Biomedical subjects

P Anthony Weil

Publications and source records attributed to P Anthony Weil.

13 recordsLinked to original sources

PubMed2006

Yeast TFIID serves as a coactivator for Rap1p by direct protein-protein interaction.

In vivo studies have previously shown that Saccharomyces cerevisiae ribosomal protein (RP) gene expression is controlled by the transcription factor repressor activator protein 1 (Rap1p) in a TFIID-dependent fashion. Here we have tested the hypothesis that yeast TFIID serves as a coactivator for RP gene transcription by directly interacting with Rap1p. We have found that purified recombinant Rap1p specifically interacts with purified TFIID in pull-down assays, and we have mapped the domains of Rap1p and subunits of TFIID responsible. In vitro transcription of a UAS(RAP1) enhancer-driven reporter gene requires both Rap1p and TFIID and is independent of the Fhl1p-Ifh1p coregulator. UAS(RAP1) enhancer-driven transactivation in extracts depleted of both Rap1p and TFIID is efficiently rescued by addition of physiological amounts of these two purified factors but not TATA-binding protein. We conclude that Rap1p and TFIID directly interact and that this interaction contributes importantly to RP gene transcription.

Binding, Competitive↗

PubMed2004

Mapping key functional sites within yeast TFIID.

The transcription factor TFIID, composed of the TATA box-binding protein (TBP) and 14 TBP-associated factors (TAFs), plays a key role in the regulation of gene expression by RNA polymerase II. The structure of yeast TFIID, as determined by electron microscopy and digital image analysis, is formed by three lobes, labelled A-C, connected by thin linking domains. Immunomapping revealed that TFIID contains two copies of the WD-40 repeat-containing TAF5 and that TAF5 contributes to the linkers since its C- and N-termini were found in different lobes. This property was confirmed by the finding that a recombinant complex containing TAF5 complexed with six histone fold containing TAFs was able to form a trilobed structure. Moreover, the N-terminal domain of TAF1 was mapped in lobe C, whereas the histone acetyltransferase domain resides in lobe A along with TAF7. TBP was found in the linker domain between lobes A and C in a way that the N-terminal 100 residues of TAF1 are spanned over it. The implications of these data with regard to TFIID function are discussed.

Acetyltransferases↗

PubMed2004

Molecular and genetic characterization of a Taf1p domain essential for yeast TFIID assembly.

Yeast Taf1p is an integral component of the multiprotein transcription factor TFIID. By using coimmunoprecipitation assays, coupled with a comprehensive set of deletion mutants encompassing the entire open reading frame of TAF1, we have discovered an essential role of a small portion of yeast Taf1p. This domain of Taf1p, termed region 4, consisting of amino acids 200 to 303, contributes critically to the assembly and stability of the 15-subunit TFIID holocomplex. Region 4 of Taf1p is mutationally sensitive, can assemble several Tafps into a partial TFIID complex, and interacts directly with Taf4p and Taf6p. Mutations in Taf1p-region 4 induce temperature-conditional growth of yeast cells. At the nonpermissive temperature these mutations have drastic effects on both TFIID integrity and mRNA synthesis. These data are consistent with the hypothesis that Taf1p subserves a critical scaffold function within the TFIID complex. The significance of these data with regard to TFIID structure and function is discussed.

Mutation↗

PubMed2004

Cluster analysis of mass spectrometry data reveals a novel component of SAGA.

The SAGA histone acetyltransferase and TFIID complexes play key roles in eukaryotic transcription. Using hierarchical cluster analysis of mass spectrometry data to identify proteins that copurify with components of the budding yeast TFIID transcription complex, we discovered that an uncharacterized protein corresponding to the YPL047W open reading frame significantly associated with shared components of the TFIID and SAGA complexes. Using mass spectrometry and biochemical assays, we show that YPL047W (SGF11, 11-kDa SAGA-associated factor) is an integral subunit of SAGA. However, SGF11 does not appear to play a role in SAGA-mediated histone acetylation. DNA microarray analysis showed that SGF11 mediates transcription of a subset of SAGA-dependent genes, as well as SAGA-independent genes. SAGA purified from a sgf11 Delta deletion strain has reduced amounts of Ubp8p, and a ubp8 Delta deletion strain shows changes in transcription similar to those seen with the sgf11 Delta deletion strain. Together, these data show that Sgf11p is a novel component of the yeast SAGA complex and that SGF11 regulates transcription of a subset of SAGA-regulated genes. Our data suggest that the role of SGF11 in transcription is independent of SAGA's histone acetyltransferase activity but may involve Ubp8p recruitment to or stabilization in SAGA.

Acetyltransferases↗

PubMed2003

High-affinity DNA binding by a Mot1p-TBP complex: implications for TAF-independent transcription.

