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Query: UNIPROT:P06889 (Mol)
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Interaction of cloned yeast, drosophila, and human transcription factor IID (yTFIID, dTFIID, and hTFIID, respectively) with the adenovirus 2 major late promoter (Ad2 MLP) confers a more limited pattern of DNase I protection than that obtained using highly purified native hTFIID (Hahn, S., Buratowski, S., Sharp, P. A. and Guarente, L. (1989) EMBO J. 8, 3379-3382; Van Dyke, M. W., and Sawadogo, M. (1990) Mol. Cell. Biol. 10, 3415-3420; Horikoshi, M., Wang, C.K., Fujii, H., Cromlish, J.A., Weil, P.A., and Roeder, R.G. (1989) Nature 341, 299-303; Peterson, M. G., Tanese, N., Pugh, B.F., and Tjian, R. (1990) Science 248, 1625-1630; Hoey, T., Dynlacht, B. D., Peterson, M.G., Pugh, B.F., and Tjian, R. (1990) Cell 61, 1179-1186). Since the mass of the cloned TFIIDs is considerably less than that of native hTFIID (27-38 kDa versus 120-140 kDa), it is considered likely that native hTFIID exists as a mixed heterodimer. We have recently identified, purified, and characterized a novel transcription factor that binds to the CAP site region (+1 to +23) of the Ad2 MLP. This CAP site binding factor, designated CBF, is required for optimal transcriptional activity. We now show that when bound to the Ad2 MLP, yTFIID and CBF interact to generate the extended pattern of DNase I protection conferred by native hTFIID. In addition, bound yTFIID and CBF interact such that the stability of the complex exceeds that of each factor bound alone. We also demonstrate the existence in nuclear extracts of a hTFIID and CBF heterodimer by the electrophoretic mobility shift analysis. CBF, therefore, may represent the first identified member of a large family of gene-specific TFIID-associated factors that are required for the regulated gene-specific expression of TFIID activity.
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PMID:Interaction of CAP sequence site binding factor and transcription factor IID preceding and following binding to the adenovirus 2 major late promoter. 204 Jun 15

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.
J Mol Biol 1990 Dec 05
PMID:Genomic footprinting of the yeast HSP82 promoter reveals marked distortion of the DNA helix and constitutive occupancy of heat shock and TATA elements. 217 61

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.
Mol Biol Med 1990 Apr
PMID:Anatomy of the rat albumin promoter. 218 62

The existence of separable functions within the human class II general transcription factor TFIID was probed for differential sensitivity to mild proteolytic treatment. Independent of whether TFIID was bound to DNA or free in solution, partial digestion with either one of a variety of nonspecific endoproteases generated a protease-resistant protein product that retained specific DNA recognition, as revealed by DNase I footprinting. However, in contrast to native TFIID, which interacts with the adenovirus major late (ML) promoter over a very broad DNA region, partially proteolyzed TFIID interacted with only a small region of the ML promoter immediately surrounding the TATA sequence. This novel footprint was very similar to that observed with the TATA factor purified from yeast cells. Partially proteolyzed human TFIID could form stable complexes that were resistant to challenge by exogenous templates. It could also nucleate the assembly of transcription complexes on the ML promoter with an efficiency comparable to that of native TFIID, yielding similar levels of transcription initiation. These results suggest a model in which the human TFIID protein is composed of at least two different regions or polypeptides: a protease-resistant "core," which by itself is sufficient for promoter recognition and basal transcriptional levels, and a protease-sensitive "tail," which interacts with downstream promoter regions and may be involved in regulatory processes.
Mol Cell Biol 1990 Jul
PMID:DNA-binding and transcriptional properties of human transcription factor TFIID after mild proteolysis. 219 57

