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Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is one of the most devastating diseases in citrus industry worldwide. Most citrus cultivars such as sweet orange are susceptible to canker disease. Here, we utilized wild citrus to identify canker-resistant germplasms, and found that Atalantia buxifolia, a primitive (distant-wild) citrus, exhibited remarkable resistance to canker disease. Although the susceptibility gene LATERAL ORGAN BOUNDARIES 1 (LOB1) could also be induced in Atalantia after canker infection, the induction extent was far lower than that in sweet orange. In addition, three of amino acids encoded by transcription factor TFIIAγ in Atalantia (AbTFIIAγ) exhibited difference from those in sweet orange (CsTFIIAγ) which could stabilize the interaction between effector PthA4 and effector binding element (EBE) of LOB1 promoter. The mutation of AbTFIIAγ did not change its interaction with transcription factor binding motifs (TFBs). However, the AbTFIIAγ could hardly support the LOB1 expression induced by the PthA4. In addition, the activity of AbLOB1 promoter was significantly lower than that of CsLOB1 under the induction by PthA4. Our results demonstrate that natural variations of AbTFIIAγ and effector binding element (EBE) in the AbLOB1 promoter are crucial for the canker disease resistance of Atalantia. The natural mutations of AbTFIIAγ gene and AbLOB1 promoter in Atalantia provide candidate targets for improving the resistance to citrus canker disease., Author summary It has been well documented that most citrus cultivars are susceptible to canker disease, while little is known about the resistance or susceptibility of primitive or wild citrus to canker disease. This study reveals that primitive citrus (Atalantia buxifolia) is highly resistant to citrus canker. Transcriptome data demonstrated that Atalantia had an active resistance response to the infection of Xcc, compared with susceptible sweet orange. Our results indicated that natural variations of AbTFIIAγ gene and AbLOB1 promoter contributed to the resistance. Hence, we propose that the natural mutations of AbTFIIAγ gene and AbLOB1 promoter could provide candidate targets for breeding canker resistant citrus.

Cell cycle progression depends on phase-specific gene expression. Here we show that the nuclear RNA degradation machinery plays a lead role in promoting cell cycle-dependent gene expression by triggering promoter-dependent co-transcriptional RNA degradation. Single molecule quantification of RNA abundance in different phases of the cell cycle indicates that relative curtailment of gene expression in certain phases is attained even when transcription is not completely inhibited. When nuclear ribonucleases are deleted, transcription of the Saccharomyces cerevisiae G1-specific axial budding gene AXL2 is detected throughout the cell cycle and its phase-specific expression is lost. Promoter replacement abolished cell cycle-dependent RNA degradation and rendered the RNA insensitive to the deletion of nuclear ribonucleases. Together the data reveal a model of gene regulation whereby RNA abundance is controlled by promoter-dependent induction of RNA degradation., Catala and Abou Elela show that the nuclear RNA degradation is executed in a promoter-specific manner, ensuring cell cycle-dependent gene expression. This study illustrates that local gene expression can be temporarily regulated without affecting overall transcription.

Transcription initiation can be reconstituted from highly purified general transcription factors (GTFs), RNA polymerase II (pol II), and promoter DNA. However, earlier biochemical reconstitution systems had a serious technical limitation, namely very poor initiation efficiency. Due to the poor efficiency of the reaction and trace amounts of proteins involved in the pre-initiation complex (PIC) assembly, detection of transcription and PIC formation was only possible by the synthesis of a radiolabeled transcript and by immunoblotting for PIC components on templates. Here we describe a transcription system that is capable of initiating transcription with >90% efficiency of template usage using homogeneous, active yeast components including TFIIA, TFIIB, TBP, TFIIE, TFIIF, TFIIH, Sub1, and pol II. The abundant specifically assembled PICs on promoter DNA can be separated from free general transcription factors (GTFs) and pol II by density gradient sedimentation, irrespective of the length of promoter DNA. The system is robust, and can be modified to accommodate many other transcription factors, and the resulting complexes can be analyzed by SDS-PAGE followed by Coomassie Blue staining. This technical advance now paves the way to conduct definitive biochemical and structural studies of the complete process of pol II initiation from the PIC, through promoter escape, and finally to productive elongation.

Transcription factor TFIIA is controlled by complex regulatory networks including proteolysis by the protease Taspase 1, though the full impact of cleavage remains elusive. Here, we demonstrate that in contrast to the general assumption, de novo produced TFIIA is rapidly confined to the cytoplasm via an evolutionary conserved nuclear export signal (NES, amino acids 21VINDVRDIFL30), interacting with the nuclear export receptor Exportin-1/chromosomal region maintenance 1 (Crm1). Chemical export inhibition or genetic inactivation of the NES not only promotes TFIIA's nuclear localization but also affects its transcriptional activity. Notably, Taspase 1 processing promotes TFIIA's nuclear accumulation by NES masking, and modulates its transcriptional activity. Moreover, TFIIA complex formation with the TATA box binding protein (TBP) is cooperatively enhanced by inhibition of proteolysis and nuclear export, leading to an increase of the cell cycle inhibitor p16INK, which is counteracted by prevention of TBP binding. We here identified a novel mechanism how proteolysis and nuclear transport cooperatively fine-tune transcriptional programs.

Head and neck cancer (HNC) is the seventh most common malignancy in the world and its prevailing form, the head and neck squamous cell carcinoma (HNSCC), is characterized as aggressive and invasive cancer type. The transcription factor II A (TFIIA), initially described as general regulator of RNA polymerase II-dependent transcription, is part of complex transcriptional networks also controlling mammalian head morphogenesis. Posttranslational cleavage of the TFIIA precursor by the oncologically relevant protease Taspase1 is crucial in this process. In contrast, the relevance of Taspase1-mediated TFIIA cleavage during oncogenesis of HNSCC is not characterized yet. Here, we performed genome-wide expression profiling of HNSCC which revealed significant downregulation of the TFIIA downstream target CDKN2A. To identify potential regulatory mechanisms of TFIIA on cellular level, we characterized nuclear-cytoplasmic transport and Taspase1-mediated cleavage of TFIIA variants. Unexpectedly, we identified an evolutionary conserved nuclear export signal (NES) counteracting nuclear localization and thus, transcriptional activity of TFIIA. Notably, proteolytic processing of TFIIA by Taspase1 was found to mask the NES, thereby promoting nuclear localization and transcriptional activation of TFIIA target genes, such as CDKN2A. Collectively, we here describe a hitherto unknown mechanism how cellular localization and Taspase1 cleavage fine-tunes transcriptional activity of TFIIA in HNSCC.

