[Detection of a specific TATA-box associated complex in nuclear extracts of cells, transformed by E1A and ras oncogenes].
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Adenovirus E1A Proteins↗
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A U-snRNA gene-specific upstream element and a -30 'TATA box' are required for transcription of the U2 snRNA gene of Arabidopsis thaliana.
The U2 and U5 snRNA genes of Arabidopsis thaliana contain in their promoter regions two elements with conserved sequence and position. To test the significance of this conservation we have made a construction in which the promoter of the U2 RNA gene is replaced by the synthetic 98 bp long sequence containing the two conserved elements: an upstream sequence element, GTCCCACATCG (USE, pos. -78 to -68), and a TATA-like sequence TATAAATA (-33 to -26), positioned approximately three helical turns apart, as in the wild-type promoter. This synthetic promoter efficiently drove transcription of the U2 gene in transfected protoplasts of Nicotiana plumbaginifolia. The importance of the individual elements and of their position within the promoter was investigated. Deletion of the USE, change of its orientation, and some single point mutations all decreased transcription 10- to 20-fold, and replacement of the TATA-like element by an unrelated sequence inactivated the promoter. Mutants in which the spacing between the USE and TATAAATA was changed were less active but no correlation was observed between promoter activity and insertion of either odd or even numbers of half helical turns. Insertion of a spacer between TATAAATA and the cap site resulted in accumulation of U2 RNA with an extended 5' end, indicating that the TATAAATA element is responsible for selection of the initiation site. The data indicate that the promoters of RNA polymerase II-specific U-snRNA genes in higher plants differ from their animal counter-parts and also from plant mRNA gene promoters. They contain two essential elements, an USE, an element found only in U-snRNA genes, and a TATA element which is indistinguishable from the TATA boxes of mRNA-coding genes.
Base Sequence↗
An archaebacterial promoter element for stable RNA genes with homology to the TATA box of higher eukaryotes.
The RNA polymerase of Methanococcus vannielii, in binary complex with two stable RNA operons, protects from exonuclease digestion the region from 32 bp upstream (-32) to 18 bp downstream (+18) of the transcription start site. Contained within this binding region, centered at -25, is an AT-rich sequence which is highly conserved upstream of 26 other archaebacterial tRNA and rRNA genes. We therefore propose the sequence TTTATAATA as a common element of promoters for stable RNA genes in archaebacteria. Both the similarity in sequence and the location of this conserved octanucleotide suggest homology to the eukaryotic TATA box preceding protein encoding genes transcribed by RNA polymerase B.
Base Sequence↗
beta-Thalassemia in American Blacks: novel mutations in the "TATA" box and an acceptor splice site.
beta-Thalassemia genes, although often mild in their effects, are common among American Blacks. We have begun a systematic molecular analysis of beta-thalassemia mutations in this group. DNA polymorphisms in the beta-globin gene cluster were examined among 22 beta-thalassemia chromosomes. Six different haplotypes were observed. beta-globin genes of two of these were cloned, and their phenotypes were examined both in heterologous cells upon transient expression and in vivo. The gene found in the most common haplotype (9 of 22 chromosomes) contained a single base substitution (A----G) at position -29 within the highly conserved proximal promoter element (the "TATA" box). This mutant gene directed beta-globin RNA at 25% of normal levels both in heterologous cells and in vivo. It was associated with a mild beta +-thalassemia phenotype. A different gene, isolated from an apparently rare haplotype (1 of 22 chromosomes), had a single base substitution (A----G) within the acceptor splice site of the second intervening sequence. This mutation abolished normal RNA splicing so that the only RNA made from the gene in vitro was an alternatively spliced RNA, which could not encode beta-globin. The mild deficit in beta-globin production attributable to the -29 A----G mutant allele most likely accounts for the frequently mild nature of beta-thalassemia among American Blacks.
Adolescent↗
Conserved TAAAT motif in vaccinia virus late promoters: overlapping TATA box and site of transcription initiation.
