Affinage

GTF2F1

General transcription factor IIF subunit 1 · UniProt P35269

Length
517 aa
Mass
58.2 kDa
Annotated
2026-06-10
32 papers in source corpus 25 papers cited in narrative 24 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 7/7 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

GTF2F1 (RAP74) is the large subunit of the general transcription factor TFIIF, which reconstitutes accurate RNA polymerase II transcription initiation in vitro together with the small subunit RAP30 (PMID:1734284, PMID:7929273). RAP74 binds RAP30 through its N-terminal domain (aa 1–172) and uses this region to recruit RNA pol II into the preinitiation complex, where it associates with promoter DNA on both sides of the TATA-box bend and induces a conformational change in the position of pol II relative to DNA (PMID:7592953, PMID:9207059, PMID:9528785). A central alpha1 helix (aa ~155–177) is critical for preinitiation complex isomerization and formation of the first phosphodiester bond, and the requirement for this region is partially relieved by negative supercoiling, indicating that RAP74 assists in untwisting promoter DNA during initiation (PMID:10523626, PMID:10567562). The same alpha1 helix drives forward translocation of pol II during elongation, suppressing backtracking and restart pathways, consistent with RAP74 supporting an active elongating conformation of the polymerase (PMID:10567562, PMID:15831464). Through its C-terminal winged-helix domain, RAP74 binds and stimulates the CTD phosphatase FCP1 to enable pol II recycling, recognizing distinct FCP1 regions through a hydrophobic groove between helices H2 and H3, with CK2 phosphorylation of FCP1 enhancing this interaction (PMID:9765293, PMID:11248041, PMID:12732728, PMID:15723518, PMID:15723517). RAP74 is itself phosphorylated by the TFIID subunit TAFII250 in a cell-cycle-dependent manner, which increases TFIIF transcription activity, and it serves as a direct binding target for transcriptional activators such as SRF (PMID:7854423, PMID:8625415, PMID:9237686). Structural studies established that the RAP30/RAP74 dimerization domains form a novel triple-barrel fold (PMID:11183778).

Mechanistic history

Synthesis pass · year-by-year structured walk · 13 steps
  1. 1992 High

    Established that RAP74 is the large TFIIF subunit and an obligate component of the pol II machinery, answering whether it is genuinely required for accurate initiation.

    Evidence In vitro transcription reconstitution with recombinant RAP74/RAP30 and biochemical fractionation

    PMID:1734284

    Open questions at the time
    • Did not resolve which RAP74 domains mediate each function
    • No structural information on the complex
  2. 1994 High

    Showed RAP74 contributes to multiple stages — stable preinitiation intermediate formation, first bond synthesis, and elongation rate — defining TFIIF as a multifunctional factor rather than an initiation-only factor.

    Evidence Highly purified in vitro transcription and kinetic elongation assays with reconstituted TFIIF

    PMID:7929273

    Open questions at the time
    • Did not map which residues drive elongation versus initiation
    • Mechanism of elongation stimulation unresolved
  3. 1995 High

    Mapped RAP74's functional architecture and identified protein partners (TAFII250, SRF), establishing that distinct domains recruit pol II, bind RAP30, and interface with activators.

    Evidence Deletion mutagenesis with in vitro binding/transcription assays; yeast two-hybrid; in vivo complementation of ts-TAFII250 cells

    PMID:7590250 PMID:7592953 PMID:7854423

    Open questions at the time
    • SRF interaction characterized in a single lab by two-hybrid and in vitro transcription only
    • No structure of any RAP74 domain yet
  4. 1996 High

    Identified TAFII250 as a serine kinase that selectively phosphorylates RAP74, linking the basal machinery to a regulatory phosphorylation event.

    Evidence In vitro kinase assay with recombinant TAFII250 and RAP74; kinase-domain deletion analysis

    PMID:8625415

    Open questions at the time
    • Phosphosite mapping incomplete
    • Functional consequence of phosphorylation not yet defined
  5. 1997 Medium

    Localized RAP74 within the preinitiation complex relative to promoter DNA and showed cell-cycle-dependent phosphorylation (aa 206–256) increases TFIIF activity, connecting RAP74 regulation to the cell cycle.

