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PubMed1992

Identification of human TFIID components and direct interaction between a 250-kDa polypeptide and the TATA box-binding protein (TFIID tau).

Previous studies have indicated that human transcription initiation factor TFIID is a large complex that contains a TATA-binding polypeptide (TFIID tau or TBP) and other components that qualitatively alter promoter interactions and are uniquely required for activator-dependent (versus basal) transcription. TFIID tau-specific antibody columns have been employed to identify a number of human TFIID polypeptides that are tightly associated with TFIID tau. These differ in size from polypeptides in known general initiation factors, including the initiator-binding factor (TFII-I) which shares some promoter binding characteristics with TFIID. The largest component (p250) identified in TFIID was shown to interact directly and tightly with TFIID tau, suggesting that it may play a major role in the assembly of the TFIID complex.

DNA-Binding Proteins↗

PubMed1992

Wild-type p53 binds to the TATA-binding protein and represses transcription.

p53 activates transcription of genes with a p53 response element, and it can repress genes lacking the element. Here we demonstrate that wild-type but not mutant p53 inhibits transcription in a HeLa nuclear extract from minimal promoters. Wild-type but not mutant p53 binds to human TATA-binding protein (TBP). p53 does not bind to yeast TBP, and it cannot inhibit transcription in a HeLa extract where yeast TBP substitutes for human TBP. These results suggest a model in which p53 binds to TBP and interferes with transcriptional initiation.

Animals↗

PubMed1992

A role for the TATA-box-binding protein component of the transcription factor IID complex as a general RNA polymerase III transcription factor.

The major class of vertebrate genes transcribed by RNA polymerase (EC 2.7.7.6) III, which includes 5S rRNA genes, tRNA genes, and the adenovirus VA genes, is characterized by split internal promoters and no absolute dependence upon specific upstream sequences. Fractionation experiments have shown that transcription of such genes requires two general RNA polymerase III-specific factors, TFIIIB and TFIIIC. We now demonstrate that a third general factor is also employed by these genes. This is the TATA-box-binding protein originally identified as being a component of the general RNA polymerase II transcription factor TFIID. This protein is involved in the transcription by RNA polymerase III of every template tested, even though the promoters of VA and most vertebrate tRNA and 5S rRNA genes do not contain recognizable TATA elements.

Base Sequence↗

PubMed1992

Cofractionation of the TATA-binding protein with the RNA polymerase III transcription factor TFIIIB.

We have investigated the requirement for TBP (TATA-binding protein) in transcription mediated by RNA polymerase III (pol III) in fractionated HeLa cell extracts. Two activities, TFIIIB and TFIIIC, found in phosphocellulose fractions PC B and PC C respectively, have been defined as necessary and sufficient, with pol III, for in vitro transcription of tRNA genes. Depletion of TBP from PC B, using antibodies raised against human TBP, is shown to inhibit the pol III transcriptional activity of the fraction. Furthermore, TBP is present in fractions with human TFIIIB activity, and a proportion of TBP cofractionates with TFIIIB over four chromatographic purification steps. TFIIIB fractions are capable of supplying TBP in the form necessary for pol III transcription, and cannot be substituted by fractions containing other TBP complexes or TBP alone. The use of a 5S RNA gene and two tRNA templates supports the general relevance of our findings for pol III gene transcription. Purified TFIIIB activity can also support pol II-mediated transcription, and is found in a complex of approximately 230kD, suggesting that TFIIIB may be the same as the previously characterized B-TFIID complex (1,2). We suggest that transcription by the three RNA polymerases is mediated by distinct TBP-TAF complexes: SL1 and D-TFIID for pol I and pol II respectively, and TFIIIB for pol III.

Chemical Fractionation↗

PubMed1992

Multiple domains of the RNA polymerase I activator hUBF interact with the TATA-binding protein complex hSL1 to mediate transcription.

