Fluorescence-based analyses of the effects of full-length recombinant TAF130p on the interaction of TATA box-binding protein with TATA box DNA.

We have used a combination of fluorescence anisotropy spectroscopy and fluorescence-based native gel electrophoresis methods to examine the effects of the transcription factor IID-specific subunit TAF130p (TAF145p) upon the TATA box DNA binding properties of TATA box-binding protein (TBP). Purified full-length recombinant TAF130p decreases TBP-TATA DNA complex formation at equilibrium by competing directly with DNA for binding to TBP. Interestingly, we have found that full-length TAF130p is capable of binding multiple molecules of TBP with nanomolar binding affinity. The biological implications of these findings are discussed.

Molecular genetic dissection of TAF25, an essential yeast gene encoding a subunit shared by TFIID and SAGA multiprotein transcription factors.

We have performed a systematic structure-function analysis of Saccharomyces cerevisiae TAF25, an evolutionarily conserved, single-copy essential gene which encodes the 206-amino-acid TAF25p protein. TAF25p is an integral subunit of both the 15-subunit general transcription factor TFIID and the multisubunit, chromatin-acetylating transcriptional coactivator SAGA. We used hydroxylamine mutagenesis, targeted deletion, alanine-scanning mutagenesis, high-copy suppression methods, and two-hybrid screening to dissect TAF25. Temperature-sensitive mutant strains generated were used for coimmunoprecipitation and transcription analyses to define the in vivo functions of TAF25p. The results of these analyses show that TAF25p is comprised of multiple mutable elements which contribute importantly to RNA polymerase II-mediated mRNA gene transcription.

Histone folds mediate selective heterodimerization of yeast TAF(II)25 with TFIID components yTAF(II)47 and yTAF(II)65 and with SAGA component ySPT7.

We show that the yeast TFIID (yTFIID) component yTAF(II)47 contains a histone fold domain (HFD) with homology to that previously described for hTAF(II)135. Complementation in vivo indicates that the yTAF(II)47 HFD is necessary and sufficient for vegetative growth. Mutation of highly conserved residues in the alpha1 helix of the yTAF(II)47 HFD results in a temperature-sensitive phenotype which can be suppressed by overexpression of yTAF(II)25, as well as by yTAF(II)40, yTAF(II)19, and yTAF(II)60. In yeast two-hybrid and bacterial coexpression assays, the yTAF(II)47 HFD selectively heterodimerizes with yTAF(II)25, which we show contains an HFD with homology to the hTAF(II)28 family We additionally demonstrate that yTAF(II)65 contains a functional HFD which also selectively heterodimerizes with yTAF(II)25. These results reveal the existence of two novel histone-like pairs in yTFIID. The physical and genetic interactions described here show that the histone-like yTAF(II)s are organized in at least two substructures within TFIID rather than in a single octamer-like structure as previously suggested. Furthermore, our results indicate that ySPT7 has an HFD homologous to that of yTAF(II)47 which selectively heterodimerizes with yTAF(II)25, defining a novel histone-like pair in the SAGA complex.

Identification of two novel TAF subunits of the yeast Saccharomyces cerevisiae TFIID complex.

Using a combination of ion exchange and immunoaffinity chromatography we have purified the general transcription initiation factor TFIID to near homogeneity from Saccharomyces cerevisiae. Yeast TFIID is composed of TBP, the TATA box binding protein, and 14 distinct TBP-associated factors (TAFs), which range in size from 17 to 150 kDa. Twelve of the TAF subunits have been previously identified, but two, TAF48p and TAF65p, are novel. TAF48p exhibits significant sequence similarity to the conserved C-terminal region of Drosophila TAF110p, human TAF130p, and human TAF105p and is encoded by a previously identified gene MPT1. TAF65p shows no significant sequence homology to any previously identified TAFp. The genes encoding TAF48p and TAF65p are single copy and essential for normal yeast cell growth. Furthermore, neither TAF48p nor TAF65p are associated with the histone acetylase Spt-Ada-Gcn5 complex or other non-TFIID TBF.TAF complexes. The significance of these results in terms of TFIID structure, function, and organization is discussed.

