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PubMed2001

Dynamic simulations of 13 TATA variants refine kinetic hypotheses of sequence/activity relationships.

The fundamental relationship between DNA sequence/deformability and biological function has attracted numerous experimental and theoretical studies. A classic prototype system used for such studies in eukaryotes is the complex between the TATA element transcriptional regulator and the TATA-box binding protein (TBP). The recent crystallographic study by Burley and co-workers demonstrated the remarkable structural similarity contrasted to different transcriptional activity of 11 TBP/DNA complexes in which the DNAs differed by single base-pairs. By simulating these TATA variants and two other single base-pair variants that were not crystallizable, we uncover sequence-dependent structural, energetic, and flexibility properties that tailor TATA elements to TBP interactions, complementing many previous studies by refining kinetic hypotheses on sequence/activity correlations. The factors that combine to produce favorable elements for TBP activity include overall flexibility; minor groove widening, as well as roll, rise, and shift increases at the ends of the TATA element; untwisting within the TATA element accompanied by large roll at the TATA element ends; and relatively low maximal water densities around the DNA. These features accompany the severe deformation induced by the minor-groove binding protein, which kinks the TATA element at the ends and displaces local water molecules to form stabilizing hydrophobic contacts. Interestingly, the preferred bending direction itself is not a significant predictor of activity disposition, although certain variants (such as wild-type AdMLP, 5'-TATA4G-3', and inactive A29, 5'-TA6G-3') exhibit large preferred bends in directions consistent with their activity or inactivity (major groove and minor groove bends, respectively). These structural, flexibility, and hydration preferences, identified here and connected to a new crystallographic study of a larger group of DNA variants than reported to date, highlight the profound influence of single base-pair DNA variations on DNA motion. Our refined kinetic hypothesis suggests the functional implications of these motions in a kinetic model of TATA/TBP recognition, inviting further theoretical and experimental research.

Base Sequence↗
PubMed1990

Sequencing and expression of complementary DNA for the general transcription factor BTF3.

The initiation of transcription of eukaryotic genes involves the ordered assembly of a multiprotein complex on proximal promoter elements such as the TATA box. In addition to RNA polymerase II (otherwise RNA pol II, RNA polymerase B), four general transcription factors are required for initiation of transcription: BTF1 (also referred to as TFIID) which has recently been cloned from yeast, BTF2, BTF3 and STF. The first step in assembly of the initiation complex is the stable binding of BTF1 to the TATA box, which is facilitated by STF. Neither BTF2 nor BTF3 bind directly to the promoter proximal elements, but BTF3 can form a stable complex with RNA pol II. We recently purified BTF3, which is a protein of relative molecular mass 27,000, but further studies have been hampered by its low abundance in cells. On the basis of sequences from peptides of BTF3, we have now cloned two complementary DNAs, one for a protein (BTF3a) with all the characteristics of purified BTF3, and one for a shorter protein (BTF3b) lacking the first 44 residues of BTF3a and which is transcriptionally inactive, despite its ability to bind RNA pol II.

Amino Acid Sequence↗
PubMed1991

E1A gene products stimulate in vitro transcription from the adenovirus early region 4 promoter by enhancing a stable preinitiation complex.

A cell-free transcription system using nuclear extracts prepared from adenovirus type 5 (Ad5)-infected and mock-infected cells were utilized to study the E1A-mediated transactivation of transcription from the Ad5 early region 4 (E4) promoter. The transcription activity of Ad-infected cell extracts was several times higher than that of mock-infected cell extracts when the E4 wild-type (WT) promoter was used. The increased activity required expression of E1A proteins. However, the transcription activities of both cell extracts were not different when the mutant promoter containing only the E4 TATA box was used. The formation of a stable preinitiation complex at the E4 WT promoter was facilitated by expression of E1A, whereas it was not facilitated at the mutant promoter. The results suggested that the E1A proteins stimulated transcription from the E4 promoter by enhancing the formation of a stable preinitiation complex through the upstream elements, together with the TATA box.

Adenovirus Early Proteins↗
PubMed1988

Simian virus 40 major late promoter: a novel tripartite structure that includes intragenic sequences.

