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Initiation factor

Initiation factors are a group of proteins that play crucial roles in the initiation phase of translation, the process by which messenger RNA (mRNA) is decoded by ribosomes to synthesize proteins in cells.^[1] These factors ensure the accurate assembly of the ribosomal initiation complex at the start codon (typically AUG) of the mRNA, facilitating the binding of the initiator transfer RNA (tRNA) carrying formylmethionine in prokaryotes or methionine in eukaryotes, and promoting the fidelity and efficiency of protein synthesis initiation.^[2] While the core function is conserved across organisms, the number and complexity of initiation factors differ significantly between prokaryotes and eukaryotes, reflecting adaptations to distinct mRNA structures and regulatory needs.^[3] In prokaryotes, such as bacteria, translation initiation relies on three primary initiation factors—IF1, IF2, and IF3—which associate with the small 30S ribosomal subunit to form a preinitiation complex.^[1] IF1 binds to the aminoacyl (A) site of the 30S subunit to prevent premature tRNA binding and stabilizes the complex; IF2, a GTPase, delivers the initiator fMet-tRNA^fMet^ to the peptidyl (P) site and promotes the joining of the large 50S subunit to form the 70S initiation complex upon GTP hydrolysis; and IF3 ensures the dissociation of ribosomal subunits post-termination, discriminates the correct start codon via base-pairing with the Shine-Dalgarno sequence on mRNA, and enhances accuracy by ejecting non-cognate tRNAs.^[3] This process is relatively simple and Shine-Dalgarno-dependent, allowing rapid initiation often on polycistronic mRNAs, with factors being recycled after release during 50S subunit association.^[3] In eukaryotes, initiation is more complex and cap-dependent, involving at least 12 eukaryotic initiation factors (eIFs) that coordinate the recruitment of the 40S small ribosomal subunit to the 5' capped end of mRNA and its scanning to the start codon.^[2] Key players include eIF2, which forms a ternary complex with GTP and Met-tRNA^i^Met^ to bind the 40S subunit's P site; eIF3, a multi-subunit scaffold that stabilizes the 43S preinitiation complex and links it to mRNA via interactions with eIF4 factors; and the eIF4F complex (comprising eIF4E for cap recognition, eIF4G as an adaptor, and eIF4A as an RNA helicase aided by eIF4B to unwind mRNA secondary structures).^[2] Additional factors like eIF1 and eIF1A aid in start codon recognition during 5' untranslated region (UTR) scanning, while eIF5 and eIF5B manage GTP hydrolysis for factor release and 60S large subunit joining to form the 80S ribosome.^[2] This elaborate machinery allows for extensive regulation, such as through phosphorylation of eIFs in response to cellular signals, enabling control of global translation rates and selective mRNA translation under stress or development.^[2]

Biological fundamentals

Definition and overview

Initiation factors are specialized proteins that play a crucial role in the initiation phase of protein synthesis, facilitating the accurate and efficient start of messenger RNA (mRNA) translation into polypeptide chains.^[4] These factors assist in assembling the necessary components at the beginning of the mRNA sequence, ensuring that translation begins at the correct start codon.^[5] In both prokaryotes and eukaryotes, they enable the binding of the small ribosomal subunit, mRNA, and initiator transfer RNA (tRNA) to form the preinitiation complex, marking the onset of ribosomal scanning or positioning for peptide bond formation.^[2] Prokaryotic initiation factors, denoted as IFs, and eukaryotic initiation factors, denoted as eIFs, represent distinct yet evolutionarily related sets of proteins adapted to the cellular environments of bacteria and higher organisms, respectively.^[6] While prokaryotes typically require only three primary IFs (IF1, IF2, and IF3) for initiation, eukaryotes employ a more complex array of over a dozen eIFs comprising more than 25 subunits.^[7] Despite these differences, core initiation factors exhibit remarkable conservation across species, reflecting their fundamental importance in the universal process of translation, with homologs traceable from bacteria to humans.^[8] The translation initiation phase, orchestrated by these factors, is widely recognized as the rate-limiting step in protein synthesis, determining the overall efficiency and specificity of gene expression in cells.^[9] In prokaryotes, this step is critical for rapid response to environmental cues, while in eukaryotes, it allows for sophisticated regulation of protein production.^[7] By recruiting and positioning ribosomes, mRNA, and initiator tRNA, initiation factors ensure fidelity in start site selection and prevent erroneous translation, thereby maintaining cellular homeostasis.^[10]