We have analyzed the DNA sequence requirements for TATA element function by assaying the transcriptional activities of 25 promoters, including those representing each of the 18 single-point mutants of the consensus sequence TATAAA, in a reconstituted in vitro system that depends on the TATA element-binding factor TFIID. Interestingly, yeast TFIID and HeLa cell TFIID were virtually identical in terms of their relative activities on this set of promoters. Of the mutated elements, only two had undetectable activity; the rest had activities ranging from 2 to 75% of the activity of the consensus element, which was the most active. In addition, mutations of the nucleotide following the TATAAA core strongly influenced transcriptional activity, although with somewhat different effects on yeast and HeLa TFIID. The activities of all these promoters depended upon TFIID, and the level of TFIID-dependent transcription in vitro correlated strongly with their activities in yeast cells. This suggests that the in vivo activities of these elements reflect their ability to functionally interact with a single TATA-binding factor. However, some elements with similar activities in vitro supported very different levels of transcriptional activation by GAL4 protein in vivo. These results extend the degree of evolutionary conservation between yeast and mammalian TFIID and are useful for predicting the level of TATA element function from the primary sequence.
Mol Cell Biol 1990 Aug
PMID:Yeast and human TATA-binding proteins have nearly identical DNA sequence requirements for transcription in vitro. 219 37

In the gal-his3 hybrid promoter his3-GG1, the yeast upstream activator protein GCN4 stimulates transcription when bound at the position normally occupied by the TATA element. This TATA-independent activation by GCN4 requires two additional elements in the gal enhancer region that are distinct from those involved in normal galactose induction. Both additional elements appear to be functionally distinct from a classical TATA element because they cannot be replaced by the TFIID-binding sequence TATAAA. One of these elements, termed Q, is essential for GCN4-activated transcription and contains the sequence GTCAC CCG, which overlaps (but is distinct from) a GAL4 binding site. Surprisingly, relatively small increases in the distance between Q and the GCN4 binding site significantly reduce the level of transcription. The Q element specifically interacts with a yeast protein (Q-binding protein [QBP]) that may be equivalent to Y, a protein that binds at a sequence that forms a constraint to nucleosome positioning. Analysis of various deletion mutants indicates that the sequence requirements for binding by QBP in vitro are indistinguishable from those necessary for Q activity in vivo, strongly suggesting that QBP is required for the function of this TATA-independent promoter. These results support the view that transcriptional activation can occur by an alternative mechanism in which the TATA-binding factor TFIID either is not required or is not directly bound to DNA. In addition, they suggest a potential role of nucleosome positioning for the activity of a promoter.
Mol Cell Biol 1990 Aug
PMID:A nucleosome-positioning sequence is required for GCN4 to activate transcription in the absence of a TATA element. 219 50

Tc is the proximal promoter element required for constitutive his3 transcription that occurs in the absence of the canonical TATA element (TR) and is initiated from the +1 site. The TC element, unlike TR, does not respond to transcriptional stimulation by the GCN4 or GAL4 activator protein. Analysis of deletion, substitution, and point mutations indicates that Tc mapped between nucleotides -54 and -83 and is a sequence-dependent element because it could not be functionally replaced by other DNA sequences. However, in contrast to the behavior of typical promoter elements, it was surprisingly difficult to eliminate Tc function by base pair substitutions. Of 15 derivatives averaging four substitutions in the Tc region and representing 40% of all possible single changes, only 1 inactivated the Tc element. Moreover, the phenotypes of mutant and hybrid elements indicated that inactivation of Tc required multiple changes. The spacing between Tc and the initiation region could be varied over a 30-base-pair range without significantly affecting the level of transcription from the +1 site. From these results, we consider it possible that Tc may not interact with TFIID or some other typical sequence-specific transcription factor, but instead might influence transcription, either directly or indirectly, by its DNA structure.
Mol Cell Biol 1990 Sep
PMID:Tc, an unusual promoter element required for constitutive transcription of the yeast HIS3 gene. 220 91