TAF25p, a non-histone-like subunit of TFIID and SAGA complexes, is essential for total mRNA gene transcription in vivo.

We demonstrate, utilizing a temperature conditional mutant allele of the gene encoding TAF25p, that this non-histone-like TBP-associated factor, which is shared between the TFIID and SAGA complexes, is required for bulk mRNA gene transcription by RNA polymerase II in vivo. Immunoblotting experiments indicate that at the restrictive temperature, inactivation of TAF25p function results in a reduction of the levels of numerous TFIID and SAGA subunits, indicating its loss of function, like the histone-like TAFs, causes degradation of the constituents of these two multisubunit complexes. These data suggest that TAF25p plays a key structural role in maintaining TFIID and SAGA complex integrity. This is the first demonstration that a non-histone-like TAF is required for continuous, high level RNA polymerase II-mediated mRNA gene transcription in living yeast cells.

A rapid technique for the determination of unknown plasmid library insert DNA sequence directly from intact yeast cells.

A polymerase chain reaction (PCR)-based technique is described which allows for the determination of library plasmid insert DNA sequence directly and rapidly from intact yeast cells. Yeast spheroplasts are used to template a PCR reaction to amplify the insert sequence. This PCR product is then purified and its sequence directly determined using thermal cycle sequencing. Readable sequence can reproducibly be obtained from multiple yeast colonies in just two days. Uses of this technique in yeast two-hybrid screening as well as other types of yeast library screens are discussed.

MOT1 can activate basal transcription in vitro by regulating the distribution of TATA binding protein between promoter and nonpromoter sites.

MOT1 is an ATPase which can dissociate TATA binding protein (TBP)-DNA complexes in a reaction requiring ATP hydrolysis. Consistent with this observation, MOT1 can repress basal transcription in vitro. Paradoxically, however, some genes, such as HIS4, appear to require MOT1 as an activator of transcription in vivo. To further investigate the function of MOT1 in basal transcription, we performed in vitro transcription reactions using yeast nuclear extracts depleted of MOT1. Quantitation of MOT1 revealed that it is an abundant protein, with nuclear extracts from wild-type cells containing a molar excess of MOT1 over TBP. Surprisingly, MOT1 can weakly activate basal transcription in vitro. This activation by MOT1 is detectable with amounts of MOT1 that are approximately stoichiometric to TBP. With amounts of MOT1 similar to those present in wild-type nuclear extracts, MOT1 behaves as a weak repressor of basal transcription. These results suggest that MOT1 might activate transcription via an indirect mechanism in which limiting TBP can be liberated from nonpromoter sites for use at promoters. In support of this idea, excess nonpromoter DNA sequesters TBP and represses transcription, but this effect can be reversed by addition of MOT1. These results help to reconcile previous in vitro and in vivo results and expand the repertoire of transcriptional control strategies to include factor-assisted redistribution of TBP between promoter and nonpromoter sites.

ADR1-mediated transcriptional activation requires the presence of an intact TFIID complex.

The yeast transcriptional activator ADR1, which is required for ADH2 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 reduced ADH2 derepression. These results indicate that ADR1 physically associates with TFIID and that its ability to activate transcription requires an intact TFIID complex.

Biochemical and genetic characterization of the dominant positive element driving transcription ofthe yeast TBP-encoding gene, SPT15.

We previously demonstrated that a combination of both positive and negative cis -acting upstream elements control the transcription of the gene encoding TBP ( SPT15 ) in Saccharomyces cerevisiae . One of these elements found in that study, resident between 5' flanking sequences -147 and -128 , and termed PED (for positive element distal), was found to play an essential positive role in driving transcription of the gene encoding TBP. In this report, we map at nucleotide-level resolution, the critical residues which comprise PED, purify and sequence the protein that binds to it and determine that this PED binding factor is Abf1p, an abundant yeast protein previously broadly implicated in both gene regulation and DNA replication. In the case of the TBP-encoding gene, however, Abf1p works through the PED element which is a non-consensus binding site. Based upon the work of others, the PED-variant ABF1 site would be predicted to be a very poor binding site for this factor yet Abf1p binds PED and a consensus ABF1 site with comparable affinity. These results are discussed in light of the broader context of Abf1p-mediated gene regulation.

