Ts, and EF-G in bacteria), GTP

  2. Steps: 1. Binding of an incoming aminoacyl-tRNA, 2. Peptide bond formation, 3. Translocation (ribosome moves on codon toward the 3' end of the mRNA)

  
• The 23S rRNA has peptidyl transferase activity that catalyzes peptide bond formation

  
• Stage 4: Termination of Polypeptide Synthesis


  1. Signaled by a termination codon in the mRNA (UAA, UAG, UGA) occupying the A site

  2. Termination factors (release factors) = the proteins RF1, RF2, and RF3 which hydrolyze the terminal peptidyl-tRNA bond, release the polypeptide and the last uncharged tRNA, and cause dissociation of the 70S ribosome into its subunits

  
• At least 4 high-energy phosphate equivalents are required to generate each peptide bond
• Ribosome may stall if mRNA is damaged/incomplete

  
• tmRNA


  Rescues stalled bacterial ribosomes in a multistep pathway

  
• Polysome


  Cluster of ribosomes

  
• Expressome


  A complex of ribosomes and RNA polymerase that begins translation during transcription

  
• Stage 5: Newly Synthesized Polypeptide Chains Undergo Folding and Processing


  Chaperones and chaperonins assist by restricting formation of unproductive aggregates and limiting the conformational space
• Puromycin


  An inhibitory antibiotic that binds to the A site and terminates polypeptide synthesis
• Diphtheria toxin


  Catalyzes the ADP-ribosylation of a diphthamide (a modified histidine) residue of eEF2 to inactivate it

  
• Ricin


  A toxic protein of the castor bean that inactivates the 60S subunit of eukaryotic ribosomes by depurinating a specific A residue in 28S rRNA

  
• Signal sequence


  A short sequence of amino acids that directs a protein to its appropriate location in the cell

  
• Posttranslational Modification of Many Eukaryotic Proteins Begins in the Endoplasmic Reticulum
• Proteolytic systems


  Contain AAA+ ATPases (ex. Lon, ClpXP, ClpAP, ClpCP, ClpYQ, FtsH) that degrade proteins
• Ubiquitin


  Protein that is covalently linked to proteins slated for destruction via an ATP-dependent pathway