Role in translation initiation

In prokaryotic translation initiation, the process begins with the binding of initiation factors to the 30S ribosomal subunit, which promotes the dissociation of 70S ribosomes and facilitates mRNA attachment via the Shine-Dalgarno sequence pairing with 16S rRNA.^[11] Initiation factors then enable the recruitment of formylmethionyl-tRNA (fMet-tRNA) to the P-site of the 30S subunit in a GTP-dependent manner, forming a 30S pre-initiation complex that positions the initiator tRNA at the start codon.^[11] Subsequently, the 50S subunit joins this complex, triggered by GTP hydrolysis, to assemble the complete 70S ribosome ready for elongation, with initiation factors being released upon subunit association.^[11] Eukaryotic translation initiation follows a more complex pathway, starting with the recognition of the mRNA 5' cap structure by the eIF4F complex, which recruits the 40S ribosomal subunit to the mRNA end.^[12] The ternary complex, consisting of eIF2 bound to GTP and methionyl-initiator tRNA (Met-tRNAi), associates with the 40S subunit to form the 43S pre-initiation complex (PIC), which then scans the 5' untranslated region (UTR) in a 5'-to-3' direction to locate the start AUG codon.^[12] Upon AUG recognition, GTP hydrolysis on eIF2 (stimulated by eIF5) leads to the release of initiation factors from the 40S subunit. Subsequently, eIF5B promotes the joining of the 60S subunit in a GTP-dependent manner to form the 80S ribosome.^[2] Initiation factors play crucial regulatory roles in ensuring the fidelity of translation initiation across both systems by discriminating against non-start codons and preventing the use of non-initiator tRNAs.^[13] For instance, they destabilize mismatched codon-anticodon pairs and block premature GTP hydrolysis or elongation until the correct start site is verified, thereby minimizing errors in protein synthesis start.^[13] In prokaryotes, this involves blocking the A-site to avoid early aminoacyl-tRNA binding, while in eukaryotes, factors monitor the Kozak context around the AUG to enhance accuracy.^[11]^[12] The formation of the pre-initiation complex (PIC) is fundamentally dependent on initiation factors, which coordinate the assembly of ribosomal subunits, mRNA, and initiator tRNA into a stable scanning-competent structure.^[2] In eukaryotes, the 43S PIC requires eIF2 for ternary complex delivery, eIF3 for stabilization, and eIF1/eIF1A for scanning promotion, ensuring efficient AUG selection before 60S joining.^[2] Prokaryotic and eukaryotic initiation differ primarily in mRNA recruitment—direct base-pairing versus cap-dependent scanning—but both rely on factors to build functional PICs for accurate translation start.^[12]

Prokaryotic initiation factors

Primary types (IF1, IF2, IF3)

In prokaryotic translation initiation, three primary initiation factors—IF1, IF2, and IF3—associate with the 30S ribosomal subunit to facilitate the accurate assembly of the initiation complex.^[14] These factors ensure the binding of initiator fMet-tRNA^fMet^ to the start codon, prevent premature elongation, and promote the joining of the 50S subunit to form the 70S ribosome.^[14] IF1, the smallest of the three factors at approximately 8 kDa, binds to the A-site of the 30S ribosomal subunit, where it blocks the entry of elongator tRNAs and enhances translational fidelity by stabilizing the subunit in a conformation conducive to correct initiator tRNA selection.^[14] Structurally, IF1 adopts an oligonucleotide/oligosaccharide-binding (OB) fold resembling an RNA helix, allowing it to mimic tRNA and occupy the A-site while interacting with ribosomal proteins S12 and S18 as well as 16S rRNA helices 44 and 34.^[14] This positioning induces long-range conformational changes in the 30S subunit, including the closure of the A-site cleft, which promotes accurate decoding during initiation.^[14] IF2 functions as a GTPase that delivers the initiator fMet-tRNA^fMet^ to the P-site of the 30S subunit, recognizing the formylated methionine and positioning the tRNA for codon-anticodon pairing.^[15] Composed of multiple domains (N, G1–G3 for GTP binding, and C1–C2), IF2 adopts a compact conformation in its GDP-bound state, with GTP binding facilitating the active form for tRNA delivery; the C2 domain specifically interacts with the formyl group of fMet-tRNA^fMet^ to increase binding affinity by fivefold.^[15] The GTP hydrolysis cycle of IF2 is triggered by 50S subunit joining, activated by the ribosomal GTPase-associated center; this event promotes IF2 dissociation, conformational rearrangement of the tRNA, and readiness for peptidyl transfer.^[15] IF3 promotes the dissociation of 70S ribosomes into free 30S and 50S subunits, ensuring availability of the small subunit for new initiation events, and it selects the correct mRNA start codon by discriminating against non-canonical codons such as those lacking proper Shine-Dalgarno (SD) complementarity.^[16] IF3 consists of an N-terminal domain (NTD) and C-terminal domain (CTD) connected by a flexible linker, with the CTD primarily responsible for 70S dissociation and initial 30S binding near the platform, while the NTD modulates fidelity and is released first during 50S joining.^[16] The CTD of IF3 interacts with the SD region of mRNA through induced conformational changes, destabilizing incorrect complexes and favoring those with canonical AUG start codons paired to fMet-tRNA^fMet^.^[16] These bacterial initiation factors exhibit evolutionary conservation in organelles derived from prokaryotic ancestors, such as mitochondria and chloroplasts, where homologs like mitochondrial IF2 (mIF2) and IF3 (mIF3) retain core functions in initiator tRNA delivery and subunit dissociation, though mIF1 is typically absent and compensated by modifications in mIF2.^[17]