Unlike most genes transcribed by RNA polymerase II, the simian virus 40 late transcription unit does not have a TATA box. To determine what sequences are required for initiation at the major late mRNA cap site of simian virus 40, clustered point mutations were constructed and tested for transcriptional activity in vitro and in vivo. Three promoter elements were defined. The first is centered 31 base pairs upstream of the cap site in a position normally reserved for a TATA box. The second is at the cap site. The third occupies a novel position centered 28 base pairs downstream of the cap site within a protein-coding sequence. The ability of RNA polymerase II to recognize this promoter suggests that there is greater variation in promoter architecture than had been believed previously.

Base Sequence↗
PubMed1989

Purification of a yeast TATA box-binding protein that exhibits human transcription factor IID activity.

By a series of conventional chromatographic procedures we have purified from whole-cell extracts of Saccharomyces cerevisiae yeast transcription factor IID (TFIID), which functionally substitutes for human TFIID in a complementation assay comprised of the adenovirus type 2 major late promoter and HeLa cell-derived RNA polymerase II, transcription factors IIA, IIB, and IIE. Similar to its human counterpart, yeast TFIID also exhibited specific binding to the adenovirus type 2 major late promoter TATA element, as shown by both DNase I footprinting and gel mobility shift assays. NaDodSO4/PAGE analyses showed that a 27-kDa polypeptide coeluted with TFIID complementing activity through each chromatographic step. In agreement with this result and also suggesting that the native protein is a monomer, gel-filtration experiments indicated a molecular mass of 28 kDa for TFIID under nondenaturing conditions. That the 27-kDa polypeptide represented TFIID was further demonstrated by the ability of an HPLC-purified protein to bind specifically after renaturation to the adenovirus type 2 major late promoter TATA sequence.

Antigens, Neoplasm↗
PubMed1982

A synthetic heat-shock promoter element confers heat-inducibility on the herpes simplex virus thymidine kinase gene.

Previous deletion analysis of the Drosophila hsp70 heat-shock promoter has identified a sequence upstream of the TATA box that is required for heat induction. This region contains homology to other heat-shock promoters, and it was proposed that the common sequence is an important element in the regulation of the heat-shock genes. We have constructed sequences similar to the consensus CT-GAA-TTC-AG from synthetic oligonucleotides and placed them upstream of the TATA box of the herpes virus thymidine kinase gene, in place of the normal upstream promoter element. The resultant genes are heat-inducible both in monkey COS cells and in Xenopus oocytes. We conclude that the transcriptional heat-shock response is mediated by some factor that interacts with this sequence.

Animals↗
PubMed1995

Repression of preprotachykinin-A promoter activity is mediated by a proximal promoter element.

The rat preprotachykinin-A promoter, which is able to direct reporter gene expression in adult dorsal root ganglia neurons grown in culture, has no detectable activity in HeLa and PC12 cells. DNAase 1 footprinting and electrophoretic mobility shift analyses with HeLa nuclear extract indicated the presence of a protein complex binding to a region of the rat preprotachykinn-A gene promoter between the TATA box and the major transcriptional start site. We demonstrate that the sequence of the preprotachykinin-A promoter spanning nucleotides -47 to +92 functions to repress reporter gene expression in HeLa and PC12 cells but not in adult rat dorsal root ganglia grown in culture, and that this repression is correlated with a protein(s) binding to the element between the TATA box and major transcription initiation site. These results indicate that the tissue-specific expression of the preprotachykinin-A gene could require the interaction of both positive and negative regulatory DNA elements.

Animals↗
PubMed1997

A negative regulatory region of the murine Hox11 gene.

The HOX11 gene was isolated from the chromosomal breakpoint of human T cell acute lymphoblastic leukemias with a chromosomal translocation t(10;14). Expression of this proto-oncogene is strictly controlled in normal tissues. However, regulatory elements of the gene have never been studied. Since the HOX11 gene is well conserved between human and murine, we sequenced 5' flanking region of the murine Hox11 gene and analyzed the elements. We identified the transcription start site (+1) of the gene using mRNA from fetal spleens by primer extension analysis. The start site was determined at 795 bp upstream from the ATG site. A typical TATA box sequence was found at 35 bp upstream from the start site. Furthermore, promoter activity of the 5' flanking region of the start site was monitored by luciferase assay. The activity mainly located within a 540-bp fragment immediately upstream from the start site (-540 to +1). The (-1240 to -540) region contained a negative regulatory element of the transcription. The TATA box sequence and the nucleotide sequence around the transcription start site were conserved in the human HOX11 gene. The transcription start site of the human HOX11 gene in normal tissues is discussed.

