Author response: Architecture of TAF11/TAF13/TBP complex suggests novel regulation properties of general transcription factor TFIID (2017)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract General transcription factor TFIID is a key component of RNA polymerase II transcription initiation. Human TFIID is a megadalton-sized complex comprising TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). TBP binds to core promoter DNA, recognizing the TATA-box. We identified a ternary complex formed by TBP and the histone fold (HF) domain-containing TFIID subunits TAF11 and TAF13. We demonstrate that TAF11/TAF13 competes for TBP binding with TATA-box DNA, and also with the N-terminal domain of TAF1 previously implicated in TATA-box mimicry. In an integrative approach combining crystal coordinates, biochemical analyses and data from cross-linking mass-spectrometry (CLMS), we determine the architecture of the TAF11/TAF13/TBP complex, revealing TAF11/TAF13 interaction with the DNA binding surface of TBP. We identify a highly conserved C-terminal TBP-interaction domain (CTID) in TAF13, which is essential for supporting cell growth. Our results thus have implications for cellular TFIID assembly and suggest a novel regulatory state for TFIID function. https://doi.org/10.7554/eLife.30395.001 Introduction Eukaryotic gene expression is a highly regulated process which is controlled by a plethora of proteins, arranged in multiprotein complexes including the general transcription factors (GTFs), Mediator and RNA polymerase II (Pol II) (Gupta et al., 2016; Thomas and Chiang, 2006). Regulated class II gene transcription is initiated by sequential nucleation of GTFs and Mediator on core promoter DNA (Buratowski and Sharp, 1990). The GTF TFIID is a cornerstone in this process and links cellular signaling events with regulatory DNA elements and the components of the transcription machinery (Albright and Tjian, 2000). A basal transcription system which supports initiation can be reconstituted with TBP and GTFs TFIIA, TFIIB, TFIIE, TFIIF and TFIIH in vitro (with TFIIA not essential if TBP repressors are not present in the system); however, TFIID is required to respond to activators (Hampsey and Reinberg, 1999). In mammalian cells, the promoters of virtually all protein-encoding genes are occupied by TFIID, and loss of TFIID components causes embryonic lethality (Gegonne et al., 2012; Kim et al., 2005; Mohan et al., 2003). TFIID subunits are thought to mediate cross-talk with epigenetic modifications on nucleosomes and regulatory DNA elements in promoter regions (Vermeulen et al., 2007; Verrijzer et al., 1995). X-ray crystallography revealed many details of TFIID components at near atomic resolution (Gupta et al., 2016). Cryo-electron microscopy (cryo-EM) provided essential insight into TFIID architecture and promoter DNA interaction (Bieniossek et al., 2013; Cianfrocco et al., 2013; Louder et al., 2016). The recent identification of a discrete TAF-containing complex in the cytoplasm of cells provided first insight into holo-TFIID assembly from preformed sub-modules, regulated by nuclear import mechanisms (Trowitzsch et al., 2015). Canonical human TFIID comprises TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs) (Matangkasombut et al., 2004; Müller and Tora, 2014). Furthermore, non-canonical TFIID and TAF-containing complexes have been identified regulating spermatogenesis and stem cell development (Goodrich and Tjian, 2010; Maston et al., 2012; Müller et al., 2010). A nuclear core-TFIID complex was identified, made up of two copies each of TAF4, 5, 6, 9 and 12 (Bieniossek et al., 2013; Wright et al., 2006). Biochemical and structural studies