We have characterized the vaccinia virus 11-kd late promoter through 5' and 3' deletions and site-directed mutagenesis. The promoter function appears to be contained within an approximately 30-bp fragment, which after translocation is able to direct RNA synthesis late in infection at a reduced level. We demonstrate that a TAAAT sequence in the proximal part of the promoter is essential for its function. This cis-acting element is highly conserved within vaccinia virus late promoters and overlaps the site of transcription initiation. Deletions or mutations within this conserved element completely inactivate the promoter. The evidence indicates that the TAAAT motif functions as a TATA box. The region immediately upstream of the TAAAT motif determines the promoter strength.
Base Sequence↗
Photofootprinting in vivo detects transcription-dependent changes in yeast TATA boxes.
To elucidate critical steps in the transcription initiation process, we have devised a protocol for obtaining information about DNA structure and DNA-protein interactions at nucleotide level resolution from intact yeast cells. Our procedure combines the ultraviolet light 'footprinting' method developed by Becker and Wang with the 'genomic sequencing' technique described by Church and Gilbert. Using this approach we were able to detect the binding of GAL 4 protein at sites within the upstream activating sequence (UASG) previously mapped using other in vivo and in vitro foot-printing procedures. We also observed transcription-dependent changes in sensitivity of DNA to ultraviolet-induced covalent modification at several positions between the upstream activating sequence and the transcription initiation sites of the GAL 1 and GAL 10 genes. The most prominent of these changes occurs at a common site within the putative 'TATA' boxes of the two genes. Ultraviolet modification at this site is enhanced only in transcriptionally active promoters.
DNA, Fungal↗
A human IFN-beta 1 gene deleted of promoter sequences upstream from the TATA box is controlled post-transcriptionally by dsRNA.
Induction of IFN-beta 1 RNA was studied in the mouse cell line SR117-21E transformed by a BPV episome containing the human IFN-beta 1 gene deleted of promoter sequences upstream from position -40. Nuclei isolated from these cells synthesize constitutively IFN-beta 1 RNA from the partially deleted promoter. The IFN-beta 1 RNA synthesized by nuclei of uninduced SR117-21E cells is similar to that made by nuclei of poly(rI):(rC)-induced cells, but does not accumulate and hence no IFN is produced unless the cells have been treated either by ds RNA or by cycloheximide. We conclude that the IFN-beta 1 gene has, in addition to the transcription control due to upstream promoter sequences, an additional post-transcriptional control acting on mRNA accumulation and linked to sequences close to the TATA box and RNA start site. Both controls are relieved by ds RNA.
Animals↗
Cell-specific expression of the human gastrin gene: evidence for a control element located downstream of the TATA box.
Fragments of 5'-flanking and noncoding exon I sequences of the human gastrin gene were analyzed in transient expression assays after transfection of a variety of cell lines with the pSVCAT vector system. In the presence of the simian virus 40 (SV40) enhancer, the gastrin gene fragment from nucleotides -250 to +57, relative to the cap site, was as efficient a promoter as the SV40 early promoter itself. In the absence of the SV40 enhancer, gastrin gene 5'-flanking sequences had no promoter activity except in the murine neuroblastoma cell line N18TG2. In this cell line, the fragment from -1300 to +57 stimulated transcription as actively as the SV40 early promoter with its enhancer. This cell-specific gastrin gene promoter activity was in accordance with the finding that gastrin is synthesized in certain neuronal cells. Promoter activity declined with decreasing distance from the 5' end to the cap site and disappeared after removal of the gastrin gene TATA box. In vector constructions containing short vector-linker sequences homologous to a functionally important region of the SV40 enhancer, the gastrin gene fragment from -17 to +57 showed considerable promoter activity, exclusively in N18TG2. It is concluded that the truncated gastrin gene promoter plus the first exon contains a cell-specific element that may act in collaboration with upstream elements to facilitate the accumulation of transcripts.
Animals↗
An exonuclease protection assay reveals heat-shock element and TATA box DNA-binding proteins in crude nuclear extracts.
