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Protein Synthesis
Chapter 17
Protein synthesis
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DNA
Responsible for hereditary information
DNA divided into genes
Gene:
Sequence of nucleotides
Determines amino acid sequence in proteins
Genes provide information to make proteins
Protein synthesis
DNA
RNA
protein
Protein Synthesis
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Gene Expression:
Process by which DNA directs the
synthesis of proteins
2 stages
Transcription
Translation
Protein synthesis
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Transcription:
DNA sequence is copied into an RNA
Translation:
Information from the RNA is turned
into an amino acid sequence
Protein synthesis
DNA
RNA
Transcription
Protein
Translation
Protein Synthesis
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Central Dogma
Mechanism of reading & expressing
genes
Information passes from the genes
(DNA) to an RNA copy
Directs sequence of amino acids to
make proteins
Protein synthesis
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Beadle & Tatum
Bread mold
3 enzymes to make arginine
Mutated mold’s DNA
Mutated code for enzymes
Unable to code for arginine
Results Table
Precursor
Enzyme A
Wild type
Minimal
medium
(MM)
(control
)
Ornithine
Enzyme B
Citrulline
Arginine
Growth:
Wild-type
cells
growing
and
dividing
Control: Minimal
medium
No growth:
Mutant
cells
cannot
grow
and divide
Condition
Enzyme C
Classes of Neurospora
crassa
Class I
Class II
mutants
mutants
Class III
mutants
MM +
ornithin
e
MM +
citrullin
e
MM +
arginine
(control
)
Summar
y
of
results
Gene
(codes
for
enzyme)
Can grow
with
or without
any
supplements
Can grow on
ornithine,
citrulline,
or arginine
Class I
mutants
(mutation in
gene A)
Precurso
r
Enzyme
Can grow
Require
only
arginine
on citrulline
to grow
or
arginine
Class II
Class III mutants
mutants
(mutation in
(mutation in
gene C)
gene B)
Precurso
Precurso
r
r
Enzyme
Enzyme
Gene A
Wild
type
Precurso
r
Enzyme
A
A
A
A
Gene B
Ornithin
e
Enzyme
Ornithin
e
Enzyme
Ornithin
e
Enzyme
Ornithin
e
Enzyme
B
B
B
B
Gene C
Citrullin
e
Enzyme
C
Arginin
e
Citrullin
e
Enzyme
C
Arginin
e
Citrullin
e
Enzyme
C
Arginin
e
Citrullin
e
Enzyme
C
Arginin
e
Protein synthesis
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Beadle & Tatum
One gene one enzyme
One gene one protein
One gene one polypeptide
An albino racoon
Cracking the code
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Codons (Triplet code)-mRNA
Each codon corresponds to an aa
20 amino acids
64 triplet codes (codons)
61 code for aa-3 are stop codons
Wobble:
Flexible base pairing in the 3rd position
3’ end
Cracking the code
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Reading frame
Reading symbols in correct groupings
1 or 2 deletions or additions
Gene was transcribed incorrectly
3 deletions
Reading frame would shift
Gene was transcribed correctly
WHYDIDTHEREDCATEATTHEFATRAT
WHYIDTHEREDCATEATTHEFATRAT
WHYDTHEREDCATEATTHEFATRAT
WHYTHEREDCATEATTHEFATRAT
Cracking the code
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Universal code
AGA codes for amino acid Arginine
Humans & bacteria
Genes from humans can be transcribed
by mRNA from bacteria
Produce human proteins
Insulin
RNA
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RNA (ribonucleic acid)
Single strand
Sugar –ribose (-OH on 2’ carbon)
Uracil instead of thymine
RNA
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mRNA:
Messenger RNA
Transcribes information from DNA
Codons
(3 nucleotides) CGU
mRNA
Codes for amino acids
rRNA:
Ribosomal RNA
Polypeptides are assembled
RNA
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tRNA:
Transfer RNA
Transports aa to build proteins
Positions aa on rRNA
Anticodons
(3 complementary nucleotides) GCA
Nuclear
envelope
TRANSCRIPTION
DNA
PreRNA PROCESSING mRNA
NUCLEUS
TRANSCRIPTION
CYTOPLASM
DNA
mRNA
Ribosome
CYTOPLASM
TRANSLATION
TRANSLATION
Ribosom
e
Polypeptide
Polypeptide
(a) Bacterial cell
mRNA
(b) Eukaryotic
cell
Transcription
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Getting the code from DNA
Triplet code
Template strand
Strand of DNA
Provides template or pattern
Transcribed or read
Transcribed RNA is complementary to
this DNA strand
Transcription
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Coding strand
DNA strand not coded
Same sequence of nucleotides as the
RNA transcript
Only T instead of U.
