{"gene":"EEFSEC","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":1990,"finding":"SELB (bacterial ortholog) was purified and shown to be an EF-Tu-like protein that specifically binds selenocysteyl-tRNA(Sec); it was found partially associated with ribosomes by cell fractionation, establishing it as the first elongation factor with specificity for a particular charged tRNA.","method":"Protein purification, limited proteolysis, immunological analysis, ribosome fractionation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct biochemical purification and characterization with multiple orthogonal methods, replicated in subsequent studies","pmids":["2140572"],"is_preprint":false},{"year":1991,"finding":"The 8-bp aminoacyl-acceptor stem of tRNA(Sec) is the key determinant for binding to SELB and for exclusion from EF-Tu; reducing the stem to 7 bp (canonical length) abolished SELB binding but allowed EF-Tu binding, irrespective of the charged amino acid.","method":"In vitro binding assays with purified SELB, EF-Tu, and tRNA(Sec) mutants; in vivo selenoprotein synthesis assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with mutagenesis, corroborated by in vivo functional assay","pmids":["1939093"],"is_preprint":false},{"year":1991,"finding":"SELB specifically complexes selenocysteyl-tRNA(Sec); interaction with the selenol group of the aminoacylated residue is required for stable SELB·tRNA complex formation, providing the biochemical basis for exclusive selection of Sec-tRNA(Sec) during UGA decoding.","method":"Biochemical characterization of purified SELB with charged tRNA variants","journal":"Biochimie","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct biochemical assay, single lab, consistent with parallel studies","pmids":["1839607"],"is_preprint":false},{"year":1993,"finding":"Bacterial SELB directly and specifically binds the mRNA hairpin SECIS element (located 3' of the UGA codon) via its loop region; in the presence of selenocysteinyl-tRNA, SELB forms a quaternary complex with charged tRNA and mRNA, positioning Sec-tRNA(Sec) at the UGA codon.","method":"Gel shift assays, RNA footprinting with nucleases and iodine, complex formation assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution of ternary/quaternary complex, footprinting, replicated across labs","pmids":["8483932"],"is_preprint":false},{"year":1994,"finding":"The SELB-GTP-Sec-tRNA(Sec) ternary complex binds to selenoprotein mRNAs fdhF and fdnG; toeprint experiments showed SELB recognizes ribosome-bound mRNA and may protrude toward the large subunit, placing the ternary complex at the selenocysteine codon.","method":"Boundary experiments, toeprinting assays","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct biochemical toeprinting on ribosome-bound mRNA, two mRNA targets tested","pmids":["8314089"],"is_preprint":false},{"year":1996,"finding":"The C-terminal domain of bacterial SELB (a ~17 kDa subdomain) specifically binds the mRNA SECIS hairpin; a truncated SELB lacking this domain still binds selenocysteyl-tRNA(Sec) but cannot bind mRNA, demonstrating that the tRNA- and mRNA-binding functions are physically separable.","method":"Domain truncation, expression of isolated fragments, RNA-binding assays, in vivo competition experiments","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — domain dissection with reconstitution, in vitro and in vivo validation, replicated","pmids":["8893853"],"is_preprint":false},{"year":1996,"finding":"Bases G23, U24, and bulge residues U17/U18 in the SECIS mRNA hairpin apical loop are directly involved in binding to SELB in both fdhF and fdnG mRNAs; SELB binding affinity for the fdhF hairpin is ~30 nM (apparent Kd).","method":"Chemical probing, NMR-based structure determination, computer modeling, binding studies","journal":"RNA (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Strong — structure-based mutagenesis with quantitative binding data, two mRNA targets","pmids":["8634916"],"is_preprint":false},{"year":1996,"finding":"Overproduction of SELB reduces selenocysteine UGA readthrough; co-overexpression of tRNA(Sec) reverses inhibition, establishing that a correct stoichiometric quaternary complex (SELB·GTP·Sec-tRNA(Sec)·mRNA) is required for selenocysteine insertion in vivo.","method":"In vivo overexpression, UGA readthrough assay (lacZ fusion), genetic complementation","journal":"Molecular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with functional readout, single lab","pmids":["8898393"],"is_preprint":false},{"year":1997,"finding":"In vitro selection (SELEX) identified novel RNA aptamers binding SELB; in vivo analysis showed that mRNA hairpin binding to SELB is necessary but not sufficient for UGA readthrough, implying SECIS element functions beyond mere tethering of Sec-tRNA(Sec) to UGA.","method":"In vitro SELEX, domain-mapping with truncated SELB, in vivo lacZ UGA readthrough assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — SELEX plus in vivo functional assay, single lab","pmids":["9192624"],"is_preprint":false},{"year":1998,"finding":"SELB possesses low intrinsic GTPase activity stimulated by 70S ribosomes; the SECIS mRNA hairpin further increases ribosome-stimulated GTPase activity 3–4 fold (via increased kcat), showing mRNA binding induces a conformational switch that promotes GTP hydrolysis.","method":"GTPase activity assays in vitro with ribosomes and mRNA hairpin, kinetic analysis","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — quantitative in vitro reconstitution with defined components, mechanistic dissection of kcat","pmids":["9454578"],"is_preprint":false},{"year":1999,"finding":"The C-terminus of SELB is the primary interaction domain for mRNA hairpin binding; the vast majority of SELEX-derived aptamers from a random pool bound specifically to the C-terminal domain of SELB.","method":"In vitro SELEX with full-length and truncated SELB, domain mapping","journal":"RNA (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — SELEX domain mapping, single lab, corroborates earlier deletion data","pmids":["10496219"],"is_preprint":false},{"year":2000,"finding":"SelB GTP affinity (Kd = 0.74 µM) and GDP affinity (Kd = 13.4 µM) are much lower than other translation factors; GDP release is very rapid (15 s⁻¹), explaining why no GDP/GTP exchange factor is needed. The SECIS RNA minihelix binds with Kd ~1 nM, affinity increasing further when Sec-tRNA(Sec) is bound to SelB.","method":"Stopped-flow kinetics using intrinsic tryptophan fluorescence and fluorescent nucleotide analogs; fluorescence resonance energy transfer","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — rigorous quantitative kinetics with multiple orthogonal fluorescence methods","pmids":["10781605"],"is_preprint":false},{"year":2000,"finding":"The archaeal SelB homolog (MJ0495/aSelB) from Methanococcus jannaschii binds guanine nucleotides and preferentially binds selenocysteyl-tRNA(Sec), but unlike bacterial SelB, does not bind the SECIS element, correlating