HUMAN ONCOPROTEIN MDM2 INTERACTS WITH THE TATA-BINDING PROTEIN IN-VITRO AND IN-VIVO

1995 ◽  
Author(s):  
P LENG ◽  
DR BROWN ◽  
S DEB ◽  
SP DEB
Keyword(s):  
Binding Protein ◽  
Tata Binding Protein

Mechanism of initiator-mediated transcription: evidence for a functional interaction between the TATA-binding protein and DNA in the absence of a specific recognition sequence

1993 ◽  
Vol 13 (7) ◽  
pp. 3841-3849
Author(s):  
B Zenzie-Gregory ◽  
A Khachi ◽  
I P Garraway ◽  
S T Smale
Keyword(s):  
Transcription Initiation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Upstream Region ◽  
Promoter Strength ◽  
Recognition Sequence ◽  
Specific Recognition ◽  
Rate Limiting

Promoters containing Sp1 binding sites and an initiator element but lacking a TATA box direct high levels of accurate transcription initiation by using a mechanism that requires the TATA-binding protein (TBP). We have begun to address the role of TBP during transcription from Sp1-initiator promoters by varying the nucleotide sequence between -14 and -33 relative to the start site. With each of several promoters containing different upstream sequences, we detected accurate transcription both in vitro and in vivo, but the promoter strengths varied widely, particularly with the in vitro assay. The variable promoter activities correlated with, but were not proportional to, the abilities of the upstream sequences to function as TATA boxes, as assessed by multiple criteria. These results confirm that accurate transcription can proceed in the presence of an initiator, regardless of the sequence present in the -30 region. However, the results reveal a role for this upstream region, most consistent with a model in which initiator-mediated transcription requires binding of TBP to the upstream DNA in the absence of a specific recognition sequence. Moreover, in vivo it appears that the promoter strength is modulated less severely by altering the -30 sequence, consistent with a previous suggestion that TBP is not rate limiting in vivo for TATA-less promoters. Taken together, these results suggest that variations in the structure of a core promoter might alter the rate-limiting step for transcription initiation and thereby alter the potential modes of transcriptional regulation, without severely changing the pathway used to assemble a functional preinitiation complex.

scholarly journals Functional Organization of the Yeast SAGA Complex: Distinct Components Involved in Structural Integrity, Nucleosome Acetylation, and TATA-Binding Protein Interaction

1999 ◽  
Vol 19 (1) ◽  
pp. 86-98 ◽  
Author(s):  
David E. Sterner ◽  
Patrick A. Grant ◽  
Shannon M. Roberts ◽  
Laura J. Duggan ◽  
Rimma Belotserkovskaya ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Structural Integrity ◽  
Functional Organization ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Saga Complex ◽  
Specific Domain ◽  
Nucleosomal Histone

ABSTRACT SAGA, a recently described protein complex in Saccharomyces cerevisiae, is important for transcription in vivo and possesses histone acetylation function. Here we report both biochemical and genetic analyses of members of three classes of transcription regulatory factors contained within the SAGA complex. We demonstrate a correlation between the phenotypic severity of SAGA mutants and SAGA structural integrity. Specifically, null mutations in the Gcn5/Ada2/Ada3 or Spt3/Spt8 classes cause moderate phenotypes and subtle structural alterations, while mutations in a third subgroup, Spt7/Spt20, as well as Ada1, disrupt the complex and cause severe phenotypes. Interestingly, double mutants (gcn5Δ spt3Δand gcn5Δ spt8Δ) causing loss of a member of each of the moderate classes have severe phenotypes, similar tospt7Δ, spt20Δ, or ada1Δmutants. In addition, we have investigated biochemical functions suggested by the moderate phenotypic classes and find that first, normal nucleosomal acetylation by SAGA requires a specific domain of Gcn5, termed the bromodomain. Deletion of this domain also causes specific transcriptional defects at the HIS3 promoter in vivo. Second, SAGA interacts with TBP, the TATA-binding protein, and this interaction requires Spt8 in vitro. Overall, our data demonstrate that SAGA harbors multiple, distinct transcription-related functions, including direct TBP interaction and nucleosomal histone acetylation. Loss of either of these causes slight impairment in vivo, but loss of both is highly detrimental to growth and transcription.

