Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We isolated a complementary DNA (cDNA) that encoded a TATA-binding protein (TBP) from a cDNA library of tobacco (Nicotiana tabacum) suspension-cultured cells (BY-2). A comparison among deduced amino acid sequences of plant TBPs revealed the presence of a long conserved region within the amino acid sequence of the TBP. Genomic Southern analysis revealed that tobacco TBP (tTBP) is encoded by only a small number of copies of a gene in the tobacco genome. Addition of recombinant tTBP to an extract of tobacco nuclei (TNE) enhanced the basal transcriptional activity in vitro. This result indicates that the level of tTBP is a rate-limiting factor for basal transcriptional activity in TNE. We subsequently succeeded in the functional complementation of TATA-dependent initiation of transcription that was associated with a plant promoter in a homologous plant system. Addition of bacterially expressed recombinant tTBP to a heat-inactivated TNE restored transcriptional activity, as did the addition of human TBP. Moreover, heating of the recombinant tTBP eliminated its ability to restore transcriptional activity. It appears that the heat inactivation of TNE was caused by the heat inactivation of tTBP in TNE.
Plant Mol Biol 1997 May
PMID:Restoration of TATA-dependent transcription in a heat-inactivated extract of tobacco nuclei by recombinant TATA-binding protein (TBP) from tobacco. 917 13

The estrogen receptor (ER) belongs to a family of ligand-inducible nuclear receptors that exert their effects by binding to cis-acting DNA elements in the regulatory region of target genes. The detailed mechanisms by which ER interacts with the estrogen response element (ERE) and affects transcription still remain to be elucidated. To study the ER-ERE interaction and transcription initiation, we employed purified recombinant ER expressed in both the baculovirus-Sf9 and his-tagged bacterial systems. The effect of high-mobility group (HMG) protein HMG-1 and purified recombinant TATA-binding protein-associated factor TAF(II)30 on ER-ERE binding and transcription initiation were assessed by electrophoretic mobility shift assay and in vitro transcription from an ERE-containing template (pERE2LovTATA), respectively. We find that purified, recombinant ER fails to bind to ERE in spite of high ligand-binding activity and electrophoretic and immunological properties identical to ER in MCF-7 breast cancer cells. HMG-1 interacts with ER and promotes ER-ERE binding in a concentration- and time-dependent manner. The effectiveness of HMG-1 to stimulate ER-ERE binding in the electrophoretic mobility shift assay depends on the sequence flanking the ERE consensus as well as the position of the latter in the oligonucleotide. We find that TAF(II)30 has no effect on ER-ERE binding either alone or in combination with ER and HMG-1. Although HMG-1 promotes ER-ERE binding, it fails to stimulate transcription initiation either in the presence or absence of hormone. In contrast, TAF(II)30, while not affecting ER-ERE binding, stimulates transcription initiation 20-fold in the presence of HMG-1. These results indicate that HMG-1 and TAF(II)30 act in sequence, the former acting to promote ER-ERE binding followed by the latter to stimulate transcription initiation.
Mol Endocrinol 1997 Jul
PMID:High-mobility group (HMG) protein HMG-1 and TATA-binding protein-associated factor TAF(II)30 affect estrogen receptor-mediated transcriptional activation. 921 49

