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At least 109 records · Page 6Linked to original sources
PubMed1995

Lens-specific activity of the mouse alpha A-crystallin promoter in the absence of a TATA box: functional and protein binding analysis of the mouse alpha A-crystallin PE1 region.

Lens-specific expression of the mouse alpha A-crystallin gene is regulated at the level of transcription. Here, we have studied the role of the PE1 region, which contains the TATA box (-31/-26) and the immediately adjacent PE1B sequence (-25/-12), in transcriptional regulation. Deletions within either the TATA box or PE1B sequence eliminated promoter activity in transfected lens cells. Surprisingly, these deletions did not eliminate lens-specific promoter activity of the transgene of transgenic mice. Transcription of the transgene with a TATA-deleted promoter initiated at multiple sites in the lenses of the transgenic mice. Footprint analysis revealed that the entire PE1 region was protected by nuclear extracts prepared from lens cells which express the alpha A-crystallin gene and from fibroblasts which do not express the gene. The -37/+3 region formed three specific EMSA complexes using lens cell nuclear extracts, while a similar but much less intense pattern was observed when a fibroblast nuclear extract was used. Competition experiments indicated that these complexes were not due to the binding of TBP to the TATA box, but rather to the binding of other nuclear proteins to the PE1B -25/-19 region. A series of co-transfection competition studies in vivo also suggested the functional importance of proteins binding in the -25/-19 region. The PE1B protein-DNA interactions appear to be conserved in the chicken, rodent and human alpha A-crystallin gene as well as within the alpha A- and alpha B-crystallin genes in the mouse. Our findings indicate that the PE1B region is important for mouse alpha A-crystallin promoter activity; the proximity of this site to the TATA box raises the possibility for cooperativity or competition between TBP and PE1B-bound proteins.

Animals↗
PubMed1994

Second-site long terminal repeat (LTR) revertants of replication-defective human immunodeficiency virus: effects of revertant TATA box motifs on virus infectivity, LTR-directed expression, in vitro RNA synthesis, and binding of basal transcription factors TFIID and TFIIA.

Second-site revertants from replication-incompetent molecular clones of human immunodeficiency virus (HIV) contain base substitutions adjacent to the TATA motif. The altered TATA box motifs were analyzed for their effect(s) on virus infectivity, long terminal repeat (LTR)-directed expression in transient transfection assays, in vitro RNA synthesis, and assembly of the TFIID-TFIIA preinitiation complex. The revertant TATA boxes accelerated the kinetics of HIV replication when present in the context of an LTR containing a Sp1 mutation (deletion or site specific); no effect was observed on the infectivity of wild-type HIV. In chloramphenicol acetyltransferase assays and in vitro transcription systems, the altered TATA box motifs led to elevated basal levels of RNA synthesis from NF-kappa B- and Sp1-mutagenized and wild-type templates, respectively, but did not increase responsiveness to Tat transactivation. The revertant TATA boxes accelerated the binding of TFIID and TFIIA to the LTR and stabilized their association with the promoter. The revertants did not assemble a more-processive elongation complex. These results suggest that in the context of an impaired enhancer/promoter (viz., three mutated Sp1 elements), a series of HIV revertants emerge which contain LTR alterations that significantly augment basal RNA synthesis. The TATA motif revertants are capable of rescuing the enhancer/promoter defect and sustain virus infectivity.

Base Sequence↗
PubMed1996

Analysis of co-crystal structures to identify the stereochemical determinants of the orientation of TBP on the TATA box.

