Abstract
Transcription factor IID (TFIID) plays a central role in regulating the expression of most eukaryotic genes. Of the 14 TBP-associated factor (TAF) subunits that compose TFIID, TAF1 is one of the largest and most functionally diverse. Yeast TAF1 can be divided into four regions including a putative histone acetyltransferase domain and TBP, TAF, and promoter binding domains. Establishing the importance of each region in gene expression through deletion analysis has been hampered by the cellular requirement of TAF1 for viability. To circumvent this limitation we introduced galactose-inducible deletion derivatives of previously defined functional regions of TAF1 into a temperature-sensitive taf1ts2 yeast strain. After galactose induction of the TAF1 mutants and temperature-induced elimination of the resident Taf1ts2 protein, we examined the properties and phenotypes of the mutants, including their impact on genome-wide transcription. Virtually all TAF1-dependent genes, which comprise approximately 90% of the yeast genome, displayed a strong dependence upon all regions of TAF1 that were tested. This finding might reflect the need for each region of TAF1 to stabilize TAF1 against degradation or may indicate that all TAF1-dependent genes require the many activities of TAF1. Paradoxically, deletion of the region of TAF1 that is important for promoter binding interfered with the expression of many genes that are normally TFIID-independent/SAGA (Spt-Ada-Gcn5-acetyltransferase)-dominated, suggesting that this region normally prevents TAF1 (TFIID) from interfering with the expression of SAGA-regulated genes.
Highlights
Transcription factor IID (TFIID) is composed of the TATA-binding protein (TBP) and 14 TBPassociated factors (TAFs),4 of which all but one are essential for cell viability
3 The abbreviations used are: TFIID, transcription factor IID; TBP, TATA box-binding protein; FHT, Flu-His6-TEV; HA, hemagglutinin; TEV, tobacco etch virus; 5-FOA, 5-fluoroorotic acid; WT, wild type; SAGA, Spt-Ada-Gcn5-acetyltransferase; TAF, TBP-associated factor; TAND, TAF1 N-terminal domain; HAT, histone acetyltransferase; gal, galactose; YPR, yeast extract, peptone, raffinose; CSM, complete synthetic medium; bis-Tris, TFIID is composed of the TATA-binding protein (TBP) and 14 TBPassociated factors (TAFs),4 of which all but one are essential for cell viability
TAF1 is considered to be a “hallmark” of TFIID in that it resides only in TFIID and not in SAGA, and it may serve as a scaffold upon which TBP and TAFs assemble, other TAFs might play a scaffolding role (12, 17–19)
Summary
YCp50 (TAF1 WT, URA3), and pRS313 taf1ts HIS3 (25) were gifts from J. To confirm the taf1ts temperature-sensitive (ts) phenotype, pJI11 and pJI12 were transformed into Y13.2 (15) and plated on CSM-LEU. 1,826-bp PCR products were transformed into yLAC3 and selected on CSM-HIS-URA (dextrose) plates containing 500 g/ml G418 (Invitrogen). Kanamycin-sensitive FHT-TAF1 strains (Table 1) were plated on CSM-HIS ϩ 5-FOA to select cells having lost pSH47 (30) and verified by replica plating on CSM-HIS and CSM-HIS-URA. Cells that lost YCp50 (TAF1 WT, URA3) were selected by plating on CSM-LEU ϩ 5-FOA. Viability—MAT␣ haploid strains carrying YCp50 (TAF1 WT, URA3) were grown at 25 °C in YPD medium (yeast extract, peptone, dextrose). Cells having lost the YCp50 plasmid were selected by growing on CSM ϩ 5-FOA with 2% dextrose or 2% galactose at 25 or 37 °C. Toxicity—MAT␣ haploid strains carrying pJI12 (TAF1 WT LEU2) plasmid were grow in CSM-LEU raffinose to mid-log phase. 0.5 A600 of cells were serially diluted 10-fold, and 5 l was plated on CSM-
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