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PubMed1999

Testing for DNA tracking by MOT1, a SNF2/SWI2 protein family member.

Proteins in the SNF2/SWI2 family use ATP hydrolysis to catalyze rearrangements in diverse protein-DNA complexes. How ATP hydrolysis is coupled to these rearrangements is unknown, however. One attractive model is that these ATPases are ATP-dependent DNA-tracking enzymes. This idea was tested for the SNF2/SWI2 protein family member MOT1. MOT1 is an essential Saccharomyces cerevisiae transcription factor that uses ATP to dissociate TATA binding protein (TBP) from DNA. By using a series of DNA templates with one or two TATA boxes in combination with binding sites for heterologous DNA binding "roadblock" proteins, the ability of MOT1 to track along DNA was assayed. The results demonstrate that, following ATP-dependent TBP-DNA dissociation, MOT1 dissociates rapidly from the DNA by a mechanism that does not require a DNA end. Template commitment footprinting experiments support the conclusion that ATP-dependent DNA tracking by MOT1 does not occur. These results support a model in which MOT1 drives TBP-DNA dissociation by a mechanism that involves a transient, ATP-dependent interaction with TBP-DNA which does not involve ATP-dependent DNA tracking.

Adenosine Triphosphatases↗
PubMed1995

The neuroendocrine protein 7B2 contains unusually potent transcriptional activating sequences.

The expression of the 7B2 protein, secreted from a variety of neural and endocrine tissues, increases dramatically in specific neuroendocrine tumors. We have recently shown that human 7B2 can act as a molecular chaperone in the deaggregation of proteins in vitro. In order to identify polypeptides which might bind 7B2 in vivo, the yeast two-hybrid system was employed. Surprisingly, mere covalent linkage of 7B2 to the DNA-binding domains of two yeast transcription activators, Ace1 and Gal4, activates transcription from the ACE1 and GAL4 operon. 7B2's ability to activate nuclear transcription surpasses that of Ace1 and compares favourably with the strong activation domain of the tumor suppressor protein, p53. Our results suggest that 7B2 must possess an activating sequence, a domain which defines all transcriptional activator proteins. Like the acidic activation domains of some transcriptional activators, 7B2 also binds the yeast TATA-box binding protein, an essential polypeptide in the basic transcription machinery. Deletion analysis of the gene encoding 7B2 reveals two independent transcriptional activating sequences in the 185 amino acid protein. It is therefore conceivable that 7B2 not only has a functional role in the secretory pathway but also in the nucleus. Moreover, these findings raise an intriguing question regarding the activation domains of 7B2 and their possible link to 7B2's oncogenic potential.

Amino Acid Sequence↗
PubMed2001

Structural studies of a yeast quaternary transcription-initiation complex.

A 96.7 kDa quaternary transcription-factor complex consisting of the conserved core domains of yeast TBP, TFIIA, TFIIB and TATA-box DNA has been assembled from purified components. Crystals of the complex were obtained by the hanging-drop vapor-diffusion method. Native data sets were collected at synchrotron sources. Crystal form I belongs to space group R32, with unit-cell parameters a = b = 173.2, c = 164.1 A, and diffracts to 2.5 resolution, but contains substoichiometric amounts of TFIIB. Crystal form II was assembled with a longer piece of DNA than that used for form I crystals and contains the complete quaternary complex. These crystals belong to space group P42(1)2, with unit-cell parameters a = b = 141.7, c = 112.0 A, and diffract to 3.6 A resolution.

Crystallization↗
PubMed1998

Inhibition of RNA polymerase II transcription in human cells by synthetic DNA-binding ligands.

Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole-imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-1, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity. The ability of small molecules to target predetermined DNA sequences located within RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication.

Base Sequence↗
PubMed1999

The p38 mitogen-activated protein kinase is required for NF-kappaB-dependent gene expression. The role of TATA-binding protein (TBP).

