TFIID is a multiprotein complex composed of TBP and several TAF II s. Small amino-terminal segments (TAF Nterminal domain (TAND)) ofYeast strains containing mutant yTAF II 145 lacking yTANDI or yTANDII showed a temperature-sensitive growth phenotype. The conserved core of dTANDII could substitute for the yTANDII core, and Phe-57 or Tyr-129 described above was critically required for the function of this segment in promoting normal cell growth at 37°C. In these respects, the impact of yTAN-DII mutations on cell growth paralleled their effects on TBP binding in vitro, strongly suggesting that the yTAF II 145-TBP interaction and its negative effects on TFIID binding to core promoters are physiologically important.Transcription of protein coding genes in eukaryotes is carried out by RNA polymerase II and a set of auxiliary initiation factors (1, 2). These factors, including TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, can be assembled in a combinatorial fashion in vitro to form a preinitiation complex. Recently, it was proposed that most of these factors are preassembled in vivo in the form of holoenzyme and recruited as a single complex to the core promoter to initiate transcription (3, 4). Barberis et al. (5) reported that recruitment of holoenzyme via a fortuitous interaction between GAL4 DNA binding domain and GAL11 or by fusing lexA to GAL11 would suffice for gene activation in Saccharomyces cerevisiae. A similar result was obtained for SRB2, another component of holoenzyme (6). On the other hand, there is evidence that TBP binding to the TATA box is also a rate-limiting step for transcriptional activation that can be accelerated by gene-specific activators (7). In fact, a physical connection of TBP to a DNA binding module bypasses the requirement for activators (8 -10). Given that TBP is a subunit of TFIID and not a component of holoenzyme (11), it appears that recruitment of either TFIID or holoenzyme will suffice for gene activation in yeast (12). However, it is notable that TFIID is required for both cases because mutation of the TATA sequence greatly decreased activation even by holoenzyme recruitment (5).In higher eukaryotes, the question of how activators stimulate transcription has been addressed mostly by biochemical approaches. Particular attention has focused on TFIID, a multiprotein complex composed of TBP and a series of TBP-associated factors (TAF II s), because TAF II s were shown to be indispensable for activated transcription in vitro (13,14). We and others cloned cDNAs encoding TAF II s from various organisms to decipher the molecular basis of transcriptional regulation (15). It is currently known that TAF II s possess some intriguing structural motifs and/or enzymatic activities. For instance, dTAF II 62/dTAF II 42 forms a histone octamer-like heterotetrameric structure (16). dTAF II 230 has multiple enzymatic activities, including a protein serine kinase activity that selectively phosphorylates RAP74 (17) and a histone acetyltransferase activity specific for histones H3 and H4 (18). Furthe...

The general transcription factor TFIID, which is composed of TATA-binding protein (TBP) and an array of TBP-associated factors (TAFs), has been shown to play a crucial role in recognition of the core promoters of eukaryotic genes. We isolated Saccharomyces cerevisiae yeast TAF145 (yTAF145) temperature-sensitive mutants in which transcription of a specific subset of genes was impaired at restrictive temperatures. The set of genes affected in these mutants overlapped with but was not identical to the set of genes affected by a previously reported yTAF145 mutant (W.-C. Shen and M. R. Green, Cell 90:615-624, 1997). To identify sequences which rendered transcription yTAF145 dependent, we conducted deletion analysis of the TUB2 promoter using a novel mini-CLN2 hybrid gene reporter system. The results showed that the yTAF145 mutations we isolated impaired core promoter recognition but did not affect activation by any of the transcriptional activators we tested. These observations are consistent with the reported yTAF145 dependence of the CLN2 core promoter in the mutant isolated by Shen and Green, although the CLN2 core promoter functioned normally in the mutants we report here. These results suggest that different promoters require different yTAF145 functions for efficient transcription. Interestingly, insertion of a canonical TATA element into the TATA-less TUB2 promoter rescued impaired transcription in the yTAF145 mutants we studied. It therefore appears that strong binding of TBP to the core promoter can alleviate the requirement for at least one yTAF145 function.In eukaryotes, transcriptional initiation by RNA polymerase II requires a set of general transcriptional factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) (reviewed in references 16, 72, and 78) and the SRB-MED complex associated with the carboxy-terminal domain of RNA polymerase II (reviewed in references 65 and 66). These factors nucleate on the core promoter of eukaryotic class II genes to form a preinitiation complex in an ordered stepwise fashion (reviewed in references 9, 23, and 78) or are recruited in a simpler sequence involving a small number of preassembled units (reviewed in references 43 and 73). In either case, the first step, which is the sequence-specific binding of TFIID (76), is thought to be a major rate-limiting step during transcription and a focal point for the activity of transcriptional activators (14,40,54).TFIID is a multiprotein complex composed of TATA-binding protein (TBP) and an array of TBP-associated factors (TAFs); in total, the complex includes 8 to 12 molecules ranging in size from 15 to 250 kDa (reviewed in references 11, 51, 91, and 93). Almost all of these TAFs are conserved among evolutionarily divergent organisms (humans, Drosophila melanogaster, and Saccharomyces cerevisiae), albeit with a few exceptions (for instance, no orthologue of Drosophila TAF110 or human TAF130 (dTAF110/hTAF130) is found in yeast). This level of conservation suggests that TAFs play a fundamental role in eukaryotic transcription (review...

