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PubMed2000

CH/pi interactions in the crystal structure of TATA-box binding protein/DNA complexes.

Crystal structures of TATA box-binding proteins (TBP) of various sources bound to their promoter DNA (TATA box) were analyzed with use of our program CHPI. A number of short CH/Csp2 contacts have been unveiled in these complexes at the boundary of TBP and the TATA box minor groove. The result was discussed in the context of the CH/pi interaction. Thus, the nature of nonpolar forces, reported in the past at the interface of the two components, has been attributed to the CH/pi interaction. Furthermore, many CH/pi contacts have been disclosed within the same strand of the promoter DNA. The structure of the TATA element, partially unwound and severely bent on complexation, seems to be stabilized by CH/pi interactions; H2' of the deoxyribose moiety and the methyl group in the thymine nucleotide play the primary role.

Binding Sites↗

PubMed2002

A single 8,5'-cyclo-2'-deoxyadenosine lesion in a TATA box prevents binding of the TATA binding protein and strongly reduces transcription in vivo.

8,5'-Cyclo-2'-deoxypurine (cPu) lesions result from the action of the hydroxyl radical on DNA. These lesions represent a unique class of oxidative DNA lesions in that they are repaired by the nucleotide excision repair (NER) pathway but not by base excision repair (BER) or direct repair. Previous work has shown that cyclopurines can block mammalian DNA and RNA polymerases. Thus, these lesions are of interest because of their potential role in the neurodegeneration as well as internal cancers observed in patients with xeroderma pigmentosum (XP) who lack the capacity to carry out NER. In the present work, we found that the S-isomer of 8,5'-cyclo-2'-deoxyadenosine (cA) can prevent binding of the TATA binding protein (TBP) to the TATA box from the CMV promoter. To assess the functional importance of this effect in living cells, we transfected constructs containing a single cA in the CMV TATA box into XP cells to determine the effect of the lesion on gene expression in vivo. Using this approach, we found that the lesion reduced gene expression by approximately 75%. This effect was comparable to the effect of an inactivating mutation of the TATA box in the same promoter. These findings identify an additional biological effect of cyclopurine lesions in mammalian cells, which is the ability to interfere with transcription by preventing transcription factor binding to cognate recognition sequences. In addition, the approach we used in this study represents a novel method for assessing the effects of DNA lesions in non-transcribed sequences on gene expression in living cells.

Cells, Cultured↗

PubMed2000

Enhanced binding of the TATA-binding protein to TATA boxes containing flanking cisplatin 1,2-cross-links.

The TATA-binding protein (TBP) is essential for transcription initiation in eukaryotes. TBP recognizes and binds to the minor groove of a consensus sequence, TATAAA, known as the TATA box or TATA element. DNA binding is affected largely by hydrophobic contacts and through the intercalation of two sets of adjacent phenylalanine residues. The resultant duplex is sharply kinked, bending toward the major groove. Inspired by prior structural information showing intercalation of a phenylalanine side chain of a high mobility group (HMG) domain into the site of a cisplatin 1, 2-intrastrand d(GpG) cross-link, a series of DNA probes was prepared with one or two such adducts flanking the TATA box positions at or near the sites of TBP intercalation. The platinum adducts bend the DNA toward the major groove and result in as much as a 175-fold increase in binding affinity of the TBP over the unmodified target sequence. Kinetic studies indicate that the enhanced binding to the modified TATA box is predominantly a consequence of a >30-fold slower dissociation rate of the protein-platinated DNA complex. This work demonstrates that it is feasible to design rationally and to synthesize an enhanced affinity-binding site for a sequence-specific DNA-binding protein by appropriate chemical modification of flanking sequences. It also has implications for the mechanism of action of cisplatin.

Binding, Competitive↗

PubMed1996

A novel form of the DNA double helix imposed on the TATA-box by the TATA-binding protein.

The structure of the TATA-box bound to the TATA-binding protein revealed a new and unexpected deformation of the double helix leading to a sharp change in the DNA trajectory. Here we show that the deformation imposed upon the TATA-box represents a novel form of the double helix--named TA-DNA--which differs from A-DNA by a single conformational parameter, namely the rotation around the glycosidic bond. This rotation causes a 50 degrees inclination of the base pairs in the TATA-box which in turn results in abrupt change in the trajectory of the flanking B-DNA segments. The observation that the TATA sequence can assume an A-DNA conformation coupled to the simplicity of the transition from A-DNA to TA-DNA may be the reason for the presence of the TATA sequence in a wide range of promoters.