with its lack of the bacterial C-terminal mRNA-binding domain.","method":"Purification of recombinant MJ0495, GTP binding assay, tRNA binding assay, SECIS binding assay","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple in vitro binding assays on purified archaeal protein, negative SECIS binding result mechanistically informative","pmids":["10860743"],"is_preprint":false},{"year":2002,"finding":"Crystal structure of the C-terminal mRNA-binding fragment of Moorella thermoacetica SelB (SelB-C) at 2.12 Å reveals four tandem winged-helix (WH) domains arranged in an L-shape; this is the first example of WH domains in RNA binding, with conserved basic residues defining the mRNA-binding site.","method":"X-ray crystallography with multiwavelength anomalous dispersion phasing","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure at 2.12 Å with functional mapping of binding residues","pmids":["12145214"],"is_preprint":false},{"year":2002,"finding":"NMR structure of the prokaryotic SECIS mRNA hairpin shows conserved nucleotides exposed for SelB recognition; binding of the SelB C-terminal domain stabilizes RNA secondary structure; a GpU sequence at the tetraloop tip and a bulge uracil 5 base pairs away are essential for SelB interaction.","method":"NMR spectroscopy, mutagenesis of RNA, SelB binding assays","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure combined with mutagenesis and quantitative binding data","pmids":["12421564"],"is_preprint":false},{"year":2003,"finding":"Inactivation of selB in Methanococcus maripaludis abolishes synthesis of all selenoproteins, proving aSelB is the archaeal translation factor specialized for selenocysteine insertion; the mutation also derepresses expression of cysteine-containing homologs and impairs growth on formate.","method":"Targeted gene knockout, metabolic labeling, proteomics","journal":"Journal of bacteriology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic knockout with defined phenotypes, multiple selenoproteins tested","pmids":["12486046"],"is_preprint":false},{"year":2005,"finding":"Crystal structure of the SelB mRNA-binding domain in complex with SECIS RNA at 2.3 Å reveals the first RNA-binding winged-helix (WH) domain; RNA recognition occurs without major conformational change in the WH motif, and the geometry allows the complex to wrap around the small ribosomal subunit.","method":"X-ray crystallography","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure of protein-RNA complex at 2.3 Å resolution","pmids":["15665870"],"is_preprint":false},{"year":2007,"finding":"Crystal structures of E. coli WH3/4 domains and M. thermoacetica WH1-4 domains each bound to SECIS RNA reveal that both WH motifs use the same structural elements for RNA binding; a salt bridge connecting WH2 to WH3 is disrupted upon mRNA binding, providing a molecular switch that may communicate between tRNA and mRNA binding sites.","method":"X-ray crystallography at 2.3 Å (E. coli WH3/4) and 2.6 Å (M. thermoacetica WH1-4), mutagenesis","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — two crystal structures with mutagenesis supporting molecular switch mechanism","pmids":["17537456"],"is_preprint":false},{"year":2007,"finding":"Crystal structure of SelB C-terminal domain complexed with RNA reveals large domain rearrangement; an interdomain region forms new interactions with phosphate backbone of a neighboring RNA in a sequence-independent manner, potentially reflecting SelB interactions with tRNA or rRNA during ribosome engagement.","method":"X-ray crystallography","journal":"Structure (London, England : 1993)","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — crystal structure observation but functional consequence of sequence-independent binding is inferred, not directly tested","pmids":["17502103"],"is_preprint":false},{"year":2009,"finding":"Sec-tRNA(Sec) binds SelB·GTP with extraordinarily high affinity (Kd = 0.2 pM); binding is enthalpically driven with ~4 ion pairs. GTP hydrolysis accelerates release of Sec-tRNA(Sec) by >10⁶-fold (from 0.3 h⁻¹ to 240 s⁻¹). Ser-tRNA(Sec) and uncharged tRNA(Sec) bind with much lower affinity (~0.5 µM) to any nucleotide-bound form.","method":"Thermodynamic and kinetic stopped-flow measurements, fluorescent tRNA analogs","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — rigorous quantitative thermodynamic and kinetic analysis with multiple orthogonal measurements","pmids":["19940162"],"is_preprint":false},{"year":2015,"finding":"Crystal structure of full-length bacterial SelB from Aquifex aeolicus in complex with GTP analog at 3.2 Å shows three EF-Tu-like domains (D1-3) followed by four WH domains; the Sec-binding site is located at the D1-D2 interface with Arg residues coordinating the Sec moiety, and is smaller and more exposed than EF-Tu's aminoacyl binding site.","method":"X-ray crystallography at 3.2 Å","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure of full-length protein with GTP analog, structural analysis of Sec-binding site","pmids":["26304550"],"is_preprint":false},{"year":2016,"finding":"Cryo-EM structures of six intermediates on the UGA recoding pathway reveal that: initial SelB-Sec-tRNA(Sec) binding induces 30S open conformation with Sec-tRNA(Sec) covering the sarcin-ricin loop (SRL); codon recognition in the decoding site causes local closure moving Sec-tRNA(Sec) away from the SRL and global 30S shoulder closure; this triggers SelB docking on the SRL, activating its GTPase.","method":"Single-particle cryo-electron microscopy, six structural intermediates","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structures of six pathway intermediates revealing mechanistic cascade, published in Nature","pmids":["27842381"],"is_preprint":false},{"year":2018,"finding":"Mammalian eEFSec folds into a chalice-like structure with three N-terminal EF-Tu-like domains and a C-terminal domain 4 that binds Sec-tRNA(Sec) and SBP2; GTP hydrolysis does not induce a canonical conformational change but promotes a slight ratchet of domains 1-2 and a lever-like movement of domain 4, which may be critical for Sec-tRNA(Sec) release on the ribosome.","method":"Structural analysis, biochemical characterization of domain functions (summary/review of published experimental work)","journal":"Biochimica et biophysica acta. General subjects","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — review of experimental structural data, but this is a review paper; the original crystal structure data it summarizes is the primary evidence","pmids":["29555379"],"is_preprint":false},{"year":2025,"finding":"Bi-allelic loss-of-function variants in EEFSEC cause reduced EEFSEC function in vitro, leading to lower selenoprotein levels in patient fibroblasts; an eEFSec-RNAi Drosophila model shows progressive motor impairment and synaptic defects, establishing that EEFSEC function is required for selenoprotein synthesis and neuronal maintenance in vivo.","method":"Patient fibroblast selenoprotein quantification, in