scholarly journals A new class of activation-defective TATA-binding protein mutants: evidence for two steps of transcriptional activation in vivo.

1996 ◽  
Vol 16 (8) ◽  
pp. 4456-4464 ◽  
Author(s):  
L A Stargell ◽  
K Struhl
Keyword(s):  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Wild Type ◽  
Protein Protein Interaction ◽  
New Class ◽  
Tata Element ◽  
Protein Mutants ◽  
Transcriptional Stimulation

Using a genetic screen, we isolated four TATA-binding protein (TBP) mutants that are specifically defective in vivo for the response to acidic activators. In contrast to previously described activation-defective TBP mutants, these TBP derivatives are not specifically defective for interactions with TATA elements or TFIIA. Three of these derivatives interact normally with a TATA element, TFIIA, TFIIB, or an acidic activation domain; presumably, they affect another protein-protein interaction important for transcriptional activation. The remaining derivative (with F-237 replaced by D) binds a TATA element with wild-type affinity, but the TBP-TATA complex has an altered electrophoretic mobility and interacts poorly with TFIIA and TFIIB; this suggests that the conformation of the TBP-TATA element complex plays a role in transcriptional activation. To determine the step at which the TBP derivatives were unable to activate transcription, we utilized an artificial recruitment assay in which TBP is targeted to the promoter via fusion to the LexA DNA-binding domain. Consistent with previous evidence that acidic activators can increase recruitment of TBP to the promoter in vivo, the activation defect of some of these TBP derivatives can be corrected by artificial recruitment. In contrast, the activation defect of the other TBP derivatives is not bypassed by artificial recruitment. Thus, these TBP mutants define two steps in the process of transcriptional stimulation by acidic activators: efficient recruitment to the TATA element and a postrecruitment interaction with a component(s) of the initiation complex.

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 Cubitus interruptus Requires DrosophilaCREB-Binding Protein To Activate wingless Expression in theDrosophila Embryo

2000 ◽  
Vol 20 (5) ◽  
pp. 1616-1625 ◽  
Author(s):  
Yang Chen ◽  
R. H. Goodman ◽  
Sarah M. Smolik
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Binding Protein ◽  
Point Mutations ◽  
Creb Binding Protein ◽  
Wild Type ◽  
Interaction Domain ◽  
Cubitus Interruptus

ABSTRACT CREB-binding protein (CBP) serves as a transcriptional coactivator in multiple signal transduction pathways. The Drosophilahomologue of CBP, dCBP, interacts with the transcription factors Cubitus interruptus (CI), MAD, and Dorsal (DL) and functions as a coactivator in several signaling pathways during Drosophiladevelopment, including the hedgehog (hh),decapentaplegic (dpp), and Tollpathways. Although dCBP is required for the expression of thehh target genes, wingless (wg) andpatched (ptc) in vivo, and potentiatesci-mediated transcriptional activation in vitro, it is not known that ci absolutely requires dCBP for its activity. We used a yeast genetic screen to identify several ci point mutations that disrupt CI-dCBP interactions. These mutant proteins are unable to transactivate a reporter gene regulated by cibinding sites and have a lower dCBP-stimulated activity than wild-type CI. When expressed exogenously in embryos, the CI point mutants cannot activate endogenous wg expression. Furthermore, a CI mutant protein that lacks the entire dCBP interaction domain functions as a negative competitor for wild-type CI activity, and the expression of dCBP antisense RNAs can suppress CI transactivation in Kc cells. Taken together, our data suggest that dCBP function is necessary forci-mediated transactivation of wg duringDrosophila embryogenesis.

scholarly journals Interdependent Recruitment of SAGA and Srb Mediator by Transcriptional Activator Gcn4p

2005 ◽  
Vol 25 (9) ◽  
pp. 3461-3474 ◽  
Author(s):  
Hongfang Qiu ◽  
Cuihua Hu ◽  
Fan Zhang ◽  
Gwo Jiunn Hwang ◽  
Mark J. Swanson ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Wild Type ◽  
General Transcription Factors ◽  
Tata Element ◽  
High Level ◽  
Target Promoters ◽  
Activation Sequence

