Affinage

RBFA

Putative ribosome-binding factor A, mitochondrial · UniProt Q8N0V3

Round 2 corrected
Length
343 aa
Mass
38.4 kDa
Annotated
2026-04-28
50 papers in source corpus 18 papers cited in narrative 18 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

RBFA is a KH-domain RNA-binding assembly factor that associates with the small ribosomal subunit during biogenesis—the bacterial 30S or the human mitoribosomal small subunit—and promotes maturation of the decoding center by chaperoning rRNA conformational changes at two sequential stages: central pseudoknot formation (including head-domain stabilization via helix 28) and positioning of helix 44 at the decoding site (PMID:34200244, PMID:17996707). In bacteria, RbfA facilitates 5′-end processing of 16S rRNA and suppresses premature entry of immature subunits into translation; it is released in a GTP-dependent manner by the GTPase RsgA/YjeQ upon helix-44 maturation and is then displaced by initiation factor IF3 (PMID:21102555, PMID:31728529). In human mitochondria, RBFA binds helices 28, 44, and 45 of 12S rRNA through its extended N-terminus, promotes TFB1M-mediated dimethylation of two conserved adenines required for monosome formation, and is ultimately outcompeted by mS37 to complete assembly and license mtIF3-dependent translation initiation (PMID:28512204, PMID:35676484, PMID:36620886).

Mechanistic history

Synthesis pass · year-by-year structured walk · 11 steps
  1. 1995 High

    Establishing RbfA as a 30S-specific ribosome assembly factor resolved how the cold-sensitive C23U 16S rRNA mutation could be suppressed: a dedicated protein facilitates a late step of small-subunit maturation involving the 5′ region of 16S rRNA.

    Evidence Genetic suppressor screen, ribosome co-sedimentation, and knockout phenotype analysis in E. coli

    PMID:7535280

    Open questions at the time
    • Precise binding site on 30S unknown
    • Molecular mechanism of suppression unresolved
    • No structural data for the protein
  2. 1998 High

    Showing that RbfA and RimM have overlapping roles in 16S rRNA processing, and that RbfA overexpression suppresses rimM deletion defects, established a network of partially redundant assembly factors converging on 30S maturation.

    Evidence Genetic suppression, rRNA processing assays, ribosome fractionation in E. coli

    PMID:8898389 PMID:9422595

    Open questions at the time
    • Physical contacts between RbfA and rRNA not mapped
    • Relationship to Era GTPase unclear
  3. 2003 High

    Determination of the NMR structure of E. coli RbfA revealed a type-II KH-domain fold and identified the RNA-binding surface, while domain-deletion and Era-suppression experiments defined the C-terminus as essential for stable 30S binding and placed RbfA in a functional pathway with Era.

    Evidence NMR structure, C-terminal deletion analysis, Era overexpression suppression, polysome profiling

    PMID:12628255 PMID:12753192 PMID:12963368

    Open questions at the time
    • No high-resolution structure of RbfA bound to 30S
    • Mechanism of C-terminus function at the structural level unknown
  4. 2007 High

    The crystal structure of T. thermophilus RbfA and cryo-EM of the 30S·RbfA complex revealed that RbfA overlaps the A/P-site region and displaces helix 44, directly explaining how it facilitates 5′-end processing and prevents premature translation on immature subunits.

    Evidence X-ray crystallography and cryo-EM 3D reconstruction of 30S·RbfA

    PMID:17996707

    Open questions at the time
    • No atomic-resolution model of the complex
    • Mechanism of RbfA release unknown
  5. 2010 High

    Demonstrating that the GTPase RsgA catalyzes GTP-dependent release of RbfA from mature 30S subunits answered how the cell distinguishes mature from immature small subunits and terminates the assembly-factor phase.

    Evidence Gain-of-function rbfA suppressors of rsgA-null, in vitro GTPase-dependent release assay

    PMID:21102555

    Open questions at the time
    • Structural basis of RsgA-mediated RbfA displacement unknown
    • Whether additional signals regulate release unclear
  6. 2015 High

    Identification of the YjeQ zinc-finger α-helix as the helix-44 conformational sensor that triggers GTP hydrolysis and RbfA release, together with genetic mapping of RbfA's role to central pseudoknot formation via S5 suppressor mutations, defined the two structural checkpoints (pseudoknot and h44) that RbfA gates.