    Evidence Site-specific protein-DNA photo-crosslinking; in vitro kinase assays with synchronized HeLa TFIID fractions and deletion mutants

    PMID:9207059 PMID:9237686

    Open questions at the time
    • Crosslinking and kinase data each from single labs
    • In vivo relevance of cell-cycle phosphorylation not established
  6. 1998 High

    Resolved the division of labor between RAP74 domains and identified FCP1 as a C-terminal-domain partner, connecting RAP74 to CTD dephosphorylation and pol II recycling.

    Evidence Deletion mutagenesis with single/multiple-round in vitro transcription; yeast two-hybrid screen and in vitro phosphatase assay

    PMID:9528785 PMID:9765293

    Open questions at the time
    • Structural basis of RAP74–FCP1 binding not yet defined
    • How CTD dephosphorylation couples to recycling unresolved
  7. 1999 Medium

    Pinpointed the alpha1 helix (T154–M177) as the element required for initiation and elongation stimulation and showed supercoiling rescue, supporting a DNA-untwisting role in initiation.

    Evidence Point mutagenesis; in vitro transcription, complex assembly, supercoiling-rescue and elongation/kinetic assays; polytene chromosome immunofluorescence with DRB

    PMID:10094404 PMID:10523626 PMID:10567562

    Open questions at the time
    • DRB/CK2alpha link to RAP74 recruitment partly inferential
    • In vivo elongation function not directly tested in mammalian cells
  8. 2000 High

    Provided the first structural views — the triple-barrel RAP30/RAP74 dimerization fold — and demonstrated RAP74 phosphorylation operates during Tat-activated HIV-1 transcription.

    Evidence X-ray crystallography at 1.7 Å with mutagenesis; biotinylated-template transcription with depletion/reconstitution and in vitro phosphorylation

    PMID:10704353 PMID:11183778

    Open questions at the time
    • Structure covers only the dimerization domains
    • Mechanism by which Tat engages RAP74 phosphorylation unresolved
  9. 2001 High

    Determined the RAP74 C-terminal winged-helix structure and located the FCP1 binding surface, giving a structural basis for the phosphatase interaction.

    Evidence X-ray crystallography of the C-terminal domain at 1.02 Å

    PMID:11248041

    Open questions at the time
    • FCP1-binding site predicted, not yet co-crystallized
    • No information on bound FCP1 conformation
  10. 2003 High

    Solved the cterRAP74–cterFCP1 complex by NMR, showing FCP1 folds upon binding into the H2/H3 groove of RAP74 via hydrophobic and electrostatic contacts.

    Evidence NMR solution structure and structure of the protein complex with chemical shift mapping

    PMID:12578358 PMID:12732728

    Open questions at the time
    • Did not address regulation of the interaction
    • Only the C-terminal FCP1 region examined
  11. 2005 High

    Linked CK2 phosphorylation of FCP1 to enhanced RAP74 binding and showed the RAP74 alpha1 helix actively promotes forward pol II translocation, mechanistically defining its elongation function.

    Evidence In vitro CK2 kinase assay, NMR chemical shift mapping and MS; transient-state kinetics with alpha1-helix deletion mutants

    PMID:15723517 PMID:15723518 PMID:15831464

    Open questions at the time
    • In vivo significance of CK2-regulated recycling not tested
    • Structural state of translocating complex not captured
  12. 2009 High

    Demonstrated RAP74 recognizes two distinct FCP1 regions through the same groove, revealing adaptability in the phosphatase interaction.

    Evidence NMR structure of the cterRAP74–phospho-centFCP1 complex with ITC

    PMID:19215094

    Open questions at the time
    • Functional consequence of dual-region recognition unresolved
    • Order of engagement of the two FCP1 regions unknown
  13. 2021 Medium

    Characterized the dynamic, fuzzy nature of the RAP74–FCP1 interface, showing hydrophobic contacts dominate binding while flanking charges tune the conformational ensemble.

