Guanine Nucleotide Exchange Factor eIF2B Research Articles

A helical fulcrum in eIF2B coordinates allosteric regulation of stress signaling

The integrated stress response (ISR) enables cells to survive a variety of acute stresses, but chronic activation of the ISR underlies age-related diseases. ISR signaling downregulates translation and activates expression of stress-responsive factors that promote return to homeostasis and is initiated by inhibition of the decameric guanine nucleotide exchange factor eIF2B. Conformational and assembly transitions regulate eIF2B activity, but the allosteric mechanisms controlling these dynamic transitions and mediating the therapeutic effects of the small-molecule ISR inhibitor ISRIB are unknown. Using hydrogen–deuterium exchange–mass spectrometry and cryo-electron microscopy, we identified a central α-helix whose orientation allosterically coordinates eIF2B conformation and assembly. Biochemical and cellular signaling assays show that this ‘switch-helix’ controls eIF2B activity and signaling. In sum, the switch-helix acts as a fulcrum of eIF2B conformational regulation and is a highly conserved actuator of ISR signal transduction. This work uncovers a conserved allosteric mechanism and unlocks new therapeutic possibilities for ISR-linked diseases.

EIF2Bδ blocks the integrated stress response and maintains eIF2B activity and cancer metastasis by overexpression in breast cancer stem cells

Breast cancer (BC) metastasis involves cancer stem cells (CSCs) and their regulation by micro-RNAs (miRs), but miR targeting of the translation machinery in CSCs is poorly explored. We therefore screened miR expression levels in a range of BC cell lines, comparing non-CSCs to CSCs, and focused on miRs that target translation and protein synthesis factors. We describe a unique translation regulatory axis enacted by reduced expression of miR-183 in breast CSCs, which we show targets the eIF2Bδ subunit of guanine nucleotide exchange factor eIF2B, a regulator of protein synthesis and the integrated stress response (ISR) pathway. We report that reduced expression of miR-183 greatly increases eIF2Bδ protein levels, preventing strong induction of the ISR and eIF2α phosphorylation, by preferential interaction with P-eIF2α. eIF2Bδ overexpression is essential for BC cell invasion, metastasis, maintenance of metastases, and breast CSC expansion in animal models. Increased expression of eIF2Bδ, a site of action of the drug ISRIB that also prevents ISR signaling, is essential for breast CSC maintenance and metastatic capacity.

Move and countermove: the integrated stress response in picorna- and coronavirus-infected cells.

Viruses, when entering their host cells, are met by a fierce intracellular immune defense. One prominent antiviral pathway is the integrated stress response (ISR). Upon activation of the ISR — typically though not exclusively upon detection of dsRNA — translation-initiation factor eukaryotic initiation factor 2 (eIF2) becomes phosphorylated to act as an inhibitor of guanine nucleotide-exchange factor eIF2B. Thus, with the production of ternary complex blocked, a global translational arrest ensues. Successful virus replication hinges on effective countermeasures. Here, we review ISR antagonists and antagonistic mechanisms employed by picorna- and coronaviruses. Special attention will be given to a recently discovered class of viral antagonists that inhibit the ISR by targeting eIF2B, thereby allowing unabated translation initiation even at exceedingly high levels of phosphorylated eIF2.

A C‐term truncated EIF2Bγ protein encoded by an intronically polyadenylated isoform introduces unfavorable EIF2Bγ–EIF2γ interactions

