scholarly journals The yeast TATA-binding protein (TBP) core domain assembles with human TBP-associated factors into a functional TFIID complex.

1995 ◽  
Vol 15 (1) ◽  
pp. 534-539 ◽  
Author(s):  
Q Zhou ◽  
A J Berk
Keyword(s):  
Rna Polymerase Ii ◽  
Associated Factors ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Stable Complex ◽  
Core Domain ◽  
Cell Extracts ◽  
Hela Cell Lines ◽  
Tfiid Complex

In mammalian and Drosophila cells, the central RNA polymerase II general transcription factor TFIID is a multisubunit complex containing the TATA-binding protein (TBP) and TBP-associated factors (TAFs) bound to the conserved TBP carboxy-terminal core domain. TBP also associates with alternative TAFs in these cells to form general transcription factors required for initiation by RNA polymerases I and III. Although extracts of human HeLa cells contain little TBP that is not associated with TAFs, free TBP is readily isolated from yeast cell extracts. However, recent studies indicate that yeast TBP can also interact with other yeast polypeptides to form multiprotein complexes. We established stable human HeLa cell lines expressing yeast TBP and several yeast-human TBP hybrids to study TBP-TAF interactions. We found that the yeast TBP core domain assembles with a complete set of human TAFs into a stable TFIID complex that can support activated transcription in vitro. The fact that the yeast TBP core, which differs from human TBP core in approximately 20% of its amino acid residues, has the structural features required to form a stable complex with human TAFs implies that Saccharomyces cerevisiae probably contains TAFs that are structurally and functionally analogous to human TAFs. Surprisingly, the non-conserved amino terminus of yeast TBP inhibited association between the yeast core domain and human TAFs.

A cell-specific factor represses stimulation of transcription in vitro by transcriptional enhancer factor 1

1994 ◽  
Vol 14 (8) ◽  
pp. 5290-5299
Author(s):  
S Chaudhary ◽  
C Brou ◽  
M E Valentin ◽  
N Burton ◽  
L Tora ◽  
...  
Keyword(s):  
Hela Cell ◽  
Binding Protein ◽  
Activation Function ◽  
Tata Binding Protein ◽  
Lymphoid Cells ◽  
Specific Factor ◽  
Transcriptional Enhancer ◽  
Cell Extracts ◽  
A Cell

Transcription in HeLa cell extracts in vitro was stimulated 8- to 10-fold by a recombinant chimera, GAL-TEF-1, consisting of the DNA-binding domain of GAL4 and the activation function of the HeLa cell activator TEF-1. In contrast, only a 2- to 3-fold stimulation was obtained with GAL-TEF-1 in extracts from BJA-B lymphoid cells. Stimulation by GAL-TEF-1 in BJA-B extracts was dramatically increased by the addition of immunopurified HeLa cell TFIID, suggesting that BJA-B TFIID lacks or contains lower quantities of a TATA-binding-protein-associated factor(s) required for the activity of the TEF-1 activation function. However, chromatography, immunopurification, and transcriptional reconstitution experiments indicated that BJA-B extracts did not lack the previously identified TATA-binding-protein-associated factors required for TEF-1 activity but rather contained a negatively acting factor(s) which inhibited transactivation by GAL-TEF-1. These results indicate that the relative lack of activity of the TEF-1 activation function in vitro in BJA-B cell extracts does not result from the absence of positively acting factors from the presence of a cell-specific negatively acting factor(s).

scholarly journals NC2α Interacts with BTAF1 and Stimulates Its ATP-Dependent Association with TATA-Binding Protein

2004 ◽  
Vol 24 (22) ◽  
pp. 10072-10082 ◽  
Author(s):  
Marcin P. Klejman ◽  
Lloyd A. Pereira ◽  
Hester J. T. van Zeeburg ◽  
Siv Gilfillan ◽  
Michael Meisterernst ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Transcriptional Activity ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Present Evidence ◽  
Cell Extracts ◽  
Human Ortholog ◽  
Rna Polymerase Ii Transcription

ABSTRACT Transcriptional activity of the TATA-binding protein (TBP) is controlled by a variety of proteins. The BTAF1 protein (formerly known as TAFII170/TAF-172 and the human ortholog of Saccharomyces cerevisiae Mot1p) and the NC2 complex composed of NC2α (DRAP1) and NC2β (Dr1) are able to bind to TBP directly and regulate RNA polymerase II transcription both positively and negatively. Here, we present evidence that the NC2α subunit interacts with BTAF1. In contrast, the NC2β subunit is not able to associate with BTAF1 and seems to interfere with the BTAF1-TBP interaction. Addition of NC2α or the NC2 complex can stimulate the ability of BTAF1 to interact with TBP. This function is dependent on the presence of ATP in cell extracts but does not involve the ATPase activity of BTAF1 nor phosphorylation of NC2α. Together, our results constitute the first evidence of the physical cooperation between BTAF1 and NC2α in TBP regulation and provide a framework to understand transcription functions of NC2α and NC2β in vivo.

