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At least 613 records · Page 34Linked to original sources
PubMed2006

Expression of the Saccharomyces cerevisiae PIS1 gene is modulated by multiple ATGs in the promoter.

The PIS1 gene encodes a key branchpoint phospholipid biosynthetic enzyme, phosphatidylinositol synthase. The PIS1 promoter contains the unusual feature of three ATG codons (ATGs1, 2, and 3) in-frame with three stop codons, located just before the authentic start codon (ATG4). Using a PIS1(promoter)-lacZ reporter expression system and site-directed mutagenesis, we investigated the role the "upstream" ATG codons play in modulation of PIS1 expression. Of the single codon changes, mutation of the first ATG (ATG1) resulted in the largest increase of the reporter gene PIS1(promoter)-lacZ expression. All combinations of altered upstream ATG codons also resulted in greater reporter expression. Reverse transcription-PCR revealed that at least some PIS1 transcripts include all AUG codons, and their synthesis is probably directed by a second TATA box upstream of the putative TATA box. These results indicate that the multiple upstream AUG codons are present in at least some PIS1 transcripts and negatively impact PIS1 expression.

Base Sequence↗
PubMedDate not supplied

Sequence-specific insertion of the Drosophila transposable genetic element 17.6.

As in the case of retrovirus proviruses, most of the Drosophila copia-like transposable elements so far examined are bounded by 5'TG...CA3' and inserted into the chromosome without obvious site-specificity. In the other copia-like elements, 297, HMS Beagle and 17.6 (refs 5-7), terminal dinucleotides (5'TG...CA3') are completely absent and, instead, 5'(A)GT is present at least at one of the termini. One important feature of these three elements may be frequent insertion into 'TATA' boxes, since the three of four insertion sites so far examined were TATA boxes, two for H3 histone genes and one for a cuticle gene. Because of the importance of this type of insertion, we extensively analysed site-specificity using 17.6 as a model. Our results, described here, suggest that insertion of 17.6 takes place in a site-specific fashion, using a target 5'ATAT corresponding to the major portion of the consensus TATA box, TATAATAAT.

Animals↗
PubMed2000

[Functional characteristics of the promoter region of the tag7/PGRP gene in KSML0, KSML100 murine mammary adenocarcinoma cell ines and VMR liver].

A study was carried out on the transcription regulation of the tag7/PGRP gene, whose product is similar to cytokines and is involved in recognizing peptidoglycane in mouse mammary adenocarcinoma cell lines KSML0, KSML100, and VMR-liver. The 3250-bp fragment of the promoter region was sequenced and tested for DNase I-hypersensitive sites (DHSs). In the KSML0 cells line, DHSs were found in the vicinity of the TATA box and approximately at position -3000 relative to the transcription start of the gene. Only a DHS cluster near the TATA box was found in the VMR-liver cell line. Regions involved in transcription regulation of the gene in the three cell lines were identified with the use of reporter constructions carrying the CAT gene under the control of deletion derivatives of the tag7/PGRP promoter region. The minimal promoter including only the TATA box with the nearest neighboring sequences was inactive in all cell lines. The elements (enhancers) positively regulating gene transcription in KSML0 and VMR-liver were mapped to region -192, -48. An element accounting for the transcriptional inactivity of the gene in KSML100 cells was assigned to region -315, -3250.

Adenocarcinoma↗
PubMed2006

Perspectives on the RNA polymerase II core promoter.

The RNA polymerase II core promoter is a critical yet often overlooked component in the transcription process. The core promoter is defined as the stretch of DNA, which encompasses the RNA start site and is typically approx. 40-50 nt in length, that directs the initiation of gene transcription. In the past, it has been generally presumed that core promoters are general in function and that transcription initiation occurs via a common shared mechanism. Recent studies have revealed, however, that there is considerable diversity in core promoter structure and function. There are a number of DNA elements that contribute to core promoter activity, and the specific properties of a given core promoter are dictated by the presence or absence of these core promoter motifs. The known core promoter elements include the TATA box, Inr (initiator), BRE(u) {BRE [TFIIB (transcription factor for RNA polymerase IIB) recognition element] upstream of the TATA box} and BRE(d) (BRE downstream of the TATA box), MTE (motif ten element), DCE (downstream core element) and DPE (downstream core promoter element). In this paper, we will provide some perspectives on current and future issues that pertain to the RNA polymerase II core promoter.

Models, Genetic↗
PubMed1999

Drosophila head segmentation factor buttonhead interacts with the same TATA box-binding protein-associated factors and in vivo DNA targets as human Sp1 but executes a different biological program.