    Evidence NMR spin relaxation and paramagnetic relaxation enhancement with charge-inversion mutagenesis and affinity measurements

    PMID:26286791 PMID:34550709

    Open questions at the time
    • Single-lab biophysical studies
    • Functional impact of interface dynamics on dephosphorylation not measured

Open questions

Synthesis pass · forward-looking unresolved questions
  • How RAP74's biochemically defined initiation, elongation, and recycling functions operate and are regulated in living human cells remains unresolved.
  • No cellular knockdown/knockout phenotype documented
  • No genome-wide occupancy data in the corpus
  • No disease association reported

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0098772 molecular function regulator activity 3 GO:0140110 transcription regulator activity 3 GO:0003677 DNA binding 2
Localization
GO:0005654 nucleoplasm 2 GO:0005634 nucleus 1
Pathway
R-HSA-74160 Gene expression (Transcription) 3
Partners
CSNK2A1FCP1GTF2F2SRFTAF1
Complex memberships
RNA polymerase II preinitiation complexTFIIF (RAP30/RAP74)

Evidence

Reading pass · 24 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1992 RAP74 (GTF2F1) is the large subunit of the TFIIF (RAP30/74) complex; recombinant RAP74 produced in E. coli, together with RAP30, reconstitutes accurate transcription initiation by RNA polymerase II in vitro, and both RAP30 and RAP74 are physical components of the preinitiation complex. In vitro transcription reconstitution with recombinant proteins; biochemical fractionation Nature High 1734284
1994 Both RAP30 and RAP74 subunits of TFIIF contribute to formation of stable preinitiation intermediates containing RNA polymerase II, synthesis of the first phosphodiester bonds during initiation, and stimulation of the rate of RNA chain elongation. Highly purified in vitro transcription system; template competition experiments; kinetic elongation assays with reconstituted TFIIF The Journal of biological chemistry High 7929273
1995 TAFII250 specifically interacts with RAP74 in vitro, and this interaction is critical for cell viability as shown by in vivo complementation of a temperature-sensitive TAFII250 cell line; the interaction domain on RAP74 was mapped by in vitro binding assays. In vitro binding assays (pulldown/co-IP); in vivo complementation of ts-TAFII250 cell line Genes & development High 7590250
1995 RAP74's central charged cluster domain binds the transcriptional activation domain of serum response factor (SRF); deletion of this domain impairs SRF-activated but not basal or SP1-activated transcription in vitro. Yeast two-hybrid interaction assay; in vitro transcription with RAP74 deletion mutants Nature Medium 7854423
1995 Deletion mutagenesis of RAP74 mapped its RAP30-binding domain to amino acids 1–172, identified a partially masked RNA polymerase II binding domain at the C-terminal region (aa 363–444), and showed that aa 1–205 are minimally sufficient to stimulate accurate transcription in vitro. Deletion mutagenesis; in vitro binding and transcription assays The Journal of biological chemistry High 7592953
1996 TAFII250 is a protein serine kinase that selectively phosphorylates RAP74 but not other basal transcription factors; TAFII250 contains two distinct kinase domains, both required for efficient transphosphorylation of RAP74, and phosphorylation occurs in the context of the complete TFIID complex. In vitro kinase assay with purified recombinant TAFII250 and RAP74; deletion analysis of TAFII250 kinase domains Cell High 8625415
1997 RAP74 associates with the preinitiation complex in close proximity to promoter DNA both upstream and downstream of a DNA bend centered on the TATA box, and binding of RAP74 induces a conformational change affecting the position of RNA pol II relative to DNA; the N-terminal region containing the RAP30-binding domain is minimally required. Site-specific protein-DNA photo-crosslinking; deletion analysis Proceedings of the National Academy of Sciences of the United States of America Medium 9207059