Transcriptional activation requires protection of the TATA-binding protein Tbp1 by the ubiquitin-specific protease Ubp3

2010 ◽  
Vol 431 (3) ◽  
pp. 391-402 ◽  
Author(s):  
Boon Shang Chew ◽  
Wee Leng Siew ◽  
Benjamin Xiao ◽  
Norbert Lehming
Keyword(s):  
Mutant Strain ◽  
Transcriptional Activation ◽  
Glutathione Transferase ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Mutant Protein ◽  
Specific Protease ◽  
Ubiquitin Specific Protease

Tbp1, the TATA-binding protein, is essential for transcriptional activation, and Gal4 and Gcn4 are unable to fully activate transcription in a Saccharomyces cerevisiae TBP1E86D mutant strain. In the present study we have shown that the Tbp1E186D mutant protein is proteolytically instable, and we have isolated intragenic and extragenic suppressors of the transcription defects of the TBP1E186D mutant strain. The TBP1R6S mutation stabilizes the Tbp1E186D mutant protein and suppresses the defects of the TBP1E186D mutant strain. Furthermore, we found that the overexpression of the de-ubiquitinating enzyme Ubp3 (ubiquitin-specific protease 3) also stabilizes the Tbp1E186D mutant protein and suppresses of the defects of the TBP1E186D mutant strain. Importantly, the deletion of UBP3 and its cofactor BRE5 lead to increased degradation of wild-type Tbp1 protein and to defects in transcriptional activation by Gal4 and Gcn4. Purified GST (glutathione transferase)–Ubp3 reversed Tbp1 ubiquitination, and the deletion of UBP3 lead to the accumulation of poly-ubiquitinated species of Tbp1 in a proteaseome-deficient genetic background, demonstrating that Ubp3 reverses ubiquitination of Tbp1 in vitro and in vivo. Chromatin immunoprecipitation showed that Ubp3 was recruited to the GAL1 and HIS3 promoters upon the induction of the respective gene, indicating that protection of promoter-bound Tbp1 by Ubp3 is required for transcriptional activation.

scholarly journals Even-skipped Represses Transcription by Binding TATA Binding Protein and Blocking the TFIID-TATA Box Interaction

1998 ◽  
Vol 18 (7) ◽  
pp. 3771-3781 ◽  
Author(s):  
Chi Li ◽  
James L. Manley
Keyword(s):  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Direct Interaction ◽  
In Vitro Transcription ◽  
Tata Binding Protein ◽  
Tata Box ◽  
Homeodomain Protein ◽  
Necessary And Sufficient

ABSTRACT The Drosophila homeodomain protein Even-skipped (Eve) is a transcriptional repressor, and previous studies have suggested that it functions by interfering with the basal transcription machinery. Here we describe experiments indicating that the mechanism of Eve repression involves a direct interaction with the TATA binding protein (TBP) that blocks binding of TBP-TFIID to the promoter. We first compared Eve activities in in vitro transcription systems reconstituted with either all the general transcription factors or only TBP, TFIIB, TFIIF30, and RNA polymerase II. In each case, equivalent and very efficient levels of repression were observed, indicating that no factors other than those in the minimal system are required for repression. We then show that Eve can function efficiently when its recognition sites are far from the promoter and that the same regions of Eve required for repression in vivo are necessary and sufficient for in vitro repression. This includes, in addition to an Ala-Pro-rich region, residues within the homeodomain. Using GAL4-Eve fusion proteins, we demonstrate that the homeodomain plays a role in repression in addition to DNA binding, which is to facilitate interaction with TBP. Single-round transcription experiments indicate that Eve must function prior to TBP binding to the promoter, suggesting a mechanism whereby Eve represses by competing with the TATA box for TBP binding. Consistent with this, excess TATA box-containing oligonucleotide is shown to specifically and efficiently disrupt the TBP-Eve interaction. Importantly, we show that Eve binds directly to TFIID and that this interaction can also be disrupted by the TATA oligonucleotide. We conclude that Eve represses transcription via a direct interaction with TBP that blocks TFIID binding to the promoter.

