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Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013.

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Mechanism of Translation Initiation in Eukaryotes

and .

The initiation of protein synthesis consists in the recruitment of a ribosome·initiator tRNA complex to the initiation codon of a messenger RNA. In prokaryotes, this process involves the direct interaction of the ribosomal RNA with the mRNA. In contrast, eukaryotes have evolved a sophisticated mechanism that relies mostly on protein-RNA and protein-protein interactions. Eukaryotes have taken advantage of the evolution of novel mRNA structures, such as the 5' cap and the poly (A) tail, to develop new mechanisms for the recruitment of the ribosome to the mRNA. As a result, the eukaryotic translation initiation apparatus is now a complex machinery comprising at least eleven factors. This complexity provides a fertile ground for enhanced regulation, and many new mechanisms have been adopted by eukaryotes to control proteins synthesis. Indeed, many translation factors are phosphoproteins whose function can be regulated by extracellular signals. We will describe here the mechanism of translation initiation in eukaryotes, with a particular emphasis on translation factors and their function.

Introduction

With the structural confirmation that the ribosome is a ribozyme (see Chapter 15 by B.T. Wimberly), translation emerges as a process driven for the most part by RNA. As such, the initiation of protein synthesis appears to be devoted to the assembly of the catalytic rRNA and an initiator tRNA at the correct AUG codon of a template mRNA. These RNAs may now be widely acknowledged as the stars but they nevertheless cannot perform alone. Indeed the translation machinery, especially in eukaryotes, has evolved to require a host of protein factors.1 The focus of this review is on the essential parts played by the eukaryotic initiation factors (eIFs) in bringing together the initiator tRNA, ribosome and mRNA.

In eukaryotes, at least eleven different initiation factors are required to properly initiate translation.1 Collectively, they ensure that the methionyl-initiator tRNA (Met-tRNAiMet) is brought in the P site of the ribosome to the initiator AUG of an mRNA. Conceptually, this process can be divided in four steps:

  1. formation of the 43S pre-initiation complex, when the Met-tRNAiMet is delivered by eIF2 to the P site of the 40S ribosomal subunit;
  2. recruitment of the 43S complex to the 5' end of the mRNA by eIF3 and the eIF4 factors;
  3. scanning of the 5' untranslated region (UTR) and recognition of the AUG codon, and
  4. assembly of the 80S ribosome (Fig. 1).

Figure 1. Scanning model of eukaryotic translation initiation.

Figure 1

Scanning model of eukaryotic translation initiation. The Met-tRNAiMet interacts with eIF2·GTP to form the ternary complex. The multifactor complex is an intermediate facilitating Met-tRNAiMet recruitment to the 40S ribosomal subunit, which generates (more...)

Only the standard “scanning” mechanism utilized by most cytoplasmic mRNAs will be considered here. The reader is directed to recent reviews by Jackson2 and Hellen & Sarnow3 for a detailed discussion of alternative initiation pathways.

The sequence of several archaeal genomes revealed that translation in archaea is a hybrid between the bacterial and eukaryotic systems.4,5 Archaeal mRNAs are uncapped, polycistronic, lack long poly (A) tails, and their coding sequences are most often preceded by a Shine-Dalgarno element. In contrast to the bacterial mechanism for initiation codon selection, archaeal genomes contain homologues of many initiation factors that were thought to be exclusively eukaryotic.68 We will not discuss translation in archaea in more detail, except to point out that archaeal factors have been utilized in the last years to derive important structural and functional insights into the mechanisms of eukaryotic translation initiation (see below).

Formation of the 43S Preinitiation Complex

Initiator tRNA

Translation is initiated by a special Met-tRNA in every organism.9 Because of its unique function, the initiator Met-tRNA (Met-tRNAiMet) presents many functional and structural features that distinguish it from the elongator Met-tRNA. In eukaryotes, the Met-tRNAiMet interacts specifically with an eIF2·GTP complex (see below), which delivers it exclusively to the ribosomal P site.10 This particular interaction can be explained by sequence divergence between the initiator and elongator form of Met-tRNA (Fig. 2). Some sequence specificities promote the activity of the Met-tRNAiMet in initiation (Fig. 2). These include the A1:U72 base pair which is critical for initiator function and cell viability in S. cerevisiae11 and, when mutated to the G1:C72 present in elongator Met-tRNA, weakens the affinity of the human Met-tRNAiMet for eIF2 by a factor of 10.12 The critical function of the A1:U72 base pair in initiation is underscored by its almost universal conservation amongst cytoplasmic eukaryotic initiator tRNAs, and its exclusion from all eukaryotic elongator tRNAs.13 In addition, the Met-tRNAiMet loop IV contains A60 in place of pyrimidine 60, and A54 instead of the T54 present in the TγC sequence found in elongator tRNAs (Fig. 2). The presence of an A at positions 54 and 60 is not absolutely required, as only the A54U substitution is lethal in yeast.11 In comparison, the double substitution A54, A60 to T54, U60 in the human tRNA has minimal effect on an in vitro initiation assay.14 A yeast Met-tRNAi Met with both G1:C72 and T54 mutations rescues an elongator tRNAMet-depleted strain, suggesting a requirement of these residues for initiator functions and discrimination against elongator tRNAMet.15 Finally, the Met-tRNAiMet contains three consecutive G:C base pairs in the anticodon stem (Fig. 2). Substituting these nucleotides with those found in the elongator tRNA reduces the initiator activity in vitro.14 Furthermore, introduction of G29:C41 and G31:C39 in a yeast elongator tRNA with the A1:U72 and A54 mutations increases its activity in initiation.15

Figure 2. Specific elements in human initiator tRNA.

