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

3. Figure 1. The translation initiation pathway
Figure 1. The translation initiation pathway. The 40S ribosomal subunit is primed for initiating translation by binding of the ternarycomplex comprising eIF2, Met-tRNAi Met, and GTP (see left side of depicted scheme). In yeast, this is aided by the multifactor complex (MFC), an intermediate with roles in several steps of translation initiation. The resulting 43S preinitiation complex is recruited to the mRNA via interactions with the eIF4 factors bound at or near the cap structure of the mRNA (see top of schematic). The 43S complex then scans the 50UTRto locate the initiator codon (centre-right of scheme).Following recognition of theAUG, involving base-pairing with the anticodon loop of Met-tRNAi Met, release of the bound factors accompanies two distinct GTP hydrolysis steps and joining of the 60S subunit to form an elongation competent 80S ribosome (see bottom of figure), poised to start the first peptide elongation cycle. GDP-bound eIF2 is recycled by eIF2B to allow further ternary complex formation (see bottom left of the diagram).For clarity, eukaryotic initiation factors are labelled with the unique portion of their respective names only, omitting the general ‘eIF’ prefix i.e., ‘4E’ instead of ‘eIF4E’). Every effort was made to design this schematic such that it accurately reflects the many interactions between factors. Due to geometric constraints, however, the figure fails to show the interaction between eIF1A, and eIF2 aswell as eIF3. The ‘?’ near the looped-out 50 UTRin the centre of the scheme indicates the speculative nature of this scanning intermediate. To simplify the bottom part of the diagram, the distinct requirements of GTP-bound eIF5B for 60S subunit joining and GTP hydrolysis after 80S ribosome formation for eIF5B release are not shown. The recently identified factor eIF4H is also not represented in the scheme (see main text for more information)

4. Passaggi dell’inizio di traduzione
Formazione complesso 43S
Reclutamento del complesso 43S sul 5’ dell’mRNA (48S)
Scanning del 5’ UTR e riconoscimento dell’AUG
Formazione del complesso 80S

5. 2

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. 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

8. Reclutamento 43S-mRNA

9. Complesso 43S-mRNA (48S)

10. eIF4F

11. 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

12. eIF4G
Figure 1. (A) 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 697–949. (B) 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.

13. Struttura di eIF4G
Figure 3. Domain structure of eIF4G and selected proteins with roles in translation initiation. Mammalian cells contain two isoforms of eIF4G (I&II), with analogous domain structure and modes of interaction with other proteins (indicated above the schematic protein sequence). Most of these interaction sites have conserved amino acid and/or structural signatures (named below the eIF4G schematic: MA3, first identified in the mouse MA3 protein; MIF4G, middle portion of eIF4G; W2, named after two conserved tryptophan residues) that are also found in other proteins involved in translation. The yeast and plant homologues of eIF4G lack some of the domains found in the mammalian factor, suggesting that eIF4G has evolved around a conserved core region. p97 is a mammalian protein with homology to the C-terminal two thirds of eIF4G. Apparently it binds eIF4A only through its MIF4G portion and it may function as a specialised initiation factor for IRES-containing mRNAsduring cellular stress. Paip1 is a further MIF4G-domain protein with two PABP-interacting motifs (PAM1 and PAM2). These motifs are also found in Paip2 and both proteins bind to overlapping regions of PABP(see Fig. 4). The 4E-BPs are molecular mimics of the eIF4E-binding region of eIF4G and regulate translation.

14. Il ribosoma scorre sul 5’ dell’mRNA fino al codone di inizio AUG
GCC CCAUGG A G _
La sequenza consenso di Kozak

15. Scansione del 5' UTR

16. Scanning

17. Formazione complesso 80S

18. “Toeprint assay”

19. 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)

21. 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 eIF4E. (Picture provided by A. Sachs and reprinted with permission.)
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.

22. Figure 4. The role of PABP and the poly(A) tail of mRNAs during translation. A: PABP consists of four conserved RRMs, and a PABC domain at the C terminus. The arrows indicate interactions with various binding partners as discussed in the main text. eIF4G and poly(A) can bind simultaneously to RRM1 and RRM2, while the available evidence suggests that only one partner can bind to the PABC at any given time. Paip2 can interact with RRM2 and RRM3 of PABP as well as with the PABC domain and forms complexes of 2:1 stochiometry with PABP. Paip2 binding to RRM2 and RRM3 of PABP can disrupt the binding of PABP to poly(A) and inhibit translation. The inhibitory effects of Paip2 are indicated by lines with a short bar at the end.Paip1 interacts withRRM1andRRM2as well as with the PABC domain in complexes of 1:1 stochiometry. B: The ribosome-recycling concept. A possible function of mRNA circularization could involve facilitation of a direct recycling of ribosomes or ribosomal subunits, after termination at the stop codon, back to the 50 region of thesamemRNA(indicated by the green arrow). This speculative model is supported by the observation of circular polyribosomes, as well as interactions between PABP, initiation factors bound to the cap structure, and the translation termination factor eRF3.

