Eukaryotic Protein Synthesis

5. Eukaryotic Protein Synthesis See Figure 33.22 for the structure of the typical mRNA transcript • Note the 5'-methyl-GTP cap and the poly A tail • Initiation of protein synthesis in eukaryotes involves a family of at least 11 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

6. Eukaryotic Initiation • Begins with formation of ternary complex of eIF-2, GTP and Met-tRNAiMet • This binds to 40S ribosomal subunit:eIF-3:eIF4C complex to form the 40S preinitiation complex • Note no mRNA yet, so no codon association with Met-tRNAiMet • mRNA then adds with several other factors, forming the initiation complex (Fig. 33.23) • Note that ATP is required! • Proteins of the initiation complex apparently scan to find the first AUG (start) codon

9. 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 • Note discussion of elongation and termination on page 1112

11. Inhibitors of Protein Synthesis Two important purposes to biochemists • These inhibitors (Figure 33.26) 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

12. Diphtheria Toxin An NAD+-dependent ADP ribosylase • One target of this enzyme is EF-2 • EF-2 has a diphthamide (see Figure 33.27) • Toxin-mediated ADP-ribosylation of EF-2 allows it to bind GTP but makes it inactive in protein synthesis • One toxin molecule ADP-ribosylates many EF-2s, so just a little is lethal!

13. Protein Translocation An essential process for membrane proteins and secretory proteins • Such proteins are synthesized with a "leader peptide", aka a "signal sequence" of about 16-26 amino acids • The signal sequence has a basic N-terminus, a central domain of 7-13 hydrophobic residues, and a nonhelical C-terminus • The signal sequence directs the newly synthesized protein to its proper destination

14. Protein Translocation II Four common features • Proteins are made as preproteins containing domains that act as sorting signals • Membranes involved in protein translocation have specific receptors on their cytosolic faces • Translocases catalyze the movement of the proteins across the membrane with metabolic energy (ATP, GTP, ion gradients) essential • Preproteins bind to chaperones to stay loosely folded

15. Prokaryotic Protein Transport All non-cytoplasmic proteins must be translocated • The leader peptide retards the folding of the protein so that molecular chaperone proteins can interact with it and direct its folding • The leader peptide also provides recognition signals for the translocation machinery • A leader peptidase removes the leader sequence when folding and targeting are assured

16. Eukaryotic Protein Sorting Eukaryotic cells contain many membrane-bounded compartments • Most (but not all) targeting sequences are N-terminal, cleaveable presequences • Charge distribution, polarity and secondary structure of the signal sequence, rather than a particular sequence, appears to target to particular organelles and membranes • Synthesis of secretory and membrane proteins is coupled to translocation across ER membrane

17. Events at the ER Membrane • As the signal sequence emerges from the ribosome, a signal recognition particle (SRP) finds it and escorts it to the ER membrane • There it docks with a docking protein or SRP receptor - see Figure 33.31 • SRP dissociates in a GTP-dependent process • Protein synthesis resumes and protein passes into ER or into ER membrane; signal is cleaved