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PubMed1996

The function of the octamer-binding site in the DRA promoter.

The octamer binding site, which is located immediately upstream of the poorly conserved DRA TATA sequence, is important for high levels of expression of this human major histocompatibility class II gene in B cells. In this study, we demonstrate that the substitution of the DRA TATA sequence with the TATA box from the adenovirus E1b promoter removes the requirement for the octamer binding site for high levels of expression from the DRA promoter. Since only the TATA box from the E1b but not the DRA promoters binds the TATA binding protein, we conclude that the octamer binding site helps to recruit TBP to the DRA promoter.

Adenovirus E1B Proteins↗
PubMed2003

Sequences downstream of the transcription initiation site are important for proper initiation and regulation of mouse ribonucleotide reductase R2 gene transcription.

Ribonucleotide reductase is essential for the synthesis of all four dNTPs required for DNA replication. The enzyme is composed of two proteins, R1 and R2, which are both needed for activity. Expression of the R1 and R2 mRNAs is restricted to the S-phase of the cell cycle, but the R1 and R2 promoters show no obvious sequence homologies that could indicate coordination of transcription. Here we study initiation of transcription at the natural mouse R2 promoter, which contains an atypical TATA-box with the sequence TTTAAA, using a combination of in vivo reporter gene assays and in vitro transcription. Our results indicate that in constructs where sequences from the R2 5'-UTR are present, the mouse R2 TATA-box is dispensable both for unregulated, basal transcription from the R2 promoter and for S-phase specific activity. Instead, initiation of R2 transcription is directed by sequences downstream from the transcription start. We report that this region contains a conserved palindrome sequence that interacts with TAFIIs. This interaction down-regulates basal transcription from the R2 promoter, both in the absence and in the presence of the TATA-box.

5' Untranslated Regions↗
PubMed1990

Promoter function and in situ protein/DNA interactions upstream of the yeast HSP90 heat shock genes.

We have mapped in vivo protein/DNA interactions within the upstream regulatory regions of the two yeast HSP90 genes, and have begun mutagenizing footprinted sequences in an effort to identify the cis-acting determinants of heat shock transcription. Genomic footprinting of the HSP82 promotor using chemical and enzymatic nucleases reveals that irrespective of transcriptional state, the most proximal of three heat shock elements, HSE1, is occupied along both sugar-phosphate backbones as well as within its major groove, while the TATA box is bound along both sugar-phosphate backbones. Distorted DNA structure is associated with each constitutively bound factor: protein binding to HSE1 appears to induce a local A-form-like helical conformation, whereas occupancy of the TATA box is associated with strand-specific nuclease hypersensitivity of an adjacent polypurine tract. In situ mutagenesis experiments indicate that HSE1 is absolutely required for both basal and induced expression, and that basal transcription can be preferentially abolished by point mutations within this sequence. In contrast, point mutations within the TATA element have the reverse effect, as induced transcription is more significantly affected. Similar to HSE1 point mutants, we have found that basal transcription is preferentially repressed by an HMRE silencer element when it is transplaced approximately 1 kb upstream of the HSP82 start site. Finally, a complementary footprinting analysis of the upstream region of the constitutively expressed HSC82 gene reveals the presence of three discrete protein complexes. These map to the TATA box, the promotor-distal heat shock element, C.HSE1, and a novel sequence upstream of C. HSE1, suggesting that the 10-fold higher basal transcription of HSC82 stems, at least in part, from a non-HSE-binding factor.

Base Sequence↗
PubMed1993

Varicella-zoster virus DNA polymerase and major DNA-binding protein genes have overlapping divergent promoters.

A detailed analysis of the transcriptionally divergent promoters of varicella-zoster virus (VZV) open reading frames (ORFs) 28 and 29, encoding the DNA polymerase and major DNA-binding proteins, respectively, was performed. We found that the 221-bp ORF 28-29 intergenic domain contains overlapping divergent promoters; these promoters have TATA boxes and cap sites arranged closely back-to-back, have highly concordant patterns of responsiveness to transactivation by VZV ORFs 4 and/or 62, and could not be separated without abolishing the effects that VZV trans activators imparted to them. Mutation of the ORF 28 TATA box rendered this promoter unresponsive to ORF 62 and the combination of ORFs 4 and 62 without altering ORF 29 promoter activity. Mutations of all potential ORF 29 TATA boxes collectively failed to abolish this promoter's responsiveness to either ORF 4 or ORF 62, suggesting a mechanism of gene regulation for ORF 29 that differs from that of ORF 28. These findings are concordant with the observation that both genes are expressed in productive infection, but only ORF 29 expression has been identified in latency.