Figure
DNA 17.4
templat
e
strand
3′
A C C A A A C C G A G T
T G G T T T G G C T C A
5′
5′
3′
TRANSCRIPTIO
N
mRNA
5′
TRANSLATIO
N
Protei
n
U G G U U U G G C U C A
Codo
n
Trp
Amino
Phe
Gly
Ser
3′
Transcription
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RNA polymerase
Enzyme
Adds nucleotides to the 3’end
5’to3’ direction
Does not need a primer to start
One polymerase in prokaryotes
Three in eukaryotes
Polymerase II makes mRNA
Transcription
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Promoters:
Sequence on DNA where transcription
starts
TATAAT
TATA box
Sequences are not transcribed
Transcription
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Stages
Initiation
Elongation
Termination
Initiation
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RNA polymerase binds promoter
Unwinds DNA
Transcription unit:
RNA polymerase, DNA & growing RNA
strand
Fig. 17-UN1
Transcription unit
Promoter
5
3
3
5
RNA polymerase
RNA transcript
3
5
Template strand
of DNA
Initiation
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Transcription factors bind first to the
promoter in Eukaryotes
RNA pol II binds DNA
Transcription Initiation Complex is
formed
Starts to transcribe
5′
3′
DNA
Promoter
Nontemplate
strand
T A T AAAA
AT AT T T T
TATA box
Transcription
factors
3′
5′
1 A eukaryotic
promoter
3′
5′
2 Several
transcription
factors bind
to DNA.
Start point Template
strand
5′
3′
RNA polymerase II
Transcription
factors
5′
3′
5′
3′
RNA
transcript
Transcription initiation
complex
3′
5′
3 Transcription
initiation
complex
forms.
Elongation
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RNA polymerase moves along DNA
Untwists DNA
Adds nucleotides to 3’ end
Fig. 17-7b
Nontemplate
strand of DNA
Elongation
RNA
polymerase
3
RNA nucleotides
3 end
5
5
Direction of
transcription
(“downstream”)
Newly made
RNA
Template
strand of DNA
Termination
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Prokaryotes
Stop signal
Sequence on DNA
RNA transcript signals polymerase to
detach from DNA
RNA strand separates from the DNA
Termination
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Eurkaryotes
Polyadenylation signal sequence on
mRNA
AAUAAA
Recognized by RNA polymerase II
mRNA is released
Transcription
Promoter
5′
3′
RNA polymerase
1 Initiation
Transcription
unit
3′
5′
Start
point
3′
5′
5′
3′
Template strand of
RNA
Unwound
transcriptDNA
DNA
2 Elongation
Rewound
DNA
3′
5′
3′
3 Termination
3′
5′
RNA
transcript
5′
Direction of
transcription
(“downstrea
m”)
3′
5′
5′
3′
5′
Completed RNA
transcript
3′
Eukaryotes
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mRNA is modified
Nucleus
RNA processing
Eukaryotes
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5’ cap
Addition of a GTP
5’ phosphate of the first base of
mRNA
Methyl group is added to the GTP
3’poly-A-tail
Several A’s on the end of the mRNA
Eukaryotes
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Introns:
non-coding sequences of nucleic acids
Exons:
coding sequences of nucleic acids
Euraryotes
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RNA splicing
Cut out introns
Reconnect exons
snRNP’s (small nuclear RNA’s)
Spliceosome:
Many snRNP’s come together & remove
introns
Translation
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Passing the code to make a polypeptide
mRNA
rRNA
ribosomes
tRNA
Translation
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Ribosome
Located in the cytoplasm
Site of translation
2 subunits composed of protein & RNA
Small (20 proteins and 1 RNA)
Large (30 proteins and 2 RNA)
3 sites on ribosome surface involved in
protein synthesis
E, P, and A sites