    Evidence YjeQ domain mutagenesis with GTPase/release assays; S5 suppressor screen with polysome profiling

    PMID:25904134 PMID:26089326

    Open questions at the time
    • Direct visualization of pseudoknot-stage intermediates lacking
    • Relationship between S5 mutations and RbfA binding site not structurally resolved
  7. 2017 High

    Discovery that human mitochondrial RBFA promotes TFB1M-mediated dimethylation of two conserved adenines in 12S rRNA and is required for monosome formation extended the RbfA paradigm to mitoribosome biogenesis and revealed a shifted functional output (rRNA modification rather than 5′-end processing).

    Evidence siRNA knockdown, co-immunoprecipitation, rRNA modification analysis, sucrose gradient sedimentation in human cells

    PMID:28512204

    Open questions at the time
    • Structural basis of RBFA–TFB1M cooperation unknown
    • Whether RBFA has additional mitoribosomal partners unclear
  8. 2020 High

    Genetic epistasis between RbfA and IF3 established that RbfA gates the transition from biogenesis to translation initiation by suppressing premature translational engagement of immature 30S subunits, with IF3 displacing RbfA on mature particles.

    Evidence rbfA-IF3 double mutants under multiple stress conditions, translation initiation assays in E. coli

    PMID:31728529

    Open questions at the time
    • Direct binding competition between RbfA and IF3 not reconstituted in vitro
    • Stress-specific regulation of the handoff not mechanistically resolved
  9. 2021 High

    High-resolution cryo-EM of pre-30S intermediates from ΔrbfA E. coli captured six sequential assembly states, demonstrating that RbfA acts at two distinct stages—early central pseudoknot/head-domain folding and later helix-44 docking—providing the most complete structural view of RbfA's biogenesis role.

    Evidence Cryo-EM at ~2.7 Å of multiple assembly intermediates from ΔrbfA strain

    PMID:34200244

    Open questions at the time
    • Structures are of ΔrbfA intermediates; RbfA-bound transition states not yet captured at atomic resolution
  10. 2022 High

    Cryo-EM of native human mitoribosomal SSU assembly intermediates revealed the ordered sequence: RBFA promotes h45 unfolding for TFB1M binding → METTL15 triggers RBFA conformational change → mtIF3 occupies the subunit interface → mS37 outcompetes RBFA, completing assembly and coupling maturation to translation initiation in mitochondria.

    Evidence Cryo-EM of native mitoribosome assembly intermediates (multiple states) in human cells

    PMID:35676484

    Open questions at the time
    • Kinetics of each transition not measured
    • Regulation of mS37 competition with RBFA not understood
  11. 2023 High

    Mapping the RNA-binding determinants of human RBFA to the entire N-terminus (beyond the KH domain alone) and solving additional bacterial structures confirmed a conserved structural mechanism while highlighting sequence/structural divergences that account for the distinct rRNA-modification-promoting role of the mitochondrial orthologue.

    Evidence In vitro RNA-binding/mutagenesis, mitochondrial function assays, X-ray/NMR/cryo-EM of S. aureus RbfA and NMR of human mtRbfA KH domain

    PMID:35666428 PMID:36620886 PMID:36768442

    Open questions at the time
    • Atomic-resolution structure of human RBFA bound to the mitoribosomal SSU not yet available
    • Contribution of N-terminal regions outside the KH domain to in vivo function not fully dissected

Open questions

Synthesis pass · forward-looking unresolved questions
  • Key unresolved questions include the atomic-resolution structure of RBFA on a native human mitoribosomal assembly intermediate, the precise signal that triggers mS37-mediated RBFA displacement, and whether RBFA dysfunction contributes to human mitochondrial disease.
  • No disease-associated mutations reported
  • No in vivo kinetic measurements of RBFA dynamics on assembling mitoribosomes
  • Potential regulatory inputs (post-translational modifications, metabolic signals) unexplored