Animals↗
PubMed2005

Conditional inhibition of cancer cell proliferation by tetracycline-responsive, H1 promoter-driven silencing of PLK1.

RNA interference (RNAi) is a powerful tool for studying gene function. We developed an inducible genetic element for short interfering RNA-mediated gene silencing. This system uses a tetracycline (Tet)-responsive derivative of the H1 promoter and the Tet repressor (TetR) for conditional expression of short hairpin RNA (shRNA) in HeLa cells. Promoter constructs were generated, which contain the Tet operator (TetO) derived from a prokaryotic Tet resistance transposon upstream and/or downstream of the TATA box. To quantify the response of controllable transcription units for shRNA expression, we examined the functional activity of polo-like kinase 1 (PLK1), a key component of mitotic progression, that is overexpressed in many human tumors. Cotransfection of plasmids for the expression of TetR and shRNA/PLK1 under the control of an H1 promoter-variant carrying TetO upstream of the TATA box did not alter PLK1 expression and proliferation properties of HeLa cells in the absence of doxycycline. Addition of the antibiotic led to marked downregulation of endogenous PLK1 accompanied by strong inhibition of cellular proliferation. Our data indicate that an inducible transcription system for shRNAs based on the human H1 promoter could be a versatile tool for controlled gene silencing in vitro.

Cell Cycle Proteins↗
PubMed1983

Sequence requirement for transcription in vitro of the human corticotropin/beta-lipotropin precursor gene.

Using HeLa whole cell extracts, we have demonstrated that transcription in vitro of the cloned human and bovine corticotropin/beta-lipotropin precursor genes is initiated accurately and efficiently. DNA sequences required for promoter function have been assessed by using a series of 5'-deletion mutants of a fusion gene that contains the 5'-flanking sequence and capping site of the human corticotropin/beta-lipotropin precursor gene and the structural sequence of the herpes simplex virus thymidine kinase gene. The results obtained have shown that the region between 22 base pairs and 35 base pairs upstream from the capping site is essential for the correct and efficient transcriptional initiation in vitro. Thus, the 'TATA box' present in this region seems to be the main promoter element for transcription of the human corticotropin/beta-lipotropin precursor gene in the HeLa cell-free system. We have also developed a transcription system in vitro from the corticotropin-producing mouse pituitary tumor cell line AtT-20 in culture. Deletion mapping of the fusion gene promoter has indicated that the 'TATA box' region is required for the accurate and efficient transcriptional initiation in this system as well. Characteristic of this system is that the deletion of the sequence lying between 53 base pairs and 59 base pairs upstream from the capping site increases the transcriptional efficiency. Because this effect is observed in the AtT-20 cell-free system, but hardly in the HeLa cell-free system, it seems reasonable to assume that the interaction of this upstream sequence with some factor(s) in the AtT-20 cell extract is responsible for the modulation of transcription of the human corticotropin/beta-lipotropin precursor gene.

Adrenocorticotropic Hormone↗
PubMed1993

An alternative pathway for transcription initiation involving TFII-I.

The minimal promoter elements required for initiation by RNA polymerase II include the TATA box and/or an initiator element (Inr) at or near the transcription start site. Studies of the adenovirus major late core promoter (containing both elements) have demonstrated an initiation pathway that involves binding of the transcription factor TFIID (or the derived subunit, the TATA-binding protein TBP (TFIID tau)) to the TATA element, which is facilitated by transcription factor TFIIA, followed by sequential interactions of other general factors. Here we describe a novel pathway that requires an intact Inr and the Inr-binding factor TFII-I (ref. 3). Sequential addition of the general factors generated TFII-I-dependent preinitiation complexes different from those formed with TFIIA. Furthermore, TBP bound cooperatively (with only TFII-I) to an Inr-containing TATA-less promoter, suggesting a means for activation of TATA-less promoters, which nonetheless require TFIID (refs 9-11). These observations provide support for functionally distinct pathways which could be subject to differential regulation by specific activators or repressors.

Adenoviridae↗
PubMed1989

DNA binding and gene regulation by the herpes simplex virus type 1 protein ICP4 and involvement of the TATA element.