The Drosophila gene buttonhead (btd) is required for the establishment of three embryonic head segments. It encodes a zinc-finger-type transcription factor expressed in the corresponding head segment anlagen in the blastoderm stage embryo. The DNA-binding properties of the btd protein (BTD) are indistinguishable from the human transcription factor Sp1. Furthermore, BTD and Sp1 are capable of activating transcription in transfected cultured cells through interaction with the same DNA target sites. Herein we show that BTD and Sp1 functionally interact with the same TATA box-binding protein-associated factors and support in vitro transcription activation through these contacts. Transgene expression of BTD results in the rescue of the head segments that fail to develop in btd mutant embryos, whereas Sp1 or Sp1 containing the zinc finger region of BTD rescues mandibular segment development. The results suggest that BTD contains functional domains other than an equivalent DNA-binding region and interaction sites of the TATA box-binding protein-associated factors, which are necessary to establish head segments that fail to develop in response to Sp1.

Animals↗
PubMed1991

In vivo stimulation of a chimeric promoter by binding sites for nuclear factor I.

Nuclear factor I (NFI) is composed of a family of site-specific DNA-binding proteins which recognize a DNA-binding site with the consensus sequence TGGC/A(N)5GCCAA. Binding sites for NFI have previously been shown to stimulate mRNA synthesis in vitro when present upstream of the TATA box of the adenovirus major late promoter (AdMLP). We have examined the effect of NFI-binding sites on transcription in vivo in transiently transfected HeLa and COS cells. An NFI-binding site isolated from the human genome activated expression from the minimal AdMLP in vivo in both the absence and presence of the simian virus 40 enhancer. A point mutation that decreased NFI binding affinity for the site in vitro reduced expression to near the basal level of the AdMLP. Several NFI-binding sites which differed in their spacer and flanking sequences were tested for their ability to activate expression in vivo. The ability of these sites to activate expression correlated with the strength of NFI binding in vitro. An NFI-binding site stimulated expression equally well when placed from 33 to 65 bp upstream of the TATA box. However, expression dropped to basal levels when the site was located from 71 to 77 bp upstream of the TATA box. These studies indicate that an NFI-binding site in this chimeric promoter activates expression in vivo only if located within a critical distance of the TATA box.

Animals↗
PubMed1999

Developmental changes in RNA polymerase I and TATA box-binding protein during early Xenopus embryogenesis.

Xenopus early embryos are transcriptionally quiescent until the midblastula transition (MBT). We have examined the question of whether the absence of rRNA synthesis is related to a deficiency in the RNA polymerase I (pol I) transcription machinery. Previously we have demonstrated that the maternally provided pol I transcription factor UBF already binds to the inactive rRNA genes of pre-MBT embryos (P. Bell et al., 1997, J. Cell Sci. 110, 2053-2063). Here we have analyzed the fate of pol I and the TATA box-binding protein (TBP) through immunofluorescence and immunoblotting experiments. Pol I stockpiled in the egg is taken up by in vitro assembled pronuclei and concentrated into numerous distinct nuclear domains. Comparable storage sites of template-free pol I are also seen in nuclei of blastula to neurula stage embryos. In contrast, the amount of TBP is relatively low in oocytes and eggs but increases dramatically during the cleavage stages. Most of the newly synthesized TBP colocalizes with the stored form of pol I in the extranucleolar domains of blastula/gastrula embryos. The amount of TBP per embryo reaches peak values at the blastula/gastrula stage and then rapidly declines to normal somatic levels. The positive correlation of maximal TBP levels with the timing of the MBT suggests that overproduction of TBP is required for the formation of productive transcription complexes.

Animals↗
PubMed1995

Overlapping TATA-dependent and TATA-independent early promoter activities in the baculovirus gp64 envelope fusion protein gene.

