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

Initiation Factors 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

Passaggi dell’inizio di traduzionePassaggi dell’inizio di traduzione

1. Formazione complesso 43S

2. Reclutamento del complesso 43S sul 5’ dell’mRNA

3. Scanning del 5’ UTR e riconoscimento dell’AUG

4. Formazione del complesso 80S

eIF2eIF2

3 subunità: Subunità aiuta attività di GTPasi e modula

il legame tRNAi-eIF2 Subunità è un regolatore della traduzione.

E’ fosforilata (ser 51) da diverse chinasi in risposta a stress

eIF2BeIF2B 5 subunità:

Fattore di scambio GDP-GTP (GEF) per eIF2

2 subcomplessi: attività catalitica attività regolativa

Complesso MFC

eIF3eIF3

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

Reclutamento 43S-mRNA

Complesso 43S-mRNA

eIF4F

eIF4FeIF4F

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

eIF4G

GCC CCAUGGAG_

La sequenza consenso di Kozak

Negli eucariotii ribosomi migrano dalla estremità 5’ dell’mRNA fino al sito di legame del ribosoma, che include un codone di inizio AUG.

60S

40S

StopAUG

scanning

structural interference

factor interference

uORF interference

FA

B BBA A

AUG

Scanning

Formazione complesso 80S

“Toeprint assay”

ScanningScanning

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)

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.

Ruolo di PABP nella traduzioneRuolo 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)

Initiation FactorInitiation Factor ActivityActivity

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

pUp UUUCCUUUU AUG

Inizio di traduzione nell’mRNA di poliovirus

AUG AUG AUG AUG

IRES= Internal ribosome entry site

Saggio dell’mRNA bicistronico

CAT luciferasicap

CAT luciferasicap IRES

CAT luciferasicap IRES

CAT luciferasicap IRES4F

+++ +++

+++ +/-

(+) +++

(+/0) +++

eIF3

40S