1997 Cell-cycle-dependent phosphorylation of RAP74 by TFIID (S/G2-phase fraction) targets amino acid residues 206–256, and this phosphorylation increases TFIIF transcription activity in vitro. In vitro kinase assay with synchronized HeLa cell TFIID fractions; RAP74 deletion mutants; in vitro transcription reconstitution FEBS letters Medium 9237686
1998 FCP1 interacts with the carboxyl-terminal evolutionarily conserved domain of RAP74 (identified by yeast two-hybrid screen); FCP1 is an essential subunit of a RAP74-stimulated phosphatase that processively dephosphorylates the CTD of RNA pol II. Yeast two-hybrid screen; biochemical phosphatase assay with purified proteins The Journal of biological chemistry High 9765293
1998 The N-terminal domain of RAP74 (aa 1–172) is sufficient to recruit RNA pol II into the preinitiation complex; the region aa 172–205 (and specifically aa 170–178) is critical for both accurate initiation and elongation; the C-terminal domain does not contribute strongly to single-round initiation or elongation but stimulates multiple-round transcription. Deletion mutagenesis; in vitro transcription assays (single-round and multiple-round); preinitiation complex assembly assays Molecular and cellular biology High 9528785
1999 The region of RAP74 between L155 and M177 is critical for initiation (formation of first phosphodiester bond) but is not required for complex assembly or polymerase II recruitment; negative DNA supercoiling partially compensates for defects in this region, suggesting TFIIF helps untwist DNA for initiation. Point mutagenesis; in vitro transcription assays; preinitiation complex assembly assays Molecular and cellular biology High 10523626
1999 RAP74 region aa T154–M177 (alpha1 helix region) is critical for isomerization of the preinitiation complex and for elongation stimulation; TFIIF has higher affinity for rapidly elongating RNA pol II than for stalled elongation complexes; TFIIF stimulates elongation by supporting an active conformational state of RNA pol II. Point mutagenesis; in vitro transcription and elongation assays; binding affinity measurements; Arrhenius analysis Molecular and cellular biology High 10567562
1999 RAP74 subunit co-localizes with productively transcribing RNA pol II on Chironomus polytene chromosomes; both RAP74 and CK2alpha are sensitive to DRB treatment, suggesting RAP74 travels with elongating pol II along the DNA template, and DRB may block transcription by interfering with TFIIF recruitment through CK2alpha-mediated phosphorylation of RAP74. Immunofluorescence on polytene chromosomes; DRB treatment; co-localization with pol II Molecular and cellular biochemistry Medium 10094404
2000 The crystal structure of the RAP30/RAP74 interaction domains at 1.7 Å resolution reveals a novel 'triple barrel' dimerization fold; mutagenesis data indicate that interactions with the transcription apparatus are mediated by the tripartite beta-barrel and via flexible loops and alpha/beta structures extending from it. X-ray crystallography at 1.7 Å; mutagenesis Journal of molecular biology High 11183778
2000 RAP74 is phosphorylated by TAFII250 during Tat-activated HIV-1 LTR transcription; depletion of RAP74 from HeLa nuclear extract inhibits both basal transcription and Tat transactivation, and reconstitution with recombinant TFIIF restores activity; exogenous RAP74 is rapidly phosphorylated in the presence of Tat. Biotinylated DNA template transcription assay; RAP74 depletion and reconstitution; in vitro phosphorylation assay Virology Medium 10704353
