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PubMed1996

Topology and reorganization of a human TFIID-promoter complex.

Transcription factor TFIID is a multiprotein complex composed of a TATA-box-binding subunit, TBP, and several tightly associated factors (TAFs). Human TFIID-promoter interactions are restricted to the TATA-box region on most core promoters but extend over a large promoter region downstream of the TATA box and the transcription start site on the Ad2ML promoter. TFIID downstream interactions are thought to be functionally relevant because they can be induced by transcriptional activators, which in some cases requires TFIIA, result in stabilization of the TFIID-promoter complex, and correlate with increased recruitment of the remaining general transcription factors. Here we examine the topological organization of human TFIID complexes bound to the Ad2ML promoter. Our data provide insight into the relative disposition of DNA and several TFIID subunits, as well as evidence for DNA wrapping by TFIID, and suggest a direct role of TFIIA in the stable positioning of promoter DNA relative to TAFs.

Crystallography, X-Ray↗
PubMed2001

MOT1-catalyzed TBP-DNA disruption: uncoupling DNA conformational change and role of upstream DNA.

SNF2/SWI2-related ATPases employ ATP hydrolysis to disrupt protein-DNA interactions, but how ATP hydrolysis is coupled to disruption is not understood. Here we examine the mechanism of action of MOT1, a yeast SNF2/SWI2-related ATPase that uses ATP hydrolysis to remove TATA binding protein (TBP) from DNA. MOT1 function requires a 17 bp DNA 'handle' upstream of the TATA box, which must be double stranded. Remarkably, MOT1-catalyzed disruption of TBP-DNA does not appear to require DNA strand separation, DNA bending or twisting of the DNA helix. Thus, TBP-DNA disruption is accomplished in a reaction apparently not driven by a change in DNA structure. MOT1 action is supported by DNA templates in which the handle is connected to the TATA box via single-stranded DNA, indicating that the upstream duplex DNA can be conformationally uncoupled from the TATA box. Combining these results with proposed similarities between SNF2/SWI2 ATPases and helicases, we suggest that MOT1 uses ATP hydrolysis to translocate along the handle and thereby disrupt interactions between TBP and DNA.

Adenosine Triphosphatases↗
PubMed1995

The far-upstream enhancer of the carbamoyl-phosphate synthetase I gene is responsible for the tissue specificity and hormone inducibility of its expression.

The role of the proximal promoter and the far-upstream enhancer in the hepatocyte-specific and hormonal regulation of the carbamoyl-phosphate synthetase I (CPS) gene was investigated in transient transfection assays using primary rat hepatocytes, hepatoma cells, and fibroblasts. These experiments revealed that the activity of the promoter is comparable in all cells tested and is, therefore, not responsible for tissue-specific expression. The 5'-untranslated region of the mRNA is a major, non-tissue specific stimulator of expression in FTO-2B hepatoma cells, acting at the post-transcriptional level. A 469-base pair DNA fragment, 6 kilobase pairs upstream of the transcription start-site in the CPS gene, confers strong hormone-dependent tissue specific expression, both in combination with the CPS promoter and a minimized viral thymidine kinase promoter. Sequences similar to a cyclic AMP-responsive element and a glucocorticosteroid-responsive element were found in the isolated enhancer. Substitutional mutations in these sites strongly affected hormone-induced expression. Analysis of the interaction between the enhancer and parts of the CPS promoter revealed that, in addition to the TATA box, the GAG box, a motif similar to the GC box near the TATA motif, is instrumental in conferring the enhancer activity.

Animals↗
PubMed2004

Deciphering the contribution of known cis-elements in the mouse cone arrestin gene to its cone-specific expression.