ABSTRACT Transcriptional activation by Gcn4p is enhanced by the coactivators SWI/SNF, SAGA, and Srb mediator, which stimulate recruitment of TATA binding protein (TBP) and polymerase II to target promoters. We show that wild-type recruitment of SAGA by Gcn4p is dependent on mediator but independent of SWI/SNF function at three different promoters. Recruitment of mediator is also independent of SWI/SNF but is enhanced by SAGA at a subset of Gcn4p target genes. Recruitment of all three coactivators to ARG1 is independent of the TATA element and preinitiation complex formation, whereas efficient recruitment of the general transcription factors requires the TATA box. We propose an activation pathway involving interdependent recruitment of SAGA and Srb mediator to the upstream activation sequence, enabling SWI/SNF recruitment and the binding of TBP and other general factors to the promoter. We also found that high-level recruitment of Tra1p and other SAGA subunits is independent of the Ada2p/Ada3p/Gcn5p histone acetyltransferase module but requires Spt3p in addition to subunits required for SAGA integrity. Thus, while Tra1p can bind directly to Gcn4p in vitro, it requires other SAGA subunits for efficient recruitment in vivo.

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 The TATA-binding protein is not an essential target of the transcriptional activators Gal4p and Gcn4p in Saccharomyces cerevisiae

2003 ◽  
Vol 370 (1) ◽  
pp. 141-147 ◽  
Author(s):  
Christine BONGARDS ◽  
Boon Shang CHEW ◽  
Norbert LEHMING
Keyword(s):  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Limiting Factors ◽  
Clear Correlation ◽  
Limiting Factor ◽  
Local Concentration ◽  
Transcriptional Activators ◽  
Transcription Process

According to the recruitment model, transcriptional activators work by increasing the local concentration of one or several limiting factors for the transcription process at the target promoter. The TATA-binding protein Tbp1 has been considered as a likely candidate for such a limiting factor. We have used a series of Gal4p and Tbp1 mutants to correlate the in vivo interaction between the two proteins with the strength of activation. We find a clear correlation between activation strength and in vivo interaction for the series of Gal4p mutants. Consistently, the weaker activator Gcn4p does not interact with Tbp1. However, a corresponding analysis of the series of Tbp1 mutants revealed that Tbp1 is not an essential target of the acidic activators Gal4p and Gcn4p. Furthermore, detailed analysis of a Tbp1 mutant deficient for transcriptional activation by Gal4p revealed that the mutant is defective in interactions with five other proteins involved in the process of transcription.

scholarly journals Recruitment of CBP/p300, TATA-Binding Protein, and S8 to Distinct Regions at the N Terminus of Adenovirus E1A

2005 ◽  
Vol 79 (9) ◽  
pp. 5594-5605 ◽  
Author(s):  
Mozhgan Rasti ◽  
Roger J. A. Grand ◽  
Joe S. Mymryk ◽  
Phillip H. Gallimore ◽  
Andrew S. Turnell
Keyword(s):  
Transcriptional Activation ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Binding Protein ◽  
Cellular Transformation ◽  
Tata Binding Protein ◽  
Transformation Process ◽  
Terminal Region ◽  
Site Directed Mutagenesis