TATA-binding protein-like protein (TLP) binds to transcription factor IIA (TFIIA) with high affinity, although the significance of this binding is poorly understood. In this study, we investigated the role of TFIIA in transcriptional regulation of the p21Waf1/Cip1 (p21) gene. It has been shown that TLP is indispensable for p53-activated transcription from an upstream TATA-less promoter of the p21 gene. We found that mutant TLPs having decreased TFIIA-binding ability exhibited weakened transcriptional activation function for the upstream promoter. Activity of the upstream promoter was enhanced considerably by an increased amount of TFIIA in a p53-dependent manner, whereas activity of the TATA-containing downstream promoter was enhanced only slightly. TFIIA potentiated the upstream promoter additively with TLP. Although TFIIA is recruited to both promoters, activity of the upstream promoter was much more dependent on TFIIA. Recruitment of TFIIA and TLP to the upstream promoter was augmented in etoposide-treated cells, in which the amount of TFIIA–TLP complex is increased, and TFIIA-reactive TLP was required for the recruitment of both factors. It was confirmed that etoposide-stimulated transcription depends on TLP. We also found that TFIIA-reactive TLP acts to decrease cell growth rate, which can be explained by interaction of the p21 promoter with the transcription factors that we examined. The results of the present study suggest that the upstream TATA-less promoter of p21 needs TFIIA and TFIIA-reactive TLP for p53-dependent transcriptional enhancement. Structured digital abstract TLP physically interacts with TFIIA beta and TFIIA alpha by anti tag coimmunoprecipitation (View interaction) TFIIA alpha/beta physically interacts with TLP by anti bait coip (View interaction)

Mammalian genomes encode two genes related to the TATA-box binding protein (TBP), TBP-related factors 2 and 3 (TRF2 and TRF3). Male Trf2−/− mice are sterile and characterized by arrested spermatogenesis at the transition from late haploid spermatids to early elongating spermatids. Despite this characterization, the molecular function of murine Trf2 remains poorly characterized and no direct evidence exists to show that it acts as a bona fide chromatin-bound transcription factor. We show here that Trf2 forms a stable complex with TFIIA or the testis expressed paralogue ALF chaperoned in the cytoplasm by heat shock proteins. We demonstrate for the first time that Trf2 is recruited to active haploid cell promoters together with Tbp, Taf7l and RNA polymerase II. RNA-seq analysis identifies a set of genes activated in haploid spermatids during the first wave of spermatogenesis whose expression is down-regulated by Trf2 inactivation. We therefore propose that Trf2 is recruited to the preinitiation complex as a testis-specific subunit of TFIIA/ALF that cooperates with Tbp and Taf7l to promote haploid cell gene expression.

Saccharomyces cerevisiae HMO1, a high mobility group B (HMGB) protein, associates with the rRNA locus and with the promoters of many ribosomal protein genes (RPGs). Here, the Sos recruitment system was used to show that HMO1 interacts with TBP and the N-terminal domain (TAND) of TAF1, which are integral components of TFIID. Biochemical studies revealed that HMO1 copurifies with TFIID and directly interacts with TBP but not with TAND. Deletion of HMO1 (Deltahmo1) causes a severe cold-sensitive growth defect and decreases transcription of some TAND-dependent genes. Deltahmo1 also affects TFIID occupancy at some RPG promoters in a promoter-specific manner. Interestingly, over-expression of HMO1 delays colony formation of taf1 mutants lacking TAND (taf1DeltaTAND), but not of the wild-type strain, indicating a functional link between HMO1 and TAND. Furthermore, Deltahmo1 exhibits synthetic growth defects in some spt15 (TBP) and toa1 (TFIIA) mutants while it rescues growth defects of some sua7 (TFIIB) mutants. Importantly, Deltahmo1 causes an upstream shift in transcriptional start sites of RPS5, RPS16A, RPL23B, RPL27B and RPL32, but not of RPS31, RPL10, TEF2 and ADH1, indicating that HMO1 may participate in start site selection of a subset of class II genes presumably via its interaction with TFIID.

Binding of the TATA-binding protein (TBP) to the promoter is the first and rate limiting step in the formation of transcriptional complexes. We show here that nuclear import of TBP is mediated by a new karyopherin (Kap) (importin) family member, Kap114p. Kap114p is localized to the cytoplasm and nucleus. A complex of Kap114p and TBP was detected in the cytosol and could be reconstituted using recombinant proteins, suggesting that the interaction was direct. Deletion of the KAP114 gene led to specific mislocalization of TBP to the cytoplasm. We also describe two other potential minor import pathways for TBP. Consistent with other Kaps, the dissociation of TBP from Kap114p is dependent on RanGTP. However, we could show that double stranded, TATA-containing DNA stimulates this RanGTP-mediated dissociation of TBP, and is necessary at lower RanGTP concentrations. This suggests a mechanism where, once in the nucleus, TBP is preferentially released from Kap114p at the promoter of genes to be transcribed. In this fashion Kap114p may play a role in the intranuclear targeting of TBP.

TFIIB recognizes DNA sequence-specific motifs that can flank the TATA elements of the promoters of protein-encoding genes. The TFIIB recognition elements (BRE(u) and BRE(d)) can have positive or negative effects on transcription in a promoter context-dependent manner. Here we show that the BREs direct the selective recruitment of TFIIA and NC2 to the promoter. We find that TFIIA preferentially associates with BRE-containing promoters while NC2 is recruited to promoters that lack consensus BREs. The functional relevance of the BRE-dependent recruitment of TFIIA and NC2 was determined by small interfering RNA-mediated knockdown of TFIIA and NC2, both of which elicited BRE-dependent effects on transcription. Our results confirm the established functional reciprocity of TFIIA and NC2. However, our findings show that TFIIA assembly at BRE-containing promoters results in reduced transcriptional activity, while NC2 acts as a positive factor at promoters that lack functional BREs. Taken together, our results provide a basis for the selective recruitment of TFIIA and NC2 to the promoter and give new insights into the functional relationship between core promoter elements and general transcription factor activity.

