Fluorescence-based Analyses of the Effects of Full-length Recombinant TAF130p on the Interaction of TATA Box-binding Protein with TATA Box DNA

Abstract

We have used a combination of fluorescence anisotropy spectroscopy and fluorescence-based native gel electrophoresis methods to examine the effects of the transcription factor IID-specific subunit TAF130p (TAF145p) upon the TATA box DNA binding properties of TATA box-binding protein (TBP). Purified full-length recombinant TAF130p decreases TBP-TATA DNA complex formation at equilibrium by competing directly with DNA for binding to TBP. Interestingly, we have found that full-length TAF130p is capable of binding multiple molecules of TBP with nanomolar binding affinity. The biological implications of these findings are discussed.Eukaryotic DNA-dependent RNA polymerase II works in concert with the six general transcription factors (GTFs) 1 TFIIA, -B, -D, -E, -F, and -H to catalyze mRNA gene transcription (1). These components act either sequentially (2) or as a part of a holoenzyme complex (3, 4) to form a multicomponent preinitiation complex on promoter DNA. A highly conserved feature of most eukaryotic mRNA promoters is the TATA box element present 25 base pairs upstream from the transcription start site. TFIID, in combination with TFIIA and TFIIB, can recognize the TATA element and form a platform for subsequent preinitiation complex formation. The TATA box-binding protein (TBP), as its name suggests, is the protein within the 15-subunit TFIID holocomplex (5) that makes primary contact with the TATA element, though several TBP-associated factor (TAF) subunits comprising TFIID contribute to promoter binding (6 -8). Binding of TFIID to TATA elements is central to the control of transcription (9, 10).Recruiting TFIID to the promoter, a process thought to be mediated in part by direct activator-TFIID interactions, is probably a key and universal mechanism of gene activation (9, 10). However, the largest subunit of metazoan TFIID, which in Drosophila (d) or humans (h) exhibits an apparent molecular mass of ϳ250 kDa, termed d-or hTAF II 250, respectively (herein termed d/hTAF250p), also contains a number of intrinsic enzymatic activities including histone acetyltransferase (11), protein kinase (12), and ubiquitin activating/conjugating activity (13). Each of these activities could be targets for transactivators, and mutation of histone acetyltransferase, protein kinase, or ubiquitin activating/conjugating activity domains decreases transcription of subsets of genes in vivo (13-15).The yeast ortholog of d/hTAF250, TAF130p, is encoded by a single-copy essential gene (TAF130/TAF145; Refs. 16 and 17). Like its metazoan counterparts, the yeast protein, TAF130p, contains a histone acetyltransferase domain as well as a number of other essential sequences (18, 19) whose exact functions remain to be defined. One highly conserved and important element found in this (family of) TAF are domains capable of directly binding the TBP subunit of TFIID (17, 18, 20 -24). Both Drosophila TAF250p and yeast TAF130p appear to contain at least two TBP binding domains (18,23,25): a high affinity N-terminal TBP binding domain and a less well de...

The Transcriptional Repressor Activator Protein Rap1p Is a Direct Regulator of TATA-binding Protein

“…3A) inhibited TBP-Rap1p interaction. As expected from our previous work (58,59,68), all of the purified proteins ( Fig. 3B) avidly bound TBP (Fig.…”

“…These include NC2, TFIIA, TFIIB (77), Mbf1p (78), Spt3p, Spt8p, and Ada1p subunits of SAGA (79,80), TFIID Taf1p and TAND (58,59,81,82), Med8p and Med20p subunits of Mediator (83), Brf1p subunit of TFIIIB (84), TAF I 48 subunit of SL1 (85), N-terminal domain of Mot1p (86), SNAP190 subunit of SNAP C (87), and the NOT complex (88). Interestingly, many of these TBP-targeted factors both activate and repress transcription (76).…”

Two-step Mechanism for Modifier of Transcription 1 (Mot1) Enzyme-catalyzed Displacement of TATA-binding Protein (TBP) from DNA

“…3A). A fluorescent donor (Atto532) was attached to a unique cysteine residue at position 61 of TBP (59,60), and a Cy5 acceptor was conjugated to the 3Јend of the bottom strand containing the TATA box of the Gap DNA template (referred to as 3Ј-Cy5-GAP). The R 0 of Atto532-Cy5 is estimated to be ϳ65 Å (see "Experimental Procedures").…”

Molecular and Genetic Characterization of a Taf1p Domain Essential for Yeast TFIID Assembly

“…Because of these critical roles, the composition, organization, assembly, structure, and function of the TFIID complex have been topics of great interest. TBP appears to be incorporated into the TFIID complex primarily through its interaction with the bipartite N-terminal domain of Taf1p, the so-called TAND domain (5,7,36,37) comprised of TAND1 and TAND2 elements. Although Taf6p and Taf12p have also been shown to interact with TBP (58,60), the exact contribution of these TBP-TAF interactions to TFIID integrity remains to be determined.…”

“…Although Taf6p and Taf12p have also been shown to interact with TBP (58,60), the exact contribution of these TBP-TAF interactions to TFIID integrity remains to be determined. TBP binds to both isolated TAND and intact Taf1p with nanomolar affinity (5,7), and the structure of the TBP-TAND complex has been solved by nuclear magnetic resonance (41). The binding of TBP to Taf1p remains the best-defined direct interaction of TBP with a subunit of the TFIID complex.…”