2001 The crystal structure of the C-terminal domain of RAP74 at 1.02 Å resolution reveals a winged-helix fold similar to linker histone H5 and HNF-3gamma; a putative FCP1 phosphatase binding site was identified within this winged-helix domain. X-ray crystallography at 1.02 Å resolution Proceedings of the National Academy of Sciences of the United States of America High 11248041
2002 The alpha1 helix of RAP74 (aa ~155–177) is critical for both initiation and elongation stimulation; mutations within the alpha1 helix decrease transcription activities without affecting RAP30 binding or TFIIF–pol II interaction; contacts between the beta4-beta5 loop and alpha1 helix are not important for alpha1 helix function. Point mutagenesis guided by crystal structure; in vitro transcription and elongation assays; binding assays The Journal of biological chemistry High 12354769
2003 The NMR solution structure of the C-terminal domain of RAP74 (cterRAP74, residues 436–517) was determined; cterFCP binds in a groove between alpha-helices H2 and H3 of cterRAP74 without altering the secondary structure of RAP74; both RAP74 and TFIIB use shallow hydrophobic/positively-charged grooves to bind FCP1's acidic C-terminal domain. NMR solution structure determination; chemical shift mapping Biochemistry High 12578358
2003 High-resolution NMR structure of the cterRAP74–cterFCP1 complex shows that the disordered cterFCP1 forms an alpha-helix (H1') upon binding; the interface relies on van der Waals contacts between hydrophobic residues of RAP74 H2/H3 helices and the FCP1 H1' helix, plus critical electrostatic interactions between FCP1 aspartate residues and RAP74 lysines. NMR structure determination of protein complex Proceedings of the National Academy of Sciences of the United States of America High 12732728
2005 CK2 phosphorylates FCP1 at T584 (central domain) and S942/S944 (C-terminal domain), which enhances binding of RAP74 to FCP1; NMR chemical shift mapping confirmed that phosphorylated FCP1 peptides interact with the same groove of cterRAP74 (H2/H3 helices); HIV-1 Tat inhibits the CK2-mediated phosphorylation and RAP74 binding to FCP1. In vitro CK2 kinase assay; NMR chemical shift mapping; FT-ICR mass spectrometry; in vitro binding assays Biochemistry High 15723517 15723518
2005 The RAP74 alpha1 helix promotes forward translocation of RNA pol II during elongation; deletion of the alpha1 helix (RAP74(1-158)) results in increased occupancy of backtracking/cleavage/restart pathways and failure to support NTP-driven translocation from a stall position. Millisecond-phase transient-state kinetics; deletion mutagenesis; elongation complex analysis Molecular and cellular biology High 15831464
2009 NMR structure of the cterRAP74–centFCP1(phosphorylated) complex shows that the central domain of FCP1 uses hydrophobic and acidic residues to recognize the same groove of RAP74 as cterFCP1, but with significant mechanistic differences, demonstrating adaptability of RAP74 in recognizing two distinct FCP1 regions. NMR structure determination; isothermal titration calorimetry Biochemistry High 19215094
2012 NMR spin relaxation analysis shows that the C-terminal tail of FCP1 undergoes local folding-upon-binding at the ~20 residues forming direct contact with RAP74, while most of FCP1 remains highly dynamic in both bound and unbound states; RAP74 shows only very limited ordering upon FCP1 binding. Carbon-detected 15N NMR spin relaxation (CON(T1)-IPAP and CON(T2)-IPAP experiments) The journal of physical chemistry letters Medium 26286791
2021 Charged residues flanking the FCP1 binding helix form transient electrostatic interactions with the winged-helix domain of RAP74; charge inversion mutations in FCP1 affect the conformational ensemble of the complex but have minimal impact on overall binding affinity, indicating hydrophobic interactions in the minimal binding motif are the primary driving force. Paramagnetic relaxation enhancement (PRE) NMR; charge inversion mutagenesis; binding affinity measurements The journal of physical chemistry. B Medium 34550709