International audience; Core promoter regions of protein-coding genes in metazoan genomes are structurally highly diverse and can contain several distinct core promoter elements, which direct accurate transcription initiation and determine basal promoter strength. Diversity in core promoter structure is an important aspect of transcription regulation in metazoans as it provides a basis for gene-selective function of activators and repressors. The basal activity of TATA box-containing promoters is dramatically enhanced by the initiator element (INR), which can function in concert with the TATA box in a synergistic manner. Here we report that a functional INR provides resistance to NC2 (Dr1/DRAP1), a general repressor of TATA promoters. INR-mediated resistance to NC2 is established during transcription initiation complex assembly and requires TBP-associated factors (TAFs) and TAF- and INR-dependent cofactor activity. Remarkably, the INR appears to stimulate TATA-dependent transcription similar to activators by strongly enhancing recruitment of TFIIA and TFIIB and, at the same time, by compromising NC2 binding.

Chromatin structure limits the accessibility of DNA sequences in eukaryotic chromosomes. Accessibility is enhanced through three major processes in vivo. First, posttranslational histone modifications either change the properties of the chromatin structure or create binding sites for other transcription factors (50). These posttranslational modifications include phosphorylation of serine residues and acetylation, methylation, and ubiquitylation of lysine residues (11). Several transcription factors that recognize specific histone modifications for their recruitment have been described. For example, the bromodomain-containing proteins recognize acetylation of histone proteins (61), and chromodomain-containing proteins are involved in recognizing methylation marks on histone proteins (6, 11). Second, ATP-dependent chromatin-remodeling factors promote accessibility by repositioning nucleosomes (9, 21, 55). These factors utilize the energy from ATP hydrolysis to establish or disrupt repressive chromatin structures (21). The third way by which the DNA sequence is made available is through ATP-independent chromatin-reorganizing factors that change the properties of nucleosomes in a localized manner (17). For example, the yFACT complex changes the properties of nucleosomes without requiring ATP hydrolysis (39, 41). The reorganization by yFACT has been shown to alter DNA accessibility both in vivo and in vitro (5, 14, 17, 30, 44, 45). The FACT complex (facilitates chromatin transcription) was first identified as a factor that promoted RNA polymerase II (Pol II) transcription in vitro using assembled chromatin as a template (33). The mammalian FACT complex is composed of two subunits, p140 and SSRP1. The homologs of p140 and SSRP1 in yeast are Spt16 and Pob3, respectively (34). The Spt16 and Pob3 proteins are always present in a heterodimer to form the SP complex in yeast (57). Although the N-terminal DNA binding domain of SSRP1 is absent in Pob3, Nhp6, a high-mobility group protein, is thought to serve as the DNA binding activity of the SP complex to form the yFACT complex (7, 18). Genetic and biochemical evidence suggests that yFACT is involved in regulating both transcription and DNA replication (1a, 3, 5, 18, 19, 27, 30, 34, 42, 56, 57). While the association of yFACT with elongation factors (27, 45) and with transcribed regions of genes (30, 42) supports an elongation role, studies also suggest that the FACT complex has a role in transcription initiation (5, 44). We have shown earlier that yFACT has a role in regulating TATA binding protein (TBP) binding during the transcriptional initiation step (5). The evidence for this included synthetic lethality between certain mutations of TBP and TFIIA and defective alleles of SPT16, reduced binding of TBP at some promoters in spt16 mutants, and enhanced binding of TBP to a TATA box within nucleosomal DNA in presence of TFIIA and yFACT. The yeast chromodomain protein (Chd1) is a member of the Snf2-like subfamily of nucleic acid-stimulated ATPases (21) and has ATP-dependent chromatin-remodeling activity in vitro (29, 40). Chd1 and other CHD proteins have two chromodomains near the N terminus, a centrally located Snf2-related helicase/ATPase domain, and a Myb-related DNA binding domain near the C terminus (59). Chd1 is thought to promote formation of inhibitory chromatin, as extracts derived from cells lacking Chd1 are unable to produce the same level of DNase I resistance at specific loci that results from similar preparations derived from normal cells (40). Genetic interactions have been reported between mutations of CHD1 and mutations in transcription elongation factors such as Spt5, Isw1, and Isw2 (45, 54). Chd1 also physically interacts with several transcription elongation factors, such as members of the Paf1 complex, the Spt4-Spt5 complex, and components of yFACT (24, 27, 45, 54). Recently Chd1 has been shown to physically associate with the SAGA/SLIK complex in yeast and to bind histone tail peptides methylated at K4 (37). However, binding of yeast Chd1 to methylated H3-K4 has not been observed by others (32, 46). Relatively little is known about the functional role of Chd1 in vivo in regulating transcription, although it was recently reported that a chd1 mutation affects chromatin structure of the ADH2 gene and the kinetics of ADH2 activation (60). In this report, we show that part of the role of Chd1 is to oppose the function of the positive transcription factor yFACT. We present evidence which suggests that Chd1 negates yFACT's ability to enhance TBP binding at promoters. We show that in a strain with a yFACT defect, deletion of CHD1 results in increased TBP binding and increased Pol II binding at promoters. Finally, we find that deletion of CHD1 suppresses synthetic lethalities between spt16 mutations and TBP mutations as well as between spt16-11 and TFIIA mutations.

Contains fulltext : 34788.pdf (Publisher’s version ) (Closed access) In higher eukaryotes, the large subunit of the general transcription factor TFIIA is encoded by the single TFIIAalphabeta gene and posttranslationally cleaved into alpha and beta subunits. The molecular mechanisms and biological significance of this proteolytic process have remained obscure. Here, we show that TFIIA is a substrate of taspase 1 as reported for the trithorax group mixed-lineage leukemia protein. We demonstrate that recombinant taspase 1 cleaves TFIIA in vitro. Transfected taspase 1 enhances cleavage of TFIIA, and RNA interference knockdown of endogenous taspase 1 diminishes cleavage of TFIIA in vivo. In taspase 1-/- MEF cells, only uncleaved TFIIA is detected. In Xenopus laevis embryos, knockdown of TFIIA results in phenotype and expression defects. Both defects can be rescued by expression of an uncleavable TFIIA mutant. Our study shows that uncleaved TFIIA is transcriptionally active and that cleavage of TFIIA does not serve to render TFIIA competent for transcription. We propose that cleavage fine tunes the transcription regulation of a subset of genes during differentiation and development.