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0003723 RNA binding 6
Localization
GO:0005840 ribosome 7 GO:0005739 mitochondrion 4
Pathway
R-HSA-392499 Metabolism of proteins 5 R-HSA-8953854 Metabolism of RNA 5 R-HSA-1852241 Organelle biogenesis and maintenance 3
Partners
ERAIF3KSGAMETTL15MS37RIMMRSGATFB1M
Complex memberships
30S ribosomal subunit assembly intermediatemitoribosomal small subunit (mt-SSU) assembly intermediate

Evidence

Reading pass · 18 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1995 RbfA (15 kDa) was identified as a high-copy suppressor of the cold-sensitive C23U mutation in 16S rRNA and found to associate specifically with free 30S ribosomal subunits but not with 70S ribosomes or polysomes; loss of RbfA produces a cold-sensitive phenotype, and allele-specificity patterns suggest RbfA interacts with the 5'-terminal helix region of 16S rRNA during a late step of 30S maturation. Genetic suppressor screen, co-sedimentation/ribosome fractionation, knockout phenotypic analysis Genes & development High 7535280
1996 RbfA is a cold-shock protein whose absence constitutively induces the cold-shock response; overproduction of RbfA accelerates cold adaptation and increases total protein synthesis, indicating RbfA converts cold-unadapted non-translatable ribosomes to cold-adapted translatable ribosomes and that its loss triggers a signal mimicking non-translatable ribosomes. rbfA deletion and overexpression strains, polysome profiling, cold-shift experiments Molecular microbiology High 8898389
1998 RbfA (and RimM) are essential for efficient processing of 16S rRNA in E. coli; overexpression of RbfA suppresses slow growth and translational deficiency caused by deletion of rimM, and both proteins associate with free 30S subunits but not 70S ribosomes. Genetic suppression, ribosome fractionation, rRNA processing assays Journal of bacteriology High 9422595
2003 Solution NMR structure of RbfA (E. coli) reveals a type-II KH-domain fold (α1-β1-β2-α2-α3-β3) with a bipolar electrostatic surface; a dynamic hot spot near Ser39 and a positive electrostatic face around the α3-loop-β3 region are identified as the putative RNA-binding site interacting with 16S rRNA. Heteronuclear NMR structure determination, 15N relaxation measurements Journal of molecular biology High 12628255
2003 RbfA's C-terminal 25 residues are required for stable association with 30S subunits and for suppression of the C23U cold-sensitive phenotype; deletion of the C-terminus (RbfAΔ25) abolishes stable 30S binding but retains ability to suppress 16S rRNA processing defects, suggesting RbfA interacts with the 30S ribosome at more than one site or acts via multiple mechanisms in 16S rRNA maturation. C-terminal deletion analysis, ribosome fractionation, cold-sensitivity suppression assays, polysome profiling Journal of molecular biology High 12963368
2003 Overexpression of Era (a GTPase with a C-terminal KH domain structurally similar to RbfA) suppresses both the cold-sensitive growth and the defective 16S rRNA maturation (17S rRNA accumulation) of rbfA-deletion cells, restoring normal polysome profiles; Era mutant E200K causes the same pre-16S rRNA accumulation phenotype as rbfA deletion, indicating overlapping functions in 30S maturation. Genetic suppression, polysome profiling, rRNA processing analysis, inducible expression systems Molecular microbiology High 12753192
2007 Crystal structure of Thermus thermophilus RbfA and cryo-EM map of the 30S·RbfA complex show that RbfA binds at the 30S subunit overlapping the A- and P-site tRNA binding sites, with its functionally important C-terminus extending toward the 5' end of 16S rRNA; RbfA binding displaces helix 44 of 16S rRNA (involved in mRNA decoding and tRNA binding), explaining how RbfA facilitates 5'-end processing during 30S maturation. X-ray crystallography, cryo-electron microscopy, 3D reconstruction of 30S·RbfA complex Molecular cell High 17996707
2010 RsgA (YjeQ), a 30S-binding GTPase, releases RbfA from the mature 30S subunit in a GTP-dependent manner; gain-of-function rbfA mutations that promote spontaneous RbfA release suppress growth and maturation defects of an rsgA-null strain, demonstrating that RsgA's function is to catalyze RbfA release during a late stage of ribosome biosynthesis. Genetic suppressor screen, gain-of-function mutagenesis, in vitro GTPase-dependent release assay, ribosome maturation analysis The EMBO journal High 21102555
2013 When RbfA is overexpressed in the absence of the KsgA methyltransferase checkpoint, KsgA becomes essential for incorporation of ribosomal protein S21 into the developing small subunit and for final rRNA maturation; imbalance between KsgA and RbfA leads to accumulation of aberrant 70S-like particles that are compositionally and functionally distinct from mature 70S ribosomes, indicating KsgA and RbfA act together in sequential SSU maturation steps. Genetic analysis (KsgA knockout + RbfA overexpression), ribosome profiling, sucrose gradient sedimentation, rRNA maturation assays Molecular microbiology High 23387871