Animals↗
PubMed2006

Investigation of non-covalent complexes of HIV-1 promoter DNA and polyamides containing N-methylpyrrole by electrospray ionization mass spectrometry.

Eight novel polyamides containing N-methylpyrrole were designed to target the sequence (5'-CTGCATATAAGCAG-3'/5'-CTGCTTATATGCAG-3') of the TATA box element of the HIV-1 promoter DNA. The non-covalent complexes of the promoter DNA and the polyamides were investigated by electrospray ionization (ESI) mass spectrometry, which provided strong evidence for the binding of the novel polyamides to the sequence of the TATA box element. It also revealed that polyamide 2 (PyPyPyPybetaDp), a potent binder of HIV-1 promoter DNA and a lead molecule for the design of new anti-HIV-1 drugs, had the highest binding affinity with the TATA box element DNA among these polyamides by examining the stoichiometry and the selectivity.

DNA, Viral↗
PubMed1989

Transcription of Xenopus selenocysteine tRNA Ser (formerly designated opal suppressor phosphoserine tRNA) gene is directed by multiple 5'-extragenic regulatory elements.

A tRNA gene whose product is aminoacylated with serine and the serine moiety is then phosphorylated to form phosphoseryl-tRNA (see Hatfield, D. (1985) Trends Biochem. Sci. 10, 201-204 for review) has now been shown to form selenocysteyl-tRNA; hence the corresponding gene is designated as selenocysteine tRNA Ser (B. J. Lee, P. J. Worland, J. N. Davis, T. C. Stadtman, and D. Hatfield (1989) J. Biol. Chem. 264, in press). In the present study, we show that the expression of this unique tRNA gene is governed by at least three upstream regulatory elements. In initial studies, the relative efficiencies of transcription of the human, rabbit, chicken, and Xenopus selenocysteine tRNA genes were compared in vivo in Xenopus oocytes and in vitro in HeLa cell extracts. The Xenopus gene was severalfold more actively expressed, both in vivo and in vitro, than the human and rabbit genes, whereas the chicken gene was poorly expressed. Exchange of the 5'-flanking regions of the Xenopus and chicken genes, which have identical gene sequences, reversed their levels of transcription, demonstrating that a regulatory site or sites exist upstream of these genes. Deletion-substitution mutants in the Xenopus gene and its 5'-flanking sequence show in in vitro assays that 1) the level of transcription is reduced substantially when a GC-rich stretch that is immediately upstream of a TATA box in the -30 region is removed; 2) the level of transcription is virtually abolished when the TATA box is removed; and 3) deletions up to and further upstream of the GC-rich region do not affect the level of transcription. The same deletions, when used in in vivo assays, demonstrate a step-down in expression with the deletion removing the GC-rich region, a further step-down in expression with the deletion removing the TATA box, but the most pronounced reduction in expression was observed with a deletion removing an AT-rich region between nucleotides -62 and -76. Thus, a regulatory site was identified in vivo which was not detected in vitro, and transcription of the selenocysteine tRNA Ser gene is determined by multiple upstream regulatory elements.

Amino Acid Sequence↗
PubMed1993

Cathepsin D gene is controlled by a mixed promoter, and estrogens stimulate only TATA-dependent transcription in breast cancer cells.