Mechanisms of action

In bacterial translation initiation, the three prokaryotic initiation factors—IF1, IF2, and IF3—bind sequentially to the 30S ribosomal subunit to form the pre-initiation complex (pre-IC). IF3 binds first, stabilizing the interaction between the 30S subunit and mRNA by positioning the Shine-Dalgarno sequence near the anti-Shine-Dalgarno helix in 16S rRNA, which ensures accurate start codon alignment.^[9] This stabilization is biphasic, with an initial association rate of approximately 1000 µM⁻¹ s⁻¹ followed by a conformational rearrangement at 34–55 s⁻¹.^[9] IF2 then associates in its GTP-bound form, with an association rate of 220–320 µM⁻¹ s⁻¹ followed by rearrangement at 2–6 s⁻¹, delivering the initiator fMet-tRNA^fMet^ to the P site in a codon-dependent manner.^[9] IF1 associates last, occupying the A site to block non-initiator tRNA binding and prevent errors such as premature peptide bond formation, with a binding rate of 10–12 µM⁻¹ s⁻¹ that locks the complex in a stable state (off-rate ~0.02 s⁻¹).^[9] This cooperative binding enhances the kinetics and fidelity of the 30S initiation complex (IC) assembly. Recent cryo-EM structures (as of 2024) have captured bacterial co-transcriptional translation initiation, showing how the pre-initiation complex assembles on nascent mRNA during transcription.^[18] Fidelity checkpoints are integral to this process, primarily enforced by IF3 and IF1 to ensure accurate start codon and tRNA selection. IF3 discriminates against non-AUG start codons (e.g., AUU or AUA) by destabilizing mismatched codon-anticodon interactions in the P site; its N-terminal domain (NTD) residues, such as R25, Q33, and R66, interact with the elbow of the initiator tRNA to scrutinize the three conserved G-C base pairs in the anticodon stem, rejecting non-cognate pairings up to fivefold more effectively for AUU.^[19] Mutations in these residues, like R25A/Q33A/R66A, impair this discrimination, allowing initiation from non-canonical codons and reducing growth fitness in Escherichia coli. Recent 2025 studies using single-molecule tracking demonstrate that IF3N dynamics are crucial for start codon decoding, with blocking IF3N delaying initiation and underscoring its fidelity role.^[20] Complementarily, IF1 prevents initiator tRNA misplacement by inducing a 30S head tilt, enabling 16S rRNA residues GA1338–9 to inspect the anticodon stem for initiator identity, thus promoting dissociation of pseudo-initiation complexes with non-initiator tRNAs.^[21] These mechanisms collectively minimize initiation errors to below 1% for non-standard codons.^[21] Upon 50S subunit joining to form the 70S IC, release of the initiation factors is triggered by GTP hydrolysis on IF2. This hydrolysis, occurring ~20–30 ms after subunit association, rotates the 70S ribosome from a transient rotated (low FRET) to a non-rotated (high FRET) conformation, facilitating dissociation of IF2, IF1, and IF3 in that order.^[22] Non-hydrolyzable GTP analogs like GDPNP trap the complex in the rotated state, inhibiting factor release and elongation entry, underscoring GTP hydrolysis as a fidelity checkpoint.^[23] Cryo-EM structures from the 2010s, resolved to 3.6 Å, have illuminated these dynamics: for instance, 11 structures of the 30S PIC reveal large-scale movements of IF3's NTD (~60 Å) and tRNA accommodation, coordinated by IF2's C2 domain in an extended conformation that shifts upon GTP hydrolysis.^[24] Earlier 2011 cryo-EM at 6.9 Å showed IF2's N-terminal domain orienting toward the 30S interface, with conformational changes in the subunit head and platform during tRNA delivery.^[25] These visualizations confirm IF2's role in sensing correct codon-anticodon pairing before committing to 70S formation.^[24]

Eukaryotic initiation factors

Core components (eIF2, eIF4F complex)

Eukaryotic translation initiation requires a more intricate array of initiation factors compared to prokaryotes, primarily due to the nuclear processing of mRNA, which includes addition of a 5' cap structure and poly(A) tail, necessitating specialized factors for ribosome recruitment and scanning.^[2]^[26] This multi-factor system enables precise control over start codon selection and mRNA unwinding, contrasting with the simpler three-factor setup in prokaryotes.^[27] A central player is eukaryotic initiation factor 2 (eIF2), a heterotrimeric GTPase composed of alpha (eIF2α), beta (eIF2β), and gamma (eIF2γ) subunits.^[28] The eIF2γ subunit directly binds GTP and the initiator methionyl-tRNA (Met-tRNAi), while the alpha and beta subunits enhance affinity for Met-tRNAi, forming the ternary complex eIF2-GTP-Met-tRNAi that delivers the initiator tRNA to the 40S ribosomal subunit.^[29] Regulation occurs via phosphorylation of a conserved serine residue (Ser51) on the eIF2α subunit, which inhibits the exchange factor eIF2B, reducing ternary complex formation and global translation while selectively promoting stress-response genes.^[30]^[31] Another key component is the eIF4F complex, a heterotrimeric assembly consisting of eIF4E, the cap-binding protein that recognizes the 5' m7G cap; eIF4A, an ATP-dependent DEAD-box RNA helicase; and eIF4G, a scaffold protein that bridges eIF4E and eIF4A while interacting with other factors like poly(A)-binding protein.^[32]^[33] This complex facilitates ribosome recruitment by binding the capped mRNA and unwinding secondary structures in the 5' untranslated region (UTR), a critical step for scanning to the start codon, which is absent in prokaryotic mRNAs lacking such caps.^[34]^[2] Additional essential factors include eIF1 and eIF1A, which promote accurate start codon selection by stabilizing the anticodon-codon interaction at AUG and preventing initiation at non-AUG sites during ribosomal scanning.^[35] eIF5 serves as the GTPase-activating protein (GAP) for eIF2, stimulating GTP hydrolysis upon start codon recognition to trigger conformational changes that commit the preinitiation complex.^[36] These components underscore the eukaryotic system's reliance on coordinated multi-factor interactions to navigate processed mRNAs, differing markedly from prokaryotic direct binding via Shine-Dalgarno sequences.^[27]