Animals↗

PubMed2000

Assembly of partial TFIID complexes in mammalian cells reveals distinct activities associated with individual TATA box-binding protein-associated factors.

The TATA box-binding protein (TBP) and TBP-associated factors (TAF(II)s) compose the general transcription factor TFIID. The TAF(II) subunits mediate activated transcription by RNA polymerase II by interacting directly with site-specific transcriptional regulators. TAF(II)s also participate in promoter recognition by contacting core promoter elements in the context of TFIID. To further dissect the contribution of individual TAF(II) subunits to mammalian TFIID function, we employed a vaccinia virus-based protein expression system to study protein-protein interactions and complex assembly. We identified the domains of human (h) TAF(II)130 required for TAF(II)-TAF(II) interactions and formation of a complex with hTBP, hTAF(II)100, and hTAF(II)250. Functional analysis of partial TFIID complexes formed in vivo indicated that hTAF(II)130 was required for transcriptional activation by Sp1 in vitro. DNase I footprinting experiments demonstrated that purified hTBP/hTAF(II)250 complex reconstituted with or without additional TAF(II)s was significantly reduced for TATA box binding (as much as 9-fold) compared with free hTBP. By contrast, hTAF(II)130 stabilized binding of hTBP to the TATA box, whereas hTAF(II)100 had little effect. Thus, our biochemical analysis supports the notion that TAF(II)s possess distinct functions to regulate the activity of TFIID.

Cell Line↗

PubMed2002

Fission yeast F-box protein Pof3 is required for genome integrity and telomere function.

The Skp1-Cullin-1/Cdc53-F-box protein (SCF) ubiquitin ligase plays an important role in various biological processes. In this enzyme complex, a variety of F-box proteins act as receptors that recruit substrates. We have identified a fission yeast gene encoding a novel F-box protein Pof3, which contains, in addition to the F-box, a tetratricopeptide repeat motif in its N terminus and a leucine-rich-repeat motif in the C terminus, two ubiquitous protein-protein interaction domains. Pof3 forms a complex with Skp1 and Pcu1 (fission yeast cullin-1), suggesting that Pof3 functions as an adaptor for specific substrates. In the absence of Pof3, cells exhibit a number of phenotypes reminiscent of genome integrity defects. These include G2 cell cycle delay, hypersensitivity to UV, appearance of lagging chromosomes, and a high rate of chromosome loss. pof3 deletion strains are viable because the DNA damage checkpoint is continuously activated in the mutant, and this leads to G2 cell cycle delay, thereby preventing the mutant from committing lethal mitosis. Pof3 localizes to the nucleus during the cell cycle. Molecular analysis reveals that in this mutant the telomere is substantially shortened and furthermore transcriptional silencing at the telomere is alleviated. The results highlight a role of the SCF(Pof3) ubiquitin ligase in genome integrity via maintaining chromatin structures.

Amino Acid Sequence↗

PubMed2006

The F-box protein Dia2 regulates DNA replication.

Ubiquitin-mediated proteolysis plays a key role in many pathways inside the cell and is particularly important in regulating cell cycle transitions. SCF (Skp1/Cul1/F-box protein) complexes are modular ubiquitin ligases whose specificity is determined by a substrate-binding F-box protein. Dia2 is a Saccharomyces cerevisiae F-box protein previously described to play a role in invasive growth and pheromone response pathways. We find that deletion of DIA2 renders cells cold-sensitive and subject to defects in cell cycle progression, including premature S-phase entry. Consistent with a role in regulating DNA replication, the Dia2 protein binds replication origins. Furthermore, the dia2 mutant accumulates DNA damage in both S and G2/M phases of the cell cycle. These defects are likely a result of the absence of SCF(Dia2) activity, as a Dia2 DeltaF-box mutant shows similar phenotypes. Interestingly, prolonging G1-phase in dia2 cells prevents the accumulation of DNA damage in S-phase. We propose that Dia2 is an origin-binding protein that plays a role in regulating DNA replication.

DNA Damage↗

PubMed2001

The archaeal TFIIEalpha homologue facilitates transcription initiation by enhancing TATA-box recognition.

Transcription from many archaeal promoters can be reconstituted in vitro using recombinant TATA-box binding protein (TBP) and transcription factor B (TFB)--homologues of eukaryal TBP and TFIIB--together with purified RNA polymerase (RNAP). However, all archaeal genomes sequenced to date reveal the presence of TFE, a homologue of the alpha-subunit of the eukaryal general transcription factor, TFIIE. We show that, while TFE is not absolutely required for transcription in the reconstituted in vitro system, it nonetheless plays a stimulatory role on some promoters and under certain conditions. Mutagenesis of the TATA box or reduction of TBP concentration in transcription reactions sensitizes a promoter to TFE addition. Conversely, saturating reactions with TBP de-sensitizes promoters to TFE. These results suggest that TFE facilitates or stabilizes interactions between TBP and the TATA box.