vitro functional assays of variants, Drosophila RNAi model with motor and synaptic phenotyping","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function validated in patient cells and animal model with defined molecular and cellular phenotypes, multiple families and variants","pmids":["39753114"],"is_preprint":false},{"year":2000,"finding":"The bulged nucleotide U17 in the E. coli SECIS upper stem-loop participates in interaction with SelB; suppressor mutations in selB that rescue SECIS bulge mutations map to a 28-amino acid stretch in SelB C-terminal subdomain 4b, identifying this region as the contact interface.","method":"Genetic suppressor analysis, in vivo UGA readthrough assay, selB sequencing","journal":"Journal of bacteriology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with allele-specific suppression mapping contact sites, single lab","pmids":["11053373"],"is_preprint":false},{"year":1999,"finding":"Genetic analysis of SelB-SECIS interaction identified suppressor mutations in selB that counteract SECIS mutations; four suppressor amino acid exchanges cluster in a 23-amino acid stretch in domain 4b (direct RNA contact sites), and a fifth mutation in domain 4a promotes allele-nonspecific readthrough, suggesting domain 4a modulates communication between tRNA- and mRNA-binding regions.","method":"In vivo suppressor selection, UGA readthrough assay, selenium incorporation, sequencing","journal":"Molecular & general genetics : MGG","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with allele-specific suppression, single lab","pmids":["10628863"],"is_preprint":false},{"year":2025,"finding":"FOXO1 and STAT3 transcription factors directly bind functional sites in the eefsec promoter (-1070 bp FOXO1 site; -428 bp STAT3 site) and regulate eefsec transcriptional activity in a selenium-dependent manner in yellow catfish.","method":"Sequential promoter deletion analysis, EMSA, chromatin immunoprecipitation","journal":"Biochimica et biophysica acta. Gene regulatory mechanisms","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — EMSA and ChIP confirm direct binding, but single lab in a fish model","pmids":["40618995"],"is_preprint":false}],"current_model":"EEFSEC (eEFSec/SELB) is a specialized GTPase translation elongation factor that delivers selenocysteinyl-tRNA(Sec) to the ribosomal A site at UGA codons: its three N-terminal EF-Tu-like domains bind GTP and Sec-tRNA(Sec) with extraordinarily high affinity (Kd ~0.2 pM in bacteria), while its C-terminal winged-helix domains recognize the downstream SECIS mRNA hairpin; codon recognition at the ribosome triggers closure of the 30S subunit, docking of SelB onto the sarcin-ricin loop, GTPase activation, and GTP hydrolysis-driven release of Sec-tRNA(Sec), with loss-of-function in humans causing selenoprotein deficiency and progressive neurodegeneration."},"narrative":{"mechanistic_narrative":"EEFSEC (eEFSec/SELB) is a specialized GTPase translation elongation factor dedicated to selenocysteine incorporation, delivering selenocysteinyl-tRNA(Sec) to the ribosomal A site for decoding of in-frame UGA codons [PMID:2140572, PMID:8483932]. It was the first elongation factor shown to recognize a single charged tRNA species: it binds Sec-tRNA(Sec) with extraordinary affinity (Kd ~0.2 pM) in a complex requiring contact with the selenol group of the aminoacylated residue, while the 8-bp acceptor stem of tRNA(Sec) is the determinant that selects this tRNA and excludes it from EF-Tu [PMID:1939093, PMID:1839607, PMID:19940162]. Architecturally, the protein combines three N-terminal EF-Tu-like GTP/tRNA-binding domains with C-terminal tandem winged-helix domains that constitute a separable mRNA-binding module recognizing the SECIS hairpin located 3' of the UGA codon, producing a quaternary SELB·GTP·Sec-tRNA(Sec)·mRNA complex that positions the charged tRNA at the recoding codon [PMID:8483932, PMID:8893853, PMID:12145214, PMID:26304550]. Unlike canonical elongation factors, SELB has low GTP/GDP affinity and very rapid GDP release, dispensing with a dedicated nucleotide-exchange factor; SECIS binding and ribosome engagement stimulate its GTPase activity, and GTP hydrolysis accelerates Sec-tRNA(Sec) release by more than a million-fold to drive insertion [PMID:9454578, PMID:10781605, PMID:19940162]. Cryo-EM of recoding intermediates shows that initial binding holds the 30S in an open state with Sec-tRNA(Sec) over the sarcin-ricin loop, and codon recognition triggers 30S closure, SELB docking on the sarcin-ricin loop, and GTPase activation [PMID:27842381]. Genetic ablation of the archaeal ortholog abolishes synthesis of all selenoproteins, confirming its essential, pathway-defining role [PMID:12486046], and bi-allelic loss-of-function variants in human EEFSEC reduce selenoprotein levels in patient fibroblasts and cause progressive motor and synaptic defects in a Drosophila model, linking the factor to selenoprotein deficiency and neurodegeneration [PMID:39753114].","teleology":[{"year":1990,"claim":"Established that selenocysteine insertion requires a dedicated elongation factor distinct from EF-Tu, answering whether Sec delivery uses the canonical translation machinery.","evidence":"Protein purification and ribosome fractionation of bacterial SELB binding selenocysteyl-tRNA(Sec)","pmids":["2140572"],"confidence":"High","gaps":["Did not define the structural basis for tRNA(Sec) discrimination","Mechanism of UGA codon recognition unresolved"]},{"year":1991,"claim":"Identified the tRNA features that route Sec-tRNA(Sec) to SELB and away from EF-Tu, explaining specificity at the level of the charged tRNA.","evidence":"In vitro binding with tRNA(Sec) stem mutants plus in vivo selenoprotein assay; biochemical characterization of selenol-group dependence","pmids":["1939093","1839607"],"confidence":"High","gaps":["Did not localize the tRNA-contact residues on SELB","Did not address mRNA recognition"]},{"year":1993,"claim":"Showed SELB directly reads the SECIS mRNA hairpin and forms a quaternary complex, establishing how Sec delivery is targeted to specific codons.","evidence":"Gel shift, RNA footprinting, and quaternary complex reconstitution on fdhF/fdnG mRNAs; toeprinting on ribosome-bound mRNA","pmids":["8483932","8314089"],"confidence":"High","gaps":["Did not separate tRNA- and mRNA-binding functions structurally","Did not define SECIS nucleotide contacts"]},{"year":1996,"claim":"Mapped the mRNA-binding function to a separable C-terminal domain and defined the SECIS nucleotides contacted, dissecting the bifunctional architecture.","evidence":"Domain truncation with RNA-binding assays, chemical probing/NMR with quantitative binding, in vivo competition and stoichiometry experiments","pmids":["8893853","8634916","8898393"],"confidence":"High","gaps":["Atomic structure of the domain still unknown","Did not explain how mRNA binding affects GTPase"]},{"year":1998,"claim":"Connected SECIS binding to catalytic activation, showing the mRNA element