Structure Function Analysis of the Phosphatidylinositol-3 ......2007/07/29 · 1 Structure Function...
Transcript of Structure Function Analysis of the Phosphatidylinositol-3 ......2007/07/29 · 1 Structure Function...
1
Structure Function Analysis of the Phosphatidylinositol-3-kinase
Domain of Yeast Tra1
A. Irina Mutiu*, Stephen M.T. Hoke*, Julie Genereaux*, Carol Hannam*1,
Katherine MacKenzie*, Olivier Jobin-Robitaille+, Julie Guzzo§, Jacques Côté+,
Brenda Andrews§, David B. Haniford* and Christopher J. Brandl*
* Department of Biochemistry
Schulich School of Medicine and Dentistry,
University of Western Ontario, London, Canada N6A5C1,
+Laval University Cancer Research Center
Hotel-Dieu de Quebec (CHUQ), Quebec City, Canada G1R-2J6
and
§Department of Medical Genetics and Microbiology and the Banting and Best Department of
Medical Research, Terrence Donnelly Centre for Cellular & Biomolecular Research,
University of Toronto, Toronto, Canada M5S 3E1
The first two authors contributed equally to this work.
1 Present address: Department of Molecular and Cellular Biology, University of Guelph, Guelph,
Ontario N1G 2W1
Microarray data from this article has been submitted to the GEO database at NCBI under accession
number GSE6847
Genetics: Published Articles Ahead of Print, published on July 29, 2007 as 10.1534/genetics.107.074476
2
Running title: Structure/function of S. cerevisiae Tra1
Keywords: Tra1, gene expression, PI3-kinase domain, SAGA complex, NuA4 complex
Running title: Structure/function of Tra1
Corresponding author: Dr. Christopher J. Brandl
Department of Biochemistry,
University of Western Ontario,
London, CANADA N6A5C1
(519) 661-2111 ext 86857
Fax: (519) 661-3175
3
Tra1 is an essential component of the Saccharomyces cerevisiae SAGA and NuA4 complexes. Using
targeted mutagenesis, we identified residues within its C-terminal phosphatidylinositol-3-kinase
(PI3K) domain that are required for function. The phenotypes of tra1-P3408A, S3463A and SRR3413-
3415AAA included temperature sensitivity and reduced growth in media containing 6% ethanol,
Calcofluor white or depleted of phosphate. These alleles resulted in ≥2-fold change in expression of
~7% of yeast genes in rich media and reduced activation of PHO5 and ADH2 promoters. Tra1-SRR3413
associated with components of both the NuA4 and SAGA complexes and with the Gal4 transcriptional
activation domain similar to wild-type protein. Tra1-SRR3413 was recruited to the PHO5 promoter in
vivo but gave rise to decreased relative amounts of acetylated histone H3 and histone H4 at SAGA and
NuA4 regulated promoters. Distinct from other components of these complexes, tra1-SRR3413 resulted
in generation dependent telomere shortening and synthetic slow growth in combination with deletions
of a number of genes with roles in membrane related processes. While the tra1 alleles have some
phenotypic similarities with deletions of SAGA and NuA4 components, their distinct nature may arise
from the simultaneous alteration of SAGA and NuA4 functions.
4
INTRODUCTION
In eukaryotic cells the post-translational modification of nucleosomes by multisubunit complexes is a
key aspect of transcriptional regulation (reviewed in Berger, 2002). Histone modifications including
acetylation, methylation, ubiquitylation and phosphorylation can directly alter chromatin structure or
act as a recruitment signal for additional factors (Strahl and Allis, 2000). As well as regulating
transcriptional initiation, nucleosome modifications affect transcriptional elongation and other nuclear
processes such as DNA replication, DNA repair and RNA export (Iizuka and Smith, 2003).
