Eucaryotic Protein Eucaryotic Protein SynthesisSynthesis

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Eukaryotic mRNAsEukaryotic mRNAs

See Figure 30.26 for the structure of the See Figure 30.26 for the structure of the typical mRNA transcript typical mRNA transcript

• Note the Note the 5'-methyl-GTP cap5'-methyl-GTP cap and the and the poly A poly A tailtail

• Cap is essential for mRNA binding and Cap is essential for mRNA binding and stabilizes mRNA by preventing degradationstabilizes mRNA by preventing degradation

• Poly A tail enhances stability and Poly A tail enhances stability and translational efficiency of mRNAstranslational efficiency of mRNAs

• Shine-Dalgarno sequence not presentShine-Dalgarno sequence not present

33

Initiation in eucaryotesInitiation in eucaryotes

•Family of at least Family of at least 14 eukaryotic 14 eukaryotic initiation factors initiation factors

•The initiator tRNA is a special one The initiator tRNA is a special one that carries only Met and functions that carries only Met and functions only in initiation - it is called only in initiation - it is called tRNAtRNAii

MetMet but it is not formylated but it is not formylated

44

55

Eukaryotic InitiationEukaryotic Initiation • Begins with formation of ternary complex of eIF-Begins with formation of ternary complex of eIF-

2, GTP and Met-tRNA2, GTP and Met-tRNA iiMetMet

• 1) This binds to 40S ribosomal subunit:eIF-1) This binds to 40S ribosomal subunit:eIF-3:eIF1A complex to form the 3:eIF1A complex to form the 43S preinitiation 43S preinitiation complex complex – Note no mRNA yet, so no codon association with Met-Note no mRNA yet, so no codon association with Met-

tRNAtRNAiiMetMet

• 2) mRNA then adds with several other factors, 2) mRNA then adds with several other factors, forming the forming the 48S48S initiation complexinitiation complex (Fig. 33.23) (Fig. 33.23) – 48S initiation complex scans to find the first AUG (start) 48S initiation complex scans to find the first AUG (start)

codoncodon

• 3) At AUG, 60S subunit adds to make 3) At AUG, 60S subunit adds to make 80S 80S initiation complexinitiation complex (GTP is hydrolyzed) (GTP is hydrolyzed)

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77

88

Regulation of InitiationRegulation of Initiation

Phosphorylation is the key, as usualPhosphorylation is the key, as usual

• At least two proteins involved in At least two proteins involved in initiation (Ribosomal protein S6 and initiation (Ribosomal protein S6 and eIF-4F) are eIF-4F) are activated by activated by phosphorylation phosphorylation

• But phosphorylation of eIF-2But phosphorylation of eIF-2 causes it causes it to bind all available eIF-2B and to bind all available eIF-2B and sequesters it, therefore translation is sequesters it, therefore translation is down-regulated by phosphorylationdown-regulated by phosphorylation

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1010

Elongation and TerminationElongation and Termination

• Elongation is similar to procaryotic Elongation is similar to procaryotic elongation: elongation: – EF1A homolog to EF-Tu, EF1B homolog EF1A homolog to EF-Tu, EF1B homolog

to EF-Ts, EF2 homolog to EF-Gto EF-Ts, EF2 homolog to EF-G

• Termination even simpler: only one Termination even simpler: only one RF, binds with GTP at the termination RF, binds with GTP at the termination codoncodon

1111

Inhibitors of Protein Inhibitors of Protein SynthesisSynthesis Two important purposes to biochemistsTwo important purposes to biochemists

• These inhibitors (Figure 30.30) have helped These inhibitors (Figure 30.30) have helped unravel the mechanism of protein synthesis unravel the mechanism of protein synthesis

• Those that affect prokaryotic but not Those that affect prokaryotic but not eukaryotic protein synthesis are effective eukaryotic protein synthesis are effective antibiotics antibiotics

• StreptomycinStreptomycin - an aminoglycoside antibiotic - - an aminoglycoside antibiotic - induces mRNA misreading. Resulting mutant induces mRNA misreading. Resulting mutant proteins slow the rate of bacterial growth proteins slow the rate of bacterial growth

• PuromycinPuromycin - binds at the A site of both - binds at the A site of both prokaryotic and eukaryotic ribosomes, prokaryotic and eukaryotic ribosomes, accepting the peptide chain from the P site, accepting the peptide chain from the P site, and terminating protein synthesisand terminating protein synthesis

1212

1313

Diphtheria ToxinDiphtheria Toxin

An NADAn NAD++-dependent ADP ribosylase-dependent ADP ribosylase

• One target of this enzyme is EF2 One target of this enzyme is EF2

• EF2 has a EF2 has a diphthamidediphthamide (see Figure (see Figure 33.27) 33.27)

• Toxin-mediated Toxin-mediated ADP-ribosylationADP-ribosylation of of EF2 allows it to bind GTP but makes it EF2 allows it to bind GTP but makes it inactive in protein synthesis inactive in protein synthesis

• One toxin molecule ADP-ribosylates One toxin molecule ADP-ribosylates many EF2s, so just a little is lethal! many EF2s, so just a little is lethal!

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1515

RicinRicin

from Ricinus communis (castor bean) from Ricinus communis (castor bean)

• One of the most deadly substances known One of the most deadly substances known

• A glycoprotein that is a disulfide-linked A glycoprotein that is a disulfide-linked heterodimer of 30 kD subunits heterodimer of 30 kD subunits

• The B subunit is a The B subunit is a lectinlectin (a class of (a class of proteins that binds specifically to proteins that binds specifically to glycoproteins & glycolipids) glycoproteins & glycolipids)

• EndocytosisEndocytosis followed by disulfide reduction followed by disulfide reduction releases A subunit, which catalytically releases A subunit, which catalytically inactivates the large subunit of ribosomesinactivates the large subunit of ribosomes

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Ricin A subunit mechanismRicin A subunit mechanism

• Ricin A chain specifically attacks a Ricin A chain specifically attacks a single, highly conserved adenosine near single, highly conserved adenosine near position 4324 in eukaryotic 28S RNA position 4324 in eukaryotic 28S RNA

• N-glycosidaseN-glycosidase activity of A chain activity of A chain removes the adenosine base removes the adenosine base

• Removal of this A (without cleaving the Removal of this A (without cleaving the RNA chain) RNA chain) inactivates the large subunit inactivates the large subunit of the ribosomeof the ribosome

• One ricin molecules can inactivate One ricin molecules can inactivate 50,000 ribosomes, killing the eukaryotic 50,000 ribosomes, killing the eukaryotic cell!cell!