AbstractEukaryotic translation initiates upon recruitment of the EIF2‐GTP·Met‐tRNAi ternary complex (TC) to the ribosomes. EIF2 (α, β, γ subunits) is a GTPase. The GDP to GTP exchange within EIF2 is facilitated by the guanine nucleotide exchange factor EIF2B (α‐ε subunits). During stress‐induced conditions, phosphorylation of the α‐subunit of EIF2 turns EIF2 into an inhibitor of EIF2B. In turn, inhibition of EIF2B decreases TC formation and triggers the internal stress response (ISR), which determines the cell fate. Deregulated ISR has been linked to neurodegenerative disorders and cancer, positioning EIF2B as a promising therapeutic target. Hence, a better understanding of the mechanisms/factors that regulate EIF2B activity is required. Here, combining transcript and protein level analyses, we describe an intronically polyadenylated (IPA) transcript of EIF2B's γ‐subunit. We show that the IPA mRNA isoform is translated into a C‐terminus truncated protein. Using structural modeling, we predict that the truncated EIF2Bγ protein has unfavorable interactions with EIF2γ, leading to a potential decrease in the stability of the nonproductive EIF2:EIF2B complex. While we discovered and confirmed the IPA mRNA isoform in breast cancer cells, the expression of this isoform is not cancer‐specific and is widely present in normal tissues. Overall, our data show that a truncated EIF2Bγ protein co‐exists with the canonical protein and is an additional player to regulate the equilibrium between productive and nonproductive states of the EIF2:EIF2B complex. These results may have implications in stress‐induced translation control in normal and disease states. Our combinatorial approach demonstrates the need to study noncanonical mRNA and protein isoforms to understand protein interactions and intricate molecular mechanisms.

Mutagenesis screen uncovers lifespan extension through integrated stress response inhibition without reduced mRNA translation

Protein homeostasis is modulated by stress response pathways and its deficiency is a hallmark of aging. The integrated stress response (ISR) is a conserved stress-signaling pathway that tunes mRNA translation via phosphorylation of the translation initiation factor eIF2. ISR activation and translation initiation are finely balanced by eIF2 kinases and by the eIF2 guanine nucleotide exchange factor eIF2B. However, the role of the ISR during aging remains poorly understood. Using a genomic mutagenesis screen for longevity in Caenorhabditis elegans, we define a role of eIF2 modulation in aging. By inhibiting the ISR, dominant mutations in eIF2B enhance protein homeostasis and increase lifespan. Consistently, full ISR inhibition using phosphorylation-defective eIF2α or pharmacological ISR inhibition prolong lifespan. Lifespan extension through impeding the ISR occurs without a reduction in overall protein synthesis. Instead, we observe changes in the translational efficiency of a subset of mRNAs, of which the putative kinase kin-35 is required for lifespan extension. Evidently, lifespan is limited by the ISR and its inhibition may provide an intervention in aging.

Dietary Protein and Amino Acid Deficiency Inhibit Pancreatic Digestive Enzyme mRNA Translation by Multiple Mechanisms.

Background & AimsChronic amino acid (AA) deficiency, as in kwashiorkor, reduces the size of the pancreas through an effect on mammalian target of rapamycin complex 1 (mTORC1). Because of the physiological importance of AAs and their role as a substrate, a stimulant of mTORC1, and protein synthesis, we studied the effect of acute protein and AA deficiency on the response to feeding.MethodsICR/CD-1 mice were fasted overnight and refed for 2 hours with 4 different isocaloric diets: control (20% Prot); Protein-free (0% Prot); control (AA-based diet), and a leucine-free (No Leu). Protein synthesis, polysomal profiling, and the activation of several protein translation factors were analyzed in pancreas samples.ResultsAll diets stimulated the Protein Kinase-B (Akt)/mTORC1 pathway, increasing the phosphorylation of the kinase Akt, the ribosomal protein S6 (S6) and the formation of the eukaryotic initiation factor 4F (eIF4F) complex. Total protein synthesis and polysome formation were inhibited in the 0% Prot and No Leu groups to a similar extent, compared with the 20% Prot group. The 0% Prot diet partially reduced the Akt/mTORC1 pathway and the activity of the guanine nucleotide exchange factor eIF2B, without affecting eIF2α phosphorylation. The No Leu diet increased the phosphorylation of eIF2α and general control nonderepressible 2, and also inhibited eIF2B activity, without affecting mTORC1. Essential and nonessential AA levels in plasma and pancreas indicated a complex regulation of their cellular transport mechanisms and their specific effect on the synthesis of digestive enzymes.ConclusionsThese studies show that dietary AAs are important regulators of postprandial digestive enzyme synthesis, and their deficiency could induce pancreatic insufficiency and malnutrition.

