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 An Array of Coactivators Is Required for Optimal Recruitment of TATA Binding Protein and RNA Polymerase II by Promoter-Bound Gcn4p

2004 ◽  
Vol 24 (10) ◽  
pp. 4104-4117 ◽  
Author(s):  
Hongfang Qiu ◽  
Cuihua Hu ◽  
Sungpil Yoon ◽  
Krishnamurthy Natarajan ◽  
Mark J. Swanson ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Pol Ii ◽  
Elongation Phase ◽  
Target Promoters

ABSTRACT Wild-type transcriptional activation by Gcn4p is dependent on multiple coactivators, including SAGA, SWI/SNF, Srb mediator, CCR4-NOT, and RSC, which are all recruited by Gcn4p to its target promoters in vivo. It was not known whether these coactivators are required for assembly of the preinitiation complex (PIC) or for subsequent steps in the initiation or elongation phase of transcription. We find that mutations in subunits of these coactivators reduce the recruitment of TATA binding protein (TBP) and RNA polymerase II (Pol II) by Gcn4p at ARG1, ARG4, and SNZ1, implicating all five coactivators in PIC assembly at Gcn4p target genes. Recruitment of Pol II at SNZ1 and ARG1 was eliminated by mutations in TBP or by deletion of the TATA box, indicating that TBP binding is a prerequisite for Pol II recruitment by Gcn4p. However, several mutations in SAGA subunits and deletion of SRB10 had a greater impact on promoter occupancy of Pol II versus TBP, suggesting that SAGA and Srb mediator can promote Pol II binding independently of their stimulatory effects on TBP recruitment. Our results reveal an unexpected complexity in the cofactor requirements for the enhancement of PIC assembly by a single activator protein.

scholarly journals SWI-SNF Complex Participation in Transcriptional Activation at a Step Subsequent to Activator Binding

1998 ◽  
Vol 18 (4) ◽  
pp. 1774-1782 ◽  
Author(s):  
Michael P. Ryan ◽  
Rachael Jones ◽  
Randall H. Morse
Keyword(s):  
Rna Polymerase Ii ◽  
Reporter Gene ◽  
Chromatin Remodeling ◽  
Transcriptional Activation ◽  
Transcriptional Activator ◽  
Major Effect ◽  
Yeast Cells ◽  
Tata Element ◽  
Promoter Recognition

ABSTRACT The SWI-SNF complex in yeast and related complexes in higher eukaryotes have been implicated in assisting gene activation by overcoming the repressive effects of chromatin. We show that the ability of the transcriptional activator GAL4 to bind to a site in a positioned nucleosome is not appreciably impaired in swimutant yeast cells. However, chromatin remodeling that depends on a transcriptional activation domain shows a considerable, although not complete, SWI-SNF dependence, suggesting that the SWI-SNF complex exerts its major effect at a step subsequent to activator binding. We tested this idea further by comparing the SWI-SNF dependence of a reporter gene based on the GAL10 promoter, which has an accessible upstream activating sequence and a nucleosomal TATA element, with that of a CYC1-lacZ reporter, which has a relatively accessible TATA element. We found that the GAL10-based reporter gene showed a much stronger SWI-SNF dependence than did theCYC1-lacZ reporter with several different activators. Remarkably, transcription of the GAL10-based reporter by a GAL4-GAL11 fusion protein showed a nearly complete requirement for the SWI-SNF complex, strongly suggesting that SWI-SNF is needed to allow access of TFIID or the RNA polymerase II holoenzyme. Taken together, our results demonstrate that chromatin remodeling in vivo can occur by both SWI-SNF-dependent and -independent avenues and suggest that the SWI-SNF complex exerts its major effect in transcriptional activation at a step subsequent to transcriptional activator-promoter recognition.

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.

Differential specificity for binding of retinoblastoma binding protein 2 to RB, p107, and TATA-binding protein

1994 ◽  
Vol 14 (11) ◽  
pp. 7256-7264
Author(s):  
Y W Kim ◽  
G A Otterson ◽  
R A Kratzke ◽  
A B Coxon ◽  
F J Kaye
Keyword(s):  
Binding Site ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Binding Activity ◽  
Reversible Binding ◽  
Complex Interactions ◽  
Differential Binding ◽  
Cellular Transcription