A subunit of the Drosophila RNA polymerase III transcription factor IIIB (TFIIIB) complex has been identified using antibodies directed against the analogous human protein, hIIIB90. This protein has an apparent molecular mass of 105 kDa and has been designated dTAFIII105. Drosophila S-2 cell extracts that were immunodepleted of dTAFIII105 were substantially reduced in their capacity to support tRNA gene transcription. A protein (far Western) blot analysis revealed that dTAFIII105, present in a TFIIIB fraction, directly interacts with TATA-binding protein (TBP). Coimmunoprecipitation assays demonstrated that this protein associates with TBP in S-2 cell extracts. Our previous studies have identified a mutation at position 332 within Drosophila TBP that changes a highly conserved arginine residue to a histidine residue, which renders it specifically defective in its ability to support RNA polymerase III transcription in S-2 cells (Trivedi, A., Vilalta, A., Gopalan, S., and Johnson, D. L. (1996) Mol. Cell. Biol. 16, 6909-6916). We further demonstrate that extracts prepared from a stable cell line expressing epitope-tagged wild-type TBP exhibit an increase in tRNA gene transcription, whereas extracts derived from cells expressing the mutant TBP protein do not. Coimmunoprecipitation assays and far Western blot analysis demonstrate that this mutation in TBP abolishes its ability to stably interact with dTAFIII105. Thus, we have identified both a Drosophila protein that is directly associated with TBP in the TFIIIB complex, dTAFIII105, and an amino acid residue within the highly conserved carboxyl-terminal region of TBP that is critical for dTAFIII105-TBP interactions.
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PMID:An RNA polymerase III-defective mutation in TATA-binding protein disrupts its interaction with a transcription factor IIIB subunit in drosophila cells. 921 40

MOT1 is an essential Saccharomyces cerevisiae protein and a member of the SNF2/SWI2 family of ATPases. MOT1 functions by removing TATA-binding protein (TBP) from DNA, and as a consequence, MOT1 can regulate transcription both in vitro and in vivo. Here we describe the in vivo and in vitro activities of MOT1 deletion and substitution mutants. The results indicate that MOT1 is targeted to TBP both in vitro and in vivo via amino acids in its nonconserved N terminus. The conserved C-terminal ATPase of MOT1 appears to contribute to TBP-DNA complex recognition in the absence of ATP, but it appears to function primarily during the actual ATP-dependent dissociation reaction. Chimeric proteins in which homologous portions of SNF2/SWI2 have been substituted for the MOT1 ATPase can bind to TBP-DNA complexes but fail to dissociate these complexes in the presence of ATP, suggesting that the specificity of action of MOT1 is also conferred by the C-terminal ATPase. ATPase assays demonstrate that the MOT1 ATPase is activated by TBP. Thus, MOT1 undergoes at least two conformational changes: (i) an allosteric effect of TBP that mediates the activation of the MOT1 ATPase and (ii) an ATP-driven "power stroke" that causes TBP-DNA complex dissociation. These results provide a general framework for understanding how members of the SNF2/SWI2 protein family use ATP to modulate protein-DNA interactions to regulate many diverse processes in cells.
Mol Cell Biol 1997 Aug
PMID:Molecular analysis of the SNF2/SWI2 protein family member MOT1, an ATP-driven enzyme that dissociates TATA-binding protein from DNA. 923 40

Saccharomyces cerevisiae transcription factor IIIB (TFIIIB) is composed of three subunits: the TATA-binding protein, the TFIIB-related protein Brf, and B". TFIIIB, which is brought to RNA polymerase III-transcribed genes indirectly through interaction with DNA-bound TFIIIC or directly through DNA recognition by the TATA-binding protein, in turn recruits RNA polymerase III to the promoter. N-terminally deleted derivatives of Brf have been examined for their ability to interact with DNA-bound TFIIIC and with the other components of TFIIIB and for participation in transcription. Brf(165-596), lacking 164 N-proximal TFIIB-homologous amino acids, is competent to participate in the assembly of TFIIIB-DNA complexes and in TFIIIC-independent transcription. Even deletion of the entire TFIIB-homologous half of the protein, as in Brf(317-596) and Brf(352-596), allows some interaction with DNA-bound TBP and with the B" component of TFIIIB to be retained. The function of Brf(165-596) in transcription has also been examined in the context of B" with small internal deletions. The ability of Brf with this sizable N-terminal segment deleted to function in TFIIIC-independent transcription requires segments of B" that are individually indispensable although required on an either/or basis, in the context of complete Brf. These findings suggest a functional complementarity and reciprocity between the Brf and B" components of TFIIIB.
Mol Cell Biol 1997 Sep
PMID:Domains of the Brf component of RNA polymerase III transcription factor IIIB (TFIIIB): functions in assembly of TFIIIB-DNA complexes and recruitment of RNA polymerase to the promoter. 927 7