EIF2B and the Integrated Stress Response: A Structural and Mechanistic View.

The eukaryotic translation initiation factor eIF2 is a GTPase, which brings the initiator Met-tRNAi to the ribosome as the eIF2-GTP·Met-tRNAi ternary complex (TC). TC regeneration is catalyzed by the guanine nucleotide exchange factor (GEF) eIF2B. eIF2 phosphorylation by several stress-induced kinases converts it into a competitive inhibitor of eIF2B. Inhibition of eIF2B activity lowers cellular TC concentrations, which in turn triggers the integrated stress response (ISR). Depending on its degree of activation and duration, the ISR protects the cell from the stress or can itself induce apoptosis. ISR dysregulation is a causative factor in the pathology of multiple neurodegenerative disorders, while ISR inhibitors are neuroprotective. The realization that eIF2B is a promising therapeutic target has triggered significant interest in its structure and its mechanisms of action and regulation. Recently, four groups published the cryo-electron microscopy structures of eIF2B with its substrate eIF2 and/or its inhibitor, phosphorylated eIF2 [eIF2(α-P)]. While all three structures of the nonproductive eIF2B·eIF2(α-P) complex are similar to each other, there is a sharp disagreement between the published structures of the productive eIF2B·eIF2 complex. One group reports a structure similar to that of the nonproductive complex, whereas two others observe a vastly different eIF2B·eIF2 complex. Here, we discuss the recent reports on the structure, function, and regulation of eIF2B; the preclinical data on the use of ISR inhibitors for the treatment of neurodegenerative disorders; and how the new structural and biochemical information can inform and influence the use of eIF2B as a therapeutic target.

Integrated stress response on the brain

Structural Biology During translation, regulation of protein synthesis by phosphorylation of eukaryotic translation initiation factor 2 (eIF2) is a common consequence of diverse stress stimuli, which leads to reprogramming of gene expression. This process, known as the integrated stress response, is one of the most fundamental mechanisms of translational control conserved throughout eukaryotes. It is also a promising therapeutic target in neurodegenerative diseases and traumatic brain injury. Kashiwagi et al. report the cryo–electron microscopy and crystal structures and Kenner et al. report the cryo–electron microscopy structure of the guanine nucleotide exchange factor eIF2B in complex with eIF2 or phosphorylated eIF2. The structures of the eIF2•eIF2B complex reveal that the single phosphorylation modification on eIF2 changes how eIF2 binds to eIF2B and locks this enzyme into an inhibited complex. Science , this issue p. [495][1], p. [491][2] [1]: /lookup/doi/10.1126/science.aaw4104 [2]: /lookup/doi/10.1126/science.aaw2922

EIF2B-catalyzed nucleotide exchange and phosphoregulation by the integrated stress response.

The integrated stress response (ISR) tunes the rate of protein synthesis. Control is exerted by phosphorylation of the general translation initiation factor eIF2. eIF2 is a guanosine triphosphatase that becomes activated by eIF2B, a two-fold symmetric and heterodecameric complex that functions as eIF2's dedicated nucleotide exchange factor. Phosphorylation converts eIF2 from a substrate into an inhibitor of eIF2B. We report cryo-electron microscopy structures of eIF2 bound to eIF2B in the dephosphorylated state. The structures reveal that the eIF2B decamer is a static platform upon which one or two flexible eIF2 trimers bind and align with eIF2B's bipartite catalytic centers to catalyze nucleotide exchange. Phosphorylation refolds eIF2α, allowing it to contact eIF2B at a different interface and, we surmise, thereby sequestering it into a nonproductive complex.

Modeling and Dynamic Behavior of eIF2 Dependent Regulatory System With Disturbances.

