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PubMed2006

The potato transcriptional co-activator StMBF1 is up-regulated in response to oxidative stress and interacts with the TATA-box binding protein.

To gain a better understanding on the function of the potato Solanum tuberosum Multiprotein Bridging Factor 1 protein (StMBF1) its interaction with the TATA box binding protein (TBP) was demonstrated. In addition we reported that StMBF1 rescues the yeast mbf1 mutant phenotype, indicating its role as a plant co-activator. These data reinforce the hypothesis that MBF1 function is also conserved among non closely related plant species. In addition, measurement of StMBF1 protein level by Western blot using anti-StMBF1 antibodies indicated that the protein level increased upon H(2)O(2) and heat shock treatments. However, the potato beta-1,3-glucanase protein level was not changed under the same experimental conditions. These data indicate that StMBF1 participates in the cell stress response against oxidative stress allowing us to suggest that MBF1 genes from different plant groups may share similar functions.

Heat-Shock Response↗

PubMedDate not supplied

Development of a system for the study of protein-protein interactions in planta: characterization of a TATA-box binding protein complex in Oryza sativa.

We describe a simple, rapid method for protein complex purification in planta. Using a biotin peptide as an affinity tag with TATA-box binding protein (TBP), 86 unique proteins present in the purified complex were identified by tandem mass spectrometry. We identified proteins known to be associated with TBP, and many other proteins involved in pre-mRNA processing and chromatin remodeling. The identification of these novel protein-protein associations will upon further investigations provide new insights into the mechanisms of mRNA transcription and pre-mRNA processing.

Amino Acid Sequence↗

PubMed1992

The transcriptional start site for a human U6 small nuclear RNA gene is dictated by a compound promoter element consisting of the PSE and the TATA box.

Transcription of vertebrate U6 snRNA genes by RNA polymerase III requires two sequence elements in the proximal promoter region: the PSE (proximal sequence element, found in snRNA promoters transcribed by RNA polymerase II) and the TATA element (found in many mRNA promoters). The locations of the PSE and the TATA box are important determinants for transcriptional start site selection in their respective RNA polymerase II promoters. In vertebrate U6 genes the PSE and the TATA elements are located in approximately the same positions as in the polymerase II transcribed genes, but their respective roles in initiation site selection are unknown. We have analyzed the effects of spacing changes between the PSE and the TATA element, and between the two elements and the normal U6 start site on human U6 gene transcription. The spacing requirement between the two elements is highly stringent, implying a possible interaction between the factors that bind them. Our results discount the possibility that the location of either the PSE or the TATA element, by itself, dictates efficient selection of a transcriptional start site. Instead, we suggest that the two elements form a compound promoter element whose location dictates the start site of transcription from the human U6 gene promoter.

Base Sequence↗

PubMed1992

The eukaryotic CCAAT and TATA boxes, DNA spacer flexibility and looping.

Regulation of RNA transcription in eukaryotic polymerase II promoters involves a complex assembly of protein factors. Some of the factors bind to their cognate DNA-sequence elements while others mediate between the DNA bound ones. In order to enable protein-protein interaction, their spatial positioning with respect to each other is critical. Here two DNA-sequence-elements are investigated, the CCAAT and the TATA boxes and their spacers. Whereas the position of the TATA is fixed at about -30, that of the CCAAT can vary substantially from -50 to -200. Despite the variable loop sizes, the CTF (CCAAT-binding) protein interacts--either directly or indirectly via a co-activator--with the general basal TATA-binding transcription factors. Sequence analysis of the spacers, as a function of their sizes, reveals that in the upstream regions of the spacers RR and YY are abundant. In the downstream, 3' region of the spacers RY and YR are very frequent. The DNA sequence elements and their intervening spacers are analyzed in terms of their geometry, anisotropic flexibility and local superhelical density. Our results indicate that the CCAAT and its vicinity is rigid, whereas the TATA and its surroundings is flexible. It is the large flexibility of this region in twist and in roll which allows DNA looping. General mechanistic implications for pol II promoters are discussed.

