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No strict alignment is required between a transcriptional activator binding site and the "TATA box" of a yeast gene.

GAL4 is a transcriptional activator of the galactose metabolism genes in the yeast Saccharomyces cerevisiae. We show that GAL4 expressed in yeast activated transcription equally well when a single GAL4 binding site was placed at any of nine positions upstream of the GAL1 (galactokinase gene) "TATA box." We chose a sufficient number of positions for the binding site to ensure that, in several of these positions, GAL4 was on the opposite side of the DNA helix with respect to the TATA box. Smaller GAL4 derivatives were similar to wild-type GAL4 in that they also activated transcription in a manner independent of the side of the DNA helix they bound with respect to the TATA box. Unlike wild-type GAL4, however, these smaller GAL4 derivatives activated transcription better when we placed a binding site progressively closer to the TATA box over a distance of 34 base pairs.

Bacterial Proteins↗

DNA sequences at and between the GC and TATA boxes: potential DNA looping and spatial juxtapositioning of the protein factors.

Regulation of gene expression in eukaryotes involves a complex assembly of DNA recognition sequence elements and their respective protein factors. The upstream promoter/enhancer sequences are position and orientation independent. Despite their variable distances from the TATA box and transcription start site, interaction between the protein activators and TATA general transcription factors takes place, enabling induced levels of transcription initiation. Here the intervening sequences between the GC and TATA boxes are examined as functions of their lengths. Regardless of the substantial differences in the spacer sizes, similar mono and dinucleotide distributions are noted. Purine-purine base pair steps, except for AA, are more frequent at and near the GC box in the 5' ends of the loops than in their 3' ends. Pyrimidine-pyrimidine base pair steps, except for TT behave similarly. AT and TA (as well as AA and TT) are more frequent in the 3' ends of the loops near the TATA. Examination of these distributions, as well as of the sequences composing the GC and TATA boxes indicates that the DNA in the upstream part of the loop is more rigid, whereas the downstream regions are far more flexible. The flexibility of the general TATA region may afford correct spatial juxtapositioning of the proteins with respect to each other, enabling interactions between the activators and the general transcription factors.

Base Composition↗

TATA-box dependent trans-activation of the human HSP70 promoter by Myb proteins.

The oncogene v-myb and its cellular progenitor c-myb encode nuclear, DNA-binding phosphoproteins that function as transcriptional regulators. Previous studies have demonstrated the existence of two distinct transactivation mechanisms for Myb-inducible promoters. One of these mechanisms, exemplified by the chicken mim-1 promoter, involves specific binding of Myb protein to Myb binding sites located upstream of the transcriptional start site. A second mechanism, exemplified by the human HSP70 promoter, does not require the presence of specific Myb binding sites. Here, we have more closely investigated transactivation of the human HSP70 promoter by Myb proteins. Mutational analysis of the human HSP70 promoter failed to show a correlation between Myb-inducibility and the presence of a particular upstream transcription factor binding site, but instead showed that Myb-inducibility was dependent on the presence of a particular type of TATA-box. This suggests that the TATA-box is involved in transactivation by v-Myb and that only a subset of TATA-boxes confers Myb-inducibility. Activation of the HSP70 promoter is dependent on the presence of most of the carboxy terminal amino acid sequences of v-Myb, including the previously identified acidic transactivation domain and a putative leucine-zipper region. Finally, we show that the HSP70 promoter is activated in a Myb binding site independent fashion not only by v-Myb, but also by chicken c-Myb and chicken B-Myb. These observations raise the possibility that the ability to transactivate in a Myb binding-site independent manner is a common property among Myb proteins. Since chicken B-Myb has been shown previously to be unable to transactivate a Myb binding-site containing promoter, such as the mim-1 promoter (Foos et al., 1992), our results also show that both modes of transactivation are independent of each other.

