Translation and post translational

1. Protein translation and post
translational modification

2. The central dogma

3. Transcription
Replication
Translation

4. A group of three nucleotides is called a codon
Protein 100 amino acids
Three nucleotides encode one amino acid
• There is a linear relationship between the information encoded
within DNA and the proteins which are synthesised using that
information
• Eukaryotic DNAcontains introns (non-coding) and exons (coding)
• Transcription of DNA-> pre-mRNA-> SPLICING (removal of
introns) -> mRNA
via mRNA
Exon DNA 300nt

5. UTR – untranslated region
polyA – protects mRNA
7MeG AAAAAn 5’cap 5’UTR coding region 3’UTR polyA
Structure of a typical mRNA
5’ “cap” (7-Methyl Guanosine) – entry site for ribosome

6. A
G
U
UUU
UUC
UUU
A
UUG
G
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
C
CUU
CUC
CUA
CUG
AUU
AUC
AU
A
AU
G
GUU
GUC
GUA
UCU
UCC
UCA
UCG
CCU
CCC
CCA
CCG
GCU
GCC
GCA
GCG
UAU
UAC
UAA
UAG
CAU
CAC
CAA
CAG
AAU
AAC
AAA
AAG
GAU
GAC
GAA
GAG
UGU
UGC
UGA
UGG
CGU
CGC
CGA
CGG
AGU
AGC
AGA
AGG
GGU
GGC
GGA
GGG
The genetic code (U=RNA, T=DNA)
first base “wobble”
position
second base
C A

7. A
G
U
U
G
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
C
Th
r
Th
r
Th
r
Th
r
There are 64 codons for 20 amino acids
second base
C A
“wobble”
position
first base

8. A
G
U
U
C A G
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
C
Start
(Met) =
AUG
Stop =
UAA
UAG
UGA
Rare
amino
fewer
codons
e.g. Met,
Trp
acids have

9. Reading a mRNA
[5’cap]CCGGACUACCUCUGGACCCCCTCCCCU
GCCACUCAAGCUGUCUUCAAAAn 3’
GUCCCCAACGCUGAGCCGAAACC AUG CAU
GGG CGC CUG AAG GUA AAG ACG UCG GCU
GAA GAG CAG GCA GAG GCC AAA AGG CUA
GAA CGA GAG AAG CUA AAG CUC UAC CAG
UGA
• Ribosome scans from 5’ end of mRNA (cap)
• Translation starts at first AUG, continues in frame immediately
succeeding triplet codon (CAU) and so on….. • Translation stops
at first in frame termination codon
, i.e. with

10. from DNA (pre-mRNA) and processed (spliced) in
the nucleus
mRNA is transported out of the nucleus
and translated in the 5’->3’ direction into
protein (N->C direction) in the
cytoplasm
and on the rough endoplasmic reticulum
The machinery for translating mRNA is
the ribosome, a large 2 subunit complex
Messenger RNA (mRNA) is transcribed

11. S = Svedberg unit of sedimentation
in a centrifuge (size)
Nobel Prize Chemistry 2009;
Yonath, Steitz and
Ramakrishnan

12. 3´
end
Anticodon
Amino acid(Met)
5´ end
UAC
AUG Codon (wobble)
transfer RNA:
ribosome
(>1
antiparallel
binding - like
DNA
for each aa) the
transporter of amino
acids to the
5’ 3’ mRNA

13. Aminoacyl tRNAsynthetases
Amino Acid
ATP
E
PPi
E-AMP-Amino Acid
AMP
Amino Acid
E
(Adenylated aa)
• important role in fidelity of translation
(selectivity for correct aa; hydrolysis of
incorrect aa-tRNA)
• one for each aa
Mutated in cancers, neuropathies, autoimmunity, metabolic disease

14. Translation: a) Initiation
Step 4: binding of 60S subunit
Step 1: dissociation of ribosome subunits (40S + 60S)
Step 2: assembly of preinitiation complex
containing Met-tRNA + eIFs + 40S subunit
Step 3: binding of mRNA to preinitiation complex
(simplified version; for details see Alberts of
the Cell, 4th ed.)
., Molecular Biology
et al

