Genteknologi

Rasmus Hartmann-Petersen

IMB, August Krogh,Protein Science Section,Room 637, 6th floorPhone: 35 32 15 02E-mail: rhpetersen@aki.ku.dk

26S proteasome

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

Robert F. WeaverMolecular 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

Concentrationof protein X Translation Degradation

Regulation of intracellularprotein levels

Transcription

Modification

Ex:PhosphorylationGlycosylationUbiquitinylationSumoylationEtc...

1981 2003

Reg

ulat

ion

(%)

Translation

Transcription

Degradation

Regulation of protein levels

100

Prokaryotes

Fig. 17.2

Fig. 17.8

The first amino acid in prokaryotic proteins is not Met, but fMet.

-Why?

-And what about eukaryotes?

50S

30S

70S ribosome(holo complex)

Peptidyl transferase activity (Chap 18)

mRNA binding (Chap 17)

Are intact 70S ribosomes stable

particles?

50S

30S

70S ribosome(holo complex)

50S + 30S

Dissociated subparticles

Fig. 17.3

Sucrose/Glycerol/CsClGradient Density Ultracentrifugation

"Light medium"C, N12 14

"Heavy medium"C, N13 15

E. coli cultured in

The Meselson & Stahl sedimentation assay

Meselson & Stahl

"Light medium"C, N12 14

"Heavy medium"C, N13 15

E. coli cultured in

Meselson sedimentation assay

After centrifugation

30S50S70S

38S

86S61S

Fig. 17.4

Fig. 17.5

← Dissociation

← No dissociation

← Negative control

Fig. 17.7

Ready for interaction with:IF2, mRNA & tRNA

50S

30S

70S ribosome(holo complex)

Peptidyl transferase activity

mRNA binding, when dissociatedfrom 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 ribosomesrecognise 5’caps instead

Scanning model

Kozak Sequence

NN NNAUGGAG

-5 -4 -3 -2 -1 +1 +2 +3 +4

Marilyn Kozak

Site Directed Mutagenesis

Fig. 5.25

Fig. 17.17

Fig. 17.18

Kozak1 Kozak2 proinsulinOOF

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

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

Gef

Ras MAPK pathway

Isolation of the CAP binding protein (CBP)

+A B A BBinding

X-linking

A B

A

B

AB

Chemical Cross-linking and Electrophoresis

Fig. 17.23

M7-GDPsensitive

GDPsensitive

Fig. 17.24

Capped Uncapped

w. CBP

w/o CBP

Luciferase

Luciferase

AAAAAALuciferase

AAAAAALuciferase

Effect of 5’ caps and polyA on mRNA stability and translatability?

Pulse chase Luciferase

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

Fig. 17.27

IRES (eukaryoticShine-Dalgarno)

Only polyA

CAP and polyA

Only CAP

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:

Heme starvation:

DNA

Transcription Translation

mRNA globin hemoglobin

Hemeincorporation

DNA

Transcription Translation

mRNA globin hemoglobin

Hemeincorporation

Fig. 17.37a

Fig. 17.37b

Will not dissociate

Inhibits translation of most mRNAs, but stimulatesthe translation of ATF4 mRNA

Robert F. WeaverMolecular 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

DNA

RNA

Nascent chain(protein) ?

5’

Ribosome

5’

Transcription and translation are two separateprocesses in eukaryotes

-Nucleous, need for mRNA transport

-Introns, need for mRNA maturation (splicing)

Fig. 19.21

5’3’

Polysomes

40S 60S 80S

polysome

ATG AAAAAAA

Nascent protein

mRNA

++ EDTA

+ Mg2+

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

Ribosome structure

Fig. 19.4

30S50S

Anti-codon armof tRNA

Exit channel

Fig. 19.1

AP

E

A: Aminoacyl (Acceptor)P: PeptidylE: Exit

Fig. 19.1

tRNA structure

Fig. 19.24

Poor primary structure similarities, but similar clover leaf secodnary structure

Fits withCUU(leucine)

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: 3rd base degeneracy

Note: Double safety

Note: Similarity safty

Fig. 18.9