Yeast Mot1p, an abundant conserved member of the Snf2p-ATPase family of proteins, both dissociates TBP from DNA in vitro using the energy of ATP and represses gene transcription in vivo, yet paradoxically, loss of Mot1p function also leads to decreased transcription of certain genes. We conducted experiments utilizing fluorescently labeled DNA, TBP, fluorescence anisotropy spectroscopy and native gel electrophoresis to study Mot1p action. We have made a number of observations, the most intriguing being that a stable Mot1p-TBP complex has the ability to bind TATA DNA with high affinity, albeit with dramatically altered specificity. We propose that this altered TBP-DNA recognition is integral to Mot1p's ability to regulate transcription, and further postulate that the Mot1p-TBP complex delivers TBP to TAF-independent mRNA encoding genes.

Adenosine Triphosphatases↗

PubMed2002

Use of a genetically introduced cross-linker to identify interaction sites of acidic activators within native transcription factor IID and SAGA.

An important goal is to identify the direct activation domain (AD)-interacting components of the transcriptional machinery within the context of native complexes. Toward this end, we first demonstrate that the multisubunit TFIID, SAGA, mediator, and Swi/Snf coactivator complexes from transcriptionally competent whole-cell yeast extracts were all capable of specifically interacting with the prototypic acidic ADs of Gal4 and VP16. We then used hexahistidine tags as genetically introduced activation domain-localized cross-linking receptors. In combination with immunological reagents against all subunits of TFIID and SAGA, we systematically identified the direct AD-interacting subunits within the AD-TFIID and AD-SAGA coactivator complexes enriched from whole-cell extracts and confirmed these results using purified TFIID and partially purified SAGA. Both ADs directly cross-linked to TBP and to a subset of TFIID and SAGA subunits that carry histone-fold motifs.

Acetyltransferases↗

PubMed2002

Mot1p is essential for TBP recruitment to selected promoters during in vivo gene activation.

Recruitment of TATA-binding protein (TBP) is central to activation of transcription by RNA polymerase II (pol II). This depends upon co-activator proteins including TBP-associated factors (TAFs). Yeast Mot1p was identified as a general transcriptional repressor in genetic screens and is also found associated with TBP. To obtain insight into Mot1p function in vivo, we determined the mRNA expression profile of the mot1-1 temperature-sensitive (Ts) strain. Unexpectedly, this indicated that Mot1p mostly plays a positive role for transcription. For one potential activation target, HXT2, we analyzed promoter recruitment of Mot1p, TBP, Taf1p (Taf130p) and pol II by chromatin immunoprecipitation assays. Whereas TBP becomes stably associated upon activation of the HXT2 and HXT4 promoters, Mot1p showed only a transient association. TBP recruitment was compromised in two different mot1 mutant strains, but was only moderately affected in a taf1 Ts strain. Together, our data indicate that Mot1p can assist in recruitment of TBP on promoters during gene activation in vivo.

Adenosine Triphosphatases↗

PubMed2002

Functional analysis of the TFIID-specific yeast TAF4 (yTAF(II)48) reveals an unexpected organization of its histone-fold domain.

Yeast TFIID comprises the TATA binding protein and 14 TBP-associated factors (TAF(II)s), nine of which contain histone-fold domains (HFDs). The C-terminal region of the TFIID-specific yTAF4 (yTAF(II)48) containing the HFD shares strong sequence similarity with Drosophila (d)TAF4 (dTAF(II)110) and human TAF4 (hTAF(II)135). A structure/function analysis of yTAF4 demonstrates that the HFD, a short conserved C-terminal domain (CCTD), and the region separating them are all required for yTAF4 function. Temperature-sensitive mutations in the yTAF4 HFD alpha2 helix or the CCTD can be suppressed upon overexpression of yTAF12 (yTAF(II)68). Moreover, coexpression in Escherichia coli indicates direct yTAF4-yTAF12 heterodimerization optimally requires both the yTAF4 HFD and CCTD. The x-ray crystal structure of the orthologous hTAF4-hTAF12 histone-like heterodimer indicates that the alpha3 region within the predicted TAF4 HFD is unstructured and does not correspond to the bona fide alpha3 helix. Our functional and biochemical analysis of yTAF4, rather provides strong evidence that the HFD alpha3 helix of the TAF4 family lies within the CCTD. These results reveal an unexpected and novel HFD organization in which the alpha3 helix is separated from the alpha2 helix by an extended loop containing a conserved functional domain.

Amino Acid Sequence↗

PubMed2002

A method for plasmid purification directly from yeast.

A rapid technique for purifying plasmids from yeast Saccharomyces cerevisiae is described that yields high-quality DNA suitable for bacterial transformation, yeast transformation, and direct DNA sequencing. The method requires only small culture volumes and proprietary bacterial plasmid miniprep kits that allow one to simultaneously prepare a large number of samples in a very short period of time while avoiding the use of toxic organic chemicals. Both yeast single-copy CEN/ARS and high-copy 2micro shuttle plasmids can be isolated using this method. This technique is useful for plasmid purification from yeast two-hybrid experiments as well as yeast genetics and molecular biology experiments.

DNA, Bacterial↗

PubMed2002

Mapping histone fold TAFs within yeast TFIID.