Endotoxin-induced cytokine gene transcription in monocytes and macrophages is regulated in part by NF-kappaB. We have previously shown that the p38 mitogen-activated protein (MAP) kinase is necessary for endotoxin-induced cytokine gene transcription. Due to the fact that most cytokine promoter sequences have active NF-kappaB sites, we hypothesized that the p38 MAP kinase was necessary for NF-kappaB-dependent gene expression. We found that NF-kappaB-dependent gene expression was reduced to near control levels with either SB 203580 or a dominant-negative p38 MAP kinase expression vector. Inhibition of the p38 MAP kinase did not alter NF-kappaB activation at any level, but it significantly reduced the DNA binding of TATA-binding protein (TBP) to the TATA box. The dominant-negative p38 MAP kinase expression vector interfered with the direct interaction of native TFIID (TBP) with a co-transfected p65 fusion protein. Likewise, this dominant-negative plasmid also interfered with the direct interaction of a co-transfected TBP fusion protein with the native p65 subunit. The p38 kinase also phosphorylated TFIID (TBP) in vitro, and SB 203580 inhibited phosphorylation of TFIID (TBP) in vivo. Thus, the p38 MAP kinase regulates NF-kappaB-dependent gene transcription, in part, by modulating activation of TFIID (TBP).

Cytokines↗
PubMed2000

Distinct classes of yeast promoters revealed by differential TAF recruitment.

The transcription factor TFIID contains the TATA box binding protein (TBP) and multiple TBP-associated factors (TAFs). Here, the association of TFIID components with promoters that either are dependent on multiple TAFs (TAFdep) or have no apparent TAF requirement (TAFind) is analyzed in yeast. At TAFdep promoters, TAFs are present at levels comparable to that of TBP, whereas at TAFind promoters, TAFs are present at levels that approximate background. After inactivation of several general transcription factors, including TBP, TAFs are still recruited by activators to TAFdep promoters. The results reveal two classes of promoters: at TAFind promoters, TBP is recruited in the apparent absence of TAFs, whereas at TAFdep promoters, TAFs are co-recruited with TBP in a manner consistent with direct activator-TAF interactions.

Cross-Linking Reagents↗
PubMed1997

Yeast TAF(II)145 functions as a core promoter selectivity factor, not a general coactivator.

In yeast, TATA box binding protein associated factors (TAF(II)s) are dispensable for transcription of most genes. Here we use differential display to identify a small subset of yeast genes whose transcription in vivo requires yTAF(II)145. Promoter-mapping studies reveal, unexpectedly, that the region of a gene that renders it yTAF(II)145-dependent is not the upstream activating sequence, which contains the activator-binding sites, but rather the core promoter. In fact, a core promoter requiring yTAF(II)145 retained that requirement when its transcription was directed by several unrelated upstream activating sequences and even in the absence of an activator. Taken together, our results indicate that yTAF(II)145 functions in recognition and selection of core promoters by a mechanism not involving upstream activators.

Cell Cycle↗
PubMed1994

Yeast TAFIIS in a multisubunit complex required for activated transcription.

In higher eukaryotes the RNA polymerase II transcription factor TFIID is composed of a TATA-box-binding protein (TBP) and a set of tightly bound polypeptides, designated TBP-associated factors (TAFIIS). One or more TAFIIS are coactivators that are required for activated but not basal transcription. The eukaryotic transcription machinery is highly conserved and it is therefore puzzling that TAFIIS have not been identified in yeast. Here we use TBP as a protein-affinity ligand to isolate from yeast a multisubunit complex that is required specifically for activated transcription by RNA polymerase II. Microsequence analysis and cloning of two subunits of this complex reveal that they are the homologues of known mammalian and Drosophila TAFIIS. The genes encoding these two yeast TAFIIS are essential, suggesting that activated transcription is required for viability of Saccharomyces cerevisiae.

Amino Acid Sequence↗
PubMed1997

Dynamic interplay of TFIIA, TBP and TATA DNA.

