Gene expression in the tra1-L3733A strain. a The promoter region of PHO5 was cloned as a LacZ reporter fusion into the LEU2 centromeric plasmid YCp87 and transformed into yeast strains CY4353 (TRA1), CY4318 (tra1-A3727S), CY4103 (tra1-L3733A), CY4324 (tra1-F3740A) and CY4350 (tra1-F3744A), containing a plasmid copy of YHR100C. β-Galactosidase activity was determined after growth in low phosphate media for 15 h at 30°. Expression is shown as a percentage of that found for CY4353. Measurements were made in triplicate with the standard deviation indicated. b Expression from stress response elements. A cassette of two STRE elements was cloned into the EcoRI and SacI sites of his3-Δ88-LacZ (Brandl et al. 1993) to give STRE-his3-LacZ. These elements replace the normal Gcn4 binding site in HIS3. STRE-his3-LacZ and his3-Δ88 were transformed into CY2706 and CY3003, and β-galactosidase assays performed after growth of cells in YPD containing 4% ethanol. c Expression levels determined by gene profiling (GP, mRNA-Seq) as compared with RNA dot blots (ADE17 and RPL4a/b) and LacZ reporter fusions (PHO5 and RPL35a). RNA was prepared from yeast strains CY3003 (tra1-L3733A) and CY2706 (TRA1_WT). mRNA-Seq libraries were constructed and sequencing performed on the Illumina/Solexa Genome Analyzer II platform. Comparisons between the strains were made as outlined in “Materials and methods”. Similarly prepared RNA was spotted onto Hybond-N membrane and probed with single-stranded DNAs complementary to ADE17 and RPL4a/b RNA. Hybridization was detected by autoradiography and quantitated using AlphaImager 3400 software and shown as a percentage of wild-type expression. Expression of PHO5-LacZ and RPL35a-LacZ fusion reporters were determined after growth of CY3003 and CY2706 in YPD. Assays were performed in triplicate

Intragenic supressors of the L3733A mutation. a Yeast strains CY3003 (tra1-L3733A), CY2706 (TRA1_WT), CY4055 (tra1-L3733A/N3677D) and CY4056 (tra1-L3733A/T3716A) were grown in minimal media and tenfold serial dilutions spotted onto a YPD plate containing 4% ethanol. bPHO5-LacZ expression. The above strains containing YCp87-PHO5-LacZ were grown to saturation in minimal media lacking leucine, diluted sixfold in YPD depleted of phosphate and grown for 15 h at 30°. β-Galactosidase activity was calculated for each strain in triplicate and plotted as a percentage of that found for CY2706

Reduced expression of Tra1 results in ethanol sensitivity. Yeast strains CY3003 (DED1 promoter: tra1-L3733A), CY2706 (DED1 promoter: TRA1_WT), and CY3021 (MET3 promoter: TRA1_WT) were grown in minimal media lacking methionine, and then serial dilutions were spotted onto minimal plates containing the indicated concentration of methionine and for the lower panel 3% ethanol

Deletion of hda1 or nhp10 does not suppress tra1-L3733A. Strains deleted for hda1 or nhp10 were generated by sporulation of diploid deletion strains obtained from Open Biosystems. Double mutants with tra1-L3733A were obtained by mating with CY4057 or CY4103 and sporulation. The wild-type strain BY4742, and the single- and double-mutant strains were grown in YPD and serial dilutions spotted onto YPD at 30°, 16° or 37°, or YPD containing 6% ethanol at 30°

FATC domain mutants. a Schematic diagram of the architecture of the Tra1/TRRAP family indicating the approximate positions of the HEAT, FAT, PI3 K, and FATC domains (Bosotti et al. 2000; Murr et al. 2007). Numbering is for the 3744 residues of S. cerevisiae Tra1. b Sequence alignment of FATC domains from PIKK family proteins. Top panel Sequences are Saccharomyces cerevisiae Tra1 (NP_011967), Schizosaccharomyces pombe Tra2 (Q10064), Schizosaccharomyces pombe Tra1 (NP_595777), Neurospora crassa Tra1 (CAC18279), Drosophila melanogaster Nipped-A (NP_001097192), Homo sapiens TRRAP (NP_003487). Lower panel Sequences from Saccharomyces cerevisiae Tra1 (NP_011967), Tor1 (CAA52849) and Homo sapiens TRRAP (NP_003487), ATM (Q13315), ATR (Q13535), DNA-PKcs (P78527) and SMG-1 (NP_055907). Residues indicated between the alignments in bold capitals are those changes that were analyzed through plasmid shuffling. Lower case letters indicate residues analyzed by gene integration, see c. Numbering is for S. cerevisiae Tra1. Alignments were performed using the default parameters of the ClustalW utility [http://www.ebi.ac.uk/clustalw/] and the terminal 49 residues of each protein. c Growth of the tra1-L3733A strain. Yeast strains containing centromeric plasmids expressing tra1-L3733A and TRA1_WT were grown to saturation and ten-fold serial dilutions plated onto YP media containing 2% glucose and grown at 30° or 16°, or at 30° on YPD containing 5 μg/ml Calcofluor white (CW), 1 nM rapamycin, 20 μg/ml geneticin, or 4% ethanol. d Analysis of integrated tra1 alleles. TRA1_WT, tra1-L3733A, tra1-A2727S, tra1-F3740A and tra1-F3744A were integrated into BY4743. Haploids containing the integrated allele were obtained after sporulation. Saturated cultures were serially diluted and plated onto YPD at 30° or 37°, or at 30° on YPD containing 5 μg/ml Calcofluor white (CW), 1 nM rapamycin, or 6% ethanol. We note that the BY4742/4741 background is less sensitive to ethanol than KY320, so ethanol sensitivity was assayed at a concentration of 6%

