Yeast TFIID Serves as a Coactivator for Rap1p by Direct Protein-Protein Interaction

Abstract

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.Eukaryotic mRNA gene transcription is controlled by the action of modular enhancer-binding transactivators, proteins composed of separable DNA binding domains (DBD) and activation domains (AD). DNA-bound transactivators functionally interact with the mRNA gene transcription machinery, the so-called general transcription factors (GTFs) TFIIA, -B, -D, -E, -F, and -H plus RNA polymerase II (RNAP II), to stimulate formation and/or function of the RNAP II preinitiation complex (PIC) (67). Activators also collaborate with one or more factors termed coactivators, proteins that serve as receptors for the transfactor-AD activation signal. Coactivators link transactivator-enhancer DNA binding to the PIC (43) and can be divided into several classes: those that are chromatin active, the mediator complex, and the individual components of the mRNA gene transcription machinery itself.One of the first and likely rate-limiting steps in mRNA gene transcription is the binding of TFIID to the promoter (36,40,47). TFIID is a multisubunit assembly composed of 15 evolutionarily conserved (86) subunits, the TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs) (72). This GTF displays high-affinity, sequence-specific promoter DNA binding activity. Two classes of yeast mRNA-encoding genes have been defined with respect to TFIID TAF function; the first, which is TAF dependent (TAF dep ), requires TAF function for transcription, while the second and smaller group is TAF independent (TAF ind ) (29,31,45,58,81,92). Both types of genes require TBP for wild-type (WT) levels of transcription, but in the case of the TAF ind genes, TBP appears to be recruited via mechanisms distinct from TFIID (4, 80).When mRNA-encoding genes are monitored for TBP and TAF occupancy by chromatin immunoprecipitation (ChIP) assay, TAF dep genes exhibit higher TAF/TBP occupancy ratios than TAF ind genes (39, 45). Three models for TAF function have been proposed (22, 23): TAFs mediate core promoter recognition; TAFs provide essential catalytic activities tha...

Selective role of Mediator tail module in the transcription of highly regulated genes in yeast

“…20 In contrast, TFIIDdependent genes, many of which may not require Swi/Snf, could depend on Mediator subunits other than those in the tail module to interact with activators or TFIID components, thus leading to the interactions with other GTFs and formation of PIC. [23][24][25] In summary, although connections between the Mediator tail, Swi/Snf and SAGA have been established, the detailed mechanisms behind these interconnections likely differ at different promoters, and an understanding of the precise molecular basis underlying these connections remains to be achieved.…”

Direct Transactivator-Transcription Factor IID (TFIID) Contacts Drive Yeast Ribosomal Protein Gene Transcription

“…Strains, and Molecular Biological Analyses-Deletion mutations in TAF4, TAF5, and RAP1 were generated using PCRbased methods (24) and verified by DNA sequencing. Taf4, Taf5, Taf12, and Rap1 were expressed in Escherichia coli and purified by chromatographic methods that varied depending upon each protein (details available on request).…”

“…In contrast to the other factors noted above (35-37), Rap1 enhancer occupancy does not change significantly with transcription rates. Importantly, a simple chimeric gene that contains just two RPG Rap1-binding sites fused to a TFIID-independent core promoter exhibits both Rap1-and TFIID-dependent transcription in vivo (21,24) and in vitro (24). These striking biochemical and genetic interactions have been attributed to direct physical contacts between Rap1 and the TFIID complex (24).…”

“…RPG transcription is coordinately regulated and highly sensitive to diverse environmental stimuli (31). Repressor activator protein 1 (Rap1)-binding sites are found within the majority of RPG enhancers, and Rap1 is the only transactivator absolutely required for RPG transcription, although Rap1 does collaborate with additional transcription factors such as Fhl1/Ifh1/Crf1, Sfp1, Hmo1, and the NuA4 coactivator to modulate RPG expression (21,24,30,(32)(33)(34). In contrast to the other factors noted above (35-37), Rap1 enhancer occupancy does not change significantly with transcription rates.…”

Yeast Est2p Affects Telomere Length by Influencing Association of Rap1p with Telomeric Chromatin

“…For the gel shift assay, oligonucleotides (5Ј-ATATACACCCATACATTGA-3Ј and 5Ј-GTCAATGTATGGGTGTATA-3Ј [11]) were annealed to create a single Rap1p binding site (in boldface) and were labeled with Klenow polymerase (Roche) in the presence of [␣-32 P]dCTP. A total of 100 fmol of 32 P-labeled DNA was mixed with increasing amounts of unlabeled telomeric DNA (0 to 80 fmol).…”