The ability to identify and purify trans-acting cellular factors that regulate eukaryotic genes is limited by the lack of a practical general assay. Current procedures using crude whole cell or nuclear extracts that restore transcriptional function in vitro or permit reconstruction of native chromatin at control sequences are effective only in select systems. I now present an exonuclease protection assay that is generally applicable for detecting sequence-specific DNA-binding proteins. The assay extends earlier work on the binding to the Drosophila heat-shock gene control element of a protein factor (HAP) present in crude nuclear extracts; the binding was shown by reconstitution of specific exonuclease resistance within a nuclease-hypersensitive site in chromatin. We show here that this same exonuclease resistance can be reconstituted on free linear DNA, despite many nonspecific binding activities present in unfractionated nuclear extracts. We have further applied this assay method to fractionate the protein factor that is bound constitutively to the heat-shock gene TATA box region in native chromatin. Exonuclease protection offers a sensitive, precise and rapid assay for any sequence-specific DNA-binding protein.
Animals↗
The control of herpes simplex virus type-1 late gene transcription: a 'TATA-box'/cap site region is sufficient for fully efficient regulated activity.
The transcriptional programme of herpes simplex virus type 1 (HSV-1) is organised into three principle phases; immediate-early (IE), early (E) and late. The appearance of IE gene products provides the switch for E transcription. Abundant expression of late genes requires viral DNA replication. There is some overlap between E and late genes according to their degree of dependence on DNA replication. The pattern of expression of gene US11 is regulated with 'true-late' kinetics (Johnson et al., 1986). In a transient assay system, regulation of a plasmid-borne US11 promoter mimics its viral counterpart, and has a similar dependence on DNA replication for abundant expression. Using plasmids which contain a functional HSV-1 origin of replication (ORIS), we have identified the sequence requirements for the expression of late genes. All DNA sequence elements necessary for fully efficient regulated expression of US11 lie within 31 bp of the RNA cap sites; therefore it appears that a late gene promoter consists only of a proximal 'TATA-box' and cap-site region. We tested this hypothesis by removing the distal upstream region of the gD promoter (which is required for its normal regulation as an early promoter) and linking this truncated promoter to ORIS. This resulted in the conversion of gD promoter regulation to late gene kinetics during virus superinfection. The implications of these results for the mechanisms of HSV gene regulation are discussed.
Base Sequence↗
Genetic tests of the role of Abf1p in driving transcription of the yeast TATA box bindng protein-encoding gene, SPT15.
In this report we describe studies which utilized yeast strains bearing gain and loss of function alleles of ABF1 in order to attempt to directly implicate Abf1p in modulating transcription of the TBP-encoding gene, SPT15, in vivo. We found that overexpression of Abf1p in a yeast cell increased transcription of the TBP-encoding gene and that this stimulation depended upon the exact sequence of the Abf1p binding site (ABF1) present in the gene. Further, in a yeast strain expressing a temperature sensitive form of Abf1p, occupancy of the chromosomal ABF1 site in the TBP-encoding gene was immediately lost following a temperature shift. Both results suggest that Abf1p drives transcription of the TBP-encoding gene. Surprisingly though we found that continuous ABF1 cis-element occupancy by Abf1p was not acutely required for normal levels of transcription of either the TBP-encoding gene or other "Abf1p-driven" genes tested. We propose a model to explain these results and suggest mechanisms by which Abf1p could activate gene transcription.
Binding Sites↗
Model of the complex formed between the H2 domain of the TATA box binding protein and the L(281-301) fragment of the human transcription factor TFIIA.