Genetic tests of the role of Abf1p in driving transcription of the yeast TATA box bindng protein-encoding gene, SPT15.

In this report we describe studies which utilized yeast strains bearing gain and loss of function alleles of ABF1 in order to attempt to directly implicate Abf1p in modulating transcription of the TBP-encoding gene, SPT15, in vivo. We found that overexpression of Abf1p in a yeast cell increased transcription of the TBP-encoding gene and that this stimulation depended upon the exact sequence of the Abf1p binding site (ABF1) present in the gene. Further, in a yeast strain expressing a temperature sensitive form of Abf1p, occupancy of the chromosomal ABF1 site in the TBP-encoding gene was immediately lost following a temperature shift. Both results suggest that Abf1p drives transcription of the TBP-encoding gene. Surprisingly though we found that continuous ABF1 cis-element occupancy by Abf1p was not acutely required for normal levels of transcription of either the TBP-encoding gene or other "Abf1p-driven" genes tested. We propose a model to explain these results and suggest mechanisms by which Abf1p could activate gene transcription.

The Gcn4p activation domain interacts specifically in vitro with RNA polymerase II holoenzyme, TFIID, and the Adap-Gcn5p coactivator complex.

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 glutathione S-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.

Structure-function analysis of TAF130: identification and characterization of a high-affinity TATA-binding protein interaction domain in the N terminus of yeast TAF(II)130.

We report structure-function analyses of TAF130, the single-copy essential yeast gene encoding the 130,000-Mr yeast TATA-binding protein (TBP)-associated factor TAF(II)130 (yTAF(II)130). A systematic family of TAF130 mutants was generated, and these mutant TAF130 alleles were introduced into yeast in both single and multiple copies to test for their ability to complement a taf130delta null allele and support cell growth. All mutant proteins were stably expressed in vivo. The complementation tests indicated that a large portion (amino acids 208 to 303 as well as amino acids 367 to 1037) of yTAF(II)130 is required to support cell growth. Direct protein blotting and coimmunoprecipitation analyses showed that two N-terminal deletions which remove portions of yTAF(II)130 amino acids 2 to 115 dramatically decrease the ability of these mutant yTAF(II)130 proteins to bind TBP. Cells bearing either of these two TAF130 mutant alleles also exhibit a slow-growth phenotype. Consistent with these observations, overexpression of TBP can correct this growth deficiency as well as increase the amount of TBP interacting with yTAF(II)130 in vivo. Our results provide the first combined genetic and biochemical evidence that yTAF(II)130 binds to yeast TBP in vivo through yTAF(II)130 N-terminal sequences and that this binding is physiologically significant. By using fluorescence anisotropy spectroscopic binding measurements, the affinity of the interaction of TBP for the N-terminal TBP-binding domain of yTAF(II)130 was measured, and the Kd was found to be about 1 nM. Moreover, we found that the N-terminal domain of yTAF(II)130 actively dissociated TBP from TATA box-containing DNA.

Molecular genetic elucidation of the tripartite structure of the Schizosaccharomyces pombe 72 kDa TFIID subunit which contains a WD40 structural motif.

BACKGROUND: The multisubunit general transcription factor termed TFIID is comprised of the TATA box DNA binding protein TBP and several TBP-associated factors termed TAFs. Current arguments regarding the mechanisms of regulation of transcription contend that TFIID makes multiple specific protein-protein interactions with numerous protein factors, and that these interactions are important for the regulation of transcriptional initiation. TAFs contain a variety of potential structural motifs and it has been speculated that these motifs participate directly in TAF function. However, to date the physiological significance of these putative structural motifs has not been systematically analysed in vivo. RESULTS: The essential gene encoding the Schizosaccharomyces pombe 72 kDa TFIID subunit is termed taf72+, which contains WD40 repeats, was cloned and sequenced. A comparison of the primary structure of this gene with its Drosophila and S. cerevisiae counterparts suggests the presence of regions that might play a role in TFIID function, due to the fact that significant portions of the sequences are highly conserved. Complementation analyses of a series of deletion mutants of this gene revealed that the most evolutionarily conserved regions of taf72+, including the WD40 repeats, are in fact indispensable for the viability. CONCLUSIONS: The 72 kDa subunit of S. pombe TFIID, which contains putative WD40 repeats, consists of three distinct functional domains separated by intervening regions. The functional significance of the WD40 repeats is demonstrated by this in vivo study.

