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1 Corinthians 1:10
10 Now 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.
©2001 Timothy G. Standish
Protein
Synthesis
Timothy G. Standish, Ph. D.
©2001 Timothy G. Standish
Introduction
The Central Dogma
of Molecular Biology
Cell
Transcription
Translation
Reverse
transcription DNA
mRNA
Ribosome
Polypeptide
(protein)
Protein Synthesis
AMINE H
H
O
N
ACID
C
C
ANYTHING
R
H
Amino Acid
Alanine
OH
H
H
Serine
H
O
N
C
OH
H
C
H
H
H
H2O
H
H
C
H
C
H
O
C
N
OH
C
HO
N
H
O
C
C
C
H
H C
H
H HO
H
OH
C
H
H
N
H
H
C
O
©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
©2001 Timothy G. Standish
Transcription And Translation
In Prokaryotes
5’
3’
3’
5’
RNA
Pol.
Ribosome
mRNA
Ribosome
5’
©2001 Timothy G. Standish
Eukaryotic Gene Expression
Cytoplasm
Packaging
Degradation
DNA
Transcription
Transportation
Modification
RNA
RNA
Processing
mRNA G
G
AAAAAA
Nucleus
Export
Degradation etc.
AAAAAA
Translation
©2001 Timothy G. Standish
Translation - Initiation
fMet
Large
subunit
E
P
A
UAC
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
3’
Small mRNA
subunit
©2001 Timothy G. Standish
Translation - Elongation
Polypeptide
Arg
Met
Phe
Leu
Ser
Aminoacyl tRNA
Gly
Ribosome
E
P
A
CCA
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
3’
mRNA
©2001 Timothy G. Standish
Translation - Elongation
Polypeptide
Met
Phe
Leu
Ser
Gly
Arg
Aminoacyl tRNA
Ribosome
E
P
A
CCA UCU
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
3’
mRNA
©2001 Timothy G. Standish
Translation - Elongation
Polypeptide
Met
Phe
Leu
Ser
Gly
Arg
Ribosome
E
P
A
CCA UCU
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
3’
mRNA
©2001 Timothy G. Standish
Translation - Elongation
Polypeptide
Met
Phe
Leu
Ala
Ser
Gly
Aminoacyl tRNA
Arg
Ribosome
E
P
A
CCA
UCU
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
3’
mRNA
©2001 Timothy G. Standish
Translation - Elongation
Polypeptide
Met
Phe
Leu
Ser
Gly
Arg
Ribosome
E
Ala
P
A
UCU CGA
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA
3’
mRNA
©2001 Timothy G. Standish
Translation - Termination
Met
Phe
Leu
Ser
Gly
Polypeptide
Arg
Ala
Ribosome
Val
E
P
A
CGA
CGA
GCA...TAAAAAA
STOP
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT
3’
mRNA
©2001 Timothy G. Standish
Translation - Termination
Met
Phe
Leu
Ser
Gly
Polypeptide
Arg
Ala
Val
5’GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 3’
mRNA
STOP
©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
©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 ShineDalgarno 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
©2001 Timothy G. Standish
Prokaryote Initiation
Initiation Factor 3 is
needed to allow specific
binding between the
small subunit and the
mRNA translation
initiation site.