established the histone-fold domain (HFD) as a key TAF-TAF interaction motif within TFIID (Gangloff et al., 2001a). TAF3, 4, 6, 8, 9, 10, 11, 12 and 13 contain HFDs and assemble specifically into heterodimers (TAF3-10, TAF4-12, TAF6-9, TAF8-10 and TAF11-13) (Birck et al., 1998; Gangloff et al., 2001b; Werten et al., 2002; Xie et al., 1996). TFIID recognizes core promoter DNA via its TATA-box-binding protein subunit, TBP. TBP is central to transcription regulation in eukaryotes and is the only subunit present in the transcription initiation complexes of each of the three RNA polymerases (Koster et al., 2015; Thomas and Chiang, 2006; Tora and Timmers, 2010). TBP consists of a highly variable N-terminal domain with less well-understood function and a conserved DNA-binding C-terminal core domain comprising two symmetrical pseudo-repeats (Thomas and Chiang, 2006; Tora and Timmers, 2010). Crystal structures of the conserved TBP core revealed a saddle-like shape with a concave DNA binding surface recognizing the minor groove of TATA-box containing DNA (Kim et al., 1993; Nikolov et al., 1996; Nikolov et al., 1992). The DNA-binding activity of TBP/TFIID is tightly regulated by gene-specific co-factors that can activate or inhibit transcription (Koster et al., 2015; Tora and Timmers, 2010). The mechanism of a number of these regulatory factors has been described in molecular detail. The TFIID component TAF1 was found to associate with the concave DNA-binding surface of TBP via its N-terminal domain (TAF1-TAND), exhibiting TATA-box mimicry (Anandapadamanaban et al., 2013). TAF1-TAND, unstructured in isolation, was found to adopt a three-dimensional structure closely resembling the TATA-element is shape and charge distribution when bound to TBP (Burley and Roeder, 1998; Liu et al., 1998). This interaction is conserved in yeast, Drosophila and human (Anandapadamanaban et al., 2013; Burley and Roeder, 1998; Liu et al., 1998; Mal et al., 2004). The recent high-resolution structure of TBP bound to yeast TAF1-TAND revealed anchoring patterns in transcriptional regulation shared by TBP interactors, providing insight into the competitive multiprotein TBP interplay critical to transcriptional regulation (Anandapadamanaban et al., 2013). Mot1 is an ATP-dependent inhibitor of TBP/TATA-DNA complex formation (Auble and Hahn, 1993). Mot1 regulates the genomic distribution of TBP and was shown to influence transcription levels both positively and negatively (Pereira et al., 2003). Recent structural analysis revealed the molecular mechanism of Mot1 wrapping around TBP resembling a bottle opener, with a 'latch helix' blocking the concave DNA-binding surface of TBP and acting as a chaperone to prevent DNA re-binding to ensure promoter clearance (Wollmann et al., 2011). Mot1 and negative co-factor 2 (NC2) are thought to cooperate in gene-specific repression of TBP activity (Hsu et al., 2008). The GTF TFIIA, on the other hand, competes with NC2 for TBP (Kamada et al., 2001; Xie et al., 2000) and promotes TBP/DNA interactions in a ternary TFIIA/TBP/DNA complex, facilitating formation of and stabilizing the preinitiation complex (PIC). Interaction of TFIIA with TBP results in the exclusion of negative factors that would interfere with PIC formation, and TFIIA acts as a coactivator assisting transcriptional activators in increasing transcription levels (Bleichenbacher et al., 2003). Genetic and biochemical experiments suggested that the TFIIA/TBP/DNA complex is further stabilized by the histone-fold containing TFIID subunits TAF11 and TAF13 conveying the formation of a TAF11/TAF13/TFIIA/TBP/DNA assembly (Kraemer et al., 2001; Lavigne et al., 1999; Robinson et