Recent evidence suggests that transcription initiation by all three eukaryotic RNA polymerases involves a complex of the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs). Here, we map the functional domains of the nucleolar HMG box protein hUBF, which binds to the human rRNA promoter and stimulates transcription by RNA polymerase I through cooperative interactions with a distinct TBP-TAF complex, hSL1. DNase I footprint analysis of mutant hUBF proteins and of a synthetic peptide of 84 amino acids reveals that HMG box 1 is necessary and sufficient for DNA sequence specificity, whereas other HMG boxes and the amino terminus modulate the binding efficiency. hUBF contains multiple activation domains that include the acidic carboxyl terminus and three HMG boxes. HMG boxes 3 and 4 and the acidic tail contribute significantly to an extended footprinting pattern in the presence of hSL1, suggestive of specific protein-protein interactions. Moreover, the inability of xUBF from Xenopus laevis to form an initiation complex with hSL1 can be overcome by hybrid proteins containing human HMG box 4 and the acidic carboxyl terminus. These results strongly suggest an important role of transcription activation domains of hUBF in mediating interactions with the TBP-TAF complex hSL1.

Animals↗

PubMed1992

Holo-TFIID supports transcriptional stimulation by diverse activators and from a TATA-less promoter.

Transcription factor IID (TFIID) binds to TATA boxes, nucleating the assembly of initiation complexes containing several general transcription factors and RNA polymerase II. Recently, TFIID was shown to be a multisubunit complex containing a TATA box-binding polypeptide (TBP) and several tightly associated polypeptides (TAFs), which are required for transcriptional stimulation by activator proteins. Here, we report the development of a human cell line expressing an epitope-tagged TBP and the immunopurification of a native, high-molecular-weight form of TFIID that supports transcriptional stimulation by several different classes of activation domains. Recovery of basal and activated TFIID transcriptional specific activity was close to approximately 100%. Electrophoretic mobility-shift analysis demonstrated a single major DNA-protein complex. This holo-TFIID contains TAFs of approximately 250, 125, 95, 78, and 50 kD and sediments at 17S. Holo-TFIID produced an extended footprint over the adenovirus major late promoter TATA box and initiator sequence and supported transcriptional activation from a promoter lacking a TATA box. These results lead us to hypothesize that a single multisubunit TFIID protein supports transcriptional stimulation by diverse activation domains and from a TATA-less promoter.

Base Sequence↗

PubMed2026

Structural Features of DNA in TATA-Containing and TATA-Less Core Promoters of RNA Polymerase II Differ.

Nucleotide motifs in the core promoters of eukaryotic protein-coding genes transcribed by RNA polymerase II (Pol II) play an important role in the transcription process. We analyzed the role of an octanucleotide located in the TATA box position. Depending on whether this octanucleotide can form a complex with the TATA-binding protein (TBP), the promoter is classified as either TATA-containing or TATA-less. We analyzed the differences in the primary and spatial structures, as well as their dynamics, in TATA-containing and TATA-less promoters of mammals and plants. We divided the complete promoter sets of six organisms (H. sapiens, M. musculus, C. familiaris, A. thaliana, Z. mays, and H. vulgare) from the EPDnew database into TATA-containing and TATA-less fractions. The sizes of the TATA-containing promoter fractions are significantly smaller than those of the TATA-less fractions in all studied organisms, except in A. thaliana, where the sizes of both fractions are approximately equal. We characterized promoter architecture using variation profiles of various base-pair step parameters, minor-groove width, and the conformational dynamics of native DNA. The architectures of TATA-containing and TATA-less promoters differ significantly. The possible mechanistic influence of DNA structural features on the formation of the pre-initiation complex (PIC) in both types of promoters is discussed.

Promoter Regions, Genetic↗

PubMed1992

TATA-binding protein activates transcription when upstream of a GCN4-binding site in a novel yeast promoter.

In the gal-his3 hybrid promoter, his3-GG1, GCN4 stimulates transcription at the position normally occupied by a TATA element. This expression requires two elements within gal1-10 sequences, a REB1-binding site and a second element, Z, which resides 20 base pairs upstream of the GCN4-binding site. No obvious TATA element is present in this promoter. To characterize the function of Z, we replaced it with short random oligonucleotides and selected for expression in vivo. Fourteen elements were identified and classified into groups based upon sequence and phenotypic similarities. Group 1 elements contained functional TATA sequences that were essential for activity. TATA elements can thus function when positioned upstream of a GCN4-binding site. The Group 2 elements activated transcription poorly when used as conventional TATA elements; however, mutational analyses demonstrated that their activity required TATA-like sequences. These TATA-like sequences bound the yeast TATA-binding protein (TBP) poorly in vitro but function in vivo as TBP interaction sites based upon two criteria. First mutations that improved their TATA character correspondingly improved function and second their activity could be enhanced in the presence of an altered binding specificity mutant of TBP. Furthermore, the Group 2 elements enabled the identification of mutations outside of the TATA-like core that contribute to transcriptional activation without adversely affecting TBP binding. The finding that low affinity TBP-binding sites can be used at unconventional positions suggests that many "TATA-less" promoters contain a cryptic interaction site for TBP.

