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Passaggi dell’inizio di traduzione Formazione complesso 43S

Passaggi dell’inizio di traduzione Formazione complesso 43S Reclutamento del complesso 43S sul 5’ dell’mRNA Scanning del 5’ UTR e riconoscimento dell’AUG Formazione del complesso 80S. eIF2 3 subunità: a, b, g

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Passaggi dell’inizio di traduzione Formazione complesso 43S

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  1. Passaggi dell’inizio di traduzione Formazione complesso 43S Reclutamento del complesso 43S sul 5’ dell’mRNA Scanning del 5’ UTR e riconoscimento dell’AUG Formazione del complesso 80S

  2. eIF2 • 3 subunità: a, b, g • Subunità b aiuta attività di GTPasi e modula il legame tRNAi-eIF2 g • Subunità a è un regolatore della traduzione. E’ fosforilata (ser 51) da diverse chinasi in risposta a stress • eIF2B • 5 subunità: a, b, g, d, e • Fattore di scambio GDP-GTP (GEF) per eIF2 • 2 subcomplessi: d, e attività catalitica a, b, g attività regolativa

  3. Complesso MFC

  4. eIF3 • 10-11 subunità • Nucleo di 5 subunità: eIF3a, b, c, i, g • In lievito forma un complesso con eIF1, eIF2, eIF5, Met-tRNAi (MFC) • Richiesto per il legame del 43S all’mRNA

  5. Reclutamento 43S-mRNA

  6. Complesso 43S-mRNA

  7. eIF4F

  8. eIF4F • Composto da 3 subunità eIF4A: elicasi, aiutato da eIF4B eIF4E: cap binding protein, regolato da fosforilazione e interazione con eIF4E-BP eIF4G: adattatore, interagisce con diversi fattori

  9. eIF4G

  10. _ A GCC CCAUGG G La sequenza consenso di Kozak Negli eucarioti i ribosomi migrano dalla estremità 5’ dell’mRNA fino al sito di legame del ribosoma, che include un codone di inizio AUG.

  11. Scanning

  12. Formazione complesso 80S

  13. “Toeprint assay”

  14. Scanning • 40S, ATP, eIF2, eIF4A, eIF4B, eIF4F, mRNA sufficienti per formare complesso I (non produttivo) • eIF1, eIF1A necessari per il complesso II (scanning fino all’AUG) • Se non ci sono strutture secondarie eIF4A, 4B, 4F non sono necessari (in vitro)

  15. Figure 2 | 3'–5' interactions: circles of mRNA.a | Visualization of circular RNA–protein complexes by atomic-force microscopy. Complexes formed on capped, polyadenylated double-stranded RNA in the presence of eIF4G, poly(A)-binding protein (PABP) and eIF4E91. (Picture provided by A. Sachs and reprinted with permission.) b | Model of messenger-RNA circularization and translational activation by PABP–eIF4G–eIF4E interactions. eIF4G simultaneously binds to eIF4E and PABP7, 9, 14, 53, 55, thereby circularizing the mRNA91 and mediating the synergistic stimulatory effect on translation of the cap and poly(A) tail by enhancing the formation of the 48S complex53, 54, 92. c | Model of mRNA circularization and translational activation by PABP–Paip1 interactions. Paip1 is a PABP-interacting protein that binds eIF4A93, acting as a translational co-activator. d | Model of mRNA circularization and translational repression by CPEB–maskin–eIF4E interactions. RNA-associated CPEB binds maskin, which in turn binds to the eIF4E. This configuration of factors precludes the binding of eIF4G to eIF4E and thus inhibits assembly of the 48S complex13. e | Model of translational repression by heterogeneous nuclear ribonucleoproteins (hnRNPs). The differentiation control element (DICE), located in the 3' UTR of 15-lipoxygenase mRNA, inhibits translation initiation by preventing the joining of the 60S ribosomal subunit to the 43S complex located at the AUG codon. This inhibition is mediated by hnRNP proteins K and E1. The inhibitory event probably targets one of the initiation factors involved in the GTP hydrolysis that releases the initiation factors and the joining of the 60S ribosomal subunit2, 94. ORF, open reading frame.

  16. Ruolo di PABP nella traduzione • In estratti “cell free” di lievito sinergismo tra cap e coda poli(A) • Interazione tra PABP e eIF4G • eIF4E, eIF4G, PABP e mRNA forma strutture circolari (in vitro) • Altre proteine che interagiscono con PABP (Paip1, 2 e eRF3)

  17. Initiation Factor Activity eIF-1 Fidelity of AUG codon recognition eIF-1A Facilitate Met-tRNAiMet binding to small subunit eIF-2 Ternary complex formation eIF-2B (GEF) GTP/GDP exchange during eIF-2 recycling eIF-3 (10 subunits) Ribosome subunit antiassociation, binding to 40S subunit eIF-4F (4E, 4A, 4G) mRNA binding to 40S, ATPase-dependent RNA helicase activity eIF-4A ATPase-dependent RNA helicase eIF-4E 5' cap recognition eIF-4G Scaffold for of eIF-4E and -4A in the eIF-4F complex eIF-4B Stimulates helicase, binds simultaneously with eIF-4F eIF-4H              Similar to eIF4B    eIF-5 Release of eIF-2 and eIF-3, ribosome-dependent GTPase eIF5B Subunit joining eIF-6 Ribosome subunit antiassociation

  18. UUUCCUUUU Inizio di traduzione nell’mRNA di poliovirus AUG AUG AUG AUG p U p A U G IRES= Internal ribosome entry site

  19. cap CAT luciferasi +/- +++ cap CAT luciferasi IRES +++ +++ IRES cap CAT luciferasi +++ (+) 4F cap CAT IRES luciferasi +++ (+/0) Saggio dell’mRNA bicistronico

  20. eIF3 40S

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