Eukaryotic initiation factor 2 (eIF2) is a central controller of the eukaryotic translational machinery. To sustain the on-going translation activity, eIF2 cycles between its GTP and GDP bound states. However, in response to cellular stresses, the phosphorylation of eIF2 takes place, which acts as an inhibitor of the guanine nucleotide exchange factor eIF2B and switches the translation activity on physiological timescales. The main objective of this paper is to investigate the stability of the regulatory system under nominal conditions, parametric fluctuations, and structural damages. In this paper, a mathematical model of eIF2-dependent regulatory system is used to identify the stability-conferring features within the system with the help of direct and indirect methods of Lyapunov stability theory. To investigate the impact of intrinsic fluctuations and structural damages on the stability of regulatory system, the mathematical model has been linearized around feasible equilibrium point and the variation of system poles has been observed. The investigations have revealed that the regulatory model is stable and able to tolerate the intrinsic stressors but becomes unstable when particular complex is targeted to override the undesirable interaction. Our analyses indicate that, the stability is a collective property and damage in the structure of the system changes the stability of the system.

Binding of ISRIB reveals a regulatory site in the nucleotide exchange factor eIF2B.

The integrated stress response (ISR) is a conserved translational and transcriptional program affecting metabolism, memory, and immunity. The ISR is mediated by stress-induced phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) that attenuates the guanine nucleotide exchange factor eIF2B. A chemical inhibitor of the ISR, ISRIB, reverses the attenuation of eIF2B by phosphorylated eIF2α, protecting mice from neurodegeneration and traumatic brain injury. We describe a 4.1-angstrom-resolution cryo-electron microscopy structure of human eIF2B with an ISRIB molecule bound at the interface between the β and δ regulatory subunits. Mutagenesis of residues lining this pocket altered the hierarchical cellular response to ISRIB analogs in vivo and ISRIB binding in vitro. Our findings point to a site in eIF2B that can be exploited by ISRIB to regulate translation.

Paradoxical Sensitivity to an Integrated Stress Response Blocking Mutation in Vanishing White Matter Cells.

The eukaryotic translation initiation factor eIF2B promotes mRNA translation as a guanine nucleotide exchange factor (GEF) for translation initiation factor 2 (eIF2). Endoplasmic reticulum (ER) stress-mediated activation of the kinase PERK and the resultant phosphorylation of eIF2’s alpha subunit (eIF2α) attenuates eIF2B GEF activity thereby inducing an integrated stress response (ISR) that defends against protein misfolding in the ER. Mutations in all five subunits of human eIF2B cause an inherited leukoencephalopathy with vanishing white matter (VWM), but the role of the ISR in its pathogenesis remains unclear. Using CRISPR-Cas9 genome editing we introduced the most severe known VWM mutation, EIF2B4A391D, into CHO cells. Compared to isogenic wildtype cells, GEF activity of cells with the VWM mutation was impaired and the mutant cells experienced modest enhancement of the ISR. However, despite their enhanced ISR, imposed by the intrinsic defect in eIF2B, disrupting the inhibitory effect of phosphorylated eIF2α on GEF by a contravening EIF2S1/eIF2αS51A mutation that functions upstream of eIF2B, selectively enfeebled both EIF2B4A391D and the related severe VWM EIF2B4R483W cells. The basis for paradoxical dependence of cells with the VWM mutations on an intact eIF2α genotype remains unclear, as both translation rates and survival from stressors that normally activate the ISR were not reproducibly affected by the VWM mutations. Nonetheless, our findings support an additional layer of complexity in the development of VWM, beyond a hyperactive ISR.

Moco biosynthesis and the ATAC acetyltransferase engage translation initiation by inhibiting latent PKR activity.

