Transcript Document

Protein Translation
• Text Ch 3, 17
• Structure
– Amino acids
– Folding
• Synthesis
–
–
–
–
Pre-initiation
Initiation
Elongation
Post-processing
Protein structure
Base
• Amino acid
H2N – CR – COOH
– Amine
– Side chain
– Carboxylic acid
• Amide backbone
• Side chains
Acid
+H N
3
– CR – COO+H
+H N
3
O- H
– CR – CO – NH – CR – COO-
– Polarity/charge
– Size
• Glycine “R” is –H
• Tryptophan “R” is C9H8N with two rings
3-D structure
• Solvent interaction – water
– Hide the hydrophobes
• Charge interaction
– Acidic side chains (-)
– Alkaline side chains (+)
– Polar
• Secondary structure
– a-helix
– b-sheet
• Tertiary structure
Protein Translation
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Assembly of 5’-cap complex
Annealing of ribosome
t-RNA decoded polypeptide elongation
Trafficking
Co-translational modification
– Sugars
– Fatty acids
– Chaperone mediated folding
80S Ribosome
• Equivalent to RNA PolII or DNA Pold
• Two major subunits: 40S & 60S
APE
60S (large) subunit structrue
40S (large) subunit structrue
tRNA docking
3 tRNA binding sites
mRNA twisted through 40S
Narrow peptide extrusion tunnel
(spinnerette)
Ban et al., 2000
Initiation
Pre-initiation complex
Fig 17-9
Transition to elongation
Elongation
eEF1 Cycle
Elongation Cycle
eEF2 cycle
(note: edited from text)
Fig 17-10
tRNA Lever
• Base complement structure
• Codon matching
– Structural amplification
Anticodon
CCA-amino acid
Pre-Initiation complex
• 40S ribosomal subunit
• eIF1A
– 80S dissociation
– Pseudo A-site tRNA
• eIF3
– 80S dissociation
– Initiation complex scaffold
• eIF2
– Met-tRNA carrier
– GTP dependent
Initiation Complex
• 43S Pre-Initiation Complex
• mRNA
– 7’methylguanosine (7mG) cap
– eIF4
• eIF4G scaffold
• eIF4E targeting
• eIF4A ATP dependent helicase
• Scanning
– 5’ UTR structure
7mG cap
eIF4E specifically binds
7mG cap
Ribosome Assembly
• 48S Initiation complex
– Scans along mRNA for AUG
– eIF5: eIF2 GAP
– eIF5B: recruits 60S subunit
• GTP hydrolysis displaces eIF5B
• 60S subunit
– Aminoacyl, peptidyl, exit docking sites
– P site initially occupied by t-Met
Elongation
• eEF1:tRNA recruitment
• eEF2 procession
(note: edited from text)
Elongation
• eEF1a (bacterial EF-Tu)
– GTP dependent
– Recruits aa-tRNA to A site
• P-protein bound to A-amino acid
– Transitional tRNA state
• eEF2 (bacterial EF-G)
– GTP dependent
– Displaces A-tRNA
• Ribosomal Release Factor (rRF)
eEF1 Function
• eEF1A: codon independent association
• Stabilized by codon recognition
– Triggers GTP hydrolysis
– 60S nuclease center
– eEF1 release as eEF1:GDP
• Codon hybridization
• Peptide binding
Translational accuracy
• AA-tRNA synthesis
• Codon matching
– Structural amplification
– 1 Å accuracy
2.5 Å H-bonds
mRNA
tRNA
Anticodon
CCA-amino acid
Ribosome procession
• eEF2
– Structurally similar to eEF1+tRNA
– Displaces A/P site tRNA to P site
– Prime A site
• GTP hydrolysis
– 60S nuclease center
Elongation
eEF1 mediated tRNA recruitment
5’
E P A
3’
5’
E P A
reset for
next cycle
ribosome mediated
peptide binding
NH3
5’
3’
NH3
E P A
3’
5’
EE PPAA
eEF2 mediated ribosome procession
NH3
NH3
3’
Elongation
eEF2
40S
eEF2
60S
PDB IDs: 2XUX, 2XUY; 2XSY, 2XTG MMDB IDs: 111552, 111555
Ratje & al., 2010
Termination
• eRF1 recruited to stop codon
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UAA, UAG, UGA
Another structural analog of tRNA
Breaks P-site peptide bond
GTPase
Mechanism of release
Barat et al., 2007
Termination
• eRF3
– eRF1 GAP
– Dissociation of eRF1 by activating GTPase
• eRF4
– 60S dissociation and recycling
• Initiation factors
– eIF3 Displaces P-site tRNA
– eIF1
Post-translational Processing
• Folding
– Chaperone proteins
– Endoplasmic reticulum
• Trafficking
– Subcellular localization
– Targeting signals
Protein folding
• Energy minimization
– Hydrophobic domains
– Charge balance
– Metallic complexes
• Ribosome holds ~40
residues denatured
• Spontaneous folding
• Assisted folding
Protein folding may be a
stochastic search for the lowest
energy configuration
Molecular Chaperones
• Heat Shock Proteins (HSP)
– HSP70 binds short hydrophobic chains
– Delay folding
– Prevent aggregation
• Chaperonins
– Receive HSP complexes
– Shield larger molecules during complex folding
Subcellular trafficking
• Posttranslational targeting to organelles
• Cotranslational targeting to compartments
– ER/Golgi
– Signal sequence (Start/Stop)
– Translocon
Glycosylation
• Co-translational addition of oligosaccharides
– ER
– Extracellular or membrane bound
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Negatively charged
Highly hydrated
Glycosaminoglycans (GAG)
Binding/recognition
– Synapse
– ECM
– Growth factor
Acylation – fatty acid transfer
• Myristic acid (C14:0)
– NH3-Met-Gly– Co-translational amide bond with Gly
• Palmitic acid (C18:0)
– N-terminal, near TM domains
– Thioester bond with Cysteine
• Isoprenoids (C15:3/C20:4)
– C-terminal CAAX box
– Thioester bond with Cysteine
– Cleavage of AAX
• Membrane association
• Acyl-chain coding of target membrane
Saturated
fatty acids
Glycophosphoinositol (GPI) Anchor
Polypeptide
• Complex membrane anchor
– Carboxy terminal
– Raft Targeting
N
C
Ethanolamine
C
• Extracellular
– Acetylcholinesterase
– “Self” recognition
Inositol
Mannose
• Paroxysmal nocturnal haemoglobinuria
– Carbonic anhydrase
• PLC cleaves PO4
PO4
Phospho
C C C
Glycosyl
Acyl