conformationally licenses GTP hydrolysis rather than merely tethering the factor.","evidence":"In vitro GTPase kinetics with ribosomes and SECIS hairpin (kcat analysis); SELEX plus in vivo readthrough indicating SECIS roles beyond tethering","pmids":["9454578","9192624"],"confidence":"High","gaps":["Structural basis of the conformational switch not yet resolved","Coupling to codon recognition unknown"]},{"year":2000,"claim":"Quantified the unusual nucleotide and RNA binding properties, explaining why SELB needs no exchange factor and how cooperative RNA binding occurs.","evidence":"Stopped-flow kinetics and FRET; archaeal ortholog binding assays; genetic suppressor mapping of SECIS contact residues","pmids":["10781605","10860743","11053373"],"confidence":"High","gaps":["Did not capture full-length structure","Pico-molar tRNA affinity and its release mechanism not yet measured"]},{"year":2005,"claim":"Determined the winged-helix fold and its RNA-recognition mode, revealing the first WH domain used for RNA binding and how the complex could wrap the small subunit.","evidence":"X-ray crystallography of SelB C-terminal domain alone and bound to SECIS RNA; further WH structures with molecular-switch mutagenesis","pmids":["12145214","15665870","17537456","12421564","17502103"],"confidence":"High","gaps":["Did not show coupling to GTPase domains in full-length context","Functional consequence of sequence-independent backbone contacts inferred only"]},{"year":2009,"claim":"Defined the thermodynamic logic of Sec-tRNA(Sec) selection and GTP-driven release, showing hydrolysis is the switch for releasing the charged tRNA.","evidence":"Thermodynamic and stopped-flow kinetic measurements with fluorescent tRNA analogs","pmids":["19940162"],"confidence":"High","gaps":["Did not place these steps in the ribosomal recoding pathway"]},{"year":2016,"claim":"Visualized the full UGA recoding cascade, establishing how codon recognition mechanically triggers SELB GTPase activation on the ribosome.","evidence":"Single-particle cryo-EM of six recoding intermediates; full-length crystal structure with GTP analog defining the Sec-binding site","pmids":["27842381","26304550"],"confidence":"High","gaps":["Mammalian eEFSec-specific conformational details only partly resolved","Role of SBP2 in human system not structurally captured here"]},{"year":2025,"claim":"Established the physiological and disease relevance of human EEFSEC, linking loss of function to selenoprotein deficiency and neurodegeneration.","evidence":"Patient fibroblast selenoprotein quantification, in vitro variant assays, Drosophila RNAi motor/synaptic phenotyping; promoter analysis of transcriptional control by FOXO1/STAT3 in fish","pmids":["39753114","40618995"],"confidence":"High","gaps":["Which selenoproteins drive neuronal phenotype not defined","Human transcriptional regulation not directly tested"]},{"year":null,"claim":"How the human eEFSec·SBP2 system and SECIS recognition differ mechanistically from the bacterial SELB single-protein paradigm, and which selenoproteins mediate neuronal maintenance, remain to be resolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No ribosome-bound structure of the mammalian eEFSec·SBP2 complex in the corpus","Tissue-specific selenoprotein requirements unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003924","term_label":"GTPase activity","supporting_discovery_ids":[9,11,19,21]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[3,5,6,13,14,16]},{"term_id":"GO:0045182","term_label":"translation regulator activity","supporting_discovery_ids":[0,1,19,23]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[26]}],"localization":[{"term_id":"GO:0005840","term_label":"ribosome","supporting_discovery_ids":[0,4,21]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,15,23]}],"complexes":[],"partners":["SBP2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P57772","full_name":"Selenocysteine-specific elongation factor","aliases":["Elongation factor sec","Eukaryotic elongation factor, selenocysteine-tRNA-specific"],"length_aa":596,"mass_kda":65.3,"function":"Translation factor required for the incorporation of the rare amino acid selenocysteine encoded by UGA codons (PubMed:27708257, PubMed:35709277). Replaces the eRF1-eRF3-GTP ternary complex for the insertion of selenocysteine directed by the UGA codon (PubMed:27708257, PubMed:35709277). Insertion of selenocysteine at UGA codons is mediated by SECISBP2 and EEFSEC: SECISBP2 (1) specifically binds the SECIS sequence once the 80S ribosome encounters an in-frame UGA codon and (2) contacts the RPS27A/eS31 of the 40S ribosome before ribosome stalling (PubMed:35709277). (3) GTP-bound EEFSEC then delivers selenocysteinyl-tRNA(Sec) to the 80S ribosome and adopts a preaccommodated state conformation (PubMed:35709277). (4) After GTP hydrolysis, EEFSEC dissociates from the assembly, selenocysteinyl-tRNA(Sec) accommodates, and peptide bond synthesis and selenoprotein elongation occur (PubMed:35709277)","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/P57772/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EEFSEC","classification":"Not Classified","n_dependent_lines":621,"n_total_lines":1208,"dependency_fraction":0.5140728476821192},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/EEFSEC","total_profiled":1310},"omim":[{"mim_id":"621102","title":"NEURODEVELOPMENTAL DISORDER WITH PROGRESSIVE SPASTICITY AND BRAIN ABNORMALITIES; NEDPSB","url":"https://www.omim.org/entry/621102"},{"mim_id":"617724","title":"TSC22 DOMAIN FAMILY, MEMBER 2; TSC22D2","url":"https://www.omim.org/entry/617724"},{"mim_id":"607695","title":"EUKARYOTIC ELONGATION FACTOR, SELENOCYSTEINE-tRNA-SPECIFIC; EEFSEC","url":"https://www.omim.org/entry/607695"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Nuclear bodies","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/EEFSEC"},"hgnc":{"alias_symbol":["SELB","EFSEC"],"prev_symbol":[]},"alphafold":{"accession":"P57772","domains":[{"cath_id":"3.40.50.300","chopping":"6-62_84-213","consensus_level":"high","plddt":85.4839,"start":6,"end":213},{"cath_id":"2.40.30.10","chopping":"226-305","consensus_level":"high","plddt":88.61,"start":226,"end":305},{"cath_id":"2.40.30.10","chopping":"311-382_407-468","consensus_level":"high","plddt":89.6661,"start":311,"end":468},{"cath_id":"2.40.10.190","chopping":"472-548_573-594","consensus_level":"high","plddt":81.0124,"start":472,"end":594}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P57772","model_url":"https://alphafold.ebi.ac.uk/files/AF-P57772-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P57772-F1-predicted_aligned_error_v6.png","plddt_mean":79.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EEFSEC","jax_strain_url":"https://www.jax.org/strain/search?query=EEFSEC"},"sequence":{"accession":"P57772","fasta_url":"https://rest.uniprot.org/uniprotkb/P57772.