The cathepsin D (cath-D) gene, coding for a ubiquitous lysosomal aspartyl protease, is overexpressed in aggressive human breast cancers, and its transcription is induced by estrogens in hormone-responsive breast cancer cells. We have determined the structure and function of the proximal 5' upstream region of the human cath-D gene from MCF7 cells. We show that the promoter has a compound structure with features of both housekeeping genes (high G+C content and potential transcription factor Sp1 sites) and regulated genes (TATAA sequence). By RNase protection assay, we show that transcription is initiated at five major transcription sites (TSSI to -V) spanning 52 base pairs. In hormone-responsive breast cancer cells, estradiol increased by 6- to 10-fold the level of RNAs initiated at TSSI, which is located about 28 base pairs downstream from the TATA box. The specific regulation by estradiol of transcription starting at site I exclusively was confirmed by primer extension. Moreover, the same estradiol effect was observed in the ZR75-1 cell line and in MDA-MB231 estrogen-resistant breast cancer cells stably transfected with the estrogen receptor. Site-directed mutagenesis indicated that the TATA box is essential for initiation of cath-D gene transcription at TSSI. In breast cancer biopsy samples, high levels of TATA-dependent transcription were correlated with overexpression of cath-D mRNA. We conclude that cath-D behaves, depending on the conditions, as a housekeeping gene with multiple start sites or as a hormone-regulated gene that can be controlled from its TATA box.

Animals↗
PubMed2006

In silico identification of putative promoter motifs of White Spot Syndrome Virus.

BACKGROUND: White Spot Syndrome Virus, a member of the virus family Nimaviridae, is a large dsDNA virus infecting shrimp and other crustacean species. Although limited information is available on the mode of transcription, previous data suggest that WSSV gene expression occurs in a coordinated and cascaded fashion. To search in silico for conserved promoter motifs (i) the abundance of all 4 through 8 nucleotide motifs in the upstream sequences of WSSV genes relative to the complete genome was determined, and (ii) a MEME search was performed in the upstream sequences of either early or late WSSV genes, as assigned by microarray analysis. Both methods were validated by alignments of empirically determined 5' ends of various WSSV mRNAs. RESULTS: The collective information shows that the upstream region of early WSSV genes, containing a TATA box and an initiator, is similar to Drosophila RNA polymerase II core promoter sequences, suggesting utilization of the cellular transcription machinery for generating early transcripts. The alignment of the 5' ends of known well-established late genes, including all major structural protein genes, identified a degenerate motif (ATNAC) which could be involved in WSSV late transcription. For these genes, only one contained a functional TATA box. However, almost half of the WSSV late genes, as previously assigned by microarray analysis, did contain a TATA box in their upstream region. CONCLUSION: The data may suggest the presence of two separate classes of late WSSV genes, one exploiting the cellular RNA polymerase II system for mRNA synthesis and the other generating messengers by a new virus-induced transcription mechanism.

Base Sequence↗
PubMed1995

Cooperation between core promoter elements influences transcriptional activity in vivo.

Core promoters for RNA polymerase II frequently contain either (or both) of two consensus sequence elements, a TATA box and/or an initiator (Inr). Using test promoters consisting of prototypical TATA and/or Inr elements, together with binding sites for sequence-specific activators, we have analyzed the function of TATA and Inr elements in vivo. In the absence of activators, the TATA element was significantly more active than the Inr, and the combination of elements was only slightly more effective than the TATA-only promoter. In the presence of any of several coexpressed activator proteins, the TATA elements was again most active, but here addition of the Inr allowed significant increases in activity, indicating a cooperative interaction between the two elements. An interesting exception was observed with the activator Sp1, which was more effective with the Inr-only promoter, and addition of a TATA box did not enhance activity. Finally, in all cases the TATA plus Inr promoters were found to be partially or completely resistant to the dominant negative effects of a transcription factor TFIIB mutant previously shown to interfere with expression from TATA-only promoters. This result strengthens the conclusion that TATA and Inr elements can cooperate in vivo.

Animals↗
PubMed1988

Construction of promoter-probe vectors for Candida maltosa, a n-alkane-assimilating yeast, using the LEU2 gene of Saccharomyces cerevisiae.