Ribosome
P site (Peptidyl-tRNA
binding site)
Exit tunnel
A site (AminoacyltRNA binding site)
E site
(Exit site)
E
P
A
mRNA
binding
site
(b) Schematic model showing binding sites
Large
subunit
Small
subunit
Ribosome
Ribsome
Growing
polypeptide
Amino end
Next amino
acid to be
added to
polypeptide
chain
E
tRNA
mRNA
5′
3′
Codons
(c) Schematic model with mRNA and tRNA
Translation
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tRNA
Aminoacyl-t-RNA synthetases
Activating enzymes
Link correct tRNA code to correct aa
One for each 20 amino acids
Some read one code, some read several
codes
45 tRNA’s
Translation
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Nonsense codes
UAA, UAG, UGA code to stop
AUG codes for start as well as
methionine
Ribosome starts at the first AUG it
comes across in the code
Translation
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mRNA binds to rRNA on the ribosome
mRNA attaches so only one codon is
exposed at a time
tRNA (anti-codon)
Complementary sequence
Binds to mRNA
tRNA carries a specific amino acid
Adds to growing polypeptide
Translation
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1. Initiation
2. Elongation
3. Termination
Initiation
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Initiation complex
1. tRNA with methionine attached binds to a
small ribosome
2. binds at the 5’ cap (Eukayotes)
3. tRNA is positioned on to the mRNA at AUG
4. Initiation factors position the tRNA on the
P site
5. Attachment of large ribosomal unit
Initiation
Requires energy
 GTP
 Forms the Initiation complex
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Initiation
3′ U A C 5′
5′ A U G 3′
P site
Pi
+
GTP GDP
Initiator
tRNA
E
mRNA
5′
Start
codon
3′
Small
ribosomal
subunit
mRNA binding
site
1 Small ribosomal subunit
binds to mRNA.
Large
ribosomal
subunit
5′
A
3′
Translation initiation
complex
2 Large ribosomal subunit
completes the initiation
complex.
Elongation
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Elongation factors
Help second tRNA bind to the A-site
Two amino acids bind (peptide bond)
Translocation:
Ribosome moves 3 more nucleotides
along mRNA in the 5’to 3’ direction
Elongation
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Initial tRNA moves to E site
Released
New tRNA moves into A site
Continues to add more aa to form the
polypeptide
Elongation
Amino end
of polypeptide
1 Codon
recognition
3′
mRNA
E
Ribosome ready
for
next aminoacyl
tRNA
P A
site site
5′
GTP
GDP + P i
E
E
PA
PA
GDP + P i
3 Translocation
2 Peptide
bond
formation
GTP
E
PA
Termination
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Release factors:
Proteins that release newly made
polypeptides
Codon (UAG, UAA, UGA)
Release factor binds to the codon
Polypeptide chain is released from A
site
Termination
Release
factor
Free
polypeptid
e
3′
3′
5′
Stop codon
(UAG, UAA, or
UGA)
1 Ribosome reaches a
stop codon on
mRNA.
5′
2 Release factor
promotes
hydrolysis.
5′
3′
2 GTP
2
+ 2 Pi
GDP
3 Ribosomal subunits
and other
components
dissociate.
Fig. 17-UN3
mRNA
Ribosome
Polypeptide
Translation
<>
Growing
polypeptides
Completed
polypeptide
Incoming
ribosomal
subunits
Start of
mRNA
(5′ end)
End of
mRNA
(3′
end)
(a) Several ribosomes simultaneously
translating
one mRNA molecule
2
1
3
Polypeptide SRP
SRP
synthesis
binds
binds to
begins.
to
receptor
signal
protein.
peptide
.
Ribosome
mRNA
Signal
peptide
ER LUMEN
5
SignalSRP
cleaving
detaches
enzyme
and
polypeptide cuts
off signal
synthesis
peptide.
resumes.
Signal
peptide
removed
SRP
CYTOSOL
4
SRP receptor
protein
Translocation complex
6
Completed
polypeptide
folds into
final
conformation.
