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
Regulation of intracellular
protein levels
Transcription  Translation 
Concentration
of protein X

Modification
Ex:
Phosphorylation
Glycosylation
Ubiquitinylation
Sumoylation
Etc...
 Degradation
Regulation of protein levels
Regulation (%)
100
Translation
Degradation
Transcription
1981
2003
Prokaryotes
Fig. 17.2
Fig. 17.8
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
Fig. 17.3
Sucrose/Glycerol/CsCl
Gradient Density Ultracentrifugation
The Meselson & Stahl sedimentation assay
E. coli cultured in
"Light medium"
12
14
C, N
"Heavy medium"
13
15
C, N
Meselson & Stahl
Meselson sedimentation assay
After
c ultured in
c olicentrifugation
E.
"Light m edium "
14
12
C, N
30S
50S
70S
"Heavy m edium "
15
13
C, N
38S
61S
86S
Fig. 17.4
Fig. 17.5
← Negative control
← No dissociation
← Dissociation
Fig. 17.7
Ready for interaction with:
IF2, mRNA & tRNA
Peptidyl transferase activity
50S
30S
70S ribosome
(holo complex)
mRNA binding, when dissociated
from 50S subcomplex
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
IF2 is a ribosome dependent GTPase
Fig. 17.15
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
A
G
NN NNAUGG
-5 -4 -3 -2 -1 +1 +2 +3 +4
Marilyn Kozak
Fig. 5.25
Site Directed Mutagenesis
Fig. 17.17
Fig. 17.18
Kozak1
OOF
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. 17.26
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
Isolation of the CAP binding protein (CBP)
Chemical Cross-linking a nd Electrophoresis
+
A
Binding
B
A
B
X-linking
A
AB
A
B
B
Fig. 17.23
GDP
sensitive
M7-GDP
sensitive
Fig. 17.24
Capped
w. CBP
w/o CBP
Uncapped
Effect of 5’ caps and polyA on mRNA stability and translatability?
Pulse chase
Luciferase
Luciferase
Luciferase
AAAAAA
Luciferase
Luciferase
AAAAAA
Table 15.1
5’ caps and polyA tails increase stability and translatability of mRNA
5’-CAP
3’-polyA
mRNA T½ (min)
Luciferase Activity (U/mg)
-
-
31
2941
-
+
44
4480
+
-
53
62595
+
+
100
1331917
Synergy
Fig. 17.27
Only CAP
IRES (eukaryotic
Shine-Dalgarno)
Only polyA
CAP and polyA
pIRES-GFP for easy expression & transfection control
Isolation of scanning promoting factors
Fig. 17.31
Toeprint:
Fig. 17.32
Most translational regulation occurs
at the initiation step
Initiation is the rate limiting step in translation
Regulation before elongation saves energy
Synthesis of hemoglobin
Heme abundance:
Transcription
Translation
mRNA
globin
DNA
Heme
incorporation
hemoglobin
Heme starvation:
Transcription
Translation
mRNA
globin
DNA
Heme
incorporation
hemoglobin
Fig. 17.37a
Fig. 17.37b
Will not dissociate
Inhibits translation of most mRNAs, but stimulates
the translation of ATF4 mRNA
Robert F. Weaver
Molecular Biology, 3rd edition
Chapters 18 & 19
The Mechanism of Translation 2
-Elongation & Termination
Ribosomes & Transfer RNA
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
Fig. 19.22
Nascent chain
(protein) ?
RNA
Ribosome
5’
5’
DNA
Transcription and translation are two separate
processes in eukaryotes
-Nucleous, need for mRNA transport
-Introns, need for mRNA maturation (splicing)
Fig. 19.21
3’
5’
Polysomes
+ Mg2+
+
+ EDTA
40S
60S
80S
Nascent protein
mRNA
AAAAAAA
ATG
polysome
80S
-EDTA
Polysomes
0.20
LDH activity (%)
90
80
70
60
50
40
Absorbance (254 nm)
100
30
0.15
0.10
60S
40S
0.05
20
10
Top
Sedimentation
Bottom
anti-Nac1
+EDTA
1.00
LDH activity (%)
90
80
70
60
50
40
30
Absorbance (254 nm)
100
0.75
60S
0.50
40S
0.25
20
10
Top
Sedimentation
Bottom
anti-Nac1
Ribosome structure
Fig. 19.4
Fig. 19.1
50S
30S
Exit channel
Anti-codon arm
of tRNA
Fig. 19.1
A: Aminoacyl (Acceptor)
P: Peptidyl
E: Exit
A
P
E
tRNA structure
Fig. 19.24
Fits with
CUU
(leucine)
Poor primary structure similarities, but similar clover leaf secodnary structure
Fig. 19.26
Tertiary tRNA structure
The genetic code
Fig. 18.6
Note: 3rd base degeneracy
Fig. 18.7
Non Watson-Crick (wobble) base pairing
Fig. 18.8
Phe
Leu
The genetic code is not a frozen accident
Fig. 18.6
Note: Similarity safty
Note: Double safety
Note: 3rd base degeneracy
Fig. 18.9