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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 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 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
Robert F. Weaver Molecular Biology, 3rd edition Chapter 17 The Mechanism of Translation 1 - Initiation
Online Translation Animation http://www.brookscole.com/chemistry_d/templates/student_resources/shared_resources/animations/protein_synthesis/protein_synthesis.html
Modification Ex: Phosphorylation Glycosylation Ubiquitinylation Sumoylation Etc... Regulation of intracellularprotein levels Concentration of protein X Transcription Translation Degradation
Regulation of protein levels 100 Regulation (%) Translation Degradation Transcription 1981 2003
The first amino acid in prokaryotic proteins is not Met, but fMet. -Why? -And what about eukaryotes?
Peptidyl transferase activity (Chap 18) 50S 30S mRNA binding (Chap 17) 70S ribosome (holo complex)
Are intact 70S ribosomes stable particles? 50S 50S + 30S 30S 70S ribosome (holo complex) Dissociated subparticles
Sucrose/Glycerol/CsCl Gradient Density Ultracentrifugation
The Meselson & Stahl sedimentation assay Meselson & Stahl
Meselson sedimentation assay After centrifugation 30S 38S 50S 61S 70S 86S
Fig. 17.5 ← Negative control ← No dissociation ← Dissociation
Fig. 17.7 Ready for interaction with: IF2, mRNA & tRNA
Peptidyl transferase activity 50S 30S mRNA binding, when dissociated from 50S subcomplex 70S ribosome (holo complex) Recognises Shine-Dalgarno sequence (AGGAGGU)
(Not curriculum) Shine-Dalgarno is poorly conserved, but 3+ bases is enough for recognition
Fig. 17.7 Ready for interaction with: IF2, mRNA & tRNA
Fig. 17.13 IF2 IF1,3
Eukaryotes Eukaryotes don’t contain Shine-Dalgarno sequences - so how do eukaryotic ribosomes recognize mRNA?
Fig. 17.16 No Shine-Dalgarno sequence, eukaryotic ribosomes recognise 5’caps instead Scanning model
Kozak Sequence NN NNAUGG A G -5 -4 -3 -2 -1 +1 +2 +3 +4 Marilyn Kozak
Fig. 5.25 Site Directed Mutagenesis
Fig. 17.18 OOF Kozak1 Kozak2 proinsulin
Fig. 17.19 Only the first Kozak sequence is efficiently utilised
Fig. 17.21 Overexpressed Strain background (his4-) Thomas Donahue
How does the ribosome deal with melting secondary mRNA structures?
Fig. 17.20 Translation - + + -
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
Ras MAPK pathway Gef
Fig. 17.23 GDP sensitive M7-GDP sensitive
Fig. 17.24 Capped Uncapped w. CBP w/o CBP
Luciferase Pulse chase Luciferase Luciferase AAAAAA Luciferase Luciferase AAAAAA Effect of 5’ caps and polyA on mRNA stability and translatability?
Table 15.1 5’ caps and polyA tails increase stability and translatability of mRNA Synergy
Fig. 17.27 Only CAP IRES (eukaryotic Shine-Dalgarno) Only polyA CAP and polyA
Fig. 17.31 Toeprint:
Most translational regulation occurs at the initiation step Initiation is the rate limiting step in translation Regulation before elongation saves energy