scholarly journals Region of Yeast TAF 130 Required for TFIID To Associate with Promoters

2001 ◽  
Vol 21 (4) ◽  
pp. 1145-1154 ◽  
Author(s):  
Mario Mencı́a ◽  
Kevin Struhl
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Thermal Inactivation ◽  
Yeast Cells ◽  
Activation Domains ◽  
Core Promoters ◽  
Transcriptional Activation Domains ◽  
Tfiid Complex

ABSTRACT TFIID, a multiprotein complex comprising the TATA-binding protein (TBP) and TBP-associated factors (TAFs), associates specifically with core promoters and nucleates the assembly the RNA polymerase II transcription machinery. In yeast cells, TFIID is not generally required for transcription, although it plays an important role at many promoters. Understanding of the specific functions and physiological roles of individual TAFs within TFIID has been hampered by the fact that depletion or thermal inactivation of individual TAFs generally results in dissociation of the TFIID complex. We describe here C-terminally deleted derivatives of yeast TAF130 that assemble into normal TFIID complexes but are transcriptionally inactive in vivo. In vivo, these mutant TFIID complexes are dramatically reduced in their ability to associate with all promoters tested. In vitro, a TFIID complex containing a deleted form of TAF130 associates poorly with DNA, but it is unaffected for interacting with transcriptional activation domains. These results suggest that the C-terminal region of TAF130 is required for TFIID to associate with promoters.

RNA polymerase II cofactor PC2 facilitates activation of transcription by GAL4-AH in vitro

1994 ◽  
Vol 14 (6) ◽  
pp. 3927-3937
Author(s):  
M Kretzschmar ◽  
G Stelzer ◽  
R G Roeder ◽  
M Meisterernst
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Activation Domains ◽  
Positive Effects ◽  
Activation Of Transcription ◽  
Transcriptional Activation Domains ◽  
Necessary And Sufficient ◽  
Specific Pathway

We have isolated from a crude Hela cell cofactor fraction (USA) a novel positive cofactor that cooperates with the general transcription machinery to effect efficient stimulation of transcription by GAL4-AH, a derivative of the Saccharomyces cerevisiae regulatory factor GAL4. PC2 was shown to be a 500-kDa protein complex and to be functionally and biochemically distinct from native TFIID and previously identified cofactors. In the presence of native TFIID and other general factors, PC2 was necessary and sufficient for activation by GAL4-AH. Cofactor function was specific for transcriptional activation domains of GAL4-AH. The repressor histone H1 further potentiated but was not required for activation of transcription by GAL4-AH. On the basis of the observation that PC2 exerts entirely positive effects on transcription, we propose a model in which PC2 increases the activity of the preinitiation complex in the presence of an activator, thereby establishing a specific pathway during activation of RNA polymerase II.

scholarly journals ADR1-Mediated Transcriptional Activation Requires the Presence of an Intact TFIID Complex

1998 ◽  
Vol 18 (10) ◽  
pp. 5861-5867 ◽  
Author(s):  
Philip B. Komarnitsky ◽  
Edward R. Klebanow ◽  
P. Anthony Weil ◽  
Clyde L. Denis
Keyword(s):  
Transcriptional Activation ◽  
Yeast Cells ◽  
Binding Studies ◽  
Whole Cell ◽  
Transactivation Domains ◽  
Cell Extracts ◽  
Genes Expression ◽  
Tfiid Complex

ABSTRACT The yeast transcriptional activator ADR1, which is required forADH2 and other genes’ expression, contains four transactivation domains (TADs). While previous studies have shown that these TADs act through GCN5 and ADA2, and presumably TFIIB, other factors are likely to be involved in ADR1 function. In this study, we addressed the question of whether TFIID is also required for ADR1 action. In vitro binding studies indicated that TADI of ADR1 was able to retain TAFII90 from yeast extracts and TADII could retain TBP and TAFII130/145. TADIV, however, was capable of retaining multiple TAFIIs, suggesting that TADIV was binding TFIID from yeast whole-cell extracts. The ability of TADIV truncation derivatives to interact with TFIID correlated with their transcription activation potential in vivo. In addition, the ability of LexA-ADR1-TADIV to activate transcription in vivo was compromised by a mutation in TAFII130/145. ADR1 was found to associate in vivo with TFIID in that immunoprecipitation of either TAFII90 or TBP from yeast whole-cell extracts specifically coimmunoprecipitated ADR1. Most importantly, depletion of TAFII90 from yeast cells dramatically reducedADH2 derepression. These results indicate that ADR1 physically associates with TFIID and that its ability to activate transcription requires an intact TFIID complex.

scholarly journals RNA polymerase II cofactor PC2 facilitates activation of transcription by GAL4-AH in vitro.

