Protein Biosynthesis II
• mRNA Binding
Shine-Dalgarno Sequence
• Initiation Complex
f-Met-tRNAfMet
Initiation factors
• Elongation
Elongation factors
• Termination
Termination factors
Dintzis Experiment
3H-Leucine
H2NH2N-
5’
3’
3H
C-terminal is richer
in radioactive 3H-leucine
Product analysis
60 min
Trypsin
COO-
8 min
H2N-
COO-
4 min
Released Protein
N
C-terminal
H2N-
Shine-Dalgarno
Purine-rich mRNA
Pyrimidine-rich rRNA complementary
fMet-tRNAfMet
H
CH3-S-CH2CH2C-COO-tRNA
N-H
C=O
H
N-Terminal Block
Blocking the N-terminal amino acid assures that
the peptide chain will grow towards the C-terminal
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
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)
(OVERALL CONT.)
The peptide now has the new amino acid
in the C-terminal…still 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
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
Initiation Complex
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
Elongation
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
Termination
COOH
Diphtheria Toxin (Cornyebacterium diphtheria with
bacteriophage cornyephage )
1. Reacts with elongation factor eEF-2 (prokaryote G)
eEF-2 + NAD+
NH2
N
N
O
N
N
O
O-P-O-P-O CH2 N
O CH2
O
O O
HO
Diphtheria toxin
CONH2
OH
Modified His in
eEF-2 is target
HO
s
+
Break glycosidic bond
to nicotinamide in reaction
s
PEPTIDECHAIN
N
N
CH2 CH2 CH
NAD+
C= O
eEf-2
s
PEPTIDECHAIN
+
N(CH3)3
N
ADP
CH2
N
O
NH2
+
N(CH3)3
A (catalytically
active)
B
(cell penetration)
CH2 CH2 CH
C= O
HO OH
(diphthamide group)
Trypsin
s
OH
NH2
ADP ribosylation
A
s
s
B
Endocytosis
Receptor
s
s
A
H
s
B Disulfide
cleavage
H
s
Active
enzyme
A
NAD+
eEF-2
ADP-ribosyl-eEF-2
(ribosylates histidine
residues with diphthamide
structure)
(See p 879)
Translation Blocking Antibiotics
Puromycin
Binds to A site and
receives peptide
from transpeptidase
Aborts peptide
prematurely
Prokaryotes and
eukaryotes
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)
Post-translational Processing
• Folding
• Proteolytic processing
• Additions to peptide chain
Carbohydrates (glycosylation)
Methyl groups
Lipid groups
Hydroxylation, deamination
• Secretion
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
Stepwise
Random Chain
Secondary helix
or  sheet
Hydrophobic
alignments
Compact folded
protein
Proteolytic Processing of Insulin
Preproinsulin
Proinsulin
Insulin
Disulfide
bonds
s s
s s
B chain
s s
s s
A chain