Possible stereochemical determinants of the orientation of TBP on the TATA box are discussed using the crystal coordinates of TBP-TATA complexes, which have been determined by other groups. The C-terminal half of the TBP beta-sheet interacts with the TATA site of the DNA, and the N-terminal half with the A-rich site, so that the two sites with distinct curvatures produce a unique fit. Although chemical contacts take place between one side of the beta-sheet and the DNA minor groove, the interaction seems to be facilitated indirectly by the characteristics of the other side of the beta-sheet and the DNA major groove. Thus, Ala71, Leu162 and Pro190 differentiate the curvature of the beta-sheet in the N- and C-halves. The methyl positions in the DNA major groove modulate the bendability of the two DNA sites by using differences in the rolling capacity of TA and AT compared with PyT, and in the shifting capacity of AT compared with TT. The deformations of the first steps (TA and PyT) in the two sites are the largest and thus are important for the overall bending of the DNA. The differences between the two DNA sites are greatest at the second steps (AT and TT) and so these are important for determining the orientation of TBP.

Amino Acid Sequence↗
PubMed1996

Localization of the 12-O-tetradecanoylphorbol-13-acetate response of the human ornithine decarboxylase promoter to the TATA box.

In a previous study, we narrowed the region of the human ornithine decarboxylase (ODC) promoter responsive to 12-O-tetradecanoylphorbol-13-acetate (TPA) to nt -42 to +54 around the transcription initiation site (Kim YJ, Pan H, Verma AK, Mol Carcinog 10:169-179, 1994). Here we report defining the role of the TATA box in TPA-induced transcription from the -42/+54 ODC promoter fragment. A transversion mutation at the third position of the TATA box (TATAAGT-->TAAAAGT) reduced TPA responsiveness of the reporter construct -42/+54 ODC-Luc by 49%. Electrophoretic mobility shift assays (EMSAs) using HeLa cell nuclear protein extracts revealed no differences in the binding pattern between the natural -42/+54 ODC promoter element and the -42/+54 ODC promoter element containing the T-->A mutation. However, antibodies to the general transcription factor TFIIB disrupted the DNA-protein complexes normally formed with the -42/+54 ODC promoter element in EMSAs. A consensus TATA box oligonucleotide formed two bands, with the faster mobility band displaying enhanced binding with nuclear protein extracts from TPA treated cells. Furthermore, incubation of HeLa cell nuclear extracts with an oligonucleotide containing the ODC TATA box also caused formation of two specific bands in EMSA. Both bands exhibited augmented binding to nuclear proteins from TPA-treated cells. Introduction of the T-->A transversion mutation in the ODC TATA oligonucleotide eliminated binding of the faster migrating band formed with the natural ODC TATA oligonucleotide. These results indicate that TPA modulation of the general transcription machinery may play a role in the TPA-activated transcription of the human ODC promoter.

Cell Nucleus↗
PubMed1990

Functional heterogeneity of mammalian TATA-box sequences revealed by interaction with a cell-specific enhancer.

A regulatory element upstream of the human myoglobin gene functions as a muscle-specific enhancer (MSE) in conjunction with core promoter elements of the myoglobin gene, but not in combination with the simian virus 40 (SV40) early promoter. These two promoters differ in the sequences of their 'TATA boxes': for the myoglobin gene, the sequence is TATAAAA, whereas for SV40, the sequence is TATTTAT. We have now tested the hypothesis that this sequence difference is responsible for the differential response of the promoters to the MSE. We found that when the TATA box sequence of the myoglobin promoter was changed to that of the SV40 promoter, responsiveness to the MSE was abolished; conversely, when the SV40 TATA box sequence was changed to that of the myoglobin promoter, the promoter became responsive to the MSE. We conclude that mammalian TATA-box elements are functionally heterogeneous, and suggest that this heterogeneity reflects differential interactions with distinctive TATA box-binding factors, only some of which can act cooperatively with MSE-binding proteins to generate an active transcriptional complex.

Chloramphenicol O-Acetyltransferase↗
PubMed2002

Targeted histone acetylation at the yeast CUP1 promoter requires the transcriptional activator, the TATA boxes, and the putative histone acetylase encoded by SPT10.