2015 The C-terminal α-helix of YjeQ's zinc-finger domain senses the mature conformation of helix 44 in the 30S subunit decoding center and catalyzes removal of RbfA; this helix is also required for mature-30S-stimulated YjeQ GTPase activity, supporting a model where conformational sensing of helix 44 triggers GTP hydrolysis and RbfA release. Domain mutagenesis, GTPase activity assays, RbfA release assays, 30S binding assays RNA (New York, N.Y.) High 25904134
2015 Suppressor mutations in the C-terminal domain of ribosomal protein S5 (at positions contacting helix 1 and helix 2 of the central pseudoknot) restore growth and increase translational capacity (polysome formation) in RbfA-lacking strains; overexpression of RimP is lethal to RbfA-lacking strains but suppressed by S5 mutations, placing RbfA function in central pseudoknot formation during 30S maturation. Genetic suppressor screen, polysome profiling, epistasis analysis RNA (New York, N.Y.) Medium 26089326
2017 Human RBFA is a mitochondrial RNA-binding protein that associates mainly with helices 44 and 45 of 12S rRNA in the mitoribosomal small subunit; RBFA promotes dimethylation of two conserved consecutive adenines in 12S rRNA by TFB1M. This modification is not required for small subunit assembly per se but is necessary for completing mt-rRNA maturation and for regulating association of the small and large subunits to form a functional monosome, implicating RBFA in quality control of mitoribosome formation. RBFA depletion (siRNA), co-immunoprecipitation, rRNA modification analysis, mitoribosome assembly/sucrose gradient sedimentation The Biochemical journal High 28512204
2020 RbfA suppresses protein synthesis by immature E. coli 30S subunits (gating step between biogenesis and translation initiation); after 30S maturation, RbfA is displaced by initiation factor IF3, which promotes translation initiation. Genetic interactions between RbfA and IF3 are important during logarithmic growth and during stress (stationary phase, low nutrition, cold, antibiotics). Genetic epistasis (rbfA-IF3 double mutants), translation initiation assays, stress phenotype analysis Nucleic acids research High 31728529
2021 Cryo-EM structures of pre-30S particles from an E. coli rbfA-deletion strain reveal six sequential assembly intermediates; RbfA acts at two distinct stages: early formation of the central pseudoknot (including head domain folding) and later positioning of helix 44 in the decoding center, with central pseudoknot formation also promoting stabilization of the head domain via RbfA-dependent maturation of neck helix 28. Cryo-electron microscopy (2.7 Å resolution structures of predominant intermediates), structural classification of assembly intermediates International journal of molecular sciences High 34200244
2022 Cryo-EM of human mitoribosomal small subunit (SSU) assembly intermediates shows that methyltransferase TFB1M binds to partially unfolded rRNA h45 promoted by RBFA while the mRNA channel is blocked; METTL15 binding then promotes further rRNA maturation and a large conformational change of RBFA, allowing initiation factor mtIF3 to occupy the subunit interface during assembly; finally, mitochondria-specific protein mS37 outcompetes RBFA to complete assembly, linking SSU maturation to translation initiation. Cryo-electron microscopy of native mitoribosome assembly intermediates, structural analysis of factor binding states Nature High 35676484
2023 Human RBFA (hsRBFA) binds double-stranded RNA through its entire N-terminus (not just the KH-like domain alone), interacting with helices 28, 44, and 45 of 12S rRNA; key residues in the N-terminus were mapped and shown to affect both RNA binding and mitoribosome maturation in vitro, with disruption of these residues impairing mitochondrial function. In vitro RNA binding assays, site-directed mutagenesis, mitoribosome maturation assays, mitochondrial function measurements Nucleic acids research High 36620886
2023 Crystal structure (2.2 Å) and NMR structure of RbfA from S. aureus, plus a 2.9 Å cryo-EM reconstruction of the S. aureus 30S·RbfA complex, confirm conserved KH-domain fold and 30S binding mode; the manner of RbfA action on the small ribosomal subunit during maturation is shared between bacteria and mitochondria. X-ray crystallography, NMR spectroscopy, cryo-electron microscopy International journal of molecular sciences High 36768442
2022 NMR solution structure of the KH domain of human mtRbfA reveals the same α1-β1-β2-α2(kinked)-β3 type-II KH topology as bacterial RbfA; structural differences in the putative RNA-binding regions (α2 helix kink geometry and the α1-β1 linking region) compared with bacterial RbfA suggest variations in the RNA-binding mode that underlie the distinct functional role of mtRbfA in promoting 12S rRNA dimethylation rather than 5'-end processing. NMR resonance assignment and solution structure determination Biomolecular NMR assignments Medium 35666428