Animals↗

PubMed1993

Functional elements DE2A, DE2B, and DE1A and the TATA box are required for activity of the chicken alpha A-crystallin gene in transfected lens epithelial cells.

alpha A-crystallin is an abundant soluble protein of the vertebrate eye lens. In addition to the TATA box, four positive cis-regulatory elements of the chicken alpha A-crystallin gene have been identified by linker scanning mutagenesis, DNase I footprinting, and gel mobility shift experiments. The regulatory elements described here have been named DE2A (at positions -144 to -134), DE2B (at positions -128 to -118), and DE1A (at positions -114 to -103). DE2A and DE2B form a dyad of symmetry between positions -141 and -118 (5'-AGACTGTCAT....AGGTCAGTCT-3'), consistent with the close similarity in the mobility of complexes formed with lens nuclear proteins by these two elements. Mutations in DE2A, DE2B, and DE1A leading to loss of promoter activity using the bacterial chloramphenicol acetyltransferase reporter gene transfected into primary embryonic chicken lens epithelial cells resulted in a corresponding loss in the ability to compete for complex formation with lens nuclear proteins in gel mobility shift assays. Mutation of the alpha A-CRYBP1-like site (-67/-57), necessary for function of the mouse alpha A-crystallin promoter, did not affect the activity of the chicken promoter. The DNase I footprinting and gel mobility shift experiments confirmed the previously noted binding of nuclear proteins to a dyad of symmetry at positions -153 to -140. In contrast to DE2A, DE2B, and DE1A, mutagenesis and gel mobility shift experiments failed to correlate function and protein binding for the -153/-140 dyad. DE2A, DE2B, and DE1A agree well with the regulatory elements alpha CE1 (-162/-134), alpha CE3 (-135/-121), and alpha CE2 (-119/-99) (Matsuo, I., and Yasuda, K. (1992) Nucleic Acids Res. 20, 3701-3712) for this gene. The present results suggest, however, that the lens enhancer activity of alpha CE1 is due to the sequence -141/-134, which forms the upper half of the DE2A/DE2B dyad of symmetry, rather than the -153/-140 dyad as previously suspected.

Animals↗

PubMed1998

A functionally distinct TATA box required for late progression through the Epstein-Barr virus life cycle.

During EBV infection, lytic DNA replication activates late gene expression in trans via an uncharacterized pathway. In this study, we mapped the target of this regulatory cascade to a variant TATA box (TATTAAA) and the 3' flanking region within the core promoter of the BcLF1 gene. The inherent late activity of this core promoter is, surprisingly, disrupted by a heterologous enhancer, suggesting that late gene expression is regulated through core promoter sequences located in a transcriptionally inert environment.

B-Lymphocytes↗

PubMed1998

Cell-type specific factors bind to regulatory elements located downstream of the TATA-box element in the mouse myelin basic protein (MBP) gene promoter.

Cell-type specific transcription of the myelin basic protein (MBP) gene in primary oligodendrocytes (OL) is regulated by cis-acting regulatory elements located at both upstream and downstream of the TATA-box region of the MBP promoter. To identify cell-type specific factors that bind to the downstream regulatory elements, we utilised DNase I footprinting analysis and gel retardation assays with nuclear extracts from myelin-forming OL as well as a non-myelin forming cell line, C6 glioma (C6) cells. Several regions of DNA were protected from DNAse I digestion by nuclear extracts of both cell types. However, two regions, from -17 to +17 and from +47 to +58 were protected specifically in OL, while three regions, from + 17 to + 22, from +43 to +49 and from +58 to +64 were protected only with C6 nuclear extracts. Inspection of the protected regions for homology with known transcription factor binding sites revealed that sequences at from +47 to +58 and from +56 to +68 showed extensive homology to the negative regulatory element (NRE1), of the mouse renin gene and to the interferon (IFN) consensus sequence of major histocompatibility complex class I genes (MHC I-ICS), respectively. Gel retardation assays using a MHC I-ICS oligonucleotide and transient transfection assays using MBP-CAT constructs were used to study the effect of IFNs on MBP promoter activity in OL and C6 cells. In OL, IFN-alpha/beta caused little induction of CAT activity, but IFN-gamma resulted in a 2-3.5-fold decrease in CAT activity. In contrast, in C6 cells both IFN-alpha/beta and IFN-gamma induced a 1.5-2.5-fold increase in CAT activity. The cooperative effects of factors binding to NREs and ICS may be responsible for the cell-type specific regulation of MBP gene transcription.