Gene expression in the tra1-L3733A strain. a Hierarchical cluster analysis. Comparisons were initially made with the compendium data set (Hughes et al. 2000), profiles from strains with deletions of NuA4 (Krogan et al. 2004) and SAGA components (Ingvarsdottir et al. 2005), and with tra1-SRR3413 (Mutiu et al. 2007a). The diagram shows those profiles clustering in closest proximity to tra1-L3733A. Gene families are indicated on the right. b Correlation between degree of Tra1-binding and average transcriptional frequency. Genome-wide localization studies were performed in triplicate for yeast strain CY2706 (myc₉-TRA1) grown at 30° in YPD, essentially as described (Yu et al. 2004). The average transcription frequency of each gene (Holstege et al. 1998) is plotted versus the relative binding of myc₉-TRA1

Expression of the tra1-L3733A and tra1-G3745 alleles. a Whole cell extracts were prepared from yeast strain KY320 (−ve, lane 1) or strains expressing myc₉-tagged versions of Tra1_WT (lanes 2, 3) or Tra1-L3733A (lanes4, 5). Extract was separated by SDS PAGE (5%) and Western blotted with anti-myc antibody. The upper panel is the Western blot with anti-myc antibody. The full length myc₉-Tra1of ~450 kDa and the mobility of a 250-kDa marker protein are indicated with an arrow and dot, respectively. The lower panel is a portion of an equivalent gel stained with Coomassie Brilliant Blue (CBB). Lanes 1, 3, and 5 contain 50 μg of protein; lanes 2 and 4, 100 μg. b Levels of Tra1-L3733A suppressors. CY2706 (myc₉-Tra1_WT, lanes 1, 2), CY3003 (myc₉-Tra1-L3733A, lanes 3, 4), CY4055 (myc₉-Tra1-N3677D/L3733A lanes5, 6), and CY4056 (myc₉-Tra1-T3716A/L3733A lanes 7, 8) were grown to A₆₀₀ = 2.0. Crude protein was isolated by bead lysis and 50 μg (even numbered lanes) and 100 μg (odd numbered lanes) was separated by SDS-PAGE and Western blotted with anti-myc or anti Mcm2 antibodies. Expression of tra1-G3745. c Quantitation of Tra1 levels. The Western blot as in B was performed in triplicate with independently grown cultures. Relative amounts of Tra1 were determined by densitometry using AlphaImager 3400 software. d Histone H4 acetylation. Chromatin was isolated from yeast strains CY2706 (TRA1_WT, lower panel) or CY3003 (tra1-L3733A, upper panel) grown to A₆₀₀ = 2.0 in YPD. Chromatin was immunoprecipitated with antibody to histone H3 (lanes2–4) and histone H4 acetylated at K8 (lanes5–7), then analyzed by PCR with primers for PHO5 promoter sequences. Consecutive samples represent twofold serial dilutions of the DNA to enable quantitation. Lane 1 is a mock experiment with no histone antibody performed with a wild-type extract at 2× concentration

Protein interactions of Tra1-L3733A and Tra1-G3745. a Interaction with Ada2. Crude extracts were prepared from an ADA2-TAP strain (Ghaemmaghami et al. 2003) expressing myc₉-Tra1_WT (lane 1), myc₉-Tra1-L3721D (lane 2), myc₉-Tra1-L3733A (lane 3) or only the genomic TRA1 (−ve, lane 4). Volumes corresponding to equal amounts of myc-tagged Tra1 were subject to tandem affinity purification and equal volumes Western blotted using anti-myc antibody (upper panel) or anti-CBP (lower panel). b Interaction with Spt7 and Esa1. Crude extracts of SPT7-TAP and ESA1-TAP strains expressing myc₉-Tra1_WT, myc₉-Tra1-L3721D, or myc₉-Tra1-L3733A were analyzed as above and scanned using AlphaImager 3400 software. The ratio of the Spt7 and Esa1-associated to input Tra1 is presented as percent of wild type. The experiments were performed in triplicate with the associated standard error indicated. c Interaction of Tra1-L3733A with GST-Gal4_AD. myc₉-Tra1_WT or myc₉-Tra1-L3733A was expressed in yeast strain CY2998 containing TAP-tagged Ada2. After CBP affinity purification approximately equal amounts of Tra1_WT (lanes 1, 2) or Tra1-L3733A (lanes 3, 4), as determined by Western blotting, were incubated with GST-Gal4_AD bound to glutathione-Sepharose. After washing, three fractions were eluted with glutathione and Western blotted for the presence of Tra1_WT (lanes5, 7 and 9) or Tra1-L3733A (lanes6, 8 and 10). The levels of bound Tra1_WT and Tra1-L3733A correlated with the elution profile of GST-Gal4_AD (not shown). d Tra1- G3745. Crude extracts were prepared from the ESA1-TAP, SPT7-TAP, or ADA2-TAP strains (Ghaemmaghami et al. 2003) expressing myc₉-TRA1_WT or myc₉-TRA1-G3745. Extracts were tandem affinity purified. Equal volumes were separated by SDS PAGE and Western blotted using anti-myc antibody to detect Tra1 or anti-CBP for Esa1, Spt7, or Ada2. AlphaImager 3400 software was used for densitometric analysis. The ratio between the purified and the input amounts is presented as percent of wild type. The experiments were performed in duplicate with the range as indicated