Assembly and regulation

The assembly of the eukaryotic 43S pre-initiation complex (PIC) begins with the binding of initiation factors eIF1, eIF1A, eIF3, and eIF5 to the 40S ribosomal subunit, forming a stable platform for subsequent recruitment.^[37] The eIF2 ternary complex (TC), consisting of eIF2 bound to GTP and methionyl-initiator tRNA (Met-tRNAi^Met), then joins this complex, positioning the initiator tRNA in the P site of the 40S subunit to prepare for mRNA interaction.^[37] eIF3 plays a central role in stabilizing the 43S PIC and facilitating its recruitment to the mRNA, while eIF1 and eIF1A maintain an open conformation of the 40S subunit's decoding center, enabling efficient scanning of the 5' untranslated region (UTR).^[37] eIF5 acts as a GTPase-activating protein (GAP) for eIF2, ensuring timely hydrolysis during start codon recognition.^[37] Following 43S PIC formation, the cap-binding complex eIF4F recruits the mRNA to the 40S subunit via interactions between eIF4G and eIF3, generating the 48S PIC that initiates scanning from the 5' cap.^[38] During scanning, the 48S PIC moves unidirectionally along the 5' UTR at approximately 10–20 nucleotides per second, as measured in recent in vivo studies, inspecting potential start codons through base-pairing between Met-tRNAi^Met anticodon and mRNA triplets.^[38]^[39] Upon recognition of the AUG start codon, eIF1 release from the 40S subunit triggers GTP hydrolysis on eIF2, promoted by eIF5, leading to a conformational shift to a closed state that stabilizes the initiation complex and commits to translation.^[38] This mechanism ensures fidelity, as eIF1 discriminates against non-AUG codons, particularly those with mismatches at the third position, preventing aberrant initiation.^[38] Regulation of assembly and scanning is tightly controlled by signaling pathways that modulate initiation factor activity. The mTORC1 pathway promotes cap-dependent initiation by phosphorylating 4E-binding proteins (4E-BPs), which releases eIF4E from inhibitory binding, allowing eIF4F complex formation and enhanced mRNA recruitment to the 43S PIC.^[40] In contrast, under stress conditions such as nutrient deprivation or endoplasmic reticulum stress, kinases like PERK or GCN2 phosphorylate eIF2α at serine-51, causing phosphorylated eIF2 (P-eIF2) to sequester eIF2B and significantly reduce TC formation, thereby inhibiting global translation initiation while selectively enhancing translation of stress-response genes like ATF4.^[41] Alternative pathways circumvent canonical assembly in certain contexts, such as viral infections. Internal ribosome entry sites (IRESs) in viral mRNAs, like those of encephalomyocarditis virus (EMCV) and hepatitis C virus (HCV), enable cap-independent initiation by directly recruiting the 40S subunit to internal sites, bypassing eIF4F and scanning; for instance, the HCV IRES binds the 40S without eIF4A or ATP, relying on eIF2 for subsequent TC delivery.^[42] This allows viruses to hijack translation machinery under conditions where cap-dependent initiation is suppressed.^[42]

Molecular structure and function

Structural features

Initiation factors exhibit diverse structural architectures adapted to their roles in translation initiation across prokaryotes and eukaryotes, often featuring conserved motifs for nucleotide and RNA binding. In prokaryotes, IF1 adopts a compact, single-domain structure characterized by a five-stranded β-barrel oligonucleotide/oligosaccharide-binding (OB) fold, which facilitates its binding to the A-site of the 30S ribosomal subunit without GTPase activity.^[43] Similarly, eukaryotic eIF1 displays a tightly packed domain with two α-helices flanking a five-stranded β-sheet, including RNA-binding elements that interact with 18S rRNA.^[44] Prokaryotic IF3 consists of two globular domains—an N-terminal domain (NTD) with an α/β fold and a C-terminal domain (CTD) forming an α/β sandwich—connected by a flexible linker, where the CTD harbors RNA-binding helices for 16S rRNA engagement.^[9] GTP-binding domains represent a prominent motif in initiation factors that deliver the initiator tRNA. Prokaryotic IF2 is a multidomain protein spanning approximately 130 Å in length, with its central G2 domain housing the GTP-binding site featuring conserved P-loop (G1), switch I/II, and G4 motifs for nucleotide coordination and hydrolysis.^[9] The eukaryotic counterpart, eIF2, is a heterotrimeric complex (α, β, γ subunits), where the γ subunit contains the GTP-binding G-domain (domain I) homologous to elongation factors, oriented inward for interaction with regulatory partners, as resolved by cryo-EM at 3.9 Å resolution.^[30] These G-domains enable GTP-dependent conformational dynamics essential for ribosomal subunit joining. The eukaryotic eIF4F complex exemplifies modularity in initiation factor architecture, serving as a scaffold for mRNA recruitment. eIF4G acts as the central scaffold with HEAT repeats in its middle domain (HEAT-1), forming binding sites for eIF4A and eIF3 to coordinate helicase activity and ribosomal attachment, while its C-terminal HEAT-2 domain provides an auxiliary eIF4A site.^[37] Complementing this, eIF4E features a concave m7G cap-binding pocket that specifically recognizes the 5′ cap structure of mRNA, stabilizing the complex through electrostatic and hydrogen-bonding interactions.^[37] This contrasts with prokaryotic simplicity, where factors like IF1 operate as standalone domains rather than multi-subunit assemblies. Conformational changes in IF2 upon GTP hydrolysis underpin the transition to elongation-competent states, as captured in cryo-EM structures from 2016 at 3.7 Å resolution (PDB: 3JCJ, 3JCN). In GTP-bound analogs, IF2's domain IV rotates approximately 180° relative to core domains I-III, positioning near the peptidyl transferase center; hydrolysis triggers 30S subunit back-rotation (from 8.4° to 3.7°), rearranging domains to release IF2 and accommodate elongation factors.^[45] These dynamics highlight IF2's role in coupling nucleotide state to ribosomal reconfiguration.^[22]