ShineDalgarno
sequence
5’
IF3
Start Codon
(May also be
GUG and UUG)
GAG...C-AGGAGG-NNNNNNNNNN-AUG---NNN---NNN---NNN---NNN---
3’
mRNA
©2001 Timothy G. Standish
Prokaryote Initiation
Initiation Factor 1 may
stabilize the initiation
complex
IF1
5’
GAG...C-AGGAGG-NNNNNNNNNN-AUG---NNN---NNN---NNN---NNN--mRNA
IF3
3’
Small subunit
©2001 Timothy G. Standish
Prokaryote Initiation
H O
O
C
C H2N
H H
C
H
S
H
H
C
H
OH
IF2
H
C
H
fMet
H
Methionine
Formyl
Methionine
UAC
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
Initiation Factor 2
binds to and mediates
the insertion of
initiator tRNA into the
initiation complex
IF1
5’
GAG...C-AGGAGG-NNNNNNNNNN-AUG---NNN---NNN---NNN---NNN--mRNA
IF3
3’
Small subunit
©2001 Timothy G. Standish
Prokaryote Initiation
Large
subunit
fMet
IF2
IF1
5’
UAC
GAG...C-AGGAGG-NNNNNNNNNN-AUG---NNN---NNN---NNN---NNN---
mRNA
IF3
3’
Small subunit
©2001 Timothy G. Standish
Prokaryote Initiation
IF2
fMet
P
A
E
5’
IF1
Large
subunit
UAC
GAG...C-AGGAGG-NNNNNNNNNN-AUG---NNN---NNN---NNN---NNN---
mRNA
3’
Small subunit
IF3
©2001 Timothy G. Standish
Prokaryote Initiation
fMet
P
A
E
5’
Large
subunit
UAC
GAG...C-AGGAGG-NNNNNNNNNN-AUG---NNN---NNN---NNN---NNN---
mRNA
3’
Small subunit
©2001 Timothy G. Standish
Met-tRNA
16 Pu
17
9
A
17:1
13 12 Py 10
X
C
1
2
3
4
5
6
U* 7
A
C
C
73
Necessary
72
A
71
70
69
68
67
Py 59A*
66
65 64 63 62 C
Py*
Pu
49 50 51 52 G T C
y
Py
G*
22 23 Pu 25
G
26
2020:120:2A
27
1
28
29
G
Signals for entry
30
G
31
G
into the P site
for formylation
47:16
47:15
43 44
42 45
41
C 46
47
40
C
47:1
39
C
38
Pu*
U
34 35 36
©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
©2001 Timothy G. Standish
Gly
P
P
Aminoacyl-tRNA
Synthetase
P
ATP
Gly
P
P
P
Aminoacyl-tRNA
Synthetase
Making
AminoacyltRNA
Pyrophosphate
Gly
P
Aminoacyl-tRNA
Synthetase
CCA
Gly
P
P
Making
AminoacyltRNA
Aminoacyl-tRNA
Synthetase
P
ATP
Gly
P
P
P
Aminoacyl-tRNA
Synthetase
Pyrophosphate
Gly
Aminoacyl-tRNA
Synthetase
Gly
Aminoacyl-tRNA
Synthetase
P
AMP
CCA
AminoacyltRNA
CCA
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
O
H
H N
C
Aminoacylation
of tRNA
O H
C
H O
H
H
R
C
H
O H
C
H
H
O H
H
C
H
C
O
N
C
3’
5’
H
H
O
O
H O
H
C
C
C
N
C
N
P
N
C
O
H
N
H
©2001 Timothy G. Standish
Class I Aminoacyl
Aminoacylation
O
tRNA Synthetases
attach amino acids to
of
tRNA
C
H
H N
Amino
acid
O
C
R
H
H
C
H
O H
C
H
H
O H
H
C
H
N
C
3’
5’
H
O
O
N
H
C
C
C
N
C
N
P
H O
tRNA
C
O
H
the 2’ carbon while
Class II attach to
the 3’carbon
C
O
H
N
H
©2001 Timothy G. Standish
Classification of AminoacyltRNA Synthetases
Aminoacyl-tRNA Class I - 2’ OH Class II - 3’ OH
Glu (a)
Gly (a b2
Synthetases
Gln (a)
Ala (a4
(ARS) may be
Arg (a)
Pro (a
mono or
Val (a)
Ser (a
multimeric.
Ile (a)
Thr (a
Two types of
Leu (a)
Asp (a ??
polypeptide
Met (a
Asn (a
chains are
Tyr (a
His (a
recognized:
(a
Lys (a
a and b.
©2001 Timothy G. Standish
©2001 Timothy G. Standish
Processing Eukaryotic mRNA
5’ Untranslated Region
3’ Untranslated Region
Protein Coding Region
5’5’ G
3’
Int. 11 Exon
Int. 23 Exon 3 AAAAA 3’
Exon 1Exon
Exon22 Exon
5’ Cap
3’ Poly A Tail
RNA processing achieves three things:
 Removal of introns
 Addition of a 5’ cap
 Addition of a 3’ tail
l
This signals the mRNA is ready to move out of
the nucleus and may control its lifespan in the
cytoplasm
©2001 Timothy G. Standish