Human transcription factor TFIID, the TATA-binding protein, was partially purified to a form capable of associating stably with the TATA motif of the adenovirus major late promoter. Binding of the human and yeast TFIID to the TATA motif was stimulated by TFIIA. TFIIA is an integral part of a complex capable of binding other transcription factors. A complex formed with human TFIID and TFIIA (DA complex) was specifically recognized by TFIIB. We found that TFIIB activity was contained in a single polypeptide of 32 kDa and that this polypeptide participated in transcription and was capable of binding to the DA complex to form the DAB complex. Formation of the DAB complex required TFIIA, TFIID, and sequences downstream of the transcriptional start site; however, the DA complex could be formed on an oligonucleotide containing only the adenovirus major late promoter TATA motif. Using anti-TFIIB antibodies and reagents that affect the stability of a transcription-competent complex, we found that yeast and human TFIID yielded DAB complexes with different stabilities.
Mol Cell Biol 1990 Dec
PMID:Factors involved in specific transcription by mammalian RNA polymerase II: role of transcription factors IIA, IID, and IIB during formation of a transcription-competent complex. 224 58

DNase I footprint analysis of the core adenovirus 2 (Ad2) major late promoter (MLP) has revealed distinct patterns of protection corresponding to the assembly of transcription components during transcriptional initiation (VanDyke, M. W., Sawadogo, M., and Roeder, R. G. (1989) Mol. Cell Biol. 7, 3371-3379). By using partially purified transcription factors, DNase I protection over the TATA box element and the CAP sequence was attributed to the binding of a single factor, TFIID. We have determined, however, that protection of the CAP region results from the binding of a novel factor, designated CAP-site binding factor (CBF), which is chromatographically and functionally distinct from TFIID. DNase I footprint analysis and gel electrophoresis mobility shift competition assays confirm that distinct polypeptides bind to the Ad2 MLP upstream promoter sequence, TATA box, and CAP sequences. When the CAP sequence is mutated, transcriptional activity of the Ad2 MLP is reduced both in vitro and in vivo. The decrease in transcriptional activity correlates with decreased CBF binding activity. Nuclear extracts depleted of CBF also exhibit reduced Ad2 MLP transcriptional activity. The addition of DNA affinity purified CBF, free of TFIID or major late transcription factor, restores the activity to control levels.
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PMID:Identification and characterization of an adenovirus 2 major late promoter CAP sequence DNA-binding protein. 235 2

We have examined the promoter sequence requirements for E1a transactivation of the human HSP70 gene by using a transient cotransfection assay. A 5' deletion study has defined a basal transcription unit extending to -74 relative to the transcription initiation site which was fully E1a responsive. Further deletion, abolishing a CCAAT element at -67, drastically reduced basal and E1a-induced expression. A linker-scanner analysis has identified four functional elements within the basal transcription unit which may interact with CTF, SP1, TFIID, and an ATF/AP1-like factor. Sequences between -100 and -188 can partially compensate for mutations in these elements. No mutation specifically abolished E1a inducibility. Any reduction in absolute E1a-induced levels was accompanied by a corresponding reduction in absolute basal levels, thereby maintaining a constant relative fold induction. We conclude that E1a transactivation of the human HSP70 promoter does not require any single basal transcription element. We also examined an HSP70 promoter fragment, containing the CCAAT element at -67 and the purine-rich element at -54, out of its normal context by fusing it upstream of a transcriptionally inactive herpes simplex virus thymidine kinase deletion construct containing only the TATA box. The resulting chimeric promoter was fully E1a responsive. Mutagenesis of this promoter fusion demonstrated that the CCAAT element was essential for detectable basal and E1a-induced expression. Mutations in the purine-rich element resulted in an approximately 10-fold elevation in basal levels and rendered the promoter nonresponsive to E1a.
Mol Cell Biol 1989 Jun
PMID:E1a transactivation of the human HSP70 promoter is mediated through the basal transcriptional complex. 247 56


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