Site-specific protein-DNA photo-cross-linking was used to show that, when bound to its cognate site at various distances upstream of the TATA element, the chimeric transcriptional activator GAL4-VP16 can physically interact with a TATA box-binding protein (TBP)- transcription factor IIA (TFIIA)-TFIIB complex assembled on the TATA element. This result implies DNA bending and looping of promoter DNA as a result of the physical interaction between GAL4-VP16 and an interface of the TBP-TFIIA-TFIIB complex. This protein-protein interaction on promoter DNA minimally requires the presence of one GAL4 binding site and the formation of a quaternary complex containing TBP, TFIIB, and TFIIA on the TATA element. Notably, the topology of the TBP-TFIIA-TFIIB-promoter complex is not altered significantly by the interaction with DNA-bound activators. We also show that the ability of GAL4-VP16 to activate transcription through a single GAL4 binding site varies according to its precise location and orientation relative to the TATA element and that it can approach the efficiency obtained with multiple binding sites. Taken together, our results indicate that the spatial positioning of the DNA-bound activation domain is important for efficient activation, possibly by maximizing its interactions with the transcriptional machinery including the TBP-TFIIA-TFIIB-promoter quaternary complex.

International audience; Thus far, no transcription factor IIIA (TFIIIA) from higher plants has been cloned and characterized. We have cloned and characterized TFIIIA and ribosomal protein L5 from Arabidopsis thaliana. Primary sequence comparison revealed a high divergence of AtTFIIIA and a relatively high conservation of AtL5 when compared with other organisms. The AtTFIIIA cDNA encodes a protein with nine Cys(2)-His(2)-type zinc fingers, a 23 amino acid spacer between fingers 1 and 2, a 66 amino acid spacer between fingers 4 and 5, and a 50 amino acid non-finger C-terminal tail. Aside from the amino acids required for proper zinc finger folding, AtTFIIIA is highly divergent from other known TFIIIAs. AtTFIIIA can bind 5S rDNA, as well as 5S rRNA, and efficiently stimulates the transcription of an Arabidopsis 5S rRNA gene in vitro. AtL5 identity was confirmed by demonstrating that this protein binds to 5S rRNA but not to 5S rDNA. Protoplast transient expression assays with green fluorescent protein fusion proteins revealed that AtTFIIIA is absent from the cytoplasm and concentrated at several nuclear foci including the nucleolus. AtL5 protein accumulates in the nucleus, especially in the nucleolus, and is also present in the cytoplasm.

Regulation of gene expression in haploid male germ cells follows a number of specific rules that differ from somatic cells. In this physiological context, transcriptional control mediated by the activator CREM (cAMP-responsive element modulator) represents an established paradigm. In somatic cells activation by CREM requires its phosphorylation at a unique regulatory site (Ser117) and subsequent interaction with the ubiquitous coactivator CBP (cAMP response element binding protein-binding protein). In testis, CREM transcriptional activity is controlled through interaction with a tissue-specific partner, ACT (activator of CREM in testis), which confers a powerful, phosphorylation-independent activation capacity. In addition to specialized transcription factors and coactivators, a variety of general factors of the basal transcriptional machinery, and their distinct tissue-specific isoforms, are highly expressed in testis, supporting the general notion that testis-specific gene expression requires specialized mechanisms. Here, we describe that CREM interacts with transcription factor IIA (TFIIA), a general transcription factor that stimulates RNA polymerase II-directed transcription. This association was identified by a two-hybrid screen, using a testis-derived cDNA library, and confirmed by coimmunoprecipitation. The interaction is restricted to the activator isoforms of CREM and does not require Ser117. Importantly, CREM does not interact with TFIIAtau-ALF, a testis-specific TFIIA homolog. CREM and TFIIA are expressed in a spatially and temporally coordinated fashion during the differentiation program of germ cells. The two proteins also colocalize intracellularly in spermatocyte and spermatid cells. These findings contribute to the understanding of the highly specialized rules of transcriptional regulation in haploid germ cells.

The transcription factor TFIIB has been identified and cloned from the yeast Schizosaccharomyces pombe. The cloned polypeptide is highly homologous to human TFIIB and to Saccharomyces cerevisiae TFIIB. S. pombe TFIIB is a 340-amino-acid-long protein and it possesses a repeated motif of 75 amino acids near the carboxy-terminal region. The purified recombinant protein is able to bind to the TBP–DNA promoter complex in gel retardation experiments. Recombinant S. pombe TFIIB is active in in vitro transcription assays, since it can complement the transcription activity of a S. pombe cell extract in which TFIIB was depleted by using antibodies.

Epstein-Barr virus (EBV) lytic cycle transcription and DNA replication require the transcriptional activation function of the viral immediate-early protein Zta. We describe a series of alanine substitution mutations in the Zta activation domain that reveal two functional motifs based on amino acid composition. Alanine substitution of single or paired hydrophobic aromatic amino acid residues resulted in modest transcription activation defects, while combining four substitutions of aromatic residues (F22/F26/W74/F75) led to more severe transcription defects. Substitution of acidic amino acid residue E27, D35, or E54 caused severe transcription defects on most viral promoters. Promoter- and cell-specific defects were observed for some substitution mutants. Aromatic residues were required for Zta interaction with TFIIA-TFIID and the CREB-binding protein (CBP) and for stimulation of CBP histone acetyltransferase activity in vitro. In contrast, acidic amino acid substitution mutants interacted with TFIIA-TFIID and CBP indistinguishably from the wild type. The nuclear domain 10 (ND10) protein SP100 was dispersed by most Zta mutants, but acidic residue mutations led to reduced, while aromatic substitution mutants led to increased SP100 nuclear staining. Acidic residue substitution mutants had more pronounced defects in transcription activation of endogenous viral genes in latently infected cells and for viral replication, as measured by the production of infectious virus. One mutant, K12/F13, was incapable of stimulating EBV lytic replication but had only modest transcription defects. These results indicate that Zta stimulates viral reactivation through two nonredundant structural motifs, one of which interacts with general transcription factors and coactivators, and the other has an essential but as yet not understood function in lytic transcription.

