Bài giảng Biochemistry 2/e - Chapter 33: Protein Synthesis and Degradation

Chapter 33Protein Synthesis and Degradationto accompanyBiochemistry, 2/ebyReginald Garrett and Charles GrishamAll 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 Outline33.1 Ribosome Structure and Assembly 33.2 Mechanics of Protein Synthesis 33.3 Protein Synthesis in Eukaryotes33.4 Inhibitors of Protein Synthesis 33.5 Protein Folding 33.6 Post-Translational Processing of Proteins 33.7 Protein Degradation 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 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 Little known of structures - these proteins are insoluble and difficult to study 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 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 propertiesMechanics 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 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) 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 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 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 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 - and note that thenomenclature for Fig. 33.15 is provided at the top of page 1103) 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 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 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 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 Phosphorylation is the key, as usualAt 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 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 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! 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 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! Protein Folding Proteins are assisted in folding by molecular chaperones - called chaperonins Hsp60 and Hsp70 are two main classes Hsp70 recognizes exposed, unfolded regions of new protein chains - especially hydrophobic regions It binds to these regions, apparently protecting them until productive folding reactions can occur The GroES-GroEL Complex The principal chaperonin in E. coliGroEL forms two stacked 7-membered rings of 60 kD subunits; GroES is a dome on the topNascent 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 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 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 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 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 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 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 Ubiquitin and Degradation Three proteins involved: E1, E2 and E3 E1 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 E2, the ubiquitin-carrier protein Ligase (E3) selects proteins for degradation. the E2-S~ubiquitin complex transfers ubiquitin to these selected proteins More than one ubiquitin may be attached to a protein target