The transcription factor TFIID is a large multiprotein complex, composed of the TATA box-binding protein (TBP) and 14 TBP-associated factors (TAFs), which plays a key role in the regulation of gene expression by RNA polymerase II. The three-dimensional structure of yeast (y) TFIID, determined at approximately 3 nm resolution by electron microscopy and image analysis, resembles a molecular clamp formed by three major lobes connected by thin linking domains. The yTFIID is structurally similar to the human factor although the clamp appears more closed in the yeast complex, probably reflecting the conformational flexibility of the structure. Immunolabelling experiments showed that nine TAFs that contain the histone fold structural motif were located in three distinct substructures of TFIID. The distribution of these TAFs showed that the previously reported pair-wise interactions between histone fold domain (HFD)-containing TAFs are likely to occur in the native yTFIID complex. Most of the HFD-containing TAFs have been found in two distinct lobes, thus revealing an unexpected and novel molecular organization of TFIID.

Amino Acid Motifs↗

PubMed2002

Proteomics of the eukaryotic transcription machinery: identification of proteins associated with components of yeast TFIID by multidimensional mass spectrometry.

The general transcription factor TFIID is a multisubunit complex of TATA-binding protein (TBP) and 14 distinct TBP-associated factors (TAFs). Although TFIID constituents are required for transcription initiation of most mRNA encoding genes, the mechanism of TFIID action remains unclear. To gain insight into TFIID function, we sought to generate a proteomic catalogue of proteins specifically interacting with TFIID subunits. Toward this end, TFIID was systematically immunopurified by using polyclonal antibodies directed against each subunit, and the constellation of TBP- and TAF-associated proteins was directly identified by coupled multidimensional liquid chromatography and tandem mass spectrometry. A number of novel protein-protein associations were observed, and several were characterized in detail. These interactions include association between TBP and the RSC chromatin remodeling complex, the TAF17p-dependent association of the Swi6p transactivator protein with TFIID, and the identification of three novel subunits of the SAGA acetyltransferase complex, including a putative ubiquitin-specific protease component. Our results provide important new insights into the mechanisms of mRNA gene transcription and demonstrate the feasibility of constructing a complete proteomic interaction map of the eukaryotic transcription apparatus.

Chromatography, Liquid↗

PubMed2002

Molecular characterization of Saccharomyces cerevisiae TFIID.

We previously defined Saccharomyces cerevisiae TFIID as a 15-subunit complex comprised of the TATA binding protein (TBP) and 14 distinct TBP-associated factors (TAFs). In this report we give a detailed biochemical characterization of this general transcription factor. We have shown that yeast TFIID efficiently mediates both basal and activator-dependent transcription in vitro and displays TATA box binding activity that is functionally distinct from that of TBP. Analyses of the stoichiometry of TFIID subunits indicated that several TAFs are present at more than 1 copy per TFIID complex. This conclusion was further supported by coimmunoprecipitation experiments with a systematic family of (pseudo)diploid yeast strains that expressed epitope-tagged and untagged alleles of the genes encoding TFIID subunits. Based on these data, we calculated a native molecular mass for monomeric TFIID. Purified TFIID behaved in a fashion consistent with this calculated molecular mass in both gel filtration and rate-zonal sedimentation experiments. Quite surprisingly, although the TAF subunits of TFIID cofractionated as a single complex, TBP did not comigrate with the TAFs during either gel filtration chromatography or rate-zonal sedimentation, suggesting that TBP has the ability to dynamically associate with the TFIID TAFs. The results of direct biochemical exchange experiments confirmed this hypothesis. Together, our results represent a concise molecular characterization of the general transcription factor TFIID from S. cerevisiae.

Animals↗

PubMed2002

Distinct mutations in yeast TAF(II)25 differentially affect the composition of TFIID and SAGA complexes as well as global gene expression patterns.

The RNA polymerase II transcription factor TFIID, composed of the TATA-binding protein (TBP) and TBP-associated factors (TAF(II)s), nucleates preinitiation complex formation at protein-coding gene promoters. SAGA, a second TAF(II)-containing multiprotein complex, is involved in transcription regulation in Saccharomyces cerevisiae. One of the essential protein components common to SAGA and TFIID is yTAF(II)25. We define a minimal evolutionarily conserved 91-amino-acid region of TAF(II)25 containing a histone fold domain that is necessary and sufficient for growth in vivo. Different temperature-sensitive mutations of yTAF(II)25 or chimeras with the human homologue TAF(II)30 arrested cell growth at either the G(1) or G(2)/M cell cycle phase and displayed distinct phenotypic changes and gene expression patterns. Immunoprecipitation studies revealed that TAF(II)25 mutation-dependent gene expression and phenotypic changes correlated at least partially with the integrity of SAGA and TFIID. Genome-wide expression analysis revealed that the five TAF(II)25 temperature-sensitive mutant alleles individually affect the expression of between 18 and 33% of genes, whereas taken together they affect 64% of all class II genes. Thus, different yTAF(II)25 mutations induce distinct phenotypes and affect the regulation of different subsets of genes, demonstrating that no individual TAF(II) mutant allele reflects the full range of its normal functions.

Base Composition↗