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