polypeptides and translation in prokaryotes and eukaryotes.ppt

lecture note on polypeptide

1. ©2001 Timothy G. Standish
1 Corinthians 1:10
10Now I beseech you, brethren, by
the name of our Lord Jesus Christ,
that ye all speak the same thing,
and that there be no divisions
among you; but that ye be perfectly
joined together in the same mind
and in the same judgement.

2. ©2001 Timothy G. Standish
Polypeptides:
Protein Synthesis
Timothy G. Standish, Ph. D.

3. ©2001 Timothy G. Standish
Ad Hominem
“The phrase arguments ad hominemtranslates literally as ‘argument
directed to the man.’ .... It is committed when, instead of trying to
disprove what is asserted one attacks the person who made the
assertion. ... This argument is fallacious, because the personal
character of an individual is logically irrelevant to the truth or
falsehood of what that individual says or the correctness or
incorrectness of that individual's argument. ... The way in which this
irrelevant argument may sometimes persuade is through the
psychological process of transference. Where an attitude of
disapproval toward a person can be evoked, it may possibly tend to
overflow the strictly emotional field and become disagreement with
what that person says. But this connection is only psychological, not
logical. Even the most wicked of men may sometimes tell the truth or
argue correctly.”
Copi I.M. 1953. Introduction to Logic," Macmillan Publishing Co: New
York NY, Seventh Edition, 1986, p 92.

4. mRNA
Transcription
Introduction
The Central Dogma
of Molecular Biology
Cell
Polypeptide
(protein)
Translation
Ribosome
Reverse
transcription
DNA

5. ©2001 Timothy G. Standish
ANYTHING
ACIDAMINE
Protein Synthesis
C
H
O
OHCN
H
H
H
C
HO H
Serine
C
H
O
OHCN
H
H
H
C
H H
Alanine
H
C
O
OHC
R
N
H
H
Amino Acid
H
2O
C
O
OHCN
H
H
H
C
HO H
C
H
O
CN
H
H
H
C
H H

6. ©2001 Timothy G. Standish
Requirements for Translation
Ribosomes -rRNA and Proteins
mRNA -Nucleotides
tRNA
–The RNA world theory might explain these three
components
Aminoacyl-tRNA Synthetase,
–A protein, thus a product of translation and cannot be
explained away by the RNA world theory
L Amino Acids
ATP -For energy
This appears to be an irreducibly complex system

7. ©2001 Timothy G. Standish
3’
5’
5’
3’
Transcription And Translation
In Prokaryotes
Ribosome
Ribosome
5’
mRNA
RNA
Pol.

8. ©2001 Timothy G. Standish
DNA
Cytoplasm
Nucleus
G AAAAAA
Export
Degradation etc.
G AAAAAA
Eukaryotic Gene Expression
G AAAAAA
RNA
Processing
mRNA
RNA
Transcription
Translation
Packaging
Modification
Transportation
Degradation

9. ©2001 Timothy G. Standish
Ribosomes
The Protein Factories
Ribosomes are the organelles in which
the mRNA nucleotide language is
translated into the protein language
The two ribosome subunits are made
up of ribosomal RNA (rRNA) and
proteins
Ribosomes in eukaryotes follow the
same basic plan as those in prokaryotes
although they are slightly larger

10. ©2001 Timothy G. Standish
Small subunit
E
AP
Large
subunit
GAG...C-AGGAGG-NNNNNNNNNN -AUG---NNN---NNN---NNN---NNN---
5’
mRNA
3’
Ribosome Structure
Aminoacyl-tRNA
binding site
Exit site
Peptidyl-tRNA
binding site

11. ©2001 Timothy G. Standish
Ribosome Structure
E. coliribosome at 25 A resolution from Frank et al. 1995. Biochem. Cell Biol. 73:757-767.
(see also Frank et al. 1995. Nature376:441-444.)
Yellow:
30S
subunit,
blue: 50S
subunit

12. ©2001 Timothy G. Standish
E. ColiRibosome In 4 D

13. ©2001 Timothy G. Standish
A
E
Large
subunit
P
Small
subunit
Translation -Initiation
fMet
UAC
GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
5’
mRNA
3’

14. ©2001 Timothy G. Standish
A
E
Ribosome P
Arg
Aminoacyl tRNA
Phe
Leu
Met
Ser
Gly
Polypeptide
CCA
Translation -Elongation
GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
5’
mRNA
3’

