Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Eukaryotic Protein Synthesis See Figure 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 tRNA i Met but it is not formylated
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Eukaryotic Initiation Begins with formation of ternary complex of eIF-2, GTP and Met-tRNA i Met 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-tRNA i Met mRNA then adds with several other factors, forming the initiation complex (Fig ) Note that ATP is required! Proteins of the initiation complex apparently scan to find the first AUG (start) codon
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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!
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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 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
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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 SRP dissociates in a GTP-dependent process Protein synthesis resumes and protein passes into ER or into ER membrane; signal is cleaved
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company
Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company