al., 2005). We therefore set out to investigate this putative pentameric complex in detail. Unexpectedly, we did not observe a stabilization of TFIIA/TBP/DNA by TAF11/TAF13, but found a marked destabilization of the TFIIA/TBP/DNA complex by TAF11/TAF13, resulting in the release of free DNA and the formation of a stable ternary complex formed by TAF11/TAF13 and TBP. We analyzed the TAF11/TAF13/TBP complex biochemically and structurally utilizing a comprehensive, integrative approach. We report a novel interaction of the TAF11/TAF13 dimer binding to the concave DNA-binding groove of TBP, thus excluding TATA-box containing DNA. Using pull-down experiments with immobilized TAF1-TAND, we demonstrate competition between TAF11/TAF13 and TAF1-TAND for TBP binding. We identify a novel C-terminal TBP-binding domain (CTID) within TAF13 which is highly conserved from yeast to man. We probe key residues within this TAF13 CTID by mutagenesis in vitro and in vivo in cell growth experiments, revealing a key role of this domain for viability. We contrast the TAF11/TAF13 interaction with other TBP DNA-binding groove interactors including Mot1 and discuss the implications of our findings in the context of TFIID assembly. Based on our results, we propose a novel, functional state of TFIID in transcription regulation. Results Identification of a novel TAF11/TAF13/TBP ternary complex We set out to analyze the structure of a putative pentameric TAF11/TAF13/TFIIA/TBP/DNA complex (Kraemer et al., 2001; Lavigne et al., 1999; Robinson et al., 2005), with the objective to better understand the possible roles of TAF11/TAF13 in TFIID function. First, we purified human TAF11/TAF13 complex and TBP to homogeneity (Figure 1A). TFIIA in human cells is made from two precursor polypeptides, TFIIAαβ and TFIIAγ, with TFIIAαβ processed in vivo into two separate polypeptides, α and β, by proteolytic cleavage mediated by the protease Taspase1 (Høiby et al., 2007). Recombinant human TFIIA is typically produced in E. coli by refolding from three separate polypeptides expressed in inclusion bodies, which correspond to the native α, β and γ chains (Bleichenbacher et al., 2003). To facilitate recombinant human TFIIA production, we designed a single-chain construct (TFIIAs-c) by connecting α, β and γ by flexible linkers, based on atomic coordinates taken from the crystal structure of human TFIIA/TBP/DNA complex (Bleichenbacher et al., 2003). TFIIAs-c could be produced in high amounts in soluble form in E. coli and purified to homogeneity without any need for refolding steps (see Materials and methods section). We stored highly purified TFIIAs-c at 4⁰C, and observed the formation of needle-shaped crystals in the storage buffer after several weeks. We improved the crystals manually and determined the 2.4 Å crystal structure of TFIIAs-c (Figure 1B, Figure 1—figure supplement 1, Table 1). The crystal structure revealed a virtually identical conformation of unliganded TFIIAs-c as compared to TFIIA in the TFIIA/TBP/DNA complex. Moreover, the crystal structure highlighted the importance of the connecting loops we had introduced in stabilizing the three-dimensional crystal lattice (Figure 1—figure supplement 1). TFIIAs-c was active in a band-shift assay with TBP and adenovirus major late promoter (AdMLP) TATA-DNA (Figure 1C) similar to purified TFIIA using the classical refolding protocol (Bleichenbacher et al., 2003). Figure 1 with 2 supplements see all Download asset Open asset TAF11/TAF13 and TBP form ternary complex. (A) Human TAF11/TAF13 complex, TBP full-length (TBPfl) and core (TBPc), and a single-chain version of TFIIA (TFIIAs-c) were purified to homogeneity as shown by