Figure 2

Specific elements in human initiator tRNA. Structure of the human elongator (A) and initiator (B) Met-tRNA. Bases important for promoting initiator function are boxed, whereas those discriminating against elongator function are circled. An asterisk denotes (more...)

Other sequence specificities are important for the discrimination of initiator and elongator tRNAMet (Fig. 2). Position 64 in plants and fungi has a unique 2'-O-phosphoribosyl group, which disrupts the structure of the TψC stem.9,16 This modification prevents the elongator function of Met-tRNAiMet,17,18 probably by inhibiting its interaction with the eEF1A·GTP complex.19,20 In vertebrates, it has been proposed that the TγC stem perturbation is due to the nature of the base pairs at the base of the stem.21 Indeed, mutation in base pairs A50:U64 and U51:A63 yield an initiator tRNA that participates in elongation and, when combined with the A1:U72 -> G1:C72 mutation, results in almost wild-type elongator function.21

eIF2

The initiator tRNA is delivered directly to the P site of the 40S ribosomal subunit as part of a ternary complex that also contains the heterotrimeric G protein eIF2 and GTP (Fig. 1). Subsequent pairing between the AUG initiation codon and the Met-tRNAiMet anticodon elicits hydrolysis of the GTP by eIF2, which requires the GTPase activating protein (GAP) eIF5 (Fig. 1; see below). The eIF2·GDP complex does not bind to the Met-tRNAiMet, and recycling of the GTP necessitates the guanine nucleotide exchange factor (GEF) eIF2B (Fig. 1; see below). An eIF2 homologue is present in archaea but not in bacteria, where IF2 delivers the fMet-tRNAfMet to the 30S subunit.7

Essential genes encode the α, β and γ subunits of eIF2 in S. cerevisiae. Mutations in all three subunits affect the accuracy of AUG initiation codon selection.22 Biochemical and genetic analyses indicate that eIF2γ likely binds to GTP and Met-tRNAiMet.10,22 Interaction between eIF2γ and the initiator tRNA is reduced by the Y142H and N135K mutations in yeast.23,24 The N135K mutant also increases GTP hydrolysis,24 whereas the K250R mutation increases the off-rate for GDP and GTP.23 These observations have recently been corroborated by the crystal structure of the archaeal eIF2γ (a-eIF2γ) from Pyrococcus abyssi.25 Sequence similarity7 suggests that a-eIF2γ is a close structural homologue of the elongation factors eEF1A and the bacterial EF1A (see Chapter 20 by P. Nissen et al and ref. 25). This is most apparent in the nucleotide-free structure, which shows that the three domains of a-eIF2γ are very similar to their equivalents in eEF1A and EF1A, although their relative orientation is somewhat different.25 Accordingly, the GDP binding pocket of a-eIF2γ is identical to that of EF1A. Superimposition of domains 2 and 3 from the EF1A·GTP·Phe-tRNAPhe structure on the corresponding domains in a-eIF2γ indicates that the Phe-tRNAPhe can readily be accommodated by the structure, except for the Phe side chain.25 This in turn suggests a binding pocket for the Met side chain of Met-tRNAiMet, which might explain why the methionyl group enhances initiation.26 The structure also shows that a-eIF2γ lacks features allowing EF1A to bind aminoacyl-tRNA nonspecifically, helping to explain the exclusion of elongator tRNA from eIF2.25 Unfortunately, no difference could be observed when comparing the GDP-bound structure of a-eIF2γ to the GMP-PNP bound form, which might indicate that eIF2β has a role in tRNAi Met binding to eIF2. Therefore, the structural explanation of how the hydrolysis of GTP causes the release of the Met-tRNAiMet will probably require a co-crystal of a-eIF2γ with a-eIF2β, a-eIF2α or both, along with the initiator tRNA. An interesting aspect of the a-eIF2γ structure is the occurrence of a conserved four cysteine cluster that was found to coordinate a zinc atom.25 The specific role of this motif is not known, but its significance has been highlighted in yeast where mutating the second cysteine (C155) to a serine causes a slow growth phenotype.27

While eIF2γ forms the structural core of eIF2, essential functions are also performed by eIF2β.10 Genetic screening in yeast identified eIF2β mutations in an essential Cys2-Cys2 zinc-finger motif found in the carboxy-terminal domain of eIF2β.22 Two of these mutants (S264Y and L254P) were biochemically characterized and confer an intrinsic GTPase activity to eIF2, implicating eIF2β in promoting eIF2γ GTPase activity.24 Independent of GTP hydrolysis, the S264Y mutant also increases initiator tRNA dissociation from eIF2.24 In addition, the zinc-finger motif is part of a larger mRNA binding region, and could therefore be important for facilitating codon-anticodon interaction in eIF2.28 mRNA binding properties were also ascribed to the three lysine repeats found at the amino-terminus of eIF2β.28 Moreover, these repeats mediate the mutually exclusive interaction of eIF5 and eIF2Bϵ with eIF2β.29,30 In agreement with the absence of eIF2B and eIF5 in archaea, a-eIF2β does not possess the region containing the lysine repeats. The solution structure of a-eIF2β from Methanococcus jannaschii was recently solved.31 a-eIF2β consists of two independent structural domains connected by a conserved helical linker.31 The N-terminal domain of a-eIF2β corresponds to the central domain of yeast eIF2β, which is responsible for the interaction with eIF2γ.32 The structure of the C-terminal domain reveals a critical role for the zinc atom in maintaining the structural integrity of the domain.31 Yeast eIF2β mutations implicated in initiation codon selection22 affect residues that are conserved in a-eIF2β. Four of these residues (Lys122, Arg125, Val 126 and Ile140) are located in close proximity in the structure of the C-terminal domain,31 and might serve as a binding site for other translational components implicated in initiation codon recognition.31