Cloning and characterization of an essential Saccharomyces cerevisiae gene, TAF40, which encodes yTAFII40, an RNA polymerase II-specific TATA-binding protein-associated factor.

In this report we describe the cloning and initial characterization of TAF40, a gene that encodes a yeast TATA-binding protein-associated factor (yTAF) of Mr = approximately 40,000. This gene has many similarities to other yTAFs described thus far in that it is present at a single copy per haploid genome, it is essential for viability, and the deduced protein sequence of yTAF40 exhibits similarity to previously described human and Drosophila TAFIIs. Immunological studies confirm that yTAF40 protein is a subunit of a large multiprotein TATA-binding protein-TAF complex that contains a subset of the total number of the yTAFs present in yeast cell extracts. Transcription reactions performed using yeast whole cell extracts reveal that of the three nuclear RNA polymerases only RNA polymerase II function is abrogated when yTAF40 and associated proteins are immunodepleted from solution, indicating that the functionality of the multiprotein complex containing yTAF40 is RNA polymerase II-specific. By these criteria yTAF40 appears to encode a bona fide RNA polymerase II-specific TAF, and thus the protein that it encodes has been termed yTAFII40.

TBP-associated factors are not generally required for transcriptional activation in yeast.

The transcription factor TFIID, a central component of the eukaryotic RNA polymerase II (Pol II) transcription apparatus, comprises the TATA-binding protein (TBP) and approximately ten TBP-associated factors (TAFs). Although the essential role of TBP in all eukaryotic transcription has been extensively analysed in vivo and in vitro, the function of the TAFs is less clear. In vitro, TAFs are dispensable for basal transcription but are required for the response to activators. In addition, specific TAFs may act as molecular bridges between particular activators and the general transcription machinery. In vivo, TAFS are required for yeast and mammalian cell growth, but little is known about their specific transcriptional functions. Using conditional alleles created by a new double-shutoff method, we show here that TAF depletion in yeast cells can reduce transcription from some promoters lacking conventional TATA elements. However, TAF depletion has surprisingly little effect on transcriptional enhancement by several activators, indicating that TAFs are not generally required for transcriptional activation in yeast.

Isolation and characterization of TAF25, an essential yeast gene that encodes an RNA polymerase II-specific TATA-binding protein-associated factor.

We describe the cloning and analysis of TAF25, a previously uncharacterized yeast gene that encodes a yeast TATA-binding protein-associated factor or yTAF of Mr = 25,000. The gene encoding yTAF25 is a single copy essential gene, and the protein sequence deduced from TAF25 exhibits sequence similarity to a metazoan hTAFII. The results from immunological studies confirm that yTAF25 is a subunit of a large multiprotein TATA-binding protein-yeast TATA-binding protein-associated factor complex that contains a subset of the total number of the yTAFs present in yeast cell extracts. Both genetic and biochemical analyses demonstrate that yTAF25 can interact directly with itself. Transcriptional data show that the activity of the multiprotein complex containing yTAF25 is RNA polymerase II-specific, thus indicating that TAF25 encodes a bona fide yeast RNA polymerase II TAF. Hence the protein encoded by TAF25 has been termed yTAFII25.

Gender and compensation in health care management.

Salary comparisons of male and female ACHE members in 1989 showed that men earned nearly $16,000 more than women. Controlling for race and region, age and experience accounted for most of the explained variation. Women earned significantly more if they had more male mentors, a spouse willing to relocate for their career advancement, or an employer whose policies accommodated families such as flextime and if they themselves socialized informally with other health care executives.