In previous studies to characterize basal and activated transcription from the early promoter of the gp64 envelope fusion protein (efp) gene of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus, the TATA box was identified as a functional element, essential for basal transcription from a minimal promoter construct. In the current study, we used discrete deletions and multiple point mutations that removed the functional TATA box from larger promoter constructs of the gp64 efp gene to reveal an overlapping TATA-independent transcriptional activity. TATA-independent transcriptional activity was inhibited in vitro by alpha-amanitin but not by tagetitoxin, demonstrating that like the TATA-dependent activity, the TATA-independent activity is mediated by RNA polymerase II. Using constructs in which the TATA box (TATATAA) was destroyed by substitution mutations, we identified four elements that are required for the TATA-independent activity. Two of the required elements, GATA (at -114) and CACGTG (at -104), were previously shown to specifically bind host transcription factors and activate transcription from the TATA-dependent wild-type gp64 efp promoter. The role of the early start site consensus CAGT sequence in TATA-independent transcription was also examined. Single-nucleotide substitution mutations in the CAGT sequence indicated that certain nucleotides within the CAGT start site were essential. In addition to the start site sequence and two upstream elements, a fourth essential element was identified in the 5' untranslated leader region (5'UTR). While the 5'UTR was not necessary for TATA-dependent transcription, deletion of a 10-bp 5'UTR sequence resulted in the loss of TATA-independent transcriptional activity. Although necessary, neither the GATA, CACGTG, start site region, nor 5'UTR element was alone sufficient for accurately initiated TATA-independent transcription from the consensus CAGT start site. Thus, the gp64 efp early promoter contains overlapping TATA-dependent and TATA-independent transcriptional activities. A number of common sequence elements (GATA, CACGTG, and start site CAGT) are involved in both of these activities, while one element (in the 5'UTR) is required only in the TATA-independent context.

Base Sequence↗
PubMed2001

Promoter-specific activation defects by a novel yeast TBP mutant compromised for TFIIB interaction.

TFIIB is an RNA polymerase II general transcription factor (GTF) that has also been implicated in the mechanism of action of certain promoter-specific activators (see, for examples, [1-11]). TFIIB enters the preinitiation complex (PIC) primarily through contact with the TATA box binding protein (TBP), an interaction mediated by three TBP residues [12-14]. To study the role of TFIIB in transcription activation in vivo, we randomly mutagenized these three residues in yeast TBP and screened for promoter-specific activation mutants. One mutant bearing a single conservative substitution, TBP-E186D, is the focus of this study. As expected, TBP-E186D binds normally to the TATA box but fails to support the entry of TFIIB into the PIC. Cells expressing TBP-E186D are viable but have a severe slow-growth phenotype. Whole-genome expression analysis indicates that transcription of 17% of yeast genes are compromised by this mutation. Chimeric promoter analysis indicates that the region of the gene that confers sensitivity to the TBP-E186D mutation is the UAS (upstream activating sequence), which contains the activator binding sites. Most interestingly, other TBP mutants that interfere with different interactions (TFIIB, TFIIA, or the TATA box) and a TFIIB mutant defective for interaction with TBP all manifest distinct and selective promoter-specific activation defects. Our results implicate the entry of TFIIB into the PIC as a critical step in the activation of certain promoters and reveal diverse mechanisms of transcription activation.

Carrier Proteins↗
PubMed1995

Promoter-proximal pausing of RNA polymerase II defines a general rate-limiting step after transcription initiation.

We have shown previously that the majority of RNA polymerase II complexes initiated at the c-myc gene are paused in the promoter-proximal region, similar to observations in the Drosophila hsp70 gene. Our analyses define the TATA box or initiator sequences in the c-myc gene as necessary components for the establishment of paused RNA polymerase II. Deletion of upstream sequences or even the TATA box does not influence significantly the degree of transcriptional initiation or pausing. Deletion of both the TATA box and sequences at the transcription initiation site, however, abolishes transcriptional pausing of transcription complexes but still allows synthesis of full-length RNA. Further analyses with synthetic promoter constructs reveal that the simple combination of upstream activator with TATA consensus sequences or initiator sequences act synergistically to recruit high levels of RNA polymerase II complexes. Only a minor fraction of these complexes escapes into regions further downstream. Several different trans-activation domains fused to GAL4-DNA-binding domains, including strong activators such as VP16, do not eliminate promoter-proximal pausing of RNA polymerase. Thus, we conclude that pausing of RNA polymerase II is a common phenomenon in eukaryotic transcription and does not require complex promoter structures. Further analyses reveal that enhancers have a modest influence on transcription initiation and on release of transcription complexes out of the pause site but may function primarily to increase the elongation competence of transcription complexes.

Animals↗
PubMed1994

Transcription of the sea urchin U6 gene in vitro requires a TATA-like box, a proximal sequence element, and sea urchin USF, which binds an essential E box.

