The Gcn4p activation domain contains seven clusters of hydrophobic residues that make additive contributions to transcriptional activation in vivo. We observed efficient binding of a glutathione S-transferase (GST) Transcription initiation by RNA polymerase II (Pol II) requires assembly of a large complex consisting of Pol II and general transcription factors (GTFs) at the promoter. It has been proposed that assembly of this complex begins when TFIID, consisting of TATA box-binding protein (TBP) and its associated factors (TAF II proteins), binds to the core promoter, followed by sequential binding of other GTFs and Pol II itself (9). In another scenario, Pol II, certain GTFs, and coactivator proteins bind to the promoter as a preformed holoenzyme complex (46). Transcriptional activators bind to the promoter, generally upstream of the TATA element, and stimulate the assembly or function of the transcription initiation complex. Binding of TFIID to the core promoter appears to be rate limiting for initiation (12,43,88), and certain activators stimulate this step in initiation complex formation (3,11,21,39,40,50,91). Several activators bind TBP in vitro in a manner that depends on amino acids in the activation domain that are critical for transcriptional activation in vivo (7,11,26,35,38,51,(61)(62)(63), suggesting that direct interactions between the activator and TBP are involved in recruiting TFIID to the core promoter. Certain activation domains also bind TFIIB in vitro in a sequence-specific manner (4,7,14,41,56,91) and may stimulate recruitment of this GTF to the initiation complex (15,41,55,56).-Other studies suggest that activator function is mediated by one or more of the TAF II coactivator proteins associated with TBP in TFIID. Different activators may require specific TAF II proteins for activation (13,(74)(75)(76), and indeed, certain activation domains bind preferentially to specific TAF II proteins in vitro (24,37,57,83). The interactions between activators and TAF II proteins may serve primarily to recruit TFIID to the promoter (75). The human TAF II 250 subunit (and its Saccharomyces cerevisiae homolog yTAF II 130) has histone acetyltransferase (HAT) activity that may also promote initiation complex formation by destabilizing a repressive nucleosome structure at the promoter (64). A yeast Pol II-TAF II complex was shown to be required for transcriptional activation of a Gcn4p-regulated promoter in vitro (44); however, recent studies indicate that yTAF II proteins are not essential for transcriptional activation in vivo by Gcn4p and by several other yeast activator proteins (65,85).

We demonstrate, utilizing a temperature conditional mutant allele of the gene encoding TAF25p, that this non-histone-like TBP-associated factor, which is shared between the TFIID and SAGA complexes, is required for bulk mRNA gene transcription by RNA polymerase II in vivo. Immunoblotting experiments indicate that at the restrictive temperature, inactivation of TAF25p function results in a reduction of the levels of numerous TFIID and SAGA subunits, indicating its loss of function, like the histone-like TAFs, causes degradation of the constituents of these two multisubunit complexes. These data suggest that TAF25p plays a key structural role in maintaining TFIID and SAGA complex integrity. This is the first demonstration that a non-histone-like TAF is required for continuous, high level RNA polymerase II-mediated mRNA gene transcription in living yeast cells.There is ongoing debate about the exact role that TAF II s, 1 a family of conserved integral protein subunits of TFIID, play in transcription by RNA polymerase II (see Refs. 1 and 2 for recent reviews). In contrast to initial studies, recently published reports from a number of laboratories indicate that at least a subset of TAF II s, the so-called histone-like TAF II s (3), are absolutely essential for ongoing high level total mRNA gene transcription in vivo (4 -7). The histone-like TAF II s examined in these experiments are of particular interest, since it was also demonstrated recently that these TAF II s, TAF61p (histone H2B-like), TAF60p (histone H4-like), and TAF17p (histone H3-like), are present in both the TFIID and SAGA complexes (8).The data from these studies has been used to formulate a model that postulates that the histone-like TAF II s form the structural "core" for both TFIID and SAGA complexes and when the integrity of these particular TAF II s is compromised, then the complexes dissociate and cease to function while the resulting free subunits are rapidly degraded. At present it is not yet clear whether the RNAP II transcription requirement for histone-like TAF II function derives from the association of these TAF II s with the TFIID, the SAGA, or both complexes. Circumstantial evidence implicates the TFIID complex though, since all of the non-TAF II -encoding genes that comprise the SAGA complex (with the exception of TRA1 (9)) are non-essential genes, while (with but one exception, TAF30) all of the TFIID TAF II s are encoded by essential genes. This question remains to be formally tested though.Having previously cloned and characterized TAF25, we decided to attempt to generate temperature-conditional alleles of the TAF25 gene, which we could use as a tool to examine the role of TAF25p in RNAP II transcription. We felt it important to test whether or not the non-histone-like TAF II , TAF25p, which is present in both TFIID and SAGA, is globally involved in total mRNA gene transcription by RNA polymerase II in vivo. We successfully generated temperature-conditional alleles of TAF25 and one particular mutant allele caused yeast cells to rapid...