Amino Acid Sequence↗

PubMed1995

Transcriptional activators differ in their responses to overexpression of TATA-box-binding protein.

We investigated how overexpression of human TATA-box-binding protein (TBP) affects the action of estrogen receptor (ER) and compared the response with that of other activators. When ER activates a simple promoter, consisting of a response element and either the collagenase or tk TATA box, TBP overexpression potentiates transcription. TBP potentiates only estrogen-induced and not basal transcription and does so independent of spacing between response element and TATA box. TBP overexpression also reduces autoinhibition by overexpressed ER, suggesting that one target of the autoinhibition may be TBP itself. Both AF-1 and AF-2 domains of ER are potentiated by TBP, and each domain binds TBP in vitro. Like ER, chimeric GAL4/VP16 and GAL4/Tat activators are also potentiated by TBP, as is the synergistic activation by ER and GAL4/VP16 on a complex promoter. Unlike ER, GAL4/Sp1 and GAL4/NF-I become less potent when TBP is overexpressed. Furthermore, synergy between ER and Sp1 or between ER and NF-I, whether these are supplied by transfected GAL4 fusions or by the endogenous genes, is inhibited by TBP overexpression. Thus, ER resembles VP16 in response to TBP overexpression and is different from Sp1 and NF-I, which predominate over ER in setting the response on complex promoters.

Animals↗

PubMed2003

A novel domain within the DEAD-box protein DP103 is essential for transcriptional repression and helicase activity.

Members of the DEAD-box family of helicases, distinguished by a core characteristic sequence of Asp-Glu-Ala-Asp, are expressed in a wide range of prokaryotes and eukaryotes and exhibit diverse cellular functions, including DNA transcription, recombination and repair, RNA processing, translation, and posttranslational regulation. Although ubiquitous, the function of most DEAD-box proteins is unknown. We and others have recently cloned DP103, which harbors conserved DEAD-box, helicase, and ATPase domains in its N terminus. DP103 (also termed Gemin3 and DDX20) interacts with SF-1, SMN, EBNA2, and EBNA3C in mammalian cells. Here we demonstrate that a discrete domain within the nonconserved C-terminal region of DP103 directly interacts with SF-1. This domain exhibits an autonomous repression function and is necessary and sufficient for repressing the transcriptional activity of SF-1. Furthermore, intact DP103 exhibits helicase activity. Importantly, the C-terminal domain is obligatory but not sufficient for this unwinding activity of DP103. Together, our results support a novel paradigm for transcriptional repression and demonstrate the bifunctional role of the C-terminal domain of DP103.

Animals↗

PubMed2006

PU.1 and a TTTAAA element in the myeloid defensin-1 promoter create an operational TATA box that can impose cell specificity onto TFIID function.

Defensins are major components of a peptide-based, antimicrobial system in human neutrophils. While packed with peptide, circulating cells contain no defensin-1 (def1) transcripts, except in some leukemia patients and in derivative promyelocytic leukemia cell lines. Expression is modulated by serum factors, mediators of inflammation, and kinase activators and inhibitors, but the underlying mechanisms are not fully understood. A minimal def1 promoter drives transcription in HL-60 cells under control of PU.1 and a def1-binding protein ("D1BP"), acting through, respectively, proximal (-22/-19) and distal (-62/-59) GGAA elements. In this study, we identify D1BP, biochemically and functionally, as GA-binding protein (GABP)alpha/GABPbeta. Whereas GABP operates as an essential upstream activator, PU.1 assists the flanking "TTTAAA" element (-32/-27), a "weak" but essential TATA box, to bring TBP/TFIID to the transcription start site. PU.1 thus imparts a degree of cell specificity to the minimal promoter and provides a potential link between a number of signaling pathways and TFIID. However, a "strong" TATA box ("TATAAA") eliminates the need for the PU.1 binding site and for PU.1, but not for GABP. As GABP is widely expressed, a strong TATA box thus alleviates promyelocytic cell specificity of the def1 promoter. These findings suggest how the myeloid def1 promoter may have evolutionarily acquired its current properties.

5' Flanking Region↗

PubMed1993

TATA box-mediated in vitro transcription by RNA polymerase III. Evidence for TATA-binding protein in a polymerase III type complex.