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P57772/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P57772"}},"corpus_meta":[{"pmid":"10512699","id":"PMC_10512699","title":"High-level expression in Escherichia coli of selenocysteine-containing rat thioredoxin reductase utilizing gene fusions with engineered bacterial-type SECIS elements and co-expression with the selA, selB and selC genes.","date":"1999","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/10512699","citation_count":193,"is_preprint":false},{"pmid":"8893853","id":"PMC_8893853","title":"Domain structure of the prokaryotic selenocysteine-specific elongation factor SelB.","date":"1996","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/8893853","citation_count":96,"is_preprint":false},{"pmid":"8483932","id":"PMC_8483932","title":"Interaction of translation factor SELB with the formate dehydrogenase H selenopolypeptide mRNA.","date":"1993","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/8483932","citation_count":93,"is_preprint":false},{"pmid":"27842381","id":"PMC_27842381","title":"The pathway to GTPase activation of elongation factor SelB on the ribosome.","date":"2016","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/27842381","citation_count":92,"is_preprint":false},{"pmid":"1939093","id":"PMC_1939093","title":"The length of the aminoacyl-acceptor stem of the selenocysteine-specific tRNA(Sec) of Escherichia coli is the determinant for binding to elongation factors SELB or Tu.","date":"1991","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/1939093","citation_count":89,"is_preprint":false},{"pmid":"10860743","id":"PMC_10860743","title":"Identification and characterisation of the selenocysteine-specific translation factor SelB from the archaeon Methanococcus jannaschii.","date":"2000","source":"Journal of molecular 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it was found partially associated with ribosomes by cell fractionation, establishing it as the first elongation factor with specificity for a particular charged tRNA.\",\n      \"method\": \"Protein purification, limited proteolysis, immunological analysis, ribosome fractionation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct biochemical purification and characterization with multiple orthogonal methods, replicated in subsequent studies\",\n      \"pmids\": [\"2140572\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"The 8-bp aminoacyl-acceptor stem of tRNA(Sec) is the key determinant for binding to SELB and for exclusion from EF-Tu; reducing the stem to 7 bp (canonical length) abolished SELB binding but allowed EF-Tu binding, irrespective of the charged amino acid.\",\n      \"method\": \"In vitro binding assays with purified SELB, EF-Tu, and tRNA(Sec) mutants; in vivo selenoprotein synthesis assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with mutagenesis, corroborated by in vivo functional assay\",\n      \"pmids\": [\"1939093\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"SELB specifically complexes selenocysteyl-tRNA(Sec); interaction with the selenol group of the aminoacylated residue is required for stable SELB·tRNA complex formation, providing the biochemical basis for exclusive selection of Sec-tRNA(Sec) during UGA decoding.\",\n      \"method\": \"Biochemical characterization of purified SELB with charged tRNA variants\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct biochemical assay, single lab, consistent with parallel studies\",\n      \"pmids\": [\"1839607\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"Bacterial SELB directly and specifically binds the mRNA hairpin SECIS element (located 3' of the UGA codon) via its loop region; in the presence of selenocysteinyl-tRNA, SELB forms a quaternary complex with charged tRNA and mRNA, positioning Sec-tRNA(Sec) at the UGA codon.\",\n      \"method\": \"Gel shift assays, RNA footprinting with nucleases and iodine, complex formation assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution of ternary/quaternary complex, footprinting, replicated across labs\",\n      \"pmids\": [\"8483932\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"The SELB-GTP-Sec-tRNA(Sec) ternary complex binds to selenoprotein mRNAs fdhF and fdnG; toeprint experiments showed SELB recognizes ribosome-bound mRNA and may protrude toward the large subunit, placing the ternary complex at the selenocysteine codon.\",\n      \"method\": \"Boundary experiments, toeprinting assays\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct biochemical toeprinting on ribosome-bound mRNA, two mRNA targets tested\",\n      \"pmids\": [\"8314089\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"The C-terminal domain of bacterial SELB (a ~17 kDa subdomain) specifically binds the mRNA SECIS hairpin; a truncated SELB lacking this domain still binds selenocysteyl-tRNA(Sec) but cannot bind mRNA, demonstrating that the tRNA- and mRNA-binding functions are physically separable.\",\n      \"method\": \"Domain truncation, expression of isolated fragments, RNA-binding assays, in vivo competition experiments\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — domain dissection with reconstitution, in vitro and in vivo validation, replicated\",\n      \"pmids\": [\"8893853\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Bases G23, U24, and bulge residues U17/U18 in the SECIS mRNA hairpin apical loop are directly involved in binding to SELB in both fdhF and fdnG mRNAs; SELB binding affinity for the fdhF hairpin is ~30 nM (apparent Kd).\",\n      \"method\": \"Chemical probing, NMR-based structure determination, computer modeling, binding studies\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — structure-based mutagenesis with quantitative binding data, two mRNA targets\",\n      \"pmids\": [\"8634916\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Overproduction of SELB reduces selenocysteine UGA readthrough; co-overexpression of tRNA(Sec) reverses inhibition, establishing that a correct stoichiometric quaternary complex (SELB·GTP·Sec-tRNA(Sec)·mRNA) is required for selenocysteine insertion in vivo.\",\n      \"method\": \"In vivo overexpression, UGA readthrough assay (lacZ fusion), genetic complementation\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with functional readout, single lab\",\n      \"pmids\": [\"8898393\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"In vitro selection (SELEX) identified novel RNA aptamers binding SELB; in vivo analysis showed that mRNA hairpin binding to SELB is necessary but not sufficient for UGA readthrough, implying SECIS element functions beyond mere tethering of Sec-tRNA(Sec) to UGA.\",\n      \"method\": \"In vitro SELEX, domain-mapping with truncated SELB, in vivo lacZ UGA readthrough assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — SELEX plus in vivo functional assay, single lab\",\n      \"pmids\": [\"9192624\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"SELB possesses low intrinsic GTPase activity stimulated by 70S ribosomes; the SECIS mRNA hairpin further increases ribosome-stimulated GTPase activity 3–4 fold (via increased kcat), showing mRNA binding induces a conformational switch that promotes GTP hydrolysis.