ER
membrane
Protein
Similarities
DNA
RNA
Transcription
Protein
Translation
Differences in gene expression
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Transcription
1. Prokaryotes one RNA polymerase
Eukaryotes 3 RNA polymerases (poli-II
mRNA synthesis)
2. Prokaryotes mRNA contain transcripts of
several genes
Eukaryotes only one gene
3. Prokaryotes no nucleus so start translation
before transcription is done
Differences in gene expression
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3. Eukaryotes complete transcription before
leaving the nucleus
4. Eukaryotes modify RNA
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Introns/exons
5. Prokaryotes Polymerase binds promoters
Eukaryotes transcription factors bind first
then enzyme
6. Termination
Differences in gene expression
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Translation
1. Prokaryotes start translation with
AUG
Eukaryotes 5’cap initiates translation
2. Prokaryotes smaller ribosomes
Nuclear
envelope
TRANSCRIPTION
DNA
PreRNA PROCESSING mRNA
NUCLEUS
TRANSCRIPTION
CYTOPLASM
DNA
mRNA
Ribosome
CYTOPLASM
TRANSLATION
TRANSLATION
Ribosom
e
Polypeptide
Polypeptide
(a) Bacterial cell
mRNA
(b) Eukaryotic
cell
Mutations
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Changes in genetic information
Point mutations:
Change in a single base pair
Sickle cell mutation
Point mutation
Wild-type β-globin
Wild-type β-globin
3′ DNA C T C
G A G
5′
5′
mRNA
G A G
Normal
hemoglobin
Glu
Sickle-cell βglobin
5′
3′
3′
3′
5′
5′
Mutant β-globin
DNA C A C
G T G
5′
3′
G U G
3′
mRNA
Sickle-cell
hemoglobin
Val
Mutations
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Two types
1. Base-pair substitution
2. Insertion or deletion
Mutations
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1. Base-pair substitution
Exchange one nucleotide and base pair
with another
A. Silent mutations
No effect on proteins
Silent mutaton
Wild type
DNA template
3′ T A C T T C
5′ A T G A A G
strand
mRNA 5′ A U G A A G
Protein
Met
Lys
Amino
end
Nucleotide-pair substitution:
silent
A A A C C G A T T 5′
T T T G G C T A A 3′
U U U G G C U A A 3′
Phe
Gly
Stop
Carboxyl
end
A instead of
AGC C A A T T 5′
3′ T A C T T C A A
5′ A T G A A G T T T G G T T A A 3′
5′ A U G A A G U U
Met
Lys
U instead of
UCG G U U A A 3′
Phe
Gly
Stop
Mutations
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B. Missense mutations:
Substitutions that change one aa for
another
Little effect
Missense
Wild type
DNA template
3′ T A C T T C
5′ A T G A A G
strand
mRNA 5′ A U G A A G
Protein
Met
Lys
Amino
end
Nucleotide-pair substitution:
missense
A A A C C G A T T 5′
T T T G G C T A A 3′
U U U G G C U A A 3′
Phe
Gly
Stop
Carboxyl
end
T instead of
A CA A T C G A
T T 5′
3′ T A C T T C
5′ A T G A A G T T T A G C T A A 3′
5′ A U G A A G
Met
Lys
A instead of
UGU U A G C U
Phe
Ser
A A 3′
Stop
Mutations
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C. Nonsense mutations
Point mutation codes for stop codon
Stops translation too soon
Shortens protein
Non-functional proteins
Mutations
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2. Insertions or deletions
Additions or losses of nucleotides
Frameshift mutations
Improperly grouped codons
Nonfuctional proteins
Fig. 17-23
Wild-type
DNA template strand 3
5
5
3
mRNA 5
3
Protein
Stop
Amino end
Carboxyl end
A instead of G
3
5
Extra A
5
3
U instead of C
5
5
3
3
5
Extra U
3
5
3
Stop
Stop
Silent (no effect on amino acid sequence)
Frameshift causing immediate nonsense (1 base-pair insertion)
T instead of C
missing
3
5
5
3
3
5
5
3
A instead of G
missing
3
5
5
3
Stop
Missense
Frameshift causing extensive missense (1 base-pair deletion)
missing
A instead of T
3
5
5
3
3
5
3
5
U instead of A
5
5
3
missing
Stop
Stop
Nonsense
(a) Base-pair substitution
3
No frameshift, but one amino acid missing (3 base-pair deletion)
(b) Base-pair insertion or deletion
Mutagens
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Chemical or physical agents
Mutations in DNA