Transcript 28_translation2

From Gene to Phenotype- part 3
DNA
TRANSCRIPTION
1 RNA is transcribed
DNA
from a DNA template.
3
5
RNA
transcript
RNA
polymerase
RNA PROCESSING
Exon
2 In eukaryotes, the
mRNA
RNA transcript (premRNA) is spliced and
modified to produce
mRNA, which moves
from the nucleus to the
cytoplasm.
RNA transcript
(pre-mRNA)
Intron
Aminoacyl-tRNA
synthetase
NUCLEUS
Amino
acid
tRNA
FORMATION OF
INITIATION COMPLEX
CYTOPLASM 3 After leaving the
nucleus, mRNA attaches
to the ribosome.
mRNA
AMINO ACID ACTIVATION
4
Each amino acid
attaches to its proper tRNA
with the help of a specific
enzyme and ATP.
Growing
polypeptide
Activated
amino acid
Ribosomal
subunits
polypeptide
5
TRANSLATION
A succession of tRNAs
add their amino acids to
the polypeptide chain
Anticodon
as the mRNA is moved
through the ribosome
one codon at a time.
(When completed, the
polypeptide is released
from the ribosome.)
5
E
A
AAA
UG GU U U A U G
Codon
Ribosome
Lecture Outline 11/9/05
• Review translation:
– Initiation, elongation, termination
– EPA model
• Post-translational modification of
polypeptides
• Signal sequences
• Mutations (again)
Exam 3 is next Monday. It will cover mitosis and
meiosis, DNA synthesis, transcription, translation,
genetics of viruses.
(chapters 12, 13, 16, 17, part of 18 (to page 345))
Translation: overview
TRANSCRIPTION
DNA
mRNA
Ribosome
TRANSLATION
Polypeptide
Amino
acids
Polypeptide
Ribosome
tRNA with
amino acid
attached
Gly
The ribosome is the
machine that builds the
polypeptide
tRNA
Anticodon
A A A
U G G U U U G G C
Codons
5
mRNA
3
tRNA serves as
an “adaptor”
that brings the
correct amino
acid to each
codon.
3A
The genetic code
Second mRNA base
C
UUU
First mRNA base (5 end)
U
UUC
U
UUA
UUG
C
CUU
CUC
C
CUA
CUG
Phe
Leu
G
A
AUC
UCC
UCA
Ser
UCG
G
U
UGU
U
Tyr
Cys
UAC
UGC
C
C
UAA Stop UGA Stop A
UAG Stop UGG Trp G
G
CCU
U
U
CAU
CGU
His
CCC
CAC
CGC
C
C
Arg
Leu CCA Pro
CAA
CGA
A
Gln
CCG
CAG
CGG
G
G
lle
ACU
AAU
ACC
AAC
AUA
ACA
AUGMet or
start
ACG
GUU
GUC
G
GUA
GUG
G
A
UAU
UCU
AUU
A
A
C
Thr
GCU
GCC
Val
Ala
GCA
GCG
AAA
AAG
Asn
Lys
AGU
AGC
AGA
Ser
AGG Arg
GAU
GGU
Asp
GAC
GGC
Gly
GAA
GGA
Glu
GAG
GGG
U
U
C
C
A
G
G
U
U
C
C
A
A
G
G
Third mRNA base (3 end)
UU
C
C
A 5
C G
G C
C G
U G
U A
A U
A U
U
G
* CC A C A G U
A* C U C A *
G
*
G
C * GU GU *
CGAG
*
AGG
UC *
* GA
G C
Hydrogen
G C
U A
bonds
* G
A
A*
C
*
U
A
AG
5’-AUGCAAUUCGGAAAC
Codon in the mRNA
An aminoacyl-tRNA synthetase joins a specific
amino acid to a tRNA
Amino acid
Aminoacyl-tRNA
synthetase (enzyme)
1 Active site binds the
amino acid and ATP.
P P P Adenosine
ATP
P Adenosine
Pyrophosphate
Pi
3
Appropriate
tRNA bonds to amino
Acid, displacing
AMP.
4
P Pi
Pi
tRNA
P Adenosine
AMP
Activated amino acid
is released by the enzyme.
Each tRNA has a
slightly different
shape
How does the ribosome find
AUG?
• Prokaryotes have a special binding
sequence upstream of the start codon.
• In Eukaryotes,the ribosome binds to the
5’ cap and “scans” the message for an
AUG.
See the Animation
• www.dnai.org
Inhibition of protein synthesis
Toxin
Mode of action
Target
forms peptidyl-puromycin, prevents
Procaryotes
translocation
blocks the A-site, prevents binding of aminoacyl
Tetracycline
Procaryotes
tRNAs
Chloramphenicol blocks peptidyl transfer
Procaryotes
Puromycin
Cycloheximide
blocks peptidyl transferase
Eucaryotes
Streptomycin
inhibits initiation at high concentrations
Procaryotes
Diphtheria toxin catalyzes ADP-ribosylation of residue in eEF2
Eucaryotes
Erythromycin
binds to 50S subunit, inhibits translocation
Procaryotes
Ricin
inactivates 60S subunit, depurinates an
adenosine in 23S rRNA
Eucaryotes
NOTE: Prokaryotes (this generally includes protein
synthesis in mitochondria and chloroplasts)
Only the anticodon of tRNA determines
which amino acid is added by a
ribosome.
