Translation of Proteins slides

1. From Gene to Phenotype- part 2
DNA
molecule
Gene 1
Gene 2
Gene 3
DNA strand
(template)
TRANSCRIPTION
mRNA
Protein
TRANSLATION
Amino acid
A C C A A A C C G A G T
U G G U U U G G C U C A
Trp Phe Gly Ser
Codon
3 5
3
5

2. Lecture Outline 11/7/05
• The central dogma:
– DNA->RNA->protein
• Various types of RNA
• The genetic code
• Mechanisms of translation
– Initiation, Elongation,Termination
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)

3. Four types of RNA
• mRNA
– Messenger RNA, encodes the amino acid sequence of a
polypeptide
• rRNA
– Ribosomal RNA, forms complexes with protein called ribosomes,
which translate mRNA to protein
• tRNA
– Transfer RNA, transports amino acids to ribosomes during protein
synthesis
• snRNA
– Small nuclear RNA, forms complexes with proteins used in
eukaryotic RNA processing

4. 3
A
C
C
A
C
G
C
U
U
A
A
G
A
C
A
C
C
U
G
C
*
GUGU
C
U
GAG
G
U
A
A
A G
U
C
A
G
A
C
C
CGAG
AG G
G
G
A
C
U
C
A
U
U
U
A
G
G
C
G
5
Hydrogen
bonds
Anticodon
*
*
*
*
*
*
*
*
*
*
*
RNA will fold to specific
shapes
• Because RNA is single-stranded,
parts of the molecule can base pair
with other parts of the same
molecule, causing it to fold into
defined shapes.
• Some RNA molecules can even act
as enzymes (ribozymes)

5. Correspondence between exons and
protein domains
Gene
DNA
Exon 1 Intron Exon 2 Intron Exon 3
Transcription
RNA processing
Translation
Domain 3
Domain 1
Domain 2
Polypeptide

6. The genetic code
Second mRNA base
U C A G
U
C
A
G
UUU
UUC
UUA
UUG
CUU
CUC
CUA
CUG
AUU
AUC
AUA
AUG
GUU
GUC
GUA
GUG
Met or
start
Phe
Leu
Leu
lle
Val
UCU
UCC
UCA
UCG
CCU
CCC
CCA
CCG
ACU
ACC
ACA
ACG
GCU
GCC
GCA
GCG
Ser
Pro
Thr
Ala
UAU
UAC
UGU
UGC
Tyr Cys
CAU
CAC
CAA
CAG
CGU
CGC
CGA
CGG
AAU
AAC
AAA
AAG
AGU
AGC
AGA
AGG
GAU
GAC
GAA
GAG
GGU
GGC
GGA
GGG
UGG
UAA
UAG Stop
Stop UGA Stop
Trp
His
Gln
Asn
Lys
Asp
Arg
Ser
Arg
Gly
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
First
mRNA
base
(5

end)
Third
mRNA
base
(3

end)
Glu

7. The code is a triplet code
– the new boy saw the big cat eat the hot dog
• The first evidence for triplet code came from
deletion experiments
– the new boy saw the big cat eat the hot dog
– the neb oys awt heb igc ate att heh otd og
• Function could be restored by an insertion
nearby
– the new boy saw the big cat eat the hot dog
– the neb oys aaw the big cat eat the hot dog

8. The code is a triplet code
• . . . or by two more deletions to get it back
into the correct “reading frame”
– the new boy saw the big cat eat the hot dog
– the neb oys awt heb igc ate att heh otd og
– the neb osa wte big cat eat the hot dog

9. Deciphering the Code
• Your book tells how people used synthetic mRNAs
and in vitro translation to determine decipher the
codons.
• E.g. UUUUUUUU -> phenylalanine only
– UCUCUCUCUCU -> mix of leucine and serine
• Why is it a mixture?

10. The code is redundant
• Several different
codons encode
the same amino
acid
Second mRNA base
U C A G
U
C
A
G
UUU
UUC
UUA
UUG
CUU
CUC
CUA
CUG
AUU
AUC
AUA
AUG
GUU
GUC
GUA
GUG
Met or
start
Phe
Leu
Leu
lle
Val
UCU
UCC
UCA
UCG
CCU
CCC
CCA
CCG
ACU
ACC
ACA
ACG
GCU
GCC
GCA
GCG
Ser
Pro
Thr
Ala
UAU
UAC
UGU
UGC
Tyr Cys
CAU
CAC
CAA
CAG
CGU
CGC
CGA
CGG
AAU
AAC
AAA
AAG
AGU
AGC
AGA
AGG
GAU
GAC
GAA
GAG
GGU
GGC
GGA
GGG
UGG
UAA
UAG Stop
Stop UGA Stop
Trp
His
Gln
Asn
Lys
Asp
Arg
Ser
Arg
Gly
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
First
mRNA
base
(5

end)
Third
mRNA
base
(3

end)

11. The code is comma free
• No punctuation between words.
– Therefore deletions cause frameshifts
• It does have start and stop signals,
however.
– Start: AUG
– Stop: UAG, UAA, UGA

