Protein Biosynthesis II

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Protein Biosynthesis II

• mRNA Binding Shine-Dalgarno Sequence
• Initiation Complex f-Met-tRNAfMet Initia
  tion factors
• Elongation Elongation factors
• Termination Termination factors

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Dintzis Experiment
3H-Leucine
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3
C-terminal is richer in radioactive 3H-leucine
Product analysis
3H
60 min
Trypsin
8 min
4 min
Released Protein
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Shine-Dalgarno
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fMet-tRNAfMet
Blocking the N-terminal amino acid assures
that the peptide chain will grow towards the
C-terminal
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(No Transcript)
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Vocabulary of Terms

• Aminoacyl-tRNA tRNA with an amino acid attached
• Peptidyl-tRNA tRNA with peptide attached
• Nascent chain peptide chain in act of being
  assembled
• P-site Site on ribosome where peptidyl-tRNA
  sits
• A-site Site on ribosome where incoming
  aminoacyl-tRNA binds
• Peptidyl transferase Enzyme that forms peptide
  bond

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OVERALL MECHANISM
Protein Biosynthesis takes place on Ribosomes
Picture the ribosomes as an assembly site
and a ratchet machine
GTP, not ATP, is the fuel that drives the machine
tRNAS are brought to the assemble site
called the A site
A peptide bond is formed by transferring
the peptide from the P site to the N-terminal
of the new amino acid (aminoacyl-tRNA)
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(OVERALL CONT.)
The peptide now has the new amino acid in the
C-terminalstill attached to the tRNA
The peptidyl tRNA is in the A site
The mRNA with the peptidyl-tRNA attached shifts
one frame bringing a new codon into the A site
Simultaneously, the peptidyl tRNA is pushed into
the P site and the uncharged tRNA is shoved into
the E site and expelled
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INITIATION
1. The machine is disassembled into its
component parts to allow the mRNA to bind
2. The starting tRNA is set into position
3. The larger 50S subunit is put back in place
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Initiation Complex
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Elongation
1. Peptidyl-tRNA sits in the P site
2. An aminoacyl-tRNA reacts with
elongation factor EF-Tu in preparation for
binding to the A site.
3. A-site binding occurs as GTP is
hydrolyzed. EF-Ts removes spent GDP.
4. A peptide bond is formed with the transfer of
the peptide from the P site to the A site
5. Elongation factor EF-G shifts the mRNA one
frame, expelling the uncharged tRNA
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Elongation
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Termination
1. Translocation results in a stop codon (UAA,
UGA, UAG) in the A site
2. A termination factor that recognizes the
codon binds to the A site
3. The peptide in the P site is transferred to
the factor
4. Because the factor cannot bind, the peptide
is released
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Termination
COOH
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Diphtheria Toxin (Cornyebacterium diphtheria with
bacteriophage cornyephage ?)
1. Reacts with elongation factor eEF-2
(prokaryote G)
eEF-2 NAD
Modified His in eEF-2 is target

NAD
eEf-2
ADP ribosylation
(diphthamide group)
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A
s
s
B
Endocytosis
Active enzyme
Receptor
A
s
A
H
s
s
B
Disulfide cleavage
NAD
eEF-2
H
s
ADP-ribosyl-eEF-2
(ribosylates histidine residues with diphthamide
structure)
(See p 879)
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Translation Blocking Antibiotics
Puromycin
Binds to A site and receives peptide from
transpeptidase
Aborts peptide prematurely
Prokaryotes and eukaryotes
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Blocks peptidyl transferase in prokaryotes
(A-site binding)
Aminoglycoside
Causes misreading and inhibits chain initiation
of prokaryote mRNA
Inhibits aminoacyl-tRNA binding to prokaryotes
blocks nutritional factor ppGpp (p867)
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Post-translational Processing

• Folding
• Proteolytic processing
• Additions to peptide chain Carbohydrates
  (glycosylation) Methyl groups Lipid
  groups Hydroxylation, deamination
• Secretion

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Folding
Rule Proteins must fold to an active
conformation. Generally, folding is
spontaneous as the protein seeks its lowest
energy level
Rule Cells have chaperones that assist in
proper folding. Typical are Hsp70 and Hsp60
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Stepwise
Random Chain
Secondary helix or ? sheet
Hydrophobic alignments
Compact folded protein
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Proteolytic Processing of Insulin
Preproinsulin
Proinsulin
Insulin