Biochemical and Genetic Characterization of a Yeast TFIID Mutant That Alters Transcription In Vivo and DNA Binding In Vitro

Arndt, Karen M.; Ricupero, S L; Eisenmann, David M.; Winston, Fred

doi:10.1128/mcb.12.5.2372-2382.1992

Cited by 11 publications

(1 citation statement)

References 65 publications

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“…Recombinant yeast and human basal transcription factors were expressed in bacteria by IPTG (1 mM) induction. Yeast TBP (Arndt et al, 1992) was partially purified by DEAE-Sephacryl and heparin sepharose chromatography (Lieberman et al, 1991). For some experiments, the eluate from the heparin sepharose column was concentrated (Centiprep-10 concentrator, Amicon) and TBP purified to near homogeneity by gel filtration on a Superose 12 column (Pharmacia).…”

Section: Methodsmentioning

confidence: 99%

Functional Interaction of the c-Myc Transactivation Domain with the TATA Binding Protein: Evidence for an Induced Fit Model of Transactivation Domain Folding

et al. 1996

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c-Myc is a member of a family of sequence specific-DNA binding proteins that are thought to regulate the transcription of genes involved in normal cell growth, differentiation, and apoptosis. In order to understand how human c-myc functions as a transcription factor, we have studied the mechanism of action and structure of the N-terminal transactivation domain, amino acids 1-143. In a protein interaction assay, c-myc1-143 bound selectively to two basal transcription factors, the TATA binding protein (TBP) and the RAP74 subunit of TFIIF. Furthermore, the isolated c-myc transactivation domain competed for limiting factors required for the assembly of a functional preinitiation complex. This squelching of basal transcription was reversed in a dose-dependent manner by recombinant TBP. Taken together, these results identify TBP as an important target for the c-myc transactivation domain, during transcriptional initiation. Similar to other transactivation domains, the c-myc1-143 polypeptide showed little or no evidence of secondary structure, when measured by circular dichroism spectroscopy (CD) in aqueous solution. However, significant alpha-helical conformation was observed in the presence of the hydrophobic solvent trifluoroethanol. Strikingly, addition of TBP caused changes in the CD spectra consistent with induction of protein conformation in c-myc1-143 during interaction with the target factor. This change was specific for TBP as a similar effect was not observed in the presence of TFIIB. These data support a model in which target factors induce or stabilize a structural conformation in activator proteins during transcriptional transactivation.

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Section: Methodsmentioning

confidence: 99%

Functional Interaction of the c-Myc Transactivation Domain with the TATA Binding Protein: Evidence for an Induced Fit Model of Transactivation Domain Folding

et al. 1996

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Rtf1 Is a Multifunctional Component of the Paf1 Complex That Regulates Gene Expression by Directing Cotranscriptional Histone Modification

Warner

Roinick

Arndt

2007

Molecular and Cellular Biology

148

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Numerous transcription accessory proteins cause alterations in chromatin structure that promote the progression of RNA polymerase II (Pol II) along open reading frames (ORFs). The Saccharomyces cerevisiae Paf1 complex colocalizes with actively transcribing Pol II and orchestrates modifications to the chromatin template during transcription elongation. To better understand the function of the Rtf1 subunit of the Paf1 complex, we created a series of sequential deletions along the length of the protein. Genetic and biochemical assays were performed on these mutants to identify residues required for the various activities of Rtf1. Our results establish that discrete nonoverlapping segments of Rtf1 are necessary for interaction with the ATP-dependent chromatin-remodeling protein Chd1, promoting covalent modification of histones H2B and H3, recruitment to active ORFs, and association with other Paf1 complex subunits. We observed transcription-related defects when regions of Rtf1 that mediate histone modification or association with active genes were deleted, but disruption of the physical association between Rtf1 and other Paf1 complex subunits caused only subtle mutant phenotypes. Together, our results indicate that Rtf1 influences transcription and chromatin structure through several independent functional domains and that Rtf1 may function independently of its association with other members of the Paf1 complex.The complex organization of eukaryotic chromosomes acts as a significant impediment to gene expression. In this environment, efficient elongation of a transcript by RNA polymerase II (Pol II) requires a multitude of accessory factors to facilitate its movement along chromatin-assembled genes. The Saccharomyces cerevisiae Paf1 complex colocalizes with Pol II during transcription elongation and is required for the normal expression of a subset of genes (22,34,35,37,55). The Paf1 complex minimally contains five subunits, Paf1, Ctr9, Cdc73, Rtf1, and Leo1, and physically associates with Pol II (22,27,58). Consistent with a role in transcription elongation, physical and genetic interactions between components of the Paf1 complex and other Pol II-associated elongation factors, including the Spt4-Spt5 (yDSIF) and Spt16-Pob3 (yFACT) complexes, have been reported (22,58). Additionally, deletion of genes encoding subunits of the Paf1 complex causes sensitivity to the base analogs 6-azauracil (6-AU) and mycophenolic acid, phenotypes associated with defects in transcription elongation (8,58).Proper elongation of a transcript by Pol II requires efficient navigation of a chromatin template. Nucleosomes, the fundamental components of chromatin, form around octamers of the histone proteins H2A, H2B, H3, and H4. Histones are subject to a myriad of posttranslational modifications, including acetylation, methylation, phosphorylation, ubiquitylation, and sumoylation (reviewed in references 17 and 51; 30, 50). Methylation of individual lysine residues within histones can occur in mono-, di-, or trimethyl states (reviewed in refer...

