1. 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

2. Control of translation General Mechanisms –Activity of GTPases –Availability of translation factors Protein specific mechanisms –mRNA structure –Sequence specific binding proteins

3. Control points in translation Cap binding structure –eIF2-GTP+tRNA (GTP exchange) –eIF4E (sequestration) Elongation –eEF2-GTP+tRNA (GTP affinity) Sequence-specific mechanisms –5’ UTR structure –Initiation complex efficiency –RNA binding proteins

4. eIF2 Regulation Met-tRNA carrier; general translation rate –0.5 eIF2a per ribosome eIF2  kinases block GDP-GTP exchange –Strengthen binding of eIF2  and eIF2B –Extremely efficient: 20-30% p-eIF2  chelates majority of eIF2B phos-eIF2  eIF2B eIF2  phospho

5. eIF2a Kinases “Stress” activated proteins –Metabolic stress –Environmental stress Reduce general translation in unhealthy conditions –Hemin-Regulated Inhibitory Kinase (HRI) –General Control of amiNo acid synth (GCN2) –Protein Kinase dsRNA activated (PKR) –PKR-like Endoplasmic Reticulum Kinase (PERK)

6. Hemin-Regulated Inhibitor kinase Constitutively active in reticulocytes & erythrocytes Inhibited by heme to allow translation in RBC precursors Balance globin synthesis to heme availability Generally suppress translation by RBC HRIeIF2aglobinHeme Hemoglobin

7. GCN2 General control of amino acid synthesis Sensor for unloaded tRNA, AA abundance Phosphorylates eIF2a, reduces protein synth Stimulates GCN4 translation –5’ upstream open reading frames –Re-initiation at GCN4 start only without eIF2 –Transcriptional activator of amino acid biosynthesis –Activation of GCN4 in anterior piriform cortex stimulates foraging behavior in mammals GCN4 mRNA ORFActive coding sequence AUG Translation

8. PKR dsRNA-activated Protein Kinase –dsRNA binding exposes ATPase –Triggers dimerization & autophosphorylation –dsRNA viruses Induces If  &NF-  B PERK (PKR ER-related kinase) –ER-Stress dependent –Slows translation in response to misfolding Translation Misfolded proteins PERK eIF2  Healthy proteins

9. eIF4 4E Binding Proteins –eIF4E cap binding protein –Compete with eIF4G –Phosphorylated after growth factor activation Release eIF4E Thr-37 & Thr-46 (PI-3K/mTOR) Ser-65 & Thr-70 (ERK/CaMK?) –Dephosphorylated by PP2A Bind eIF4E eIF4EeIF443S4EBP Translation

10. eEF phosphorylation eEF1B is the eEF1  GEF –Phosphorylation increases activity PKC MSK6 –Increases eEF1  recycle rate & availability of tRNA eEF2 –Needs no GEF –Phosphorylated in GTP binding domain CaMKIII = eEF2 Kinase PKA dependent activation of eEF2 –Blocks activity

11. eEF1B phosphorylation eEF1B phosphorylation increases eEF1a recycle rate Increases tRNA availability

12. eEF2 phosphorylation eEF2 phosphorylation blocks GTP binding Decreases ribosome procession

13. PI-3K cascade GFR mediated activation of PI3K Generation of PIP 3 PH binding –PKB/Akt –PDK1 mTOR Translational Machinery

14. PI3K targets in translational control 4EBP1 –Releases eIF4E to promote initiation eIF4E –Facilitates binding to eIF4G eEF2 Kinase –Blocks calmodulin binding –Reduces phosphorylation of eEF2B p70S6 Kinase –Increases 5’-TOP translation

15. Specific Targeting by S6 phosphorylation 5’ terminal oligopyrimidine (CU) structure S6 protein of 40S subunit –Phoshporylation increases affinity for 5’TOP Ribosomal proteins eIFs, eEFs

16. Regulation of Termination Stop codon recognition depends on context E coli RF2 –In-frame, premature UGA stop –Low RF2 gives 1-base frameshift readthrough –RF2 translationally autoregulated RF association with eIF4

17. Poly(A) binding protein Translation efficiency –In vivo, (competitive) using electroporation 5x faster with poly(A) 5x faster with 7mG 250-10,000x faster with poly(A) and 7mG –Not in reconstituted systems Kessler & Sachs –Pab1 eIF4G binding poly(A) binding

18. Poly(A) binding protein Pab1:eIF4G association –Loop formation, steric facilitation 3’UTR –Conformational facilitation No apparent change in IP complexes –Inhibition of inhibitors

19. Evaluation of translational efficiency Comparison of protein and mRNA –RT-PCR/PCR/Northern Blot –ELISA/Western Blot Polysome profiles –Sedimentation rate by HPLC mRNA protein Transcriptional Translational Faster sedimenting Heavier

20. 5’ UTR structure control 50-70 nt; longer is better Scanning model Upstream open reading frame Stem-loop structures –Self-complimentary sequences Internal Ribosome Entry Site (IRES) 2 0 structure of HCV RNA 5’ Residue 330

21. mRNA Binding Elements Iron response element: block 40S binding 5’ TOP: promote 40S binding Bruno: spatial repression of oskar by eIF4G competition Micro RNA

22. Iron Response Element Stereotypical hairpin-loop Iron Response Protein –Low iron allows binding 5’ block 40S binding –eg ferritin iron buffer 3’ shield vs nuclease –eg transferrin receptor to import Fe Fe-IRP is part of the Kreb’s cycle

23. Developmental regulation by oskar Little transcription early in development Oskar expression defines the posterior pole of flies –Anatomical axes defined during oogenesis –Propagated by subcellular localization Bruno suppresses oskar translation –Begins phenotypic specialization Bru1 localization in zebrafish embryo (Hashimoto et al. 2006) Single cell Multi-cell

24. Summary Regulatory elements in untranslated regions of mRNA –Analogous to promoter/enhancer elements of DNA General translational efficiency controls –Metabolic status –Growth controls Mechanisms –GTP turnover –Co-factor availability