We demonstrate that the yeast flocculation gene, FLO1, is representative of a distinct subset of subtelomeric genes that are robustly repressed by the Cyc8–Tup1 complex. We have examined Cyc8–Tup1 localisation, histone acetylation and long-range chromatin remodelling within the extensive FLO1 upstream region. We show that Cyc8–Tup1 is localised in a DNase I hypersensitive site within an ordered array of strongly positioned nucleosomes around − 700 base pairs upstream of the transcription start site. In cyc8 deletion mutant strains, Tup1p localisation is absent, with concomitant histone hyperacetylation of adjacent regions at the FLO1 promoter. This is accompanied by extensive histone depletion across the upstream region and gene activation. The yeast histone deacetylases, Hda1p and Rpd3p, occupy the repressed FLO1 promoter region in a Cyc8–Tup1 dependent manner and coordinate histone deacetylation, nucleosome stabilisation and gene repression. Moreover, we show that the ATP-dependent chromatin remodelling complex Swi–Snf occupies the site vacated by Cyc8–Tup1 in a cyc8 mutant. These data suggest that distinctly bound Cyc8–Tup1 cooperates with Hda1p and Rpd3p to establish or maintain an extensive array of strongly positioned, deacetylated nucleosomes over the FLO1 promoter and upstream region which inhibit histone acetylation, block Swi–Snf binding and prevent transcription.

The general transcription factor TFIID has been shown to be involved in both core promoter recognition and the transcriptional activation of eukaryotic genes. We recently isolated TAF145 (one of TFIID subunits) temperature-sensitive mutants in yeast, in which transcription of the TUB2 gene is impaired at restrictive temperatures due to a defect in core promoter recognition. Here, we show in these mutants that the transcription of the RPS5 gene is impaired, mostly due to a defect in transcriptional activation rather than to a defect in core promoter recognition, although the latter is slightly affected as well. Surprisingly, the RPS5 core promoter can be activated by various activation domains fused to a GAL4 DNA binding domain, but not by the original upstream activating sequence (UAS) of the RPS5 gene. In addition, a heterologous CYC1 core promoter can be activated by RPS5-UAS at normal levels even in these mutants. These observations indicate that a distinct combination of core promoters and activators may exploit alternative activation pathways that vary in their requirement for TAF145 function. In addition, a particular function of TAF145 that is deleted in our mutants appears to be involved in both core promoter recognition and transcriptional activation.In eukaryotes, transcriptional initiation by RNA polymerase II requires a set of general transcriptional factors (reviewed in Refs. 1-3). These factors are assembled in a stepwise manner to form a preinitiation complex on the core promoter (1) or are recruited as a few preassembled units (4 -6). In either case, the first step in preinitiation complex assembly is the binding of a protein complex called TFIID to the core promoter, which in turn provides a structural platform for the remainder of the general transcriptional factors to be incorporated (6). Previous studies have shown that TFIID-promoter interactions are a major rate-limiting step during transcriptional initiation and therefore are one of the most important molecular targets for transcriptional activators (7-9).TFIID is a multiprotein complex composed of the TATAbinding protein (TBP) 1 and ϳ10 -12 phylogenetically conserved TBP-associated factors (TAFs) (reviewed in Refs. 9 and 10). A number of biochemical studies have revealed coactivator and core promoter recognition activities to be two important functions for TAFs (reviewed in Refs. 9 -11). Earlier experiments using in vitro transcription systems demonstrated that TBP can mediate basal transcription but is unable to support activated transcription by itself. In contrast, TFIID, even when reconstituted with recombinant TBP and TAFs (12), mediates both basal and activated transcription, supporting the idea that TAFs are essential cofactors for transcriptional activation (reviewed in Refs. 9 and 10). More recent studies have begun to address how core promoters of eukaryotic genes are recognized by TFIID (reviewed in Refs. 13 and 14). The three classes of core promoter elements that are currently known are the TATA element, the initiator, and ...