25. Interazioni 5'-3' nell'mRNA

26. 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)

28. 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

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

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

32. Complessi d'inizio
Figure 1 (a) Assembly of the 43S preinitiation complex onto the mRNA. EIF4G acts as a scaffold and binds to eIF4E (cap-binding protein), eIF4A (dead box helicase which is required to unwind secondary structure, the activity of this protein is stimulated by its interaction with eIF4B), eIF3 which in turn interacts with the ribosome and Mnk (Map-integrating kinase which phosphorylates eIF4E when bound in this complex). The interaction of poly Abinding protein (PABP) with eIF4G allow circularisation of the mRNA. (b) Schematic representation of a picornaviral IRES. Picornaviral messages are uncapped and recruit ribosomes directly to the initiation codon via an IRES. This binding is facilitated by primary sequences such as the polypyrimidine tract and secondary structural elements. Host encoded IRES-trans-acting factors (X and Y) may also be required to stabilize the structures or facilitate ribosome binding (adapted from Belsham and Jackson, 2000)

33. IRES di EMCV
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.

34. IRES di HCV
eIF3
40S
Figure 2. Protein–RNA interactions on the HCV IRES. The secondary structures of domains II, III, and IV of the HCV IRES are shown schematically (adapted from Honda et al. 1999). Binding sites for eIF3 (yellow) and 40S subunits (red) are shown. A subdomain that is required for binding of eIF3 and the 40S subunit is shown in orange, and elements of the IRES that interact with 40S subunits and are required for accurate placement of the coding region in the decoding site of the 40S subunit are colored light blue. The toeprints detected at the leading edge of bound eIF3 are indicated by arrows. The toeprints at the leading edge 40S subunits in binary IRES/40S subunit complexes are indicated by unfilled diamonds; toeprints formed on eIF2/GTP/Met-tRNAi/40S subunits are indicated by filled diamonds.

35. Fattori necessari per la traduzione

36. IRES di CrPV

37. Sequenze IRES virali
Internal ribosome entry sites (IRESs) are RNA elements that mediate end-independent ribosomal recruitment to internal
locations in mRNA. Structurally related viral IRESs use distinct mechanisms, based on non-canonical interactions with
eukaryotic initiation factors (eIFs) and/or 40S subunits (see the figure). Initiation on type 1 and type 2 IRESs involves their
specific binding to the p50 domain of eIF4G (FIG.3a), which is enhanced by eIF4A112–114, on type 3 IRESs involves their
interaction with the eIF3 and 40S subunit components of 43S complexes8,115 and on type 4 IRESs involves their binding to
40S subunits7,116. The eIF4G–eIF4A complex recruits 43S complexes to type 1 and type 2 IRESs without the involvement
of eIF4E. Type 3 IRESs directly attach 43S complexes to the initiation codon independently of eIF4F, eIF4B, eIF1 and
eIF1A, whereas type 4 IRESs initiate without eIFs or tRNAi
Met (the P-site of the 40S subunit is occupied by an IRES domain
that mimics codon–anticodon base pairing). Hence, IRES-mediated initiation might be resistant to cellular regulatory
mechanisms, such as eIF2 phosphorylation (type 4 IRESs) and/or eIF4E sequestration (all types of IRESs)116. Initiation on
some IRESs also requires IRES trans-acting factors (ITAFs) — RNA-binding proteins that are thought to stabilize the
optimal three-dimensional IRES conformation117.
The list of cellular mRNAs that are thought to contain IRESs is growing and, although a recent stringent test has
questioned some of these claims118, it would be prudent to presume that many are still valid. Cellular IRESs show little
structural relationship to each other and their underlying mechanism remains largely unknown but probably follows the
picornavirus paradigm of binding the eIF4G–eIF4A complex. Importantly, cellular IRES-containing mRNAs can also be
translated by the scanning mechanism, which raises the crucial question of what regulates the switch between these
modes of initiation. One key parameter might be the intracellular concentration of eIF4G. The concentration of eIF4G
(but not eIF4E) is highly elevated in many advanced breast cancers, and in inflammatory breast cancer this results in
efficient IRES-dependent translation of p120 catenin and vascular endothelial growth factor (VEGF) mRNAs119. In other
breast cancer cell lines with high eIF4G levels, overexpression of eIF4E-binding protein 1 (4E-BP1), to sequester eIF4E,
coupled with hypoxia, activates VEGF and hypoxia-inducible factor 1α (HIF1A) IRESs120. Another parameter that may
determine which mechanism predominates is the intracellular concentration of ITAFs.
REVIEWS
118 | FEBRUARy 2010 | vOlUME 10

