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Chapter 33

Chapter 33. Biochemistry 432/832 Protein Synthesis and Degradation December 10 and 12. Announcements. 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

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Chapter 33

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  1. Chapter 33 Biochemistry 432/832 Protein Synthesis and Degradation December 10 and 12

  2. Announcements

  3. 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

  4. Location of tRNA binding sites in a ribosome

  5. Peptide chain elongation

  6. Reaction of the tRNA-linked peptidyl chain with the a-amino group of an adjacent aminoacyl-tRNA - no energy is required for activation

  7. Peptidyl Transferase • This is the central reaction of protein synthesis • 23S rRNA is the peptidyl transferase! • The "reaction center" of 23S rRNA - the catalytic bases are among the most highly conserved in all of biology. • Translocation of peptidyl-tRNA from the A site to the P site follows

  8. Ribosome is a ribozyme (catalytic rRNA) Catalytic center is located in the 50S particle The peptidyl transferase center of 23S rRNA

  9. Ribosome is ribozyme Puglisi JD, Blanchard SC, Green R, Nat Struct Biol 2000 Oct;7(10):855 Approaching translation at atomic resolution. Atomic resolution structures of 50S and 30S ribosomal particles have recently been solved by X-ray diffraction In the 50S structure, the active site for peptide bond formation was localized and found to consist of RNA. The ribosome is thus a ribozyme In the 30S structure, tRNA binding sites were located The 30S subunit particle has three globular domains, and relative movements of these domains may be required for translocation of the ribosome during protein synthesis

  10. Ribosome structure Yusupov MM, Yusupova GZ, Baucom A, Lieberman K, Earnest TN, Cate JH, Noller HF. Science 2001 Mar 29 Crystal Structure of the Ribosome at 5.5 A Resolution. We describe the crystal structure of the complete Thermus thermophilus 70S ribosome containing bound mRNA and tRNAs at 5.5 A resolution. All of the 16S, 23S and 5S rRNA chains, the A-, P- and E-site tRNAs, and most of the ribosomal proteins can be fitted to the electron density map. The core of the interface between the 30S small subunit and the 50S large subunit, where the tRNA substrates are bound, is dominated by RNA, with proteins located mainly at the periphery, consistent with ribosomal function being based on rRNA. In each of the three tRNA binding sites, the ribosome contacts all of the major elements of tRNA, providing an explanation for the conservation of tRNA structure. The tRNAs are closely juxtaposed with the intersubunit bridges, in a way that suggests coupling of the 20 to 50 A movements associated with tRNA translocation with intersubunit movement.

  11. Movement of tRNAs during translation

  12. Relative positions of tRNA molecules in a ribosome during peptidyl transfer and translocation

  13. 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

  14. Termination of protein synthesis

  15. The ribosome life cycle

  16. Polyribosomes (polysomes) - multiple ribosomes translating the same mRNA molecule

  17. The E.coli trp operon Transcription 4 min Translation of the first two genes 2.5 min

  18. Transcription and translation occur at the same time

  19. Eukaryotic Protein Synthesis the structure of the typical mRNA transcript • the 5'-methyl-GTP cap - essential for ribosomal binding, also enhances stability of mRNA • and the poly A tail - enhances stability, enhances translation efficiency • 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

  20. Structure of eukaryotic mRNA Cap 5’-UTR Coding region 3’-UTR Poly-A Termination (AUG, UGA, UAA) Initiation (AUG)

  21. Eukaryotic Initiation • No Shine-Dalgarno sequences • 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 43S preinitiation complex • No mRNA yet, so no codon association with Met-tRNAiMet • mRNA then adds with several other factors, forming the initiation complex • ATP is required!

  22. Eukaryotic Initiation • Proteins of the initiation complex apparently scan to find the first AUG (start) codon • Not all AUG codons can serve as initiators • Kozak consensus sequence: Pu-Py-Py-A-U-G-Pu (the best at ACCAUGG)

  23. Initiation of translation in eukaryotes

  24. Initiation of translation in eukaryotes - role of factor eIF4G as a multipurpose adapter to organize 40S, cap, polyA and other factors

  25. 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

  26. eIF2 is regulated through reversible phosphorylation of a Ser residue

  27. Elongation of peptide chain in eukaryotes Very similar to prokaryotes Elongation factor EF1A is equivalent to EF-Tu binds aminoacyl-tRNA and GTP Elongation factor EF1B is equivalent to EF-Ts exchanges GDP with GTP in EF1A Elongation factor EF2 is equivalent to EF-G involved in ribosome translocation (GTP-dependent)

  28. Termination of protein synthesis in eukaryotes Only one release factor (RF), homodimer of 55 kDa subunits (three RFs are in prokaryotes) Binds at the A site GTP-dependent

  29. Inhibitors of Protein Synthesis Two important purposes to biochemists • These inhibitors 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

  30. Protein synthesis inhibitors Inhibitor Organism Mode of action Initiation Kasugamycin P Inhibits f-Met-tRNA binding Streptomycin P Prevents initiation Elongation (aminoacyltRNA binding) Tetracycline P Inhibits aminoacyl-tRNA binding Streptomycin P Codon misreading Elongation (peptide bond formation) Chloramphenicol P Binds 50S, blocks peptidyl transf Cycloheximide E Inhib. transloc. of peptidyl-tRNA

  31. Protein synthesis inhibitors Inhibitor Organism Mode of action Elongation (translocation) Diphteria toxin E Inact. eIF2 thru ADP-ribosylati Premature termination Puromycin P, E Aminoacyl-tRNA analog Acts as a peptidyl acceptor Prevents further peptide elong. Ribosome inactivation Ricin E Catalytic inactiv. of 28S rRNA

  32. Structures of common antibiotics

  33. Diphtheria Toxin An NAD+-dependent ADP ribosylase • One target of this enzyme is EF-2 • EF-2 has a diphthamide • 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!

  34. Diphtheria toxin - catalysis of NAD+-dependent ADP-ribosylation of proteins

  35. Ricin from Ricinus communis (castor bean) • One of the most deadly substances known • A glycoprotein that is a disulfide-linked heterodimer of 30 kDa 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

  36. 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!

  37. Protein Folding • Translation is linked to transcription, proteins begin to fold while being synthesized by ribosomes • 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

  38. Pathways of protein folding

  39. The GroES-GroEL Complex • The principal chaperonin in E. coli • GroEL forms two stacked 7-membered rings of 60 kDa 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

  40. GroEL-GroES complex GroES GroEL

  41. Protein Translocation An essential process for membrane proteins and secretory proteins • Such proteins are synthesized with a "leader peptide", 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

  42. Protein Translocation 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

  43. 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

  44. 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

  45. Eukaryotic Signal Peptides Postulated in 1970 by Hunter Blobel (1999 Nobel prize) • N-terminal ER signal peptide (C-terminal ER-retention signal) • N-terminal mitochondrial signal peptide (chloroplast signals are similar) • Nuclear localization signal (not cleaved)

  46. 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 • SRP dissociates in a GTP-dependent process • Protein synthesis resumes and protein passes into ER or into ER membrane; signal is cleaved

  47. Synthesis of a eukaryotic integral membrane protein and its translocation via the ER

  48. 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 kDa) protein found widely in eukaryotes • Proteins are committed to degradation by conjugation with ubiquitin

  49. 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

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