The relationship between chromatin remodeling and histone acetylation at the yeast CUP1 gene was addressed. CUP1 encodes a metallothionein required for cell growth at high copper concentrations. Induction of CUP1 with copper resulted in targeted acetylation of both H3 and H4 at the CUP1 promoter. Nucleosomes containing upstream activating sequences and sequences farther upstream were the targets for H3 acetylation. Targeted acetylation of H3 and H4 required the transcriptional activator (Ace1p) and the TATA boxes, suggesting that targeted acetylation occurs when TATA-binding protein binds to the TATA box or at a later stage in initiation. We have shown previously that induction results in nucleosome repositioning over the entire CUP1 gene, which requires Ace1p but not the TATA boxes. Therefore, the movement of nucleosomes occurring on CUP1 induction is independent of targeted acetylation. Targeted acetylation of both H3 and H4 also required the product of the SPT10 gene, which encodes a putative histone acetylase implicated in regulation at core promoters. Disruption of SPT10 was lethal at high copper concentrations and correlated with slower induction and reduced maximum levels of CUP1 mRNA. These observations constitute evidence for a novel mechanism of chromatin activation at CUP1, with a major role for the TATA box.

Acetylation↗
PubMed1995

TATA box and initiator functions in the accurate transcription of a plant minimal promoter in vitro.

The functional architecture of the proximal region of a rice phenylalanine ammonia-lyase (PAL) promoter was analyzed by transcription of PAL-beta-glucuronidase (GUS) templates by whole-cell extracts of rice cell suspension cultures. The promoter 5' truncated to position -35 was sufficient for accurate initiation of basal transcription. Substitution of the TATTTAA sequence between positions -35 and -28 with GCGGGTT or 2-bp substitutions to give TCGTTAA and TATGGAA inactivated the minimal promoter. Moreover, the function of the TATTTAA sequence was dependent on its position relative to the initiation site; hence, this element is an authentic TATA box essential for RNA polymerase II-dependent transcription. Substitutions in the TCCAAG initiator cis element (-3 to +3) at the -1 (C to A or G) and +1 (A to C or T) residues caused inaccurate initiation, whereas mutations at the other residues of this conserved element or sequence substitutions between the TATA box and initiator had little effect. TATA box and initiator functions were confirmed by analysis of the effects of promoter mutations on expression in stably transformed rice cell suspensions and plants. We concluded that the proximal region of the PAL promoter has a simple functional architecture involving a TATA box appropriately positioned upstream of the initiator. Transcription of derivatives of such minimal promoters by highly active cell extracts should allow molecular analysis of functional interactions between specific cis elements and cognate trans factors.

Base Sequence↗
PubMed1994

Binding of TFIID to the CYC1 TATA boxes in yeast occurs independently of upstream activating sequences.

Functional transcription initiation complexes can be assembled in vitro without the aid of regulatory factors that bind to upstream activating sequences. However, promoters that lack upstream activating sequences are transcribed poorly if at all in vivo, suggesting that regulatory factors are necessary for the assembly of transcription initiation complexes in cells. To test this possibility, we asked whether the general transcription factor TFIID can bind to a promoter in yeast that lacks upstream activating sequences and is transcriptionally inactive. Analysis of an inactive CYC1 core promoter by high-resolution genomic footprinting revealed efficient binding of TFIID to either of two TATA box elements. Addition of a heat shock element rendered this promoter highly responsive to induction of transcription by heat shock but did not alter the TATA box footprints in the core promoter. Inactivation of all but one TATA box by site-directed mutagenesis did not prevent TFIID from binding to the remaining wild-type TATA box independently of regulatory sequences. These results indicate that upstream regulatory factors are not required for the in vivo binding of TFIID to the CYC1 promoter and that binding of TFIID to DNA is not necessarily a rate-limiting step in the activation of transcription in cells. Differences in chromatin structure may account for why regulatory transcription factors are required for the binding of TFIID to some promoters but not to others.

Base Sequence↗
PubMed1996

Transcription of the transforming growth factor-beta2 gene is dependent on an E-box located between an essential cAMP response element/activating transcription factor motif and the TATA box of the gene.