\",\n      \"method\": \"GTPase activity assays in vitro with ribosomes and mRNA hairpin, kinetic analysis\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — quantitative in vitro reconstitution with defined components, mechanistic dissection of kcat\",\n      \"pmids\": [\"9454578\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The C-terminus of SELB is the primary interaction domain for mRNA hairpin binding; the vast majority of SELEX-derived aptamers from a random pool bound specifically to the C-terminal domain of SELB.\",\n      \"method\": \"In vitro SELEX with full-length and truncated SELB, domain mapping\",\n      \"journal\": \"RNA (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — SELEX domain mapping, single lab, corroborates earlier deletion data\",\n      \"pmids\": [\"10496219\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"SelB GTP affinity (Kd = 0.74 µM) and GDP affinity (Kd = 13.4 µM) are much lower than other translation factors; GDP release is very rapid (15 s⁻¹), explaining why no GDP/GTP exchange factor is needed. The SECIS RNA minihelix binds with Kd ~1 nM, affinity increasing further when Sec-tRNA(Sec) is bound to SelB.\",\n      \"method\": \"Stopped-flow kinetics using intrinsic tryptophan fluorescence and fluorescent nucleotide analogs; fluorescence resonance energy transfer\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — rigorous quantitative kinetics with multiple orthogonal fluorescence methods\",\n      \"pmids\": [\"10781605\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The archaeal SelB homolog (MJ0495/aSelB) from Methanococcus jannaschii binds guanine nucleotides and preferentially binds selenocysteyl-tRNA(Sec), but unlike bacterial SelB, does not bind the SECIS element, correlating with its lack of the bacterial C-terminal mRNA-binding domain.\",\n      \"method\": \"Purification of recombinant MJ0495, GTP binding assay, tRNA binding assay, SECIS binding assay\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple in vitro binding assays on purified archaeal protein, negative SECIS binding result mechanistically informative\",\n      \"pmids\": [\"10860743\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Crystal structure of the C-terminal mRNA-binding fragment of Moorella thermoacetica SelB (SelB-C) at 2.12 Å reveals four tandem winged-helix (WH) domains arranged in an L-shape; this is the first example of WH domains in RNA binding, with conserved basic residues defining the mRNA-binding site.\",\n      \"method\": \"X-ray crystallography with multiwavelength anomalous dispersion phasing\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure at 2.12 Å with functional mapping of binding residues\",\n      \"pmids\": [\"12145214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"NMR structure of the prokaryotic SECIS mRNA hairpin shows conserved nucleotides exposed for SelB recognition; binding of the SelB C-terminal domain stabilizes RNA secondary structure; a GpU sequence at the tetraloop tip and a bulge uracil 5 base pairs away are essential for SelB interaction.\",\n      \"method\": \"NMR spectroscopy, mutagenesis of RNA, SelB binding assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure combined with mutagenesis and quantitative binding data\",\n      \"pmids\": [\"12421564\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Inactivation of selB in Methanococcus maripaludis abolishes synthesis of all selenoproteins, proving aSelB is the archaeal translation factor specialized for selenocysteine insertion; the mutation also derepresses expression of cysteine-containing homologs and impairs growth on formate.\",\n      \"method\": \"Targeted gene knockout, metabolic labeling, proteomics\",\n      \"journal\": \"Journal of bacteriology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic knockout with defined phenotypes, multiple selenoproteins tested\",\n      \"pmids\": [\"12486046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Crystal structure of the SelB mRNA-binding domain in complex with SECIS RNA at 2.3 Å reveals the first RNA-binding winged-helix (WH) domain; RNA recognition occurs without major conformational change in the WH motif, and the geometry allows the complex to wrap around the small ribosomal subunit.\",\n      \"method\": \"X-ray crystallography\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure of protein-RNA complex at 2.3 Å resolution\",\n      \"pmids\": [\"15665870\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Crystal structures of E. coli WH3/4 domains and M. thermoacetica WH1-4 domains each bound to SECIS RNA reveal that both WH motifs use the same structural elements for RNA binding; a salt bridge connecting WH2 to WH3 is disrupted upon mRNA binding, providing a molecular switch that may communicate between tRNA and mRNA binding sites.\",\n      \"method\": \"X-ray crystallography at 2.3 Å (E. coli WH3/4) and 2.6 Å (M. thermoacetica WH1-4), mutagenesis\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — two crystal structures with mutagenesis supporting molecular switch mechanism\",\n      \"pmids\": [\"17537456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Crystal structure of SelB C-terminal domain complexed with RNA reveals large domain rearrangement; an interdomain region forms new interactions with phosphate backbone of a neighboring RNA in a sequence-independent manner, potentially reflecting SelB interactions with tRNA or rRNA during ribosome engagement.\",\n      \"method\": \"X-ray crystallography\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — crystal structure observation but functional consequence of sequence-independent binding is inferred, not directly tested\",\n      \"pmids\": [\"17502103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Sec-tRNA(Sec) binds SelB·GTP with extraordinarily high affinity (Kd = 0.2 pM); binding is enthalpically driven with ~4 ion pairs. GTP hydrolysis accelerates release of Sec-tRNA(Sec) by >10⁶-fold (from 0.3 h⁻¹ to 240 s⁻¹). Ser-tRNA(Sec) and uncharged tRNA(Sec) bind with much lower affinity (~0.5 µM) to any nucleotide-bound form.\",\n      \"method\": \"Thermodynamic and kinetic stopped-flow measurements, fluorescent tRNA analogs\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — rigorous quantitative thermodynamic and kinetic analysis with multiple orthogonal measurements\",\n      \"pmids\": [\"19940162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Crystal structure of full-length bacterial SelB from Aquifex aeolicus in complex with GTP analog at 3.2 Å shows three EF-Tu-like domains (D1-3) followed by four WH domains; the Sec-binding site is located at the D1-D2 interface with Arg residues coordinating the Sec moiety, and is smaller and more exposed than EF-Tu's aminoacyl binding site.