scholarly journals A Human TATA Binding Protein-Related Protein with Altered DNA Binding Specificity Inhibits Transcription from Multiple Promoters and Activators

1999 ◽  
Vol 19 (11) ◽  
pp. 7610-7620 ◽  
Author(s):  
Paul A. Moore ◽  
Josef Ozer ◽  
Moreh Salunek ◽  
Gwenael Jan ◽  
Dennis Zerby ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcriptional Repression ◽  
Transcription Initiation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Related Protein ◽  
Multiple Promoters ◽  
Binding Surface

ABSTRACT The TATA binding protein (TBP) plays a central role in eukaryotic and archael transcription initiation. We describe the isolation of a novel 23-kDa human protein that displays 41% identity to TBP and is expressed in most human tissue. Recombinant TBP-related protein (TRP) displayed barely detectable binding to consensus TATA box sequences but bound with slightly higher affinities to nonconsensus TATA sequences. TRP did not substitute for TBP in transcription reactions in vitro. However, addition of TRP potently inhibited basal and activated transcription from multiple promoters in vitro and in vivo. General transcription factors TFIIA and TFIIB bound glutathioneS-transferase–TRP in solution but failed to stimulate TRP binding to DNA. Preincubation of TRP with TFIIA inhibited TBP-TFIIA-DNA complex formation and addition of TFIIA overcame TRP-mediated transcription repression. TRP transcriptional repression activity was specifically reduced by mutations in TRP that disrupt the TFIIA binding surface but not by mutations that disrupt the TFIIB or DNA binding surface of TRP. These results suggest that TFIIA is a primary target of TRP transcription inhibition and that TRP may modulate transcription by a novel mechanism involving the partial mimicry of TBP functions.

scholarly journals Mechanism of initiator-mediated transcription: evidence for a functional interaction between the TATA-binding protein and DNA in the absence of a specific recognition sequence.

1993 ◽  
Vol 13 (7) ◽  
pp. 3841-3849 ◽  
Author(s):  
B Zenzie-Gregory ◽  
A Khachi ◽  
I P Garraway ◽  
S T Smale
Keyword(s):  
Transcription Initiation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Upstream Region ◽  
Promoter Strength ◽  
Recognition Sequence ◽  
Specific Recognition ◽  
Rate Limiting

Promoters containing Sp1 binding sites and an initiator element but lacking a TATA box direct high levels of accurate transcription initiation by using a mechanism that requires the TATA-binding protein (TBP). We have begun to address the role of TBP during transcription from Sp1-initiator promoters by varying the nucleotide sequence between -14 and -33 relative to the start site. With each of several promoters containing different upstream sequences, we detected accurate transcription both in vitro and in vivo, but the promoter strengths varied widely, particularly with the in vitro assay. The variable promoter activities correlated with, but were not proportional to, the abilities of the upstream sequences to function as TATA boxes, as assessed by multiple criteria. These results confirm that accurate transcription can proceed in the presence of an initiator, regardless of the sequence present in the -30 region. However, the results reveal a role for this upstream region, most consistent with a model in which initiator-mediated transcription requires binding of TBP to the upstream DNA in the absence of a specific recognition sequence. Moreover, in vivo it appears that the promoter strength is modulated less severely by altering the -30 sequence, consistent with a previous suggestion that TBP is not rate limiting in vivo for TATA-less promoters. Taken together, these results suggest that variations in the structure of a core promoter might alter the rate-limiting step for transcription initiation and thereby alter the potential modes of transcriptional regulation, without severely changing the pathway used to assemble a functional preinitiation complex.