1994 ◽  
Vol 14 (6) ◽  
pp. 3927-3937 ◽  
Author(s):  
M Kretzschmar ◽  
G Stelzer ◽  
R G Roeder ◽  
M Meisterernst
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Activation Domains ◽  
Positive Effects ◽  
Activation Of Transcription ◽  
Transcriptional Activation Domains ◽  
Necessary And Sufficient ◽  
Specific Pathway

We have isolated from a crude Hela cell cofactor fraction (USA) a novel positive cofactor that cooperates with the general transcription machinery to effect efficient stimulation of transcription by GAL4-AH, a derivative of the Saccharomyces cerevisiae regulatory factor GAL4. PC2 was shown to be a 500-kDa protein complex and to be functionally and biochemically distinct from native TFIID and previously identified cofactors. In the presence of native TFIID and other general factors, PC2 was necessary and sufficient for activation by GAL4-AH. Cofactor function was specific for transcriptional activation domains of GAL4-AH. The repressor histone H1 further potentiated but was not required for activation of transcription by GAL4-AH. On the basis of the observation that PC2 exerts entirely positive effects on transcription, we propose a model in which PC2 increases the activity of the preinitiation complex in the presence of an activator, thereby establishing a specific pathway during activation of RNA polymerase II.

scholarly journals Transcriptional Activation in Yeast Cells Lacking Transcription Factor IIA

Genetics ◽  
1999 ◽  
Vol 153 (4) ◽  
pp. 1573-1581 ◽  
Author(s):  
Susanna Chou ◽  
Sukalyan Chatterjee ◽  
Mark Lee ◽  
Kevin Struhl
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Large Subunit ◽  
Yeast Cells ◽  
Specific Cell ◽  
Wild Type ◽  
Activation Domains ◽  
Cycle Arrest ◽  
General Transcription Factor

Abstract The general transcription factor IIA (TFIIA) forms a complex with TFIID at the TATA promoter element, and it inhibits the function of several negative regulators of the TATA-binding protein (TBP) subunit of TFIID. Biochemical experiments suggest that TFIIA is important in the response to transcriptional activators because activation domains can interact with TFIIA, increase recruitment of TFIID and TFIIA to the promoter, and promote isomerization of the TFIID-TFIIA-TATA complex. Here, we describe a double-shut-off approach to deplete yeast cells of Toa1, the large subunit of TFIIA, to <1% of the wild-type level. Interestingly, such TFIIA-depleted cells are essentially unaffected for activation by heat shock factor, Ace1, and Gal4-VP16. However, depletion of TFIIA causes a general two- to threefold decrease of transcription from most yeast promoters and a specific cell-cycle arrest at the G2-M boundary. These results indicate that transcriptional activation in vivo can occur in the absence of TFIIA.

Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain

1994 ◽  
Vol 14 (9) ◽  
pp. 6056-6067
Author(s):  
M Tanaka ◽  
W Herr
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Pou Domain