Phosphorylation of the conserved serine residue (Ser51) in eIF2α converts eIF2·GDP from a substrate to a competitive inhibitor of eIF2B.10 Whereas eIF2α is required for yeast survival under normal conditions, it is not essential as long as the β and γ subunits as well as the initiator tRNA are overexpressed.33 Removal of the α subunit of eIF2 causes an 18-fold increase in the Km of eIF2B catalyzed nucleotide exchange, indicating that eIF2α is required for interactions between eIF2 and eIF2B that promote wild-type rates of nucleotide exchange.34 Consistent with this, phosphorylation of eIF2α on Ser51 increases its affinity for the regulatory subcomplex of eIF2B (α, β, δ), in a way that hampers the function of the eIF2B catalytic subcomplex (γ, ϵ).35 Taken together, these data clearly indicate that the a subunit of eIF2 has evolved to become first and foremost a regulator of translation initiation. The X-ray structure of the N-terminal two-thirds of human eIF2α has been solved.36 This structure contains two domains: an N-terminal oligonucleotide binding (OB) domain similar to that in IF137 and eIF1A,38 and a C-terminal helical domain.36 The Ser51 residue is located in an unstructured loop connecting β3 and β4 of the OB domain. The putative RNA binding site of the OB domain lacks the cluster of positive charges that is observed in other members of the OB family, which is consistent with eIF2α not interacting with RNA.36 Protein-protein interactions involving eIF2α could be mediated by a highly conserved groove that is observed between the OB and helical domains.36

eIF2B

eIF2B is a heteropentameric GEF that is required to recycle the inactive eIF2·GDP complex into eIF2·GTP (Fig. 1). eIF2B is present in cells in lower amounts than eIF2.39 Phosphorylation of eIF2 on Ser51 of its α subunit sequesters eIF2B, thus repressing translation initiation.10 Genetic and biochemical methods indicate that eIF2B is divided into a catalytic subcomplex comprising eIF2Bγ and eIF2Bϵ, and a regulatory subcomplex consisting of eIF2Bα, eIF2Bβ and eIF2Bδ.40 The catalytic subcomplex can perform the guanylate exchange reaction even when eIF2α is phosphorylated, unless the regulatory subcomplex is present to modulate its activity.40

eIF2Bϵ and eIF2γ display sequence homology throughout their length, and both subunits are essential in yeast.10 eIF2Bγ does not exhibit catalytic activity, but it enhances the weak intrinsic guanylate exchange activity of eIF2Bϵ.40,41 Indeed, the catalytic subcomplex exhibits a higher activity than the holoenzyme, probably owing to the absence of regulation from the other subunits.40 The catalytic site is located in the C-terminal region of eIF2Bϵ, and is independent from the bipartite motif that is required for binding to eIF2β.30,42 The N-terminus of eIF2Bϵ contains elements that are necessary for the assembly of eIF2Bϵ and eIF2Bγ into the eIF2B complex.42

eIF2Bα, eIF2Bβ and eIF2Bδ also display sequence homology to each other. These three subunits are required for the regulatory subcomplex to bind to eIF2α and differentiate between its phosphorylated and nonphosphorylated forms.40 eIF2Bβ and eIF2Bδ are essential in yeast, but eIF2Bα is dispensable.10 However, the absence of eIF2Bα renders eIF2B insensitive to the phosphorylation status of eIF2α, both with yeast and rat factors.40,43 In summary, eIF2B appears to have evolved to function in two roles: a GEF to recycle eIF2·GDP, and a regulator to respond to eIF2α phosphorylation.

Interestingly, mutations in the five subunits of human eIF2B are associated with leukoencephalopathy with vanishing white matter (VWM), a rare brain disease usually affecting children.44,45

This is the first documented disease to be associated with mutations in a translation factor. Many missense mutations were observed in the different eIF2B subunits, but nonsense mutations were always found to be heterozygous.44,45 These data suggest that mutations completely inactivating eIF2B are lethal when homozygous, in agreement with the essential role of eIF2B.10 The central nervous system specificity of the disease is surprising. Translational regulation by eIF2B involves the phosphorylation of eIF2 on the a subunit in response to various stress signals (see below). Thus, it is possible that the brain is more sensitive to an altered stress response brought about by mutated eIF2B. Indeed, neurological deterioration is observed during or after episodes of fever in VWM patients.44,45

eIF3

The largest and most complex initiation factor, eIF3, is composed of up to 11 nonidentical subunits in mammals, 6 of which are conserved in yeast (Table 1).1,10 In agreement with its elaborate constitution, eIF3 has multiple functions in translation initiation:

  1. it is thought to promote the dissociation of 40S and 60S ribosomal subunits,
  2. it participates in the recruitment of the ternary complex to the 40S subunit, and
  3. it is required for mRNA recruitment to the preinitiation complex.1,10

Table 1. eIF3 subunit composition.