Animals↗

Methylation status of a single CpG locus 3 bases upstream of TATA-box of receptor activator of nuclear factor-kappaB ligand (RANKL) gene promoter modulates cell- and tissue-specific RANKL expression and osteoclastogenesis.

Receptor activator of nuclear factor-kappaB ligand (RANKL) expression is tissue specific and limited to certain subsets of T-lymphocytes and stromal/osteoblastic cells. Even among osteoblasts, RANKL is expressed on about 20% of osteoblasts of the normal mouse. To clarify the mechanism of population-specific RANKL expression, we analyzed the effect of CpG methylation on its transcription, mRNA and protein expression as well as on osteoclastogenesis. Subpopulations of ST2 cells were used: P9, which expresses RANKL and supports osteoclastogenesis, and P16, which does not. By sodium bisulfite mapping, the rate of CpG methylation of the -65/+350 region, especially of CpG locus no. 1 three bases upstream of the TATA-box, was higher in P16 than in P9 ST2 cells. ChIP and gel shift assay showed that methylated CpG locus no. 1 was a target of MeCP2 binding that, in turn, blocked the binding of the TATA-box binding protein to the TATA-box. In vitro methylation by SssI of the promoter construct reduced its transcriptional activity at the steady state and its response to 1alpha,25(OH)2 vitamin D3. Conversely, treatment with DNA methylase inhibitor, 5-aza-2'-deoxycytidine, significantly restored RANKL expression and osteoclastogenesis in P16 cells. Except for primary cultured osteoblasts, CpG locus no. 1 was frequently methylated in various normal mouse tissues. We propose that the methylation status of the CpG locus three bases upstream of the TATA-box modulates the control of cell- and tissue-specific expression of RANKL gene and osteoclastogenesis. The heterogeneity of stromal/ osteoblastic cells in response to bone-resorbing stimuli may be attributed, in part, to the methylation status of the RANKL gene promoter.

Animals↗

Conformational properties of the TATA-box binding sequence of DNA.

Nanosecond scale molecular dynamics simulations in water demonstrate that the DNA oligomer, GCGTATATAAAACGC, which contains a target site for the TATA-box binding protein (TBP), has an intrinsic preference to adopt an A-like conformation in the region of the TATA-box and undergoes bending related to that seen within in the TBP complex. This result is obtained from two independent simulations using different starting structures. In line with earlier suggestions of Guzikevich-Guerstein and Shakked, these simulations imply that an A-DNA conformation may be an important intermediate step in forming the strongly distorted DNA observed within the crystallographically determined complex with TBP. These results also support modeling studies by Lebrun et al. which suggest that the TBP binding mechanism can be broken down into a backbone transition to an A-like form coupled with a mechanical distortion which locally stretches and unwinds the DNA.

Binding Sites↗

A role of the TATA box and the general co-activator hTAF(II)130/135 in promoter-specific trans-activation by simian virus 40 small t antigen.

The small t antigen (st-ag) of simian virus 40 can exert pleiotropic effects on biological processes such as DNA replication, cell cycle progression and gene expression. One possible mode of achieving these effects is through stimulation of NFkappaB-responsive genes encoding growth factors, cytokines, transcription factors and cell cycle regulatory proteins. Indeed, a previous study has shown that st-ag enhanced NFkappaB-mediated transcription. This study demonstrates that promoters possessing a consensus TATA box (i.e. TATAAAAG) in the context of either NFkappaB- or Sp1-binding sites are trans-activated by st-ag. Overexpressing the general transcription factor hTAF(II)130/135, but not hTAF(II)28 or hTAF(II)80, stimulated the activity of promoters in a consensus TATA box-dependent mode. Converting the consensus TATA motif into a non-consensus TATA box strongly impaired activation by st-ag and hTAF(II)130/135. Conversely, mutating a non-consensus TATA motif into the consensus TATA box rendered the mutated promoter inducible by st-ag and hTAF(II)130/135. Mutation of the TATA box had no effect on TNFalpha- or RelA/p65-mediated induction of NFkappaB-responsive promoters, indicating a specific st-ag effect on hTAF(II)130/135. St-ag stimulated the intrinsic transcriptional activity of hTAF(II)130/135. Substitutions in the conserved HPDKGG motif in the N-terminal region or a mutation that impaired the interaction with protein phosphatase 2A abrogated the ability of st-ag to activate hTAF(II)130/135-mediated transcription. These results indicate that trans-activation of promoters by st-ag may depend on a consensus TATA motif and suggest that such promoters recruit the general transcription factor hTAF(II)130/135.