scholarly journals Expression of Human Herpesvirus 6B repwithin Infected Cells and Binding of Its Gene Product to the TATA-Binding Protein In Vitro and In Vivo

2000 ◽  
Vol 74 (13) ◽  
pp. 6096-6104 ◽  
Author(s):  
Yasuko Mori ◽  
Panadda Dhepakson ◽  
Takuya Shimamoto ◽  
Keiji Ueda ◽  
Yasuyuki Gomi ◽  
...  
Keyword(s):  
Gene Product ◽  
Binding Protein ◽  
Protein Biosynthesis ◽  
Human Herpesvirus ◽  
Tata Binding Protein ◽  
Northern Blotting ◽  
Its Gene ◽  
Infected Cells

ABSTRACT We have characterized the human herpesvirus 6B (HHV-6B)rep gene, which is a homologue of the adeno-associated virus type 2 rep and is unique in the herpesvirus family. Three transcripts, 9.0, 5.0, and 2.7 kb (the major transcript), were detected by Northern blotting using an HHV-6B rep probe under late conditions. We investigated the expression kinetics of therep gene using cycloheximide (CHX) and phosphonoformic acid (PFA), which are inhibitors of protein synthesis and viral DNA synthesis, respectively. The 5.2-kb transcript was mainly detected in the absence of protein biosynthesis upon infection, and none of the 9.0-, 5.0-, and 2.7-kb transcripts detected under the late conditions were detected in the presence of CHX and PFA. Sequences obtained from a cDNA library showed that the 5.0- and 2.7-kb transcripts were spliced from two and three exons, respectively, and the 2.7-kb transcript was more abundant. Immunohistochemistry using an antibody raised against the HHV-6 rep gene product (REP) revealed that REP was mainly present in the nucleus of MT-4 cells within 24 h after infection with HHV-6B. Using pull-down assays, coimmunoprecipitation, and a mammalian two hybrid system, we showed that HHV-6 REP binds to a transcription factor, human TATA-binding protein, through its N-terminal region.

scholarly journals The transcriptional repressor even-skipped interacts directly with TATA-binding protein.

1995 ◽  
Vol 15 (9) ◽  
pp. 5007-5016 ◽  
Author(s):  
M Um ◽  
C Li ◽  
J L Manley
Keyword(s):  
Protein Interactions ◽  
Transcriptional Repression ◽  
Binding Protein ◽  
Transcriptional Repressor ◽  
Direct Interaction ◽  
Tata Binding Protein ◽  
Homeodomain Protein ◽  
Repression Domain

The Drosophila homeodomain protein Even-skipped (Eve) has previously been shown to function as a sequence-specific transcriptional repressor, and in vitro and in vivo experiments have shown that the protein can actively block basal transcription. However, the mechanism of repression is not known. Here, we present evidence establishing a direct interaction between Eve and the TATA-binding protein (TBP). Using cotransfection assays with minimal basal promoters whose activity can be enhanced by coexpression of TBP, we found that Eve could efficiently block, or squelch, this enhancement. Squelching did not require Eve DNA-binding sites on the reporter plasmids but was dependent on the presence of the Eve repression domain. Further support for an in vivo interaction between the Eve repression domain and TBP was derived from a two-hybrid-type assay with transfected cells. Evidence that Eve and TBP interact directly was provided by in vitro binding assays, which revealed a specific protein-protein interaction that required an intact Eve repression domain and the conserved C terminus of TBP. The Eve homeodomain was also required for these associations, suggesting that it may function in protein-protein interactions. We also show that a previously characterized artificial repression region behaves in a manner similar to that of the Eve repression domain, including its ability to squelch TBP-enhanced expression in vivo and to bind TBP specifically in vitro. Our results suggest a model for transcriptional repression that involves an interaction between Eve and TBP.