Alleles↗

PubMed1992

Recombinant TBP, transcription factor IIB, and RAP30 are sufficient for promoter recognition by mammalian RNA polymerase II.

Initiation of transcription by RNA polymerase II is a complex, multistep process which requires several accessory factors in addition to the polymerase itself. A critical event in transcription initiation is the specific association of RNA polymerase II with promoter DNA. In this report we show that three eukaryotic polypeptides, produced in Escherichia coli and purified to near homogeneity, constitute a minimal set of general transcription factors both necessary and sufficient for specific and stable promoter binding by RNA polymerase II. These polypeptides are the yeast TATA box binding protein TBP, the human general initiation factor TFIIB, and human RAP30, the small subunit of RAP30/74 (or transcription factor IIF). Formation of the polymerase-containing complex required only the TATA box, and not the initiator element (Inr), of the adenovirus major late promoter which was used in these experiments.

Base Sequence↗

PubMed1992

Construction of a tetracycline-inducible promoter in Schizosaccharomyces pombe.

We have developed a tightly repressed Schizosaccharomyces pombe promoter which can be efficiently induced by Tetracycline. This promoter is a derivative of the plant viral cauliflower mosaic virus 35S promoter which normally functions as a strong constitutive promoter in S. pombe. Location of three binding sites for the Tn10-encoded Tet repressor in the vicinity of the TATA-box of the CaMV 35S promoter led to a tight repression of promoter activity in the presence of the Tet repressor protein. Up to a 400-fold induction was observed after addition of the inducer Tetracycline, which inactivates the operator-binding capacity of the repressor.

Base Sequence↗

PubMed1992

Zmhox1a, the product of a novel maize homeobox gene, interacts with the Shrunken 26 bp feedback control element.

A new maize homeobox gene was isolated by screening a lambda gt11 expression library with the 26 bp Shrunken feedback control element. Zmhox1a (Zea mays homeobox) is an unidentified maize gene mapping to the long arm of chromosome 8. It is a member of a new class of maize homeobox genes only distantly related to the Knotted class. The 3.1 kb Zmhox1a transcript can be detected in different maize tissues and encodes a polypeptide of 719 amino acids. Western blotting experiments detect the native 112 or 115 kDa protein in nuclear protein extracts, the nuclear localization being compatible with a function in transcriptional control. No Zmhox1a protein is detected in maize roots despite the presence of the Zmhox1a transcript; this may indicate a post-transcriptional control mechanism. A highly acidic central region of the Zmhox1a polypeptide implies a transcriptional activator function. The carboxy-terminal part of the maize homeodomain protein is related to the human Oct2 transcription factor, but homology to the POU specific domain is restricted to the POU-B subdomain. It was confirmed by DNase I footprinting experiments that DNA binding of the Zmhox1a homeodomain was at three sites flanking the TATA-box of the Shrunken promoter.

Amino Acid Sequence↗

PubMed1992

Transcription factor eUSF is an essential component of isolated transcription complexes on the duck histone H5 gene and it mediates the interaction of TFIID with a TATA-deficient promoter.