Molybdenum cofactor (Moco) biosynthesis is linked to c-Jun N-terminal kinase (JNK) signaling in Drosophila through MoaE, a molybdopterin (MPT) synthase subunit that is also a component of the Ada Two A containing (ATAC) acetyltransferase complex. Here, we show that human MPT synthase and ATAC inhibited PKR, a double-stranded RNA-dependent protein kinase, to facilitate translation initiation of iron-responsive mRNA. MPT synthase and ATAC directly interacted with PKR and suppressed latent autophosphorylation of PKR and its downstream phosphorylation of JNK and eukaryotic initiation factor 2α (eIF2α). The suppression of eIF2α phosphorylation via MPT synthase and ATAC prevented sequestration of the guanine nucleotide exchange factor eIF2B, which recycles eIF2-GDP to eIF2-GTP, resulting in the promotion of translation initiation. Indeed, translation of the iron storage protein, ferritin, was reduced in the absence of MPT synthase or ATAC subunits. Thus, MPT synthase and ATAC regulate latent PKR signaling and link transcription and translation initiation.

Stress responses. Mutations in a translation initiation factor identify the target of a memory-enhancing compound.

The integrated stress response (ISR) modulates messenger RNA translation to regulate the mammalian unfolded protein response (UPR), immunity, and memory formation. A chemical ISR inhibitor, ISRIB, enhances cognitive function and modulates the UPR in vivo. To explore mechanisms involved in ISRIB action, we screened cultured mammalian cells for somatic mutations that reversed its effect on the ISR. Clustered missense mutations were found at the amino-terminal portion of the delta subunit of guanine nucleotide exchange factor (GEF) eIF2B. When reintroduced by CRISPR-Cas9 gene editing of wild-type cells, these mutations reversed both ISRIB-mediated inhibition of the ISR and its stimulatory effect on eIF2B GEF activity toward its substrate, the translation initiation factor eIF2, in vitro. Thus, ISRIB targets an interaction between eIF2 and eIF2B that lies at the core of the ISR.

A new function and complexity for protein translation initiation factor eIF2B

eIF2B is a multisubunit protein that is critical for protein synthesis initiation and its control. It is a guanine nucleotide exchange factor (GEF) for its GTP-binding protein partner eIF2. eIF2 binds initiator tRNA to ribosomes and promotes mRNA AUG codon recognition. eIF2B is critical for regulation of protein synthesis via a conserved mechanism of phosphorylation of eIF2, which converts eIF2 from a substrate to an inhibitor of eIF2B GEF. In addition, inherited mutations affecting eIF2B subunits cause the fatal disorder leukoencephalopathy with Vanishing White Matter (VWM), also called Childhood Ataxia with Central nervous system Hypomyelination (CACH). Here we review findings which reveal that eIF2B is a decameric protein and also define a new function for the eIF2B. Our results demonstrate that the eIF2Bγ subunit is required for eIF2B to gain access to eIF2•GDP. Specifically it displaces a third translation factor eIF5 (a dual function GAP and GDI) from eIF2•GDP/eIF5 complexes. Thus eIF2B is a GDI displacement factor (or GDF) in addition to its role as a GEF, prompting the redrawing of the eIF2 cycling pathway to incorporate the new steps. In structural studies using mass spectrometry and cross-linking it is shown that eIF2B is a dimer of pentamers and so is twice as large as previously thought. A binding site for GTP on eIF2B was also found, raising further questions concerning the mechanism of nucleotide exchange. The implications of these findings for eIF2B function and for VWM/CACH disease are discussed.

Toll-like receptor activation suppresses ER stress factor CHOP and translation inhibition through activation of eIF2B

Activation of toll-like receptors (TLRs) induces the endoplasmic reticulum (ER) Unfolded Protein Response (UPR) to accommodate essential protein translation1,2. However, despite increased p-eIF2α, a TLR-TRIF-dependent pathway assures that the cells avoid CHOP induction, apoptosis, and translational suppression of critical proteins3. Because p-eIF2α decreases the functional interaction of eIF2 with eIF2B, a guanine nucleotide exchange factor (GEF), we explored the hypothesis that TLR-TRIF signaling activates eIF2B-GEF activity to counteract the effects of p-eIF2α. We now show that TLR-TRIF signaling activates eIF2B-GEF through PP2A-mediated Ser-dephosphorylation of the eIF2B ε-subunit. PP2A itself is activated by decreased Src-family-kinase-induced Tyr-phosphorylation of its catalytic subunit. Each of these processes are required for TLR-TRIF-mediated CHOP suppression in ER-stressed cells in vitro and in vivo. Thus, in the setting of prolonged, physiologic ER stress, a unique TLR-TRIF-dependent translational control pathway enables cells to carry out essential protein synthesis and avoid CHOP-induced apoptosis while still benefitting from the protective arms of the UPR.