SDS-PAGE. (B) TFIIA subunits α(ΑΑ2–59), β(ΑΑ302–376) and γ(ΑΑ2–110)(Bleichenbacher et al., 2003) were connected with linkers L1(-DGKNTANSANTNTV-) and L2(-SRAVDGELFDT-) as indicated (top). TFIIAs-c crystallized during purification (Figure 1—figure supplement 1). The 2.4 Å X-ray structure is shown in a cartoon representation with a section of electron density at 1σ(bottom). (C) TFIIAs-c was assayed by band-shift for activity. DNA, Adenovirus major late promoter (AdMLP) DNA. (D) Probing formation of putative pentameric TAF11/TAF13/TBPTFIIA/DNA complex by band-shift assay (Kraemer et al., 2001; Robinson et al., 2005). TAF11/TAF13 titration to the TFIIA/TBP/DNA complex results in DNA release. (E) SEC analysis reveals a stable TAF11/TAF13/TBP ternary complex. Elution fractions (1-7) were analyzed by SDS-PAGE (inset). IN, equimolar mixture of TAF11/TAF13 and TBP. No interactions were found between TAF11/TAF13/TBP complex and TFIIA (Figure 1—figure supplement 2). (F) TAF11/TAF13 competes with DNA for TBP binding, evidenced by SEC. Elution fractions (1-6, 11-16) were analyzed by SDS-PAGE and ethidium-bromide stained agarose gel (inset). IN, preformed TAF11/TAF13/TBP complex; DNA, AdMLP DNA; M, protein molecular weight marker and DNA ladder, respectively. (G) Immobilized human TAF1 N-terminal domain (TAF1-TAND) (Anandapadamanaban et al., 2013) efficiently depletes TBP from preformed TAF11/TAF13/TBP complex. RSIN, amylose resin with MBP-tagged TAF1-TAND bound; IN, preformed TAF11/TAF13/TBP; M: protein marker; FT, flow-through fraction; W, wash fraction; E, maltose elution fraction. https://doi.org/10.7554/eLife.30395.002 Table 1 X-ray data collection and refinement statistics. https://doi.org/10.7554/eLife.30395.005 TFIIAs-cData collectionSpace groupP65Cell dimensions a, b, c (Å)123.3, 123.3, 34.8 a, b, g (°)90, 90, 120Resolution (Å)53.4–2.4Last resolution bin (Å)2.52–2.38Rmeasure (%)12.9 (64.8)I / σI11.5 (2.72)Completeness (%)99.8 (99.9)Multiplicity6.7 (6.8)RefinementResolution (Å)40.36–2.38 (2.44–2.38)No. reflections Work set11859 Free set601Rwork0.18 (0.27)Rfree0.24 (0.36)No. of atoms Protein1689 Water50r.m.s deviations Bond lengths (Å)0.0223 Bond angles (°)2.088 *Values in parentheses are for highest resolution shell. Next, we attempted to reconstitute the TAF11/TAF13/TFIIA/TBP/DNA complex following published procedures (Kraemer et al., 2001; Robinson et al., 2005). Titrating TAF11/TAF13 dimer to a preformed TFIIA/TBP/DNA complex had been reported to stabilize TFIIA/TBP/DNA in band-shift assays using AdMLP TATA-DNA (Robinson et al., 2005). Surprisingly, in our titration experiments, TAF11/TAF13 did not stabilize the preformed TFIIA/TBP/DNA complex but, in marked contrast, resulted in TAF11/TAF13-dependent release of free promoter-containing DNA in band-shift assays (Figure 1D). We analyzed the possible underlying intermolecular interactions that may be formed between the individual components TAF11/TAF13, TFIIAs-c, TBP and AdMLP TATA-DNA. We observed that TFIIAs-c and TAF11/TAF13 did not interact in our experiments (Figure 1—figure supplement 2). Combining the TAF11/TAF13 dimer with TBP, in contrast, revealed a novel TAF11/TAF13/TBP complex that was stable in size exclusion experiments (Figure 1E). We concluded that human TAF11/TAF13 did not further stabilize the preformed TFIIA/TBP/DNA complex, but rather sequestered TBP from this complex giving rise to a novel assembly comprising TAF11, TAF13 and TBP. TATA-DNA and TAF1-TAND compete with TAF11/TAF13 for TBP binding We tested competition between TAF11/TAF13/TBP formation and TBP binding to AdMLP DNA and showed that the ternary TAF11/TAF13/TBP complex remained stable in the presence of excess