The growth suppressor activities of the RB and p107 products are believed to be mediated by the reversible binding of a heterogeneous family of cellular proteins to a conserved T/E1A pocket domain that is present within both proteins. To study the functional role of these interactions, we examined the properties of cellular retinoblastoma binding protein 2 (RBP2) binding to RB, p107, and the related TATA-binding protein (TBP) product. We observed that although RBP2 bound exclusively to the T/E1A pocket of p107, it could interact with RB through independent T/E1A and non-T/E1A domains and with TBP only through the non-T/E1A domain. Consistent with this observation, we found that a mutation within the Leu-X-Cys-X-Glu motif of RBP2 resulted in loss of ability to precipitate p107, while RB- and TBP-binding activities were retained. We located the non-T/E1A binding site of RBP2 on a 15-kDa fragment that is independent from the Leu-X-Cys-X-Glu motif and encodes binding activity for RB and TBP but does not interact with p107. Despite the presence of a non-T/E1A binding site, however, recombinant RBP2 retained the ability to preferentially precipitate active hypophosphorylated RB from whole-cell lysates. In addition, we found that cotransfection of RBP2 can reverse in vivo RB-mediated suppression of E2F activity. These findings confirm the differential binding specificities of the related RB, p107, and TBP proteins and support the presence of multifunctional domains on the nuclear RBP2 product which may allow complex interactions with the cellular transcription machinery.

scholarly journals Three functional classes of transcriptional activation domain.

1996 ◽  
Vol 16 (5) ◽  
pp. 2044-2055 ◽  
Author(s):  
J Blau ◽  
H Xiao ◽  
S McCracken ◽  
P O'Hare ◽  
J Greenblatt ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Type I ◽  
Rnase Protection ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Transactivation Domains ◽  
Functional Classes

We have studied the abilities of different transactivation domains to stimulate the initiation and elongation (postinitiation) steps of RNA polymerase II transcription in vivo. Nuclear run-on and RNase protection analyses revealed three classes of activation domains: Sp1 and CTF stimulated initiation (type I); human immunodeficiency virus type 1 Tat fused to a DNA binding domain stimulated predominantly elongation (type IIA); and VP16, p53, and E2F1 stimulated both initiation and elongation (type IIB). A quadruple point mutation of VP16 converted it from a type IIB to a type I activator. Type I and type IIA activators synergized with one another but not with type IIB activators. This observation implies that synergy can result from the concerted action of factors stimulating two different steps in transcription: initiation and elongation. The functional differences between activators may be explained by the different contacts they make with general transcription factors. In support of this idea, we found a correlation between the abilities of activators, including Tat, to stimulate elongation and their abilities to bind TFIIH.

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 TATA-Binding Protein Mutants That Are Lethal in the Absence of the Nhp6 High-Mobility-Group Protein

2004 ◽  
Vol 24 (14) ◽  
pp. 6419-6429 ◽  
Author(s):  
Peter Eriksson ◽  
Debabrata Biswas ◽  
Yaxin Yu ◽  
James M. Stewart ◽  
David J. Stillman
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Synthetic Lethality ◽  
Binding Protein ◽  
High Mobility ◽  
Tata Binding Protein ◽  
High Mobility Group ◽  
Multicopy Plasmid ◽  
Pol Ii ◽  
Pol Iii

ABSTRACT The Saccharomyces cerevisiae Nhp6 protein is related to the high-mobility-group B family of architectural DNA-binding proteins that bind DNA nonspecifically but bend DNA sharply. Nhp6 is involved in transcriptional activation by both RNA polymerase II (Pol II) and Pol III. Our previous genetic studies have implicated Nhp6 in facilitating TATA-binding protein (TBP) binding to some Pol II promoters in vivo, and we have used a novel genetic screen to isolate 32 new mutations in TBP that are viable in wild-type cells but lethal in the absence of Nhp6. The TBP mutations that are lethal in the absence of Nhp6 cluster in three regions: on the upper surface of TBP that may have a regulatory role, near residues that contact Spt3, or near residues known to contact either TFIIA or Brf1 (in TFIIIB). The latter set of mutations suggests that Nhp6 becomes essential when a TBP mutant compromises its ability to interact with either TFIIA or Brf1. Importantly, the synthetic lethality for some of the TBP mutations is suppressed by a multicopy plasmid with SNR6 or by an spt3 mutation. It has been previously shown that nhp6ab mutants are defective in expressing SNR6, a Pol III-transcribed gene encoding the U6 splicing RNA. Chromatin immunoprecipitation experiments show that TBP binding to SNR6 is reduced in an nhp6ab mutant. Nhp6 interacts with Spt16/Pob3, the yeast equivalent of the FACT elongation complex, consistent with nhp6ab cells being extremely sensitive to 6-azauracil (6-AU). However, this 6-AU sensitivity can be suppressed by multicopy SNR6 or BRF1. Additionally, strains with SNR6 promoter mutations are sensitive to 6-AU, suggesting that decreased SNR6 RNA levels contribute to 6-AU sensitivity. These results challenge the widely held belief that 6-AU sensitivity results from a defect in transcriptional elongation.

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 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.

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