The Saccharomyces cerevisiae SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex modifies chromatin
and provides an interface between DNA binding transcriptional regulators and the basal transcriptional
machinery (reviewed in Green, 2005). The structural core of SAGA is composed of a subset of the
TBP-associated factors (TAFs) (Grant et al., 1998a; Wu et al., 2004), with Spt7, Ada1 and Spt20 also
being required for the integrity of the complex (Horiuchi et al., 1997; Roberts and Winston, 1997;
Sterner et al., 1999). The histone acetyltransferase Gcn5/Ada4 activates and represses transcription by
modifying histones H3 and H2B (Brownell et al., 1996; Grant et al., 1997; Kuo et al., 1998; Wang et
al., 1998, Ricci et al., 2002). In turn, the Ada proteins, Ada2 and Ngg1/Ada3 regulate the activity and
substrate preference of Gcn5 (Balasubramanian et al., 2001). Further regulation is provided by the
interaction of Spt3 and Spt8 with the TATA-binding protein (Eisenmann et al., 1992, 1994; Dudley et
al., 1999). Recruitment of SAGA to promoters is mediated by Tra1, an essential 437 kDa protein
(Saleh et al., 1998; Grant et al., 1998b) that interacts directly with transcriptional activators (Brown et
al., 2001; Bhaumik et al., 2004; Fishburn et al., 2005; Reeves and Hahn, 2005). The mammalian
ortholog of Tra1, TRRAP is required for transcriptional regulation by myc, p53, E2F and E1A (Ard et
al., 2002; Bouchard et al., 2001, Deleu et al., 2001; Kulesza et al., 2002; McMahon et al., 1998). Its
deletion results in defects in cell cycle progression and early embryonic lethality (Herceg et al., 2001).
5
Tra1 is also a component of the multisubunit NuA4 complex (Allard et al., 1999) that preferentially
acetylates histones H4 and H2A, the catalytic subunit being the essential protein Esa1 (Clarke et al.,
1999; Smith et al., 1998). NuA4 associates with acidic activation domains, probably through Tra1
mediated interactions, and activates transcription in an acetylation-dependent manner (Vignali et al.,
2000; Nourani et al., 2004). Acetylation by NuA4 is also critical for nonhomologous end-joining of
DNA double stand breaks and for replication coupled repair (Bird et al., 2002; Choy and Kron, 2002;
Downs et al., 2004). The role of NuA4 in repair was highlighted by the finding that it acetylates the
histone variant Htz1 which is intimately involved with these processes (Keogh et al., 2006).
Aside from its length, a distinguishing feature of Tra1 is its C-terminal domain of approximately 300
amino acids that is related to the phosphatidylinositol-3-kinase (PI3K) domain found in several key
cellular regulators including ATM, DNA-PK, and FRAP (Keith and Schreiber, 1999). The group also
shares less well-defined sequences flanking the PI3K domain called the FAT (FRAP-ATM-TRRAP)
and C-FAT domains (Bosotti et al., 2000). Unlike other members of the family, Tra1 and TRRAP lack
the signature motifs of kinases and kinase activity (Bosotti et al., 2000). The exact role of the PI3K
domain is thus unclear; though in human cells, the PI3K domain of TRRAP is required for cellular
transformation by myc and E1A (Park et al., 2001).
We have used a mutagenesis approach to determine the structure/function relationships of Tra1,
focusing in particular on the PI3K domain. We have identified eight mutations in the PI3K domain that
result in cellular inviability and three that result in temperature sensitive growth and reduced growth on
media containing 6% ethanol. Characterization of these temperature sensitive alleles at the permissive
temperature confirms a role for the PI3K domain in transcriptional regulation. These mutations confer
altered expression of ~7% of the yeast genome and were distinct from those affecting individual
components of either SAGA or NuA4 complexes.
6
MATERIALS AND METHODS
Yeast strains and growth. Yeast strains are listed in Table 1. TRA1 alleles contained on TRP1
centromeric plasmids were transformed into CY1021 and the wild-type copy displaced by plasmid
shuffling (Sikorski and Boeke, 1991). CY1896 is a derivative of Y5565 that has been gene replaced
with tra1-SRR3413 and selected for through placement of Tn10LUK at the downstream BstB1 site.
CY2437 and CY2438 are derivatives of KY320 and similarly contain integrations of wild-type TRA1
and tra1-SRR3413, respectively, in a background lacking BamHI and SalI restriction sites within TRA1.
Growth comparisons were performed on selective plates after three to five days at 30o unless stated
otherwise. Assays were performed in duplicate on independently constructed strains. Scoring for PI3K
domain mutations was done in relation to CY1524, which contains TRA1SB, the background allele used
to construct mutations within the PI3K domain. TRA1SB is Flag-tagged and contains a BamHI site that
converts N3580A and a SalI site of nucleotides A9714G and T9717G. Scoring for the viability of non-PI3K
domain mutations was relative to CY1020 (Saleh et al., 1998).