Transforming growth factor-beta2 (TGF-beta2) is an important regulator of cell proliferation and differentiation; however, its transcriptional regulation is not well understood. Here we report characterization of an essential E-box motif, positioned at -50/-45 between a previously described functional cAMP response element/activating transcription factor site and the TATA box of the human TGF-beta2 promoter. By site-directed mutagenesis, we demonstrate that this E-box motif is necessary for the promoter activity, not only in differentiated cells derived from embryonal carcinoma cells, but also in choriocarcinoma cells and in MCF-7 breast carcinoma cells. We also demonstrate that the transcription factors USF1 and USF2 bind to this E-box motif in vitro when nuclear extracts from each of these cell lines are examined by gel retardation assays. Moreover, using a dominant-negative USF2 protein, we show that USF proteins are critical for TGF-beta2 promoter activity in vivo. The importance of the E-box motif described in this study is supported by the presence of an E-box motif in the same position in the chicken TGF-beta2 gene promoter.

Cell Line↗
PubMed1988

A regulatory region responsible for proline-specific induction of the yeast PUT2 gene is adjacent to its TATA box.

Deletion analysis of the promoter of the PUT2 gene that functions in the proline utilization pathway of Saccharomyces cerevisiae identified a PUT2 upstream activation site (UAS). It is contained within a single 40-base-pair (bp) region located immediately upstream of the TATA box and is both necessary and sufficient for proline induction. When placed upstream of a CYC7-lacZ gene fusion, the 40-bp sequence conferred proline regulation on CYC7-lacZ. A 35-bp deletion within the PUT2 UAS in an otherwise intact PUT2 promoter resulted in noninducible expression of a PUT2-lacZ gene fusion. When a plasmid bearing this UAS-deleted promoter was placed in a strain carrying a constitutive mutation in the positive regulatory gene PUT3, expression of PUT2-lacZ was not constitutive but occurred at levels below those found under noninducing conditions. In heterologous as well as homologous gene fusions, the PUT2 UAS appeared to be responsible for uninduced as well as proline-induced levels of expression. Although located immediately adjacent to the PUT2 UAS, the TATA box did not appear to play a regulatory role, as indicated by the results of experiments in which it was replaced by the CYC7 TATA box. A 26-bp sequence containing this TATA box was critical to the expression of PUT2, since a deletion of this region completely abolished transcriptional activity of the gene under both inducing and noninducing conditions. Our results indicate that the PUT2 promoter has a comparatively simple structure, requiring UAS and TATA sequences as well as the PUT3 gene product (directly or indirectly) for its expression.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1992

The TATA box promoter region of maize Adh1 affects its organ-specific expression.

We have isolated two lineage-related Mutator (Mu3) transposon-induced Adh1 promoter mutants in maize: Adh1-3F1124 carries a duplicated TATA box and its revertant, Adh1-3F1124r17, bears a deleted TATA box. Both alterations lead to unique patterns of organ-specific ADH1 enzyme expression. Enzyme activity in Adh1-3F1124 sporophytic organs (scutellum and roots) is greatly reduced, while activity levels remain normal in the male gametophyte (pollen). Conversely, enzyme activity in Adh1-3F1124r17 roots and scutellum is partially restored, but is concomitantly reduced in pollen. Transcript analysis suggests (i) that the TATA box region of the Adh1 gene influences post-transcriptional processes in the male gametophyte but not in roots and (ii) that organ-specific transcription signals in the promoter are distinct from the previously identified anaerobic environment-specific cis-acting transcription signals. Different organs appear to provide surrogate TATA function in different ways, leading to organ-specific differences in the length of the Adh1 message 5' leader.

Alcohol Dehydrogenase↗
PubMed1998

Solution structure of a TBP-TAF(II)230 complex: protein mimicry of the minor groove surface of the TATA box unwound by TBP.