Functional interactions

Initiation factors engage in precise physical contacts with ribosomal components to ensure accurate positioning during translation initiation. In prokaryotes, initiation factor IF3 binds to the platform domain of the 30S ribosomal subunit, interacting with the 16S rRNA, including regions near the anti-Shine-Dalgarno sequence, to prevent premature mRNA binding and stabilize the subunit in a conformation suitable for initiator tRNA recruitment.^[24] Similarly, in eukaryotes, the gamma subunit of eIF2 (eIF2γ) contacts the 40S ribosomal subunit at the P-site by binding to helix h44 of 18S rRNA and the acceptor stem of Met-tRNAi^Met, thereby promoting the delivery and positioning of the initiator tRNA within the ribosomal decoding center.^[46] These factors also form synergistic interactions with one another to coordinate their activities. For instance, eIF4G acts as a scaffold in the eIF4F complex, bridging eIF4E—which recognizes the mRNA 5' cap—to eIF4A, thereby enhancing the RNA helicase activity of eIF4A to unwind secondary structures in the mRNA 5' untranslated region and facilitate ribosomal scanning.^[47] In a conserved mechanism across domains, prokaryotic IF2 and its eukaryotic homolog eIF5B coordinate GTPase activity during ribosomal subunit joining; IF2/eIF5B interacts with the large subunit to trigger GTP hydrolysis, which releases the factor and stabilizes the initiator tRNA in the P-site of the complete 70S/80S ribosome.^[48] Allosteric regulation further refines these interactions upon start codon recognition. In eukaryotes, eIF1 binds to the 40S subunit near the P-site and is displaced following accurate AUG codon selection, an event that stabilizes the 48S preinitiation complex by allowing eIF5-mediated GTP hydrolysis on eIF2 and commitment to translation initiation.^[49] Inhibitory interactions provide a layer of control to prevent inappropriate initiation. The eukaryotic 4E-binding proteins (4E-BPs) compete with eIF4G for binding to eIF4E, thereby sequestering eIF4E and inhibiting its recruitment of capped mRNAs to the ribosome, which represses translation under conditions such as nutrient limitation.^[50]

Clinical and pathological roles

Involvement in cancer

Dysregulation of eukaryotic initiation factors plays a pivotal role in oncogenesis by altering translational control to favor the synthesis of pro-tumorigenic proteins. Overexpression of eIF4E, a key component of the eIF4F complex, is frequently observed in various cancers, including breast and prostate malignancies, where it enhances cap-dependent translation of oncogenes such as c-Myc and cyclin D1, thereby promoting cell proliferation and tumor progression.^[51]^[52] This overexpression correlates with advanced disease stages and poor prognosis in these cancers, as eIF4E preferentially translates mRNAs with complex 5' untranslated regions that encode growth-promoting factors.^[53]^[54] Phosphorylation of eIF2α, mediated by kinases like PERK during cellular stress responses such as hypoxia, also contributes to tumor adaptation and survival. In hypoxic tumor microenvironments, eIF2α phosphorylation inhibits global protein synthesis while selectively allowing translation of stress-response genes, including those regulated by hypoxia-inducible factor (HIF), which supports angiogenesis, metabolic reprogramming, and resistance to apoptosis.^[55]^[56] This mechanism enhances tumor cell viability under nutrient deprivation and oxidative stress, facilitating metastasis and therapy resistance in solid tumors.^[57]^[58] Therapeutic strategies targeting initiation factors, particularly through mTOR inhibitors like rapamycin and its analogs (everolimus, temsirolimus), have shown promise by disrupting eIF4F assembly and cap-dependent translation. These agents block mTORC1 signaling, reducing eIF4E activity and oncogene translation, with clinical trials demonstrating activity in hematologic malignancies.^[59]^[60] However, single-agent rapalogs have shown limited efficacy in relapsed/refractory acute lymphoblastic leukemia (ALL), with response rates around 10% or less; combinations with chemotherapy may improve outcomes. As of 2025, ongoing trials evaluate rapalogs and novel mTOR inhibitors, such as bi-steric mTORC1-selective agents, in B-ALL and other leukemias, though challenges like feedback activation of PI3K/AKT pathways limit monotherapy outcomes.^[61]^[62] While prokaryotic initiation factors have limited direct involvement in eukaryotic cancers, vulnerabilities in translational machinery have motivated research into ribosome-targeting therapies for oncology.