Table 1

eIF3 subunit composition.

eIF3 and eIF1A are thought to promote ribosome dissociation, but the mechanism is still poorly characterized.1 Recent work suggests that eIF3 prevents 60S subunits from disrupting the 43S preinitiation complex.46 This is consistent with earlier observations of eIF3 being associated with native 40S subunits,47 which implies that the ternary complex binds to a preformed 40S·eIF3 complex. Indeed, a ternary complex bound to 40S subunits in the absence of eIF3 was readily dissociated by 60S subunits.46 Thus, eIF3 appears to be required for the stabilization of ternary complex binding to the small ribosomal subunit.

The study of eIF3 in yeast has recently clarified its role in the recruitment of the ternary complex to 40S subunits. eIF3 from S. cerevisiae is composed of a core of five essential subunits that are conserved in mammals (Table 1): eIF3a, eIF3b, eIF3c, eIF3g and eIF3i.10,48,49 The core complex co-purifies with eIF5,48 an interaction that has also been observed in mammalian cells.50 This interaction is mediated by the amino-terminus of eIF3c,30,51 and is important for bridging eIF3 to eIF2, since both can concurrently bind to the C-terminal bipartite motif of eIF5 (Fig. 3).51 eIF1 can also interact with eIF3c,49,52 and a large multifactor complex consisting of eIF1/eIF2/eIF3/eIF5/ Met-tRNAiMet is observed in vivo in the absence of 40S subunits (Fig. 1).51 Since eIF1, eIF5 and the three eIF2 subunits promote stringent AUG selection,10,22,24 eIF3 could potentially have a structural function in AUG recognition. Thus, the multiple protein-protein interactions mediated by eIF3 appear to be critical in the recruitment or stable binding of the ternary complex to 40S subunits (Fig. 3).

Figure 3. eIF3 and eIF4G coordinate protein-protein interactions among initiation factors.

Figure 3

eIF3 and eIF4G coordinate protein-protein interactions among initiation factors. (A) Summary of the protein-protein interactions within yeast eIF3.,,, (B) The assembly of the mammalian translation initiation complex is coordinated by eIF3 and eIF4G. eIF4G (more...)

A yeast homologue of the mammalian eIF3j is not associated with the purified eIF3 core complex. 48 However, it appears that at least a fraction of eIF3j is incorporated into eIF3.53 Like its mammalian counterpart,54 yeast eIF3j interacts with eIF3a.53 Interestingly, the amino acid sequence of eIF3j is similar to the middle region of eIF3a, and forms a motif that is sufficient for binding to the RNA recognition motif (RRM) of eIF3b. Deletion of eIF3j is not lethal but causes a destabilization of eIF3 and its associated factors, suggesting that it is important in stabilizing the interaction of eIF3a with eIF3b.53 The latter interaction is critical in yeast, since deletion of the eIF3b RRM releases eIF3a and induces the breakdown of the multifactor complex, with the remaining eIF3b-eIF3g-eIF3i being unable to bind 40S subunits.53 In support of these observations, a purified subcomplex of eIF3 containing eIF3a, eIF3b and eIF3c is found to integrate eIF3j, eIF1 and eIF5, and can rescue translation in vitro almost as efficiently as purified eIF3 core complex.55

Finally, eIF3 is required for the recruitment of mRNA to the 43S preinitiation complex. In mammals, this is thought to be due to the interaction of eIF3 with eIF4F, which acts as a bridge to the mRNA through the interaction of eIF4E with the cap structure.56,57 The subunit of eIF3 responsible for this interaction has not been identified yet, and there is no report of a similar interaction in yeast. However, it was recently reported that yeast eIF5 can interact with the carboxy-terminal half of eIF4G, and could act as a link between eIF3 and the mRNA (Fig. 3).58 Another factor that could be involved in recruiting the 43S preinitiation complex to the mRNA is eIF4B (see below), which interacts with eIF3a in mammals,59 and with eIF3g in S. cerevisiae.60

Recruitment of the 43S Complex to the mRNA

Cap Structure

Eubacterial and archaeal mRNAs possess a Shine-Dalgarno sequence that facilitates ribosome recruitment by interacting directly with the complementary sequence at the 3' end of the 16S ribosomal RNA. Despite having a very similar 3' end, the eukaryotic 18S rRNA lacks such an element.2 To identify the 5' end of mRNAs, eukaryotic cells have instead opted for a specific structure called a cap. This m7GpppX sequence (where X is any nucleotide) is present at the 5' end of all nuclear transcribed mRNAs from eukaryotes,61 and has possibly evolved to stabilize mRNAs. The cap facilitates translation initiation, but also participate in other processes such as mRNA splicing and nucleocytoplasmic transport.62 The recruitment of the 43S complex to the mRNA is mediated by the eIF4 group of factors. The function of these factors has been thoroughly reviewed elsewhere,63 and thus only a summary of our current understanding will be presented here.

eIF4E

eIF4E interacts with the cap structure and, together with eIF4G and eIF4A, forms the eIF4F complex (Fig. 1). eIF4E is an essential factor that is conserved across all eukaryotic kingdoms. The structures of yeast and mouse eIF4E bound to m7GDP were solved,64,65 and demonstrate the conservation of the cap-binding mode. Indeed, all the amino acids involved in cap recognition are conserved from yeast to humans, which strongly suggests a common ancestral origin. In light of the fact that an eIF4E homologue is absent from archaea, it will be very interesting to address the question of the origin of this factor.66 Why did the eukaryotes select to use the cap structure to initiate translation? What selective advantage is achieved by adopting such a radically different ribosome recruitment mode?