For the purpose of isolation of promoter regions which are regulated by a carbon source in the medium in an n-alkane-assimilating yeast, Candida maltosa, two promoter-probe vectors were constructed. Each of them consists of the LEU2 gene of Saccharomyces cerevisiae whose 5'-noncoding region was trimmed with BAL31, an autonomously replicating sequence isolated from C. maltosa genome (the TRA region) which we have previously isolated, and the pBR322 sequence. One of them, pPLC2, having the TATA box, lacks the regulatory sequence ("sequence L") of the LEU2 gene, and the other, pPLC1, lacks both the TATA box and sequence L. Using pPLC1 as a short-gun cloning vector in C. maltosa, many promoter regions which were active when glucose was present in the medium as a carbon source were obtained from the genome of C. maltosa. The sizes of the inserted fragments of two of them were determined. (In this paper, a promoter region refers to a promoter which includes a TATA box, plus a regulatory sequence such as an UAS (upstream activating sequence)-like sequence).

Candida↗
PubMed1993

Recognition of a palindromic DNA sequence by ESF-1, a factor stimulating transcription of the adenovirus type 12 E1A gene.

We previously identified a factor, designated E1A-stimulating factor 1 (ESF-1), that stimulates transcription of the adenovirus type 12 E1A gene in vitro by binding to a region next to a TATA box. We report here on the partial characterization of the mode of ESF-1 action. Point mutations within the nucleotide sequence 5'-TCAGCTGA-3' abolished the binding of ESF-1. Stimulation of transcription of the adenovirus type 12 E1A gene in vitro was lost when a 7 base pair insertion was introduced between the ESF-1-binding site and a TATA box. These results suggest that ESF-1 stimulates adenovirus type 12 E1A gene transcription by interacting with a palindromic DNA sequence present in a region immediately upstream from a TATA box.

Adenovirus E1A Proteins↗
PubMed1996

Different arrangement of human papillomavirus E2 binding sites distinguishes cutaneous types from those associated with mucosal lesions.

High-risk-type human papillomavirus DNA sequences are found in a high percentage of carcinomas from the uterine-cervix, with the viral E1-E2 gene region usually disrupted and the E6 and E7 oncoproteins consistently expressed. The E2 protein is known to repress early transcription from genital HPV promoters having a proximal E2 binding site (E2BS) close to the TATA box. On the contrary, the E2 protein activates cutaneous early promoters having a longer distance between these sites. Using an in vivo approach we analyzed the regulation, by the BPV-1E2 protein, of a natural HPV-18 promoter where proximal E2BS were placed at variable positions relative to the TATA box, and of heterologous promoters where E2BS was placed upstream of any other known DNA-binding elements. Our results confirm that the E2 protein represses or activates HPV early gene transcription depending on the distance between the TATA box and the proximal E2BS.

Animals↗
PubMed2001

YY1 is a positive regulator of transcription of the Col1a1 gene.

Both cell-specific and ubiquitous transcription factors in fibroblasts have been identified as critical for expression of the Col1a1 gene, which encodes the alpha1 chain of type I collagen. Here, we report that Yin Yang 1 (YY1) binds to the Col1a1 promoter immediately upstream of the TATA box, and we examine the functional implications of YY1 binding for regulation of Col1a1 gene expression in BALBc/3T3 fibroblasts. The Col1a1 promoter region spanning base pairs (bp) -56 to -9 bound purified recombinant YY1 and the corresponding binding activity in nuclear extracts was supershifted using a YY1-specific antibody. Mutation of the TATA box to TgTA enhanced YY1 complex formation. Mutation analysis revealed two YY1 core binding sites at -40/-37 bp (YY1A) and, on the reverse strand, at -32/-29 bp (YY1B) immediately adjacent to the TATA box. In transfections using Col1a1-luciferase constructs, mutation of YY1A decreased activity completely (wild-type p350 (p350wt), -222/+113 bp) or partially (p130wt, -84 bp/+13 bp), whereas mutation of YY1B blocked the expression of both promoter constructs. Cotransfection with pCMV-YY1 increased p350wt and p130wt activities by as much as 10-fold, whereas antisense YY1 decreased constitutive expression and blocked the increased activity due to pCMV-YY1 overexpression. The mTgTA constructs were devoid of activity, arguing for a requirement for cognate binding of the TATA box-binding protein (TBP). Electrophoretic mobility shift assays performed under conditions permitting TBP binding showed that recombinant TBP/TFIID and YY1 could bind to the -56/-9 bp fragment and that YY1B was the preferred site for YY1 binding. Our results indicate that YY1 binds to the Col1a1 proximal promoter and functions as a positive regulator of constitutive activity in fibroblasts. Although YY1 is not sufficient for transcriptional initiation, it is a required component of the transcription machinery in this promoter.