PURPOSE: Cone arrestin (CAR) is highly expressed in all cone photoreceptors of the retina and a subset of pinealocytes in the pineal gland. This study was initiated to examine the cis-elements responsible for the cell-specific expression pattern of CAR. METHODS: Mutagenesis and specific deletions of known cis-elements in the proximal promoter of the mouse CAR (mCAR) gene were introduced and analyzed in vitro and in vivo. A series of mCAR promoter-luciferase reporter constructs were transiently transfected into COS-7 or Weri-Rb-1 retinoblastoma cells and tested in in vitro promoter assays. Transgenic Xenopus laevis were created with deletional or mutated promoter fragments driving an enhanced green fluorescent protein (EGFP) reporter gene. The resultant EGFP expression pattern in the transgenic animals was analyzed by fluorescence microscopy and immunocytochemistry. RESULTS: A significant decrease in in vitro transcriptional activities was observed when the minimal 215-bp promoter fragment was mutated in each of the four cone-rod homeobox (CRX)-binding elements (CBEs) or in either of the two TATA-elements. The 215-bp mCAR proximal promoter drove EGFP expression to cone photoreceptors and pinealocytes in transgenic Xenopus laevis; however, the truncated 147-bp fragment drove expression to both cone and rod photoreceptors. Transgenic tadpoles carrying a single mutation in either the TATA-box, the TATA-element or the proximal CBE had undetectable EGFP expression in the retina. However, when one of the other three CBEs was mutated, EGFP expression was observed in muscle and brain tissues, in addition to the eyes. Also, when both TATA elements were mutated, transgenic animals had EGFP expression in all photoreceptors. Because no reporter activity was observed when either a 3.2-kb 5' extended region or the first intron of the mCAR gene was tested in Weri-Rb-1 cells, neither construct was examined in vivo. CONCLUSIONS: The data demonstrate that the regulatory functions of the known cis-elements in the mCAR promoter are highly dependent on location and nucleotide sequence conservation. The TATA elements and CBEs are crucial for driving both basal transcriptional activity and tissue specificity to cone photoreceptors and pinealocytes.

Animals↗
PubMed1997

Curvature and sequence analysis of eukaryotic promoters.

We have calculated the curvature of 504 eukaryotic promoters predicted by the bent A-tract model of Bolshoy et al. (Proc. Natl. Acad. Sci. USA, 88(6), pp. 2312-16) and the bent non-A-tract models of Calladine et al. (J. Mol. Biol., 201, pp. 127-37) and Satchwell et al. (J. Mol. Biol., 191, pp. 659-75) and found in each case a correlation between TBP binding sites and DNA curvature. Characterizing the TBP binding sites revealed that in addition to the classical TATA box (TATAAA) five more elements occur significantly often in the promoters, nearly all of them being one point mutations of the classical TATA box element. Separate curvature calculations for promoters with canonical and non-canonical TATA boxes have shown that in both cases the strong curvature of the helix axes in the domain of the binding sites is maintained (classical TBP binding sites: + 64-135%, non-classical TBP binding sites: + 27-49%). These results support the proposition that beside DNA flexibility and DNA-protein interactions intrinsic curvature of DNA is one further important criterion for the recognition of different DNA elements by TBP.

Animals↗
PubMed1996

The TATA motif is a target for efficient transcriptional activation and nerve growth factor induction of the peripherin gene.

Three proximal elements, PER1, PER2, and PER3, have been implicated in the regulation of peripherin gene expression. PER1 contains the TATA motif and was identified as the principal mediator of neuronal specificity. Here, we demonstrate by transfection of constructs mutated in PER1 that the in vitro protein binding activity of PER1 is irrelevant to its function. However, mutations or substitutions in the TATA box decreased promoter activity by up to 80%. We have investigated this unusual preference for a particular TATA sequence in PC12 cells. In these cells, nerve growth factor induces neuronal differentiation, increasing peripherin gene expression 3-4-fold, while dexamethasone elicits chromaffin differentiation and a 3-fold decrease in peripherin mRNA. Experiments with stably transfected PC12 cells revealed that the specific TATA box of the peripherin gene was crucial for nerve growth factor response. However, it did not affect dexamethasone down-regulation. Therefore, nerve growth factor acts through an element essential for neuronal peripherin gene expression. The results predict that proteins interacting in the vicinity of the TATA box, by inference factors associated with the preinitiation complex, are important for peripherin gene regulation and provide new insights into the mechanisms underlying neuronal differentiation.

Animals↗
PubMed1993

DNA melting on yeast RNA polymerase II promoters.

Transcription-dependent DNA melting on the yeast GAL1 and GAL10 promoters was found to be more closely correlated with the TATA box than the transcription start site. On both these genes, melting begins about 20 base pairs downstream of the TATA box. Physical and genetic analyses suggest that RNA polymerase II associates with this region. Thus, the distance between promoter melting and the TATA box in yeast may be similar to that in higher eukaryotes, even though transcription initiates in a region about 10 to 90 base pairs farther downstream in yeast.

Base Sequence↗
PubMed1995

Evidence for functional binding and stable sliding of the TATA binding protein on nonspecific DNA.

