Abstract
Transcription factor IID (TFIID) plays a key role in regulating eukaryotic gene expression by directly binding promoters and enhancer-bound transactivator proteins. However, the precise mechanisms and outcomes of transactivator-TFIID interaction remain unclear. Transcription of yeast ribosomal protein genes requires TFIID and the DNA-binding transactivator Rap1. We have previously shown that Rap1 directly binds to the TFIID complex through interaction with its TATA-binding protein-associated factor (Taf) subunits Taf4, -5, and -12. Here, we identify and characterize the Rap1 binding domains (RBDs) of Taf4 and Taf5. These RBDs are essential for viability but dispensable for Taf-Taf interactions and TFIID stability. Cells expressing altered Rap1 binding domains exhibit conditional growth, synthetic phenotypes when expressed in combination or with altered Rap1, and are selectively defective in ribosomal protein gene transcription. Taf4 and Taf5 proteins with altered RBDs bind Rap1 with reduced affinity. We propose that collectively the Taf4, Taf5, and Taf12 subunits of TFIID represent the physical and functional targets for Rap1 interaction and, furthermore, that these interactions drive ribosomal protein gene transcription.
Highlights
Grant GM52461. □S The on-line version of this article contains supplemental Figs
We previously demonstrated that yeast Repressor activator protein 1 (Rap1) directly binds Transcription factor IID (TFIID) subunits Taf4, Taf5, and Taf12 and mapped the Taf12 Rap1 binding domain to Taf12 N-terminal sequences that are conserved in sensu strictu yeast strains [24]
RPG Transcription in taf4 and taf5 Cells—Given the growth defects associated with alteration of Taf4 and Taf5 Rap1 binding domains (RBDs), the known roles of Rap1 and TFIID in RPG transcription, and the importance of ongoing ribosome synthesis to cellular growth, we examined the effect of temperature up-shift on RPG transcription in wild type (WT) and taf RBDϪ Tsϩ cells
Summary
Grant GM52461. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 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 Rap and the TFIID complex [24]. Our studies have firmly established an important model genetic system with which to dissect the mechanisms of transactivator-TFIID interactions in the context of mRNA gene transcription activation
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