Construction of DNA molecules. lacZ reporter constructs were cloned as his3-lacZ fusions into the
LEU2 centromeric plasmid YCp87 (Brandl et al., 1993). ADH2-lacZ contains promoter sequences (-
440 to -150; Saleh et al., 1998). PHO5 (-452 to +47), INO1 (-517 to +40) and HIS4 (-545 to + 29),
were engineered by PCR as BamHI-HindIII fragments and cloned into YCp87 (see supplemental Table
1 for a listing of oligonucleotides).
Construction of TRA1 alleles: Mutations of DSP171AAA, EFS184AAA, KEL345AAA, VLL837AAA,
VRL867AAA, KKK1686AAA, GLW2677AAA, GYH2939AAA, TLP2972AAA, HFQ3168AAA,
SRR3413AAA, and PFR3621AAA were engineered into myc-TRA1-Ycplac111 (Saleh et al., 1998). Each
allele was synthesized in two PCR reactions. Reactions contained a listed oligonucleotide and the
appropriate outside flanking primer with a unique cloning site (oligonucleotides 842, 2542-3, 2337-1,
7
2323-2, 2323-1, 2542-4, 2337-2 and 2346). Mutagenized codons were replaced with a NotI site such
that 5’ and 3’ halves could be ligated sequentially into pUC vectors. Fragments were moved into myc-
TRA1-Ycplac111 using the outside restriction sites. Mutations within the coding region of the PI3K
domain (including additional alleles of EER3416AAA, and PFR3621AAA) were constructed in TRA1SB.
Flag epitopes were introduced at an N-terminal NotI site. Mutagenized alleles were constructed by
PCR and ligated as SalI-BamHI (for alterations N-terminal to N3580) or BamHI-SacI (for alterations C-
terminal to N3580). Flanking primers used in the PCR reactions were 4293-1 and 4225-3 or 4249-1 and
2346/4479-1 depending on the placement relative to N3580. YNG2 and GCN5 were similarly cloned into
this molecule.
TAP-tagged molecules were cloned into YCpDed-TAP, a derivative of the URA3-containing
centromeric plasmid YCplac33 (Gietz and Sugino, 1988) that contains a DED1 promoter driving
expression of a TAP epitope. The DED1 promoter was synthesized by PCR using oligonucleotides
4149-1 and 4149-2 and cloned as a PstI-BamHI fragment into YCplac33. The TAP epitope was
subsequently cloned as a BamHI to SacI fragment into this molecule after PCR using oligonucleotides
4149-4 and 4168-1 and pFA6a as the template (kindly provided by Kathy Gould). The coding
sequence for the Flag-epitope was cloned into this molecule at the engineered NotI site to give
YCpDed-TAP-Flag. TRA1 was introduced into this vector as a NotI-Sac1 fragment.
TAP purification. Tandem affinity purification was carried out as described by Rigaut et al. (1999).
One liter of cells grown in YPD to an A600 ~ 1.5 were ground in liquid nitrogen (Saleh et al., 1997).
Further steps were at 4°. Cell extract suspended in 5 ml of IPP150 with protease inhibitors (1 mM
phenylmethylsulfonylflouride, 0.1 mM benzamidine hydrochloride, 2 μg/ml pepstatin A, 2 μg/ml
leupeptin and 0.1 mg/ml trypsin inhibitor) was cleared by centrifugation at 147,000 g and incubated
with 600 μl of IgG-Agarose (Sigma-Aldrich Canada Ltd) for 2 h. After washing with 30 ml of IPP150
and 10 ml of TEV cleavage buffer, beads were suspended in 1 ml of cleavage buffer and incubated
8
with 100 U TEV protease for 2 h. 3 μl of 1 M CaCl2 was added to the eluent, then diluted with 3 ml of
calmodulin binding buffer. The protein was incubated with 600 μl of Calmodulin-Sepharose
(Stratagene Inc) for 1 h, washed with 30 ml of calmodulin binding buffer and protein eluted.
