Eucaryotic Protein Synthesis

2 Eukaryotic mRNAs See Figure for the structure of the typical mRNA transcript Note the 5'-methyl-GTP cap and the poly A tail Note the 5'-methyl-GTP cap and the poly A tail Cap is essential for mRNA binding and stabilizes mRNA by preventing degradation Cap is essential for mRNA binding and stabilizes mRNA by preventing degradation Poly A tail enhances stability and translational efficiency of mRNAs Poly A tail enhances stability and translational efficiency of mRNAs Shine-Dalgarno sequence not present Shine-Dalgarno sequence not present

3 Initiation in eucaryotes Family of at least 14 eukaryotic initiation factors Family of at least 14 eukaryotic initiation factors The initiator tRNA is a special one that carries only Met and functions only in initiation - it is called tRNA i Met but it is not formylated The initiator tRNA is a special one that carries only Met and functions only in initiation - it is called tRNA i Met but it is not formylated

4

5 Eukaryotic Initiation Begins with formation of ternary complex of eIF-2, GTP and Met-tRNA i Met Begins with formation of ternary complex of eIF-2, GTP and Met-tRNA i Met 1) This binds to 40S ribosomal subunit:eIF-3:eIF1A complex to form the 43S preinitiation complex 1) This binds to 40S ribosomal subunit:eIF-3:eIF1A complex to form the 43S preinitiation complex –Note no mRNA yet, so no codon association with Met- tRNA i Met 2) mRNA then adds with several other factors, forming the 48S initiation complex (Fig ) 2) mRNA then adds with several other factors, forming the 48S initiation complex (Fig ) –48S initiation complex scans to find the first AUG (start) codon 3) At AUG, 60S subunit adds to make 80S initiation complex (GTP is hydrolyzed) 3) At AUG, 60S subunit adds to make 80S initiation complex (GTP is hydrolyzed)

6

7

8 Regulation of Initiation Phosphorylation is the key, as usual At least two proteins involved in initiation (Ribosomal protein S6 and eIF-4F) are activated by phosphorylation At least two proteins involved in initiation (Ribosomal protein S6 and eIF-4F) are activated by phosphorylation But phosphorylation of eIF-2  causes it to bind all available eIF-2B and sequesters it, therefore translation is down-regulated by phosphorylation But phosphorylation of eIF-2  causes it to bind all available eIF-2B and sequesters it, therefore translation is down-regulated by phosphorylation

9

10 Elongation and Termination Elongation is similar to procaryotic elongation: Elongation is similar to procaryotic elongation: –EF1A homolog to EF-Tu, EF1B homolog to EF- Ts, EF2 homolog to EF-G Termination even simpler: only one RF, binds with GTP at the termination codon Termination even simpler: only one RF, binds with GTP at the termination codon

11 Inhibitors of Protein Synthesis Two important purposes to biochemists These inhibitors (Figure 30.30) have helped unravel the mechanism of protein synthesis These inhibitors (Figure 30.30) have helped unravel the mechanism of protein synthesis Those that affect prokaryotic but not eukaryotic protein synthesis are effective antibiotics Those that affect prokaryotic but not eukaryotic protein synthesis are effective antibiotics Streptomycin - an aminoglycoside antibiotic - induces mRNA misreading. Resulting mutant proteins slow the rate of bacterial growth Streptomycin - an aminoglycoside antibiotic - induces mRNA misreading. Resulting mutant proteins slow the rate of bacterial growth Puromycin - binds at the A site of both prokaryotic and eukaryotic ribosomes, accepting the peptide chain from the P site, and terminating protein synthesis Puromycin - binds at the A site of both prokaryotic and eukaryotic ribosomes, accepting the peptide chain from the P site, and terminating protein synthesis

12

13 Diphtheria Toxin An NAD + -dependent ADP ribosylase One target of this enzyme is EF2 One target of this enzyme is EF2 EF2 has a diphthamide (see Figure 33.27) EF2 has a diphthamide (see Figure 33.27) Toxin-mediated ADP-ribosylation of EF2 allows it to bind GTP but makes it inactive in protein synthesis Toxin-mediated ADP-ribosylation of EF2 allows it to bind GTP but makes it inactive in protein synthesis One toxin molecule ADP-ribosylates many EF2s, so just a little is lethal! One toxin molecule ADP-ribosylates many EF2s, so just a little is lethal!

14

15 Ricin 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 heterodimer of 30 kD subunits A glycoprotein that is a disulfide-linked heterodimer of 30 kD subunits The B subunit is a lectin (a class of proteins that binds specifically to glycoproteins & glycolipids) The B subunit is a lectin (a class of proteins that binds specifically to glycoproteins & glycolipids) Endocytosis followed by disulfide reduction releases A subunit, which catalytically inactivates the large subunit of ribosomes Endocytosis followed by disulfide reduction releases A subunit, which catalytically inactivates the large subunit of ribosomes

16 Ricin A subunit mechanism Ricin A chain specifically attacks a single, highly conserved adenosine near position 4324 in eukaryotic 28S RNA Ricin A chain specifically attacks a single, highly conserved adenosine near position 4324 in eukaryotic 28S RNA N-glycosidase activity of A chain removes the adenosine base N-glycosidase activity of A chain removes the adenosine base Removal of this A (without cleaving the RNA chain) inactivates the large subunit of the ribosome Removal of this A (without cleaving the RNA chain) inactivates the large subunit of the ribosome One ricin molecules can inactivate 50,000 ribosomes, killing the eukaryotic cell! One ricin molecules can inactivate 50,000 ribosomes, killing the eukaryotic cell!