The tandemly repeated gene set encoding the sea urchin U6 gene has been cloned from the sea urchin Strongylocentrotus purpuratus. The U6 gene is transcribed by RNA polymerase III in a sea urchin nuclear extract. Like that of the vertebrate U6 genes, transcription of the sea urchin U6 gene does not require any internal sequences or 3' sequences but requires only 5' flanking sequences. Only 88 nucleotides of 5' flanking sequence are required for maximal expression in vitro. Mutagenesis experiments demonstrated the requirement for three elements, a CACGTG element at -80, a proximal sequence element at about -55, and the TATA-like box at -25. The major protein in sea urchin extracts that interacts with the CACGTG element is sea urchin USF, and immunodepletion of sea urchin USF greatly reduces transcription. The USF binding site in the U6 gene is highly homologous (11 of 13 nucleotides) with the USF binding sites found in the promoter of the S. purpuratus spec genes.

Animals↗
PubMed1991

Identification of 5'-regions affecting the expression of the human CR2 gene.

Human CR2 has a restricted cellular distribution, being expressed on B lymphocytes, dendritic cells of the spleen, pharyngeal epithelial cells, and at low levels on some T lymphocytes. CR2 is expressed by mature B lymphocytes, but not by pre-B cells or by plasma cells, suggesting that mechanisms exist for positive and negative regulation of CR2 gene expression during B cell development. S1 nuclease digestion and primer extension analysis positioned the transcriptional start site between 92 and 94 bp upstream of the ATG codon. Nucleotide sequence analysis identified several sequences within the CR2 promoter region with homology to other known promoter sequences. These included a site similar to an AP-1 site, a sequence with 10 of 13 nucleotides identical to the X box of class II genes, and a TATA box. Genomic DNA starting immediately 5' of the sequence encoding the CR2 signal peptide was subcloned upstream of the bacterial chloramphenicol acetyltransferase gene for analysis of functional promoter and enhancer sites. The functional boundaries of the CR2 promoter were determined by deletion analysis, with both the X box-like sequences and the TATA box required for CR2 expression. This analysis revealed sequences with regulatory effects on CR2 gene expression, however, these transcriptional controlling sequences did not act in a tissue specific fashion.

Antigens, Differentiation, B-Lymphocyte↗
PubMed2004

The equine herpesvirus 1 EICP27 protein enhances gene expression via an interaction with TATA box-binding protein.

The mechanism(s) by which the early EICP27 gene product cooperates with other equine herpesvirus 1 (EHV-1) regulatory proteins to achieve maximal promoter activity remains unknown. Transient transfection assays revealed that deletion of residues 93-140 of the 470-aa EICP27 protein substantially diminished its activation of the immediate-early (IE) promoter, whereas deletion of residues 140-470 that contain a zinc-finger motif abolished this activity. Fluorescence microscopy of cells expressing the full-length EICP27 protein or portions of this protein revealed that an arginine-rich sequence spanning residues 178-185 mediates nuclear entry. Experiments employing the mammalian Gal4 two-plasmid system revealed that the EICP27 protein does not possess an independent trans-activation domain (TAD). Protein-protein interaction assays using purified proteins revealed that residues 124-220 of the EICP27 protein mediate its direct interaction with TATA box-binding protein (TBP). Partial deletion of this TBP-binding domain attenuated the ability of the EICP27 protein to stimulate the IE and early EICP0 promoters by 68% and 71%, respectively, indicating the importance of this protein-protein interaction.

Amino Acid Sequence↗
PubMed1991

Baculovirus gp64 gene expression: analysis of sequences modulating early transcription and transactivation by IE1.

Expression of the baculovirus major envelope glycoprotein gene (gp64) is regulated by transcription from both early and late promoters. To characterize the early promoter and identify sequences involved in the regulation of gp64 early transcription, promoter-reporter gene fusions were generated from the Orygia pseudotsugata nuclear polyhedrosis virus gp64 promoter and were analyzed by transient expression in uninfected insect cells. For these analyses, 5' deletion mutations were constructed in the gp64 upstream regulatory region. Larger promoter constructs were functional in uninfected Lymantria dispar cells, indicating that transcription from the gp64 early promoter required no additional viral gene products. Deletion analysis of the gp64 upstream region revealed several regulatory regions. These included a putative negative regulatory element between -319 and -166 nucleotides (nt) and multiple positive regulatory elements between -166 and -77 nt. Deletion of the TATA box located between -77 and -62 nt resulted in the loss of transcriptional activity. Cotransfections of reporter constructs and a plasmid containing a baculovirus transcriptional transactivator gene (Autographa californica nuclear polyhedrosis virus IE1) resulted in transcriptional transactivation of all constructs containing an intact TATA box. These data demonstrate that sequences upstream of the gp64 TATA box are not essential for IE1 transactivation and that only 34 nt upstream of the early transcription start site were necessary for basal levels of transcription and for transactivation by IE1. Function of the gp64 early promoter was also examined in cell lines from Spodoptera frugiperda and Drosophila melanogaster.