15. Translation:
Initiation
Factors:
eIF-2
40S
a) Initiation
Small Ribosomal
Subunit
mRNA
eIF4E, G
Step 2: assembly of pre-initiation complex
GTP
[5’cap]
• Only
3
’
Met-tRNA can bind to 40S subunit alone
initiator
• 40S subunit is primarily involved in tRNA and mRNA recognition

16. Translation:
eIF4E, G
eIF-2
40S
a) Initiation
mRNA
Step 3: binding of mRNA to preinitiation complex;
initiator Met binding sets the frame of the translation
• eIF4E and G bind to cap and are recognised by 40S/Met-
GTP
tRNA/eIF2
[5’cap] 3
’

17. eIF-2
40S
60S
a) Initiation
mRNA
GTP GDP
Translation:
Step 4: binding of 60S subunit
GTP GDP + Pi (ensures correct base pairing)
3
’

18. Translation:
40S
GTP GDP + Pi
a) Initiation
eIF-2
mRNA
Step 4: binding of 60S subunit
60S
GDP
3
’

19. Translation: b) Elongation
Step 1: binding of new tRNA carrying second
Step 2: catalysis of peptide bond between
the two amino acids by peptidyl transferase
Step 3: translocation of tRNA to P (peptidyl) site
and dissociation of first tRNA
amino acid to A (amino acyl) site

20. Translation: b) Elongation
mRNA
P siteA site
Step 1: binding of new tRNA to immediately adjacent A site in
frame with initiator Met
3
’

21. Translation: b) Elongation
Step 2: catalysis of peptide bond between the two
amino acids by peptidyl transferase on 60S subunit
mRNA
P site A site
P
T
3
’

22. Translation: b) Elongation
mRNA
P siteA site
Met
Arg
U A C G C U
Step 2 continued:
3
’

23. Translation: b) Elongation
mRNA
P siteA site
Met
Arg
U A C G C U
U G C G G A U G C G A U G G A A A U U C
movement of ribosome along mRNA using GTP
Step 3: translocation of peptidyl tRNA to P site
3
’
• Elongation Factors (EFs) are proteins that promote

24. Translation: b) Elongation
mRNA
P siteA site
U G C G G A U G C G A U G G A A A U U C
Met
Arg
G C U
Step 3 (continued): dissociation of first tRNA
3
’

25. Translation: b) Elongation
mRNA
P siteA site
U G C G G A U G C G A U G G A A A U U C
Met
Arg
G C U
New cycle with new step 1
3
’

26. Translation: b) Elongation
mRNA
P siteA site
U G C G G A U G C G A U G G A A A U U C
A C C
Met
Arg
Tr
p
New step 2
3
’

27. Translation: b) Elongation
3’mRNA
P site A site
A C C
Met
Arg
Tr
p
U G C G G A U G C G A U G G A A A U U C
New step 3

28. Translation: b) Elongation
mRNA
P site A site
A C C
Met
Arg
Tr
p
U G C G G A U G C G A U G G A A A U U C
Etc., etc.
3
’
EFs use the energy of GTP to enhance the efficiency and accuracy of
translation by providing “pauses” (e.g. GTP hydrolysis) that allow incorrect
base pairs to dissociate

29. Translation: c) Termination
Step 1: recognition of stop codon
Step 3: dissociation of release factors and ribosomes
Step 2: release of peptide chain

30. Translation:
mRNA
c) Termination
C G U G A A U C A G U A A U U C G A A U
Release factors (proteins,
not tRNAs) bind to empty
A site
Step 1: recognition of stop codon
RF
3
’
Out of frame UGA

31. Translation: c) Termination
mRNA
C G U G A A U C A G U A A U U C G A A U
RF
Step 2: release of peptide chain
Peptidyl transferase catalyses transfer of the completed protein
chain to water and releases it from the ribosome
3
’

