Protein Synthesis

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Protein Synthesis

• Chapter 17

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Protein synthesis

• 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

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Protein synthesis

• DNA RNA protein

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Protein Synthesis

• Gene Expression
• Process by which DNA directs the synthesis of
  proteins
• 2 stages
• Transcription
• Translation

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Protein synthesis

• Transcription
• DNA sequence is copied into an RNA
• Translation
• Information from the RNA is turned into an amino
  acid sequence

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Protein synthesis

• DNA RNA Protein
• Transcription Translation

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Protein Synthesis

• 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

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Protein synthesis

• Beadle Tatum
• Bread mold
• 3 enzymes to make arginine
• Mutated molds DNA
• Mutated code for enzymes
• Unable to code for arginine

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Results Table
Classes of Neurospora crassa
Precursor
Enzyme A
Wild type
Class I mutants
Class II mutants
Class III mutants
Minimal medium (MM) (control)
Ornithine
Enzyme B
Citrulline
Enzyme C
MM ornithine
Arginine
Condition
MM citrulline
MM arginine (control)
No growth Mutant cells cannot grow and divide
Growth Wild-type cells growing and dividing
Can grow on ornithine, citrulline, or arginine
Can grow with or without any supplements
Can grow only on citrulline or arginine
Summary of results
Require arginine to grow
Control Minimal medium
Gene (codes for enzyme)
Class I mutants (mutation in gene A)
Class II mutants (mutation in gene B)
Class III mutants (mutation in gene C)
Wild type
Precursor
Precursor
Precursor
Precursor
Enzyme A
Enzyme A
Enzyme A
Enzyme A
Gene A
Ornithine
Ornithine
Ornithine
Ornithine
Enzyme B
Enzyme B
Enzyme B
Enzyme B
Gene B
Citrulline
Citrulline
Citrulline
Citrulline
Enzyme C
Enzyme C
Enzyme C
Enzyme C
Gene C
Arginine
Arginine
Arginine
Arginine
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Protein synthesis

• Beadle Tatum
• One gene one enzyme
• One gene one protein
• One gene one polypeptide

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An albino racoon
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Cracking the code

• 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

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Cracking the code

• 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

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• WHYDIDTHEREDCATEATTHEFATRAT
• WHYIDTHEREDCATEATTHEFATRAT
• WHYDTHEREDCATEATTHEFATRAT
• WHYTHEREDCATEATTHEFATRAT

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Cracking the code

• Universal code
• AGA codes for amino acid Arginine
• Humans bacteria
• Genes from humans can be transcribed by mRNA from
  bacteria
• Produce human proteins
• Insulin

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RNA

• RNA (ribonucleic acid)
• Single strand
• Sugar ribose (-OH on 2 carbon)
• Uracil instead of thymine

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RNA

• mRNA
• Messenger RNA
• Transcribes information from DNA
• Codons
• (3 nucleotides) CGU
• mRNA
• Codes for amino acids
• rRNA
• Ribosomal RNA
• Polypeptides are assembled

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RNA

• tRNA
• Transfer RNA
• Transports aa to build proteins
• Positions aa on rRNA
• Anticodons
• (3 complementary nucleotides) GCA

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Nuclear envelope
DNA
TRANSCRIPTION
Pre-mRNA
RNA PROCESSING
mRNA
NUCLEUS
DNA
TRANSCRIPTION
CYTOPLASM
CYTOPLASM
mRNA
TRANSLATION
Ribosome
Ribosome
TRANSLATION
Polypeptide
Polypeptide
(b) Eukaryotic cell
(a) Bacterial cell
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Transcription

• 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

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Transcription

• Coding strand
• DNA strand not coded
• Same sequence of nucleotides as the RNA
  transcript
• Only T instead of U.

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Figure 17.4
DNA template strand
5'
3'
C
C
C
C
G
G
T
A
A
A
A
A
A
C
T
T
T
T
C
G
G
G
G
T
3'
5'
TRANSCRIPTION
U
G
G
U
U
U
G
G
C
C
U
A
mRNA
5'
3'
Codon
TRANSLATION
Protein
Gly
Ser
Phe
Trp
Amino acid
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Transcription

• RNA polymerase
• Enzyme
• Adds nucleotides to the 3end
• 5to3 direction
• Does not need a primer to start
• One polymerase in prokaryotes
• Three in eukaryotes
• Polymerase II makes mRNA

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Transcription

• Promoters
• Sequence on DNA where transcription starts
• TATAAT
• TATA box
• Sequences are not transcribed

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Transcription

• Stages
• Initiation
• Elongation
• Termination

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Initiation

• RNA polymerase binds promoter
• Unwinds DNA
• Transcription unit
• RNA polymerase, DNA growing RNA strand

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Fig. 17-UN1
Transcription unit
Promoter
5?
3?
3?
5?
3?
5?
Template strand of DNA
RNA polymerase
RNA transcript
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Initiation

