riconoscimento UAA, UAG riconoscimento UGA, UAA

Presentazione sul tema: "riconoscimento UAA, UAG riconoscimento UGA, UAA"— Transcript della presentazione:

1 riconoscimento UAA, UAG riconoscimento UGA, UAA
procarioti eucarioti Fattori di allungamento EF-Tu eEF1A trasporto aa-tRNA EF-Ts eEF1B riciclo EF-G eEF2 traslocazione Fattori di terminazione RF1 eRF1 riconoscimento UAA, UAG RF2 “ riconoscimento UGA, UAA RF3 eRF3 GTPase RRF rilascio

2 Initiation Factors prokaryotes eukaryotes
Activity prokaryotes eukaryotes IF3 eIF-1 Fidelity of AUG codon recognition IF1 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 (12 subunits) Ribosome antiassociation, binding to 40S eIF-4F (4E, 4A, 4G) mRNA binding to 40S, RNA helicase activity eIF-4A ATPase-dependent RNA helicase eIF-4E 5' cap recognition eIF-4G Scaffold for of eIF-4E and -4A eIF-4B Stimulates helicase, binds with eIF-4F eIF-4H Similar to eIF4B eIF-5 Release of eIF-2 and eIF-3, GTPase IF2 eIF5B Subunit joining eIF-6 Ribosome subunit antiassociation

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4 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

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6 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

7 Complesso MFC

8 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

9 Reclutamento 43S-mRNA

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11 Complesso 43S-mRNA

12 eIF4F

13 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

14 eIF4G Schematic diagram of human eIF4G1 protein. Shown are the binding sites for PABP, eIF4E, eIF4A, and eIF3 and the target site for picornaviral proteinase 2Apro. The minimum eIF4G1 fragment that binds specifically to the EMCV IRES and supports 48S complex formation corresponds to amino acid residues

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

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18 Scanning

19 Formazione complesso 80S

20 “Toeprint assay”

21 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)

22 Figure 2 | 3'–5' interactions: circles of mRNA
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.

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28 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)

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30 Initiation Factor Activity
eIF Fidelity of AUG codon recognition eIF-1A Facilitate Met-tRNAiMet binding to small subunit eIF 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 Release of eIF-2 and eIF-3, ribosome-dependent GTPase eIF5B Subunit joining eIF Ribosome subunit antiassociation

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

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

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35 Model for assembly of 48S complexes on EMCV-like IRESs
Model for assembly of 48S complexes on EMCV-like IRESs. Structural domains of the IRES and regions of contact with the following factors as determined by footprinting are shown: eIF4G/4A complex (blue/green), ITAF 45 (diamonds), PTB (gray). PTB contains four RRM domains and binds multiple sites on EMCV-like IRESs; such binding (indicated by a dotted line) therefore may stabilize a specific conformation of the IRES. The recruitment of a 40S ribosomal subunit (red) carrying initiator tRNA and eIF3 (yellow) is shown. See text for details.

36 eIF3 40S

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39 c-Myc IRES, b) L-myc IRES – strutture verificate con reagenti chimici e con Rnasi VI

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