The TATA binding protein (TBP) is required at RNA polymerase I, II, and III promoters that either contain or lack a TATA box. In an effort to understand how TBP might function at such a wide variety of promoters, we have investigated the specific and nonspecific DNA binding properties of human TBP. We show that TBP has less than a 10(3)-fold preference for binding a TATA box (TATAAAAG) than for an average nonspecific site. In contrast to TBP, which binds to the minor groove of DNA, major groove binding proteins typically display binding specificities in the range of 10(6). Once TBP is bound to DNA, whether it be a TATA box or nonspecific DNA, binding is quite stable with a t1/2 of dissociation in the range of 20-60 min for a 300-base pair DNA fragment. In this binding state, TBP appears to be capable of stable one-dimensional sliding along the DNA. Sequence-specific binding can be accounted for, in part, by different rates of sliding. Additional findings demonstrate that specific and nonspecific DNA impart upon TBP an enormous and equivalent degree of thermal stability, suggesting that the TBP-DNA interface on non-specific DNA is not radically different from that on TATA. Consistent with this notion, we find that nonspecifically bound TBP is competent in establishing pol II transcription complexes on DNA. Together, these finding provide a plausible mechanistic explanation for the ability of TBP to function at TATA-containing and TATA-less promoters.

Base Sequence↗
PubMed1990

Activity of herpes simplex virus type 1 latency-associated transcript (LAT) promoter in neuron-derived cells: evidence for neuron specificity and for a large LAT transcript.

By using chloramphenicol acetyltransferase (CAT) assays in neuron-derived cell lines, we show here that promoter activity associated with the herpes simplex virus type 1 latency-associated transcript (LAT) had neuronal specificity. Promoter activity in these transient CAT assays coincided with a DNA region containing excellent RNA polymerase II promoter consensus sequences. Primer extension analysis in a LAT promoter-CAT plasmid construct placed the start of transcription about 28 nucleotides from the first T in the consensus TATA box sequence. Neuronal specificity of this promoter was suggested by examining the effect of sequences upstream of the promoter on CAT activity in neuronal versus nonneuronal cells. In nonneuronal cells, promoter activity was decreased 3- to 12-fold with the addition of upstream sequences. In contrast, in neuron-derived cells, the addition of upstream sequences did not decrease promoter activity. The LAT promoter predicted by our transient CAT assays was located over 660 nucleotides upstream from the 5' end of the previously mapped 2-kilobase (kb) LAT. This unusual location was explained by in situ and Northern (RNA) blot hybridization analyses that suggested that LAT transcription began near the promoter detected in our CAT assays, rather than near the 5' end of the 2-kb LAT. In situ hybridization with neurons from latently infected rabbits detected small amounts of LAT RNA within 30 nucleotides of the consensus TATA box sequence. This suggested that LAT transcription began near this TATA box. Northern blot hybridization of RNA from ganglia of latently infected rabbits revealed a faint 8.3-kb band of the same sense as LAT. We conclude that (i) the LAT promoter has neuronal specificity, (ii) the LAT promoter is located over 660 nucleotides upstream of the 5' end of the previously characterized stable 2-kb LAT, (iii) LAT transcription begins about 28 nucleotides from the first T of the consensus TATA box sequence and extends to near the first available polyadenylation site approximately 8.3 kb away, and (iv) this 8.3-kb RNA may be an unstable precursor of the more stable 2- and 1.3-kb LATs.

Animals↗
PubMed1996

Analysis of human interferon-alpha gene promoters by multiple sequence alignment.

A map of conserved motifs within 11 human interferon-alpha gene promoters was developed using multiple sequence alignment. Multiple sequence alignment revealed the highly conserved structure of the virus-response element and a second region immediately upstream of the tata box. Homolog analysis indicated six families of potential promoter elements: (1) tata boxes, (2) GTATGT motifs, (3) virus-response elements, (4) distal putative interferon regulatory factor binding sites, (5) proximal putative interferon regulatory factor binding sites, and (6) TG sequences. On the basis of conserved promoter elements, a theoretical model of human interferon-alpha gene promoter organization was advanced. Transcriptional regulation of human interferon-alpha gene expression is controlled by a tata box and four regulatory domains. Experimental verification is necessary to confirm the predicted transcriptional control sequences. Proposed experimentation will generate insight into the differential transcriptional regulation of human interferon-alpha gene expression.

Amino Acid Sequence↗
PubMed1984

Identification of sequences involved in the transcriptional control of a Drosophila heat-shock gene.

We have used stable transformation of a cloned Drosophila hsp70 gene into mouse Ltk- cells as an assay system to determine which sequences are involved in the temperature-induced transcription of the gene. We have analyzed the effect of different external deletions on the ability of the gene to be transcribed after temperature elevation. We have also constructed chimeric genes containing different sequences of the hsp70 promoter region joined to the herpes simplex virus thymidine kinase gene, and we have studied the heat-shock inducibility of the thymidine kinase gene after transformation into mouse cells. Our results suggest that the heat-shock genes are under negative control and that their activation is the result of a derepression of the gene. In addition, our experiments indicate that the heat-shock consensus sequence is necessary but not sufficient for temperature-induced transcription, and its role seems to be functionally similar to other transcription signals (i.e. the CAT box) located upstream from the TATA box in several eukaryotic genes. We are proposing that there are two additional regulatory elements besides the consensus sequence which must be present for proper temperature-dependent transcription to occur. Either one of these elements, which are located on opposite sides of the TATA box, may serve at any one time to ensure transcription; that is, only one at a time is needed for proper expression of the heat-shock gene.