Implications in other diseases

Initiation factors play significant roles in neurodegenerative disorders beyond their canonical functions in protein synthesis. In Alzheimer's disease (AD), hyperphosphorylation of eIF2α has been implicated in synaptic dysfunction and memory impairment. This phosphorylation event, mediated by kinases such as PERK, elevates the translation of ATF4, a transcription factor that promotes stress responses but impairs long-term memory consolidation by reducing general protein synthesis. Studies in AD mouse models demonstrate that inhibiting eIF2α phosphorylation restores spatial memory and synaptic plasticity, highlighting its causal role in cognitive deficits. In human AD brains, elevated phospho-eIF2α and ATF4 levels correlate with disease progression and neuronal degeneration.^[63] Viruses exploit initiation factors to hijack host translation machinery, contributing to pathogenesis in infectious diseases. Picornaviruses, such as poliovirus and foot-and-mouth disease virus, encode proteases (e.g., 2A^pro or L^pro) that cleave eIF4G, disrupting cap-dependent translation of host mRNAs while enabling internal ribosome entry site (IRES)-mediated translation of viral RNA. This cleavage separates the eIF4E-binding domain from the eIF4A and PABP-interacting regions of eIF4G, favoring viral replication and suppressing antiviral responses. In HIV-1 infection, the Tat protein activates the Akt/mTORC1 pathway, which indirectly enhances eIF4E activity by phosphorylating 4E-BP1, thereby promoting cap-dependent translation of viral transcripts and contributing to persistent inflammation and immune dysregulation in infected cells.^[64]^[65]^[66] Mutations in initiation factors underlie certain genetic disorders, particularly leukodystrophies. Vanishing white matter disease (VWMD), a severe childhood-onset leukodystrophy, arises from biallelic mutations in genes encoding subunits of eIF2B, the guanine nucleotide exchange factor for eIF2. These mutations impair eIF2 recycling, leading to hypersuppression of translation under stress and oligodendrocyte dysfunction, resulting in myelin loss and cerebral white matter rarefaction. Over 200 mutations across the five eIF2B subunits (EIF2B1–5) have been identified, with severity linked to residual eIF2B activity; for instance, hypomorphic variants in EIF2B2 or EIF2B5 cause stress-induced foam cell accumulation and neurodegeneration. Adult-onset forms are rarer but share similar neuropathology, emphasizing eIF2B's role in glial cell resilience.^[67]^[68]^[69] Recent research has linked initiation factors to inflammatory responses in viral pandemics. In severe COVID-19 cases, phosphorylation of eIF4E (p-eIF4E) has been implicated in SARS-CoV-2 pathogenesis. A 2023–2024 study showed that p-eIF4E, activated via the ERK-MNK pathway, promotes selective translation of host cell membrane-residential factors (such as TSPAN3, CD63, and ITGB2), facilitating viral entry and replication in infected cells; this suggests potential therapeutic targeting of the pathway to inhibit SARS-CoV-2 infection without broadly impairing translation.^[70]