Protein-protein interactions between human heat shock transcription factor 1 (hHSF1) and general transcription factors TFIIA-gamma, TFIIB, TBP, TAF(II)32, and TAF(II)55 and positive coactivator PC4 were characterized in order to identify potential targets of contact in the transcriptional preinitiation complex. These contacts represent one of the final steps in the signal transfer of heat stress to the transcriptional apparatus. TATA-binding protein (TBP) and transcription factor IIB (TFIIB) were identified as major targets for HSF1 transcriptional activation domains AD1 and AD2 based on in vitro interaction assays. TBP showed affinity for AD2 and a fragment containing AD1, while the core domain of TFIIB interacted primarily with the AD1 fragment. Interactions were also detected between full-length HSF1 and the small subunit (gamma) of TFIIA. PC4 interacted weakly with HSF2 and showed even less affinity for HSF1. Coimmunoprecipitation of transiently expressed TBP in HeLa cells demonstrated that HSF1 AD2 and AD1+AD2 are able to bind TBP in vivo. Assays based on transcriptional interference confirmed predictions that both TBP and TFIIB can interact with HSF1 activation domains in HeLa cells. The negative regulatory region (NR) of HSF1 did not interact with any general factors tested in vitro but did bind TFIID in nuclear extracts through contacts that probably involve TATA associated proteins (TAFs). These results suggest a model for transcriptional regulation by HSF1 that involves a shift between formation of dysfunctional TFIID complexes with the NR and transcriptionally competent complexes with the C-terminal activation domains.

Negative cofactor 2 (NC2) is a dimeric histone-fold complex that represses RNA polymerase II transcription through binding to TATA-box-binding protein (TBP) and inhibition of the general transcription factors TFIIA and TFIIB. Here we study molecular mechanisms of repression by human NC2 in vivo in yeast. Yeast NC2 genes are essential and can be exchanged with human NC2. The physiologically rele– vant regions of NC2 have been determined and shown to match the histone-fold dimerization motif. A suppressor screen based upon limiting concentrations of NC2β yielded a cold-sensitive mutant in the yeast TFIIA subunit Toa1. The single point mutation in Toa1 alleviates the requirement for both subunits of NC2. Biochemical characterization indicated that mutant (mt)-Toa1 dimerizes well with Toa2; it supports specific recognition of the TATA box by TBP but forms less stable TBP–TFIIA–DNA complexes. Wild-type but not the mt-Toa1 can relieve NC2 effects in purified trans– cription systems. These data provide evidence for a dimeric NC2 complex that is in an equilibrium with TFIIA after the initial binding of TBP to promoter TATA boxes.

Adenovirus E1B 55,000-molecular-weight protein (55K) binds to host cell p53, stabilizing it, greatly increasing its affinity for its cognate DNA-binding site, and converting it from a regulated activator to a constitutive repressor. Here we analyzed the mechanism of repression by the p53-E1B 55K complex. E1B 55K repression requires that 55K be tethered to the promoter by binding directly to DNA-bound p53. Transcription from an assembled, p53-activated preinitiation complex was not repressed by the subsequent addition of E1B 55K, suggesting that either sites of 55K interaction with p53 or targets of 55K in the preinitiation complex are blocked. Specific E1B 55K repression was observed in reactions lacking TFIIA and with recombinant TATA-binding protein in place of TFIID, conditions under which p53 does not activate transcription. Thus, E1B 55K does not simply inhibit a p53-specific activation mechanism but rather blocks basal transcription. As a consequence, E1B 55K may repress transcription from any promoter with an associated p53-binding site, no matter what other activators associate with the promoter. E1B 55K did not repress basal transcription in reactions with recombinant and highly purified general transcription factors and RNA polymerase II but rather required a corepressor that copurifies with the polymerase.

Potential pathways that could account for observed rapid rates of transcription reinitiation were explored. A nuclear extract system was established in which reinitiation rates were observed to be kinetically facilitated and in which the rate was sensitive to TATA box mutation. Kinetic facilitation of functional complex formation could be mimicked by pre-assembling activator and certain general transcription factors on the promoter and then adding nuclear extract. The minimal activated complex with this characteristic contained general factors TFIID and TFIIA. The ability of the TFIID:TFIIA complex to complete assembly rapidly was reduced by the same TATA box mutation that reduced reinitiation rate. Band shift experiments also showed that this same mutation lowered the stability of the TBP:TFIIA complex on the DNA. The results suggest that TATA-dependent variations in retention of the TFIID:TFIIA complex after release of the polymerase could be a primary determinant of reinitiation rate, allowing diversity in promoter strength to be related to diversity in TATA element sequences.

The TFIID complex interacts with at least three types of core promoter elements within protein-coding genes, including TATA, initiator (Inr), and downstream promoter elements. We have begun to explore the mechanism by which the TFIID–Inr interaction leads to functional synergy between TATA and Inr elements during both basal and activated transcription. In DNase I footprinting assays, GAL4–VP16 recruited TFIID–TFIIA to core promoters containing either a TATA box, an Inr, or both TATA and Inr elements, with synergistic interactions apparent on the TATA–Inr promoter. Appropriate spacing between the two elements was essential for the synergistic binding. Despite the sequence-specific TFIID–Inr interactions, gel shift experiments revealed that TFIID alone possesses similar affinities for the TATA–Inr and TATA promoters. Interestingly, however, recombinant TFIIA strongly and selectively enhanced TFIID binding to the TATA–Inr promoter, with little effect on binding to the TATA promoter. Studies of the natural adenovirus major late promoter confirmed these findings, despite the existence of specific but nonfunctional TFIID interactions downstream of the Inr in that promoter. These results suggest that a TFIIA-induced conformational change is essential for the sequence-specific TFIID–Inr interaction to occur with sufficient affinity to support the functional synergism between TATA and Inr elements.