β-galactosidase assays. For HIS4-lacZ cells grown in minimal media to an A600 of ~1.5 were pelleted,
washed in LacZ buffer and concentrated ~5-fold. β-galactosidase was determined using ο-nitrophenol-
β-D-galactosidase as substrate, standardizing to cell density (Ausubel et al., 1998). Analysis of PHO5-
lacZ, INO1-lacZ and ADH2-lacZ were performed similarly except that overnight cultures were washed
3-times in water then grown to an A600 ~1.5 in YPD depleted of phosphate (Han et al., 1988), depleted
of inositol or minimal media containing 3% ethanol (v/v) and 0.1% glucose.
Gal4 affinity chromatography. Amino acid residues 764 to 882 of Gal4 were expressed as a GST
fusion in the vector pGEX1. One liter of E. coli cells was induced with 0.6 mM isopropyl β-D-
thiogalactopyranoside for 6 hours at 37o. Harvested cells were resuspended in lysis buffer (40 mM
TRIS [pH 8.5], 20 mM NaCl, 10% glycerol, 0.08% NP-40, 0.2 mM EDTA [pH 8.0], 1 mM DTT) with
protease inhibitors, treated with 1 mg/ml lysozyme on ice for 1 h and broken by sonication. Cleared
extract was incubated on ice with one ml Glutathione Sepharose 4B (GE Healthcare), washed three
times with 10 ml of 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 and once with 10
ml of lysis buffer. TAP-Tra1 purified on IgG-Agarose from 1.0 liter cultures of CY1962 and CY1963
containing HA-tagged Ada2 (Saleh et al., 1997) was applied to Gal4-containing Glutathione Sepharose
in 3 ml of lysis buffer and rotated at 4o for 60 minutes, washed with lysis buffer and bound protein
eluted with 10 mM reduced glutathione. 30 μl of the eluent was separated on a 10% SDS-PAGE gel,
transferred to PVDF membrane and blotted with anti-HA antibody.
9
Genetic analyses. Identification of an intragenic suppressor of tra1-SRR3413. Approximately 2x106
cells of yeast strain CY1531 (tra1-SRR3413) were plated onto YPD containing 6% ethanol. Two
colonies were isolated after 5 days of growth and Tra1 expressing plasmids recovered (Hoffman and
Winston, 1987). The SalI-SacI fragment was re-cloned into TRA1SB and found sufficient for
suppression. Systematic genetic array analysis on yeast strain CY1896 (tra1-SRR3413) was performed
as described by Tong and Boone (2006).
RNA purification and microarray analysis. Yeast cells were grown in YP media containing 2%
glucose to an A600nm=2.0 at 30o. RNA was purified from 108 cells after glass bead disruption as
described previously (Mutiu and Brandl, 2005; Mutiu et al., 2007). Microarray analyses were
performed using the Agilent Yeast Oligo Array Kit at MOgene LC (St. Louis, MO). Comparisons were
made with wild-type strain CY1524 and performed in duplicate with dye reversal. Array data for the
confirmed set of protein coding genes was log transformed and averages obtained. Genes displaying
an expression change greater than 1.5-fold, where the direction of the response differed between
replicates were discarded. Profiles were normalized by subtracting the mean response of all genes
from the response of each gene, then dividing by the standard deviation of all responses within a
profile. An initial cluster analysis using a subset of the compendium data (Hughes et al., 2000) that
included deletion strains with gene ontology terms indicating gene expression, identified microarray
profiles of strains with deletions of cka2, ckb2, gcn4, hat2, isw1, mbp1, rpd3, rtg1, sap30, sin3, ste12
and yap3 as being most similar to tra1-SRR3413. Agglomerative hierarchical clustering based on the
average linkage of uncentered correlations was performed on the profiles from these strains and strains
with deletions of SAGA (Ingvarsdottir et al, 2005) and NuA4 (Krogan et al., 2004) components using
CLUSTER 3.0 software (Eisen et al., 1998). Genes not appearing in at least two of the microarray
profiles were excluded. The data was visualized using MAPLETREE
(http://rana.lbl.gov/EisenSoftware.htm). Gene ontology terms associated with gene clusters were
determined using FunSpec (Robinson et al., 2002).