HeLa cell nuclear extracts were used to study the mechanism of activation of RNA polymerase II-mediated transcription by the N-terminal transactivation domain (tau1) of the glucocorticoid receptor in vitro. When fused to the Gal4 DNA-binding domain, the tau1 domain activated transcription approximately 9-fold in HeLa nuclear extracts. Using heat treatment to inactivate transcription factor IID (TFIID) in the extract, it was shown that the addition of purified TFIID complex, but not the TATA-binding protein alone, was sufficient to restore this level of activation. The tau1 domain was shown to interact directly with the TFIID complex. This interaction was markedly reduced by a mutation in the tau1 domain that reduces its activity. Furthermore, the interaction was specific for the TFIID complex, since no interaction was seen with TFIIIB, an analogous protein complex involved in RNA polymerase III transcription. The tau1 domain was further shown to interact with the TATA-binding protein subunit of the TFIID complex. These results suggest a mechanism by which the GR tau1 domain might contribute to gene activation by recruitment of the TFIID complex to target promoters.
Mol Endocrinol 1997 Sep
PMID:Involvement of the transcription factor IID protein complex in gene activation by the N-terminal transactivation domain of the glucocorticoid receptor in vitro. 928 62

The general transcription factor IIB (TFIIB) plays an essential role in transcription of protein-coding genes by RNA polymerase II. We have used site-directed mutagenesis to assess the role of conserved amino acids in several important regions of yeast TFIIB. These include residues in the highly conserved amino-terminal region and basic residues in the D1 and E1 core domain alpha-helices. Acidic substitutions of residues K190 (D1) and K201 (E1) resulted in growth impairments in vivo, reduced basal transcriptional activity in vitro, and an inability to form stable TFIIB-TATA-binding protein-DNA (DB) complexes. Significantly, these mutants retained the ability to respond to acidic activators in vivo and to the Gal4-VP16 activator in vitro, supporting the view that these basic residues play a role in basal transcription. In addition, 14 single-amino-acid substitutions were introduced in the conserved amino-terminal region. Three of these mutants, the L50D, R64E, and R78L mutants, displayed altered growth properties in vivo and were compromised for supporting transcription in vitro. The L50D mutant was impaired for RNA polymerase II interaction, while the R64E mutant exhibited altered transcription start site selection both in vitro and in vivo and, surprisingly, was more active than the wild type in the formation of stable DB complexes. These results support the view that the amino-terminal domain is involved in the direct interaction between yeast TFIIB and RNA polymerase II and suggest that this domain may interact with DNA and/or modulate the formation of a DB complex.
Mol Cell Biol 1997 Dec
PMID:Mutational analysis of the D1/E1 core helices and the conserved N-terminal region of yeast transcription factor IIB (TFIIB): identification of an N-terminal mutant that stabilizes TATA-binding protein-TFIIB-DNA complexes. 937 9

Biochemical experiments indicate that the general transcription factor IIB (TFIIB) can interact directly with acidic activation domains and that activators can stimulate transcription by increasing recruitment of TFIIB to promoters. For promoters at which recruitment of TFIIB to promoters is limiting in vivo, one would predict that transcriptional activity should be particularly sensitive to TFIIB mutations that decrease the association of TFIIB with promoter DNA and/or with activation domains; i.e., such TFIIB mutations should exacerbate a limiting step that occurs in wild-type cells. Here, we describe mutations on the DNA-binding surface of TFIIB that severely affect both TATA-binding protein (TBP)-TFIIB-TATA complex formation and interaction with the VP16 activation domain in vitro. These TFIIB mutations affect the stability of the TBP-TFIIB-TATA complex in vivo because they are synthetically lethal in combination with TBP mutants impaired for TFIIB binding. Interestingly, these TFIIB derivatives support viability, and they efficiently respond to Gal4-VP16 and natural acidic activators in different promoter contexts. These results suggest that in vivo, recruitment of TFIIB is not generally a limiting step for acidic activators. However, one TFIIB derivative shows reduced transcription of GAL4, suggesting that TFIIB may be limiting at a subset of promoters in vivo.
Mol Cell Biol 1997 Dec
PMID:Transcriptional activation by TFIIB mutants that are severely impaired in interaction with promoter DNA and acidic activation domains. 937 10