The POU domain activator Oct-2 contains an N-terminal glutamine-rich transcriptional activation domain. An 18-amino-acid segment (Q18III) from this region reconstituted a fully functional activation domain when tandemly reiterated and fused to either the Oct-2 or GAL4 DNA-binding domain. A minimal transcriptional activation domain likely requires three tandem Q18III segments, because one or two tandem Q18III segments displayed little activity, whereas three to five tandem segments were active and displayed increasing activity with increasing copy number. As with natural Oct-2 activation domains, in our assay a reiterated activation domain required a second homologous or heterologous activation domain to stimulate transcription effectively when fused to the Oct-2 POU domain. These results suggest that there are different levels of synergy within and among activation domains. Analysis of reiterated activation domains containing mutated Q18III segments revealed that leucines and glutamines, but not serines or threonines, are critical for activity in vivo. Curiously, several reiterated activation domains that were inactive in vivo were active in vitro, suggesting that there are significant functional differences in our in vivo and in vitro assays. Reiteration of a second 18-amino-acid segment from the Oct-2 glutamine-rich activation domain (Q18II) was also active, but its activity was DNA-binding domain specific, because it was active when fused to the GAL4 than to the Oct-2 DNA-binding domain. The ability of separate short peptide segments derived from a single transcriptional activation domain to activate transcription after tandem reiteration emphasizes the flexible and modular nature of a transcriptional activation domain.

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.

scholarly journals Yeast NC2 Associates with the RNA Polymerase II Preinitiation Complex and Selectively Affects Transcription In Vivo

2001 ◽  
Vol 21 (8) ◽  
pp. 2736-2742 ◽  
Author(s):  
Joseph V. Geisberg ◽  
Frank C. Holstege ◽  
Richard A. Young ◽  
Kevin Struhl
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Negative Regulator ◽  
Preinitiation Complex ◽  
Positive Role ◽  
Pol Ii ◽  
Basal Transcription Factors

ABSTRACT NC2 (Dr1-Drap1 or Bur6-Ydr1) has been characterized in vitro as a general negative regulator of RNA polymerase II (Pol II) transcription that interacts with TATA-binding protein (TBP) and inhibits its function. Here, we show that NC2 associates with promoters in vivo in a manner that correlates with transcriptional activity and with occupancy by basal transcription factors. NC2 rapidly associates with promoters in response to transcriptional activation, and it remains associated under conditions in which transcription is blocked after assembly of the Pol II preinitiation complex. NC2 positively and negatively affects approximately 17% of Saccharomyces cerevisiaegenes in a pattern that resembles the response to general environmental stress. Relative to TBP, NC2 occupancy is high at promoters where NC2 is positively required for normal levels of transcription. Thus, NC2 is associated with the Pol II preinitiation complex, and it can play a direct and positive role at certain promoters in vivo.

scholarly journals The Gcn4p Activation Domain Interacts Specifically In Vitro with RNA Polymerase II Holoenzyme, TFIID, and the Adap-Gcn5p Coactivator Complex

1998 ◽  
Vol 18 (3) ◽  
pp. 1711-1724 ◽  
Author(s):  
Connie M. Drysdale ◽  
Belinda M. Jackson ◽  
Richard McVeigh ◽  
Edward R. Klebanow ◽  
Yu Bai ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Adapter Proteins ◽  
Activation Domain ◽  
Hydrophobic Residues ◽  
Cell Extracts ◽  
Tata Box Binding Protein ◽  
Rna Polymerase Ii Holoenzyme

ABSTRACT The Gcn4p activation domain contains seven clusters of hydrophobic residues that make additive contributions to transcriptional activation in vivo. We observed efficient binding of a glutathioneS-transferase (GST)–Gcn4p fusion protein to components of three different coactivator complexes in Saccharomyces cerevisiae cell extracts, including subunits of transcription factor IID (TFIID) (yeast TAFII20 [yTAFII20], yTAFII60, and yTAFII90), the holoenzyme mediator (Srb2p, Srb4p, and Srb7p), and the Adap-Gcn5p complex (Ada2p and Ada3p). The binding to these coactivator subunits was completely dependent on the hydrophobic clusters in the Gcn4p activation domain. Alanine substitutions in single clusters led to moderate reductions in binding, double-cluster substitutions generally led to greater reductions in binding than the corresponding single-cluster mutations, and mutations in four or more clusters reduced binding to all of the coactivator proteins to background levels. The additive effects of these mutations on binding of coactivator proteins correlated with their cumulative effects on transcriptional activation by Gcn4p in vivo, particularly with Ada3p, suggesting that recruitment of these coactivator complexes to the promoter is a cardinal function of the Gcn4p activation domain. As judged by immunoprecipitation analysis, components of the mediator were not associated with constituents of TFIID and Adap-Gcn5p in the extracts, implying that GST-Gcn4p interacted with the mediator independently of these other coactivators. Unexpectedly, a proportion of Ada2p coimmunoprecipitated with yTAFII90, and the yTAFII20, -60, and -90 proteins were coimmunoprecipitated with Ada3p, revealing a stable interaction between components of TFIID and the Adap-Gcn5p complex. Because GST-Gcn4p did not bind specifically to highly purified TFIID, Gcn4p may interact with TFIID via the Adap-Gcn5p complex or some other adapter proteins. The ability of Gcn4p to interact with several distinct coactivator complexes that are physically and genetically linked to TATA box-binding protein can provide an explanation for the observation that yTAFII proteins are dispensable for activation by Gcn4p in vivo.