38. IRES negli mRNA cellulari

39. Strutture secondarie delle IRES
Figure 2 Secondary structural models of c-myc (a) and L-myc IRESs (b). c-myc or L-myc RNA was treated with chemicals (CMCT, kethoxal, DMS) to detect single-stranded regions or with RNase VI to detect double-stranded regions. This information was then used to add constraints to the m-fold algorithm to obtain the structural model shown. The c-myc IRES has a more open and flexible structure than the L-myc IRES. However, interestingly both IRES contain potential pseudoknots close to the region where the ribosome lands

40. Fattori trans-agenti delle IRES

41. Ruolo delle IRES

42. Meccanismi di traduzione negli eucarioti
Figure 1: Schematic representation of eukaryotic mRNAs. (a) Features of a conventional mRNA. The red circle at the 5_end depicts the cap (m7Gppp); A(n) depicts the poly(A) tail at the 3_end. (b) (c) Schematic of atypical RNA structures, stem loops (black hairpin), or uAUGs, respectively, located in mRNAs translated via cap-dependent initiation. (d) RNA structural elements located in the 3_untranslated region of viral RNAs mediating cap-independent translation (3_CITE). Different types of IRES elements found in the viral RNA of picornaviruses (e), hepatitis C (f), and dicistroviruses (g) are schematically depicted in red.

43. Meccanismi di reclutamento della 40S
Figure 1. Prokaryotic and eukaryotic (cap- and IRES-dependent) strategies for the recruitment of the small ribosomal subunit by the mRNA during initiation of translation. For simplicity, only some components of the translation machinery are shown. Blue boxes represent open reading frames. (a) In prokaryotic initiation of translation, the small 30S ribosomal subunit (yellow) is positioned near or very near the authentic start codon by a direct interaction of the ribosomal 16S rRNA with the Shine-Dalgarno (S-D) sequence (black bar) in the mRNA without the requirement for translation factors [21]. (b) Most eukaryotic mRNAs are translated by the cap-dependent mechanism, which requires recognition by eIF4E (green crescent) complexed with eIF4G (red) and eIF4A (light green) – the so called eIF4F complex – of the cap structure (m7G) at the 50 end. A 43S pre-initiation complex – consisting of a 40S ribosomal subunit loaded with eIF3 (pink), eIF1 and eIF1A (grey), initiator Met-tRNAi Met (blue clover), eIF2 (dark green) and GTP – binds the eIF4F–mRNA complex and scans along the 50-UTR of the mRNA (black line) to reach the start codon, which is usually an AUG triplet. During the scanning, eIF4A, stimulated by eIF4B (dark blue), unwinds the secondary RNA structure in an ATP-dependent manner. The placement of the 43S complex on the authenticable start codon forms a 48S pre-initiation complex [1]. (c) Dicistrovirus IRESs, like that of cricket paralysis virus (CrPV), bind directly to the 40S ribosomal subunit without the requirement for any initiation factors, initiator Met-RNAi Met, ATP or GTP. CrPV mRNA possesses a stem-loop structure that contains an anti-codon triplet that is paired with the start codon GCU, thus mimicking the base-paired AUG codon-Met-tRNAi Met of conventional 48S complexes. (d) The IRES from hepatitis C virus (HCV, a flavivirus) also binds directly to the 40S ribosomal subunit without the requirement for host canonical initiation factors. It requires, however, the participation of factors eIF3 and eIF2 bound to GTP and initiator tRNAMet to form functional initiation complexes. ITAFs (purple) are involved in this process. Dicistrovirus and HCV IRES-dependent initiation of translation are furthermore considered ’prokaryotic-like’ mechanisms that occur in their eukaryotic hosts. By contrast, to recruit the 40S ribosomal subunit and form functional initiation complexes, the IRES of encephalomyocarditis virus (EMCV, a picornavirus) (e) requires eIF2, GTP, ATP, initiator tRNAMet eIF3, eIF4A, eIF4B and the central domain of eIF4G, but not eIF4E, eIF1 or eIF1A [2,4,6]. According to the complexity of the secondary structure, two types of eukaryotic cellular IRES have been described (f,g). The first type are A-rich, low structured IRESs (f) such as those of Drosophila hsp70 and some proapoptotic mRNAs [12,13] as well as the IRESs of yeast PABP and some starvation-response genes [14]. These Drosophila IRESs are active in the absence of eIF4E and, at least for one of them, the activity of ITAFs is involved [42]. In (f), the IRES of yeast YMR181c binds PABP in complex with eIF4G to recruit the 40S ribosomal subunit [14]. This mechanism can functionally substitute for a cap and eIF4E and, although not yet proven, might be a general mechanism in the functioning of A-rich IRESs. The second type are cellular IRESs with a highly structured secondary structure (g), such as those of FGF-1, FGF-2, c-myc, L-myc, Apaf-1, and Cat-1 mRNAs [5,7]. These IRESs also require the activity of ITAFs [3–5,7]. Note, because the requirement for the canonical initiation factors of most of the cellular IRESs is not known they are not shown (indicated by ‘?’). Additionally, the secondary structures shown illustrate only the relative complexity of each and are not intended to represent the actual structures.