Animals↗
PubMed1995

Relationship between TATA-binding protein and herpes simplex virus type 1 ICP4 DNA-binding sites in complex formation and repression of transcription.

The herpes simplex virus (HSV) regulatory protein, infected-cell polypeptide 4 (ICP4), represses the transcription of promoters that have binding sites for ICP4 located near the transcription start site. It also been shown that ICP4 binds such promoter DNA cooperatively with the TATA-binding protein (TBP) and TFIIB to form a tripartite protein-DNA complex (C. Smith, P. Bates, R. Rivera-Gonzales, B. Gu, and N. A. DeLuca, J. Virol. 67:4676-4687, 1993). In this study, we analyzed the effects of position and orientation of the ICP4-binding site relative to the TATA box in the ICP4 promoter on transcriptional repression by ICP4 and on the ability of ICP4 to form tripartite complexes with TBP and TFIIB. The results of theis parallel study provide a strong correlation between tripartite complex formation and repression. Both tripartite-complex formation and transcriptional repression were efficient when the ICP4-binding site was downstream of the TATA box, within a short distance and in proper orientation. In addition, both tripartite-complex formation and repression were partially sensitive to the stereoaxial positioning of the ICP4-binding site relative to the TATA box. As a preliminary characterization of the tripartite complex, circular permutation analysis was performed to assess the distortion of the proximal promoter region in the tripartite complex. As previously reported, both TBP and ICP4 independently could bend DNA and the relative magnitude by which each of these proteins bent DNA in the tripartite complex was preserved. The results of this study suggest that the formation of tripartite complexes on a promoter is part of the mechanism of repression and that simple blocking as a sole result of ICP4 binding is not sufficient for full repression.

Base Sequence↗
PubMed1999

High mobility group protein 1 interacts specifically with the core domain of human TATA box-binding protein and interferes with transcription factor IIB within the pre-initiation complex.

The high mobility group (HMG) box domain has defined a family of proteins, mostly transcription factors, that specifically interacts with DNA on the minor groove and sharply bends it. The founding member of the family, HMG1, does not specifically recognize regular B-DNA but is recruited to DNA by interaction with other transcription factors and TATA box-binding protein (TBP). However, conflicting effects of HMG1 on transcription have been reported. We show that the interaction between HMG1 and TBP is species-specific. This interaction in turn affects the interaction of TBP with transcription factor (TF) IIB and is competed by TFIIA. A primary binding site was mapped to the H2' alpha-helix in the highly conserved core domain of human TBP. On HMG1, the primary binding site was only in the HMG box A, and HMG box A was also sufficient to interact with native TFIID. Both HMG boxes efficiently repressed transcription in vitro as fusions to the Gal4-DNA binding domain. Additionally, HMG box B showed a weak level of activation at very low amounts. These results suggest a general involvement of HMG1 at the early stages of polymerase II transcription that may result in subtle activation or repression of individual genes.

Carrier Proteins↗
PubMed1993

Genomic footprinting of the promoter regions of STE2 and STE3 genes in the yeast Saccharomyces cerevisiae.

Dimethyl sulfate, DNase I and micrococcal nuclease DNA cleavage were combined with the ligation-mediated polymerase chain reaction to obtain high resolution maps of the promoter regions for two cell-type-specific genes: the a-specific STE2 gene and the alpha-specific STE3 gene. We find that MCM1 binds in vivo in a-cells to a 16 bp P-box sequence located in the STE2 UAS. In alpha-cells, the footprint pattern is extended relative to a-cells, consistent with the additional binding of MAT alpha 2 to the sequences flanking each end of the P-box. A nucleosome was found adjacent to the P-box of the transcriptionally repressed a-specific STE2 UAS in alpha-cells, positioned so that the nucleosome overlaps the TATA-box. In contrast, such well-positioned nucleosomes were not found for the transcriptionally active STE2 UAS in a-cells, where instead the TATA box appears to be bound to the general transcription factor TFIID. These observations support the hypothesis that MAT alpha 2 repression of a-specific genes is mediated by nucleosomes, perhaps by exclusion of TFIID from the TATA-box.