We analysed the formation of transcription complexes on the H5 gene of the duck which is efficiently transcribed in HeLa cell extracts in vitro. Upon deletion of its TATA-box, the fidelity of transcription of the H5 gene is maintained, although the efficiency of this process is significantly reduced. Selective inactivation of TFIID in whole cell extracts and reconstitution experiments either with human recombinant TFIID or a protein fraction from duck erythrocytes enriched in TFIID show that transcription of the TATA-less H5 promoter nevertheless requires the protein TFIID. Screening of promoter elements which could indirectly mediate the interaction of TFIID with a TATA-less H5 promoter led to the identification of a sequence element located about 40 base-pairs downstream from the H5 initiation site that shows partial homology to the USF consensus sequence. In electrophoretic mobility shift and footprinting studies we demonstrated a specific interaction of the erythroid factor USF (eUSF) with this downstream element. By isolating active transcription complexes we found that all components required for correct initiation remain stably associated with the H5 promoter irrespective of the presence or absence of the TATA box. Moreover, the reconstitution of eUSF and TFIID-depleted transcription complexes with purified protein fractions demonstrate that not only TFIID but also eUSF essentially participates in complex formation even on H5 promoter mutations lacking the TATA-box. Mutual interactions between eUSF and TFIID appear to stabilize the binding of TFIID in the presence or absence of its proper binding site.

Animals↗

PubMed1992

During negative regulation of the human papillomavirus-16 E6 promoter, the viral E2 protein can displace Sp1 from a proximal promoter element.

The principal early promoter of human papillomaviruses (HPVs), designated P97 in the case of HPV-16, contains four characteristically aligned cis-responsive elements, namely one binding site for Sp1, two for the viral E2 proteins, and the TATA box. The Sp1 binding site is needed to mediate activation of P97 by the remote epithelial-specific enhancer, and the two E2 binding sites contribute to a negative feedback-loop of viral gene expression. The Sp1 consensus motif and the TATA-box distal E2 binding site are spaced in all genital papillomaviruses by a single nucleotide. We show here that at physiological concentrations, the binding of E2 proteins and Sp1 are mutually exclusive events, since a bandshift analysis with nuclear extracts from ID13, a mouse cell line transformed by BPV-1, showed only the E2 or the Sp1 bandshift, but no complex indicative of the concomitant binding of both factors. Increasing concentrations of in vitro translated E2 protein compete efficiently with the Sp1 factor for binding to an oligonucleotide containing both binding sites. Interference between Sp1 and E2 protein binding is apparently relevant for P97 repression in vivo, since a mutational analysis revealed that both E2 binding sites are necessary for negative transcriptional regulation: Alone, neither the distal site, where E2 protein can induce Sp1 displacement, nor the proximal site, where E2 protein interferes with formation and function of the pre-initiation complex, have a significant effect, but two functional E2 binding sites lead to repression of P97.

Animals↗

PubMed1992

A novel intestinal trans-factor (NF-LPH1) interacts with the lactase-phlorizin hydrolase promoter and co-varies with the enzymatic activity.

The promoter of the pig lactase-phlorizin hydrolase was cloned and showed to be functional in the human intestinal cell line Caco2. The proximal promoter was analyzed for binding of nuclear proteins from small intestine and liver. DNase I footprinting and electrophoretic mobility shift assays show, that an intestinal nuclear factor (NF-LPH1) binds to a sequence (-40 to -54) located close to the TATA-box. Enterocytes from newborn pigs with high lactase activity contain high amounts of NF-LPH1, whereas enterocytes from adult pigs with low lactase activity contain low amounts of NF-LPH1. The liver does not contain lactase activity, and NF-LPH1 is not present in liver nuclear extracts in detectable amounts. This indicates that NF-LPH1 is involved in the decline of lactase at weaning and may be of importance for the molecular explanation of hypolactasia in humans. It was demonstrated by transfection of two different promoter-reporter gene constructs into Caco2 cells, that there are additional cis-element(s) in the region -142 to approximately -980, which are important for the transcription of the lactase-phlorizin hydrolase gene.

Animals↗

PubMed1992

Activation of the weakly regulated PHO8 promoter in S. cerevisiae: chromatin transition and binding sites for the positive regulatory protein PHO4.

