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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

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Eukaryotic Protein Synthesis

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eukaryotic protein synthesis
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
eukaryotic initiation
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
regulation of initiation
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
inhibitors of protein synthesis
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
diphtheria toxin
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!
protein translocation
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
protein translocation ii
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
prokaryotic protein transport
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
eukaryotic protein sorting
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
events at the er membrane
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