Transcriptional repression of eukaryotic genes is essential for many cellular and developmental processes, yet the precise mechanisms of repression remain poorly understood. The Dorsal Switch Protein (DSP1) was identified in a genetic screen for activities which convert Dorsal into a transcriptional repressor. DSP1 shares structural homology with the HMG-1/2 family and inhibits activation by the rel transcription factors Dorsal and NF-kappaB in transfection studies. Here we investigate the mechanism of transcriptional repression by DSP1. We found that DSP1 protein can act as a potent transcriptional repressor for multiple activator families in vitro and in transfection studies. DSP1 bound directly to the TATA binding protein (TBP), and formed a stable ternary complex with TBP bound to DNA. DSP1 preferentially disrupted the DNA binding of TBP complexes containing TFIIA and displaced TFIIA from binding to TBP. Consistent with the inhibition of TFIIA-bound complexes, DSP1 was shown to inhibit activated but not basal transcription reactions in vitro. The ability of DSP1 to interact with TBP and to repress transcription was mapped to the carboxy-terminal domain which contains two HMG boxes. Our results support the model that DSP1 represses activated transcription by interfering with the binding of TFIIA, a general transcription factor implicated in activated transcription pathways.

We have previously reported the direct physical interaction between the human immunodeficiency virus (HIV) type I Tat protein and the basal transcription factor TBP/TFIID. Affinity chromatography demonstrated that wild-type Tat, but not a transactivation mutant of Tat, was capable of depleting TBP/TFIID from cell extracts. These experiments represented the first demonstration of a basal transcription factor that binds, in an activation-dependent manner, to Tat. We now report that the Tat-TBP interaction can be detected in HIV type 1-infected cells. The domain of TBP interacting with Tat has been mapped from amino acids 163 to 196 by using deletion and site-specific mutants of TBP. This domain of TBP, which includes the HI and S2 domains, is distinct from the H2 binding site for other activator proteins, such as E1A. The interaction of Tat with TFIID regulates the binding of accessory proteins to TFIID. Tat stabilizes the interaction of TFIID with TFIIA in a gel shift assay. In addition, Tat competes for Dr1 interaction with TBP. Our results suggest that the basal transcription factor TBP/TFIID represents an important regulatory molecule in HIV transcription.

Two quite different types of research have been invaluable in elucidating principles in the field of molecular biology: biochemistry and genetics. When those two approaches intersect, as they have in recent work on eukaryotic transcription control, the resulting reverberation has the ring of truth. In a recent issue of these Proceedings, Li et al. (1) presented the latest example of the convergence of biochemical and genetic studies that have led to the recent discovery of a eukaryotic RNA polymerase holoenzyme. The holoenzyme is a multiprotein complex containing the multimeric RNA polymerase II protein that transcribes protein coding genes, most of the general transcription factors required for the polymerase to locate eukaryotic promoters and initiate transcription, and a newly discovered multimeric protein complex called mediator required for the proper temporal regulation of transcription in response to regulatory transcription factors. Together with the transcription initiation factors TFIID and TFIIA, the holoenzyme forms an approximately ribosome-sized complex of more than 50 polypeptides that assembles on eukaryotic promoters. This huge preinitiation complex integrates signals from regulatory transcription factors bound at enhancer and silencer DNA sequences to determine how frequently RNA polymerase II initiates transcription of the neighboring gene. Even though RNA polymerase II is a complex multimeric protein of 14 subunits (2), by itself it is incapable of starting transcription at the specific initiation sites used in intact cells. Specification of the transcription initiation site is a critical aspect of transcription control, since it determines which portions of genomic DNA are transcribed into mRNA. Consequently, the initial efforts of biochemists went toward identifying a set of protein factors called general transcription factors (named TFIIA, -B, -D, -E, and -H) that direct the polymerase to initiate transcription at sites in DNA that are used in the cell (3-5). However, although this complex set of general transcription factors can act together to specify the correct starts for RNA polymerase II, they are not sufficient to reproduce the regulation of transcription initiation observed in cells. A combination of genetic and biochemical studies established that, in vivo, activator proteins bind to regulatory DNA sequences called enhancers and increase the frequency of transcription initiation at neighboring promoters, even when the closest promoter is 50 kb away in the linear DNA sequence (6). Activators stimulate transcription in cell-free in vitro reactions using complex extracts prepared from isolated cell nuclei. However, they fail to stimulate transcription in reactions with the purified general transcription factors. Working in the budding yeast system, Roger Kornberg and co-workers went back to the complex nuclear extract to search for an activity that would allow activators to stimulate transcription in the in vitro reaction with purified general transcription factors. They succeeded in identifying such an activity, calling it mediator (7). As they purified and analyzed mediator (8), it became apparent that they were characterizing the same multiprotein complex that was concurrently discovered by Richard Young and colleagues (24) through what was initially a genetic strategy for the analysis of transcription control. This was the initial intersection of the biochemical and genetic studies that led to the discovery of the holoenzyme. Young and colleague's genetic strategy began with an analysis of a unique structural aspect of RNA polymerase II, a specific C-terminal domain (CTD) associated with its largest subunit (9, 10). The CTD is composed of 26-52 tandem copies (depending on the organism) of nearly perfect repeats of a seven amino acid residue sequence, Tyr-Ser-Pro-Thr-SerPro-Ser. When RNA polymerase II is assembled into the massive preinitiation complex at eukaryotic promoters, its CTD is unmodified. As the polymerase initiates transcription and then transcribes away from the initiation site on the template, the CTD is phosphorylated at multiple sites (1 1-14). A number of protein kinases have been identified that can phosphorylate the CTD, but the Cdk7 subunit of TFIIH appears to be responsible for most CTD phosphorylations in in vitro transcription reactions (15). In these in vitro reactions, Cdk7 phosphorylation of the CTD is required for transcription from some promoters (16) but not others (15). Genetic experiments using the methods of site-directed mutagenesis revealed that the CTD is essential for the viability of budding yeast, Drosophila, and mammalian cells (17-20). To explore the function of the CTD through genetic methods, Young and his colleagues (17) identified the minimal number of heptapeptide repeats that would allow a single cell to grow into a colony on a plate of rich medium, 1O, whereas the number found in wild-type Saccharomyces cerevisiae is about 26. They found that, unlike cells with the full-length CTD, cells with this mutant form of RNA polymerase II were unable to form colonies at low temperature, most likely because of generalized defects these mutants exhibit in regulating transcription of multiple genes. This cold sensitivity of cells with a partially deleted CTD allowed Young and his colleagues to take advantage of the genetic technique of identifying suppressing mutations. Such suppressors can result from mutations in additional proteins involved in the same process. The identification of suppressing mutations that allow the CTD partial deletion mutant to grow at low temperature (2123) led to the discovery of nine genes called SRB2, -4, . . . -11 (for suppressor of RNA polymerase B). When the Young group isolated these genes and used them to prepare specific antibody against the encoded Srb proteins, they discovered that all the Srb proteins were part of the same, very large, macromolecular complex that included the subunits of RNA polymerase II and most of the subunits of the general transcription factors (22, 24). [However, the critical TFIID factor that binds to the TATA-box sequence of eukaryotic promoters was not present in the complex (24)]. The multiprotein complex they purified from yeast nuclear extracts was very similar to the complex identified by Kornberg and co-workers during their purification of mediator activity (8). Both groups agreed to call the complex the holoenzyme. While there are potentially important differences between the composition of the holoenzymes described by the two groups, the many similarities seem more significant than the differences. In experiments with cells having temperature-sensitive srb4 or srb6 mutations, the transcription of multiple genes shut down rapidly when the cells were shifted to the nonpermissive temperature (25). This important observation argues that the holoenzyme is required for the transcription of most, if not all, yeast genes.