\",\n      \"method\": \"X-ray crystallography at 3.2 Å\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure of full-length protein with GTP analog, structural analysis of Sec-binding site\",\n      \"pmids\": [\"26304550\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Cryo-EM structures of six intermediates on the UGA recoding pathway reveal that: initial SelB-Sec-tRNA(Sec) binding induces 30S open conformation with Sec-tRNA(Sec) covering the sarcin-ricin loop (SRL); codon recognition in the decoding site causes local closure moving Sec-tRNA(Sec) away from the SRL and global 30S shoulder closure; this triggers SelB docking on the SRL, activating its GTPase.\",\n      \"method\": \"Single-particle cryo-electron microscopy, six structural intermediates\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structures of six pathway intermediates revealing mechanistic cascade, published in Nature\",\n      \"pmids\": [\"27842381\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Mammalian eEFSec folds into a chalice-like structure with three N-terminal EF-Tu-like domains and a C-terminal domain 4 that binds Sec-tRNA(Sec) and SBP2; GTP hydrolysis does not induce a canonical conformational change but promotes a slight ratchet of domains 1-2 and a lever-like movement of domain 4, which may be critical for Sec-tRNA(Sec) release on the ribosome.\",\n      \"method\": \"Structural analysis, biochemical characterization of domain functions (summary/review of published experimental work)\",\n      \"journal\": \"Biochimica et biophysica acta. General subjects\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — review of experimental structural data, but this is a review paper; the original crystal structure data it summarizes is the primary evidence\",\n      \"pmids\": [\"29555379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Bi-allelic loss-of-function variants in EEFSEC cause reduced EEFSEC function in vitro, leading to lower selenoprotein levels in patient fibroblasts; an eEFSec-RNAi Drosophila model shows progressive motor impairment and synaptic defects, establishing that EEFSEC function is required for selenoprotein synthesis and neuronal maintenance in vivo.\",\n      \"method\": \"Patient fibroblast selenoprotein quantification, in vitro functional assays of variants, Drosophila RNAi model with motor and synaptic phenotyping\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function validated in patient cells and animal model with defined molecular and cellular phenotypes, multiple families and variants\",\n      \"pmids\": [\"39753114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The bulged nucleotide U17 in the E. coli SECIS upper stem-loop participates in interaction with SelB; suppressor mutations in selB that rescue SECIS bulge mutations map to a 28-amino acid stretch in SelB C-terminal subdomain 4b, identifying this region as the contact interface.\",\n      \"method\": \"Genetic suppressor analysis, in vivo UGA readthrough assay, selB sequencing\",\n      \"journal\": \"Journal of bacteriology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with allele-specific suppression mapping contact sites, single lab\",\n      \"pmids\": [\"11053373\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Genetic analysis of SelB-SECIS interaction identified suppressor mutations in selB that counteract SECIS mutations; four suppressor amino acid exchanges cluster in a 23-amino acid stretch in domain 4b (direct RNA contact sites), and a fifth mutation in domain 4a promotes allele-nonspecific readthrough, suggesting domain 4a modulates communication between tRNA- and mRNA-binding regions.\",\n      \"method\": \"In vivo suppressor selection, UGA readthrough assay, selenium incorporation, sequencing\",\n      \"journal\": \"Molecular & general genetics : MGG\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with allele-specific suppression, single lab\",\n      \"pmids\": [\"10628863\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FOXO1 and STAT3 transcription factors directly bind functional sites in the eefsec promoter (-1070 bp FOXO1 site; -428 bp STAT3 site) and regulate eefsec transcriptional activity in a selenium-dependent manner in yellow catfish.\",\n      \"method\": \"Sequential promoter deletion analysis, EMSA, chromatin immunoprecipitation\",\n      \"journal\": \"Biochimica et biophysica acta. Gene regulatory mechanisms\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — EMSA and ChIP confirm direct binding, but single lab in a fish model\",\n      \"pmids\": [\"40618995\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EEFSEC (eEFSec/SELB) is a specialized GTPase translation elongation factor that delivers selenocysteinyl-tRNA(Sec) to the ribosomal A site at UGA codons: its three N-terminal EF-Tu-like domains bind GTP and Sec-tRNA(Sec) with extraordinarily high affinity (Kd ~0.2 pM in bacteria), while its C-terminal winged-helix domains recognize the downstream SECIS mRNA hairpin; codon recognition at the ribosome triggers closure of the 30S subunit, docking of SelB onto the sarcin-ricin loop, GTPase activation, and GTP hydrolysis-driven release of Sec-tRNA(Sec), with loss-of-function in humans causing selenoprotein deficiency and progressive neurodegeneration.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"EEFSEC (eEFSec/SELB) is a specialized GTPase translation elongation factor dedicated to selenocysteine incorporation, delivering selenocysteinyl-tRNA(Sec) to the ribosomal A site for decoding of in-frame UGA codons [#0, #3]. It was the first elongation factor shown to recognize a single charged tRNA species: it binds Sec-tRNA(Sec) with extraordinary affinity (Kd ~0.2 pM) in a complex requiring contact with the selenol group of the aminoacylated residue, while the 8-bp acceptor stem of tRNA(Sec) is the determinant that selects this tRNA and excludes it from EF-Tu [#1, #2, #19]. Architecturally, the protein combines three N-terminal EF-Tu-like GTP/tRNA-binding domains with C-terminal tandem winged-helix domains that constitute a separable mRNA-binding module recognizing the SECIS hairpin located 3' of the UGA codon, producing a quaternary SELB·GTP·Sec-tRNA(Sec)·mRNA complex that positions the charged tRNA at the recoding codon [#3, #5, #13, #20]. Unlike canonical elongation factors, SELB has low GTP/GDP affinity and very rapid GDP release, dispensing with a dedicated nucleotide-exchange factor; SECIS binding and ribosome engagement stimulate its GTPase activity, and GTP hydrolysis accelerates Sec-tRNA(Sec) release by more than a million-fold to drive insertion [#9, #11, #19]. Cryo-EM of recoding intermediates shows that initial binding holds the 30S in an open state with Sec-tRNA(Sec) over the sarcin-ricin loop, and codon recognition triggers 30S closure, SELB docking on the sarcin-ricin loop, and GTPase activation [#21]. Genetic ablation of the archaeal ortholog abolishes synthesis of all selenoproteins, confirming its essential, pathway-defining role [#15], and bi-allelic loss-of-function variants in human EEFSEC reduce selenoprotein levels in patient fibroblasts and cause progressive motor and synaptic defects in a Drosophila model, linking the factor to selenoprotein deficiency and neurodegeneration [#23].