• Transcription factors bind first to the promoter
  in Eukaryotes
• RNA pol II binds DNA
• Transcription Initiation Complex is formed
• Starts to transcribe

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Promoter
Nontemplate strand
DNA
5'
3'
T
A
T
A
A
A
A
A eukaryotic promoter
1
3'
5'
T
A
A
T
T
T
T
TATA box
Start point
Template strand
Transcription factors
5'
3'
Several transcription factors bind to DNA.
2
3'
5'
RNA polymerase II
Transcription factors
3'
5'
Transcription initiation complex forms.
3'
3
5'
5'
3'
RNA transcript
Transcription initiation complex
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Elongation

• RNA polymerase moves along DNA
• Untwists DNA
• Adds nucleotides to 3 end

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Fig. 17-7b
Nontemplate strand of DNA
Elongation
RNA nucleotides
RNA polymerase
3?
3? end
5?
Direction of transcription (downstream)
5?
Template strand of DNA
Newly made RNA
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Termination

• Prokaryotes
• Stop signal
• Sequence on DNA
• RNA transcript signals polymerase to detach from
  DNA
• RNA strand separates from the DNA

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Termination

• Eurkaryotes
• Polyadenylation signal sequence on mRNA
• AAUAAA
• Recognized by RNA polymerase II
• mRNA is released

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Transcription
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Promoter
Transcription unit
3'
5'
3'
5'
Start point
RNA polymerase
Initiation
1
5'
3'
3'
5'
Template strand of DNA
RNA transcript
Unwound DNA
Elongation
2
Rewound DNA
5'
3'
3'
3'
5'
5'
Direction of transcription (downstream)
RNA transcript
Termination
3
3'
5'
3'
5'
5'
3'
Completed RNA transcript
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Eukaryotes

• mRNA is modified
• Nucleus
• RNA processing

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Eukaryotes

• 5 cap
• Addition of a GTP
• 5 phosphate of the first base of mRNA
• Methyl group is added to the GTP
• 3poly-A-tail
• Several As on the end of the mRNA

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Eukaryotes

• Introns
• non-coding sequences of nucleic acids
• Exons
• coding sequences of nucleic acids

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Euraryotes

• RNA splicing
• Cut out introns
• Reconnect exons
• snRNPs (small nuclear RNAs)
• Spliceosome
• Many snRNPs come together remove introns

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Translation

• Passing the code to make a polypeptide
• mRNA
• rRNA
• ribosomes
• tRNA

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Translation

• 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

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Ribosome
P site (Peptidyl-tRNA binding site)
Exit tunnel
A site (Aminoacyl- tRNA binding site)
E site (Exit site)
E
P
A
Large subunit
mRNA binding site
Small subunit
(b) Schematic model showing binding sites
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Ribosome
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Ribsome
Growing polypeptide
Amino end
Next amino acid to be added to polypeptide chain
E
tRNA
mRNA
3'
Codons
5'
(c) Schematic model with mRNA and tRNA
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Translation

• 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 tRNAs

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Translation

• 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

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Translation

• 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

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Translation

• 1. Initiation
• 2. Elongation
• 3. Termination

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Initiation

• 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

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Initiation

• Requires energy
• GTP
• Forms the Initiation complex

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Initiation
Large ribosomal subunit
3'
5'
U
C
A
P site
Met
3'
5'
A
G
U
Met
P
i
Initiator tRNA

GTP
GDP
E
A
mRNA
5'
5'
3'
3'
Start codon
Small ribosomal subunit
mRNA binding site
Translation initiation complex
Large ribosomal subunit completes the
initiation complex.
Small ribosomal subunit binds to mRNA.
1
2
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Elongation

• 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 5to 3 direction

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Elongation

• Initial tRNA moves to E site
• Released
• New tRNA moves into A site
• Continues to add more aa to form the polypeptide

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Elongation
Amino end of polypeptide
Codon recognition
1
3'
E
mRNA
Ribosome ready for next aminoacyl tRNA
P
A
5'
site
site
GTP
P
GDP
i
E
E
P
A
P
A
GDP
P
i
Peptide bond formation
Translocation
2
3
GTP
E
P
A
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Termination

• 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

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Termination
Release factor
Free polypeptide
5'
3'
3'
3'
5'
5'
2
GTP
Stop codon (UAG, UAA, or UGA)
2 GDP

2
P
i
Ribosome reaches a stop codon on mRNA.
Release factor promotes hydrolysis.
Ribosomal subunits and other components dissociate
.
2
3
1
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Fig. 17-UN3
Ribosome
mRNA
Polypeptide
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Translation
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Second nucleotide

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ltgt
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Completed polypeptide
Growing polypeptides
Incoming ribosomal subunits
Polyribosome
Start of mRNA (5' end)
End of mRNA (3' end)
Several ribosomes simultaneously translating one
mRNA molecule
(a)
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4
5
6
1
2
3
Polypeptide synthesis begins.
Signal- cleaving enzyme cuts off signal peptide.
SRP binds to signal peptide.
SRP binds to receptor protein.
SRP detaches and polypeptide synthesis resumes.
Completed polypeptide folds into final conformatio
n.
Ribosome
mRNA
Signal peptide
ER membrane
Signal peptide removed
SRP
Protein
SRP receptor protein
CYTOSOL
ER LUMEN
Translocation complex
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Similarities