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Cap Binding Complex - an overview | ScienceDirect Topics
Cap-binding protein (one of the subunits of eIF4) binds to the cap of the mRNA. Eukaryotes also have more initiation factors than prokaryotes and the order of ...
[25]
All Things Must Pass: Contrasts and Commonalities in Eukaryotic ...
Differences in the structure and translation of bacterial and eukaryotic mRNAs. ... A bacterial mRNA element that guides translation initiation at a ...
[26]
Role of Eukaryotic Initiation Factors during Cellular Stress and ...
eIF2 is a heterotrimeric protein, composed of α, β, and γ subunits [70]. eIF2 is a required element of the ternary complex that delivers met-tRNAi to the 40S ...4.1. Eif2 · 4.2. Eif3 · 4.3. Eif4f Complex
[27]
The Mechanism of Eukaryotic Translation Initiation: New Insights ...
eIF2γ binds directly to both GTP and Met-tRNAi and it appears that the α and β subunits each increase the affinity of the eIF2 complex for Met-tRNAi by ∼100- ...
[28]
The structural basis of translational control by eIF2 phosphorylation
May 13, 2019 · Phosphorylation of the translation initiation factor eIF2 alpha at a conserved serine residue mediates translational control at the ISR core. To ...
[29]
Fail-safe control of translation initiation by dissociation of eIF2α ...
Mar 18, 2017 · Phosphorylation of eIF2α controls translation initiation by restricting the levels of active eIF2-GTP/Met-tRNAi ternary complexes (TC).
[30]
Cap-dependent eukaryotic initiation factor-mRNA interactions ...
eIF4F is a heterotrimeric complex composed of three subunits: eIF4E, a 7-methyl guanosine cap binding protein; eIF4A, a DEAD-box RNA helicase; and eIF4G. The ...
[31]
Dynamic Regulation of the Translation Initiation Helicase Complex ...
The heterotrimeric complex eIF4F, comprised of the m7GTP cap-binding protein eIF4E, the ATP-dependent helicase eIF4A, and the scaffold eIF4G, recruits ribosomal ...
[32]
Human eukaryotic initiation factor 4E (eIF4E) and the nucleotide ...
The eIF4F complex functions to also unwind secondary structure in the mRNA 5′ UTR, an activity that is attributed to the eIF4A helicase component (3, 4, 5).
[33]
Enhanced eIF1 binding to the 40S ribosome impedes ... - NIH
Coordinated movements of eukaryotic translation initiation factors eIF1, eIF1A, and eIF5 trigger phosphate release from eIF2 in response to start codon ...
[34]
Eukaryotic translation initiation factor eIF5 promotes the accuracy of ...
Abstract. eIF5 is the GTPase activating protein (GAP) for the eIF2·GTP·Met-tRNAiMet ternary complex with a critical role in initiation codon selection.
[35]
The structure of a human translation initiation complex reveals two ...
Jan 29, 2024 · Here we report the structure of a human 48S that shows that, in addition to the eIF4A that is part of eIF4F, there is a second eIF4A helicase bound at the mRNA ...
[36]
Selection of start codon during mRNA scanning in eukaryotic ...
Jun 15, 2022 · The study highlights how the scanning conformation of ribosomal 48S PIC acts as a coarse selectivity checkpoint for start codon selection and scans long 5′ ...
[37]
Normalizing translation through 4E-BP prevents mTOR-driven ...
Sep 19, 2016 · To begin filling that void, this study looked at the role of one of the many cellular processes regulated by mTORC1, cap-dependent translation.
[38]
Role of eIF2α Kinases in Translational Control and Adaptation to ...
eIF2α kinases, activated by stress, regulate translation by phosphorylating eIF2α, which reduces global translation and promotes specific gene translation.Family Of Eif2α Kinases... · Uorfs In Isr Translational... · Isr And Disease
[39]
Internal ribosome entry sites in eukaryotic mRNA molecules
In examples discussed above, the EMCV IRES is able to bypass the requirement for eIF4E, and the HCV IRES does not require eIF4E, eIF4A, eIF4B, or eIF4F.
[40]
The structure of the translational initiation factor IF1 from E.coli ...
In addition, by binding to the A‐site of the 30S ribosomal subunit, IF1 may ... Equilibrium binding studies have shown that IF1 binds to the 30S ribosomal subunit ...
[41]
Structure and interactions of the translation initiation factor eIF1
eIF1 has a new fold but resembles ribosomal and RNA‐binding proteins. Residues 29–113 of the native sequence form a tightly folded domain with two α‐helices ...Eif1 Has A New Fold But... · The His Tag And First 28... · Yeast Mutations And...
[42]
Structures of ribosome-bound initiation factor 2 reveal the ... - Science
Mar 4, 2016 · We determine the structure of IF2 complexed with a nonhydrolyzable guanosine triphosphate analog and initiator fMet-tRNAiMet in the context of ...Missing: delivery | Show results with:delivery
[43]
Initiation Factor eIF2γ Promotes eIF2–GTP–Met-tRNAiMet Ternary ...
eIF2γ promotes eIF2 binding to the 40S ribosome by binding to 18S rRNA helix h44 and contacting the acceptor stem of Met-tRNAiMet. eIF2 binds to the 40S ...
[44]
Structural basis for the enhancement of eIF4A helicase activity by ...
The two-domain RNA helicase eIF4A is a very weak helicase by itself, but the activity is enhanced upon interaction with the scaffolding protein eIF4G.
[45]
Initiation factor eIF5B catalyzes second GTP-dependent step in ... - NIH
eIF5B, the eukaryotic ortholog of IF2, is a GTPase that promotes ribosomal subunit joining. Here we show that eIF5B GTPase activity is required for protein ...Missing: coordination | Show results with:coordination
[46]
[PDF] Translational initiation factor eIF5 replaces eIF1 on the 40S ... - eLife
Nov 30, 2018 · formation of the 48S might also facilitate dissociation of eIF1 after AUG recognition and subsequent. eIF5-NTD binding. The path of mRNA and ...
[47]
The 4E-BP Caf20p Mediates Both eIF4E-Dependent and ... - PubMed
May 14, 2015 · A family of binding proteins, termed the 4E-BPs, interact with eIF4E to hinder ribosome recruitment. Mechanisms underlying mRNA specificity for 4E-BP control ...
