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Structural Analysis and Dimerization Potential of the Human TAF5 Subunit of TFIID

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Structural Analysis and Dimerization Potential of the Human TAF5 Subunit of TFIID Structural analysis and dimerization potential of the human TAF5 subunit of TFIID Suparna Bhattacharya, Shinako Takada, and Raymond H. Jacobson* Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center and the Program in Genes and Development at the University of Texas, Graduate School in Biochemical Sciences, 1515 Holcombe Boulevard, Unit 1000, Houston, TX 77030 Communicated by Brian W. Matthews, University of Oregon, Eugene, OR, November 21, 2006 (received for review October 12, 2006) TFIID is an essential factor required for RNA polymerase II tran- analysis have provided visualizations of the TFIID complex scription but remains poorly understood because of its intrinsic (19–21). These studies showed that TFIID forms a three lobed, complexity. Human TAF5, a 100-kDa subunit of general transcrip- asymmetric structure. Further immunomapping studies have tion factor TFIID, is an essential gene and plays a critical role in begun to reveal the relative positions of different TAFs within assembling the 1.2 MDa TFIID complex. We report here a structural the complexes and point to key structural roles for the TAF5 and analysis of the TAF5 protein. Our structure at 2.2-Å resolution of TAF1 subunits in forming the characteristic three lobed molec- the TAF5-NTD2 domain reveals an ␣-helical domain with distant ular assembly. structural similarity to RNA polymerase II CTD interacting factors. TAF5, a 100-kDa polypeptide in humans, is a key TAF subunit The TAF5-NTD2 domain contains several conserved clefts likely to that seems to play a major role in forming the scaffold critical for be critical for TFIID complex assembly. Our biochemical analysis of TFIID complex formation. TAF5 proteins from all eukaryotes the human TAF5 protein demonstrates the ability of the N-terminal contain three highly conserved sequence motifs in their N- and half of the TAF5 gene to form a flexible, extended dimer, a key C-terminal regions. The C-terminal portion of TAF5 contains six property required for the assembly of the TFIID complex. WD40 repeats that are likely to form a closed beta propeller structure. Beta propeller domains have more generally been inititation complex ͉ transcription ͉ x-ray crystallography ͉ protein–protein shown to have functions in mediating protein-protein interac- interaction tions among a variety of different proteins and might be impor- tant for TAF–TAF interactions (22, 23). Unlike the WD40 BIOCHEMISTRY he general transcription factor TFIID plays a central role in motifs present in the C terminus of TAF5, the N-terminal region Tthe recognition of core promoter elements and is used for of the TAF5 sequence contains two conserved motifs for which accurate transcription initiation by RNA Pol II for a large class little is known. The EM studies had implicated the N-terminal of genes. Recent studies in yeast indicate that the majority of portion of TAF5 as playing a role in dimerization and forming genes present are TFIID dependent (1). TFIID is a multiprotein a scaffold upon which other TFIID subunits could assemble. To complex composed of the TATA box-binding protein (TBP) and further clarify the structural organization and function of TAF5, 14 other TBP-associated factors (TAFs) which have been highly we have determined the crystal structure to a resolution of 2.2Å conserved during eukaryotic evolution (2). TFIID is the only of the larger of the two N-terminal conserved motifs general transcription factor with specific TATA box binding (hTAFII100) (residues 189–343) and characterized the relation- activity and has been shown to initiate recruitment of the other ship between this domain and the smaller, most N-terminal general transcription factors (TFIIA, TFIIB, TFIIE, TFIIF, conserved region of the TAF5 sequence (TAF5-NTD1/LisH TFIIH) along with RNA Pol II into a functional preinitiation domain residues 90–124). complex (PIC) that forms at the start site of Pol II genes. Our studies show that the N-terminal half of the TAF5 TAF subunits seem to serve multiple functions within TFIID sequence is capable of forming a dimeric assembly in the absence holocomplex. For instance, hTAF6 and hTAF9 have been of other TAFs. Our crystal structure shows that the second reported to interact with the downstream promoter element, conserved TAF5 motif (TAF5-NTD2) adopts a mostly ␣-helical whereas TAF1 and TAF2 have been shown to bind to the domain of novel structure that forms a calcium dependent dimer initiator. The specific contacts with the promoter DNA by both in the crystal lattice and in solution. However, direct TFIID support basal transcription from promoters containing measurement Ca2ϩ binding revealed only weak affinity of this these elements and have revealed the role of several TAFs on domain for