Animals↗

PubMed2003

Frequent co-occurrence of the TATA box mutation associated with Gilbert's syndrome (UGT1A128) with other polymorphisms of the UDP-glucuronosyltransferase-1 locus (UGT1A62 and UGT1A7*3) in Caucasians and Egyptians.

Polymorphisms of drug metabolizing enzymes are frequently associated with diseases and side effects of drugs. Recently, a TATA box mutation of UGT1A1 (UGT1A1*28), a common genotype leading to Gilbert's syndrome, and several missense mutations of other UDP-glucuronosyltransferase 1 (UGT1) family members have been described. Furthermore, co-occurrence of UGT1A1*28 and UGT1A6*2 has been observed. In order to elucidate the basis for co-occurrence of UGT1 mutations, fluorescence resonance energy transfer techniques were developed for rapid determination of polymorphisms of three UGT isoforms (UGT1A1*28, 1A6*2, and 1A7*2/*3). Hundred healthy Caucasians and 50 Egyptians were genotyped. All genotypes followed the Hardy-Weinberg equilibrium. Only three major haplotypes were found, including a haplotype consisting of allelic variants of all three isoforms (29% in Caucasians and 22% in Egyptians), all leading to reduced UGT activity. Frequent haplotypes containing several UGT1 allelic variants should be taken into account in studies on the association between diseases, abnormal drug reactions, and UGT1 family polymorphisms.

Egypt↗

PubMed1998

Dysregulation through the NF-kappaB enhancer and TATA box of the human immunodeficiency virus type 1 subtype E promoter.

The global diversity of human immunodeficiency virus type 1 (HIV-1) genotypes, termed subtypes A to J, is considerable and growing. However, relatively few studies have provided evidence for an associated phenotypic divergence. Recently, we demonstrated subtype-specific functional differences within the long terminal repeat (LTR) region of expanding subtypes (M. A. Montano, V. A. Novitsky, J. T. Blackard, N. L. Cho, D. A. Katzenstein, and M. Essex, J. Virol. 71:8657-8665, 1997). Notably, all HIV-1E isolates were observed to contain a defective upstream NF-kappaB site and a unique TATA-TAR region. In this study, we demonstrate that tumor necrosis factor alpha (TNF-alpha) stimulation of the HIV-1E LTR was also impaired, consistent with a defective upstream NF-kappaB site. Furthermore, repair of the upstream NF-kappaB site within HIV-1E partially restored TNF-alpha responsiveness. We also show, in gel shift assays, that oligonucleotides spanning the HIV-1E TATA box displayed a reduced efficiency in the assembly of the TBP-TFIIB-TATA complex, relative to an HIV-1B TATA oligonucleotide. In transfection assays, the HIV-1E TATA, when changed to the canonical HIV-1B TATA sequence (ATAAAA-->ATATAA) unexpectedly reduces both heterologous HIV-1B Tat and cognate HIV-1E Tat activation of an HIV-1E LTR-driven reporter gene. However, Tat activation, irrespective of subtype, could be rescued by introducing a cognate HIV-1B TAR. Collectively, these observations suggest that the expanding HIV-1E genotype has likely evolved an alternative promoter configuration with altered NF-kappaB and TATA regulatory signals in contradistinction with HIV-1B.

Base Sequence↗

PubMed1995

Expression of a yeast gene can be blocked by insertion of short yeast DNA fragments between a UAS and the TATA box.

We have constructed a plasmid, pOV10, which facilitates the introduction of putative upstream activating sequences (UAS) or upstream repressing sequences (URS) from yeast genes into plasmids containing CYCl-lacZ fusions. We have observed that the insertion of yeast sequences from 155 to 195 bp between the UAS and the TATA box of a CYCl-lacZ fusion gene can block beta-galactosidase expression. It is suggested that this block is related to the formation of nucleosomes on the DNA.

Cloning, Molecular↗

PubMed1991

Thirty-three nucleotides of 5' flanking sequence including the 'TATA' box are necessary and sufficient for efficient U2 snRNA transcription in Schizosaccharomyces pombe.