3T3 Cells↗
PubMed1990

Anatomy of the rat albumin promoter.

The sequences preceding the albumin mRNA start site are able to direct efficient transcription only upon introduction into cells expressing the endogenous albumin gene. In transient expression assays, the activity of a reporter gene (CAT) linked to this promoter is 100-fold higher in H4II differentiated hepatoma cells than in H5 dedifferentiated cells which no longer express their albumin gene. This tissue specificity depends on the very proximal promoter region, composed of a CCAAT box, the proximal element and a TATA box. Deletion of the CCAAT box leads to a two- to threefold decrease in activity, deletion of the proximal element (PE) results in loss of activity. The PE is a high-affinity binding site for HNF1/APF, a strictly liver specific trans-acting factor. When the affinity of this factor for PE is decreased by bacterial methylation (PE includes a dam methylase site), by mutation, or by its replacement with the homologous element from the alpha-fetoprotein gene (AFP), the activity of the short promoter (PE plus the TATA box) is abolished. This activity can be rescued in the presence of the more upstream elements: DEII, DEI and the CCAAT box (recognized, respectively, by the NF1/CTF, C/EBP and NFY/ACF factors) which are then absolutely required. Our results suggest that the upstream elements contribute to promoter activity by stabilizing the HNF1-PE complex and not by direct interaction with TFIID or the RNA polymerase. It is probable that these elements, essentially dispensable in already differentiated hepatoma cells, play a crucial role during development or differentiation to activate the promoter in cells that contain a low concentration of HNF1 and/or an HNF1 unable to open inactive chromatin alone.

Animals↗
PubMed1990

Genomic footprinting of the yeast HSP82 promoter reveals marked distortion of the DNA helix and constitutive occupancy of heat shock and TATA elements.

We describe here for the first time successful application of the hydroxyl radical technique for genomic footprinting. In combination with two complementary techniques, DNase I footprinting and dimethyl sulfate methylation protection, we have obtained a high-resolution map of the promoter region of the yeast HSP82 heat shock gene, which resides within a constitutive nuclease hypersensitive site. We find that irrespective of transcriptional state, basal or induced, only one of three putative heat shock elements, HSE1, and the TATA box are tightly bound by proteins, presumably heat shock factor (HSF) and TFIID, respectively. Whereas the HSE1-associated factor binds tightly within the major groove of DNA, as discerned by protection of guanine residues from methylation by dimethyl sulfate in intact cells, the TATA factor appears to bind principally to the sugar-phosphate backbone, as revealed by strong protection from hydroxyl radical cleavage in whole-cell lysates. In addition, while HSE1 is strongly footprinted by DNase I in lysates, the TATA box is only weakly footprinted. Strikingly, both elements are associated with marked distortion of the DNA double helix in chromatin. Protein binding to HSE1 appears to cause a non-B-conformation, on the basis of a local 12 base-pair periodicity of hydroxyl radical protection and the presence of multiple DNase I hyperreactive cleavages flanking HSE1, whose pattern changes following heat shock. Similarly, helix distortion is evident in the vicinity of the TATA box, since hydroxyl radical detects a lower strand-specific hypersensitive site at the dyad center of an adjacent polypurine tract. Finally, the absence of discernable modulation in the DNase I cleavage pattern argues against the presence of a specifically positioned nucleosome within the IISP82 promoter region.

Base Sequence↗
PubMed1994

Identification of two promoter regions in the rat B-50/GAP-43 gene.