Our previous studies have shown that the hepatitis B virus protein, X, activates all three classes of RNA polymerase III (pol III)-dependent promoters by increasing the cellular level of TATA-binding protein (TBP) (H.-D. Wang et al., Mol. Cell. Biol. 15:6720-6728, 1995), a limiting transcription component (A. Trivedi et al., Mol. Cell. Biol. 16:6909-6916, 1996). We have investigated whether these X-mediated events are dependent on the activation of the Ras/Raf-1 signaling pathway. Transient expression of a dominant-negative mutant Ras gene (Ras-ala15) in a Drosophila S-2 stable cell line expressing X (X-S2), or incubation of the cells with a Ras farnesylation inhibitor, specifically blocked both the X-dependent activation of a cotransfected tRNA gene and the increase in cellular TBP levels. Transient expression of a constitutively activated form of Ras (Ras-val12) in control S2 cells produced both an increase in tRNA gene transcription and an increase in cellular TBP levels. These events are not cell type specific since X-mediated gene induction was also shown to be dependent on Ras activation in a stable rat 1A cell line expressing X. Furthermore, increases in RNA pol III-dependent gene activity and TBP levels could be restored in X-S2 cells expressing Ras-ala15 by coexpressing a constitutively activated form of Raf-1. These events are serum dependent, and when the cells are serum deprived, the X-mediated effects are augmented. Together, these results demonstrate that the X-mediated induction of RNA pol III-dependent genes and increase in TBP are both dependent on the activation of the Ras/Raf-1 signaling cascade. In addition, these studies define two new and important consequences mediated by the activation of the Ras signal transduction pathway: an increase in the central transcription factor, TBP, and the induction of RNA pol III-dependent gene activity.
Mol Cell Biol 1997 Dec
PMID:Hepatitis B virus X protein induces RNA polymerase III-dependent gene transcription and increases cellular TATA-binding protein by activating the Ras signaling pathway. 937 15

Artificial recruitment of TATA-binding protein (TBP) to many eukaryotic promoters bypasses DNA-bound activator function. The human immunodeficiency virus type 1 (HIV-1) Tat is an unconventional activator that up-regulates transcription from the HIV-1 long terminal repeat (LTR) through binding to a nascent RNA sequence, TAR. Because this LTR and its cognate activator have atypical features compared to a standard RNA polymerase II (RNAP II) transcriptional unit, the precise limiting steps for HIV-1 transcription and how Tat resolves these limitations remain incompletely understood. We thus constructed human TBP fused to the DNA-binding domain of GAL4 to determine whether recruitment of TBP is one rate-limiting step in HIV-1 LTR transcription and whether Tat functions to recruit TBP. As a control, we compared the activity of the adenovirus E1b promoter. Our findings indicate that TBP tethering to the E1b promoter fully effected transcription to the same degree achievable with the potent GAL4-VP16 activator. By contrast, TBP recruitment to the HIV-1 LTR, although necessary for conferring Tat responsiveness, did not bypass a physical need for Tat in achieving activated transcription. These results document that the HIV-1 and the E1b promoters are transcriptionally limited at different steps; the major rate-limiting step for E1b is recruitment of TBP, while activation of the HIV-1 LTR requires steps in addition to TBP recruitment. We suggest that Tat acts to accelerate rate-limiting steps after TBP recruitment.
Mol Cell Biol 1997 Dec
PMID:Promoter activity of Tat at steps subsequent to TATA-binding protein recruitment. 937 21


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