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.

Interspersion of an unusual GCN4 activation site with a complex transcriptional repression site in Ty2 elements of Saccharomyces cerevisiae

1993 ◽  
Vol 13 (4) ◽  
pp. 2091-2103
Author(s):  
S Türkel ◽  
P J Farabaugh
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Physiological Control ◽  
Reading Frame ◽  
Negative Region ◽  
Activation Site

Transcription of the Ty2-917 retrotransposon of Saccharomyces cerevisiae is modulated by a complex set of positive and negative elements, including a negative region located within the first open reading frame, TYA2. The negative region includes three downstream repression sites (DRSI, DRSII, and DRSIII). In addition, the negative region includes at least two downstream activation sites (DASs). This paper concerns the characterization of DASI. A 36-bp DASI oligonucleotide acts as an autonomous transcriptional activation site and includes two sequence elements which are both required for activation. We show that these sites bind in vitro the transcriptional activation protein GCN4 and that their activity in vivo responds to the level of GCN4 in the cell. We have termed the two sites GCN4 binding sites (GBS1 and GBS2). GBS1 is a high-affinity GCN4 binding site (dissociation constant, approximately 25 nM at 30 degrees C), binding GCN4 with about the affinity of a consensus UASGCN4, this though GBS1 includes two differences from the right half of the palindromic consensus site. GBS2 is more diverged from the consensus and binds GCN4 with about 20-fold-lower affinity. Nucleotides 13 to 36 of DASI overlap DRSII. Since DRSII is a transcriptional repression site, we tested whether DASI includes repression elements. We identify two sites flanking GBS2, both of which repress transcription activated by the consensus GCN4-specific upstream activation site (UASGCN4). One of these is repeated in the 12 bp immediately adjacent to DASI. Thus, in a 48-bp region of Ty2-917 are interspersed two positive and three negative transcriptional regulators. The net effect of the region must depend on the interaction of the proteins bound at these sites, which may include their competing for binding sites, and on the physiological control of the activity of these proteins.

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 A yeast ARS-binding protein activates transcription synergistically in combination with other weak activating factors.

1990 ◽  
Vol 10 (3) ◽  
pp. 887-897 ◽  
Author(s):  
A R Buchman ◽  
R D Kornberg
Keyword(s):  
Dna Sequences ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Essential Gene ◽  
Specific Binding ◽  
Binding Protein ◽  
Point Mutations ◽  
Yeast Genes

ABFI (ARS-binding protein I) is a yeast protein that binds specific DNA sequences associated with several autonomously replicating sequences (ARSs). ABFI also binds sequences located in promoter regions of some yeast genes, including DED1, an essential gene of unknown function that is transcribed constitutively at a high level. ABFI was purified by specific binding to the DED1 upstream activating sequence (UAS) and was found to recognize related sequences at several other promoters, at an ARS (ARS1), and at a transcriptional silencer (HMR E). All ABFI-binding sites, regardless of origin, provided weak UAS function in vivo when examined in test plasmids. UAS function was abolished by point mutations that reduced ABFI binding in vitro. Analysis of the DED1 promoter showed that two ABFI-binding sites combine synergistically with an adjacent T-rich sequence to form a strong constitutive activator. The DED1 T-rich element acted synergistically with all other ABFI-binding sites and with binding sites for other multifunctional yeast activators. An examination of the properties of sequences surrounding ARS1 left open the possibility that ABFI enhances the initiation of DNA replication at ARS1 by transcriptional activation.

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 Translation of Maternal TATA-Binding Protein mRNA Potentiates Basal but Not Activated Transcription in Xenopus Embryos at the Midblastula Transition

1999 ◽  
Vol 19 (12) ◽  
pp. 7972-7982 ◽  
Author(s):  
Gert Jan C. Veenstra ◽  
Olivier H. J. Destrée ◽  
Alan P. Wolffe
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Translational Regulation ◽  
Binding Protein ◽  
Cell Extract ◽  
Tata Binding Protein ◽  
Nucleosome Assembly ◽  
Midblastula Transition ◽  
Egg Extract

ABSTRACT Early embryonic development in Xenopus laevis is characterized by transcriptional repression which is relieved at the midblastula stage (MBT). Here we show that the relative abundance of TATA-binding protein (TBP) increases robustly at the MBT and that the mechanism underlying this increase is translation of maternally stored TBP RNA. We show that TBP is rate-limiting in egg extract under conditions that titrate nucleosome assembly. Precocious translation of TBP mRNA in Xenopus embryos facilitates transcription before the MBT, without requiring TBP to be prebound to the promoter before injection. This effect is transient in the absence of chromatin titration and is sustained when chromatin is titrated. These data show that translational regulation of TBP RNA contributes to limitations on the transcriptional capacity before the MBT. Second, we examined the ability of trans-acting factors to contribute to promoter activity before the MBT. Deletion of cis-acting elements does not affect histone H2B transcription in egg extract, a finding indicative of limited trans-activation. Moreover, in the context of the intact promoter, neither the transcriptional activator Oct-1, nor TBP, nor TFIID enable transcriptional activation in vitro. HeLa cell extract, however, reconstitutes activated transcription in mixed extracts. These data suggest a deficiency in egg extract cofactors required for activated transcription. We show that the capacity for activated H2B transcription is gradually acquired at the early gastrula transition. This transition occurs well after the blastula stage when the basal transcription machinery can first be complemented with TBP.

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 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.

Sign in / Sign up

Export Citation Format

Share Document