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Chapter 33

Chapter 33. Protein Synthesis and Degradation to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham.

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Chapter 33

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  1. Chapter 33 Protein Synthesis and Degradation to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777

  2. Outline • 33.1 Ribosome Structure and Assembly • 33.2 Mechanics of Protein Synthesis • 33.3 Protein Synthesis in Eukaryotes • 33.4 Inhibitors of Protein Synthesis • 33.5 Protein Folding • 33.6 Post-Translational Processing of Proteins • 33.7 Protein Degradation

  3. Ribosome Structure and Assembly • E. coli ribosome is 25 nm diameter, 2520 kD in mass, and consists of two unequal subunits that dissociate at < 1mM Mg2+ • 30S subunit is 930 kD with 21 proteins and a 16S rRNA • 50S subunit is 1590 kD with 31 proteins and two rRNAs: 23S rRNA and 5S rRNA • These ribosomes and others are roughly 2/3 RNA • 20,000 ribosomes in a cell, 20% of cell's mass

  4. Ribosomal Proteins • One of each per ribosome, except L7/L12 with 4 • L7/L12 identical except for extent of acetylation at N-terminus • Four L7/L12 plus L10 makes "L8" • Only one protein is common to large and small subunits: S20 = L26 • Variety of structures, still being characterized

  5. Ribosome Assembly/Structure • If individual proteins and rRNAs are mixed, functional ribosomes will assemble • Gross structures of large and small subunits are known - see Figure 33.3 • A tunnel runs through the large subunit • Growing peptide chain is thought to thread through the tunnel during protein synthesis

  6. Eukaryotic Ribosomes • Mitochondrial and chloroplast ribosomes are quite similar to prokaryotic ribosomes, reflecting their supposed prokaryotic origin • Cytoplasmic ribosomes are larger and more complex, but many of the structural and functional properties are similar • See Table 33.2 for properties

  7. Mechanics of Protein Synthesis • All protein synthesis involves three phases: initiation, elongation, termination • Initiation involves binding of mRNA and initiator aminoacyl-tRNA to small subunit, followed by binding of large subunit • Elongation: synthesis of all peptide bonds - with tRNAs bound to acceptor (A) and peptidyl (P) sites. See Figure 33.5 • Termination occurs when "stop codon" reached

  8. Prokaryotic Initiation • The initiator tRNA is one with a formylated methionine: f-Met-tRNAfMet • It is only used for initiation, and regular Met-tRNAmMet is used instead for Met addition • N-formyl methionine is first aa of all E.coli proteins, but this is cleaved in about half • A formyl transferase adds the formyl group (see Figure 33.8)

  9. More Initiation • Correct registration of mRNA on ribosome requires alignment of a pyrimidine-rich sequence on 3'-end of 16S RNA with a purine-rich part of 5'-end of mRNA • The purine-rich segment - the ribosome-binding site - is known as the Shine-Dalgarno sequence (see Figure 33.9) • Initiation factor proteins, GTP, N-formyl-Met- tRNAfMet, mRNA and 30S ribosome form the 30S initiation complex

  10. Events of Initiation • 30S subunit with IF-1 and IF-3 binds mRNA, IF-2, GTP and f-Met-tRNAfMet (Figure 33.10) • IF-2 delivers the initiator tRNA in a GTP-dependent process • Loss of the initiation factors leads to binding of 50S subunit • Note that the "acceptor site" is now poised to accept an incoming aminoacyl-tRNA

  11. The Elongation Cycle • The elongation factors are vital to cell function, so they are present in significant quantities (EF-Tu is 5% of total protein in E. coli • EF-Tu binds aminoacyl-tRNA and GTP • Aminoacyl-tRNA binds to A site of ribosome as a complex with 2EF-Tu and 2GTP • GTP is then hydrolyzed and EF-Tu:GDP complexes dissociate • EF-Ts recycles EF-Tu by exchanging GTP for GDP

  12. Peptidyl Transferase • This is the central reaction of protein synthesis • 23S rRNA is the peptidyl transferase! • The "reaction center" of 23S rRNA is shown in Figure 33.14 - these bases are among the most highly conserved in all of biology. • Translocation of peptidyl-tRNA from the A site to the P site follows (see Figures 33.12 & 33.15 ) catalyzed by EF-G

  13. The Role of GTP Hydrolysis • Three GTPs are hydrolyzed for each amino acid incorporated into peptide. • Hydrolysis drives essential conformation changes • Total of five high-energy phosphate bonds are expended per amino acid residue added - three GTP here and two in amino acid activation via aminoacyl-tRNA synthesis

  14. Peptide Chain Termination • Proteins known as "release factors" recognize the stop codon at the A site • Presence of release factors with a nonsense codon at A site transforms the peptidyl transferase into a hydrolase, which cleaves the peptidyl chain from the tRNA carrier

  15. 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

  16. 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)

  17. 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

  18. 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

  19. 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

  20. 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!

  21. 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

  22. 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!

  23. Protein Folding • Proteins are assisted in folding by molecular chaperones • Hsp60 (chaperonins) and Hsp70 are two main classes • Hsp70 recognizes exposed, unfolded regions of new protein chains - especially hydrophobic regions • It binds to these regions, protecting them until productive folding reactions can occur

  24. The GroES-GroEL Complex • The principal chaperonin in E. coli • GroEL forms two stacked 7-membered rings of 60 kD subunits; GroES is a dome on the top • Nascent protein apparently binds reversibly many times to the walls of the donut structure, each time driven by ATP hydrolysis, eventually adopting its folded structure, then being released from the GroES-GroEL complex • Rhodanese (as one example) requires hydrolysis of 130 ATP to reach fully folded state

  25. 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

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