AdMLP DNA (Figure 1F). Thus, our results indicate that TAF11/TAF13 and TATA-DNA compete for at least parts of the same binding interface within TBP, and that once TAF11/TAF13 is bound to TBP, TATA-DNA binding is precluded. TAF1 had been shown previously to bind to the DNA-binding surface of TBP via its TAND domain (Anandapadamanaban et al., 2013; Burley and Roeder, 1998; Liu et al., 1998). We produced human TAF1-TAND fused to maltose-binding protein (MBP) and immobilized highly purified fusion protein to an amylose column (Materials and methods). We added preformed, purified TAF11/TAF13/TBP complex in a pull-down assay using immobilized TAF1-TAND. We found that TAF1-TAND effectively sequestered TBP from the TAF11/TAF13/TBP complex, evidenced by TAF11/TAF13 eluting in the flow-through fraction. Elution by maltose, in contrast, revealed a TAF1-TAND/TBP complex (Figure 1G). Together, these findings substantiate our view that TAF11/TAF13, TAF1-TAND and AdMLP TATA-DNA all interact with the concave DNA-binding surface of TBP, and that the interactions are mutually exclusive. TAF11, TAF13, TBP form a 1:1:1 complex Next, we set out to determine the subunit stoichiometry within the TAF11/TAF13/TBP complex, by using two complementary methods, analytical ultra-centrifugation (AUC) and native mass-spectrometry (native MS). Both methods were consistent in revealing a 1:1:1 complex (Figure 2A,B; Figure 2—figure supplement 1). Collision-induced dissociation (CID) in native MS, evidenced a TBP monomer and a TAF11/TAF13 dimer (Figure 2B). Of note, our control experiment using highly purified TBP evidenced a dimer in the native MS (Figure 2—figure supplement 1). In aggregate, our results are consistent with a ternary assembly which accommodates one copy each of TAF11, TAF13 and TBP in the complex. Figure 2 with 1 supplement see all Download asset Open asset TAF11/TAF13/TBP interactions. (A) Sedimentation velocity analytical ultracentrifugation (AUC) experiments reveal a major peak consistent with a 1:1:1 TAF11/TAF13/TBP ternary complex. The second, smaller peak corresponds to excess TAF11/TAF13. (B) Native mass spectrometry of the TAF11/TAF13/TBP complex confirms 1:1:1 stoichiometry. Collision induced dissociation (CID) results in TBP monomer and TAF11/TAF13. Experimental masses are provided (inset). Calculated masses: 20659 Da (TBP); 40691 Da (TAF11/TAF13); 61351 Da (TAF11/TAF13/TBP). Mass spectra of the TAF11/TAF13/TBP complex and TBP dimer are shown in Figure 2—figure supplement 1. (C) TAF11/TAF13/TBP and TAF11/TAF13 were analyzed by hydrogen-deuterium exchange/mass spectrometry (HDX-MS) (Rajabi et al., 2015). Changes in the deuteration level of selected peptides in TAF11, TAF13 or TBP are depicted in diagrams (top row). Peptides protected upon ternary complex formation are coloured in red in cartoon representations of TAF11/TAF13 and TBP (bottom row). One peptide in TBP (grey, far right) becomes more accessible, hinting at disassembly of a TBP dimer when TAF11/TAF13 binds (Figure 2—figure supplement 1). All peptides implicated in TAF11/TAF13-binding map to the concave DNA-binding surface of TBP. (N) and (C) indicate N- and C-terminal TBP stirrups, respectively. https://doi.org/10.7554/eLife.30395.006 TAF11/TAF13 interacts with the concave DNA-binding surface of TBP With the objective to elucidate the nature of the TAF11/TAF13 interaction with TBP, and to provide direct evidence that TAF11/TAF13 indeed engages the DNA-binding concave surface of TBP, we performed hydrogen-deuterium exchange/mass spectrometry (HDX-MS) (Rajabi et al., 2015) experiments (Figure 2C, Table 2). We analyzed unbound TAF11/TAF13 and TAF11/TAF13/TBP, and compared changes in