Additional interactions possibly involving the well-exposed H2 helical domain of hTBP and the acidic fragment L(281-301) of the non-conserved domain of hTFIIA have been proposed to account for the apparent discrepancies between the results of mutagenesis experiments on human proteins and the structure of the ternary complex TBP/TATA box/TFIIA established from yeast proteins by X-ray crystallography. To verify this hypothesis both peptides were synthesized and their structures studied by circular dichroism (CD), NMR and molecular modelling. These peptides exist preferentially under helical conformations in solution (30% TFE in H2O). An interaction between the two peptides was observed by fluorescence (Kapp 170 microM), CD and NMR techniques. Molecular modelling studies indicate that this complex could be stabilized by electrostatic interactions involving the glutamate Glu287 and aspartates (Asp290, Asp294, Asp297 and Asp298) of L(281-301)TFIIA and lysine residues (Lys133, Lys138 and Lys145) and arginine residues (Arg137, Arg140) of H2(TBP) in agreement with mutagenesis experiments. Similar studies could now be carried out with human proteins to demonstrate the biological relevance of this interaction.
Amino Acid Sequence↗
Cyclosporine A inhibits the activity of a TATA box-binding protein that is required for transcription from the adenovirus major late promoter.
Nuclear extracts from P1798 lymphoma cells support transcription from the adenovirus major late promotor (AdMLP) and the human histone H4 promoter. Nuclear extracts prepared from P1798 cells treated with 1 microgram/ml cyclosporine A for 24 h fail to support transcription from AdMLP, whereas transcription from the histone H4 promoter is unimpaired. Both control and cyclosporine-treated extracts contain proteins that interact with synthetic deoxyoligonucleotides that correspond to the CAAT box, TATA box, and upstream stimulatory element of AdMLP. Cyclosporine had no discernible qualitative or quantitative effect upon such DNA-protein interactions, as observed by gel mobility shift assays. Analysis of 5' deletion mutants of AdMLP indicates that deletion of sequences upstream of the TATA box reduces AdMLP transcription by only 50%. This observation suggests that cyclosporine A, which inhibits AdMLP transcription by > 90%, is unlikely to act through changes in the amount or activity of upstream activators such as upstream stimulatory factor- or CAAT box-binding proteins. On the other hand, deletion of TATA box sequences between -50 and -11 base pairs virtually eliminates transcription from AdMLP in vitro. A partially purified TFIID fraction was obtained from control P1798 nuclear extracts. The TFIID fraction reconstitutes transcription from AdMLP when added to extracts from cyclosporine A-treated cells. Recombinant TATA box-binding protein also reconstitutes transcription from AdMLP in cyclosporine A-treated extracts. These results are consistent with the hypothesis that cyclosporine A regulates the activity of a subset of general transcription factors which are required for initiation from some promoters (such as AdMLP) but not from others (such as histone H4).
Adenoviridae↗
The erythroid-specific protein cGATA-1 mediates distal enhancer activity through a specialized beta-globin TATA box.
The erythroid-specific protein cGATA-1 regulates the chick beta-globin gene through GATA sequences present at the canonical TATA location in the promoter as well as the distal 3' enhancer. We have analyzed beta-globin transcription in transfected erythroid cells and in erythroid extracts to determine whether cGATA-1 binding at -30 regulates promoter or enhancer activity. The interaction of both cGATA-1 and TFIID at different times with the -30 GATA site is required for efficient beta-globin expression in vivo, and the GATA enhancer site can functionally replace the TATA element in the beta-globin promoter. TFIID initiates transcription in vitro by complexing with adaptor proteins and displacing cGATA-1 from the -30 GATA site. Mutations that abolish TFIID binding to the -30 GATA box inactivate the promoter, whereas elimination of cGATA-1 binding to this site selectively diminishes enhancer-dependent transcription. We propose that interaction of cGATA-1 with the distal 3' enhancer and the specialized TATA box confers erythroid specificity to the initiation complex by mediating promoter-enhancer communication. Thus, one mechanism of action for tissue-specific proteins that recognizes noncanonical TATA motifs is to enable TFIID to be regulated by distal control elements. In this way, the initiation complex can be responsive to specific regulators that may not recognize a canonical TFIID-TATA structure.
Animals↗
Adenovirus E1A activation domain binds the basic repeat in the TATA box transcription factor.