We report the results of fine mapping the sequences responsible for negative regulation of immediate-early (IE) gene 3 by its own gene product, ICP4. Affinity-purified ICP4 binds the transcriptional start site of IE gene 3 and protein-protein interactions induce a secondary mobility shift that footprints exactly as the primary complex. Since these DNA-protein complexes contain ICP4, it is likely that the two differ only in stoichiometry of protein. Additional data show that the DNA-binding domain recognized by ICP4 can be embedded as a cassette in foreign DNA and that native ICP4 will recognize and bind the resulting DNA. In two different immediate-early promoters, the ICP4 binding site can be located either 3' or 5' of the TATA box; however, the ICP4 site is rotationally displaced from the transcription factor IID (TFIID) site by a roughly one-half helical turn, suggesting that ICP4 and TFIID are on the opposite helical face when bound at their respective sites. In the IE1 and IE3 promoters, binding of ICP4 causes an alteration in the helical geometry of the minor groove of the TATA region as visualized by copper footprinting. In contrast, TATA hypersensitivity was not detected in the glycoprotein D promoter (an early gene promoter containing the ICP4 site separated from TATA by eight helical turns) or in an artificial IE3 promoter construct in which the TATA-A4 separation was increased from 2.5 to roughly 5 helical turns. Such stereospecific and distance-dependent conformational alterations in the TATA box under the influence of ICP4 binding may be important in the repression of immediate-early genes.

DNA↗
PubMed1999

Comparison between site-specific DNA binding proteins of male and female Schistosoma mansoni.

Several amplicons with approximately 120 bp each, obtained from the upstream domain of Schistosoma mansoni female-specific gene F-10, were coupled to Dynabeads M-280 streptavidin. The beads were used as a matrix for affinity purification of nuclear proteins obtained from mixed populations of adult worms. A protein of approximately 12 kDa, bound to the DNA in a sequence-independent manner. In contrast, when the DNA matrix was narrowed down to smaller synthetic oligonucleotides, bearing sequences corresponding to the TATA box and the CAAT box, band-shift assays revealed that different nuclear proteins from either adult male or female worms formed complexes with the DNA adduct. In order to characterise the bound proteins, the same oligonucleotides were UV cross-linked to the male and female protein extracts. Whilst the band shift experiments showed that the proteins from each sex produced a distinct mobility pattern when the TATA box sequences were tested and a similar one when the CAAT box sequences were added to the proteins, UV cross-linking experiments revealed clear qualitative differences between both, male and female proteins and also between the proteins binding to the two motifs. These results are compatible with a model in which the differential expression of the F-10 gene might depend on individual sub-sets of proteins.

Animals↗
PubMed2006

Interactions between the cytomegalovirus promoter and the estrogen response element: implications for design of estrogen-responsive reporter plasmids.

We aimed to produce an estrogen-responsive reporter plasmid that would permit monitoring of estrogen receptor function in the uterus in vivo. The plasmid pBL-tk-CAT(+)ERE was induced by estrogen in bovine endometrial stromal cells. When the CAT gene was replaced by the secreted alkaline phosphatase SeAP, the resulting construct pBL-tk-SeAP(+)ERE remained estrogen responsive. However when the tk promoter was replaced by the cytomegalovirus (cmv) promoter, the resulting plasmid (pBL-cmv-SeAP(+)ERE) was not estrogen responsive. Inhibition of ERE function was not due to an effect in trans or due to lack of estrogen receptor. It was not due to an interaction between the cmv promoter and the SeAP gene. cmv promoter function was dependent on NF-kappaB, and mutagenesis in the NF-kappaB sites reduced basal reporter expression without imparting responsiveness to estrogen. A mutation in the TATA box also failed to impart estrogen responsiveness. Modeling of DNA accessibility indicated the ERE was inserted at a site accessible to transcription factors. We conclude that the cmv promoter inhibits ERE function in cis when the two sequences are located in the same construct, and that this effect does not involve an interaction between cmv and reporter gene, NF-kappaB sites or the TATA box, or DNA inaccessibility.

Animals↗
PubMed1984

A Drosophila RNA polymerase II transcription factor binds to the regulatory site of an hsp 70 gene.

