Protein Translation

• Text Ch 3, 17

• Structure– Amino acids– Folding

• Synthesis– Pre-initiation– Initiation– Elongation– Post-processing

Protein structure

• Amino acid– Amine– Side chain– Carboxylic acid

• Amide backbone

• Side chains– Polarity/charge– Size

• Glycine “R” is –H

• Tryptophan “R” is C9H8N with two rings

+H3N – CR – COO-

H2N – CR – COOHAcidBase

+H3N – CR – CO – NH – CR – COO-

+H O- H

3-D structure

• Solvent interaction – water– Hide the hydrophobes

• Charge interaction– Acidic side chains (-)– Alkaline side chains (+)– Polar

• Secondary structure– -helix– -sheet

• Tertiary structure

Protein Translation

• 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 Pol• Two major subunits: 40S & 60S

3 tRNA binding sites

Narrow peptide extrusion tunnel(spinnerette)

60S

(la

rge)

sub

unit

stru

ctru

e

Ban et al., 2000

40S

(la

rge)

sub

unit

stru

ctru

e

A P E

mRNA twisted through 40S

tRNA docking

Initiation

Pre-initiation complex Transition to elongation

Fig 17-9

Elongation

Elongation CycleeEF1 Cycle

(note: edited from text)Fig 17-10

eEF2 cycle

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 eIF4E specifically binds

7mG cap

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

• eEF1 (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

Anticodon

CCA-amino acid

70 Å

tRNA

mRNA2.5 Å H-bonds

9 Å

Ribosome procession

• eEF2– Structurally similar to eEF1+tRNA– Displaces A/P site tRNA to P site– Prime A site

• GTP hydrolysis– 60S nuclease center

3’5’P AE

NH3

3’5’P AE

NH3

Elongation

3’5’P AE

NH3

3’5’P AE

NH3

P AE

eEF1 mediated tRNA recruitment

ribosome mediatedpeptide binding

eEF2 mediated ribosome procession

reset fornext cycle

Elongation

Ratje & al., 2010

eEF2

eEF2

40S

60S

PDB IDs: 2XUX, 2XUY; 2XSY, 2XTG MMDB IDs: 111552, 111555

Termination

• eRF1 recruited to stop codon– 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 foldingProtein 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

• 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

• Complex membrane anchor– Carboxy terminal– Raft Targeting

• Extracellular– Acetylcholinesterase– “Self” recognition

• Paroxysmal nocturnal haemoglobinuria

– Carbonic anhydrase

• PLC cleaves PO4

C C C Glycosyl

Phospho

Acyl

InositolMannose

C

C

N

PO4

Ethanolamine

Polypeptide