scholarly journals Remodeling of Yeast CUP1 Chromatin Involves Activator-Dependent Repositioning of Nucleosomes over the Entire Gene and Flanking Sequences

2001 ◽  
Vol 21 (2) ◽  
pp. 534-547 ◽  
Author(s):  
Chang-Hui Shen ◽  
Benoit P. Leblanc ◽  
Jennifer A. Alfieri ◽  
David J. Clark
Keyword(s):  
Rna Polymerase Ii ◽  
Chromatin Structure ◽  
Yeast Cells ◽  
Chromatin Domain ◽  
Sliding Mechanism ◽  
Flanking Sequences ◽  
Flanking Regions ◽  
Entire Gene

ABSTRACT The yeast CUP1 gene is activated by the copper-dependent binding of the transcriptional activator, Ace1p. An episome containing transcriptionally active or inactiveCUP1 was purified in its native chromatin structure from yeast cells. The amount of RNA polymerase II on CUP1 in the purified episomes correlated with its transcriptional activity in vivo. Chromatin structures were examined by using the monomer extension technique to map translational positions of nucleosomes. The chromatin structure of an episome containing inactive CUP1 isolated from ace1Δ cells is organized into clusters of overlapping nucleosome positions separated by linkers. Novel nucleosome positions that include the linkers are occupied in the presence of Ace1p. Repositioning was observed over the entire CUP1 gene and its flanking regions, possibly over the entire episome. Mutation of the TATA boxes to prevent transcription did not prevent repositioning, implicating a chromatin remodeling activity recruited by Ace1p. These observations provide direct evidence in vivo for the nucleosome sliding mechanism proposed for remodeling complexes in vitro and indicate that remodeling is not restricted to the promoter but occurs over a chromatin domain including CUP1 and its flanking sequences.

scholarly journals ADA3: a gene, identified by resistance to GAL4-VP16, with properties similar to and different from those of ADA2.

1993 ◽  
Vol 13 (10) ◽  
pp. 5981-5989 ◽  
Author(s):  
B Piña ◽  
S Berger ◽  
G A Marcus ◽  
N Silverman ◽  
J Agapite ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Gene Disruption ◽  
Temperature Sensitive ◽  
Yeast Gene ◽  
Activation Domains ◽  
General Transcription Factors ◽  
General Transcription Factor ◽  
Selection Of

We describe the isolation of a yeast gene, ADA3, mutations in which prevent the toxicity of GAL4-VP16 in vivo. Toxicity was previously proposed to be due to the trapping of general transcription factors required at RNA polymerase II promoters (S. L. Berger, B. Piña, N. Silverman, G. A. Marcus, J. Agapite, J. L. Regier, S. J. Triezenberg, and L. Guarente, Cell 70:251-265, 1992). trans activation by VP16 as well as the acidic activation domain of GCN4 is reduced in the mutant. Other activation domains, such as those of GAL4 and HAP4, are only slightly affected in the mutant. This spectrum is similar to that observed for mutants with lesions in ADA2, a gene proposed to encode a transcriptional adaptor. The ADA3 gene is not absolutely essential for cell growth, but gene disruption mutants grow slowly and are temperature sensitive. Strains doubly disrupted for ada2 and ada3 grow no more slowly than single mutants, providing further evidence that these genes function in the same pathway. Selection of initiation sites by the general transcriptional machinery in vitro is altered in the ada3 mutant, providing a clue that ADA3 could be a novel general transcription factor involved in the response to acidic activators.

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