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 Regulation of rRNA Synthesis by TATA-Binding Protein-Associated Factor Mot1

2007 ◽  
Vol 27 (8) ◽  
pp. 2886-2896 ◽  
Author(s):  
Arindam Dasgupta ◽  
Rebekka O. Sprouse ◽  
Sarah French ◽  
Pavel Aprikian ◽  
Robert Hontz ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Rrna Synthesis ◽  
Direct Role ◽  
Pol Ii ◽  
Associated Factor ◽  
Pol I

ABSTRACT Mot1 is an essential, conserved, TATA-binding protein (TBP)-associated factor in Saccharomyces cerevisiae with well-established roles in the global control of RNA polymerase II (Pol II) transcription. Previous results have suggested that Mot1 functions exclusively in Pol II transcription, but here we report a novel role for Mot1 in regulating transcription by RNA polymerase I (Pol I). In vivo, Mot1 is associated with the ribosomal DNA, and loss of Mot1 results in decreased rRNA synthesis. Consistent with a direct role for Mot1 in Pol I transcription, Mot1 also associates with the Pol I promoter in vitro in a reaction that depends on components of the Pol I general transcription machinery. Remarkably, in addition to Mot1's role in initiation, rRNA processing is delayed in mot1 cells. Taken together, these results support a model in which Mot1 affects the rate and efficiency of rRNA synthesis by both direct and indirect mechanisms, with resulting effects on transcription activation and the coupling of rRNA synthesis to processing.

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 A cell-specific factor represses stimulation of transcription in vitro by transcriptional enhancer factor 1.

1994 ◽  
Vol 14 (8) ◽  
pp. 5290-5299 ◽  
Author(s):  
S Chaudhary ◽  
C Brou ◽  
M E Valentin ◽  
N Burton ◽  
L Tora ◽  
...  
Keyword(s):  
Hela Cell ◽  
Binding Protein ◽  
Activation Function ◽  
Tata Binding Protein ◽  
Lymphoid Cells ◽  
Specific Factor ◽  
Transcriptional Enhancer ◽  
Cell Extracts ◽  
A Cell

Transcription in HeLa cell extracts in vitro was stimulated 8- to 10-fold by a recombinant chimera, GAL-TEF-1, consisting of the DNA-binding domain of GAL4 and the activation function of the HeLa cell activator TEF-1. In contrast, only a 2- to 3-fold stimulation was obtained with GAL-TEF-1 in extracts from BJA-B lymphoid cells. Stimulation by GAL-TEF-1 in BJA-B extracts was dramatically increased by the addition of immunopurified HeLa cell TFIID, suggesting that BJA-B TFIID lacks or contains lower quantities of a TATA-binding-protein-associated factor(s) required for the activity of the TEF-1 activation function. However, chromatography, immunopurification, and transcriptional reconstitution experiments indicated that BJA-B extracts did not lack the previously identified TATA-binding-protein-associated factors required for TEF-1 activity but rather contained a negatively acting factor(s) which inhibited transactivation by GAL-TEF-1. These results indicate that the relative lack of activity of the TEF-1 activation function in vitro in BJA-B cell extracts does not result from the absence of positively acting factors from the presence of a cell-specific negatively acting factor(s).

scholarly journals Phosphorylation of TFIIA Stimulates TATA Binding Protein-TATA Interaction and Contributes to Maximal Transcription and Viability in Yeast

1999 ◽  
Vol 19 (4) ◽  
pp. 2846-2852 ◽  
Author(s):  
Steven P. Solow ◽  
Larissa Lezina ◽  
Paul M. Lieberman
Keyword(s):  
Binding Protein ◽  
Large Subunit ◽  
Tata Binding Protein ◽  
Binding Activity ◽  
Single Amino Acid ◽  
Stable Complex ◽  
Alanine Substitution ◽  
High Level