Source papers

Stage 0 corpus · 50 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2002 Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences. Proceedings of the National Academy of Sciences of the United States of America 1479 12477932
2010 Network organization of the human autophagy system. Nature 1286 20562859
2015 The BioPlex Network: A Systematic Exploration of the Human Interactome. Cell 1118 26186194
2017 Architecture of the human interactome defines protein communities and disease networks. Nature 1085 28514442
2014 A proteome-scale map of the human interactome network. Cell 977 25416956
2020 A reference map of the human binary protein interactome. Nature 849 32296183
2003 Complete sequencing and characterization of 21,243 full-length human cDNAs. Nature genetics 754 14702039
2021 Dual proteome-scale networks reveal cell-specific remodeling of the human interactome. Cell 705 33961781
2004 The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC). Genome research 438 15489334
2021 A proximity-dependent biotinylation map of a human cell. Nature 339 34079125
2021 Quantitative high-confidence human mitochondrial proteome and its dynamics in cellular context. Cell metabolism 239 34800366
2018 Mapping the Genetic Landscape of Human Cells. Cell 225 30033366
2007 hORFeome v3.1: a resource of human open reading frames representing over 10,000 human genes. Genomics 222 17207965
1996 RbfA, a 30S ribosomal binding factor, is a cold-shock protein whose absence triggers the cold-shock response. Molecular microbiology 160 8898389
2020 A High-Density Human Mitochondrial Proximity Interaction Network. Cell metabolism 148 32877691
1995 Suppression of a cold-sensitive mutation in 16S rRNA by overexpression of a novel ribosome-binding factor, RbfA. Genes & development 132 7535280
1998 RimM and RbfA are essential for efficient processing of 16S rRNA in Escherichia coli. Journal of bacteriology 108 9422595
2003 Suppression of defective ribosome assembly in a rbfA deletion mutant by overexpression of Era, an essential GTPase in Escherichia coli. Molecular microbiology 99 12753192
2017 Cell cycle-dependent phosphorylation regulates RECQL4 pathway choice and ubiquitination in DNA double-strand break repair. Nature communications 89 29229926
2013 Interaction proteome of human Hippo signaling: modular control of the co-activator YAP1. Molecular systems biology 84 24366813
2007 Structural aspects of RbfA action during small ribosomal subunit assembly. Molecular cell 77 17996707
2022 Mechanism of mitoribosomal small subunit biogenesis and preinitiation. Nature 75 35676484
2003 The role of RbfA in 16S rRNA processing and cell growth at low temperature in Escherichia coli. Journal of molecular biology 67 12963368
2003 Solution NMR structure of ribosome-binding factor A (RbfA), a cold-shock adaptation protein from Escherichia coli. Journal of molecular biology 66 12628255
2010 RsgA releases RbfA from 30S ribosome during a late stage of ribosome biosynthesis. The EMBO journal 50 21102555
2013 RBF1, a plant homolog of the bacterial ribosome-binding factor RbfA, acts in processing of the chloroplast 16S ribosomal RNA. Plant physiology 43 24214533
2018 ATG5 is required for B cell polarization and presentation of particulate antigens. Autophagy 42 30196744
2022 In-Depth In Vivo Crosslinking in Minutes by a Compact, Membrane-Permeable, and Alkynyl-Enrichable Crosslinker. Analytical chemistry 38 35575683
2017 The human RNA-binding protein RBFA promotes the maturation of the mitochondrial ribosome. The Biochemical journal 38 28512204