12. Translation: the basic concept
TRANSCRIPTION
TRANSLATION
DNA
mRNA
Ribosome
Polypeptide
Polypeptide
Amino
acids
tRNA with
amino acid
attached
Ribosome
tRNA
Anticodon
mRNA
Gly
A A A
U G G U U U G G C
Codons
5 3

13. The structure of transfer RNA (tRNA)
3
A
C
C
A
C
G
C
U
U
A
A
G
A
C
A
C
C
U
G
C
*
G U G U
C
U
GAG
G
U
A
A
A G
U
C
A
G
A
C
C
C G A G
A G G
G
G
A
C
U
C
A
U
U
U
A
G
G
C
G
5
Amino acid
attachment site
Hydrogen
bonds
Anticodon
*
*
*
*
*
*
*
*
*
*
*

14. tRNA
Amino acid
attachment site
Hydrogen
bonds
Anticodon
Anticodon
(b) Three-dimensional structure
A A G
5
3
3 5
Symbol used
in this book
(c)

15. TRANSCRIPTION
TRANSLATION
DNA
mRNA
Ribosome
Polypeptide
E
P A
mRNA
5
3
Ribosomes
Growing
polypeptide
tRNA
molecules Large
subunit
Small
subunit
RNA + protein.
We now think that RNA
catalyzes the reactions.

16. P site (Peptide site)
E site (Exit site)
mRNA
binding site
Large
subunit
Small
subunit
E P A
A site (Acceptor site)
EPA model of a ribosome

17. Amino end Growing polypeptide
Next amino acid
to be added to
polypeptide chain
tRNA
mRNA
Codons
3
5
New amino acids are added to the
carboxyl end of the polypeptide

18. The initiation of translation
2
Initiator tRNA
mRNA
mRNA binding site
Small
ribosomal
subunit
Large
ribosomal
subunit
Translation initiation complex
P site
GDP
GTP
Start codon
1
U A C
A U G
E A
3
5
5
3
3
5 3
5
Small subunit binds just
upstream of start
Special initiator methionine
binds to the start codon (AUG)
The large subunit can now
bind to make the active
ribosome

19. Elongation
Amino end
of polypeptide
mRNA
tRNA with
appropriate
anticodon binds at A
site
Ribosome ready for
next aminoacyl tRNA
Translocaton.
Everything moves left
one notch. Polypeptide
is now at P. Empty
tRNA leaves the E site.
E
P A
E
P A
E
P A
E
P A
GDP
GTP
GTP
GDP
2
2
site site
5
3
3
2
1
TRANSCRIPTION
TRANSLATION
DNA
mRNA
Ribosome
Polypeptide
Peptide bond
formed. Growing
chain is now at A

20. Termination
Release
factor
Free
polypeptide
Stop codon
(UAG, UAA, or UGA)
1 2 3
5
3
5
3
5
3
Stop codon has
no matching
tRNA
Release factor
binds and
separates the
polypeptide
from the tRNA
The ribosome
separates
from mRNA

21. 4
An aminoacyl-tRNA synthetase joins a specific
amino acid to a tRNA
Amino acid
ATP
Adenosine
Pyrophosphate
Adenosine
Adenosine
tRNA
P P P
P
P Pi
Pi
Pi
P
AMP
Appropriate
tRNA bonds to amino
Acid, displacing
AMP.
Active site binds the
amino acid and ATP.
1
3
Aminoacyl-tRNA
synthetase (enzyme)
Activated amino acid
is released by the enzyme.

22. See the Animation
• www.dnai.org

23. Inhibition of protein synthesis
Toxin Mode of action Target
Puromycin
forms peptidyl-puromycin, prevents
translocation
Procaryotes
Tetracycline
blocks the A-site, prevents binding of aminoacyl
tRNAs
Procaryotes
Chloramphenicol blocks peptidyl transfer Procaryotes
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)

24. Summary of transcription and
translation in a eukaryotic cell
TRANSCRIPTION
RNA is transcribed
from a DNA template.
DNA
RNA
polymerase
RNA
transcript
RNA PROCESSING
In eukaryotes, the
RNA transcript (pre-
mRNA) is spliced and
modified to produce
mRNA, which moves
from the nucleus to the
cytoplasm.
Exon
RNA transcript
(pre-mRNA)
Intron
NUCLEUS
FORMATION OF
INITIATION COMPLEX
After leaving the
nucleus, mRNA attaches
to the ribosome.
CYTOPLASM
mRNA Growing
polypeptide
Ribosomal
subunits
Aminoacyl-tRNA
synthetase
Amino
acid
tRNA
AMINO ACID ACTIVATION
Each amino acid
attaches to its proper tRNA
with the help of a specific
enzyme and ATP.
Activated
amino acid
TRANSLATION
A succession of tRNAs
add their amino acids to
the polypeptide chain
as the mRNA is moved
through the ribosome
one codon at a time.
(When completed, the
polypeptide is released
from the ribosome.)
Anticodon
A A A
UG GUU UA U G
E A
Ribosome
1
5
5
3
Codon
2
3 4
5