Source papers

Stage 0 corpus · 32 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
1996 TAFII250 is a bipartite protein kinase that phosphorylates the base transcription factor RAP74. Cell 184 8625415
1998 FCP1, the RAP74-interacting subunit of a human protein phosphatase that dephosphorylates the carboxyl-terminal domain of RNA polymerase IIO. The Journal of biological chemistry 128 9765293
1992 A cDNA encoding RAP74, a general initiation factor for transcription by RNA polymerase II. Nature 96 1734284
1994 Roles for both the RAP30 and RAP74 subunits of transcription factor IIF in transcription initiation and elongation by RNA polymerase II. The Journal of biological chemistry 87 7929273
1995 Interaction with RAP74 subunit of TFIIF is required for transcriptional activation by serum response factor. Nature 79 7854423
2000 Novel dimerization fold of RAP30/RAP74 in human TFIIF at 1.7 A resolution. Journal of molecular biology 72 11183778
1995 Human TAFII250 interacts with RAP74: implications for RNA polymerase II initiation. Genes & development 54 7590250
1993 Production of human RAP30 and RAP74 in bacterial cells. Protein expression and purification 54 8390879
1997 RAP74 induces promoter contacts by RNA polymerase II upstream and downstream of a DNA bend centered on the TATA box. Proceedings of the National Academy of Sciences of the United States of America 52 9207059
1995 Functional domains of human RAP74 including a masked polymerase binding domain. The Journal of biological chemistry 46 7592953
2001 Crystal structure of the C-terminal domain of the RAP74 subunit of human transcription factor IIF. Proceedings of the National Academy of Sciences of the United States of America 42 11248041
1998 Functions of the N- and C-terminal domains of human RAP74 in transcriptional initiation, elongation, and recycling of RNA polymerase II. Molecular and cellular biology 41 9528785
1994 Importance of codon preference for production of human RAP74 and reconstitution of the RAP30/74 complex. Protein expression and purification 40 7827505
2003 NMR structure of a complex containing the TFIIF subunit RAP74 and the RNA polymerase II carboxyl-terminal domain phosphatase FCP1. Proceedings of the National Academy of Sciences of the United States of America 36 12732728
2012 Carbon-Detected (15)N NMR Spin Relaxation of an Intrinsically Disordered Protein: FCP1 Dynamics Unbound and in Complex with RAP74. The journal of physical chemistry letters 28 26286791
1999 The RAP74 subunit of human transcription factor IIF has similar roles in initiation and elongation. Molecular and cellular biology 26 10567562
2005 Human RNA polymerase II elongation in slow motion: role of the TFIIF RAP74 alpha1 helix in nucleoside triphosphate-driven translocation. Molecular and cellular biology 24 15831464
1999 A region within the RAP74 subunit of human transcription factor IIF is critical for initiation but dispensable for complex assembly. Molecular and cellular biology 23 10523626
2013 RNA polymerase III-specific general transcription factor IIIC contains a heterodimer resembling TFIIF Rap30/Rap74. Nucleic acids research 22 23921640
1999 The binding of the alpha subunit of protein kinase CK2 and RAP74 subunit of TFIIF to protein-coding genes in living cells is DRB sensitive. Molecular and cellular biochemistry 21 10094404
2005 Enhanced binding of RNAP II CTD phosphatase FCP1 to RAP74 following CK2 phosphorylation. Biochemistry 20 15723518
2002 A key role for the alpha 1 helix of human RAP74 in the initiation and elongation of RNA chains. The Journal of biological chemistry 18 12354769
2011 Atomistic simulations reveal structural disorder in the RAP74-FCP1 complex. The journal of physical chemistry. B 17 21988473
2005 Interactions of the HIV-1 Tat and RAP74 proteins with the RNA polymerase II CTD phosphatase FCP1. Biochemistry 16 15723517
2003 Solution structure of the carboxyl-terminal domain of RAP74 and NMR characterization of the FCP1-binding sites of RAP74 and human TFIIB. Biochemistry 16 12578358
2009 NMR structure of a complex formed by the carboxyl-terminal domain of human RAP74 and a phosphorylated peptide from the central domain of the FCP1 phosphatase. Biochemistry 13 19215094
2000 Phosphorylation of the RAP74 subunit of TFIIF correlates with Tat-activated transcription of the HIV-1 long terminal repeat. Virology 13 10704353
2013 Native-based simulations of the binding interaction between RAP74 and the disordered FCP1 peptide. The journal of physical chemistry. B 11 23387368
1997 Cell-cycle-dependent phosphorylation of the basal transcription factor RAP74. FEBS letters 7 9237686
1993 Assignment of the human GTF2F1 gene to chromosome 19p13.3. Genomics 5 8486367
2021 Transient Electrostatic Interactions between Fcp1 and Rap74 Bias the Conformational Ensemble of the Complex with Minimal Impact on Binding Affinity. The journal of physical chemistry. B 2 34550709
1994 Enhancement of bacterial transcription initiation in vitro by the 74 kDa subunit of human general transcription factor IIF (RAP74). Biochimica et biophysica acta 2 7948016

Missed literature

Know a paper Affinage missed for GTF2F1? Flag it for the maintainers and the community.

No submissions yet.