10
Chromatin Immunoprecipitation Assays. Assays were performed essentially as described by Kuo
and Allis, (1999). For assays comparing acetylated histone H3, 50 ml of culture was grown to an
A600nm ~ 2.0. To equalize amounts of input DNA, cell extract was reverse cross-linked and PCR
reactions performed on serial dilutions. For immunoprecipitations, DNA equal to that contained in 50
μg (protein) of cell extract from the wild-type sample, was incubated with Protein A Sepharose 4 Fast
Flow (Pharmacia Inc), transferred to clean tubes, and rotated overnight with one μl of antibody (anti-
H3: Abcam Inc., ab1791; anti-AcH3, Lys9: Upstate Biotechnology, 07-352). Precipitate was removed
by centrifugation and the supernatant incubated with Protein A beads. After washing and reversal of
crosslinks products were analyzed by PCR (Ricci et al., 2002) using primers for the PHO5 (-550 to -
300), ADE1 (-505 to 51), PHO84 (-199 to 74), PGK1 (-249 to 28) promoters or PHO5 gene (1035 to
1322). PCR conditions were 5 minutes at 95o, followed by 25 cycles of 30 seconds at 95o, 52o, and 72o,
then 5 minutes at 72o. Assays of acetylated histone H4 were performed with 250 ml cultures grown to
A600nm ~ 2.0 in YPD, 0.5 mg of chromatin (in a total volume 0.5 ml), and Protein A/G plus 2 μl of anti-
AcH4, Lys8 (Abcam Inc, ab1760). For immunoprecipitations using TAP-Flag-Tra1, chromatin was
prepared in RIPA buffer with washing and elution as described by Alekseyenko et al. (2006).
Western blotting. Yeast extract prepared by glass bead disruption (Brandl et al., 1993) or grinding in
liquid nitrogen (Saleh et al., 1997) was separated by SDS-PAGE, transferred to PVDF membranes
(Roche Applied Science) using a wet transfer system in 48mM Tris, 40mM glycine, 0.0375% SDS and
20% methanol for 1 h at 100V. Anti-Flag antibody (M2, Sigma-Aldrich Canada Ltd) and Anti-HA
antibody (ascites fluid from the 12CA5 cell line) were used at a ratio of 1:4000. Secondary antibody
(Anti-Mouse IgG HRP, Promega) used at a ratio of 1:10000 was detected using a 20% solution of
Immobilon Western (Millipore Corp). Densitometric scanning of films was performed using
AlphaImager 3400 software (Alpha Innotech, Inc).
Telomere length assays - Plasmid linearization assay. Strains were transformed with pVL106
(Lundblad and Szostak, 1989). Leu+ Ura+ colonies were grown to saturation in media lacking uracil
11
then YPD. 10 μl was spotted onto leucine-depleted plates containing 5-FOA. Values reported are the
percent of 5-FOA resistant, Leu+ colonies in the mutant strain as compared to wild-type, normalized to
the number of cells plated. Terminal restriction fragment determination. TRA1 alleles were
transformed into CY1021 and plasmid shuffling performed (generation zero). 10 ml saturated cultures
were grown from these colonies at 30˚ or 34˚ and DNA prepared (Ausubel et al., 1998). Each culture
was also diluted 1:1000 into YPD and grown again to saturation, then repeated. Genomic DNA was
digested with XhoI and Southern blotting performed (Teng and Zakian, 1999).
Fractionation of Tra1 containing complexes on HiTrap Q Sepharose Fast Flow. 18 mg of whole
cell extract containing HA-Ada2p and Tra1SB or Tra1-SRR3413 was prepared in 40 mM Tris-HCl (pH
7.7) buffer containing 20 mM NaCl, 10% glycerol, 0.08% Nonidet P-40, 0.2 mM EDTA, and 0.1 mM
dithiothreitol. Extracts were treated with protamine sulphate (0.2%), rotated at 4o for 30 minutes,
cleared by centrifugation at 147,000 g and applied to a HiTrap Q Sepharose Fast Flow column (1.0 ml;
flow rate of 1.0 ml/min ; Amersham Pharmacia Biotech). After washing with 4 ml of extraction buffer,
protein was eluted with a 16-ml gradient of 0-1.0 M NaCl. 20-µl aliquots of 200-µl fractions was
separated by SDS-PAGE and analyzed by Western blotting with anti-HA antibody. 25 mg of whole
cell extract containing TAP-Flag-Yng2 and Tra1SB or Tra1-SRR3413 was blotted using anti-Flag
antibody.
