From Gene to Phenotype part 3

1
From Gene to Phenotype- part 3
DNA
mRNA
polypeptide
2
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))
3
Translation overview
tRNA serves as an adaptor that brings the
correct amino acid to each codon.
The ribosome is the machine that builds the
polypeptide
4
The genetic code
3?
A
C
C
5?
A
C
G
C
G
C
G
U
G
U
A
A
U
A
U
U
C
G

G
C
A
U
A
C
A

A
C
U
C
G


U
G
U
G
G

G
A
C
C
G
C


A
G
U
G


G
A
G
C
Hydrogen bonds
G
C
U
A
G

A

A
C

U
A
G
A
5-AUGCAAUUCGGAAAC
Codon in the mRNA
5
An aminoacyl-tRNA synthetase joins a specific
amino acid to a tRNA
Amino acid
Aminoacyl-tRNA synthetase (enzyme)
Adenosine
P
P
P
ATP
Each tRNA has a slightly different shape
Adenosine
P
Pyrophosphate
P
Pi
Pi
Pi
tRNA
Appropriate tRNA bonds to amino Acid,
displacing AMP.
Adenosine
P
AMP
Activated amino acid is released by the enzyme.
6
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.

7
See the Animation

• www.dnai.org

8
Inhibition of protein synthesis
NOTE Prokaryotes (this generally includes
protein synthesis in mitochondria and
chloroplasts)
9
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

10
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--gtAGG)
• 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.

11
Alananine tRNA synthetase
Aminoacyl tRNA synthetase enzyme is specific to a
particular amino acid and a particular tRNA
Glycine doesnt fit . .
12
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

13
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!

14
Transcription and translation can occur
simultaneously
RNA polymerase
DNA
mRNA
Polyribosome
Direction of transcription
0.25 ?m
RNA polymerase
DNA
Polypeptide
Ribosome
mRNA (5? end)
15
Post translational modifications and sorting
Glycosylation
Signal directs protein to the right compartment
16
The signal mechanism for targeting proteins to
the ER
Signal peptide removed
Translation begins in the cytosol
Polypeptide synthesized into the ER
Folds to final shape
Attaches to translocation pore in ER membrane
SRP binds to the signal peptide,
2
3
1
4
5
6
Ribosome
mRNA
Signal peptide
ER membrane
Signal peptide removed
Signal- recognition particle (SRP)
Protein
SRP receptor protein
CYTOSOL
Translocation complex
17
Signal peptide determines where it goes
Destined for cytosol or other organelles
Destined for ER
Imported during translation
Imported after translation
Stays within the membrane system
Brooker Figure 13.22
18
(No Transcript)
19
Chaperones help fold proteins
Hsp 70 covers exposed hydrophobic patches until
the protein can fold
Hsp60 is like an isolation chamber
20
Mis-folded proteins are marked for destruction
with ubiquitin
Ubiquitin tail
Proteosome acts as garbage disposal
21
Mutations (again)
22
The molecular basis of sickle-cell disease a
point mutation
23
Base-pair substitution
24
Base-pair insertion or deletion
25
Mutations in the 3rd position of a codon are
often silent
Second mRNA base
U
C
A
G
U
UAU
UUU
UCU
UGU
For amino acids that have only two codons, the
3rd base will either both be purines or both be
pyrimidines
Tyr
Cys
Phe
UAC
UUC
UCC
UGC
C
U
Ser
UUA
UCA
UAA
Stop
Stop
UGA
A
Leu
UAG
UUG
UCG
Stop
UGG
Trp
G
CUU
CCU
U
CAU
CGU
His
CUC
CCC
CAC
CGC
C
C
Arg
Pro
Leu
CUA
CCA
CAA
CGA
A
Gln
CUG
CCG
CAG
CGG
G
Third mRNA base (3? end)
First mRNA base (5? end)
U
AUU
ACU
AAU
AGU
Asn
Ser
C
lle
AUC
ACC
AAC
AGC
A
Thr
A
AUA
ACA
AAA
AGA
Lys
Arg
Met or start
AUG
ACG
AAG
AGG
G
U
GUU
GCU
GAU
GGU
Asp
C
GUC
GCC
GAC
GGC
G
Val
Ala
Gly
GUA
GCA
GAA
GGA
A
Glu
GUG
GCG
GAG
GGG
G
26
Wobble in 3rd position