44. Evoluzione del reclutamento della 40S
Figure 2. A model for the evolutionary stages of the initiation of translation. The image illustrates my proposed course from the mechanism found in ancestral archaeal cells to those found in contemporary eukaryotes. For simplification, only the most relevant factors and structures for the hypothesis discussed here are shown. (a) Ancestral archaeal cells had polycistronic (blue boxes) mRNAs with a Shine-Dalgarno (SD)-based recognition (black bar) of the 50-UTR of the mRNA by the ancestral 40S ribosomal subunit (yellow). Therefore, the initiation of translation at this time was prokaryotic-like. However, an eIF4A-like factor (green) performing RNA unwinding was probably present and probably evolved from the RNA-helicases found at that time in the ribosome or splicing complexes. (b) The evolutionary transition from the ancestral archaeal cell to early eukaryotes is represented by a dotted line. This delineation is due to the appearance of the nuclear membrane (dark blue). In addition, monocistronic and probably polyadenylated mRNAs and introns also arose at this time. Loss of cistrons in the transcripts was probably attributable to the incorporation of introns into the genome and, therefore, transcripts. Consequently, it is likely that splicing mechanisms also emerged at this time. Owing to the emergence of a nuclear structure, a temporal and spatial separation occurred between transcription and translation. The transport (dotted arrow) of mRNAs being spliced (by spliceosomal proteins that became ITAFs, purple) occurred from the nucleus to the cytoplasm. Once exported, initiation of translation occurred via the direct recruitment of the 40S ribosomal subunit by non-spliced introns at the 50-UTR of mRNAs. Spliceosomal proteins (purple) and eIF4A possibly facilitated the initiation of translation from these ribosomal landing pads. (c) Further on in the evolutionary process, the 50-cap (black dot) and the cap-binding protein eIF4E (green crescent) emerged to protect the transcripts against degradation during, for example, viral invasions of the host cell or mRNA transport. The evolution of eIF4G (red), possibly from other metabolic routes, improved the delivery of the 40S ribosomal subunit to the early IRESs that evolved from non-spliced introns. No contact between eIF4E and eIF4G occurred in these early stages as the initiation of translation was still IRES-driven. (d) An interaction between eIF4G and eIF4E developed, as well as between other translation initiation factors, leading to the canonical cap-dependent mechanism of today (i) in which the 40S ribosomal subunit is loaded onto the mRNA at the very 50-end and then scans the RNA to find the authenticable start codon. This mechanism became dominant, and the usefulness of IRESs was diminished and they disappeared from most transcripts. IRES-dependent initiation of translation (ii) remains mostly for those transcripts that are still translated during stress conditions, in which the IRESs have remained as part of a less structured 50-UTR. An increase in RNA structure complexity in some cellular IRESs has also occurred. Although not shown, viral IRESs probably arose several independent times after the emergence of the cap-dependent mechanism of initiation and not directly from the ancestral IRESs of the proto-eukaryotes.

46. Complesso MFC