In mouse and human, eIF4E is phosphorylated on one major site, serine 209.67 The function of this phosphorylation in not completely understood. Phosphorylation of eIF4E is correlated with increased translation rates, but it is still disputed whether phosphorylation increases the cap binding affinity of eIF4E.6870 The structure suggests that phosphorylated Ser209 could form a salt bridge with Lys159, which would act as a clamp above the proposed trajectory of the mRNA path.64 However, a more recent structural analysis of the full-length eIF4E complexed with m7GpppA indicates that the distance between Ser209 and Lys159 would be too long to form a salt bridge.68 In fact, the C-terminal loop region of eIF4E, which encompasses Ser209, interacts with the second nucleotide of the mRNA. Hence, phosphorylation of Ser209 could still have an effect on the affinity of eIF4E for capped mRNA.68

Notwithstanding its structural basis, the biological significance of eIF4E phosphorylation is not immediately clear. Studies on eIF4E phosphorylation mutants have demonstrated that they can restore translation as well as the wild-type protein in an in vitro assay.71 These eIF4E variants can also rescue the lethal phenotype of eIF4E deletion in yeast.71 In contrast, transgenic Drosophila melanogaster expressing an eIF4EI phosphorylation mutant (S251A, analogous to S209A) displays reduced viability (65% survival) in an eIF4EI mutant background, with the surviving flies having growth retardation defects.72 These defects were not observed when a S251D mutant was used to mimic a constitutively phosphorylated residue.

eIF4E is phosphorylated in vivo by the kinase Mnk1.73,74 In order to phosphorylate eIF4E, Mnk1 needs to be recruited to the C-terminal part of eIF4G (Fig. 3).75 It appears highly unlikely that such a sophisticated mechanism would have evolved if it did not have a purpose. Therefore we conclude that eIF4E phosphorylation must play an important regulatory role in vivo, at least in higher eukaryotes. This will probably be more fully appreciated by performing in vivo studies of a mouse knock-in bearing an eIF4E Ser209Ala mutation.

eIF4G

Two related proteins, eIF4GI and eIF4GII, function as modular adaptor proteins that mediate the series of protein-protein interactions culminating in the recruitment of the 43S complex to the mRNA 5' end (Fig. 3).63,76,77 eIF4G has evolved around a phylogenetically conserved central core (Fig. 4).66 This core is contained within the middle fragment of the protein and possesses two interaction domains: the first interacts with the eIF4A RNA helicase,78,79 and the second is an RNA binding domain.80,81 In addition, this fragment of mammalian eIF4G also contains an eIF3 interaction domain.56,78 The X-ray structure of the middle portion of human eIF4GII has been solved, and exhibits a crescent-shaped domain consisting of ten alpha helices organized into five HEAT repeats. 82 Proteins containing HEAT repeats are involved in the assembly of large multiprotein complexes, which is probably facilitated by the large and accessible surface area of the domain.83

Figure 4. eIF4G evolved around a conserved core domain.

Figure 4

eIF4G evolved around a conserved core domain. Mammalian eIF4G contains five distinct structural elements. Its amino-terminal third contains a motif for PABP binding, and an eIF4E binding site. The middle fragment of eIF4G (MIF4G) is a HEAT domain and (more...)

Extension of the central core of eIF4G to include the eIF4E-binding site generates an eIF4G domain that is necessary and sufficient for cap-dependent translation initiation.84 The 4E-binding site is apparently only required for targeting eIF4G to the mRNA. Indeed cap-independent translation of an mRNA containing an iron response element (IRE) can be directed by the central core of eIF4G, if this domain is fused to the IRE-binding protein IRP-1.85,86

The amino-terminal portion of eIF4G also contains an interaction domain for the poly(A) binding protein (PABP).87,88 PABP is a cytoplasmic protein that binds to the poly(A) tail present at the 3'-end of messenger RNAs. Its interaction with eIF4G provides a physical link between the cap and the poly(A) tail (Fig. 3). It also explains the observation that the poly(A) tail can operate as an enhancer of translation initiation.89,90 The significance of the cap-poly(A) tail interaction is discussed in more detail in Chapter 12.

