scholarly journals Mutations on the DNA-binding surface of TATA-binding protein can specifically impair the response to acidic activators in vivo.

1995 ◽  
Vol 15 (10) ◽  
pp. 5461-5469 ◽  
Author(s):  
M Lee ◽  
K Struhl
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Convex Surface ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Protein Protein Interactions ◽  
Activator Proteins ◽  
Tata Element ◽  
Binding Surface

The TATA-binding protein (TBP) contains a concave surface that interacts specifically with TATA promoter elements and a convex surface that mediates protein-protein interactions with general and gene-specific transcription factors. Biochemical experiments suggest that interactions between activator proteins and TBP are important in stimulating transcription by the RNA polymerase II machinery. To gain insight into the role of TBP in mediating transcriptional activation in vivo, we implemented a genetic strategy in Saccharomyces cerevisiae that involved the use of a TBP derivative with altered specificity for TATA elements. By genetically screening a set of TBP mutant libraries that were biased to the convex surface that mediates protein-protein interactions, we identified TBP derivatives that are impaired in the response to three acidic activators (Gcn4, Gal4, and Ace1) but appear normal for constitutive polymerase II transcription. A genetic complementation assay indicates that the activation-defective phenotypes reflect specific functional properties of the TBP derivatives rather than an indirect effect on transcription. Surprisingly, three of the four activation-defective mutants affect residues that directly contact DNA. Moreover, all four mutants are defective for TATA element binding, but they interact normally with an acidic activation domain and TFIIB. In addition, we show that a subset of TBP derivatives with mutations on the DNA-binding surface of TBP are also compromised in their responses to acidic activators in vivo. These observations suggest that interactions at the TBP-TATA element interface can specifically affect the response to acidic activator proteins in vivo.

scholarly journals Recruiting TATA-binding protein to a promoter: transcriptional activation without an upstream activator.

1995 ◽  
Vol 15 (10) ◽  
pp. 5757-5761 ◽  
Author(s):  
H Xiao ◽  
J D Friesen ◽  
J T Lis
Keyword(s):  
Dna Binding ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activator Proteins ◽  
Tata Element

The binding of TATA-binding protein (TBP) to the TATA element is the first step in the initiation of RNA polymerase II transcription from many promoters in vitro. It has been proposed that upstream activator proteins stimulate transcription by recruiting TBP to the promoter, thus facilitating the assembly of a transcription complex. However, the role of activator proteins acting at this step to stimulate transcription in vivo remains largely speculative. To test whether recruitment of TBP to the promoter is sufficient for transcriptional activation in vivo, we constructed a hybrid protein containing TBP of the yeast Saccharomyces cerevisiae fused to the DNA-binding domain of GAL4. Our results show that TBP recruited by the GAL4 DNA-binding domain to promoters bearing a GAL4-binding site can interact with the TATA element and direct high levels of transcription. This finding indicates that binding of TBP to promoters in S. cerevisiae is a major rate-limiting step accelerated by upstream activator proteins.

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 Evidence that TAF-TATA Box-Binding Protein Interactions Are Required for Activated Transcription in Mammalian Cells

2002 ◽  
Vol 22 (8) ◽  
pp. 2788-2798 ◽  
Author(s):  
Lisa S. Martel ◽  
Helen J. Brown ◽  
Arnold J. Berk
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Convex Surface ◽  
Binding Protein ◽  
Tata Box ◽  
Binding Surface ◽  
Substitution Mutations ◽  
Residue Substitution ◽  
Tata Box Binding Protein

ABSTRACT Surfaces of human TATA box-binding protein (hsTBP) required for activated transcription in vivo were defined by constructing a library of surface residue substitution mutations and assaying them for their ability to support activated transcription in transient-transfection assays. In earlier work, three regions were identified where mutations inhibited activated transcription without interfering with TATA box DNA binding. One region is on the upstream surface of the N-terminal TBP repeat with respect to the direction of transcription and corresponds to the TBP surface that interacts with TFIIA. A second region on the stirrup of the C-terminal TBP repeat corresponds to the TFIIB-binding surface. Here we report that the third region where mutations inhibit activated transcription in mammalian cells, the convex surface of the N-terminal repeat, corresponds to a surface on TBP that interacts with hsTAF1, the major scaffold subunit of TFIID. Since mutations at the center of the hsTAF1-interacting region inhibit the ability of the protein to support activated transcription in vivo, these results are consistent with the conclusion that an interaction between hsTBP and TAFIIs is required for activated transcription in mammalian cells.

scholarly journals A severely defective TATA-binding protein-TFIIB interaction does not preclude transcriptional activation in vivo.