Evaluation of the endoplasmic reticulum-stress response in eIF2B-mutated lymphocytes and lymphoblasts from CACH/VWM patients

BackgroundEukaryotic translation initiation factor 2B (eIF2B), a guanine nucleotide exchange factor (GEF) and a key regulator of translation initiation under normal and stress conditions, causes an autosomal recessive leukodystrophy of a wide clinical spectrum. EBV-immortalised lymphocytes (EIL) from eIF2B-mutated patients exhibit a decrease in eIF2B GEF activity. eIF2B-mutated primary fibroblasts have a hyper-induction of activating transcription factor 4 (ATF4) which is involved in the protective unfolded protein response (UPR), also known as the ER-stress response. We tested the hypothesis that EIL from eIF2B-mutated patients also exhibit a heightened ER-stress response.MethodsWe used thapsigargin as an ER-stress agent and looked at polysomal profiles, rate of protein synthesis, translational activation of ATF4, and transcriptional induction of stress-specific mRNAs (ATF4, CHOP, ASNS, GRP78) in normal and eIF2B-mutated EIL. We also compared the level of stress-specific mRNAs between EIL and primary lymphocytes (PL).ResultsDespite the low eIF2B GEF activity in the 12 eIF2B-mutated EIL cell lines tested (range 40-70% of normal), these cell lines did not differ from normal EIL in their ATF4-mediated ER-stress response. The absence of hyper-induction of ATF4-mediated ER-stress response in eIF2B-mutated EIL in contrast to primary fibroblasts is not related to their transformation by EBV. Indeed, PL exhibited a higher induction of the stress-specific mRNAs in comparison to EIL, but no hyper-induction of the UPR was noticed in the eIF2B-mutated cell lines in comparison to controls.ConclusionsTaken together with work of others, our results demonstrate the absence of a major difference in ER-stress response between controls and eIF2B-mutated cells. Therefore, components of the ER-stress response cannot be used as discriminantory markers in eIF2B-related disorders.

HIGHLIGHTS FROM THE LITERATURE

HIGHLIGHTS FROM THE LITERATURE

Differential Phosphorylation of Plant Translation Initiation Factors by Arabidopsis thaliana CK2 Holoenzymes

A previously described wheat germ protein kinase (Yan, T. F., and Tao, M. (1982) J. Biol. Chem. 257, 7037-7043) was identified unambiguously as CK2 using mass spectrometry. CK2 is a ubiquitous eukaryotic protein kinase that phosphorylates a wide range of substrates. In previous studies, this wheat germ kinase was shown to phosphorylate eIF2alpha, eIF3c, and three large subunit (60 S) ribosomal proteins (Browning, K. S., Yan, T. F., Lauer, S. J., Aquino, L. A., Tao, M., and Ravel, J. M. (1985) Plant Physiol. 77, 370-373). To further characterize the role of CK2 in the regulation of translation initiation, Arabidopsis thaliana catalytic (alpha1 and alpha2) and regulatory (beta1, beta2, beta3, and beta4) subunits of CK2 were cloned and expressed in Escherichia coli. Recombinant A. thaliana CK2beta subunits spontaneously dimerize and assemble into holoenzymes in the presence of either CK2alpha1 or CK2alpha2 and exhibit autophosphorylation. The purified CK2 subunits were used to characterize the properties of the individual subunits and their ability to phosphorylate various plant protein substrates. CK2 was shown to phosphorylate eIF2alpha, eIF2beta, eIF3c, eIF4B, eIF5, and histone deacetylase 2B but did not phosphorylate eIF1, eIF1A, eIF4A, eIF4E, eIF4G, eIFiso4E, or eIFiso4G. Differential phosphorylation was exhibited by CK2 in the presence of various regulatory beta-subunits. Analysis of A. thaliana mutants either lacking or overexpressing CK2 subunits showed that the amount of eIF2beta protein present in extracts was affected, which suggests that CK2 phosphorylation may play a role in eIF2beta stability. These results provide evidence for a potential mechanism through which the expression and/or subcellular distribution of CK2 beta-subunits could participate in the regulation of the initiation of translation and other physiological processes in plants.