15. ©2001 Timothy G. Standish
A
E
Ribosome P
Phe
Leu
Met
Ser
Gly
Polypeptide
Arg
Aminoacyl tRNA
UCUCCA
Translation -Elongation
GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
5’
mRNA
3’

16. ©2001 Timothy G. Standish
A
E
Ribosome P
CCA
Arg
UCU
Phe
Leu
Met
Ser
Gly
Polypeptide
Translation -Elongation
GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
5’
mRNA
3’

17. ©2001 Timothy G. Standish
A
E
Ribosome P
Translation -Elongation
Aminoacyl tRNA
Ala
CCA
Arg
UCU
Phe
Leu
Met
Ser
Gly
Polypeptide
GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
5’
mRNA
3’

18. ©2001 Timothy G. Standish
A
E
Ribosome P
Translation -Elongation
Arg
UCU
Phe
Leu
Met
Ser
Gly
Polypeptide
CGA
Ala
GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
5’
mRNA
3’

19. ©2001 Timothy G. Standish
A
E
Ribosome P
Translation -Termination
CGA
Phe
Leu
Met
Ser
Gly
Polypeptide
Ala
Arg
Val
CGA
GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
5’
mRNA
3’
STOP

20. ©2001 Timothy G. Standish
Translation -Termination
Phe
Leu
Met
Ser
Gly
Polypeptide
Ala
Arg
Val
GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
5’
mRNA
3’
STOP

21. ©2001 Timothy G. Standish
Initiation
The small ribosome subunit binds to
the 5’ untranslated region of mRNA
The small ribosomal subunit slides
along the mRNA 5’ to 3’ until it finds a
start codon (AUG)
The initiator tRNA with methionine
binds to the start codon
The large ribosomal subunit binds with
the initiator tRNA in the P site

22. ©2001 Timothy G. Standish
Prokaryotic Initiation
Prokaryotic initiation involves the recognition of a
conserved sequence 10 bases upstream from the
start codon on mRNA
This conserved sequence is called the Shine-
Dalgarno sequence -5’…AGGAGG…3’
This sequence is complimentary to a highly
conserved sequence near the 16S rRNA 3’ end -
3’…UCCUCC…5’
The start codon is usually AUG, but less often
GUG and (least often) UUG are used

23. ©2001 Timothy G. Standish
Prokaryote Initiation
Shine-
Dalgarno
sequence
Start Codon
(May also be
GUG and UUG)
GAG...C-AGGAGG-NNNNNNNNNN -AUG---NNN---NNN---NNN---NNN---
5’
mRNA
3’
Initiation Factor 3 is
needed to allow specific
binding between the
small subunit and the
mRNA translation
initiation site.
IF3

24. ©2001 Timothy G. Standish
Initiation Factor 1 may
stabilize the initiation
complex
IF1
Small subunit
Prokaryote Initiation
GAG...C-AGGAGG-NNNNNNNNNN -AUG---NNN---NNN---NNN---NNN---
5’
mRNA
3’
IF3

25. ©2001 Timothy G. Standish
IF1
IF2
Small subunit
Prokaryote Initiation
fMet
UAC
GAG...C-AGGAGG-NNNNNNNNNN -AUG---NNN---NNN---NNN---NNN---
5’
mRNA
3’
IF3
Initiation Factor 2
binds to and mediates
the insertion of
initiator tRNA into the
initiation complex
Formyl Methionine is
modified with a formyl
group on the amine
group so that a peptide
bond can only be formed
at the carboxyl group
OH
H
H
O
H
H
H
2N
C
C
H
H
C
H
H
C
S
Methionine
H
H
H
H
H
CH
O
C
H
H
C
H
H
C
S
Formyl
Methionine
N
OH
O
C

26. ©2001 Timothy G. Standish
Large
subunit
IF1
IF2
Small subunit
fMet
UAC
GAG...C-AGGAGG-NNNNNNNNNN -AUG---NNN---NNN---NNN---NNN---
5’
mRNA
3’
IF3
Prokaryote Initiation

27. ©2001 Timothy G. Standish
Small subunit
E
AP
Large
subunit
fMet
UAC
GAG...C-AGGAGG-NNNNNNNNNN -AUG---NNN---NNN---NNN---NNN---
5’
mRNA
3’
IF1
IF2
IF3
Prokaryote Initiation

28. ©2001 Timothy G. Standish
Small subunit
E
AP
Large
subunit
fMet
UAC
GAG...C-AGGAGG-NNNNNNNNNN -AUG---NNN---NNN---NNN---NNN---
5’
mRNA
3’
Prokaryote Initiation