A Drosophila RNA polymerase II transcription factor that is specific for at least one of the heat-shock genes has been isolated (designated HSTF for heat-shock transcription factor). This factor is required for active transcription of an hsp 70 gene in addition to RNA polymerase II and another general transcription factor, the A factor. Footprint analysis of the HSTF on the hsp 70 gene reveals that it binds specifically to a 55 bp region upstream from the TATA box. Both coding and noncoding DNA strands are completely protected from DNAase I cleavage by the HSTF . HSTF binding occurs in the apparent absence of RNA polymerase II. The HSTF is present in both heat-shocked and nonshocked cells, although it is more transcriptionally active when isolated from heat-shocked cells. The previously described B factor (an RNA polymerase II transcription factor that binds to the TATA box), isolated from nonshocked cells, is significantly reduced in both binding and transcriptional activity in heat-shocked cells. The potential role of the HSTF and the B factor in the activation of heat-shock gene transcription is discussed.

Actins↗
PubMed1993

TFIIIC relieves repression of U6 snRNA transcription by chromatin.

The U6 small nuclear (sn)RNA gene (SNR6) from the yeast Saccharomyces cerevisiae is transcribed by RNA polymerase III in vivo. This gene is unusual in having a TATA box at position -30, and an essential B-block element located downstream of the T-rich termination signal. The B block is one of the two intragenic promoter elements of transfer RNA genes that are recognized by transcription factor (TF)IIIC (ref. 4). But accurate in vitro transcription of yeast U6 snRNA gene by PolIII in a purified system requires only TFIIIB components, including the TATA-box binding protein TBP. Here we report that, after nucleosome reconstitution or chromatin assembly, U6 snRNA synthesis becomes dependent on TFIIIC and on the integrity of the B-block element. This observation resolves an apparent paradox between in vitro and in vivo results concerning the necessity of the downstream B-block element and sheds light on a new role of TFIIIC in gene activation.

Animals↗
PubMed1995

Transcription factor TFIID is a direct functional target of the adenovirus E1A transcription-repression domain.

The 243-amino acid adenovirus E1A oncoprotein both positively and negatively modulates the expression of cellular genes involved in the regulation of cell growth. The E1A transcription repression function appears to be linked with its ability to induce cellular DNA synthesis, cell proliferation, and cell transformation, as well as to inhibit cell differentiation. The mechanism by which E1A represses the transcription of various promoters has proven enigmatic. Here we provide several lines of evidence that the "TATA-box" binding protein (TBP) component of transcription factor TFIID is a cellular target of the E1A repression function encoded within the E1A N-terminal 80 amino acids. (i) The E1A N-terminal 80 amino acids [E1A-(1-80)protein] efficiently represses basal transcription from TATA-containing core promoters in vitro. (ii) TBP reverses completely E1A repression in vitro. (iii) TBP restores transcriptional activity to E1A-(1-80) protein affinity-depleted nuclear extracts. (iv) The N-terminal repression domain of E1A interacts directly and specifically with TBP in vitro. These results may help explain how E1A represses a set of genes that lack common upstream promoter elements.

Adenovirus E1A Proteins↗
PubMed1988

Transcriptional activation of heat-shock genes in eukaryotes.

Prokaryotes and eukaryotes respond to thermal or various chemical stresses by the rapid induction of a group of genes collectively referred to as the heat shock genes. In eucaryotes, the expression of these genes is primarily regulated at the transcriptional level. The early observations that transfected heat shock genes were inducible in heterologous systems suggested the existence of common regulatory elements in these ubiquitous genes. Sequence analysis of cloned Drosophila heat shock genes revealed a conserved 14 base pair (bp) inverted repeat, which is essential for heat induction. This regulatory sequence, referred to as the heat shock element (HSE), is found in multiple imperfect copies upstream of the TATA box of all heat shock genes. While studies in heterologous systems indicated that a single copy of HSE was sufficient for inducibility, further analysis in homologous assays suggests that multiple HSE can act in a cooperative way and that the efficiency of transcriptional activation is related, within limits, to the number of HSE. Comparative analysis of heat shock genes reveals that HSE can be positioned at different distances from the TATA box in either orientation, a behavior reminiscent of enhancer elements. However, the presence of HSE does not necessarily confer heat inducibility, as shown by their presence in the constitutively expressed but non-heat-inducible homologous cognate genes. Footprinting and nuclease mapping have been used to show that a protein factor (HSTF: heat shock transcription factor) binds to the HSE element, activating heat shock gene transcription in a dose-dependent manner. The recent progress in the isolation and characterization of HSTF in Drosophila, yeast, and human cells is reviewed. Finally, different models suggested to account for the positive regulation of heat shock genes by the HSTF are presented.

Animals↗