Amino Acid Sequence↗
PubMed2002

The vasopressin gene non-canonical Hogness box: effect on protein binding and promoter function.

Comparison of the promoter sequences of the genes encoding the neuropeptide hormone vasopressin from a number of organisms has revealed that they do not contain a classical Hogness box. In all vertebrate species examined, the canonical TATA box is replaced with a CATA sequence. We hypothesised that this conserved modified sequence may play a role in the regulation of vasopressin promoter activity. We used electrophoretic mobility shift assays to show that TATA and CATA sequences generate different complexes with SON nuclear proteins. Further, the transfection of wild-type (CATA) and mutated (TATA) VP promoter-reporter constructs into a heterologous cell line demonstrated a sequence-specific effect on transcriptional activity. The CATA sequence contributes to weaker promoter activity than a TATA box, but is able to interact with the upstream elements to increase the efficacy of an enhancer. The CATA box may thus be involved in the cell-specific and physiological regulation of the VP gene.

Animals↗
PubMed1990

Identification of cis-acting regulatory elements in the promoter region of the rat brain creatine kinase gene.

The functional organization of the rat brain creatine kinase (ckb) promoter was analyzed by deletion, linker scanning, and substitution mutagenesis. Mutations were introduced into the ckb promoter of hybrid ckb/neo (neomycin resistance gene) genes, and the mutant genes were expressed transiently in HeLa cells. Expression was assayed by primer extension analysis of neo RNA, which allowed the transcription start sites and the amount of transcription to be determined. Transfections and primer extension reactions were internally controlled by simultaneous analysis of transcription from the adenovirus VA gene located on the same plasmid as the hybrid ckb/neo gene. We demonstrate that 195 bp of the ckb promoter is sufficient for efficient in vivo expression in HeLa cells. A nonconsensus TTAA element at -28 bp appears to provide the TATA box function for the ckb promoter in vivo. Two CCAAT elements, one at -84 bp and the other at -54 bp, and a TATAAA TA element (a consensus TATA box sequence) at -66 bp are required for efficient transcription from the TTAA element. In addition, we present evidence that the consensus beta-globin TATA box responds to the TATAAATA element in the same way as the ckb nonconsensus TTAA element.

Animals↗
PubMed2006

HESR1/CHF2 suppresses VEGFR2 transcription independent of binding to E-boxes.

The bHLH transcription factor HESR1 (CHF2) acts downstream of notch to regulate cardiovascular development and angiogenesis, at least in part through down-regulation of the VEGF receptor, VEGFR2. Surprisingly, we find that HESR1 interacts with the promoter in endothelial cells (EC) not through direct binding to the E-boxes, but through intermediary interactions with GC-box-binding proteins. The bHLH and orange domains of HESR1 are sufficient for repression in EC, likely through recruitment of co-repressors, however, the C-terminal YRPW motif is not required. The VEGFR2 promoter contains a functional initiator element but no TATA box, however, addition of a TATA sequence renders the promoter resistant to inhibition by HESR1. In agreement with this finding, the NrCAM, TK, and CMV promoters, which have TATA boxes, cannot be repressed. Thus, HESR1 represses VEGFR2 through interactions with SP-1-like factors and requires an Inr element in the absence of a TATA box. Our findings illuminate an important mechanism for notch/HESR1 regulation of VEGF-induced angiogenesis.

Base Sequence↗
PubMed1996

A cell-free transcription system for the hyperthermophilic archaeon Pyrococcus furiosus.

We describe here the establishment of a cell-free transcription system for the hyperthermophilic Archaeon Pyrococcus furiosus using the cloned glutamate dehydrogenase (gdh) gene as template. The in vitro system that operated up to a temperature of 85 degrees C initiated transcription 23 bp downstream of a TATA box located 45 bp upstream of the translational start codon of gdh mRNA, at the same site as in Pyrococcus cells. Mutational analyses revealed that this TATA box is essential for in vitro initiation of transcription. Pyrococcus transcriptional components were separated into at least two distinct transcription factor activities and RNA polymerase. One of these transcription factors could be functionally replaced by Methanococcus aTFB and Thermococcus TATA bind- ing protein (TBP). Immunochemical analyses demonstrated a structural relationship between Pyrococcus aTFB and Thermococcus TBP. These findings indicate that a TATA box and a TBP are essential components of the Pyrococcus transcriptional machinery.

Archaea↗