Base Sequence↗
PubMed2001

DNA bends in TATA-binding protein-TATA complexes in solution are DNA sequence-dependent.

The TATA-binding protein (TBP) initiates assembly of transcription preinitiation complexes on eukaryotic class II promoters, binding to and restructuring consensus and variant "TATA box" sequences. The sequence dependence of the DNA structure in TBP-TATA complexes has been investigated in solution using fluorescence resonance energy transfer. The mean 5'dye-3'dye distance varies significantly among oligomers bearing the adenovirus major late promoter sequence (AdMLP) and five single-site variants bound to Saccharomyces cerevisiae TBP, consistent with solution bend angles for AdMLP of 76 degrees and for the variants ranging from 30 degrees to 62 degrees. These solution bends contrast sharply with the corresponding co-crystal structures, which show approximately 80 degrees bends for all sequences. Transcription activities for these TATA sequences are strongly correlated with the solution bend angles but not with TBP-DNA binding affinities. Our results support a model in which transcription efficiency derives primarily from the sequence-dependent structure of the TBP-TATA binary complex. Specifically, the distance distribution for the average solution structure of the TBP-TATA complex may reflect the sequence-dependent probability for the complex to assume a conformation in which the TATA box DNA is severely bent. Upon assumption of this geometry, the binary complex becomes a target for binding and correctly orienting the other components of the preinitiation complex.

Base Sequence↗
PubMed2000

TATA-binding protein and the Gal4 transactivator do not bind to promoters cooperatively.

The yeast Gal4 protein, like many activators, binds TATA-binding protein (TBP) directly in vitro. It has been speculated that this protein-protein interaction is important for Gal4p-mediated activation of transcription, but little work has been done to test specific models involving this interaction. In this study, the effect of Gal4p on TBP-TATA binding is addressed. Specifically, it is asked if the Gal4p-TBP interaction can support cooperative binding of the two factors to promoters. It is easy to see how such an event could stimulate transcription, particularly from promoters with a non-consensus TATA box. In vitro, however, a derivative of Gal4p (Gal4-(1-93+768-881)) containing the DNA-binding, dimerization, and activation domains does not bind to promoter DNA cooperatively with either recombinant, purified TBP, or with protein from a yeast crude extract. In vivo, reporter gene experiments using promoters with differing TBP affinities reveal no major Gal4p-mediated stimulation of TBP function from weak TATA boxes, as would be predicted if the proteins bind cooperatively. Furthermore, native Gal4p and a potent Gal4p-based artificial activator lacking a TBP-binding activation domain support similar ratios of transcription from a series of promoters identical except for mutations in the TATA box. It is concluded that Gal4p and TBP do not bind cooperatively to promoters and that this mechanism does not contribute substantially to Gal4p-mediated transcriptional activation.

DNA↗
PubMed1992

Calculated enhancement of open base pair probability downstream of a (TATA)2 box.

The modified self-consistent phonon approximation is generalized to calculate the open base pair probability of a (TATA)2 insert between two semi-infinite poly(dA-dC) . poly(dG-dT) helices. An iterative method based entirely on the Green's function method is developed to compute the open interbase hydrogen bond probability of the insert and near bases versus temperature. The open interbase hydrogen bond probability is calculated for two temperatures and compared with perfect homopolymer open base pair probabilities at room temperature. The probability of opening is enhanced by a factor of two for the major groove bond of the AT pair at the transcriptional downstream end of the (TATA)2 insert. The total base pair opening probability of that pair is enhanced by 54%. The probability of the next inline GC base pair to be open is increased by a factor of ten.

Base Composition↗
PubMed1995

A unique role for enhancers is revealed during early mouse development.

Transcription and replication of genes in mammalian cells always requires a promoter or replication origin, respectively, but the ability of enhancers to stimulate these regulatory elements and the interactions that mediate this stimulation are developmentally acquired. The primary function of enhancers is to prevent repression, which appears to result from particular components of chromatin structure. Factors responsible for this repression are present in the maternal nucleus of oocytes and its descendant, the maternal pronucleus of mouse 1-cell embryos and in mouse 2-cell embryos, but are absent in the paternal pronucleus. Thus, enhancers are not needed to achieve efficient transcription and replication in paternal pronuclei. However, enhancers, even in the presence of their specific activation protein, are inactive prior to formation of a 2-cell embryo, suggesting that a coactivator essential for enhancer function is not available until zygotic gene expression begins. Furthermore, enhancer stimulation of transcription appears to be mediated through a promoter transcription factor, but this interaction can change as cells undergo differentiation, switching from a TATA-box independent to a TATA-box dependent mode.

Animals↗