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Genteknologi

Genteknologi. Rasmus Hartmann-Petersen. IMB, August Krogh, Protein Science Section, Room 637, 6th floor Phone: 35 32 15 02 E-mail: rhpetersen@aki.ku.dk. 26S proteasome. Bachelor, Master’s & PhD student positions available.

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Genteknologi

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  1. Genteknologi Rasmus Hartmann-Petersen IMB, August Krogh, Protein Science Section, Room 637, 6th floor Phone: 35 32 15 02 E-mail: rhpetersen@aki.ku.dk 26S proteasome

  2. Bachelor, Master’s & PhD student positions available The Protein Science Section at the Institute of Molecular Biology, August Krogh Building 1 Professor 4 Associate Professors 5 Laboratory Technicians 4 Post Docs 7 PhD students 5 Master’s Students People from: Denmark, Germany, Sweden, USA, Portugal, Switzerland, Russia

  3. Robert F. Weaver Molecular Biology, 3rd edition Chapter 17 The Mechanism of Translation 1 - Initiation

  4. Online Translation Animation http://www.brookscole.com/chemistry_d/templates/student_resources/shared_resources/animations/protein_synthesis/protein_synthesis.html

  5.  Modification Ex: Phosphorylation Glycosylation Ubiquitinylation Sumoylation Etc... Regulation of intracellularprotein levels Concentration of protein X Transcription  Translation   Degradation

  6. Regulation of protein levels 100 Regulation (%) Translation Degradation Transcription 1981 2003

  7. Prokaryotes

  8. Fig. 17.2

  9. Fig. 17.8

  10. The first amino acid in prokaryotic proteins is not Met, but fMet. -Why? -And what about eukaryotes?

  11. Peptidyl transferase activity (Chap 18) 50S 30S mRNA binding (Chap 17) 70S ribosome (holo complex)

  12. Are intact 70S ribosomes stable particles? 50S 50S + 30S 30S 70S ribosome (holo complex) Dissociated subparticles

  13. Fig. 17.3

  14. Sucrose/Glycerol/CsCl Gradient Density Ultracentrifugation

  15. The Meselson & Stahl sedimentation assay Meselson & Stahl

  16. Meselson sedimentation assay After centrifugation 30S 38S 50S 61S 70S 86S

  17. Fig. 17.4

  18. Fig. 17.5 ← Negative control ← No dissociation ← Dissociation

  19. Fig. 17.7 Ready for interaction with: IF2, mRNA & tRNA

  20. Peptidyl transferase activity 50S 30S mRNA binding, when dissociated from 50S subcomplex 70S ribosome (holo complex) Recognises Shine-Dalgarno sequence (AGGAGGU)

  21. (Not curriculum) Shine-Dalgarno is poorly conserved, but 3+ bases is enough for recognition

  22. Fig. 17.7 Ready for interaction with: IF2, mRNA & tRNA

  23. Fig. 17.13 IF2 IF1,3

  24. IF2 is a ribosome dependent GTPase

  25. Fig. 17.15

  26. Eukaryotes Eukaryotes don’t contain Shine-Dalgarno sequences - so how do eukaryotic ribosomes recognize mRNA?

  27. Fig. 17.16 No Shine-Dalgarno sequence, eukaryotic ribosomes recognise 5’caps instead Scanning model

  28. Kozak Sequence NN NNAUGG A G -5 -4 -3 -2 -1 +1 +2 +3 +4 Marilyn Kozak

  29. Fig. 5.25 Site Directed Mutagenesis

  30. Fig. 17.17

  31. Fig. 17.18 OOF Kozak1 Kozak2 proinsulin

  32. Fig. 17.19 Only the first Kozak sequence is efficiently utilised

  33. Fig. 17.21 Overexpressed Strain background (his4-) Thomas Donahue

  34. How does the ribosome deal with melting secondary mRNA structures?

  35. Fig. 17.20 Translation - + + -

  36. Fig. 17.26

  37. Fig. 17.22 G-protein: GTPase, GTP=active, GDP=inactive (eIF2) GAP: GTPase activating protein (eIF5) Inactivates G-protein GEF: GTP exchange factor (eIF2B) Activates G-protein GAP eIF2-GTP GEF (eIF2B) eIF2-GDP GDP GTP

  38. Ras MAPK pathway Gef

  39. Isolation of the CAP binding protein (CBP)

  40. Fig. 17.23 GDP sensitive M7-GDP sensitive

  41. Fig. 17.24 Capped Uncapped w. CBP w/o CBP

  42. Luciferase Pulse chase Luciferase Luciferase AAAAAA Luciferase Luciferase AAAAAA Effect of 5’ caps and polyA on mRNA stability and translatability?

  43. Table 15.1 5’ caps and polyA tails increase stability and translatability of mRNA Synergy

  44. Fig. 17.27 Only CAP IRES (eukaryotic Shine-Dalgarno) Only polyA CAP and polyA

  45. pIRES-GFP for easy expression & transfection control

  46. Isolation of scanning promoting factors

  47. Fig. 17.31 Toeprint:

  48. Fig. 17.32

  49. Most translational regulation occurs at the initiation step Initiation is the rate limiting step in translation Regulation before elongation saves energy

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