3T3 Cells↗

A synthetic early promoter from a baculovirus: roles of the TATA box and conserved start site CAGT sequence in basal levels of transcription.

Many baculovirus early genes and insect genes transcribed by RNA polymerase II have a conserved transcription start site sequence (CAGT) located downstream of a consensus TATA box. To examine the functions and interactions of these two motifs in initiating accurately positioned basal transcription, a 43-nt synthetic promoter was synthesized from the TATA box and start site sequences of the gp64 early promoter from the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV). The synthetic promoter initiated accurately and was also transactivated by the baculovirus transcriptional activator, IE1. To determine the roles of sequences within the 43-nt synthetic promoter, a series of linker-scanning and spacing mutations were analyzed for transcriptional activity, start site selection, and transactivation. Linker-scanning mutations were examined in vivo by transient expression and reporter gene assays. To examine transcription start site selection, promoter constructs were used for in vitro transcription in nuclear extracts from uninfected Spodoptera frugiperda (Sf9) cells. In vivo and in vitro analyses show that the TATA box, and not the start site CAGT, is the primary element controlling start site selection. Substitution of the conserved start site CAGT sequence resulted in a reduction of both reporter gene activity and in vitro transcripts, although transcripts initiated accurately. Data from linker-scanning and spacing mutations indicate that the conserved start site CAGT sequences are not required for accurate initiation but sequences at the start site play an important role in initiation efficiency.

Baculoviridae↗

Saturation mutagenesis of the TATA box and upstream activator sequence in the haloarchaeal bop gene promoter.

Degenerate oligonucleotides were used to randomize 21 bp of the 53-bp minimal bop promoter in three 7-bp segments, including the putative TATA box and the upstream activator sequence (UAS). The mutagenized bop promoter and the wild-type structural gene and transcriptional terminator were inserted into a shuttle plasmid capable of replication in the halophilic archaeon Halobacterium sp. strain S9. Active promoters were isolated by screening transformants of an orange (Pum- bop) Halobacterium mutant for purple (Pum+ bop+) colonies on agar plates and analyzed for bop mRNA and/or bacteriorhodopsin content. Sequence analysis yielded the consensus sequence 5'-tyT(T/a)Ta-3', corresponding to the promoter TATA box element 30 to 25 bp 5' of the transcription start site. A putative UAS, 5'-ACCcnactagTTnG-3', located 52 to 39 bp 5' of the transcription start site was found to be conserved in active promoters. This study provides direct evidence for the requirement of the TATA box and UAS for bop promoter activity.

Bacteriorhodopsins↗

The downstream promoter element DPE appears to be as widely used as the TATA box in Drosophila core promoters.

The downstream promoter element (DPE) functions cooperatively with the initiator (Inr) for the binding of TFIID in the transcription of core promoters in the absence of a TATA box. We examined the properties of sequences that can function as a DPE as well as the range of promoters that use the DPE as a core promoter element. By using an in vitro transcription assay, we identified 17 new DPE-dependent promoters and found that all possessed identical spacing between the Inr and DPE. Moreover, mutational analysis indicated that the insertion or deletion of a single nucleotide between the Inr and DPE causes a reduction in transcriptional activity and TFIID binding. To explore the range of sequences that can function as a DPE, we constructed and analyzed randomized promoter libraries. These experiments yielded the DPE functional range set, which represents sequences that contribute to or are compatible with DPE function. We then analyzed the DPE functional range set in conjunction with a Drosophila core promoter database that we compiled from 205 promoters with accurately mapped start sites. Somewhat surprisingly, the DPE sequence motif is as common as the TATA box in Drosophila promoters. There is, in addition, a striking adherence of Inr sequences to the Inr consensus in DPE-containing promoters relative to DPE-less promoters. Furthermore, statistical and biochemical analyses indicated that a G nucleotide between the Inr and DPE contributes to transcription from DPE-containing promoters. Thus, these data reveal that the DPE exhibits a strict spacing requirement yet some sequence flexibility and appears to be as widely used as the TATA box in Drosophila.