The human T-cell lymphotropic virus type 1 (HTLV-1) is the etiological agent for adult T-cell leukemia and tropical spastic paraparesis/HTLV-1-associated myelopathy. The HTLV-1 Tax1 gene product has been shown to transactivate transcription of viral and cellular promoters. To examine the biochemical mechanism of Tax1 transactivation, we have developed an in vitro transactivation assay in which wild-type Tax1 is able to specifically transactivate a polymerase II promoter through upstream Tax1-responsive elements. The in vitro system utilizes the HTLV-1 21-bp repeats cloned upstream of the ovalbumin promoter and G-free cassette. Purified Tax1 specifically transactivates this template 5- to 10-fold in a concentration-dependent manner. No transactivation of the ovalbumin promoter (pLovTATA) template control was observed. Tax1 transactivation was inhibited by low concentrations of alpha-amanitin and was effectively neutralized by anti-Tax1 but not control sera. Consistent with in vivo transactivating activity, Tax1 NF-kappa B mutant M22, but not cyclic AMP-responsive element-binding protein mutant M47, transactivated the template containing the tandem 21-bp repeat. In a reconstituted in vitro transcription assay, Tax1 transactivation was dependent upon basal transcription factors TFIIB, TFIIF, Pol II, TFIID, and TFIIA. TATA-binding protein did not functionally substitute for TFIID in the transactivation assay by Tax1 but was sufficient for basal transcription. Finally, we have used anti-TFIIA antibody (p55) to ask if Tax1 transactivation required TFIIA activity. Addition of TFIIA antibody to in vitro transcription reactions, as well as depletion of TFIIA by preclearing with antibody, showed that TFIIA was required for Tax1 transactivation. Only a slight (twofold) drop of basal transcription was observed. Tax1 transactivation was restored when purified HeLa TFIIA was added back into the reconstituted system. We propose that Tax1 utilizes a transactivation pathway involving the activator regulated basal transcription factors TFIID and TFIIA.

We have analyzed the mechanisms underlying stimulation of transcription by the activator GAL4-AH and the recombinant coactivator p15 (PC4). We show that p15 binds to both double-stranded and single-stranded DNA. Analyses of deletion mutants correlates binding to double-stranded DNA with the ability to mediate activator-dependent transcription. Consistent with this finding, phosphorylation of p15 by casein kinase II inhibits binding to double-stranded DNA and the activity of p15. The functional characterization suggests interactions of p15 with both DNA and components of the TFIID complex. GAL4-AH functions in concert with p15 during formation of TFIIA-TFIID-promoter (DA) complexes, as concluded from order-of-addition experiments. At limiting TFIID concentrations, the number of DA complexes is enhanced. The activator also stimulates transcription moderately after DA complex formation, independently of the concentrations of general transcription factors.

TATA boxes are common structural features of eucaryal class II and archaeal promoters. In addition, a gene encoding a polypeptide with sequence similarity to eucaryal TATA-binding protein (TBP) has recently been detected in Archaea, but its relationship to the archaeal transcription factors A (aTFA) and B (aTFB) was unclear. Here, we demonstrate that yeast and human TBP can substitute for aTFB in a Methanococcus-derived archaeal cell-free transcription system. Template-commitment studies show that eucaryal TBP is stably sequestered at the archaeal promoter and that this interaction is further stabilized in combination with aTFA. Binding studies revealed that recognition of an archaeal promoter by TBP involves specific binding to the TATA box. These findings demonstrate a common function of TBP and aTFB and imply a common evolutionary origin of eucaryal and archaeal transcriptional machinery.

A basal repressor of class II gene transcription was identified, purified, and found to be identical to nonhistone chromosomal protein HMG2. HMG2 was shown to inhibit basal transcription under conditions in which transcription templates form soluble complexes with HMG2. Order-of-addition experiments clearly revealed that HMG2 acted after assembly of a TBP-TFIIA-promoter complex and before formation of the fourth phosphodiester bond by RNA polymerase II. Subsequently, an activity that efficiently counteracted repression of transcription by HMG2 in both TBP- and TFIID-containing transcription systems was isolated. Several lines of evidence suggested that antirepression was mediated by a TFIIH-associated factor. The antirepressor first coeluted with TFIIH, was depleted from this fraction by antibodies directed against the TFIIH subunit p62, was dependent on either ATP or dATP, and then was inhibited by the ATP analogs AMP-PNP and ATP gamma S. Relief of HMG2-mediated repression as well as basal promoter function of TFIIH may involve a helicase that coelutes with TFIIH and displays similar nucleotide specificities. Taken together, these data suggest novel consequences of chromatin-associated HMG proteins and they provide direct evidence for a role of TFIIH-associated enzymes in ATP-dependent antirepression of nonhistone chromosomal proteins.

