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Chapter 33

Chapter 33. Biochemistry 432/832 Protein Synthesis and Degradation December 05. Announcements. Central dogma of biochemistry: DNA --> RNA --> protein. Transcription Translation. Protein Synthesis and Degradation. Outline. 33.1 Ribosome Structure and Assembly

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Chapter 33

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  1. Chapter 33 Biochemistry 432/832 Protein Synthesis and Degradation December 05

  2. Announcements

  3. Central dogma of biochemistry: DNA --> RNA --> protein Transcription Translation Protein Synthesis and Degradation

  4. 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

  5. Proof that polypeptides (hemoglobin) grow by addition of new amino acid residues to the carboxyl end Direction of chain growth 0 min 4 min 7 min 16 min 60 min unlabeled labeled N-terminus C-terminus Dintzis experiment, 1961, rabbit reticulocytes labeled with leucine

  6. Ribosomes • Ribonucleoprotein particles • Found in the cytosol, mitochondria and chloroplasts of all cells • Move along mRNAs and synthesize proteins • Bind and orient mRNAs and aminoacyl-tRNAs • Organize interactions between codons and anticodons in aminoacyl-tRNAs • Catalyze the formation of peptide bonds between adjacent amino acid residues

  7. Ribosome Structure and Assembly • E. coli ribosome is 25 nm diameter, 2520 kDa in mass, and consists of two unequal subunits that dissociate at < 1mM Mg2+ • 30S subunit is 930 kDa with 21 proteins and a 16S rRNA • 50S subunit is 1590 kDa 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

  8. Organization of E.coli ribosomes • Ribosome Small Large • subunit subunit • Sedimentation 70S 30S 50S coefficient • Mass (kDa) 2520 930 1590 • Major RNAs 16S (1542 b) 23S (2904 b) • Minor RNAs 5S (120 b) • RNA mass 1664 560 1104 • RNA proportion 66% 60% 70% • Proteins 21 31 • Protein mass 857 370 487 • Protein 34% 40% 30%

  9. Ribosomal RNA operons in Escherichia coli. Precursor RNA is cleaved to generate 23S, 16S and 5S rRNA as well as tRNA

  10. The 16S rRNA fits within the 30S ribosomal subunit The rest of space (peripheral) is occupied by proteins

  11. Ribosomal Proteins • One of each per ribosome, except L7/L12 (same proteins that differ at N-terminus) 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 • No similarity (Lys, Arg-rich). The largest is S1 (557 aa) , the smallest is L34 (46 aa) • Overall fold of proteins was established less than a year ago

  12. Ribosome Assembly/Structure • If individual proteins and rRNAs are mixed, functional ribosomes will assemble • Structures of large and small subunits have been determined in 2000/2001 • A tunnel runs through the large subunit • Growing peptide chain is thought to thread through the tunnel during protein synthesis

  13. A 3D model for the E.coli ribosome Two views 30S 70S 50S

  14. Comparison of ribosomes and tRNAs (two tRNAs may be bound to a ribosome) E.coli ribosome Image reconstruction is based on cryoelectron microscopy

  15. 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 • Complexity and size of ribosomes are increased from prokaryotes to lower eukaryotes to higher eukaryotes • Conservation of overall RNA structure as well as specific segments of primary sequences

  16. Properties of Eukaryotic Ribosomes • Ribosome Small Large • subunit subunit • Sedimentation 80S 40S 60S coefficient • Mass (kDa) 4220 1400 2820 • Major RNAs 18S (1874 b) 28S (4718 b) • Minor RNAs 5.8S (160 b) 5S (120 b) • RNA mass 2520 700 1820 • RNA proportion 60% 50% 65% • Proteins 33 49 • Protein mass 1700 700 1000 • Protein 40% 50% 35%

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

  18. Basic steps in protein syn-thesis Two binding sites Transfer of a polypeptide to the amino group of amino acid carried by the tRNA in the A site Aminoacyl tRNA is at the A site; polypeptidyl-tRNA is at the P site One codon translocation; tRNA expulsion

  19. Location of tRNA binding sites in a ribosome

  20. 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

  21. Structure of N-formyl-methionyl-tRNA[Met] Differences with other tRNAs

  22. 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 • Initiation factor proteins, GTP, N-formyl-Met- tRNAfMet, mRNA and 30S ribosome form the 30S initiation complex

  23. Shine-Dalgarno sequences recognized by E.coli ribosomes

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

  25. Peptide chain initiation 30S subunit (IF-3:IF-1) binds mRNA IF-2 delivers the initiator f-Met-tRNA to the P site IF-2 dissociates from 30S subunit GTP hydrolysis is accompanied by IFs release and binding of the 50S subunit

  26. A structure of non-hydrolyzable analog of GTP Allowed separation of GTP binding from GTP hydrolysis

  27. 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-T recycles EF-Tu by exchanging GTP for GDP

  28. Elongation factors Factor Mass Molecules/Cell Function • EF-Tu 43 kDa 70,000 Binds tRNA-GTP • EF-Ts 74 kDa 10,000 Displaces GDP from EF-Tu • EF-G 77 kDa 20,000 Binds GTP, promotes translocation of ribosome

  29. Peptide chain elongation

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

  31. 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

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

  33. 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

  34. 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.

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

  36. Movement of tRNAs during translation

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

  38. 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

  39. Termination of protein synthesis

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