We have shown previously that under specific conditions, a TATA box will mediate efficient in vitro transcription by RNA polymerase (pol) III in the absence of a PSE or other promoter elements. The reaction requires a HeLa cell phosphocellulose protein fraction, fraction B, which must be preincubated with the template DNA. Fraction B does not contain any detectable pol II type transcription factor IID (TFIID) activity. In this report, the relationship between fraction B and TFIID was further examined. Purified human TATA-binding protein (TBP) can substitute for fraction B to mediate TATA-dependent pol III transcription. Both TBP and fraction B prefer a reverse TATA box for pol III transcription, yet TBP bound to a reverse TATA box functions poorly for pol II transcription. Like TFIID, fraction B forms a template-committed complex with TATA-containing promoters. TBP, however, will not template commit for pol III transcription unless premixed with phosphocellulose fraction C. TBP-mediated pol III transcription is also more sensitive to the detergent Sarkosyl (N-lauroylsarcosine, Sigma) than is the fraction B reaction unless it is premixed with fraction C. Together, the data suggest that TBP can complex with a component of fraction C, and this complex is then functionally equivalent to fraction B. We propose that fraction B contains TBP in a complex with some other component(s) of the pol III transcription machinery and that this B complex TBP may be specific for pol III transcription.

Base Sequence↗

PubMed1997

Cross-competition for TATA-binding protein between TATA boxes of the selenocysteine tRNA[Ser]Sec promoter and RNA polymerase II promoters.

In this study we show that the TATA-binding protein (TBP) interacts with the selenocysteine tRNA[Ser]Sec TATA element in a fashion analogous to the TBP-TATA interaction in RNA polymerase (Pol) II-transcribed genes even though the gene is transcribed by Pol III. Recombinant TBPs expressed in Escherichia coli bound to the tRNA[Ser]Sec TATA element. A factor was detected in Xenopus oocyte extracts which contain TBP and bind to the TATA boxes of the tRNA[Ser]Sec gene and various class II genes. Transcription of the microinjected tRNA[Ser]Sec gene was inhibited in Xenopus oocytes by coinjection with the TATA box of the adenovirus major late promoter (AdMLP). Transcription of a 5S gene was not affected under these conditions. These results suggest that the tRNA[Ser]Sec gene recruits TBP in a manner similar to that of TATA-dependent Pol II-transcribed genes and differently from that of Pol III-transcribed genes lacking a TATA box.

Animals↗

PubMed2004

Behavioral disorder, dementia, ataxia, and rigidity in a large family with TATA box-binding protein mutation.

BACKGROUND: Spinocerebellar ataxia type 17 is an autosomal dominant cerebellar ataxia caused by a CAG repeat expansion in the TATA box-binding protein gene. Ataxia is typically the first sign whereas behavioral symptoms occur later. OBJECTIVE: To characterize the unusual phenotypic expression of a large spinocerebellar ataxia type 17 kindred. DESIGN: Clinical, neuropathological, and molecular genetic characterization of a 4-generation family with 16 affected patients. RESULTS: Behavioral symptoms and frontal impairment dominated the early stages preceding ataxia, rigidity, and dystonic movements. Neuropathological examination showed cortical, subcortical, and cerebellar atrophy. Purkinje cell loss and gliosis, pseudohypertrophic degeneration of the inferior olive, marked neuronal loss and gliosis in the caudate nucleus, and in the medial thalamic nuclei were salient features together with neuronal intranuclear inclusions stained with anti-TATA box-binding protein and antipolyglutamine antibodies. The disease was caused by a stable 52 CAG repeat expansion of the TATA box-binding protein gene, although there was apparent variability in the age of onset. CONCLUSION: The characteristics of this family broaden the clinical picture of spinocerebellar ataxia type 17: initial presenile dementia with behavioral symptoms should be added to ataxia, rigidity, and dystonic movements, which are more commonly encountered.

Adult↗

PubMed1999

An ascidian T-box gene As-T2 is related to the Tbx6 subfamily and is associated with embryonic muscle cell differentiation.