The TATA binding protein (TBP) binds to the -30 region of eukaryotic and archaea promoters, where it assembles a transcription complex. For those genes transcribed by RNA polymerase II, transcription factor TFIIA binds TBP and positively regulates its activity, including enhancing TBP/ TATA interactions. Since little is known about the dynamic interplay among TFIIA, TBP and DNA, we set out to examine the stability of these interactions. Using the nitrocellulose filter binding assay, the koff of recombinant human TBP from TATA and non-specific DNA was determined to be 5.5(+/-0.1) x 10(-5) s-1 (t1/2 = 210 minutes) and 5.8(+/-0.1) x 10(-4) s-1 (t1/2 = 20 minutes), respectively. TFIIA/TBP complexes, containing either HeLa-derived or recombinant human TFIIA, possessed a nearly tenfold lower koff when bound to TATA. Interactions of TFIIA with DNA upstream of the TATA box did not appear to play a major role in stabilizing TBP/TATA interactions. Instead, the upstream DNA contacts appeared to be important for stabilizing the association of TFIIA with the TBP/TATA complex as measured in electrophoretic mobility shift assays: koff of TFIIA decreased from 1.4(+/-0.1) x 10(-3) s-1 (t1/2 = eight minutes) to 2.4(+/-0.2) x 10(-4) s-1 (t1/2 = 49 minutes) when upstream DNA contacts were allowed. The stability of TFIIA/TBP interactions was measured using a rapid "pull-down" assay, which employed-nickel agarose and polyhistidine-tagged TFIIA. In the absence of DNA, TFIIA dissociated from TBP with a koff = 4.9(+/-0.6) x 10(-3) s-1 (t1/2 = 2.4 minutes), which varied with solution conditions.

Base Composition↗
PubMed1994

Stimulation of the adenovirus major late promoter in vitro by transcription factor USF is enhanced by the adenovirus DNA binding protein.

Previous studies have shown that the sequence-independent adenovirus DNA binding protein (DBP) increases transcription from several promoters, notably from the adenovirus major late promoter (MLP) and the adeno-associated virus P5 promoter, both of which contain a USF/MLTF binding site. In order to study this mechanism, we have investigated the effects of DBP on the binding of USF/MLTF to MLP and on transcription from MLP by a reconstituted in vitro system. As shown by gel retardation and DNase I footprinting, upon saturation of DNA, DBP enhances the binding affinity of USF43 to the promoter three- to fourfold without changing the footprint pattern. In contrast, the binding of the TATA box binding protein to the promoter is not influenced by DBP. No protein-protein interactions between DBP and USF43 could be observed in the absence of DNA, suggesting that enhanced binding is caused by a change in DNA structure induced by the DBP-DNA complex. Employing a transcription system reconstituted with purified general transcription factors, we show that USF43 enhances basal transcription and that USF43-dependent transcription is further increased by DBP, while DBP alone does not have an effect on basal transcription. Our results suggest that transcription enhancement by DBP is based on a specific increase in the binding of a transcription factor to a promoter through subtle changes in DNA structure, similar to the mechanism by which DBP stimulates the initiation of DNA replication.

Adenoviridae↗
PubMed1996

Transcription activation in cells lacking TAFIIS.

The general transcription factor TFIID is composed of the TATA-box-binding protein (TBP) and a set of TBP-associated factors (TAFIIs). In vitro, TAFIIs are required for activated transcription, and have been proposed to be obligatory targets of transcriptional activator proteins (activators)2. The function of TAFIIs has not been investigated systematically in vivo. A Saccharomyces cerevisiae TAFII complex (yTAFII complex) has been identified that shares functional and structural similarities with higher eukaryotic TFIID. In particular, most yTAFIIs are the homologue of a higher eukaryotic TAFII. Here we report that inactivation or depletion of six different yTAFIIs, including the core yTAFII, that contacts TBP, does not compromise transcriptional activation. We conclude that in vivo, activated transcription of many genes can occur in the absence of functional yTAFIIS, and that in these instances another transcription component(s) must be the target of the activator.

Antigens, Neoplasm↗
PubMed2000

TFIIA-TAF regulatory interplay: NMR evidence for overlapping binding sites on TBP.

TATA box binding protein (TBP)-promoter interaction nucleates assembly of the RNA polymerase II transcription initiation complex. Transcription factor IIA (TFIIA) stabilizes the TBP-promoter complex whereas the N-terminal domain of the largest TAF(II) inhibits TBP-promoter interaction. We have mapped the interaction sites on TBP of Drosophila TAF(II)230 and yeast TFIIA (comprising two subunits, TOA1 and TOA2), using nuclear magnetic resonance (NMR), and also report structural evidence that subdomain II of the TAF(II)230 N-terminal inhibitory domain and TFIIA have overlapping binding sites on the convex surface of TBP. Together with previous mutational and biochemical data, our NMR results indicate that subdomain II augments subdomain I-mediated inhibition of TBP function by blocking TBP-TFIIA interaction.