12
RESULTS
The size of Tra1 presents a significant challenge for analyses of structure and function using molecular
genetic approaches. Because of the difficulty in identifying and characterizing random mutations, we
undertook a targeted mutagenesis approach, initially constructing 12 triple alanine scanning mutations
scattered in regions that might be relevant for function (Table 2). These alleles were expressed on a
TRP1-containing centromeric plasmid and transformed into yeast strain CY1021 that contains a
disruption of the genomic copy of TRA1 complemented by wild-type TRA1 expressed on a URA3-
containing centromeric plasmid (Saleh et al., 1998). Each allele was analyzed for growth properties
after shuffling out wild-type TRA1 on media containing 5-fluoroorotic acid (5-FOA). Phenotypes
examined included: mating efficiency, temperature sensitivity (37o), growth on media depleted for
inositol, amino acids and phosphate, fermentation of galactose and raffinose, sensitivity to
hydroxyurea, methylmethanesulfonate (MMS), ultraviolet irradiation, γ-irradiation, high osmolarity,
cycloheximide and tunicamycin. Two of the alleles tested, DSP171AAA and EFS184AAA altered
potential phosphorylation sites. (The number indicates the first residue of the three. We will not
include the AAA for each allele.) VLL837 altered a putative LXXLL motif. Many of the other altered
residues are conserved in yeast and/or human Tra1 homologs. Only two of the 12 alleles, both with
mutations within the PI3K domain, had discernible phenotypes. tra1-PFR3621 would not support
growth on media containing 5-FOA and therefore is a nonfunctional allele. tra1-EER3416 showed
reduced growth on all media tested, as well as slight temperature sensitivity.
Targeted mutation of the PI3K-domain. Based on the above results, we directed additional
mutations to the PI3K domain. As we were concerned that mutations might simply alter the fold of the
PI3K domain, we focused on mutagenizing codons for amino acid residues conserved in the
Tra1/TRRAP family of molecules but not in the broader PI3K domain family. In the absence of a
structure for the Tra1 PI3K domain, the structure of PI3K-γ (Walker et al., 1999; see supplemental
Figure 1) allows an initial prediction of the positions of these residues. The PI3K domain consists of
13
N-terminal and C-terminal lobes connected by a loop between β7 and β8. For PI3K-γ, ATP
interactions are found with the β3-β4 loop (P-loop), the end of β5 and the β7-β8 loop. The region
between α6 and β9 corresponds to the catalytic loop. The C-terminal lobe also contains an activation
loop that is required for substrate binding of the PI3K molecules (Bondeva et al., 1998) and likely
binds phosphatidylinositols in PI3K-γ.
The phenotypes of strains containing the mutagenized tra1 alleles are shown in Table 3. Of the
nineteen alleles with targeted mutations in the PI3K domain, eight did not support viability. Two of the
eight, R3456A and R3567A, were single amino acid substitutions; the others, WRR3317, DIE3351, RFL3374,
FRK3544, PFR3621 and RDE3668 were triple alanine scanning mutations. Five of the eight mutations were
within predicted loop regions (WRR3317, DIE3351, RFL3374, R3567 and PFR3621). F3443, which aligns with
the α3-β6 loop can be considered in this group since strains containing tra1- F3443A were marginally
viable in rich media but inviable under conditions of stress. If their positioning within loops is correct,
it is not likely that these residues are important for structure but instead may be involved in protein-
protein interactions or have regulatory roles. In agreement with this, R3567 of Tra1 aligns with R947, an
essential residue within the catalytic loop of PI3K-γ (Dhand et al., 1994; Stack and Emr, 1994).
Likewise it is required for the activity of Tra1, though for Tra1/TRRAP the size of the residue may be
the critical factor since human TRRAP contains a leucine at this position. Similarly, PFR3621 aligns
with the activation loop and despite the lack of kinase activity, these residues are required for the
activity of Tra1. (We note that the phenotype of PFR3621 may in part be due to reduced expression
levels, not shown). Three mutations giving rise to inviability were not in predicted loops. R3456 is in a
comparable position to G877 of PI3K-γ, which is found within β7 and conserved in many of the lipid
kinases (Walker et al., 1999). FRK3544 and RDE3668 align with α6 and α9, respectively, and are
uniquely conserved in the Tra1/TRRAP molecules.