\",\n  \"teleology\": [\n    {\n      \"year\": 1990,\n      \"claim\": \"Established that selenocysteine insertion requires a dedicated elongation factor distinct from EF-Tu, answering whether Sec delivery uses the canonical translation machinery.\",\n      \"evidence\": \"Protein purification and ribosome fractionation of bacterial SELB binding selenocysteyl-tRNA(Sec)\",\n      \"pmids\": [\"2140572\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the structural basis for tRNA(Sec) discrimination\", \"Mechanism of UGA codon recognition unresolved\"]\n    },\n    {\n      \"year\": 1991,\n      \"claim\": \"Identified the tRNA features that route Sec-tRNA(Sec) to SELB and away from EF-Tu, explaining specificity at the level of the charged tRNA.\",\n      \"evidence\": \"In vitro binding with tRNA(Sec) stem mutants plus in vivo selenoprotein assay; biochemical characterization of selenol-group dependence\",\n      \"pmids\": [\"1939093\", \"1839607\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not localize the tRNA-contact residues on SELB\", \"Did not address mRNA recognition\"]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Showed SELB directly reads the SECIS mRNA hairpin and forms a quaternary complex, establishing how Sec delivery is targeted to specific codons.\",\n      \"evidence\": \"Gel shift, RNA footprinting, and quaternary complex reconstitution on fdhF/fdnG mRNAs; toeprinting on ribosome-bound mRNA\",\n      \"pmids\": [\"8483932\", \"8314089\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not separate tRNA- and mRNA-binding functions structurally\", \"Did not define SECIS nucleotide contacts\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Mapped the mRNA-binding function to a separable C-terminal domain and defined the SECIS nucleotides contacted, dissecting the bifunctional architecture.\",\n      \"evidence\": \"Domain truncation with RNA-binding assays, chemical probing/NMR with quantitative binding, in vivo competition and stoichiometry experiments\",\n      \"pmids\": [\"8893853\", \"8634916\", \"8898393\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic structure of the domain still unknown\", \"Did not explain how mRNA binding affects GTPase\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Connected SECIS binding to catalytic activation, showing the mRNA element conformationally licenses GTP hydrolysis rather than merely tethering the factor.\",\n      \"evidence\": \"In vitro GTPase kinetics with ribosomes and SECIS hairpin (kcat analysis); SELEX plus in vivo readthrough indicating SECIS roles beyond tethering\",\n      \"pmids\": [\"9454578\", \"9192624\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the conformational switch not yet resolved\", \"Coupling to codon recognition unknown\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Quantified the unusual nucleotide and RNA binding properties, explaining why SELB needs no exchange factor and how cooperative RNA binding occurs.\",\n      \"evidence\": \"Stopped-flow kinetics and FRET; archaeal ortholog binding assays; genetic suppressor mapping of SECIS contact residues\",\n      \"pmids\": [\"10781605\", \"10860743\", \"11053373\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not capture full-length structure\", \"Pico-molar tRNA affinity and its release mechanism not yet measured\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Determined the winged-helix fold and its RNA-recognition mode, revealing the first WH domain used for RNA binding and how the complex could wrap the small subunit.\",\n      \"evidence\": \"X-ray crystallography of SelB C-terminal domain alone and bound to SECIS RNA; further WH structures with molecular-switch mutagenesis\",\n      \"pmids\": [\"12145214\", \"15665870\", \"17537456\", \"12421564\", \"17502103\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not show coupling to GTPase domains in full-length context\", \"Functional consequence of sequence-independent backbone contacts inferred only\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined the thermodynamic logic of Sec-tRNA(Sec) selection and GTP-driven release, showing hydrolysis is the switch for releasing the charged tRNA.\",\n      \"evidence\": \"Thermodynamic and stopped-flow kinetic measurements with fluorescent tRNA analogs\",\n      \"pmids\": [\"19940162\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not place these steps in the ribosomal recoding pathway\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Visualized the full UGA recoding cascade, establishing how codon recognition mechanically triggers SELB GTPase activation on the ribosome.\",\n      \"evidence\": \"Single-particle cryo-EM of six recoding intermediates; full-length crystal structure with GTP analog defining the Sec-binding site\",\n      \"pmids\": [\"27842381\", \"26304550\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mammalian eEFSec-specific conformational details only partly resolved\", \"Role of SBP2 in human system not structurally captured here\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established the physiological and disease relevance of human EEFSEC, linking loss of function to selenoprotein deficiency and neurodegeneration.\",\n      \"evidence\": \"Patient fibroblast selenoprotein quantification, in vitro variant assays, Drosophila RNAi motor/synaptic phenotyping; promoter analysis of transcriptional control by FOXO1/STAT3 in fish\",\n      \"pmids\": [\"39753114\", \"40618995\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which selenoproteins drive neuronal phenotype not defined\", \"Human transcriptional regulation not directly tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the human eEFSec·SBP2 system and SECIS recognition differ mechanistically from the bacterial SELB single-protein paradigm, and which selenoproteins mediate neuronal maintenance, remain to be resolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No ribosome-bound structure of the mammalian eEFSec·SBP2 complex in the corpus\", \"Tissue-specific selenoprotein requirements unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003924\", \"supporting_discovery_ids\": [9, 11, 19, 21]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [3, 5, 6, 13, 14, 16]},\n      {\"term_id\": \"GO:0045182\", \"supporting_discovery_ids\": [0, 1, 19, 23]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [26]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005840\", \"supporting_discovery_ids\": [0, 4, 21]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-72766\", \"supporting_discovery_ids\": [0, 3, 23]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 15, 23]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"SBP2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}