lecture 26: RIbosome and Translation

Question 1

Eubacterial Ribosome Subunit structure

Answer 1

• ribosomes: two subunits
  
  • 70S=50S + 30S
    
    • 50S = 5S rRNA + 23S rRNA + 34 proteins
      
      • 23S rRNA contacts tRNA
    • 30S = 16S rRNA + 21 proteins
      
      • 16s RNA attracts mRNA (Shine Delgarno) and contacts tRNA
  • separated by differential centrifugation of cell free extract
• eukaryotic ribosome: more complex + larger

Question 2

shine delgarno complement

Answer 2

• at 3’ end of 16S rRNA (30S)

Question 3

Ribosomal subunit function

Answer 3

• small subunit: mediates interaction between mRNA, codons, and tRNA anticodons
• large subunit: catalyzes peptide bond formation
  
  • binding sites for G-proteins that assist in initation, elongation, termination

Question 4

Translation Overview Process

Answer 4

1. form initiation complex on RNA
   
   • ​IF1 and IF2 bind at A site
   • IF3 binds at E site
   • initiator tRNA binds to AUG codon at P site
2. large ribosomal subunit binds
3. elongation
   
   • aminoacyl tRNA binds
     
     • tRNA selection
   • peptide bond forms
     
     • peptidyl transfer: amino terminus of AA attacks carboxyl C of peptide chain - cov bond to rRNA
   • translocation
4. termination

• ​RF1 recognizes UAG and UAA
• RF2 recognizes UGA and UAA
• RF3 stimulates release of RF1 and RF2
• release polypeptide
• dissociate ribosomes

Question 5

Bacterial Translation Factors

Answer 5

• Initiation: IF-1, IF-2, IF-3
• Elongation: EF-Tu, EF-G
  
  • EF-Tu: binds aminoacyl t-RNA to A site
  • EF-G + GTP: translocation - large subunit moves tRNA in Asite to P site
• Termination: RF-1, RF-2, RF-3-GTP
• proteins - not part of ribosome, but required for translation

Question 6

ribosome tRNA binding sites

Answer 6

• A site (aminoacyl tRNA): aminoacyl tRNA
  
  • contacts mRNA
• P site (peptidyl tRNA): new peptide bonds formed
  
  • contacts mRNA
• E site (exit site): transiently occupied by tRNA that is leaving ribosome

Question 7

Prokaryotic Initiation of translation

Answer 7

• bind mRNA so AUG is in p site bound to initiator tRNA
• IF1 and IF2 bind to A site
• IF3 binds to E site
• after factors bind, larg subunit is associated

Question 8

Initiator AUG vs. normal AUG

Answer 8

• in eubacteria, initiation only occurs at AUGs with nearby ribosome binding sites
  
  • shine Dalgarno sequence - 5-10 nt upstream on mRNA allows binding to 16S rRNA

Question 9

Initiator tRNA

Answer 9

• two types of tRNAMet in bacteria
• tRNAmet: incorporate methionine into protein chain
• tRNA_i^fMet: initiate protein synthesis
  
  • modified with additional formyl group on methionine
    
    • after protein synthesis, formyl group usually removed.
    • N-formyl methioinine resembles peptide bond

Question 10

Polyribosome

Answer 10

• mRNA translated by multiple ribosomes at same time
  *

Question 11

Eukaryotic Translation Initiation

Answer 11

• initiation requires mRNA cap binding protein: eIF4E
  
  • recruits a small subunit with preloaded rRNA_i-Met
  • ribosome scanning finds first AUG
    *

Question 12

antibiotic inhibitors of protein synthesis

Answer 12