ABSTRACT The N-terminal region of the adenovirus (Ad) 12S E1A gene product targets several cellular proteins that are essential for the induction of S phase, cellular immortalization, cellular transformation, transcriptional repression, and transcriptional activation. The precise binding sites for these proteins, however, remain to be resolved. We therefore undertook an extensive site-directed mutagenesis approach to generate specific point mutants and to precisely map the binding sites for CBP, p300, TATA-binding protein (TBP), S4, S8, hGcn5, P/CAF, and Ran within the first 30 amino acids of the Ad5 12S E1A protein. We determined that although common residues within the N-terminal region can form partial binding sites for these proteins, point mutants were also generated that could discriminate between binding sites. These data indicate that AdE1A can target each of these proteins individually through distinct binding sites. It was evident, however, that the mutation of specific hydrophobic residues typically had the greatest effect upon AdE1A's ability to bind individual partners. Indeed, the mutation of L at positions 19 and 20 eliminated the ability of AdE1A to interact with any of the N-terminal binding proteins studied here. Interestingly, although TBP and S8 or CBP/p300 can exist as functional complexes, RNA interference revealed that the recruitment of either TBP, S8, or CBP/p300 to AdE1A was not dependent upon the expression of the other proteins. These data further indicate that AdE1A can target individual partner proteins in vivo and that it does not necessarily recruit these proteins indirectly as components of larger macromolecular complexes. Finally, we took advantage of the fine-mapping data to ascertain which proteins were targeted during the transformation process. Consistent with previous studies, CBP/p300 was found to be targeted by AdE1A during this process, although our data suggest that binding to other N-terminal proteins is also important for transformation.

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 SPT20/ADA5 encodes a novel protein functionally related to the TATA-binding protein and important for transcription in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (6) ◽  
pp. 3206-3213 ◽  
Author(s):  
S M Roberts ◽  
F Winston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Missense Mutations ◽  
Poor Growth ◽  
New Gene ◽  
Novel Protein

Mutations selected as suppressors of Ty and solo delta insertion mutations is Saccharomyces cerevisiae have identified a number of genes important for transcription initiation. One of these gens, SPT15, encodes the TATA-binding protein, and three others, SPT3, SPT7, and SPT8, encode proteins functionally related to the TATA-binding protein. To identify additional related functions, we have selected for new spt mutations. This work has identified one new gene, SPT20. Null mutations in SPT20 cause poor growth and a set of severe transcriptional defects very similar to those caused by null mutations in SPT3, SPT7, and SPT8 and also very similar to those caused by certain missense mutations in SPT15. Consistent with its having an important function in transcription in vivo, SPT20 was also recently identified as ADA5 and has been shown to be important for transcriptional activation (G.A. Marcus, J. Horiuchi, N. Silverman, and L. Guarente, Mol. Cell. Biol. 16:3197-3205, 1996.

scholarly journals Artificially Recruited TATA-Binding Protein Fails To Remodel Chromatin and Does Not Activate Three Promoters That Require Chromatin Remodeling

2000 ◽  
Vol 20 (16) ◽  
pp. 5847-5857 ◽  
Author(s):  
Michael P. Ryan ◽  
Grace A. Stafford ◽  
Liuning Yu ◽  
Randall H. Morse
Keyword(s):  
Binding Site ◽  
Rna Polymerase Ii ◽  
Reporter Gene ◽  
Chromatin Remodeling ◽  
Chromatin Structure ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Activation Domains

ABSTRACT Transcriptional activators are believed to work in part by recruiting general transcription factors, such as TATA-binding protein (TBP) and the RNA polymerase II holoenzyme. Activation domains also contribute to remodeling of chromatin in vivo. To determine whether these two activities represent distinct functions of activation domains, we have examined transcriptional activation and chromatin remodeling accompanying artificial recruitment of TBP in yeast (Saccharomyces cerevisiae). We measured transcription of reporter genes with defined chromatin structure by artificial recruitment of TBP and found that a reporter gene whose TATA element was relatively accessible could be activated by artificially recruited TBP, whereas two promoters, GAL10 and CHA1, that have accessible activator binding sites, but nucleosomal TATA elements, could not. A third reporter gene containing theHIS4 promoter could be activated by GAL4-TBP only when a RAP1 binding site was present, although RAP1 alone could not activate the reporter, suggesting that RAP1 was needed to open the chromatin structure to allow activation. Consistent with this interpretation, artificially recruited TBP was unable to perturb nucleosome positioning via a nucleosomal binding site, in contrast to a true activator such as GAL4, or to perturb the TATA-containing nucleosome at theCHA1 promoter. Finally, we show that activation of theGAL10 promoter by GAL4, which requires chromatin remodeling, can occur even in swi gcn5 yeast, implying that remodeling pathways independent of GCN5, the SWI-SNF complex, and TFIID can operate during transcriptional activation in vivo.

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.

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