Translation factor control of ribosome conformation during start codon selection

Eukaryotic translation initiation factors (eIFs) function at multiple steps. They enable the small 40S ribosome subunit to bind to initiator tRNA and mRNA, and scan to and select an initiation codon on the mRNA. They facilitate joining of the large 60S ribosome subunit, at which point the initiation phase of translation ends with the initiator tRNA in the P (peptidyl) site, and the ribosome poised to accept a tRNA into its A (aminoacyl) site (Kapp and Lorsch 2004; Pestova et al. 2007). There are at least 10 eIFs, and many of them (eIF1, eIF1A, eIF2, eIF3, eIF4F, eIF5, and eIF5B) are direct components of the ribosomal preinitiation complex (PIC). eIF2 and eIF3 are well-characterized multimeric factors that bind directly to Met-tRNAi Met and the 40S subunit, respectively. While considered relatively large, the 40S subunit has only a limited functional space to bind translation factors. eIF1 and eIF1A are small single-polypeptide factors that bind directly to the ribosome at or near the decoding site. They are thought to directly regulate ribosome conformation. An elegant combination of genetic and biochemical studies reported in the previous issue of Genes & Development from the Hinnebusch, Lorsch, and Pestova laboratories (Cheung et al. 2007) provides substantial evidence that these factors indeed regulate the ribosome conformational rearrangement in response to start codon selection. This report and other recent reports on the structures and functions of yeast initiation factors lead to a deeper understanding of how eIFs bind the ribosome productively, communicate with each other to enable initiation, and regulate the ribosome’s conformation and activity to maintain initiation fidelity. The salient points concerning translation initiation via scanning (outlined in Fig. 1) that are important for considering the functions of eIFs in start site selection can be summarized as follows. The ternary complex (TC) is composed of eIF2, GTP, and Met-tRNAi . It associates with the 40S ribosome subunit with the assistance of eIF1, eIF1A, and eIF3. TC, eIF1, eIF3, and eIF5 together can be isolated as a multifactor complex (MFC) in yeast (Asano et al. 2000); a similar complex may be present in mammals (LeFebvre et al. 2006). There are at least two possible pathways for 43S assembly as depicted in Figure 1, one of which involves preformed MFC. The 43S PIC that is formed by these interactions is composed of the 40S subunit, MFC, and eIF1A. The 43S PIC is recruited to the mRNA by eIF4F, which is associated with the mRNA mG-cap and poly(A) tail through association with poly(A)-binding protein; the PIC in association with mRNA becomes the 48S PIC [eIF4F, poly(A), and PABP are not shown in Fig. 1]. eIF3 also is important for PIC binding to mRNA (Kolupaeva et al. 2005; Siridechadilok et al. 2005; Hinnebusch 2006; Jivotovskaya et al. 2006). The PIC scans the mRNA; upon determining it has reached a start codon, it releases the Pi formed by hydrolysis of GTP in the TC, and also releases eIF2·GDP, leaving Met-tRNAi Met positioned at the start codon. As discussed below, eIF1 also dissociates from the ribosome during selection of the start codon. A second GTP-binding factor, eIF5B, then facilitates 60S subunit joining. The guanine nucleotide exchange factor eIF2B recycles eIF2•GDP to eIF2•GTP, because only the latter binds Met-tRNAi .