The adenovirus large E1A protein is a potent activator of transcription. We use several different experimental approaches to demonstrate that the large E1A protein binds specifically and stably to the TATA box-binding factor (TFIID), the general polymerase II transcription factor that initiates assembly of transcription complexes. Sedimentation velocity centrifugation revealed that TFIID and E1A form a heterodimer in vitro. We demonstrate that the activation domain of E1A (conserved region 3) binds to TFIID. E1A interacts with a 51 residue region from the conserved C-terminal domain of TFIID that includes a repeat of basic residues between the homologous direct repeats of TFIID. Analysis of TFIID binding by various E1A mutants indicates that TFIID binding is necessary, although not sufficient, for E1A transactivation.
Adenoviridae↗
Fluorescence-based analyses of the effects of full-length recombinant TAF130p on the interaction of TATA box-binding protein with TATA box DNA.
We have used a combination of fluorescence anisotropy spectroscopy and fluorescence-based native gel electrophoresis methods to examine the effects of the transcription factor IID-specific subunit TAF130p (TAF145p) upon the TATA box DNA binding properties of TATA box-binding protein (TBP). Purified full-length recombinant TAF130p decreases TBP-TATA DNA complex formation at equilibrium by competing directly with DNA for binding to TBP. Interestingly, we have found that full-length TAF130p is capable of binding multiple molecules of TBP with nanomolar binding affinity. The biological implications of these findings are discussed.
DNA↗
No evidence for association of the TATA-box binding protein glutamine repeat sequence or the flanking chromosome 6q27 region with type 1 diabetes.
Susceptibility to the autoimmune disease type 1 diabetes has been linked to human chromosome 6q27 and, moreover, recently associated with one of the genes in the region, TATA box-binding protein (TBP). Using a much larger sample of T1D families than those studied by others, and by extensive re-sequencing of nine other genes in the proximity, in which we identified 279 polymorphisms, 83 of which were genotyped in up to 725 T1D multiplex and simplex families, we obtained no evidence for association of the TBP CAG/CAA (glutamine) microsatellite repeat sequence with disease, or for nine other genes, PDCD2, PSMB1, KIAA1838, DLL1, dJ894D12.4, FLJ25454, FLJ13162, FLJ11152, PHF10 and CCR6. This study also provides an exon-based tag single nucleotide polymorphism map for these 10 genes that can be used for analysis of other diseases.
Chromosomes, Human, Pair 6↗
Mutational analysis of an archaebacterial promoter: essential role of a TATA box for transcription efficiency and start-site selection in vitro.
By using a recently developed in vitro transcription assay, the 16S/23S rRNA-encoding DNA promoter from the archaebacterium Sulfolobus sp. B12 was dissected by deletion and linker substitution mutagenesis. The analysis of 5' and 3' deletion mutants defined a core promoter region between positions -38 and -2 containing all information for efficient and specific transcription. Further characterization of this region by linker substitution mutagenesis indicated two sequence elements important for promoter function--one located between positions -38 and -25 (distal promoter element) and the other one located between positions -11 and -2 (proximal promoter element). The distal promoter element encompassed the TATA-like "box A" element located approximately 26 nucleotides upstream of the majority of transcription start sites in archaebacteria (Archaeobacteria). All mutations within this box A motif virtually abolished promoter function. Complete inactivation of the proximal promoter element was dependent on extensive mutagenesis; this element is not conserved between archaebacterial promoters except for a high A + T content in stable RNA gene promoters from Sulfolobus. Mutants containing insertions or deletions between the distal and proximal promoter elements were only slightly affected in their transcription efficiency but displayed a shift in their major initiation site, retaining an essentially fixed distance between the distal promoter element and the transcription start site. Thus, efficient transcription and start-site selection were dependent on a conserved TATA-like sequence centered approximately 26 nucleotides upstream of the initiation site, a situation unlike that of eubacterial promoters but resembling the core structure of most eukaryotic RNA polymerase II (and some RNA polymerase III) promoters. This finding suggests a common evolutionary origin of these promoters consistent with the known similarities between archaebacterial and eukaryotic RNA polymerases.
Archaea↗