ABSTRACT Posttranslational modification of general transcription factors may be an important mechanism for global gene regulation. The general transcription factor IIA (TFIIA) binds to the TATA binding protein (TBP) and is essential for high-level transcription mediated by various activators. Modulation of the TFIIA-TBP interaction is a likely target of transcriptional regulation. We report here that Toa1, the large subunit of yeast TFIIA, is phosphorylated in vivo and that this phosphorylation stabilizes the TFIIA-TBP-DNA complex and is required for high-level transcription. Alanine substitution of serine residues 220, 225, and 232 completely eliminated in vivo phosphorylation of Toa1, although no single amino acid substitution of these serine residues eliminated phosphorylation in vivo. Phosphorylated TFIIA was 30-fold more efficient in forming a stable complex with TBP and TATA DNA. Dephosphorylation of yeast-derived TFIIA reduced DNA binding activity, and recombinant TFIIA could be stimulated by in vitro phosphorylation with casein kinase II. Yeast strains expressing thetoa1 S220/225/232A showed reduced high-level transcriptional activity at the URA1, URA3, andHIS3 promoters but were viable. However, S220/225/232A was synthetically lethal when combined with an alanine substitution mutation at W285, which disrupts the TFIIA-TBP interface. Phosphorylation of TFIIA could therefore be an important mechanism of transcription modulation, since it stimulates TFIIA-TBP association, enhances high-level transcription, and contributes to yeast viability.

scholarly journals Functional interaction between p53, the TATA-binding protein (TBP), andTBP-associated factors in vivo.

1996 ◽  
Vol 16 (8) ◽  
pp. 4295-4304 ◽  
Author(s):  
G Farmer ◽  
J Colgan ◽  
Y Nakatani ◽  
J L Manley ◽  
C Prives
Keyword(s):  
Transcriptional Activation ◽  
Associated Factors ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Mutant P53 ◽  
Functional Interaction ◽  
General Transcription Factor ◽  
First Time

The transcriptional activator p53 is known to interact with components of the general transcription factor TFIID in vitro. To examine the relevance of these associations to transcriptional activation in vivo, plasmids expressing a p53-GAL4 chimera and Drosophila TATA-binding protein (dTBP) were transfected into Drosophila Schneider cells. p53-GAL4 and dTBP displayed a markedly synergistic effect on activated transcription from a GAL4 site-containing reporter that was at least 10-fold greater than observed with other activators tested. A mutant p53 previously shown to be defective in both transcriptional activation in vivo and in binding to TBP-associated factors (TAFs) in vitro, although still capable of binding dTBP, did not cooperate with dTBP, suggesting that TAFs may contribute to this synergy. Providing further support for this possibility, transfected dTBP assembled into rapidly sedimenting complexes and could be immunoprecipitated with anti-TAF antibodies. While overexpression of any of several TAFs did not affect basal transcription, in either the presence or the absence of cotransfected dTBP, overexpression of TAFII230 inhibited transcriptional activation mediated by p53-GAL4 as well as by GAL4-VP16 and Sp1. Overexpression of TAFII40 and TAFII60 also inhibited activation by p53-GAL4 but had negligible effects on activation by GAL4-VP16 and Sp1, while TAFII110 did not affect any of the activators. TAF-mediated inhibition of activated transcription could be rescued by high levels of exogenous dTBP, which also restored full synergy. These data demonstrate for the first time that functional interactions can occur in vivo between TBP, TAFs, and p53.

scholarly journals Multiple Functions of the Nonconserved N-Terminal Domain of Yeast TATA-Binding Protein

Genetics ◽  
2001 ◽  
Vol 158 (1) ◽  
pp. 87-93
Author(s):  
Mark Lee ◽  
Kevin Struhl
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Protein S ◽  
Tata Binding Protein ◽  
Terminal Region ◽  
Core Domain ◽  
Terminal Domain ◽  
Two Factors

Abstract The TATA-binding protein (TBP) is composed of a highly conserved core domain sufficient for TATA-element binding and preinitiation complex formation as well as a highly divergent N-terminal region that is dispensable for yeast cell viability. In vitro, removal of the N-terminal region domain enhances TBP-TATA association and TBP dimerization. Here, we examine the effects of truncation of the N-terminal region in the context of yeast TBP mutants with specific defects in DNA binding and in interactions with various proteins. For a subset of mutations that disrupt DNA binding and the response to transcriptional activators, removal of the N-terminal domain rescues their transcriptional defects. By contrast, deletion of the N-terminal region is lethal in combination with mutations on a limited surface of TBP. Although this surface is important for interactions with TFIIA and Brf1, TBP interactions with these two factors do not appear to be responsible for this dependence on the N-terminal region. Our results suggest that the N-terminal region of TBP has at least two distinct functions in vivo. It inhibits the interaction of TBP with TATA elements, and it acts positively in combination with a specific region of the TBP core domain that presumably interacts with another protein(s).

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