scholarly journals TATA-Binding Protein Mutants That Increase Transcription from Enhancerless and Repressed Promoters In Vivo

2000 ◽  
Vol 20 (5) ◽  
pp. 1478-1488 ◽  
Author(s):  
Joseph V. Geisberg ◽  
Kevin Struhl
Keyword(s):  
Binding Protein ◽  
Tata Binding Protein ◽  
Negative Regulator ◽  
Unusual Structure ◽  
Enhancer Element ◽  
Pol Ii ◽  
Activator Proteins ◽  
Pol Iii

ABSTRACT Using a genetic screen, we isolated three TATA-binding protein (TBP) mutants that increase transcription from promoters that are repressed by the Cyc8-Tup1 or Sin3-Rpd3 corepressors or that lack an enhancer element, but not from an equivalently weak promoter with a mutated TATA element. Increased transcription is observed when the TBP mutants are expressed at low levels in the presence of wild-type TBP. These TBP mutants are unable to support cell viability, and they are toxic in strains lacking Rpd3 histone deacetylase or when expressed at higher levels. Although these mutants do not detectably bind TATA elements in vitro, genetic and chromatin immunoprecipitation experiments indicate that they act directly at promoters and do not increase transcription by titration of a negative regulatory factor(s). The TBP mutants are mildly defective for associating with promoters responding to moderate or strong activators; in addition, they are severely defective for RNA polymerase (Pol) III but not Pol I transcription. These results suggest that, with respect to Pol II transcription, the TBP mutants specifically increase expression from core promoters. Biochemical analysis indicates that the TBP mutants are unaffected for TFIID complex formation, dimerization, and interactions with either the general negative regulator NC2 or the N-terminal inhibitory domain of TAF130. We speculate that these TBP mutants have an unusual structure that allows them to preferentially access TATA elements in chromatin templates. These TBP mutants define a criterion by which promoters repressed by Cyc8-Tup1 or Sin3-Rpd3 resemble enhancerless, but not TATA-defective, promoters; hence, they support the idea that these corepressors inhibit the function of activator proteins rather than the Pol II machinery.

scholarly journals TATA-Binding Protein (TBP)-Like Factor (TLF) Is a Functional Regulator of Transcription: Reciprocal Regulation of the Neurofibromatosis Type 1 and c-fos Genes by TLF/TRF2 and TBP

2005 ◽  
Vol 25 (7) ◽  
pp. 2632-2643 ◽  
Author(s):  
Jayhong A. Chong ◽  
Magdalene M. Moran ◽  
Martin Teichmann ◽  
J. Stefan Kaczmarek ◽  
Robert Roeder ◽  
...  
Keyword(s):  
Neurofibromatosis Type 1 ◽  
Binding Protein ◽  
Neurofibromatosis Type ◽  
Tata Binding Protein ◽  
Related Factor ◽  
Reciprocal Regulation ◽  
In Cells

ABSTRACT The lack of direct targets for TATA-binding protein (TBP)-like factors (TLFs) confounds the understanding of their role in gene expression. Here we report that human TLF (also called TBP-related factor 2 [TRF2]) activates a number of different genes, including the neurofibromatosis type 1 (NF1) gene. The overexpression of TLF increases the amount of NF1 mRNA in cells. In vivo, TLF binds to and upregulates transcription from a fragment of the NF1 promoter. In vitro, purified TLF-TFIIA binds directly to the same NF1 promoter fragment that is required for TLF responsiveness in cells. Furthermore, targeted deletion of TLF in mice reduces NF1 levels. In contrast, TLF inhibits transcription driven by a fragment from the TATA-containing c-fos promoter by sequestering TFIIA. TBP affects the NF1 and c-fos promoters in a manner reciprocal to that of TLF, stimulating the c-fos promoter and inhibiting NF1 transcription. We conclude that TLF is a functional regulator of transcription with targets distinct from those of TBP.

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