Eucaryotic Protein Synthesis

1. Eucaryotic Protein Synthesis

2. Eukaryotic mRNAs See Figure 30.26 for the structure of the typical mRNA transcript • Note the 5'-methyl-GTP cap and the poly A tail • Cap is essential for mRNA binding and stabilizes mRNA by preventing degradation • Poly A tail enhances stability and translational efficiency of mRNAs • Shine-Dalgarno sequence not present

3. Initiation in eucaryotes • Family of at least 14 eukaryotic initiation factors • The initiator tRNA is a special one that carries only Met and functions only in initiation - it is called tRNAiMet but it is not formylated

5. Eukaryotic Initiation • Begins with formation of ternary complex of eIF-2, GTP and Met-tRNAiMet • 1) This binds to 40S ribosomal subunit:eIF-3:eIF1A complex to form the 43S preinitiation complex • Note no mRNA yet, so no codon association with Met-tRNAiMet • 2) mRNA then adds with several other factors, forming the 48Sinitiation complex (Fig. 33.23) • 48S initiation complex scans to find the first AUG (start) codon • 3) At AUG, 60S subunit adds to make 80S initiation complex (GTP is hydrolyzed)

8. Regulation of Initiation Phosphorylation is the key, as usual • At least two proteins involved in initiation (Ribosomal protein S6 and eIF-4F) are activated by phosphorylation • But phosphorylation of eIF-2a causes it to bind all available eIF-2B and sequesters it, therefore translation is down-regulated by phosphorylation

10. Elongation and Termination • Elongation is similar to procaryotic elongation: • EF1A homolog to EF-Tu, EF1B homolog to EF-Ts, EF2 homolog to EF-G • Termination even simpler: only one RF, binds with GTP at the termination codon

11. Inhibitors of Protein Synthesis Two important purposes to biochemists • These inhibitors (Figure 30.30) have helped unravel the mechanism of protein synthesis • Those that affect prokaryotic but not eukaryotic protein synthesis are effective antibiotics • Streptomycin - an aminoglycoside antibiotic - induces mRNA misreading. Resulting mutant proteins slow the rate of bacterial growth • Puromycin - binds at the A site of both prokaryotic and eukaryotic ribosomes, accepting the peptide chain from the P site, and terminating protein synthesis

13. Diphtheria Toxin An NAD+-dependent ADP ribosylase • One target of this enzyme is EF2 • EF2 has a diphthamide (see Figure 33.27) • Toxin-mediated ADP-ribosylation of EF2 allows it to bind GTP but makes it inactive in protein synthesis • One toxin molecule ADP-ribosylates many EF2s, so just a little is lethal!

15. Ricin from Ricinus communis (castor bean) • One of the most deadly substances known • A glycoprotein that is a disulfide-linked heterodimer of 30 kD subunits • The B subunit is a lectin (a class of proteins that binds specifically to glycoproteins & glycolipids) • Endocytosis followed by disulfide reduction releases A subunit, which catalytically inactivates the large subunit of ribosomes

16. Ricin A subunit mechanism • Ricin A chain specifically attacks a single, highly conserved adenosine near position 4324 in eukaryotic 28S RNA • N-glycosidase activity of A chain removes the adenosine base • Removal of this A (without cleaving the RNA chain) inactivates the large subunit of the ribosome • One ricin molecules can inactivate 50,000 ribosomes, killing the eukaryotic cell!