32. Translation:
mRNA
40S
c) Termination
60S
C G U G A A U C A G U A A U U C G A A U
RF
3
’

33. Protein Translation animation
http://www.youtube.com/watch?v=5bLEDd-
PSTQ

34. Polyribosomes
Ribosomes do not work singly on a mRNA but in multiple
copies on the mRNA – a polyribosome – like a string of
beads
http://www.sumanasinc.com/webcontent/animations/content
/
polyribosomes.html

35. Translation speed
• Translation speed of each ribosome = 15 amino
• Multiple ribosomes processing simultaneously a of
synthesized protein - the number of protein
molecules produced in 1 min is ~4000
acids/sec
300 a.a. long protein, i.e. one ribosome every 30a.a.

36. Many antibiotics inhibit protein
synthesis in prokaryotes
• Translational machinery is complex, easily disrupted –
common target for antibiotics
• Antibiotics exploit differences between prokaryotic and
eukaryotic ribosomes and translation factors
• Antibiotics selectively inhibit prokaryotes
• Antibiotics are natural products of bacteria or fungi to
give them a selective advantage over other microbes

37. Antibiotics inhibiting protein synthesis
•Streptomycin
•Tetracycline
•Erythromycin
•Chloramphenicol
•Puromycin
Inhibits initiation
Inhibits aa-tRNA binding
Inhibits translocation
Inhibits peptidyl transferase
Terminates elongation
prematurely

38. INTRACELLULAR
COMPARTMENTS
Most cellular
compartments are bounded
by a membrane so the cell
needs a mechanism to
transfer proteins across
membranes
Protein synthesis takes
place in the cytoplasm

39. Secretory and transmembrane proteins are synthesised in the
Rough Endoplasmic Reticulum
Same ribosomes
on RER as for
cytoplasmic
proteins

40. Protein synthesis on
Rough Endoplasmic Reticulum (RER):
secreted and transmembrane proteins
First 20-24 amino acids = “signal sequence”
(hydrophobic amino acids, e.g. Leu, Ile, Phe, Trp, Tyr, Ala)
40S
60S
mRNA

41. Protein synthesis on RER
Step 1: recognition of signal
“Signal-Recognition Particle” (SRP),
halting translation
SRP
RER
lumen
mRNA
sequence by a protein-RNA complex
cytoplasm
Receptor
SRP
40S
60S

42. Protein synthesis on RER
Step 2: binding of SRP to a receptor at the
RER surface, translation resumes
SRP
RER
Receptor
SRP
40S
60S

43. Protein synthesis on RER
RER
Step 3: translocation
into the lumen of RER
Protein channel
40S
60S
SRP

44. Protein synthesis on RER
Cytoplasm
RER lumen
60S
40S
Transmembrane proteins
have an extra hydrophobic
sequence holding
them in the
membrane
Secreted protein

45. Protein synthesis on RER
Step 4: cleavage of
signal sequence by
signal peptidase (co-
translational) and
folding

46. Post-translational modification
•Hydroxylation (e.g. Collagen; Leitinger lecture)
- After synthesis most proteins are modified further before
they are fully functional
- Only 20 amino acids – cell uses post-translational
modifications (over 200) to increase diversity, including:
•Disulphide bond formation (e.g. insulin)
•Proteolytic cleavage (e.g. insulin -> A and B chains)
•Addition of carbohydrate (Glycosylation)
•Addition of phosphate (Phosphorylation)
•Addition of lipid groups (Prenylation, Acylation)

47. Insulin biosynthesis in
pancreatic β cells
Insulin undergoes extensive post-
translational modification along the
production pathway, including disulphide
bond formation in the ER and proteolytic
cleavage in the secretory vesicle to
produce active insulin

48. Glycosylation in the RER and Golgi complex
RER Golgi
PM
Initial N-glycosylation
Pre-assembled carbohydrate
chains N-linked to Asn of
(AsnXSer/Thr) “sequon”
Transport
vesicles
Glycan processing ( )
Carbohydrate chains
processed by trimming
followed by extension
Glycan on
lipid-linked
precursor