ATP-Binding Cassette Transporters↗

Cell-type specificity of human CYP11A1 TATA box.

Expression of the CYP11A1 (SCC) genes, which encode the enzyme important for the first step of steroid biosynthesis, occurs in the adrenal gland and gonads, and is stimulated by cAMP. Transfection of serial deletions of the SCC promoter, which drives reporter gene expression, showed that a minimal promoter containing only the TATA box could direct cAMP-dependent transcription. Transcription factor SF1, which binds to a site next to the TATA box, can stimulate basal transcription but not cAMP response, either in adrenal cell lines or in COS-1 co-transfected with the SF1 expression plasmid. These data lead to the conclusion that the minimal promoter containing only the TATA box can drive cell type-specific, cAMP-dependent transcription. Additional experiments replacing the TATA sequence of SCC with other TATA sequences suggested that the TATA sequence itself is important for this cAMP-dependent transcription.

Animals↗

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↗

The TATA-box binding protein of Entamoeba histolytica: cloning of the gene and location of the protein by immunofluorescence and confocal microscopy.

A 309 bp DNA fragment from Entamoeba histolytica was amplified by PCR using primers derived from the Acanthamoeba castellanii consensus TATA-box binding protein amino acid sequence. The amplified fragment was used to isolate cDNA and genomic DNA clones containing an ORF encoding the complete E. histolytica TATA-box binding protein (Ehtbp, 702 bp, 234 aa, molecular mass 26 kDa). The EhTBP functional domain showed 55% sequence identity to that of Homo sapiens, 54% to A. castellanii and 37% to Plasmodium falciparum TBPs. In Southern blot experiments we detected a single Ehtbp band, which was transcribed as a 1.3 kb mRNA containing a 420 nt 5' untranslated region. However, the probe hybridized with the 0.8 and 1.5 Mb chromosomes, suggesting that this sequence is diploid. In situ PCR assays showed two signals in 95% of trophozoites, one located in the nucleus and another in EhkO, the novel DNA-containing organelle recently reported. The recombinant E. histolytica TATA-box binding protein was expressed in Escherichia coli. Antibodies against it recognized two proteins of 26 and 29 kDa in E. histolytica nuclear extracts. Confocal microscopy immunofluorescence analysis located the protein in both the nucleus and EhkO.

Amino Acid Sequence↗

Factors responsible for the higher transcriptional activity of extracts of adenovirus-infected cells fractionate with the TATA box transcription factor.

