Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 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 Mg 2+ 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

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 5 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 6 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

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 9 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 10 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

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 12

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 13 Prokaryotic Initiation The initiator tRNA is one with a formylated methionine: f-Met-tRNA f Met It is only used for initiation, and regular Met-tRNA m Met 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)

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 15 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

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 17 Events of Initiation 30S subunit with IF-1 and IF-3 binds mRNA, IF-2, GTP and f-Met-tRNA f Met (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

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 20 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

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 22

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 23 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 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 & ) catalyzed by EF-G

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 28 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 29 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

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 32 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

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 34 Eukaryotic Initiation Begins with formation of ternary complex of eIF-2, GTP and Met-tRNA i Met 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- tRNA i Met 2) mRNA then adds with several other factors, forming the 48S initiation complex (Fig ) –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)

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 37 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-2  causes it to bind all available eIF-2B and sequesters it

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 39 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 40 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

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 42 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!

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 44 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

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 45 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!

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 46 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

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 48 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

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 50 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 51 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 52 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 53 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 54 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

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Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 56 Mitochondrial protein import Mitochondria have two membranes, and two spaces in between the membranes Signal sequences are N-terminal, positively charged regions of aa Form amphiphilic  -helices, positive on one side and uncharged, hydrophobic on the other Mitochondrial receptor will bind to preprotein

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 57 Protein Degradation Some protein degradation pathways are nonspecific - randomly cleaved proteins seem to be rapidly degraded However, there is also a selective, ATP- dependent pathway for degradation - the ubiquitin-mediated pathway Ubiquitin is a highly-conserved, 76 residue (8.5 kD) protein found widely in eukaryotes Proteins are committed to degradation by conjugation with ubiquitin

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 58 Ubiquitin and Degradation Three proteins involved: E 1, E 2 and E 3 E 1 is the ubiquitin-activating enzyme - it forms a thioester bond with C-terminal Gly of ubiquitin Ubiquitin is then transferred to a Cys-thiol of E 2, the ubiquitin-carrier protein Ligase (E 3 ) selects proteins for degradation. the E 2 -S~ubiquitin complex transfers ubiquitin to these selected proteins More than one ubiquitin may be attached to a protein target

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