The C-terminus of eIF4G has been considerably extended during the expansion of eukaryotes.66 A second eIF4A interaction domain is present in human eIF4G.78 This domain is absent from yeast eIF4G, but can be found in Arabidopsis and Drosophila eIF4G homologues. The related protein p97/NAT1/DAP-5 has a similar domain,66 which on the other hand does not bind to eIF4A.78 In eIF4G, the C-terminal eIF4A binding domain is not essential for translation, but instead plays a role in modulating the function of the core domain.84 There is no agreement as to whether eIF4G interacts with one or two eIF4A molecules.84,91,92 The extreme C-terminus of eIF4G from metazoans displays another conserved motif which, in mammals, is necessary for the interaction with the eIF4E kinase Mnk1.75 It is intriguing that this motif is conserved in the C-termini of eIF5 and eIF2Bϵ,93 and participates in their interaction with eIF2β.30 This may represent a conserved module important for protein-protein interactions regulating the assembly of the translational apparatus.66

eIF4A

eIF4A is a member of the large family of RNA helicases termed DEAD-box, after one of their 8 conserved motifs.94 In yeast, eIF4A is encoded by two genes whose simultaneous inactivation is lethal.95 There are three eIF4A isoforms in mammals, but only eIF4AI and eIF4AII appear to be involved in translation initiation.96 Interestingly, mammalian eIF4A can interact with yeast eIF4G1 in vitro,79 even if it cannot complement yeast mutants defective in eIF4A.97 In fact mammalian eIF4A appears to act as a translational inhibitor in yeast,79 much like eIF4AIII inhibits translation in mammals.96 eIF4A is the only eIF4 factor to have a homologue in archaea.6

eIF4A is an RNA dependent ATPase and can unwind RNA duplexes by itself or, much more efficiently, as part of the eIF4F complex.63 The intrinsic helicase activity of eIF4A is relatively weak, but it is strongly stimulated by eIF4B, although the two proteins do not appear to interact directly.98 Recent studies suggest that eIF4A is a nonprocessive helicase that is limited to a single round of unwinding.99,100 The presence of eIF4B (or eIF4H) may limit RNA re-association and allow the unwinding of longer duplexes.99,100 It is noteworthy that eIF4A functions primarily as part of the eIF4F complex, but that it apparently needs to be exchanged with free eIF4A to effect unwinding.101 It is possible that this recycling process is what conveys processivity to the complex. An unanswered question is how does eIF4A facilitate ribosome bindingγ eIF4F is generally believed to target eIF4A to the mRNA, which then unwinds secondary structures to allow ribosome recruitment. The role of eIF4A could be to utilize the energy from ATP hydrolysis to rearrange RNA structures, or maybe RNA-protein complexes, thus allowing 40S subunit binding to the mRNA.102,103

eIF4B

The function of eIF4B in translation initiation is not completely clear. In vitro, eIF4B enhances the helicase activity of eIF4A and eIF4F,98 and promotes 48S complex assembly.84,104 However, eIF4B is not required in ribosome binding assays,84,104 and yeast strains with a disrupted eIF4B gene display translational defects and a slow-growth phenotype, but are viable.105,106

eIF4B homodimerizes and interacts with RNA through a N-terminal RRM and a C-terminal arginine-rich motif (ARM).59,107,108 Since eIF4B also demonstrates an RNA-annealing activity, it was proposed that it could play a role in mediating interactions between the rRNA and mRNA.109 Consistent with this hypothesis, eIF4B can interact with the 18S rRNA in vitro, and can simultaneously bind to two RNA molecules.110

eIF4H

eIF4H is a recently identified factor that shares functional and sequence homology with eIF4B, mostly with the RRM domain.111 It weakly binds RNA and stimulates protein synthesis in vitro, as well as the ATPase and helicase activity of eIF4A and eIF4F.99,100,111,112 A recent report shows that the virion host shutoff protein (Vhs) of herpes simplex virus interacts with eIF4H.113 This protein accelerates mRNA degradation, which modulates the levels of viral and cellular gene expression. It appears that Vhs is targeted to the mRNA 5' end through its interaction with eIF4H.113

Scanning and Localization of the Initiator AUG

mRNA 5' UTR

After binding to the 5' end of the mRNA, the preinitiation complex must locate the AUG codon. It is believed that the ribosome and associated factors migrate through the 5' UTR until the Met-tRNAiMet can form a productive base pairing with an initiation codon. This has been defined as the scanning model,114,115 and is consistent with the experimental data. For example, the insertion of an upstream AUG usually creates a new translation start site. In addition, insertion of a stable secondary structure in the 5' UTR dramatically reduces translational efficiency, probably by hampering ribosome movement.116 The minimal set of factors required for 48S complex assembly at an initiation codon has been better defined in recent years (see below).117,118 Nevertheless, scanning has never been biochemically assayed and little is known about its mechanism.

eIF1

Sequence homologues of eIF1 have been identified in archaea and bacteria, indicating an ancient origin for this factor.8 In yeast, eIF1 is essential for viability,119 and genetic screenings have identified a number of mutations affecting the fidelity of initiation codon selection.22 These mutations affect residues that are conserved between eukaryotes, archaea and bacteria,8 and the NMR structure of eIF1 suggest that they represent an interaction surface for other molecules.52 A fraction of eIF1 from S. cerevisiae copurifies with eIF3, through an interaction with eIF3c.48,49,120 This might explain how eIF5 mutants can suppress the effect of eIF1 mutations in yeast,22 because eIF3c can interact simultaneously with eIF1 and eIF5 (Fig. 3).51 Thus, eIF1 is in close proximity to eIF5 and could affect its GTPase activating function. Toe-printing analysis of pre-initiation complexes bound to mRNA have provided evidence that eIF1 is required for the formation of 48S complexes.117 In this assay, which also requires eIF2, eIF3, eIF4A, eIF4B, eIF4F and Met-tRNAiMet, eIF1 can weakly promote the correct positioning of 43S complex at the initiation codon. This activity is strongly enhanced by eIF1A (see below).117 The molecular mechanism remains however unclear, and thus the exact function of eIF1 could be to participate in scanning, destabilize incorrectly positioned complexes, or stabilize correctly positioned ones.