We describe the cloning and analysis of TAF25, a previously uncharacterized yeast gene that encodes a yeast TATA-binding protein-associated factor or yTAF of Mr = 25,000. The gene encoding yTAF25 is a single copy essential gene, and the protein sequence deduced from TAF25 exhibits sequence similarity to a metazoan hTAFII. The results from immunological studies confirm that yTAF25 is a subunit of a large multiprotein TATA-binding protein-yeast TATA-binding protein-associated factor complex that contains a subset of the total number of the yTAFs present in yeast cell extracts. Both genetic and biochemical analyses demonstrate that yTAF25 can interact directly with itself. Transcriptional data show that the activity of the multiprotein complex containing yTAF25 is RNA polymerase II-specific, thus indicating that TAF25 encodes a bona fide yeast RNA polymerase II TAF. Hence the protein encoded by TAF25 has been termed yTAFII25.

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 yeast transcriptional activator ADR1, which is required for ADH2 and other genes' expression, contains four transactivation domains (TADs). While previous studies have shown that these TADs act through GCN5 and ADA2, and presumably TFIIB, other factors are likely to be involved in ADR1 function. In this study, we addressed the question of whether TFIID is also required for ADR1 action. In vitro binding studies indicated that TADI of ADR1 was able to retain TAF II 90 from yeast extracts and TADII could retain TBP and TAF II 130/145. TADIV, however, was capable of retaining multiple TAF II s, suggesting that TADIV was binding TFIID from yeast whole-cell extracts. The ability of TADIV truncation derivatives to interact with TFIID correlated with their transcription activation potential in vivo. In addition, the ability of LexA-ADR1-TADIV to activate transcription in vivo was compromised by a mutation in TAF II 130/145. ADR1 was found to associate in vivo with TFIID in that immunoprecipitation of either TAF II 90 or TBP from yeast whole-cell extracts specifically coimmunoprecipitated ADR1. Most importantly, depletion of TAF II 90 from yeast cells dramatically reduced ADH2 derepression. These results indicate that ADR1 physically associates with TFIID and that its ability to activate transcription requires an intact TFIID complex.The derepression of the glucose-repressible ADH2 gene from Saccharomyces cerevisiae requires the transcriptional activator ADR1 (16). ADR1 binds to a 22-bp palindromic sequence-UAS1-located 215 bp upstream of the transcription start site of the ADH2 gene (47). ADR1 also regulates the transcription of genes involved in glycerol metabolism (5, 29) and peroxisome biogenesis, and sequences similar to UAS1 of the ADH2 gene are found in the promoters of these genes (7,36). Four regions of ADR1 have been identified that are required for its efficient activation of ADH2 transcription: transcription activation domain I (TADI) (residues 76 to 172), TADII (residues 263 to 357), TADIII (residues 420 to 462), and TADIV (residues 642 to 704) (5,9,12,14,39). TADII and TADIII are functionally redundant in the context of full-length ADR1 (12), suggesting that they may affect the same step in the process of transcriptional activation of the ADH2 gene. TADIV seems most important to the protein in that deletion of it reduces ADR1 function dramatically (9). In our earlier report, we had shown that individual activation domains of ADR1 can contact TFIIB, ADA2, and the histone acetyltransferase GCN5 in vitro (9). However, the deletion of ADA2 or GCN5 had only a moderate effect on the derepression of the ADH2 gene (9), suggesting the existence of additional activation mechanisms.There are a number of potential targets for ADR1 activation domains among the core transcription factors. TFIID, TFIIF, TFIIB, RNA polymerase II (polII), TFIIH, and TFIIE have been implicated in mammalian and drosophila systems as being direct contacts for various transcription activators (48). For example, the glutamine-rich activation...