[48]
Targeting Eukaryotic Translation Initiation Factor 4E (eIF4E) in Cancer
eIF4E overexpression is common in multiple cancer types, including malignancies of the prostate, breast, stomach, colon, lung, skin, and the hematopoietic ...Eif4e And Translation... · Eif4e In Cancer Initiation... · Clinical-Translational...
[49]
Phosphorylation of the mRNA cap-binding protein eIF4E and cancer
eIF4E overexpression can lead to increased levels of Cyclin D1 and c-Myc proteins, which are key mediators of cell proliferation[40]. Cyclin D1 is required ...2.1. The Mapk-Mnk Signaling... · 3. Eif4e Phosphorylation In... · 4. Targeting Eif4e...
[50]
eIF4E expression in tumors: its possible role in progression of ...
Furthermore, eIF4E is elevated in carcinomas of the breast, head and neck (HNSCC) and prostate, but not in typical benign lesions.
[51]
Signalling to eIF4E in cancer - PMC - NIH
Oct 9, 2015 · Thus, translational control via eIF4E acts as a convergence point for hyperactive signalling pathways to promote tumorigenesis. Consequently, ...Introduction · Figure 1. Model Of Cap... · Strategies For Targeting...
[52]
Control of HIF-1α expression by eIF2α phosphorylation mediated ...
Hypoxia inducible factor 1alpha (HIF-1α) plays a central role in regulating tumor angiogenesis via its effects on vascular endothelial growth factor (VEGF) ...
[53]
PERK/eIF2α signaling protects therapy resistant hypoxic cells ...
By using isogenic models, we find that HIF and eIF2α signaling contribute to the survival of hypoxic cells in vitro and in vivo. However, the eIF2α-dependent ...
[54]
“Translating” Tumor Hypoxia: Unfolded Protein Response (UPR)
Jul 18, 2006 · These results indicate that PERK and eIF2α phosphorylation contribute to cellular survival under prolonged hypoxia most likely by increasing the ...
[55]
Cancer-specific PERK signaling drives invasion and metastasis ...
Oct 20, 2017 · Upon its activation, PERK phosphorylates eIF2a, which reduces global protein synthesis while selectively increasing the translation of ATF4. In ...
[56]
Targeting eIF4F translation complex sensitizes B-ALL cells to ... - NIH
Nov 4, 2021 · However, TOR-KIs have not shown substantial efficacy at tolerated doses in blood cancer clinical trials. Selective inhibition of mTORC1 or ...
[57]
Targeting mTOR for cancer therapy
Jul 5, 2019 · In this review, we discuss the roles of mTOR in human cancer and the rationales and challenges for developing mTOR inhibitors to treat cancer.
[58]
A phase II trial of the oral mTOR inhibitor everolimus in relapsed ...
Dec 7, 2010 · Everolimus has single-agent activity in relapsed/refractory aggressive NHL and provides proof-of-concept that targeting the mTOR pathway is clinically relevant.
[59]
Targeting mTOR Kinase for Cancer Treatment: A Comprehensive ...
Oct 5, 2025 · Additionally, we summarize the findings from major clinical trials, including FDA-approved mTOR inhibitors like everolimus and temsirolimus, and ...
[60]
Efficacy of a Novel Bi-Steric mTORC1 Inhibitor in Models of B-Cell ...
Aug 1, 2021 · Here we report the activity of the mTORC1-selective bi-steric inhibitor, RMC-4627, in BCR-ABL-driven models of B-cell acute lymphoblastic leukemia (B-ALL).
[61]
Bacterial Protein Synthesis as a Target for Antibiotic Inhibition - NIH
There are a number of antibiotics that are commonly referred to as translation initiation inhibitors, namely, kasugamycin, pactamycin, edeine, GE81112, which ...
[62]
Ribosome-Targeting Antibiotics: Modes of Action, Mechanisms of ...
Jun 20, 2018 · In this review, we describe the modes of action of many ribosome-targeting antibiotics, highlight the major resistance mechanisms developed by ...
[63]
Basal levels of eIF2alpha phosphorylation determine cellular ...
Jan 9, 2009 · Furthermore, in human brains, ATF4 and phospho-eIF2alpha levels are tightly correlated and up-regulated in Alzheimer disease, most probably ...Missing: hyperphosphorylation | Show results with:hyperphosphorylation
[64]
GADD34 suppresses eIF2α phosphorylation and improves cognitive ...
Feb 28, 2023 · In summary, GADD34 suppresses eIF2α phosphorylation in the brain and prevents memory loss. As quercetin feeding increases GADD34 expression, it ...Missing: hyperphosphorylation | Show results with:hyperphosphorylation
[65]
The proteolytic cleavage of eukaryotic initiation factor (eIF) 4G is ...
Feb 17, 1997 · A common feature of viral infection is the subversion of the host cell machinery towards the preferential translation of viral products.Missing: cleaving | Show results with:cleaving
[66]
Translation Directed by Hepatitis A Virus IRES in the ... - PubMed
Translation directed by several picornavirus IRES elements can usually take place after cleavage of eIF4G by picornavirus proteases 2A(pro) or L(pro).
[67]
HIV-1 Tat Activates Akt/mTORC1 Pathway and AICDA Expression by ...
We found that HIV-1 Tat activated the Akt/mTORC1 signaling pathway; this leads to aberrant activation of activation-induced cytidine deaminase (AICDA).
[68]
EIF2B2 mutations in vanishing white matter disease hypersuppress ...
Apr 9, 2018 · Mutations in eIF2B genes cause vanishing white matter disease (VWMD), a fatal leukodystrophy that can manifest following physical trauma or ...
[69]
EIF2B5 mutations compromise GFAP+ astrocyte generation in ...
Feb 20, 2005 · Vanishing white matter disease (VWM) is a heritable leukodystrophy linked to mutations in translation initiation factor 2B (eIF2B).
[70]
Leukodystrophy Due to eIF2B Mutations in Adults - PubMed
Sep 2, 2021 · Vanishing white matter disease (VWMD) due to eIF2B mutations is a common leukodystrophy characterised by childhood onset, autosomal recessive inheritance.
[71]
Phospho-eIF4E stimulation regulates coronavirus entry by selective ...
Feb 1, 2024 · Previous studies have shown that p-eIF4E is involved in the selective translation of mRNAs that are linked to the inflammation, extracellular ...