the deuteration levels in proteolytic peptides by MS indicating the level of protection of the respective regions in TAF11, TAF13 or TBP, respectively, upon ternary complex formation. The HDX-MS results underscored that the DNA-binding surface of TBP was recognized by TAF11/TAF13, evidenced by decrease in deuteration levels which corresponds to increased protection from the solvent of peptides located in the concave surface of TBP upon TAF11/TAF13 binding. The extent of protection within TBP further indicates that the binding of TAF11/TAF13 engages both symmetric pseudo-repeats in TBP, thus spanning the entire concave interface (Figure 2C). Interestingly, we identified one peptide (AA residues 157–167) in TBP which evidenced an increased level of deuteration upon TAF11/TAF13/TBP complex formation in the HDX-MS experiments (Figure 2C). This peptide is located at the dyad relating the two pseudo-symmetric repeats in TBP. We interpret this result as an indication, that this particular region within TBP is more protected in a presumed TBP dimer which dissociates when TAF11/TAF13 is binding and the 1:1:1 complex is formed. Our HDX-MS experiments provide direct evidence that TAF11/TAF13 engage the concave DNA-binding surface of TBP, in excellent agreement with our above described biochemical experiments involving TAF11/TAF13, TBP, AdMLP DNA and TAF1-TAND. Table 2 Peptide deuteration level changes upon complex formation in HDX-MS https://doi.org/10.7554/eLife.30395.008 TAF11Residues numbersSequenceΔ%D (15 s)†Δ%D (120 s)†93–96EKKQ−8.465−7.309105–109KMQIL−7.014150–153VVIA−7.295TAF13Residues numbersSequenceΔ%D (15 s)†Δ%D (120 s)†14–31NEEIGGGAEGGQGKRKRL−7.507−7.19232–35FSKE−8.79436–40LRCMM−7.086–88IVF−8.59886–89IVFL−10.36697–104FARVKDLL−7.223116–124AFDEANYGS10.898.721TBPResidues numbersSequenceΔ%D (15 s)†Δ%D (120 s)†157–167*IVPQLQNIVST9.038167–174TVNLGCKL−9.675−10.171193–197FAAVI−14.068−12.267197–208IMRIREPRTTAL−7.785199–208RIREPRTTAL−9.661209–215IFSSGKM−8.655−8.75232–244KYARVVQKLGFPA−21.337−18.382233–242YARVVQKLGF−7.128247–252LDFKIQ−7.271250–256KIQNMVG−8.913259–266DVKFPIRL−11.932−10.109259–268DVKFPIRLEG−13.79−11.479260–266VKFPIRL−10.393260–268VKFPIRLEG−16.066−13.002281–287PELFPGL−7.811285–289PGLIY−8.061−7.339326–335PILKGFRKTT−7.077−7.902 *TBP peptide with increasing deuteration level upon complex formation. †Peptides exhibiting changes in deuteration level ≥7% are shown. Architecture of the TAF11/TAF13/TBP complex We proceeded to determine the architecture of the TAF11/TAF13/TBP complex by using a comprehensive, integrative multi-parameter approach. We utilized the available crystal structure of TBP (Nikolov et al., 1992) as well as the crystal structure of the globular histone-fold containing domains of the TAF11/TAF13 dimer (Birck et al., 1998) and combined these atomic coordinates with our results from biochemical and biophysical experiments, including small angle X-ray scattering (SAXS) experiments (Figure 3—figure supplement 1, Table 3). We acquired distance constraints to define our structural model by carrying out cross-linking/mass-spectrometry (CLMS) experiments using two different approaches. We first carried out a series of CLMS experiments using the cross-linker bis(sulfosuccinimidyl)-suberate (BS3) (Figure 3—figure supplement 2; Table 4). BS3 cross-links primary amines on the side chain of lysine residues and the N-terminus of polypeptide chains. Inclusion of the BS3 CLMS derived distance constraints into our calculation already evidenced that the TAF11/TAF13 engaged the concave DNA-binding surface of TBP. In addition, we carried out site-specific UV-induced CLMS experiments utilizing MultiBacTAG (Koehler et al., 2016), a method