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  1. 1. TranslationChapter 17: p.313-321MechanismTraffickingModification
  2. 2. Contents Players: codons, tRNA, ribosome Translation mechanism: initiation, elongation, termination Protein Trafficking Post-translational modifications
  3. 3. Overview of Protein SynthesisAnimation: Fig. 17.2b Fig. 17.3
  4. 4. RNA Exists in 3 forms:  mRNA (messenger) made by eukaryotic RNAP II  tRNA (transfer) made by eukaryotic RNAP III  rRNA (ribosomal) made by eukaryotic RNAP I made (transcribed) in the nucleus, used in the cytoplasm Single stranded Examples of RNA in a helix:  DNA-mRNA helix during transcription  tRNA structure allows H-bonding between nucleotides within the same tRNA strand
  5. 5. mRNA RNA nucleotides transcribed from DNA Codons: mRNA nucleotide triplets that code for amino acids
  6. 6. tRNA tRNA carries the amino acid to make the polypeptide chain Anticodon: complementary sequence on tRNA Codon Fig. 17.12
  7. 7. tRNA Structure 3’ end 3’ end Hydrogen bond base pairing between nucleotides of the same single strand of RNA (80 Cloverleaf nucleotides) L-shape Anticodon loop: H-bonds with mRNA codonFig. 17.13
  8. 8. tRNA Activation Enzyme aminoacyl-tRNA synthetase  attach appropriate amino acid to tRNA  catalyze covalent joining of amino acid to tRNA  tRNA + amino acid = aminoacyl- tRNA (aatRNA) 20 different aatRNA synthetases for each of the 20 different Fig. 17.14 amino acids tRNA molecule is reactivated many times (recycled)
  9. 9. Ribosome Structure  Ribonucleoprotein: composed of ribosomal RNA (rRNA) and protein  From picture:  RNA double stranded helix in turquoise, grey, orange, indigo  protein alpha helix in violet and navy blue
  10. 10. Ribosome Structure  made in nucleolus (eukaryote)  2 subunits:  large (turquoise colour)  small (lime green colour)
  11. 11. Ribosome Structure 2 subunits:  large (60S) and small (40S)  Final size is 80S S (Svedberg): a unit of measure of size based on how quickly an object sediments in a centrifuge Fig. 17.15b and c
  12. 12. Ribosome Binding Sites 1 mRNA binding site 3 tRNA binding sites  E, P, A Fig. 17.15b and c
  13. 13. Ribosome tRNA binding sites A site:  aminoacyl-tRNA site  holds the aatRNA carrying the next amino acid to be added P site:  peptidyl-tRNA site  holds the tRNA molecule carrying the growing polypeptide chain E site:  Exit site  where tRNA molecules leave the ribosome Fig. 17.15b and c
  14. 14. Translation Mechanism Initiation Elongation Termination Post-translational modifications Video clip: Translation overview
  15. 15. Initiation step 1: Ribosome finds and binds to the mRNA strandProkaryotes: mRNA transcript has a Shine-Dalgarno sequence rRNA on ribosome small subunit has a complementary section: anti Shine-Dalgarno sequenceEukaryotes Ribosome small subunit recognizes and bind to mRNA at 5’ cap Fig. 17.8
  16. 16. Initiation step 2: Ribosome locates translation start site Ribosome small subunit moves along 5’ leader of mRNA until reach translation start site (start codon AUG) Factors that help ribosome small subunit find start codon:  Prokaryotes: Initiation factors  Eukaryotes: Kozak sequence on the mRNA Fig. 17.8
  17. 17. Initiation step 3: Initator tRNA binds  Once ribosome small subunit reach AUG, initiator tRNA attaches AUG  H bonds forms between the mRNA codon and tRNA anticodonFig. 17.17
  18. 18. Initiation step 3: Initator tRNAbindsStarting amino acid: Prokaryotes: formyl-methionine (fmet) Eukaryotes: regular methionine (met)
  19. 19. Initiation step 4: Ribosome largesubunit binds Final position of initiator tRNA is in P site Forming complex requires = complete ribosome energy + initiator tRNA + mRNA at AUG
  20. 20. Elongation Overview N-terminus C-terminus (methionine) (is growing) Fig. 17.18
  21. 21. Elongation Step 1:Codon recognition Incoming aatRNA to A site H bonds form between the mRNA codon and tRNA anticodon Energy is required
  22. 22. Elongation Step 2: Peptide bond formation Ribosome catalyzes the formation of a peptide bond  between the amino acid in the P-site to the amino acid in the A-site  involves the carboxyl end of the polypeptide chain Result:  polypeptide chain is longer by one amino acid  polypeptide chain is transferred to tRNA at the A site
  23. 23. Elongation Step 3: Translocation Translocation requires energy Ribosome moves:  tRNA from P site to E site: leaves ribosome  tRNA from A site to P site: polypeptide returns to P site, ready for next polymerization A site is now empty  next aatRNA can bind