General transcription factor TFIID consists of TATA box-binding protein (TBP) and TBP-associated factors (TAF(II)s), which together play a central role in both positive and negative regulation of transcription. The N-terminal region of the 230 kDa Drosophila TAF(II) (dTAF(II)230) binds directly to TBP and inhibits TBP binding to the TATA box. We report here the solution structure of the complex formed by dTAF(II)230 N-terminal region (residues 11-77) and TBP. dTAF(II)230(11-77) comprises three alpha helices and a beta hairpin, forming a core that occupies the concave DNA-binding surface of TBP. The TBP-binding surface of dTAF(II)230 markedly resembles the minor groove surface of the partially unwound TATA box in the TBP-TATA complex. This protein mimicry of the TATA element surface provides the structural basis of the mechanism by which dTAF(II)230 negatively controls the TATA box-binding activity within the TFIID complex.

Amino Acid Sequence↗
PubMed1990

A downstream initiation element required for efficient TATA box binding and in vitro function of TFIID.

The gfa gene encodes glial fibrillary acidic protein, an intermediate filament protein expressed in glial cells. In vitro transcription analysis has shown that the human gfa promoter contains two initiation elements that can independently specify the transcription startpoint. One of the elements is a TATA box 25 base pairs (bp) upstream from the transcription startpoint; the other is located between 10 and 50 bp downstream from the transcription initiation site. We have now shown by transfection that both elements are required for efficient transcription in cultured cells. A partially purified natural human TATA box-binding factor (TFIID) from HeLa cells gave footprints that extended from upstream of the TATA box through the downstream initiator. Deletion of the downstream initiator inhibited both TFIID binding to the TATA box and transcription in vitro. In contrast to natural human TFIID, clone human and yeast TFIIDs expressed in bacteria gave footprints covering only the TATA box region, although hypersensitive sites were observed in the downstream region. The cloned TFIIDs also showed less dependence than natural human TFIID on the downstream initiator for both TATA box binding and in vitro transcription. These results suggest that natural human TFIID contains an additional component(s) that contribute(s) to stable TFIID binding and effective transcription by interacting with the downstream initiator.

Base Sequence↗
PubMed1995

Genomic structure of the human TATA-box-binding protein (TBP).

The gene encoding the human TATA-box-binding protein (hTBP) is contained within a 20-kb DNA fragment and is split into eight exons. The coding sequence is interrupted by six introns and the 5'-untranslated region (5'-UTR) of the gene by a 2.5-kb intron. A comparison of the hTBP exon/intron organization with the various TBP cloned to date is presented.

Amino Acid Sequence↗
PubMed1998

TATA-box mutant in the promoter of the uridine diphosphate glucuronosyltransferase gene in Italian patients with Gilbert's syndrome.

BACKGROUND: A mutation in the promoter of the uridine diphosphate glucuronosyltransferase gene has been described in patients with Gilbert's syndrome from Northern Europe. AIMS: To assess the frequency of this mutation in Italian patients with Gilbert's syndrome and in normal controls, in order to establish the molecular basis and molecular epidemiology of the syndrome in Italy. PATIENTS: Forty-six patients with a clinical diagnosis of Gilbert's syndrome and 44 individuals from the general population unselected for bilirubin levels. METHODS: Polymerase chain reaction amplification of the TATA-box element in the promoter of uridine diphosphate glucuronosyltransferase and identification of wild-type and variant alleles by high-resolution polyacrylamide gel electrophoresis. RESULTS: A TATA-box variant in the promoter of uridine diphosphate glucuronosyltransferase was found on 93% of chromosomes from patients with Gilbert's syndrome. The same variant was present on 44% of chromosomes from controls, unselected for bilirubin levels. Only 55% of controls homozygous for the TATA-box variant, however, had increased bilirubin levels. CONCLUSIONS: The TATA-box variant in the promoter of uridine diphosphate glucuronosyltransferase is strongly associated with the phenotype of Gilbert's syndrome in Italy. The incomplete penetrance of the mutation observed in controls indicates that other acquired or inherited conditions affecting bilirubin production, uptake, cellular transport or excretion may contribute to the hyperbilirubinaemia of Gilbert's syndrome.

Adolescent↗
PubMed1995

Electrostatic analysis of DNA binding properties in lysine to leucine mutants of TATA-box binding proteins.