1997 ◽  
Vol 17 (3) ◽  
pp. 1336-1345 ◽  
Author(s):  
M Lee ◽  
K Struhl
Keyword(s):  
Dna Binding ◽  
Complex Formation ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Yeast Cells ◽  
Synthetic Lethal ◽  
Binding Surface

In yeast cells, mutations in the TATA-binding protein (TBP) that disrupt the interaction with the TATA element or with TFIIA can selectively impair the response to acidic activator proteins. We analyzed the transcriptional properties of TBP derivatives in which residues that directly interact with TFIIB were replaced by alanines. Surprisingly, a derivative with a 50-fold defect in TBP-TFIIB-TATA complex formation in vitro (E188A) supports viability and responds efficiently to activators in vivo. The E186A derivative, which displays a 100-fold defect in TBP-TFIIB-TATA complex formation, does not support viability, yet it does respond to activators. Conversely, the L189A mutation, which has the mildest effect on the interaction with TFIIB (10-fold), can abolish transcriptional activation and cell viability when combined with mutations on the DNA-binding surface. This "synthetic lethal" effect is not observed with E188A, suggesting that the previously described role of L189 in transcriptional activation may be related to its location on the DNA-binding surface and not to its interaction with TFIIB. Finally, when using TBP mutants defective on multiple interaction surfaces, we observed synthetic lethal effects between mutations on the TFIIA and TFIIB interfaces but found that mutations implicated in association with polymerase II and TFIIF did not have significant effects in vivo. Taken together, these results argue that, unlike the TBP-TATA and TBP-TFIIA interactions, the TBP-TFIIB interaction is not generally limiting for transcriptional activation in vivo.

c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein

1994 ◽  
Vol 14 (9) ◽  
pp. 6021-6029
Author(s):  
R Metz ◽  
A J Bannister ◽  
J A Sutherland ◽  
C Hagemeier ◽  
E C O'Rourke ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Box ◽  
Binding Motif ◽  
Protein Protein Interactions ◽  
High Concentrations ◽  
Tata Box Binding Protein

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

scholarly journals Evidence for two modes of cooperative DNA binding in vivo that do not involve direct protein–protein interactions

1998 ◽  
Vol 8 (8) ◽  
pp. 452-458 ◽  
Author(s):  
Sanjay Vashee ◽  
Karsten Melcher ◽  
W.Vivianne Ding ◽  
Stephen Albert Johnston ◽  
Thomas Kodadek
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Protein Protein Interactions

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 Redox Potential Regulates Binding of Universal Minicircle Sequence Binding Protein at the Kinetoplast DNA Replication Origin

2004 ◽  
Vol 3 (2) ◽  
pp. 277-287 ◽  
Author(s):  
Itay Onn ◽  
Neta Milman-Shtepel ◽  
Joseph Shlomai
Keyword(s):  
Dna Binding ◽  
Redox Potential ◽  
Protein Interactions ◽  
Replication Origin ◽  
Binding Protein ◽  
Binding Activity ◽  
Kinetoplast Dna ◽  
Minicircle Sequence

ABSTRACT Kinetoplast DNA, the mitochondrial DNA of the trypanosomatid Crithidia fasciculata, is a remarkable structure containing 5,000 topologically linked DNA minicircles. Their replication is initiated at two conserved sequences, a dodecamer, known as the universal minicircle sequence (UMS), and a hexamer, which are located at the replication origins of the minicircle L- and H-strands, respectively. A UMS-binding protein (UMSBP), binds specifically the conserved origin sequences in their single stranded conformation. The five CCHC-type zinc knuckle motifs, predicted in UMSBP, fold into zinc-dependent structures capable of binding a single-stranded nucleic acid ligand. Zinc knuckles that are involved in the binding of DNA differ from those mediating protein-protein interactions that lead to the dimerization of UMSBP. Both UMSBP DNA binding and its dimerization are sensitive to redox potential. Oxidation of UMSBP results in the protein dimerization, mediated through its N-terminal domain, with a concomitant inhibition of its DNA-binding activity. UMSBP reduction yields monomers that are active in the binding of DNA through the protein C-terminal region. C. fasciculata trypanothione-dependent tryparedoxin activates the binding of UMSBP to UMS DNA in vitro. The possibility that UMSBP binding at the minicircle replication origin is regulated in vivo by a redox potential-based mechanism is discussed.

scholarly journals TFIIA Interacts with TFIID via Association with TATA-Binding Protein and TAF40

2001 ◽  
Vol 21 (5) ◽  
pp. 1737-1746 ◽  
Author(s):  
Susan M. Kraemer ◽  
Ryan T. Ranallo ◽  
Ryan C. Ogg ◽  
Laurie A. Stargell
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
Functional Role ◽  
Associated Factors ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Functional Interaction ◽  
General Transcription Factor ◽  
Tata Element

ABSTRACT TFIIA and TATA-binding protein (TBP) associate directly at the TATA element of genes transcribed by RNA polymerase II. In vivo, TBP is complexed with approximately 14 TBP-associated factors (TAFs) to form the general transcription factor TFIID. How TFIIA and TFIID communicate is not well understood. We show that in addition to making direct contacts with TBP, yeast TAF40 interacts directly and specifically with TFIIA. Mutational analyses of the Toa2 subunit of TFIIA indicate that loss of functional interaction between TFIIA and TAF40 results in conditional growth phenotypes and defects in transcription. These results demonstrate that the TFIIA-TAF40 interaction is important in vivo and indicate a functional role for TAF40 as a bridging factor between TFIIA and TFIID.

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.

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