Genteknologi
description
Transcript of Genteknologi
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Genteknologi
Rasmus Hartmann-Petersen
IMB, August Krogh,Protein Science Section,Room 637, 6th floorPhone: 35 32 15 02E-mail: [email protected]
26S proteasome
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Bachelor, Master’s & PhDstudent positions available
The Protein Science Section at the Institute of Molecular Biology, August Krogh Building
1 Professor4 Associate Professors5 Laboratory Technicians4 Post Docs7 PhD students5 Master’s Students
People from: Denmark, Germany, Sweden, USA, Portugal, Switzerland, Russia
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Robert F. WeaverMolecular Biology, 3rd edition
Chapter 17
The Mechanism of Translation 1- Initiation
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Online Translation Animation
http://www.brookscole.com/chemistry_d/templates/student_resources/shared_resources/animations/protein_synthesis/protein_synthesis.html
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Concentrationof protein X Translation Degradation
Regulation of intracellularprotein levels
Transcription
Modification
Ex:PhosphorylationGlycosylationUbiquitinylationSumoylationEtc...
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1981 2003
Reg
ulat
ion
(%)
Translation
Transcription
Degradation
Regulation of protein levels
100
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Prokaryotes
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Fig. 17.2
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Fig. 17.8
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The first amino acid in prokaryotic proteins is not Met, but fMet.
-Why?
-And what about eukaryotes?
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50S
30S
70S ribosome(holo complex)
Peptidyl transferase activity (Chap 18)
mRNA binding (Chap 17)
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Are intact 70S ribosomes stable
particles?
50S
30S
70S ribosome(holo complex)
50S + 30S
Dissociated subparticles
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Fig. 17.3
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Sucrose/Glycerol/CsClGradient Density Ultracentrifugation
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"Light medium"C, N12 14
"Heavy medium"C, N13 15
E. coli cultured in
The Meselson & Stahl sedimentation assay
Meselson & Stahl
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"Light medium"C, N12 14
"Heavy medium"C, N13 15
E. coli cultured in
Meselson sedimentation assay
After centrifugation
30S50S70S
38S
86S61S
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Fig. 17.4
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Fig. 17.5
← Dissociation
← No dissociation
← Negative control
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Fig. 17.7
Ready for interaction with:IF2, mRNA & tRNA
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50S
30S
70S ribosome(holo complex)
Peptidyl transferase activity
mRNA binding, when dissociatedfrom 50S subcomplex
Recognises Shine-Dalgarno sequence(AGGAGGU)
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(Not curriculum)
Shine-Dalgarno is poorly conserved, but 3+ bases is enough for recognition
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Fig. 17.7
Ready for interaction with:IF2, mRNA & tRNA
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Fig. 17.13
IF2
IF1,3
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IF2 is a ribosome dependent GTPase
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Fig. 17.15
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Eukaryotes
Eukaryotes don’t contain Shine-Dalgarno sequences- so how do eukaryotic ribosomes recognize mRNA?
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Fig. 17.16
No Shine-Dalgarno sequence, eukaryotic ribosomesrecognise 5’caps instead
Scanning model
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Kozak Sequence
NN NNAUGGAG
-5 -4 -3 -2 -1 +1 +2 +3 +4
Marilyn Kozak
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Site Directed Mutagenesis
Fig. 5.25
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Fig. 17.17
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Fig. 17.18
Kozak1 Kozak2 proinsulinOOF
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Fig. 17.19
Only the first Kozak sequence is efficiently utilised
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Fig. 17.21
Overexpressed
Strain background (his4-)
Thomas Donahue
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How does the ribosome deal with
melting secondary mRNA structures?
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Fig. 17.20
+
+
-
-Translation
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Fig. 17.26
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Fig. 17.22
GAP
GEF (eIF2B)
eIF2-GDP
GTP
GDP
G-protein: GTPase, GTP=active, GDP=inactive (eIF2)GAP: GTPase activating protein (eIF5) Inactivates G-proteinGEF: GTP exchange factor (eIF2B) Activates G-protein
eIF2-GTP
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Gef
Ras MAPK pathway
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Isolation of the CAP binding protein (CBP)
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+A B A BBinding
X-linking
A B
A
B
AB
Chemical Cross-linking and Electrophoresis
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Fig. 17.23
M7-GDPsensitive
GDPsensitive
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Fig. 17.24
Capped Uncapped
w. CBP
w/o CBP
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Luciferase
Luciferase
AAAAAALuciferase
AAAAAALuciferase
Effect of 5’ caps and polyA on mRNA stability and translatability?
Pulse chase Luciferase
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5’-CAP 3’-polyA mRNA T½ (min) Luciferase Activity (U/mg)
- - 31 2941
- + 44 4480
+ - 53 62595
+ + 100 1331917
Table 15.1
5’ caps and polyA tails increase stability and translatability of mRNA
Synergy
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Fig. 17.27
IRES (eukaryoticShine-Dalgarno)
Only polyA
CAP and polyA
Only CAP
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pIRES-GFP for easy expression & transfection control
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Isolation of scanning promoting factors
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Fig. 17.31
Toeprint:
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Fig. 17.32
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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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Synthesis of hemoglobin
Heme abundance:
Heme starvation:
DNA
Transcription Translation
mRNA globin hemoglobin
Hemeincorporation
DNA
Transcription Translation
mRNA globin hemoglobin
Hemeincorporation
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Fig. 17.37a
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Fig. 17.37b
Will not dissociate
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Inhibits translation of most mRNAs, but stimulatesthe translation of ATF4 mRNA
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Robert F. WeaverMolecular Biology, 3rd edition
Chapters 18 & 19
The Mechanism of Translation 2-Elongation & Termination
Ribosomes & Transfer RNA
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Transcription and translation are coupled in prokaryotes
-No nucleous, i.e. ribosomes and RNA polymerase in same compartment
-No introns, i.e. primary transcript = mature mRNA
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Fig. 19.22
DNA
RNA
Nascent chain(protein) ?
5’
Ribosome
5’
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Transcription and translation are two separateprocesses in eukaryotes
-Nucleous, need for mRNA transport
-Introns, need for mRNA maturation (splicing)
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Fig. 19.21
5’3’
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Polysomes
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40S 60S 80S
polysome
ATG AAAAAAA
Nascent protein
mRNA
++ EDTA
+ Mg2+
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40S
60S
Abs
orb
ance
(2
54 n
m)
SedimentationTop Bottom
LDH
act
ivity
(%
)
10
20
30
40
50
60
70
80
90
100
0.25
0.50
0.75
1.00+EDTA
anti-Nac1
80S
60S40S
Polysomes
Abs
orb
ance
(2
54 n
m)
SedimentationTop Bottom
0.05
0.10
0.15
0.20
LDH
act
ivity
(%
)
10
20
30
40
50
60
70
80
90
100
-EDTA
anti-Nac1
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Ribosome structure
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Fig. 19.4
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30S50S
Anti-codon armof tRNA
Exit channel
Fig. 19.1
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AP
E
A: Aminoacyl (Acceptor)P: PeptidylE: Exit
Fig. 19.1
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tRNA structure
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Fig. 19.24
Poor primary structure similarities, but similar clover leaf secodnary structure
Fits withCUU(leucine)
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Fig. 19.26
Tertiary tRNA structure
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The genetic code
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Fig. 18.6
Note: 3rd base degeneracy
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Fig. 18.7
Non Watson-Crick (wobble) base pairing
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Fig. 18.8
Phe Leu
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The genetic code is not a frozen accident
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Fig. 18.6
Note: 3rd base degeneracy
Note: Double safety
Note: Similarity safty
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Fig. 18.9