We have sequenced the 5' flanking region of the U2 gene and compared this with the 5' flanking sequences of other snRNA genes from Schizosaccharomyces pombe. This revealed no regions of clear homology 5' to a region surrounding the 'TATA' box at -32 to -29. Deletion analysis shows that a 5' flanking region extending to only -33 is sufficient for accurate and efficient transcription of U2 in Schizosaccharomyces pombe.

Base Sequence↗

PubMed1997

Repression of the HSV-1 latency-associated transcript (LAT) promoter by the early growth response (EGR) proteins: involvement of a binding site immediately downstream of the TATA box.

During herpes simplex virus (HSV) latency, in neurons of the nervous system, a single family of viral transcripts (the Latency-Associated Transcripts or LATs) are synthesized. Within the LAT promoter region, we have identified a consensus sequence for the EGR proteins in an unusual position immediately downstream of the TATA box. The early growth response (EGR) proteins are rapidly induced in cells by stimuli which also induce HSV to reactivate from latency. In order to determine if EGR proteins play any role in control of LAT transcription, we have analyzed the interactions between EGR proteins and the LAT promoter. Gel retardation and DNase I protection assays demonstrated that EGR1 zinc finger protein bound specifically to the LAT promoter region EGR consensus sequence. To determine if EGR proteins could modulate transcription through the LAT promoter, cotransfection assays were performed using chloramphenicol acetyltransferase (CAT) reporter constructs driven by either the wild-type LAT promoter or a LAT promoter with a mutated EGR binding site. Contransfection of the wild-type LAT promoter construct with EGR expression plasmids resulted in inhibition of the basal level of CAT activity with EGR-2 but not EGR-1 or 3. However, normal levels of CAT activity were observed in cotransfections using the mutant LAT promoter CAT construct suggesting that repression was mediated by the binding of EGR-2 proteins to the LAT promoter. Furthermore, data from combination binding assays using EGR1 and TATA binding protein (TBP) in vitro support the hypothesis that binding of EGR proteins to the LAT promoter prevents binding of TBP and thus suppresses transcription. These results may provide a link between stress responses in neurons of the CNS which activate the EGR family of proteins and HSV reactivation from latency due to the same stress response.

Animals↗

PubMed1997

RAP74 induces promoter contacts by RNA polymerase II upstream and downstream of a DNA bend centered on the TATA box.

RAP74, the large subunit of transcription factor IIF, associates with a preinitiation complex containing RNA polymerase II (pol II) and other general initiation factors. We have mapped the location of RAP74 in close proximity to promoter DNA at similar distances both upstream and downstream of a DNA bend centered on the TATA box. Binding of RAP74 induces a conformational change that affects the position of pol II relative to that of the DNA. This reorganization of the preinitiation complex minimally requires the N-terminal region of RAP74 containing both its RAP30-binding domain and another region necessary for accurate transcription in vitro. We propose a role for RAP74 in controlling the topological organization of the pol II preinitiation complex.

Base Sequence↗

PubMed1990

The binding site for the liver-specific transcription factor Tf-LF1 and the TATA box of the human transferrin gene promoter are the only elements necessary to direct liver-specific transcription in vitro.

We have studied the liver-specific transcriptional activity of the human transferrin gene promoter. Results of competition experiments, site-directed mutagenesis, and 5' deletion analysis have demonstrated that a TATA box and a binding site for the liver-specific protein Tf-LF1 are the only elements needed to direct hepatic-specific transcription in vitro. Thus, Tf-LF1 behaves as other previously described proteins, HNF-1, DBP and LF-A1, in that it is sufficient to confer liver-specific transcriptional activity to a promoter in vitro. This results contrast with observations made in transient expression experiments, in which Tf-LF1 binding alone cannot direct hepatic-specific expression, and the binding of at least one more protein, similar to C/EBP, is needed. Thus, as described for other hepatic genes, the number of elements necessary to confer tissue specificity is different in vivo and in vitro.