2020 RbfA and IF3 couple ribosome biogenesis and translation initiation to increase stress tolerance. Nucleic acids research 37 31728529
2019 The RNA-mediated estrogen receptor α interactome of hormone-dependent human breast cancer cell nuclei. Scientific data 32 31527615
2022 UBE2O promotes lipid metabolic reprogramming and liver cancer progression by mediating HADHA ubiquitination. Oncogene 27 36273042
2013 Overexpression of RbfA in the absence of the KsgA checkpoint results in impaired translation initiation. Molecular microbiology 27 23387871
2006 Era and RbfA have overlapping function in ribosome biogenesis in Escherichia coli. Journal of molecular microbiology and biotechnology 27 16825789
2021 RbfA Is Involved in Two Important Stages of 30S Subunit Assembly: Formation of the Central Pseudoknot and Docking of Helix 44 to the Decoding Center. International journal of molecular sciences 24 34200244
2023 Multisite phosphorylation dictates selective E2-E3 pairing as revealed by Ubc8/UBE2H-GID/CTLH assemblies. Molecular cell 21 38113892
2020 Wnt regulation: exploring Axin-Disheveled interactions and defining mechanisms by which the SCF E3 ubiquitin ligase is recruited to the destruction complex. Molecular biology of the cell 19 32129710
2018 Identification of proteins interacting with the mitochondrial small heat shock protein Hsp22 of Drosophila melanogaster: Implication in mitochondrial homeostasis. PloS one 13 29509794
2021 Temporal modulation of the NF-κB RelA network in response to different types of DNA damage. The Biochemical journal 12 33438746
2015 The C-terminal helix in the YjeQ zinc-finger domain catalyzes the release of RbfA during 30S ribosome subunit assembly. RNA (New York, N.Y.) 11 25904134
2022 TRABID targets DDB2 for deubiquitination to promote proliferation of hepatocellular carcinoma cells. Biochemical and biophysical research communications 9 35944360
2024 TRIM40 interacts with ROCK1 directly and inhibits colorectal cancer cell proliferation through the c-Myc/p21 axis. Biochimica et biophysica acta. Molecular cell research 8 39357549
2023 The recognition mode between hsRBFA and mitoribosome 12S rRNA during mitoribosomal biogenesis. Nucleic acids research 7 36620886
2023 Yet Another Similarity between Mitochondrial and Bacterial Ribosomal Small Subunit Biogenesis Obtained by Structural Characterization of RbfA from S. aureus. International journal of molecular sciences 6 36768442
2015 Mutations of ribosomal protein S5 suppress a defect in late-30S ribosomal subunit biogenesis caused by lack of the RbfA biogenesis factor. RNA (New York, N.Y.) 6 26089326
2020 The folding and aggregation properties of a single KH-domain protein: Ribosome binding factor A (RbfA) from Pseudomonas aeruginosa. Biochimica et biophysica acta. General subjects 4 33157160
2020 In vitro Reconstitution of the S. aureus 30S Ribosomal Subunit and RbfA Factor Complex for Structural Studies. Biochemistry. Biokhimiia 3 32571184
2018 Backbone and side chain NMR assignments for the ribosome binding factor A (RbfA) from Staphylococcus aureus. Biomolecular NMR assignments 3 30225569
2022 1H, 13C, and 15N resonance assignments and solution structures of the KH domain of human ribosome binding factor A, mtRbfA, involved in mitochondrial ribosome biogenesis. Biomolecular NMR assignments 1 35666428
2020 Backbone and sidechain NMR assignments for the ribosome maturation factor RbfA from Escherichia coli. Biomolecular NMR assignments 1 32671633