• DNA RNA Protein
• Transcription Translation

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Differences in gene expression

• 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

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Differences in gene expression

• 3. Eukaryotes complete transcription before
  leaving the nucleus
• 4. Eukaryotes modify RNA
• Introns/exons
• 5. Prokaryotes Polymerase binds promoters
• Eukaryotes transcription factors bind first then
  enzyme
• 6. Termination

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Differences in gene expression

• Translation
• 1. Prokaryotes start translation with AUG
• Eukaryotes 5cap initiates translation
• 2. Prokaryotes smaller ribosomes

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Nuclear envelope
DNA
TRANSCRIPTION
Pre-mRNA
RNA PROCESSING
mRNA
NUCLEUS
DNA
TRANSCRIPTION
CYTOPLASM
CYTOPLASM
mRNA
TRANSLATION
Ribosome
Ribosome
TRANSLATION
Polypeptide
Polypeptide
(b) Eukaryotic cell
(a) Bacterial cell
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Mutations

• Changes in genetic information
• Point mutations
• Change in a single base pair
• Sickle cell mutation

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Point mutation
Wild-type ß-globin
Sickle-cell ß-globin
Mutant ß-globin DNA
Wild-type ß-globin DNA
3'
5'
3'
5'
A
C
C
C
C
T
5'
5'
3'
3'
G
G
T
G
G
A
mRNA
mRNA
5'
5'
3'
3'
U
G
G
G
G
A
Normal hemoglobin
Sickle-cell hemoglobin
Val
Glu
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Mutations

• Two types
• 1. Base-pair substitution
• 2. Insertion or deletion

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Mutations

• 1. Base-pair substitution
• Exchange one nucleotide and base pair with
  another
• A. Silent mutations
• No effect on proteins

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Silent mutaton
Wild type
DNA template strand
T
T
T
A
C
C
A
A
A
C
C
G
A
T
T
5'
3'
A
A
A
A
A
T
G
G
T
T
T
G
G
C
T
5'
3'
3'
mRNA Protein
5'
A
A
U
C
G
G
U
U
U
G
A
A
G
A
U
Met
Lys
Phe
Gly
Stop
Amino end
Carboxyl end
Nucleotide-pair substitution silent
A instead of G
5'
T
T
T
A
C
C
A
A
A
C
C
T
T
A
A
3'
3'
5'
A
A
A
T
G
G
T
T
T
G
G
T
A
A
T
U instead of C
3'
5'
G
G
U
U
U
G
A
A
G
A
U
U
A
A
U
Met
Lys
Phe
Gly
Stop
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Mutations

• B. Missense mutations
• Substitutions that change one aa for another
• Little effect

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Missense
Wild type
DNA template strand
T
T
T
A
C
C
A
A
A
C
C
G
A
T
T
3'
5'
5'
3'
A
A
A
A
A
T
G
G
T
T
T
G
G
C
T
3'
5'
mRNA Protein
A
A
U
C
G
G
U
U
U
G
A
A
G
A
U
Met
Lys
Phe
Gly
Stop
Amino end
Carboxyl end
Nucleotide-pair substitution missense
T instead of C
3'
5'
T
A
C
C
C
A
A
A
A
T
T
T
T
T
G
5'
3'
A
T
G
A
A
A
A
T
C
G
T
T
T
G
A
A instead of G
5'
3'
A
U
G
A
A
G
U
U
U
U
A
A
C
G
A
Met
Lys
Phe
Ser
Stop
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Mutations

• C. Nonsense mutations
• Point mutation codes for stop codon
• Stops translation too soon
• Shortens protein
• Non-functional proteins

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Mutations

• 2. Insertions or deletions
• Additions or losses of nucleotides
• Frameshift mutations
• Improperly grouped codons
• Nonfuctional proteins

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Fig. 17-23
Wild-type
3?
DNA template strand
5?
3?
5?
5?
3?
mRNA
Protein
Stop
Amino end
Carboxyl end
A instead of G
Extra A
3?
5?
5?
3?
3?
5?
5?
3?
U instead of C
Extra U
5?
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?
3?
5?
3?
5?
3?
5?
A instead of G
missing
3?
5?
5?
3?
Stop
Missense
Frameshift causing extensive missense (1
base-pair deletion)
missing
A instead of T
5?
3?
3?
5?
5?
3?
3?
5?
U instead of A
missing
5?
3?
5?
3?
Stop
Stop
Nonsense
No frameshift, but one amino acid missing (3
base-pair deletion)
(a) Base-pair substitution
(b) Base-pair insertion or deletion
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Mutagens

• Chemical or physical agents
• Mutations in DNA