Eleven alleles supported viability in rich media. Their growth characteristics under a variety of
conditions that reflect ada, spt or NuA4 phenotypes are shown in Table 3. Other conditions and assays
14
tested but without substantial effects were growth on MMS (also see below), tert-butylhydroperoxide,
galactose, raffinose, 6-azauracil, 1.0 M NaCl, and tunicamycin. In addition, none of these alleles
significantly changed the rate of loss of a URA3-containing centromeric plasmid, after growth on
nonselective conditions or resulted in a significant defect in mating (not shown). K3571A and N3580A
had little effect on growth under any of the conditions tested. Four of the alleles, P3446A, DKL3491,
P3662A and N3617A, showed a very weak classic ada phenotype (Berger et al., 1992), being partially
resistant to overexpression of VP16. (Note that a deletion of gcn5 would score as ++++ on this scale.)
Some of these alleles were also slightly sensitive to 6% ethanol, characteristic of an ada2 disruption
(Takahashi et al., 2001).
Three alleles, tra1-SRR3413, tra1-P3408A and tra1-S3463A, resulted in a common and distinct phenotype.
(EER3416 had a similar phenotype but to a lesser extent.) Strains containing these alleles were
temperature sensitive for growth at 37o, had dramatically reduced growth in media containing elevated
concentrations of ethanol and grew poorly in media lacking phosphate. Plates showing the growth of
strains containing these alleles under some of the conditions are shown in Figure 1a. The two alleles
with the greatest temperature sensitivity (tra1-SRR3413 and tra1-P3408A) resulted in somewhat reduced
growth on media depleted of inositol or containing benomyl, a microtubule-destabilizing agent. These
latter two phenotypes are shared with deletions of some Spt components of SAGA (Roberts and
Winston, 1997) and for components of the NuA4 complex (LeMasson et al., 2003; Bittner et al., 2004;
Krogan et al., 2004), respectively. The tra1 alleles displayed similar sensitivity to ethanol as deletion
of ada2 (not shown) and like ada2 had reduced growth on Calcofluor white (Figure 1a) suggesting that
the effect results from changes in cell wall properties or the ability of the strains to respond to changes
in cell wall integrity (Takahashi et al., 2001). We examined the growth properties of strains containing
these alleles in more detail (Figure 1b). At 34o, the tra1-SRR3413, tra1-P3408A and tra1-S3463A alleles
resulted in a reduced growth rate in rich media relative to the TRA1SB allele. Upon heat shock the
mutant strains showed an immediate growth arrest but did recover; however, four hours after the shift
to 37o growth stopped. To ensure that the effects of these mutations was not due to expression on a
15
plasmid or the background N3580A mutation from the BamHI site within TRA1SB, tra1-SRR3413 lacking
the N3580A mutation was integrated into the genome. As shown in Figure 1c, the integrated allele
showed similar slow growth at elevated temperature and in the presence of Calcofluor white as did the
plasmid version.
Since the NuA4 complex has a role in DNA double strand break repair (Bird et al., 2002), we
examined strains containing Tra1-SRR3413, P3408A and S3463A more closely for sensitivity to MMS
comparing to a deletion of the NuA4 component Vid21/Eaf1. tra1-SRR3413 showed a slight sensitivity
to MMS but dramatically less so than deletion of vid21/eaf1 (not shown). In support of only minor
changes in double strand break repair, none of the three alleles resulted in enhanced sensitivity to γ-
irradiation at 30o (not shown).
To evaluate whether the growth phenotypes might result from reduced expression of the altered forms
of Tra1, the levels of Flag3x-tagged Tra1-P3408A, Tra1-S3463A and Tra1-SRR3413 were examined by
Western blotting of crude yeast extracts (Figure 2a). Each of these forms was expressed at a level
approximately equivalent to Tra1SB, though we observed slightly elevated levels of Tra1-S3463A. We
focused additional studies on tra1-SRR3413 since it displayed the most dramatic phenotypes. To
determine if Tra1-SRR3413 associated with components of NuA4 and SAGA complexes, a TAP-tagged
version of the molecule was purified by tandem affinity purification and the presence of Spt7 and Flag-
tagged Yng2 determined by Western blotting (Figure 2b). SRR3413 interacted with Flag-Yng2 and with
two forms of Spt7 suggesting its presence in NuA4, SAGA and SAGA-like (SLIK) complexes. To
further examine if Tra1-SRR3413 is found within SAGA and NuA4 complexes, crude extracts
containing Tra1-SRR3413 were fractionated on a HiTrap Q Sepharose column and the elution profile of
HA-Ada2 and Flag-Yng2 compared to that found in a strain containing Tra1SB (Figure 2c and 2d). The
elution profiles for Tra1-SRR3413 varied little with Tra1SB suggesting that it associates in the
appropriate high molecular mass complexes.