The structures of the complexes between TATA-box binding proteins (TBPs) and DNA solved recently with X-ray crystallography identify both direct and indirect readout interactions. Examples of indirect readout mechanisms in these complexes are DNA bending and non-local electrostatic complementarity. An intriguing question arising from these structures is the role that a series of lysine residues may have in DNA binding. Thus, in the yeast complex, seven lysines are found to be close to the phosphate backbone, but they appear to form hydrogen bonds to the protein and not to be involved in any direct (or water-mediated) interactions with the DNA. The proposal based on the crystal structure, that these residues set up a delocalized electrostatic potential that stabilizes the complex with DNA, is evaluated here from calculations of the electrostatic potentials generated by the wild-type TBP and various lysine to leucine mutants. The results suggest a grouping of these mutants into three classes, based on their phenotypes and electrostatic profiles. As these groups are affected differently by specific measures taken to rescue DNA binding and transcription functions, the mechanistic inferences from the analysis can be probed experimentally in a manner that also reveals possible binding sites for transcription factors IIA and IIB to the TBP-DNA complex in the transcription preinitiation complex.

Base Sequence↗
PubMed1986

The effect of changing the distance between the TATA-box and cap site by up to three base pairs on the selection of the transcriptional start site of a cloned eukaryotic gene in vitro and in vivo.

We have studied how small changes in the distance between the TATA-box and cap site affect transcription of a eukaryotic gene in vitro and in vivo. The trout protamine gene TPG-3 [Gregory et al. (1982) Nucl. Acids Res. 10, 7581-7592] is a good model for such a study as it has (i) a consensus TATA-box 32 base pairs (bp) upstream from an A-residue which is the natural cap site (designated +1) (ii) two further A-residues at -5 and +5, providing alternative transcriptional start sites which are in significantly different sequence environments and (iii) a unique AvaII restriction site immediately downstream from the TATA-box which is ideal for the insertion or deletion of up to 3bp. Transcripts of the wild type and mutant genes were generated in vitro using a HeLa whole cell extract or 'in vivo' by transient expression following their transfection into HeLa cells. These 'spacer' mutations did not affect the efficiency of transcription of the gene in vitro but they did affect the selection of transcriptional start site both in vitro and 'in vivo'. Analysis of 5'-ends by S1-mapping and primer extension showed that the A-residue(s) selected are those which, by insertion or deletion, come to lie on the same face of the DNA double helix as the TATA-box, although the DNA sequence in the immediate vicinity of the potential start sites influences their utilisation. Comparison of the TPG-3 wild type transcripts in these experimental systems with natural mRNA suggests that cap site selection is more stringent in the developing trout testis.

Animals↗
PubMed2003

A major role for the TATA box in recruitment of chromatin modifying complexes to a globin gene promoter.

The developmentally regulated mammalian beta-globin genes are activated by a distant locus control region/enhancer. To understand the role of chromatin remodeling complexes in this activation, we used stably replicated chromatin templates, in which transcription activation of the human embryonic epsilon-globin gene depends on the tandem Maf-recognition elements (MAREs) within the beta-globin locus control region HS2 enhancer, to which the erythroid factor NF-E2 binds. The HS2 MAREs are required for nucleosome mobilization and histone hyperacetylation at the distant promoter. Nucleosome mobilization also requires the promoter TATA box, and is independent of histone hyperacetylation. In contrast, promoter hyperacetylation requires the promoter GATA-1, and CACC-factor activator motifs, as well as the TATA box. ChIP analysis reveals that NF-E2 is associated with the active epsilon-globin promoter, which lacks an NF-E2 binding sequence, in a TATA box and HS2/MARE-dependent fashion. NF-E2 association with the epsilon-globin promoter coincides with that of RNA polymerase II at both regulatory sites. The results emphasize MARE-TATA box interactions in the recruitment of complexes modifying promoter chromatin for transcription activation and imply close physical interaction between widely separated regulatory sequences mediated through these sites.

Acetylation↗