Amino Acid Sequence↗
PubMed1996

Transcriptional activation domain of the herpesvirus protein VP16 becomes conformationally constrained upon interaction with basal transcription factors.

The transcriptional activation domain of the herpesvirus protein VP16 resides in the carboxyl-terminal 78 amino acids (residues 413-490). Fluorescence analyses of this domain indicated that critical amino acids are solvent-exposed in highly mobile segments. To examine interactions between VP16 and components of the basal transcriptional machinery, we incorporated (at position 442 or 473 of VP16) tryptophan analogs that can be selectively excited in complexes with other Trp-containing proteins. TATA-box binding protein (TBP) (but not transcription factor B (TFIIB)) caused concentration-dependent changes in the steady-state anisotropy of VP16, from which equilibrium binding constants were calculated. Quenching of the fluorescence from either position (442 or 473) was significantly affected by TBP, whereas TFIIB affected quenching only at position 473. 7-aza-Trp residues at either position showed a emission spectral shift in the presence of TBP (but not TFIIB), indicating a change to a more hydrophobic environment. In anisotropy decay experiments, TBP reduced the segmental motion at either position; in contrast, TFIIB induced a slight change only at position 473. Our results support models of TBP as a target protein for transcriptional activators and suggest that ordered structure in the VP16 activation domain is induced upon interaction with target proteins.

Binding Sites↗
PubMed1997

Transcription factor TFIID recruits factor CPSF for formation of 3' end of mRNA.

Initiation of transcription by RNA polymerase II from a promoter region on DNA requires the assembly of several initiation factors to form a preinitiation complex. Assembly of this complex is initiated by the binding of the transcription factor TFIID, composed of the TATA-box binding protein (TBP) and TBP-associated factors (TAF[II]s), to the promoter. We have now characterized an immunopurified TFIID complex which we unexpectedly find contains the cleavage-polyadenylation specificity factor (CPSF), one of the factors required for formation of the 3' end of messenger RNA. CPSF is brought to the preinitiation complex by TFIID, but after transcription starts, CPSF dissociates from TFIID and becomes associated with the elongating polymerase. We also show that overexpression of recombinant TBP in HeLa cells decreases polyadenylation without affecting the correct initiation of transcription of the reporter gene. This indicates that, owing to incomplete assembly of TFIID on recombinant TBP, CPSF is not brought to the promoter and therefore polyadenylation becomes less efficient. Our observations have thus revealed a link between transcription initiation and elongation by RNA polymerase II and processing of the 3' end of mRNA.

Amino Acid Sequence↗
PubMed1998

Nucleosome structure of the yeast CHA1 promoter: analysis of activation-dependent chromatin remodeling of an RNA-polymerase-II-transcribed gene in TBP and RNA pol II mutants defective in vivo in response to acidic activators.

The Saccharomyces cerevisiae CHA1 gene encodes the catabolic L-serine (L-threonine) dehydratase. We have previously shown that the transcriptional activator protein Cha4p mediates serine/threonine induction of CHA1 expression. We used accessibility to micrococcal nuclease and DNase I to determine the in vivo chromatin structure of the CHA1 chromosomal locus, both in the non-induced state and upon induction. Upon activation, a precisely positioned nucleosome (nuc-1) occluding the TATA box and the transcription start site is removed. A strain devoid of Cha4p showed no chromatin alteration under inducing conditions. Five yeast TBP mutants defective in different steps in activated transcription abolished CHA1 expression, but failed to affect induction-dependent chromatin rearrangement of the promoter region. Progressive truncations of the RNA polymerase II C-terminal domain caused a progressive reduction in CHA1 transcription, but no difference in chromatin remodeling. Analysis of swi1, swi3, snf5 and snf6, as well as gcn5, ada2 and ada3 mutants, suggested that neither the SWI/SNF complex nor the ADA/GCN5 complex is involved in efficient activation and/or remodeling of the CHA1 promoter. Interestingly, in a sir4 deletion strain, repression of CHA1 is partly lost and activator-independent remodeling of nuc-1 is observed. We propose a model for CHA1 activation based on promoter remodeling through interactions of Cha4p with chromatin components other than basal factors and associated proteins.