• Experimental evidence:
– Convert cystein to alanine chemically, after
it is attached to tRNA (remove SH group)
– Alanine shows up in Cystein sites
The amino acid carried by a tRNA is
independent of the anticodon sequence
• Determined by the amino-acyl tRNA
synthetase enzyme
– tRNA with mutations in the anticodon still
have their normal amino acid at the 3’
end.
– Experiment:. mutate anticodon of tRNAthr
(AGU-->AGG)
• Now binds to proline codon instead (CCU).
• Those tRNA still carry threonine, but now
bind to proline sites.
• Threonine inserted into polypeptide where
proline normally goes.
Alananine tRNA synthetase
Aminoacyl tRNA
synthetase
enzyme is
specific to a
particular amino
acid and a
particular tRNA
Glycine doesn’t fit . .
Quality control
• Both cap and tail bind to initiation factors to
start translation
– Ensures that mRNA is intact
• Small subunit can detect mis-paired tRNA
and remove them
– Needs a short delay before peptide bond is
formed (to give time for proofreading)
• Error rate: about 10-4
Cost of protein biosynthesis
• Synthesis of aminoacyl tRNAs 2 ATPs
• Formation of 1 peptide bond 2 GTPs
– 1 for codon recognition; 1 for translocation
• Proofreading
1 ATP/error
• Construction of a specific amino acid sequence is
much more costly than formation of a random
peptide bond!
Transcription and translation can
occur simultaneously
RNA polymerase
DNA
mRNA
Polyribosome
RNA
polymerase
Direction of
transcription
0.25 m
DNA
Polypeptide
Ribosome
mRNA (5 end)
Post translational
modifications and sorting
Glycosylation
Signal directs protein to the right compartment
The signal mechanism for targeting
proteins to the ER
2
SRP binds3 Attaches to
Translation
begins in to the signal translocation
pore in ER
the cytosol peptide,
membrane
1
Polypeptide
synthesized
into the ER
4
Signal 6 Folds
peptide to final
removed shape
5
Ribosome
mRNA
Signal
peptide
Signalrecognition
particle
(SRP) SRP
receptor
CYTOSOL protein
Translocation
complex
Signal
peptide
removed
ER
membrane
Protein
Signal peptide
determines where it
goes
Destined for ER
Destined for cytosol
or other organelles
Imported
during
translation
Stays within the
membrane
system
Brooker Figure 13.22
Imported
after
translation
Chaperones help fold proteins
Hsp 70 covers exposed
hydrophobic patches until
the protein can fold
Hsp60 is like an isolation chamber
Mis-folded proteins are
marked for destruction with
ubiquitin
Ubiquitin tail
Proteosome acts as garbage disposal
Mutations (again)
The molecular basis of sickle-cell
disease: a point mutation
Wild-type hemoglobin DNA
3
Mutant hemoglobin DNA
5
C
T
T
In the DNA, the
mutant template
strand has an A where
the wild-type template
has a T.
A
The mutant mRNA has
a U instead of an A in
one codon.
3
5
T
C
mRNA
A
mRNA
G
A
A
5
G
3
U
5
3
Normal hemoglobin
Sickle-cell hemoglobin
Glu
Val
The mutant (sickle-cell)
hemoglobin has a valine
(Val) instead of a glutamic
acid (Glu).
Base-pair substitution
Wild type
mRNA
A U G A A G U U U G G C U A A
5
Protein
Met
3
Lys
Phe
Amino end
Gly
Stop
Carboxyl end
Base-pair substitution
No effect on amino acid sequence
U instead of C
A U G A A G U U U G G U U A A
Met
Lys
Missense
Phe
Gly
Stop
A instead of G
A U G A A G U U U A G U U A A
Met
Lys
Phe
Ser
Stop
Nonsense
U instead of A
A U G U A G U U U G G C U A A
Met
Stop
Base-pair insertion or deletion
Wild type
mRNA 5
Protein
A UG A A GU U U GG C U A A
Met
Lys
Gly
Phe
3
Stop
Amino end
Carboxyl end
Base-pair insertion or deletion
Frameshift causing immediate nonsense
Extra U
A U GU A A G U U U GG C U A
Met
Stop
Frameshift causing
extensive missense
U Missing
A U G A A G U U G G C U A A
Met
Lys
Leu
Ala
Insertion or deletion of 3 nucleotides:
no frameshift but extra or missing amino acid
A A G Missing
A U G U U UG G C U A A
Met
Phe
Gly
Stop
Mutations in the 3rd position
of a codon are often silent
CUU
CUC
C
CUA
CUG
A
lle
ACU
AAU
ACC
AAC
AUA
ACA
AUGMet or
ACG
start
GUU
GUC
G
GUA
GUG
UGU
U
Cys
UGC
C
UGA Stop A
UGG Trp G
CCU
U
CAU
CGU
His
CCC
CAC
CGC
C
Arg
Leu CCA Pro
CAA
CGA
A
Gln
CCG
CAG
CGG
G
AUU
AUC
G
Thr
GCU
GCC
Val
Ala
GCA
GCG
AAA
AAG
Asn
Lys
AGU
AGC
AGA
U
Ser C
A
AGG Arg G
U
GAU
GGU
Asp
C
GAC
GGC
Gly
GAA
GGA
A
Glu
GAG
GGG
G
Third mRNA base (3 end)
First mRNA base (5 end)
For amino acids that
have only two codons,
the 3rd base will either
both be purines or both
be pyrimidines
Second mRNA base
U
C
A
UAU
UUU
UCU
Tyr
Phe
UAC
UUC
UCC
U
UCA Ser UAA Stop
UUA
Leu
UAG Stop
UCG
UUG
Wobble in 3rd position