Base Sequence↗

PubMed1997

C/EBP binds over the TATA box and can activate the M promoter of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The gene A coding for 6-phosphofructo-2-kinase gives rise to three mRNA that originate from distinct promoters called F (fetal), M (muscle) and L (liver). The regulation of the M promoter is ill-understood and its TATA box region binds an unidentified factor. The aim of this work was to identify this factor and to investigate its activity. In vitro protein-DNA binding assays and transfection experiments showed that this factor is C/EBP and that C/EBP can stimulate the M promoter despite its potential interference with TFIID binding. The effect of C/EBP was abolished by a dominant negative variant of C/EBP. These data and other work lead to the conclusion that C/EBP may participate to the regulation of promoter switching in 6-phosphofructo-2-kinase gene A.

Animals↗

PubMed2003

Sequence and spacing of TATA box elements are critical for accurate initiation from the beta-phaseolin promoter.

The beta-phaseolin (phas) gene, which encodes one of the major seed storage proteins of P. vulgaris, is tightly regulated at the transcription level resulting in strict tissue-specific and spatial expression during embryonic development. The phas proximal promoter contains a complex arrangement of core promoter elements including three TATA boxes as well as several putative initiator elements. To delineate the respective contributions of the core promoter elements to transcription initiation we have performed site-directed mutagenesis of the phas promoter. In vivo expression studies were performed on transgenic Arabidopsis harboring phas promoter mutants driving expression of the beta-glucuronidase (gus) reporter gene. Quantitative assessment of GUS activity in seeds bearing the promoter mutants indicated that both sequence and spacing of the TATA elements influenced the efficiency of transcription. Substitution, insertion or deletion mutations had no effect on histochemical staining patterns indicating that strict spacing requirements are not essential for correct spatial expression of phas during embryogenesis. Further evaluation of the phas promoter by in vitro transcription analysis revealed the presence of multiple TATA-dependent transcription initiation start sites. The distance between TATA elements and transcription start sites was maintained in insertion and deletion mutants through the creation of novel initiation sites, indicating that positioning of the TATA elements rather than DNA sequence was the primary determinant of start site location. We conclude that, while dispensable for proper spatial distribution, the complex architecture of the phas promoter is required to ensure high levels of accurate phas transcription initiation in the developing embryo.

Arabidopsis↗

PubMed1997

Molecular variation of the human angiotensinogen core promoter element located between the TATA box and transcription initiation site affects its transcriptional activity.

Recent genetic studies indicate that several molecular variants discovered in angiotensinogen (AG), the precursor of vasoactive octapeptide angiotensin II, could potentially be responsible for inherited predisposition to human blood pressure variation. We have previously shown that a ubiquitously expressed nuclear factor, AGCF1, bound to AGCE1 (AG core promoter element 1 including the core nucleotides, CTCGTG, CTC-type) located between the TATA box and transcription initiation site (positions -25 to -1) is an authentic regulator of human AG transcription. In the present study, we showed that AGCF1 has biologically and immunologically similar properties to those of a helix-loop-helix nuclear factor USF1 and examined the effects of two other naturally occurring molecular variants (ATCGTG, ATC-type and ATTGTG, ATT-type) found in the AGCE1 position on the human AG transcriptional activity. Competitive gel-shift and transfection experiments demonstrated that the transcriptional activity for the CTC- and ATC-type promoters was 2.5 times higher than that for the ATT-type through the alteration of AGCF1-binding affinity. These results suggest the possible involvement of USF1 as a component in AGCF1 formation and the potential importance of AGCE1 variation in blood pressure regulation through human AG expression.

Angiotensinogen↗

PubMed1994

Evidence for the presence of a functional TATA box (ATAAAA) sequence in the gene for bovine elastin.

The wild type sequence (wt; ATAAAA) was mutated and the effects of the mutants were determined by assaying the expression of the chloramphenicol acetyltransferase (CAT)-encoding gene (cat) cloned downstream. The negative mutant (neg; ACGAAA) practically abolished the activities of the 129 bp and the 416 bp elastin promoters in NIH 3T3 and neonatal rat aortic smooth muscle cells, respectively. However, when the positive mutant (pos; TATAAAA) was assayed in parallel experiments, there was enhancement of activity. The TATA box-binding protein (TBP) was shown to bind to the ATAAAA sequence and the retardation of the band was abolished by competition assay using unlabeled wt and pos sequences, but not by the neg mutant. These results provide evidence for the presence of a functional TATA box in the gene for elastin.

Animals↗