In the process of characterizing cellular proteins that modulate basal transcription by RNA polymerase II, we identified a novel repressor activity specific for promoters containing consensus TATA boxes. This activity strongly represses TATA-binding protein (TBP)-dependent transcription initiation from core promoter elements containing a consensus TATA sequence, but activates TBP-dependent transcription from core promoter elements lacking a consensus TATA sequence. Purification of this activity to near homogeneity from rat liver nuclear extracts led to the surprising discovery that it co-purifies closely with mammalian transcription factor IIA (TFIIA). The close association of TATA sequence-dependent transcriptional repressor activity with TFIIA adds a new and unexpected dimension to the already complex picture of this factor's function in transcription by RNA polymerase II.

A systematic analysis of the primary sequences of the polymerase II initiation complex has revealed unusual charge features in the TFII family proteins. In particular, the proteins TFIIA alpha, TFIIE alpha, and TFIIF carry multiple charge clusters and hyper charge runs, sequence features occurring in < 4% of all (available) eukaryotic proteins. Possible implications for these charge structures are discussed in relation to the assembly and function of the polymerase II transcriptional complex.

Avian histone H5 and alpha A-globin genes are transcribed much more efficiently in whole cell extracts derived from immature polychromatic erythrocytes than in extracts from mature duck erythrocytes. We found that these differential activities are detectable only if assayed with promoters containing a functional TATA box. The addition of either highly purified human or recombinant yeast transcription factor IIA (TFIIA) to extracts from mature erythrocytes resulted in a significant increase in transcription from TATA-containing promoters, whereas transcription from TATA-less promoters remained unaffected. Moreover, the activity of TFIIA was found to be reduced in extracts from mature erythrocytes. These data support the proposition that inactivation of TFIIA may contribute to a general repression of gene activity in avian erythrocytes, and only those genes with alternative mechanisms of initiation complex formation continue to be expressed in these cells. In the case of the histone H5 gene, such an alternative mechanism could be mediated via the interaction between duck erythrocyte upstream stimulating factor and TFIID.

We have developed a high-throughput protein binding microarray (PBM) assay to systematically investigate transcription regulatory protein complexes binding to DNA with varied specificity and affinity. Our approach is based on the novel coupling of total internal reflectance fluorescence (TIRF) spectroscopy, swellable hydrogel double-stranded DNA microarrays and dye-labeled regulatory proteins, making it possible to determine both equilibrium binding specificities and kinetic rates for multiple protein:DNA interactions in a single experiment. DNA specificities and affinities for the general transcription factors TBP, TFIIA and IIB determined by TIRF-PBM are similar to those determined by traditional methods, while simultaneous measurement of the factors in binary and ternary protein complexes reveals preferred binding combinations. TIRF-PBM provides a novel and extendible platform for multi-protein transcription factor investigation.

Human transcription factor TFIID, the TATA-binding protein, was partially purified to a form capable of associating stably with the TATA motif of the adenovirus major late promoter. Binding of the human and yeast TFIID to the TATA motif was stimulated by TFIIA. TFIIA is an integral part of a complex capable of binding other transcription factors. A complex formed with human TFIID and TFIIA (DA complex) was specifically recognized by TFIIB. We found that TFIIB activity was contained in a single polypeptide of 32 kDa and that this polypeptide participated in transcription and was capable of binding to the DA complex to form the DAB complex. Formation of the DAB complex required TFIIA, TFIID, and sequences downstream of the transcriptional start site; however, the DA complex could be formed on an oligonucleotide containing only the adenovirus major late promoter TATA motif. Using anti-TFIIB antibodies and reagents that affect the stability of a transcription-competent complex, we found that yeast and human TFIID yielded DAB complexes with different stabilities.

Summary The evolution of tissue-specific general transcription factors (GTFs), such as testis-specific TBP-related factor 2 (TRF2), enables the spatiotemporal expression of highly specialized genetic programs. Taspase1 is a protease that cleaves nuclear factors MLL1, MLL2, TFIIAα-β, and ALFα-β (TFIIAτ). Here, we demonstrate that Taspase1-mediated processing of TFIIAα-β drives mammalian spermatogenesis. Both Taspase1 −/− and noncleavable TFIIAα-βnc/nc testes release immature germ cells with impaired transcription of Transition proteins ( Tnp ) and Protamines ( Prm ), exhibiting chromatin compaction defects and recapitulating those observed with TRF2 −/− testes. Although the unprocessed TFIIA still complexes with TRF2, this complex is impaired in targeting and thus activating Tnp1 and Prm1 promoters. The current study presents a paradigm in which a protease (Taspase1) cleaves a ubiquitously expressed GTF (TFIIA) to enable tissue-specific (testis) transcription, meeting the demand for sophisticated regulation of distinct subsets of genes in higher organisms.

Protein kinase CK2 is a ubiquitous Ser/Thr kinase which phosphorylates a large number of proteins including several transcription factors. Recombinant Xenopus laevis CK2 phosphorylates both recombinant Saccharomyces cerevisiae and Schizosaccharomyces pombe TATA binding protein (TBP). The phosphorylation of TBP by CK2 reduces its binding activity to the TATA box. CK2 copurifies with the transcription factor IID (TFIID) complex from HeLa cell extracts and phosphorylates several of the TBP-associated factors within TFIID. Taken together these findings argue for a role of CK2 in the control of transcription by RNA polymerase II through the modulation of the binding activity of TBP to the TATA box.

Transcription in eukaryotic cells requires the remodeling of chromatin and the assembly of functional preinitiation complexes (PICs), which contain the general transcription factors (GTFs), RNA polymerase II (Pol II), and coactivators [1, 2]. Genetic and biochemical studies have implicated the multisubunit Mediator coactivator complex (Med) as a critical component of the PIC [3–5], a direct target of activators [6, 7], and a checkpoint for regulated gene expression during differentiation [8], development (reviewed in [9]), signaling [10, 11], and oncogenesis [12]. In this report, we show that a complex containing the activator GAL4-VP16, Med, and TFIID/TFIIA (DA) recruits pol II and the remaining GTFs to a model promoter in vitro. A preassembled DAMed complex bypasses the requirement for an activator. We also demonstrate that coordinated assembly of DAMed is essential to establishing a functional PIC. We conclude that the DAMed complex generates a platform that supports activated levels of PIC assembly and transcription.