The transcription initiation factor TFIID is a multimeric protein complex composed of TATA box-binding protein (TBP) and many TBP-associated factors (TAF(II)s). TAF(II)s are important cofactors that mediate activated transcription by providing interaction sites for distinct activators. Here, we present evidence that human TAF(II)250 and its homologs in Drosophila and yeast have histone acetyltransferase (HAT) activity in vitro. HAT activity maps to the central, most conserved portion of dTAF(II)230 and yTAF(II)130. The HAT activity of dTAF(II)230 resembles that of yeast and human GCN5 in that it is specific for histones H3 and H4 in vitro. Our findings suggest that targeted histone acetylation at specific promoters by TAF(II)250 may be involved in mechanisms by which TFIID gains access to transcriptionally repressed chromatin.

A complex of two TFIID TATA box-binding protein-associated factors (TA FIIs) is described at 2.0A resolution. The amino-terminal portions of dTAFII42 and dTAFII62 from Drosophila adopt the canonical histone fold, consisting of two short alpha-helices flanking a long central alpha-helix. Like histones H3 and H4, dTAFII42 and dTAFII62 form an intimate heterodimer by extensive hydrophobic contacts between the paired molecules. In solution and in the crystalline state, the dTAFII42/dTAFII62 complex exists as a heterotetramer, resembling the (H3/H4)2 heterotetrameric core of the histone octamer, suggesting that TFIID contains a histone octamer-like substructure.

The Drosophila 230-kDa TFIID subunit (dTAF230) interacts with the DNA binding domain of TATA boxbinding protein (TBP) which exists in the same complex. Here, we characterize the inhibitory domain in the yeast TAF145 (yTAF145), which is homologous to dTAF230. Mutation studies show that the N-terminal inhibitory region (residues 10 to 71) can be divided into two subdomains, I (residues 10 to 37) and II (residues 46 to 71). Mutations in either subdomain significantly impair function. Acidic residues in subdomain II are important for the interaction with TBP. In addition, yTAF145 interaction is impaired by mutating the basic residues on the convex surface of TBP, which are crucial for interaction with TFIIA. Consistently, TFIIA and yTAF145 bind competitively to TBP. A deletion of the inhibitory domain of yTAF145 leads to a temperaturesensitive growth phenotype. Importantly, this phenotype is suppressed by overexpression of the TFIIA subunits, indicating that the yTAF145 inhibitory domain is involved in TFIIA function.Transcription factor TFIID is a multisubunit protein complex found in various organisms including Drosophila melanogaster (17, 40), human (12,59,61,74), and more recently, the budding yeast Saccharomyces cerevisiae (23,(52)(53)(54). Holo-TFIID is composed of the highly conserved TATA box-binding polypeptide protein (TBP) and a number of associated polypeptides (TBP-associated factors [TAFs]). In vitro transcription studies revealed an important functional difference between holo-TFIID and TBP. Holo-TFIID mediates activator regulated transcription, whereas TBP itself mediates only basal levels of transcription. Thus, at least one or more TAFs included in holo-TFIID are essential for transmitting signals from various activators to the basal transcriptional machinery. Within the past 5 years, cDNAs encoding TAFs have been cloned from

In-cell NMR is an application of solution NMR that enables the investigation of protein conformations inside living cells. We have measured in-cell NMR spectra in oocytes from the African clawed frog Xenopus laevis. (15)N-labeled ubiquitin, its derivatives and calmodulin were injected into Xenopus oocytes and two-dimensional (1)H-(15)N correlation spectra of the proteins were obtained. While the spectrum of wild-type ubiquitin in oocytes had rather fewer cross-peaks compared to its in vitro spectrum, ubiquitin derivatives that are presumably unable to bind to ubiquitin-interacting proteins gave a markedly larger number of cross-peaks. This observation suggests that protein-protein interactions between ubiquitin and ubiquitin-interacting proteins may cause NMR signal broadening, and hence spoil the quality of the in-cell HSQC spectra. In addition, we observed the maturation of ubiquitin precursor derivative in living oocytes using the in-cell NMR technique. This process was partly inhibited by pre-addition of ubiquitin aldehyde, a specific inhibitor for ubiquitin C-terminal hydrolase (UCH). Our work demonstrates the potential usefulness of in-cell NMR with Xenopus oocytes for the investigation of protein conformations and functions under intracellular environmental conditions.

HMO1 is a high-mobility group B protein that plays a role in transcription of genes encoding rRNA and ribosomal proteins (RPGs) in Saccharomyces cerevisiae. This study uses genome-wide chromatin immunoprecipitation to study the roles of HMO1, FHL1, and RAP1 in transcription of these genes as well as other RNA polymerase II-transcribed genes in yeast. The results show that HMO1 associates with the 35S rRNA gene in an RNA polymerase I-dependent manner and that RPG promoters (138 in total) can be classified into several distinct groups based on HMO1 abundance at the promoter and the HMO1 dependence of FHL1 and/or RAP1 binding to the promoter. FHL1, a key regulator of RPGs, binds to most of the HMO1-enriched and transcriptionally HMO1-dependent RPG promoters in an HMO1-dependent manner, whereas it binds to HMO1-limited RPG promoters in an HMO1-independent manner, irrespective of whether they are transcribed in an HMO1-dependent manner. Reporter gene assays indicate that these functional properties are determined by the promoter sequence.The yeast ribosome is composed of four rRNAs and 79 ribosomal proteins (RPs) (58, 73). Yeast rRNA genes occur as a tandem repeat of approximately 150 copies. The 25S, 18S, and 5.8S RNAs are transcribed by RNA polymerase I (Pol I), 5S RNA is transcribed by RNA Pol III, and the 138 RP genes (RPGs) are transcribed by RNA Pol II (58,73). In a rapidly growing cell, transcription of rRNA and RPGs accounts for approximately 60% of total transcription and 50% of Pol IImediated transcription, respectively (73), representing a large fraction of the total energy consumption of the cell. Little is known about the regulatory mechanisms involved in coordinating the transcription of rRNA and RPGs under various growth conditions. Recent studies showed that the TOR complex 1 (TORC1) plays a central role in regulating transcription of rRNA and RPGs in response to changes in the abundance of extracellular nutrients (44). Under favorable nutrient conditions, TORC1 is localized to the nucleus and directly binds to the 35S rDNA promoter to activate transcription by Pol I (36). TORC1 also indirectly regulates Pol II-mediated RPG transcription by recruiting IFH1, a coactivator for FHL1 (45,58,60,72). FHL1 was originally identified as a suppressor of a Pol III mutant (24) and was later shown to be important for RPG transcription (27,34,45,58,60,72). Under poor nutrient conditions, TORC1 is exported from the nucleus to the cytoplasm, so that the synthesis of 35S rRNA is substantially diminished (36). Concurrently, CRF1, a corepressor for FHL1, displaces IFH1 from RPG promoters to inhibit RPG transcription (45).HMO1 is a member of the high-mobility group B (HMGB) protein family, which include nonhistone proteins that bind to and have diverse roles in eukaryotic chromatin. HMGB proteins contain one or more distinctive DNA-binding motifs known as "HMG boxes" (11, 69). The HMG box is a conserved protein structural motif, in which three alpha helices are arranged in an L shape (55, 74). As the HMG box domain binds...

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...

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).