To determine cis-acting elements controlling the rat B-50/GAP-43 gene expression, the genomic DNA encoding exon 1 and the 5' flanking sequence was isolated. Sequence analysis of 1 kb 5' untranslated region (UTR) revealed the presence of a (GA)-repeat and a (GT)-repeat. The size of the (GA)-repeat varied due to both an instability of phage lambda lambda DNA in E. coli and genomic variation between rats. Transcription initiation sites were mapped in 8-day-old rat brain poly(A)+ mRNA. Primer extension indicated multiple transcription start sites at -159 and -339/-342 nt upstream of the translation start site; reverse transcriptase coupled PCR showed that the most 5' transcription start site is located between -465 and -440. Northern blotting demonstrated that approximately 90% of the B-50 mRNAs initiates at approximately -50. Promoter analysis by transient transfection assays in undifferentiated and retinoic acid-differentiated P19-EC cells revealed that the rat B-50 gene contains two promoters. P1 (located between -750 and -407) contains commonly observed promoter elements such as a TATA box and CCAAT boxes. P2 (located between -233 and -1) neither contains TATA boxes, CCAAT boxes nor consensus sequences of house-keeping gene promoters like GC-boxes. The activity of P1 is inhibited at neuroectodermal differentiation of P19-EC cells whereas the activity of P2 is stimulated. In 8 day old rat brain the majority of the B-50 mRNA transcripts are derived from P2. It is concluded that at this developmental stage P2 is the most important promoter.

Animals↗
PubMed1997

An Ssn6-Tup1-dependent negative regulatory element controls sporulation-specific expression of DIT1 and DIT2 in Saccharomyces cerevisiae.

Sporulation of the yeast Saccharomyces cerevisiae is a process of cellular differentiation that occurs in MATa/MAT alpha diploid cells in response to starvation. The sporulation-specific genes DIT1 and DIT2, which are required for spore wall formation, are activated midway through the sporulation program, with maximal transcript accumulation occurring at the time of prospore enclosure. In this study, we have identified a negative regulatory element, termed NREDIT, that is located between the start sites of transcription of these divergently transcribed genes. This element, which prevents expression of the DIT1 and DIT2 genes during vegetative growth, reduces expression of a CYC1-lacZ reporter gene more than 1,000-fold and acts in an orientation- and position-independent manner. We found that the ability of NREDIT to turn of expression of the reporter gene and the chromosomal DIT1 and DIT2 genes in vegetative cells requires the Ssn6-Tup1 repression complex. Interestingly, NREDIT-mediated repression of the reporter gene is maintained during sporulation. Derepression during sporulation requires complex interactions among several cis-acting elements. These are present on an approximately 350-bp DNA fragment extending from NREDIT to the TATA box and an approximately 125-bp fragment spanning the TATA box of DIT1. Additionally, a region of NREDIT which is very similar in sequence to UASSPS4, an element that activates gene expression midway through sporulation, contributes both to vegetative repression and to sporulation-specific induction of DIT1. We propose a model to explain the requirement for multiple elements in overcoming NREDIT-mediated repression during sporulation.

Base Sequence↗
PubMed1984

Contact points between transcription machinery and the fibroin gene promoter deduced by functional tests of single-base substitution mutants.

An efficient method for oligonucleotide-directed mutagenesis was developed to construct a set of site-specific mutations by using a mixture of oligodeoxyribonucleotides. With this method, as high as 40% of the tested clones turned out to be desired mutants. Seven single-point mutants were isolated in the "TATA" box region of the fibroin gene. In vitro transcription experiments showed that single-base transversions at the TATA box (A----T at position -29, T----A or G at -28, A----T at -27, and A----T at -26) resulted in decreased promoter activities, whereas A----G transitions at positions -24 and -23 had no effect. The initiation site of transcription was normal in three down-promoter mutants at positions -28 and -26, but the A----T transversions at positions -29 and -27 induced an additional transcription start from position +4. Using 10 single-point mutants obtained as above or by nitrous acid-induced mutagenesis, we have prepared a pair of heteroduplex DNAs consisting of a mutant strand and the wild-type one. The molecules heterozygous at positions -30, -21, and -20 showed reduced transcription activities when the noncoding strand bears the mutation, whereas that at position -26 gave a low activity when the coding strand carries the mutation. Both types of heteroduplex at positions -29, -28, -27, and -17 exhibited decreased activities. These results suggest a transcription machinery contact to a major groove of the DNA helix at the TATA box and region of position -20.

Animals↗