The T-box genes, including Brachyury, encode a novel family of transcription factors that play critical roles in various processes of development, in particular, mesoderm formation in chordate embryos. In the case of the ascidian Halocynthia roretzi, the Brachyury (As-T) is expressed exclusively in notochord cells, whereas another T-box gene (As-T2) is expressed in the muscle cells and in the tip of the tail of tailbud embryos. In a previous study, we suggested that the combined pattern of the spatial expressions of As-T and As-T2 appears to correspond to that of a single vertebrate Brachyury gene (Yasuo et al., Dev Biol 1996;180:773-779). The present molecular phylogenetic analysis suggested that As-T2 is a divergent member of the T-box family with a similarity to the Tbx6 subfamily. Vertebrate members of this subfamily are expressed in the posterior paraxial mesoderm, and the mouse Tbx6 is essential for the specification of posterior somite. To investigate the function of As-T2, we examined an ectopic- and/or overexpression of this gene by injecting synthetic mRNA into fertilized eggs. The results showed that the injection of As-T2 mRNA induced an ectopic expression of muscle-specific myosin heavy-chain gene and actin gene, especially in presumptive epidermal cells. This ectopic muscle-specific expression was accompanied by the partial suppression of an epidermis-specific gene expression. The overexpression of As-T2, however, rarely affected the expression of As-T (Brachyury) and genes that are expressed in the tailbud.

Amino Acid Sequence↗

PubMed1992

The TATA box promoter region of maize Adh1 affects its organ-specific expression.

We have isolated two lineage-related Mutator (Mu3) transposon-induced Adh1 promoter mutants in maize: Adh1-3F1124 carries a duplicated TATA box and its revertant, Adh1-3F1124r17, bears a deleted TATA box. Both alterations lead to unique patterns of organ-specific ADH1 enzyme expression. Enzyme activity in Adh1-3F1124 sporophytic organs (scutellum and roots) is greatly reduced, while activity levels remain normal in the male gametophyte (pollen). Conversely, enzyme activity in Adh1-3F1124r17 roots and scutellum is partially restored, but is concomitantly reduced in pollen. Transcript analysis suggests (i) that the TATA box region of the Adh1 gene influences post-transcriptional processes in the male gametophyte but not in roots and (ii) that organ-specific transcription signals in the promoter are distinct from the previously identified anaerobic environment-specific cis-acting transcription signals. Different organs appear to provide surrogate TATA function in different ways, leading to organ-specific differences in the length of the Adh1 message 5' leader.

Alcohol Dehydrogenase↗

PubMed2002

Microarray analysis of E-box binding-related gene expression in young and replicatively senescent human fibroblasts.

An E-box (CACGTG) designer microarray was developed to monitor a group of genes whose expressions share a particular regulatory mode. Sensitivity and specificity of microarray hybridization, as well as variability of microarray data, were evaluated. This designer microarray was used to generate expression profiles of E-box binding-related genes in WI-38 fibroblast cultures at three different growth states: low-passage replicating, low-passage contact-inhibited quiescent, and replicatively senescent. Microarray gene screening reveals that quiescent and senescent cells, in comparison with replicating ones, are characterized by downregulation of Pam, a protein associated with c-Myc, and upregulation of Mad family genes, Max dimerization proteins. Moreover, quiescence and senescence can be distinguished by increased expression of Irlb, c-Myc transcription factor, and Miz-1, c-Myc-interacting Zn finger protein 1, only in the former state. Senescence is characterized by downregulation of Id4, inhibitor of DNA binding 4, and Mitf, microphthalmia-associated transcription factor, in comparison with young replicating and quiescent states. Differential expression of genes detected by microarray hybridization was independently confirmed by reverse transcription polymerase chain reaction technique. Alterations in the expression of E-box-binding transcription factors and c-Myc-binding proteins demonstrate the importance of these genes in establishing the contact-inhibited quiescent or senescent phenotypes.

Cell Division↗

PubMed1997

alpha B-crystallin TATA sequence mutations: lens-preference for the proximal TATA box and the distal TATA-like sequence in transgenic mice.

The mouse alpha B-crystallin promoter is active in lens (preferentially), heart and skeletal muscle, and contains a proximal (-28/-22) and distal (-76/-69) TATA sequence. The present investigation explores by site-specific mutagenesis of alpha B-crystallin promoter-chloramphenicol acetyltransferase (cat) reporter gene constructs the function of these two potential TATA boxes in transfected lens cells and transgenic mice. Unexpectedly, mutagenesis of either or both TATA sequences had no effect on promoter activity in transfected lens cells. By contrast, in transgenic mice mutagenesis of the proximal, distal or both TATA sequences preferentially reduced promoter activity in the lens, with minimal effect in the heart or muscle. 5' RACE analysis of lens and muscle RNA of transgenic mice showed that elimination of the proximal TATA box led to transcription initiation at position -48. This upstream initiation site was apparently not due to the utilization of the distal TATA sequence, since the transgene carrying mutations in both TATA sequences also initiated at position -48. The preferential function of the distal TATA sequence in the lens is probably due to the binding of a transcription factor unrelated to transcription initiation, while the preferential lens function of the proximal TATA box appears to involve transcription initiation.

Animals↗