Extracts of adenovirus-infected HeLa cells have 5- to 10-fold-higher activity for transcription from the major late promoter in vitro than do extracts of mock-infected or E1A mutant-infected cells (K. Leong and A. J. Berk, Proc. Natl. Acad. Sci. USA 83:5844-5848, 1986). In this study, we analyzed extracts from mock-infected cells and from cells infected with an E1A mutant, pm975, which expresses principally the large E1A protein responsible for the stimulation of transcription. These extracts were fractionated by phosphocellulose chromatography, a procedure which separates factors required for transcription from this promoter (J. D. Dignam, B. S. Shastry, and R. G. Roeder, Methods Enzymol. 101:582-589, 1983), allowing the quantitative assay of individual factors (M. Samuels, A. Fire, and P. A. Sharp, J. Biol. Chem. 257:14419-14427, 1982). Fractions eluted with 0.04, 0.35, and 0.6 M KCl, which contained RNA polymerase II, the upstream factor MLTF, and three general polymerase II transcription factors, had similar activities when prepared from virus-infected or from mock-infected cells. The sequence-specific DNA-binding activity of MLTF was also similar in the virus-infected- and mock-infected-cell extracts. In contrast, the 1.0 M KCl fraction prepared from virus-infected cells consistently exhibited activity severalfold higher than that of the equivalent fraction prepared in parallel from mock-infected cells. E1A protein eluted principally (greater than 80%) in the 0.35 M KCl fraction. Results of others (M. Sawadogo and R. G. Roeder, Cell 43:165-175, 1985) have shown that the 1.0 M KCl fraction, containing 2 to 5% of the unfractionated protein extract, contains a factor which binds specifically to the major late promoter TATA box. These results, together with a recent genetic analysis of the E1B promoter which demonstrated that the TATA box was required for its efficient transcriptional activation (transactivation) by E1A (L. Wu, D. S. E. Rosser, M. Schmidt, and A. J. Berk, Nature (London) 326:512-515, 1987), are consistent with the model that E1A protein indirectly activates the TATA box transcription factor. Consistent with this model was the finding that mutants of the major late promoter containing only the TATA box and cap site region were transcribed at higher rates with extracts from virus-infected cells than with extracts from mock-infected cells. Other models consistent with the results are also discussed.

Adenovirus Early Proteins↗

Precise nucleosome positioning and the TATA box dictate requirements for the histone H4 tail and the bromodomain factor Bdf1.

Acetylation of histone tails plays a key role in chromatin dynamics and is associated with the potential for gene expression. We show here that a 2-3 bp mispositioning of the nucleosome covering the TATA box at PHO5 induces a dependency on the acetylatable lysine residues of the histone H4 N-terminal region and on the TFIID-associated bromodomain factor Bdf1. This dependency arises either through fusion of the PHO5 promoter to a lacZ reporter or by mutation of the TATA box in the natural gene. The results suggest that promoters in which the TATA box is either absent or poorly accessible on the surface of a nucleosome may compensate by using Bdf1 bromodomains and acetylated H4 tails to anchor TFIID to the promoter during the initial stages of transcription activation. We propose that nucleosome positioning at the nucleotide level provides a subtle, but highly effective, mechanism for gene regulation.

Acetylation↗

The TATA-box-binding protein (TBP) of Halobacterium salinarum. Cloning of the tbp gene, heterologous production of TBP and folding of TBP into a native conformation.

The TATA-box binding protein (TBP) is a basal transcription factor involved in transcription initiation in Eucarya and Archaea. Using a tbp-specific probe, a 4.5-kbp genomic fragment from Halobacterium salinarum was cloned and sequenced. It contained the tbp gene and the 5'-ends of two additional open reading frames, but surprisingly, 70% of the cloned fragment (3.2 kbp) was devoid of coding capacity or similarity to database sequences. The deduced halobacterial TBP exhibits sequence similarities to other archaeal (41-43%) as well as to eucaryal (27-38%) TBP. A comparative analysis showed that the archaeal and eucaryal TBP form two related monophylic protein families, and the archaeal TBP possess features which separate them from eucaryal TBP. Compared with the other TBP, the halobacterial TBP is unique in having a high excess of negatively charged residues. A histidine-tagged version of the halobacterial TBP was produced in Escherichia coli in a denatured conformation and purified by means of Ni-chelating chromatography. CD spectroscopy was used to monitor TBP secondary structure and the conditions necessary for folding it into a native conformation. In the absence of denaturating agents, the folded as well as the unfolded state were found to be stable over a wide range of salt concentrations. Properly folded TBP was shown to bind to a halobacterial TATA-box-containing DNA fragment, indicating that the fusion protein can be used to characterize DNA recognition by the halobacterial TBP.

