Structure-Function Analysis of TAF130: Identification and Characterization of a High-Affinity TATA-Binding Protein Interaction Domain in the N Terminus of Yeast TAF_II130

Abstract

TFIID, one of the multiple eukaryotic general transcription factors (GTFs), plays a key role in DNA-dependent RNA polymerase II (RNAP II)-mediated transcription initiation and regulation. The form and function of TFIID have been extensively studied by using in vitro approaches (see references 7, 22, and 57 for recent reviews). TFIID exists as a stable multisubunit complex in a variety of distinct eukaryotic systems (7,17,22,47,57,92), and in vitro transcription assays have shown that one subunit of TFIID, the TATA-binding protein (TBP), is sufficient for TATA element DNA recognition and subsequent incorporation of the other GTFs and RNAP II into the preinitiation complex (PIC). A TBP-assembled PIC can catalyze basal transcription in vitro (6, 67). However, these same in vitro assays also demonstrate that activation of transcription by sequence-specific DNA binding transactivator proteins can be observed only from a PIC formed by utilizing the multisubunit TBP-containing complex, TFIID, and not with TBP (32, 64). This observation suggested that the other subunits of TFIID are essential for its regulatory functions and ultimately led to the identification of the TBP-associated factors (TAFs), which in combination with TBP comprise the TFIID complex (17,47,64,92).At least 8 to 10 RNAP II-specific TAFs (or TAF II s) associate with TBP to form eukaryotic TFIID. These TAF II s exhibit molecular masses ranging from 250 to 15 kDa, depending on the organism analyzed (human, Drosophila melanogaster, and Saccharomyces cerevisiae) (17,47,61,66,92; see reference 7 for a recent review). A comparison of the amino acid sequences of TAF II s from these evolutionarily divergent organisms shows that there is a striking conservation of TAF II sequences (7). The results of various biochemical analyses have led to the hypothesis that TAF II s participate in a variety of protein-protein and protein-DNA interactions. These interactions range from the facilitation of the formation and/or stabilization of the PIC by TAF II binding to DNA (78,82,83) to TAF II s interacting with each other (7, 29, 30, 33, 34, 37, 41-46, 53, 71, 79, 80, 83, 86, 87, 89, 91) or with basal transcription factors (7,23,26,30,42,90) to the direct interaction of TAF II s with the activation domains of transcriptional regulatory molecules (9, 11, 12, 19-21, 23, 31, 37, 42, 51, 72, 75, 80, 81, 86). Clearly, TAF II s are involved in a large number of critical regulatory events in RNAP II transcription, and detailed analyses of TAF II molecules will shed light on the molecular mechanisms of RNAP II transcriptional regulation.The largest subunit of metazoan TFIID has been termed either hTAF II 250 or dTAF II 250, depending on whether it is a human or Drosophila protein. The human protein, hTAF II 250, contains an acidic N terminus, a central region including a high-mobility-group (HMG) homology box, and two bromodomain-like direct repeats, as well as a glycine-and serine-rich C terminus (29, 71). The Drosophila homolog, dTAF II 250, has Ͼ90% similarity and Ͼ50% iden...

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

“…Second, Taf1p contains separate domains for binding TBP and various Tafps. Third, within the TFIID complex, Taf1p subserves more than a simple structural role because mutants ⌬8Taf1p to ⌬16Taf1p formed TFIID "holocomplexes" with normal or nearly normal apparent stoichiometry, yet these "TFIID complexes" were unable to support cell growth (5).…”

“…For co-IP assays, yeast cells were grown either in YPAD medium or in appropriate synthetic complete (SC) selective medium lacking either histidine (SC-H), leucine (SC-L), or uracil (SC-U) as indicated. Co-IP experiments were performed as described previously (5). Table 1 lists the strains used in our experiments.…”

“…The PCR-generated library of region 4 fragments was digested with NotI and ligated into the NotI site of the HIS3-marked pRS313-HA 3 -TAF1⌬4 plasmid; this plasmid carries an allele of TAF1 that has a NotI site introduced at the site of deletion (5). This construct was modified to remove the NotI site at the multiple cloning site of pRS313 by site-directed mutagenesis prior to reinsertion of region 4 sequences.…”

“…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.…”