Chromatin↗
PubMed1995

Induction of Sp1 phosphorylation and NF-kappa B-independent HIV promoter domain activity in T lymphocytes stimulated by okadaic acid.

In contrast to the purely enhancer-dependent effect of cytokines such as TNF on the activity of the HIV regulatory region (LTR), we observed that okadaic acid (OKA) activates HIV transcription through both the enhancer, responding to the factor NF-kappa B, and the promoter domain of the LTR. The inducibility of HIV LTR-driven luciferase expression constructs in lymphoblastoid cells stimulated by OKA depended on both functional Sp1 binding elements and the ability of the TATA box to bind the protein TBP. In both transformed and normal lymphocytes, OKA stimulation induced intense phosphorylation of the constitutively expressed Sp1 protein in the nucleus, a property of OKA not shared by TNF, phorbol ester, or PHA and interleukin 2. Responsiveness of LTR constructs deleted of kappa B elements to HIV Tat expression was increased upon OKA but not TNF stimulation. Our results suggest that SP1 phosphorylation induced by OKA, a selective inhibitor of the serine-threonine phosphatase PP2A, facilitates the formation of a transcription complex involving general transcription factors, HIV Tat, and Sp1 proteins. The formation of this complex would increase, independently of an in synergy with NF-kappa B, the low basal activity of the HIV LTR observed in normal T lymphocytes.

Base Sequence↗
PubMed1994

The POU domains of the Oct1 and Oct2 transcription factors mediate specific interaction with TBP.

We had previously shown that the ubiquitous Oct1 and the lymphoid-specific Oct2 transcription factors stimulate transcription at the level of stable preinitiation complex formation. We have therefore investigated whether the octamer binding proteins might physically interact with TBP, the TATA box binding protein component of the TFIID factor. By using several different experimental systems we show that TBP efficiently associates with Oct1 and Oct2. The interaction is direct and does not depend on the presence of DNA or additional proteins. N- and C-terminal deletions of the different proteins were used to localize the domains involved in the interaction. We show that the POU homeodomain of Oct2 and the evolutionarily conserved C-terminal core domain of TBP are both required and sufficient for the interaction. The Oct1 POU domain, which is highly homologous to the Oct2 POU domain, likewise mediates interaction with TBP. The interaction can also be observed in vivo, as TBP can be co-precipitated with Oct2 from co-transfected Cos1 cells and TBP co-immunoprecipitates with the endogenous Oct1 from HeLa cells. Co-transfection of human TBP and Oct2 expression vectors into B cells resulted in a synergistic activation of an octamer motif containing promoter.

Binding Sites↗
PubMed1998

Recruitment of the TATA-binding protein to the HIV-1 promoter is a limiting step for Tat transactivation.

OBJECTIVES: To examine the functional interaction between HIV-1 Tat protein and the TATA-binding protein (TBP). DESIGN: HIV long terminal repeat reporter plasmids were cotransfected into mammalian and Drosophila insect cells with expression vectors encoding Tat and vectors encoding TBP either alone or linked to an heterologous DNA-binding domain. METHODS: The activity of the different reporters was compared in the presence or absence of Tat or TBP, or both, upon cotransfections into human and Drosophila insect cell lines. RESULTS: Tat protein is unable to transactivate enhancerless HIV-1 minimal promoter bearing only the TATA box and TAR. Artificial recruitment of human TBP (hTBP) to the enhancerless HIV minimal promoter was found to trigger gene expression and coexpression of Tat resulted in a marked synergy. Tat protein cooperated with DNA-bound hTBP by inducing high levels of processive viral transcripts. Synergy between Tat and hTBP was also observed when both factors were targeted to a promoter DNA template. The functional cooperation between TBP and Tat was further demonstrated using the Drosophila Schneider SL2 cells. In these cells Tat protein alone was ineffective; however, coexpression of Drosophila TBP and Tat resulted in a trans-activating response region-dependent synergistic transactivation of basal transcription. CONCLUSION: The strong synergy between TBP and Tat in the absence of any DNA-bound activator suggests that Tat stimulates transcription in an activator-independent manner most likely by a functional interaction with general transcription factors that occurs after TBP recruitment. Thus, efficient recruitment of TBP represents a limiting step for Tat transactivation.

Animals↗