PHO8 encodes an alkaline phosphatase in Saccharomyces cerevisiae whose transcription is regulated by the phosphate concentration in the medium. This occurs through the action of several positive and negative regulatory proteins, also involved in the regulation of other members of the phosphatase gene family. A central role is played by PHO4, the gene encoding a DNA binding regulatory protein. Digestion experiments with DNasel, micrococcal nuclease and 20 different restriction nucleases show that under conditions of PHO8 repression, there is a highly ordered chromatin structure at the promoter consisting of three hypersensitive regions, approximately 820 to 690, 540 to 510, and 230 to 160 bp upstream of the initiation codon. These hypersensitive sites are surrounded by DNA organized in nucleosomes. Gel shift analysis and in vitro footprinting revealed the presence of two PHO4 binding sites at the PHO8 promoter: a low affinity site at -728 and a high affinity site at -532. Each one is located within a hypersensitive site. Upon derepression of PHO8, the chromatin structure changes significantly: The two upstream hypersensitive sites containing the PHO4 binding sites merge, resulting in a long region of hypersensitivity. This transition is PHO4 dependent. However, not all of the promoter becomes nucleosome free. Instead, as a novel feature, regions of intermediate accessibility are generated upstream and downstream of the third hypersensitive site, the latter region encompassing the TATA-box. The available data fit best into a concept that these regions are organized in unstable or partly unfolded nucleosomes.

Alkaline Phosphatase↗

PubMed1992

Structure and expression of the nuclear gene coding for the plastid CS1 ribosomal protein from spinach.

The chloroplast ribosomal protein CS1 is an essential component of the plastids translational machinery involved in translation initiation. Southern analysis suggests that the corresponding nuclear gene is present in one copy in the spinach genome. We have isolated and sequenced the gene (rps1) to study its expression at the transcriptional level. The gene consists of 7 exons and 6 introns including an unusually large intron in the 5' coding region. No canonical TATA-box is found in the 5' upstream region of the gene. rps1 transcripts are detected early during germination and a significant accumulation is observed after the protrusion of the radicle. CS1 mRNAs are present in all organs of young seedlings although there are dramatic differences in the steady state level of the mRNAs between leaves and roots tissues. Transcripts accumulate independently of the presence or absence of light. Band shift analysis shows that the +1, -400 bp region of the gene can bind different sets of proteins isolated from roots and leaves nuclei. We suggest that the expression of the housekeeping plastid-related rps1 gene is regulated in a tissue-specific manner by transcriptional trans-acting factors.

Amino Acid Sequence↗

PubMed1992

Positive and negative regulation of collagenase gene expression.

Transcription of the human collagenase I gene is induced by phorbol esters and repressed by glucocorticoids. Both types of regulation are mediated by the major enhancer element of the gene, which is localized between positions -73 and -65. The enhancer suffices to transmit positive and negative signals to a heterologous promoter or to a minimal promoter carrying only the TATA-box, both detected by the appearance of chloramphenicol-acetyl-transferase transcribed from the reporter gene linked to the promoters. Sequences 5' of the enhancer modulate its activity. Up- and down-regulation of gene constructs which contain only the collagenase enhancer linked to a heterologous promoter, are independent of ongoing protein synthesis, suggesting posttranslational modification of the transcription factor binding to the enhancer. Repression by glucocorticoids depends on an activated glucocorticoid receptor; a tenfold lower glucocorticoid concentration is needed for repression of the collagenase gene as compared to the activation of the mouse mammary tumor virus long terminal repeat. Immunoprecipitates of the dexamethasone receptor contain AP-1, suggesting a direct interaction of both transcription factors; this interaction may lead to the inactivation of AP-1. The action mechanism of phorbol esters and dexamethasone confirms the central role of AP-1 in proliferation control and tumor promotion. It appears that the most effective tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and the most effective anti-tumor promoter dexamethasone exert their action through the modulation of the same transcription factor.

Base Sequence↗

PubMed1992

A cyclic AMP response element is involved in retinoic acid-dependent RAR beta 2 promoter activation.

Activation of the retinoic acid receptor (RAR) beta 2 promoter is known to be mediated by a RA response element located in the proximity of the TATA-box. By deletion studies in P19 embryonal carcinoma cells we have analyzed the RAR beta 2 promoter for the presence of additional regulatory elements. We found that the cyclic AMP response element-related motif, TGATGTCA at position -99 to -92, is able to enhance RA-dependent RAR beta 2 promoter activation. In addition we demonstrate that this element, designated CRE-beta 2, is functionally active as a CRE since it can bind members of the CREB/ATF transcription factor family and, moreover, mediates the stimulatory effect of cAMP on RA-dependent RAR beta 2 promoter activation in human foetal kidney 293 cells.

Base Sequence↗