Protein Biosynthesis II
• mRNA BindingShine-Dalgarno Sequence
• Initiation Complexf-Met-tRNAf
Met
Initiation factors
• ElongationElongation factors
• TerminationTermination factors
• mRNA BindingShine-Dalgarno Sequence
• Initiation Complexf-Met-tRNAf
Met
Initiation factors
• ElongationElongation factors
• TerminationTermination factors
Dintzis Experiment
COO-
5’
Trypsin
H2N-
H2N-COO-
C-terminal is richerin radioactive 3H-leucine
C-terminal is richerin radioactive 3H-leucine
3H-Leucine
H2N-
H2N-
3’
Released Protein4 min
8 min
60 min
3H
C-terminalN
Product analysis
Shine-Dalgarno
Purine-rich mRNA
Pyrimidine-rich rRNA complementary
fMet-tRNAfMet
CH3-S-CH2CH2C-COO-tRNA
H
N-H
C=OH
N-Terminal Block
Blocking the N-terminal amino acid assures thatthe peptide chain will grow towards the C-terminal
Blocking the N-terminal amino acid assures thatthe 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
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 MECHANISMOVERALL MECHANISM
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 newcodon into the A site
Simultaneously, the peptidyl tRNA is pushedinto the P site and the uncharged tRNA isshoved into the E site and expelled
(OVERALL CONT.)(OVERALL CONT.)
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 inplace
INITIATIONINITIATION
Initiation Complex
ElongationElongation
2. An aminoacyl-tRNA reacts with elongationfactor EF-Tu in preparation for bindingto the A site.
1. Peptidyl-tRNA sits in the P site
3. A-site binding occurs as GTP is hydrolyzed.EF-Ts removes spent GDP.
4. A peptide bond is formed with the transferof the peptide from the P site to the A site
5. Elongation factor EF-G shifts the mRNAone frame, expelling the uncharged tRNA
Elongation
TerminationTermination
2. A termination factor that recognizes the codon binds to the A site
3. The peptide in the P site is transferredto the factor
1. Translocation results in a stopcodon (UAA, UGA, UAG) in the A site
4. Because the factor cannot bind,the peptide is released
Termination COOH
Diphtheria Toxin (Cornyebacterium diphtheria with
1. Reacts with elongation factor eEF-2 (prokaryote G)
eEF-2 + NAD+
bacteriophage cornyephage )
N
CONH2
O
OH
CH2OO-P-O-P-
OO
OOO CH2
N
N
N
N
NH2
OHHO HO
+
Break glycosidic bond to nicotinamide in reaction
ss
Trypsin
ss
A (catalytically active)
B
Diphtheria toxin
(cell penetration)NH2
N
N
CH2CH2 CH
C=O
N(CH3)3+
PEPTIDE CHAIN
NH2
N
N
CH2CH2 CH
C=O
N(CH3)3+
PEPTIDE CHAIN
O
OHHO
CH2ADPNAD+
eEf-2(diphthamide group)
Modified His in eEF-2 is target
ADP ribosylation
ss
A
B
Receptor
ss
A
BsH
Endocytosis
Disulfide cleavage
s
A
NAD+
eEF-2
ADP-ribosyl-eEF-2
Active enzyme
H
(ribosylates histidineresidues with diphthamide structure)
(See p 879)
Translation Blocking Antibiotics
Puromycin
Binds to A site andreceives peptidefrom transpeptidase
Aborts peptideprematurely
Prokaryotes and eukaryotes
Aminoglycoside
Causes misreading and inhibits chain initiation of prokaryote mRNA
Blocks peptidyl transferase inprokaryotes (A-site binding)
Inhibits aminoacyl-tRNA bindingto prokaryotes: blocks nutritionalfactor ppGpp (p867)
Post-translational Processing
• Folding
• Proteolytic processing
• Additions to peptide chainCarbohydrates (glycosylation)Methyl groups Lipid groups
Hydroxylation, deamination
• Secretion
FoldingRule: 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
Random ChainRandom Chain
Secondary helix or sheet
Secondary helix or sheet
Hydrophobic alignments
Hydrophobic alignments
Compact folded protein
Compact folded protein
StepwiseStepwise
Proteolytic Processing of Insulin
A chain
Preproinsulin Proinsulin Insulin
B chainDisulfide bonds
sss
ss s
s s