eIF1A

eIF1A is the eukaryotic homologue of the bacterial IF1 and the archaeal a-eIF1A.8 The 3D structures of eIF1A shows that it contains an OB domain similar to that of IF1.37,38 eIF1A is essential in S. cerevisiae and appears to have multiple roles in translation initiation.1 Pestova et al reported that eIF1A acts in synergy with eIF1 to promote the assembly of 48S complex at the initiation codon.117 However, eIF1A without eIF1 can only promote the formation of an aberrant complex located close to the cap. This cap-proximal complex is not a translational intermediate, and it is unable to reach the AUG codon.117 eIF1A also promotes the binding of the ternary complex to isolated 40S subunits.46,121 In addition, eIF1A interacts with eIF5B.122 Along with structural studies of a 30S·IF1 complex,123 this suggests that eIF1A occupies the A site of the small ribosomal subunit and, in conjunction with eIF5B, directs the initiator tRNA to the P site.124

60S Ribosomal Subunit Joining

Once the preinitiation complex has reached an initiator codon, base pairing between the AUG and the Met-tRNAiMet anticodon elicits a series of events that culminate in the joining of the 60S ribosomal subunit to form an active ribosome that is competent for elongation. This involves the release of the initiation factors bound to the 40S subunits, which requires GTP hydrolysis. Recruitment of the 60S subunit is not spontaneous after the factors release, and it necessitates additional initiation factors.

eIF5

Once the 48S complex is correctly assembled at the initiation codon, the bound initiation factors must be displaced to allow the joining of the 60S subunit. This step requires the hydrolysis of the GTP molecule bound to eIF2 (Fig. 1), since its substitution with the nonhydrolyzable GMP-PNP arrests initiation at the 48S stage.125 Activation of the GTPase function of eIF2 requires its association with eIF5 and the 40S subunit.10 eIF5 is essential in yeast, and an activated eIF5 mutant (G31R) can initiate translation at non-AUG codons.22 This mutant demonstrates a two-fold stimulation of GTP hydrolysis. Combined with the studies on eIF2 mutants showing intrinsic GTPase activity, it appears that eIF2 and eIF5 act in concert to maintain the accuracy of initiation codon recognition in eukaryotes. Intriguingly, eIF5 has not been found to interact with the GTP-bound eIF2γ, but rather with eIF2β, at the same site which was demonstrated to interact with eIF2Bϵ.29,30 This argues that GTP hydrolysis must be triggered by a conformational change in eIF2, when the initiator tRNA is correctly paired with the AUG codon. The amino-terminus of eIF5 is similar to the carboxy-terminus of eIF2β, including the Cys2-Cys2 zinc-finger motif, and is expected to adopt a similar fold.31

Recent observations suggest that eIF5 acts as a bridge between eIF3 and eIF2, and would therefore participate in the recruitment of the ternary complex to the 40S subunit (see above).51 It has also been demonstrated that yeast eIF5 can interact simultaneously with eIF3 and eIF4G, and could thus take part in 43S complex recruitment to the mRNA.58 These interactions are all mediated by the C-terminal domain of eIF5, while the GTPase activating region apparently resides in the N-terminus.58 Interestingly, the amino-terminus of eIF5 shares sequence homology with the carboxy-terminal fragment of eIF2β.29,31

eIF5B

eIF5B was first identified as a translation factor in yeast, and termed yeast IF2 (yIF2) for its homology to the prokaryotic IF2.126 Human,127,128 Drosophila,129 and archaeal7 homologues were also identified, making IF2/eIF5B a universally conserved translation factor. Disruption of the eIF5B gene (FUN12) in yeast causes a severe slow-growth phenotype, associated with a defect in translation initiation.126 It was later demonstrated that eIF5B has a function analogous to prokaryotic IF2 in mediating the joining of the 60S ribosomal subunit.130 The requirement for both eIF2 and eIF5B strongly suggest that translation initiation in eukaryotes requires the hydrolysis of two GTP molecules (Fig. 1).125 This is consistent with the kinetic analysis of translation initiation in vitro,131 and offers additional possibilities for regulation.

The structure of eIF5B has revealed an unusual architecture.132 It consists of three N-terminal domains (I, II, III) connected by a long a helix to domain IV. Domain I is a G domain, domains II and IV are β-barrels and domain III has a novel a/β/a sandwich fold.132 The G domain and the β-barrel domain II display a similar structure and arrangement to the homologous domains in EF1A, eEF1A and a-eIF2γ.25,132 This suggests that they form a core structure present in all GTPases involved in translation.124 GTP-bound eIF5B facilitates 60S subunit joining, but GTP hydrolysis occurs after 80S formation and is required for the release of eIF5B.130 The comparison of eIF5B·GTP and eIF5B·GDP demonstrates that, like other GTPases, GTP hydrolysis induces a modest conformational switch in the G domain. This small modification is however amplified via a coordinated rearrangement of domains II-IV, resulting in the movement of domain IV.132 Domain IV is essential for the in vivo function of eIF5B and interacts with eIF1A, suggesting that the release of eIF1A and eIF5B from the ribosome could be coupled.122

