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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 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 Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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 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 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 Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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 Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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! Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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 1626 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 Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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 Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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 Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham Eukaryotic Protein Sorting Eukaryotic cells contain many membrane-bounded compartments • Most (but not all) targeting sequences are Nterminal, 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 Copyright © 1999 by Harcourt Brace & Company Biochemistry 2/e - Garrett & Grisham 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 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