Amino Acid Sequence↗

Insensitivity of the present hsp26 chromatin structure to a TATA box mutation in Drosophila.

The role of the TATA element in establishing the chromatin structure and inducible transcription of the Drosophila melanogaster hsp26 gene has been analyzed. An hsp26/lacZ fusion gene with a mutant promoter, in which the TATA box sequence TATAAA was changed to CCCAAA, was introduced into Drosophila by P-element transformation. The mutation had little effect on formation of the preset chromatin structure observed prior to induction. However, the mutation dramatically reduced transcription levels following heat shock. Northern analysis indicated that weak, inducible expression of the mutant promoter occurred within the same period of heat shock as for the normal promoter, suggesting that TFIID was associated with the mutant promoter prior to heat shock. Biochemical analysis showed that the mutant promoter still bound TFIID in vitro, but with 3-5-fold less affinity than the normal promoter. DNase I footprinting revealed that the conformation of the TFIID-DNA complex differed significantly from that of the normal promoter. These results indicate that alterations in the conformation or the stability of the TFIID-DNA complex drastically reduce the level of induction, but do not dramatically affect chromatin structure formation. Formation of the requisite chromatin structure is either independent of, or highly tolerant of, changes in the TFIID-DNA complex.

Animals↗

Topological effects of the TATA box binding protein on minicircle DNA and a possible thermodynamic linkage to chromatin remodeling.

DNA ring closure experiments on short restriction fragments ( approximately 160 bp) bound by the TATA box binding protein (TBP) have demonstrated the formation of negative topoisomers, consistent with crystallographically observed TBP-induced DNA untwisting but in contrast to most previous results on topological effects in plasmid DNA. The difference may be due to the high free energy cost of substantial writhe in minicircles. A speculative mechanism for the loss of TBP-induced writhe suggests that TBP is capable of inducing DeltaTw between 0 and -0.3 in minicircles, via loss of out-of-plane bending upon retraction of intercalating Phe stirrups, and that TBP can thus act as a "supercoil shock absorber". The proposed biological relevance of these observations is that they may model the behavior of DNA in constrained chromatin environments. Irrespective of the detailed mechanism of TBP-induced supercoiling, its existence suggests that chromatin remodeling and enhanced TBP binding are thermodynamically linked. Remodeling ATPases or histone acetylases release some of the negative supercoiling previously restrained by the nucleosome. When TBP takes up the supercoiling, its binding should be enhanced transiently until the unrestrained supercoiling is removed by diffusion or topoisomerases. The effect is predicted to be independent of local remodeling-induced changes in TATA box accessibility.

Chromatin↗

The exchange of cognate TATA boxes results in a corresponding change in the strength of two HSV-1 early promoters.

Previous analysis of two Herpes Simplex Virus Type-1 (HSV-1) promoters controlling expression of mRNA encoding early genes (U(L)37 and U(L)50) showed that the U(L)50 (dUTPase) promoter is at least 6-fold stronger both in its normal genomic location and in the non-essential gC locus. In the present report we demonstrate that the TATA element of either promoter is the major determinant of promoter strength. When the U(L)37 TATA element (CGTATAAC) was mutated with two base changes to the U(L)50 TATA sequence (CATAAAAC) in recombinant viruses, the activity of the U(L)37 promoter was increased to that of the U(L)50 promoter. Conversely, when the U(L)50 TATA element was changed to that of the U(L)37 promoter, U(L)50 promoter activity was reduced to the level of the U(L)37 promoter. In addition, we investigated the spacing of the TATA box with respect to upstream promoter elements. We found that re-positioning the U(L)37 TATA box to a location equivalent to that of the U(L)50 promoter relative to the transcript start site; i.e. three bases upstream of its cognate location, significantly diminished activity. Substitution of the U(L)50 TATA box at the new position could only partially restore promoter activity. Thus, we also conclude that the spacing of TATA elements vis-à-vis upstream promoter elements is also a critical determinant of promoter strength.

Animals↗