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Identification and Analysis of Mot3, a Zinc Finger Protein That Binds to the Retrotransposon Ty Long Terminal Repeat (δ) in Saccharomyces cerevisiae

Madison

Dudley

Winston

1998

Molecular and Cellular Biology

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Spt3 and Mot1 are two transcription factors of Saccharomyces cerevisiae that are thought to act in a related fashion to control the function of TATA-binding protein (TBP). Current models suggest that while Spt3 and Mot1 do not directly interact, they do function in a related fashion to stabilize the TBP-TATA interaction at particular promoters. Consistent with this model, certain combinations of spt3 and mot1 mutations are inviable. To identify additional proteins related to Spt3 and Mot1 functions, we screened for high-copy-number suppressors of the mot1 spt3 inviability. This screen identified a previously unstudied gene, MOT3, that encodes a zinc finger protein. We show that Mot3 binds in vitro to three sites within the retrotransposon Ty long terminal repeat (␦) sequence. One of these sites is immediately 5 of the ␦ TATA region. Although a mot3 null mutation causes no strong phenotypes, it does cause some mild phenotypes, including a very modest increase in Ty mRNA levels, partial suppression of transcriptional defects caused by a mot1 mutation, and partial suppression of an spt3 mutation. These results, in conjunction with those of an independent study of Mot3 (A. Grishin, M. Rothenberg, M. A. Downs, and K. J. Blumer, Genetics, in press), suggest that this protein plays a varied role in gene expression that may be largely redundant with other factors.RNA polymerase II (pol II) transcription involves a balance of positive and negative regulators to properly control gene expression. The study of pol II transcription has identified several general factors that are required to initiate and transcribe mRNA from most promoters (77). Other studies have focused on gene-specific activators, such as the Saccharomyces cerevisiae activators Gal4 or Gcn4, or coactivators, such as the TATA-binding protein (TBP)-associated factors, and the mechanisms by which these two groups of proteins help the general transcription apparatus (31, 34). Repression of transcription, by both gene-specific repressors and general repressors, is also important for the proper regulation of pol II transcription. The mechanisms and targets of repression appear as varied as the mechanisms that lead to activation of transcription (19,63). One target of repression is TBP, whose binding to the promoter TATA sequence represents one of the early rate-limiting steps in the formation of a pol II preinitiation complex (11,12).To understand the complex interplay of the many diverse proteins and mechanisms of transcriptional regulation, we have isolated and analyzed a number of mutations that affect pol II transcription initiation in vivo. The mutations were isolated as suppressors of insertion mutations caused by Ty elements or Ty long terminal repeats, ␦ sequences (29,71,72). This analysis has identified a number of genes, called SPT genes, for suppressor of Ty (70). Although identified by this specific selection, most SPT genes have now been shown to be generally important or essential for growth and transcription in vivo. One related group of SPT genes...

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Biochemical and Genetic Characterization of a Yeast TFIID Mutant That Alters Transcription In Vivo and DNA Binding In Vitro

Cited by 11 publications

References 65 publications

Functional Interaction of the c-Myc Transactivation Domain with the TATA Binding Protein: Evidence for an Induced Fit Model of Transactivation Domain Folding

Functional Interaction of the c-Myc Transactivation Domain with the TATA Binding Protein: Evidence for an Induced Fit Model of Transactivation Domain Folding

Rtf1 Is a Multifunctional Component of the Paf1 Complex That Regulates Gene Expression by Directing Cotranscriptional Histone Modification

Identification and Analysis of Mot3, a Zinc Finger Protein That Binds to the Retrotransposon Ty Long Terminal Repeat (δ) in Saccharomyces cerevisiae

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