29. ©2001 Timothy G. Standish
Met-tRNA
U
*
9
26
2223Pu
16
12Py10
25
20:1
G
*
17:1
Pu
A
20:2
17
13
20
G
A
5051
656463
G
62
52
C
Pu
59
y
A
*
C
Py
T
49
39
41
42
31
29
28
Pu
*
43127
U
35
38
36
Py
*
34
4030
47:1
47:15
46
Py
47:16
45
44
47
73
C
C
A
70
71
72
66
67
68
69
3
2
1
7
6
5
4
C
C
G
G
CG
ACX
Signals for entry
into the P site
Necessary for formylation

30. ©2001 Timothy G. Standish
Aminoacyl-tRNA Synthetase
Aminoacyl-tRNA Synthetase enzymes
attach the correct amino acids to the
correct tRNA
This is an energy-consuming process
Aminoacyl-tRNA Synthetases recognize
tRNAs on the basis of their looped
structure, not by direct recognition of the
anticodon

31. Gly
Amino-
acyl-tRNA
Synthetase
Gly
Amino-
acyl-tRNA
Synthetase
P
Making
Aminoacyl-
tRNA
P
P
Pyrophosphate
P
P
P
ATP
Amino-
acyl-tRNA
Synthetase
P
Gly
CCA

32. Amino-
acyl-tRNA
Synthetase
Making
Aminoacyl-
tRNA
Gly
CCA
Aminoacyl-
tRNA
Note that the amino acid is not paired with the
tRNA on the basis of the anticodon. The correct
tRNA for a given amino acid is recognized on
the basis of other parts of
the molecule.
©1998 Timothy G. Standish
Gly
P
P
Pyrophosphate
P
P
P
ATP
Amino-
acyl-tRNA
Synthetase
Gly
Amino-
acyl-tRNA
Synthetase
P
P
AMP
Amino-
acyl-tRNA
Synthetase
Gly
CCA

33. ©2001 Timothy G. Standish
Aminoacylation
of tRNA
H
H
C
O
C
N
C
O
N
C
C
C
C
C
H
HO
H
H
O
P
OH
O
N
N
C
C
O
H H
H
N
H
H
3’
5’
H
CN
C
O
H
R
H
HO
H
OH

34. ©2001 Timothy G. Standish
Amino
acid
tRNA
Aminoacylation
of tRNA
3’
5’
H
H
C
O
C
N
C
O
N
C
C
C
C
C
H
HO
H
H
O
P
OH
O
N
N
C
C
O
H
H
N
H
H
H
CN
C
O
H
R
H
H
OH
Class I Aminoacyl
tRNA Synthetases
attach amino acids to
the 2’ carbon while
Class II attach to
the 3’carbon

35. ©2001 Timothy G. Standish
Classification of Aminoacyl-
tRNA Synthetases
Class I -2’ OH
Glu (a)
Gln (a)
Arg (a)
Val (a)
Ile (a)
Leu (a)
Met (a

Tyr (a
(a
Class II -3’ OH
Gly (ab
2
Ala (a
4
Pro (a
Ser (a
Thr (a
Asp (a??
Asn (a
His (a
Lys (a
Aminoacyl-tRNA
Synthetases
(ARS) may be
mono or
multimeric.
Two types of
polypeptide
chains are
recognized:
aand b.

36. ©2001 Timothy G. Standish

37. ©2001 Timothy G. Standish
3’5’ Exon 2 Exon 3Int. 2Exon 1Int. 1
Processing Eukaryotic mRNA
Protein Coding Region
3’ Untranslated Region5’ Untranslated Region
3’AAAAA
3’ Poly A Tail
5’G
5’ Cap
Exon 2Exon 3Exon 1
RNA processing achieves three things:
Removal of introns
Addition of a 5’ cap
Addition of a 3’ tail
lThis signals the mRNA is ready to move out of
the nucleus and may control its lifespan in the
cytoplasm

38. Gly
Amino-
acyl-tRNA
Synthetase
Gly
CCA
Amino-
acyl-tRNA
Synthetase
Gly
Amino-
acyl-tRNA
Synthetase
P
Amino-
acyl-tRNA
Synthetase
Making
Aminoacyl-
tRNA
P
P
Pyrophosphate
P
AMP
Gly
CCA
Aminoacyl-
tRNA
Note that the amino acid is not paired with the
tRNA on the basis of the anticodon. The correct
tRNA for a given amino acid is recognized on
the basis of other parts of
the molecule.
P
P
P
ATP
©1998 Timothy G. Standish