  24. 24. Unidirectional Translocation mRNA bound to tRNA Translocates by 1 codon (3 nucleotides) Ribosome reads mRNA 5’  3’ mRNA moved through ribosome unidirectionally
  25. 25. Termination At stop codon a protein called release factor binds to A site (no tRNA for stop codon, thus no aatRNA) Release factor:  adds of water molecule instead of amino acid to polypeptide  polypeptide hydrolyzed from tRNA in P site and released Translation complex disassembles Fig. 17.19
  26. 26. Video Clips PBS DNA: The Secret of Life (translation starts at 2:00)
  27. 27. Tutorials: Protein Synthesis
  28. 28. Polyribosomes  A single strand of mRNA can be used to make many copies of a polypeptide simultaneously  Polyribosomes: when 1 molecule of mRNA has multiple ribosomes simultaneously translating the mRNAFig. 17.20
  29. 29. Protein Synthesis in Prokaryotes  Prokaryotes don’t have a nucleus  How does that change protein synthesis? Fig. 17.22
  30. 30. Simultaneous transcription & translation in E. coli genes  The long fiber running from top to bottom is a segment of the E. coli chromosome.  Extending from it are polysomes (red arrow), each consisting of a backbone of mRNA to which the ribosomes are attached.  Each polysome is attached to the DNA fiber by RNA polymerase.  DNA is transcribed by RNAP molecules moving from top to bottom, and the growing mRNA molecules are translated by ribosomes moving in a proximal to distal direction.  In E. coli, then, and probably in all prokaryotes, the transcription of DNA into mRNA and the translation of mRNA into polypeptides (not visible here) are closely coordinated in both time and space. (Electron micrograph courtesy of O. L. Miller, Jr., B. A. Hamkalo, and C. A. Thomas, Jr.),Hamkalo.html
  31. 31. Schematic: Prokaryotic proteinsynthesis DNA = blue RNA = green Ribosome = red Which direction is transcription occurring? Explain how you know. Label the 5’ and 3’ end of one of the RNA strands. Given 3 ribosomes (A, B, C) that are translating off an RNA strand, which ribosome would have the A B longest polypeptide chain? C
  32. 32. Protein Trafficking
  33. 33. Protein TraffickingRibosome Type Free (unbound) BoundRibosome location Cytosolic side of the Cytoplasmin cell rough ERType of proteins Cytoplasmic proteins Secretory proteinssynthesized Plasma membrane Cytosol (transmembrane Organelles proteins / integral)Destination Secreted from cell (mitochondria, chloroplast, nucleus) (exocytosis) Lysosome
  34. 34. Cytosolic Proteins Water soluble Folding aided by chaperone proteins
  35. 35. Signal peptides: intracellularpostal codes All translation begins with free ribosomes Free ribosome synthesizes polypeptide revealing a signal in the N-terminus (signal peptide) that will direct the translation complex to the ER Signal peptide: 20 amino acids at the leading end (N-terminus) of a polypeptide that destines the polypeptide for the ER
  36. 36. Trafficking Secretory Proteins
  37. 37. Trafficking Secretory Proteins Signal recognition particle (SRP):  binds to signal peptide, halts translation  brings translation complex to SRP receptor on ER membrane  orient signal peptide into translocon SRP released, translation resumes Translocon:  acts as a channel protein  polypeptide threaded into the lumen (interior space) of the ER Signal peptide cleaved by signal peptidase When translation is complete, polypeptide released into ER
  38. 38. Endomembrane System  Proteins secreted into the ER leave the ER in vesicles  Vesicles transport to the Golgi body where post- translational modifications occurFig. 7.12, page 120.
  39. 39. Post-Translational Modifications Addition: of sugars, lipids, or phosphate groups Removal (Cleavage): of some amino acids (such as the methionine) or whole polypeptide chains. Polymerization: Two or more polypeptides may join to form a protein. Example: hemoglobin Folding
  40. 40. Example: Insulin
  41. 41. HW Questions Why does DNA need to be transcribed and then translated into proteins at all? What is the purpose behind the central dogma?
  42. 42. Summary and Comparisons Transcription versus Translation RNA Types
  43. 43. Comparison Transcription Translation InputGenetic Code Output Location Molecules used
  44. 44. Comparison Transcription Translation Input DNA mRNAGenetic Code Triplets Codons Output mRNA Polypeptide Ribosomes Location Nucleus (RER/cytoplasm) Ribosomes Molecules RNAP tRNA used RNA nucleotides Amino acids
  45. 45. Types of RNA