we recently developed to unlock protein complex chemical space (Figure 3—figure supplement 3). MultiBacTAG relies on genetic code expansion (GCE) and employs a modified MultiBac baculoviral genome into which we engineered expression cassettes encoding for the orthogonal pyrolysine tRNA (tRNAPyl)/tRNA synthetase (PylRS) pair from Methanosarcina mazei. Infection of insect cell cultures with a MultiBacTAG virus containing TAF11/TAF13 mutants harboring an AMBER stop codon resulted in efficient incorporation of the lysine provided in the to site-specific cross-links upon of the reconstituted complex (Koehler et al., (Figure 3—figure supplement 3). Table Data collection and refinement collection size at size at mass mass for Table observed by BS3 of TAF13 of TAF11 of Our three-dimensional TAF11/TAF13/TBP ternary complex model more of all constraints and evidenced a of the TAF11/TAF13 histone-fold regions with the concave surface of TBP, giving rise to a structure (Figure Figure 3—figure supplement 4). To our we carried out using we TBP by around its to the surface to TAF11/TAF13, to the of the N- and C-terminal of TBP were far in and distance in to with our biochemical thus our TAF11/TAF13/TBP structural Figure with supplements see all Download asset Open asset Architecture of TAF11/TAF13/TBP complex. TAF11/TAF13/TBP complex architecture was determined by using an integrative multi-parameter approach. We utilized the crystal structure of TBP (Nikolov et al., as well as the crystal structure of the TAF11/TAF13 dimer (Birck et al., 1998) combined with our native MS, and HDX-MS results as well as distance constraints from CLMS experiments (Figure 3—figure supplements The structure of the TAF11/TAF13/TBP ternary complex is shown in a cartoon representation in and as a space model of unstructured in three as the between the TAF11 is in TAF13 in and TBP in This model of the (Figure 3—figure supplement 4, 2 and 4). A highly conserved domain within TAF13 is required for TAF11/TAF13 interaction with TBP and for supporting cell growth In our TAF11/TAF13/TBP complex, the domains of TAF11 and TAF13 the concave DNA-binding surface of TBP. Moreover, the structural model that the C-terminal the histone fold domain of TAF13 may a role in stabilizing the interaction with TBP. We analyzed TAF13 primary from yeast to human (Figure 4). revealed a high of in this C-terminal TAF13 with key residues virtually identical in all TAF13 analyzed (Figure Based on our we these residues in the human TAF13 C-terminal region and analyzed the of the on TAF11/TAF13/TBP complex formation. In we analyzed two TAF13 A and (Figure 4). In we with conserved residues located in the of the TAF11/TAF13/TBP in we conserved residues located more to the (Figure supplement 1A). Both TAF13 mutants formed with SEC experiments evidenced of TBP interaction with both but with In of TBP interaction was and the TAF11/TAF13/TBP complex was not In of on the other hand, TBP interaction was however, TAF11/TAF13/TBP complex formation was observed (Figure Based on we mutants containing identical in CTID as the human TAF13 A and proteins, respectively. With these mutants A and we carried out in vivo experiments in yeast, using previously described et al., and 2000). or growth at the on and in cell at to A did not the at that the conserved in this effectively cell growth (Figure Figure with 1 supplement see all Download asset Open asset conserved C-terminal TBP-interaction domain (CTID) in TAF13 required for (A) reveal a highly conserved C-terminal TBP interaction domain (CTID) in TAF13 comprising virtually identical residues in TAF13 from yeast to that were in the CTID of TAF13 are indicated by