Ca2ϩ. Subsequent studies by using fragments en- transcription (3–6). TAF1, the largest subunit of TFIID is known compassing both TAF5-NTD1 and TAF5-NTD2 revealed that to harbor multiple enzymatic activities (7–9) and is also involved the small LisH homology domain at N terminus of TAF5 in chromatin transactions by using its double bromodomains to cooperates with TAF5-NTD2 to induce dimer formation of contact acetylated histone tails (10, 11). Furthermore, many of hTAF5 in a calcium independent manner. Our studies are the TAFs have been shown to be involved in interactions with gene specific activators and other general transcription factors either to stabilize the preinitiation complex (12) or to induce Author contributions: S.B. and R.H.J. designed research; S.B., S.T., and R.H.J. performed structural changes in them (13). TAFs are not only restricted to research; S.B., S.T., and R.H.J. contributed new reagents/analytic tools; R.H.J. analyzed data; the TFIID complex but also can be found in the yeast SAGA and S.B. and R.H.J. wrote the paper. (Spt-Ada-GCN5-acetyltransferase complex), human STAGA The authors declare no conflict of interest. (Spt3-TAF9-Ada-GCN5-acetyltransferase complex), PCAF Abbreviations: TBP, TATA box-binding protein; TAF, TBP-associated factor; TF, transcrip- tion factor; MAD, multiwavelength anomalous dispersion; NCS, noncrystallographic sym- (p300/CBP-associated factor), or TFTC (TBP-free TAFII- metry; CID, RNA polymerase CTD-interacting domain; DLS, dynamic light scattering; SEC, containing complex) (14–16). The TAFs within these multipro- size exclusion chromatography; SLS, static light scattering. tein complexes are involved in extensive protein-protein inter- Data deposition: The atomic coordinates and structure factors have been deposited in the actions and various studies indicate the emerging role of TAFs Protein Data Bank, www.rcsb.org (PDB ID code 2NXP). as cofactors with important functional properties required for *To whom correspondence should be addressed. E-mail: [email protected]. transcription (17, 18). This article contains supporting information online at www.pnas.org/cgi/content/full/ Low-resolution three-dimensional structures of the yeast and 0610297104/DC1. human TFIID complexes from electron microscopy and digital © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0610297104 PNAS ͉ January 23, 2007 ͉ vol. 104 ͉ no. 4 ͉ 1189–1194 Downloaded by guest on September 29, 2021 1 91 124 194 340 460 739 800 N NTD2 WD40 C a b NTD1/ LisH Fig. 1. Primary sequence organization of hTAF5. Schematic diagram of the domain structure present in human TAF5 where NTD1/LisH corresponds to LIS1 homology domain (29), NTD2 corresponds to the ␣-helical domain reported here, and WD40 repeats are predicted to form a closed ␤-propeller structure. consistent with previous EM analyses that suggested that the N terminus of TAF5 might play a critical role in organizing the c 205 To α2 three-lobed TFIID structure. We have also identified several 198 1 230 α surface pockets that may serve as binding sites for other TAFs 227 328 268 302 305 or transcriptional regulators. 273 η3 336 α7 Results 299 307 339 323 α4 β1 β2 The primary sequence of human TAF5 is shown schematically in α2 α3 α5 η2 α6 Fig. 1. All TAF5 proteins contain two evolutionary conserved 295 310 342 motifs in the N-terminal half (TAF5-NTD1 and TAF5-NTD2, 311 257 283 290 for the human TAF5 residues 90–124 and 194–340, respectively). 211 η1 From α1 The N-terminal region of TAF5 proteins from Drosophila and 253 288 humans share 31% sequence identity, whereas yeast and human Helical-Bundle Helical-Sheet TAF5 protein share 22% sequence identity. Several subfrag- ments of TAF5 N terminus were generated, purified and sub- Fig. 2. Structure of the hTAF5-NTD2 domain. (a) Diagram of the hTAF5- jected to limited proteolysis. Digestion of a fragment containing NTD2. (b) Top view of the ␣-helical domain of the hTAF5-NTD2 showing the residues 1–500 of the human TAF5 protein produced a proteo- arrangement of the other helices around the central helix (␣3) in the crystal lytically resistant fragment with an observed mass by SDS/PAGE structure. All of the helices and the ␤ strands are labeled. Two views in a and of Ϸ19 kDa. Liquid chromatography (LC)-MS unambiguously b are related by rotation of 90° around a horizontal axis. (c) Topology diagram identified this tryptic fragment as the TAF5-NTD2 region of the secondary structural elements of the hTAF5-NTD2 domain to show the arrangement of the helical bundle (front view) and the helical sheet (back (194–340). view) of the crystal structure. A construct corresponding to TAF5-NTD2 (residues 189–343) was expressed, purified, and crystallized as described in Materials and Methods. A multiwavelength anomalous dispersion (MAD) A key feature of the TAF5-NTD2 motif is the presence of a experiment was performed by using SeMet-labeled crystals dif- long central helix (␣3) that forms the core of the domain.
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