Regulation of Translation Initiation

As with any multi-step process, eukaryotic translation can be regulated at various levels. However, it is generally more efficient to regulate complex pathways at their initiation step, and this is where translational control most often occurs.133 Two factors are known to play a critical role in the regulation of translation initiation: eIF2 and eIF4E. The activity of eIF2 is controlled by the rate of GTP recycling, and thus by the availability of eIF2B.10 The function of eIF4E is regulated by its incorporation into eIF4F, which is inhibited by the eIF4E-Binding Proteins (4E-BPs).63 Both of these mechanisms are subjected to regulatory phosphorylation and can thus provide rapid and reversible means to maintain cellular homeostasis in response to environmental signals. We will give a brief outline of how the activity of eIF2 and eIF4E can be regulated. The reader is directed to a recent review by Dever134 for a more detailed discussion of how gene-specific regulation can be achieved by modulating the activity of general translation factors. An in depth review of the multiple translational control mechanisms can be found in reference 135.

eIF2α Kinases

As mentioned above, the phosphorylation of eIF2α on Ser51 results in the conversion of eIF2 from a substrate to a competitive inhibitor of eIF2B. The critical importance and versatility of this regulatory event is emphasized by the successive appearance of the four known eIF2• kinases through the eukaryotic lineages: mammals acquired the double-stranded RNA-activated protein kinase (PKR); vertebrates, the hemin-regulated inhibitor kinase (HRI); metazoans, the PKR-like ER kinase (PERK); while all eukaryotes possess the kinase GCN2.10,136138 These four kinases are activated by environmental stress.139 PKR is part of the anti-viral response, since its expression is induced by interferon and its kinase function is activated by double-stranded RNA.138 HRI regulates protein synthesis in erythroid cells in response to heme deprivation, heat shock and oxidative and osmotic stress.137,140 PERK is activated as part of the unfolded protein response, which is caused by stress to the endoplasmic reticulum.136 Finally the activity of GCN2 can be stimulated by a variety of conditions, the best characterized being amino acid starvation.10,141

eIF4E-Binding Proteins

The complex m7GpppX·eIF4E·eIF4G directs the 43S preinitiation complex to the mRNA 5' end. The 4E-BPs specifically inhibit cap-dependent translation initiation by preventing the interaction of eIF4E with eIF4G.63 Three 4E-BPs were identified and found to have similar functions,142,143 but 4E-BP1 has been more thoroughly characterized. The binding of 4E-BP1 to eIF4E is reversible, and is regulated by the phosphorylation state of 4E-BP1. Upon stimulation of cells with serum, growth factors or hormones, 4E-BP1 becomes phosphorylated on specific serine/threonine residues and dissociates from eIF4E to relieve translational inhibition.63,144 Conversely, cellular stresses such as nutrient deprivation or viral infections will cause a decrease in 4E-BP1 phosphorylation and increase its affinity for eIF4E.63,67 The phosphorylation events leading to the dissociation of 4E-BP1 from eIF4E are mediated by the FKBP12-rapamycin associated protein/mammalian target of rapamycin (FRAP/mTOR), and probably other kinase(s) that have yet to be identified.144146 The activity of FRAP/mTOR is modulated by the availability of nutrients such as amino acids, and by the intracellular concentration of ATP.146148 Given that FRAP/mTOR signals not only to 4E-BP1 but to other translational targets (eIF4B, eIF4GI, eEF2 and S6K1), it emerges as a molecular sensor capable of integrating diverse inputs to modulate protein synthesis in response to a cell's nutritional and energetic status.146,148

Conclusion

The structural and functional analysis of eukaryotic translation initiation factors reveals that they have most likely evolved to take advantage of the many RNA structural elements that are specific to eukaryotes. Archaeal translation illustrates the evolution of the protein synthesis machinery from eubacteria to eukaryotes, exhibiting characteristics from both systems. For example, archaeal mRNAs have a Shine-Dalgarno sequence but utilize a factor homologous to eIF2 for Met-tRNAiMet delivery to the ribosome. Many of the eukaryotic translation initiation factors do not have counterparts in prokaryotes, and must have evolved together with the unique features of eukaryotic mRNA (cap structure, poly(A) tail). Archaea lack a 5' cap or long poly(A) tail and, accordingly, are devoid of factors related to eIF3, eIF4G or eIF4E. It can therefore be envisaged that eIF4G has evolved through the accretion of various interaction domains around a conserved central core. An eIF4E-binding site could thus have been added after the 5' cap structure has evolved, and a PABP interaction domain after the poly(A) tail was developed. Thus, RNA structural elements appear to have played a critical role in directing the evolution of the eukaryotic translational machinery. The increase in complexity that has resulted served to develop new regulatory mechanisms to control gene expression, such as the sequestration of eIF4E by the 4E-BPs or the phosphorylation of eIF2α. Once evolved, regulatory mechanisms could be subjected to further refinement. For instance, mammals have evolved four different eIF2α kinases. As a consequence of the increased regulatory capabilities, translation initiation has become a very dynamic process that can be delicately and rapidly modulated in response to environmental changes. Thus, the ability to better control translation is likely to have provided eukaryotes with increased adaptive flexibility.

Acknowledgements

We would like to thank Thomas Dever, Emmanuel Petroulakis, Mathieu Miron and Park Cho-Park for critical reading of the manuscript. F.P. is supported by a Doctoral Award from the Canadian Institutes of Health Research (CIHR). N.S. is a CIHR Distinguished Investigator and a Howard Hughes Medical Institute International Scholar.

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