16
Gene expression in the conditional tra1 strains. To determine if the tra1-SRR3413, tra1-P3408A and
tra1-S3463A alleles resulted in changes in transcriptional regulation, we examined expression of PHO5,
HIS4, INO1 and ADH2 using lacZ-reporter fusions (Figure 3a). Assays were performed at 30o under
conditions that would induce each specific promoter. Strains deleted for gcn5, spt7 and yng2 were
examined for comparison. The tra1 mutations resulted in expression of PHO5 at a level from 3-17%
that seen in the wild-type (TRA1SB), the extent paralleling the severity of the growth defects.
Expression of HIS4 and INO1 were relatively unchanged in the tra1 backgrounds. ADH2 was
decreased on average to 60% wild-type levels for the three tra1 mutants with the extent again
paralleling the growth defect. The pattern of expression seen in the tra1 strains most closely resembled
that seen upon disruption of gcn5; expression of HIS4 is reduced upon disruption of spt7 and yng2,
unlike the tra1 strains and INO1 is reduced upon disruption of spt7. Elevated temperature did result in
enhanced effects of tra1-SRR3413 as expression of INO1-lacZ was reduced to ~ 50% that seen in
TRA1SB when cells were grown at 35o (Figure 3b).
A more detailed analysis of gene expression in the tra1-SRR3413, tra1-P3408A and tra1-S3463A strains
was obtained by performing microarray analysis. The Agilent Yeast Oligo array representing all
independent yeast ORFs was probed with cRNA produced from cells grown logarithmically at 30o in
YP media containing 2% glucose (note that this differs from the data shown in Figure 4 which were
performed in inducing conditions). Two-dye experiments were performed comparing expression in the
mutant strains to that found in a strain containing TRA1SB. For each mutant, the experiment was
performed in duplicate with dye-reversal. The tra1-SRR3413 allele resulted in a change in expression of
two-fold or greater of ~7% of the protein encoding genes with ~70% having decreased expression. The
25 genes showing the greatest increase and decrease in expression for each mutant is shown in Table 4
(the full data set has been submitted to the GEO database at NCBI under accession number GSE6847).
The genes affected in the three strains were highly similar, consistent with the mutant alleles having a
common defect. Gene ontology of those most affected indicate a group of upregulated genes are
17
involved with ion transport and cell cycle regulation, while a number of the down-regulated genes have
roles in nucleotide and amino acid metabolism.
Hierarchical cluster analysis was performed with the array data for the tra1-SRR3413 strain, strains with
deletions of SAGA and NuA4 components and a subset of the compendium data set (Hughes et al.,
2000) including those genes linked to transcription. tra1-SRR3413 clustered in a leaf with a deletion of
ada2, though the depth of the branch is indicative of only weak similarity (Figure 4). Profiles of strains
with deletions of the NuA4 components (eaf5, yaf9, eaf1/vid21 and yng2) showed less similarity with
tra1-SRR3413, than ada2, spt3 or spt8. Overall, the relatively low level of similarity indicates that the
transcription pattern of tra1-SRR3413 results from the cumulative effects of this allele on both SAGA
and NuA4 complexes, and/or there being one or more independent roles for Tra1.
Brown et al. (2000) previously identified a tra1 allele that resulted in decreased binding to the Gal4
transcriptional activation domain. To address whether the transcriptional changes observed for tra1-
SRR3413 were due to a defect in its interaction with transcriptional activation domains, we compared the
ability of Tra1-SRR3413 and Tra1SB to associate with the Gal4 activation domain (Gal4AD). Tandem
affinity purified Tra1SB and Tra1-SRR3413, prepared from strains containing HA-tagged Ada2 were
chromatographed on GST-Gal4AD columns. Association of SAGA with Gal4 was detected by Western
blotting for HA-Ada2 (Figure 5a). SAGA containing Tra1-SRR3413 bound Gal4AD to the same extent as
SAGA-containing Tra1SB (compare lanes 4 and 6), suggesting that the mutation does not decrease
association with transcriptional activators. This is consistent with the region required for activator
interaction being contained within amino acid residues 2233 to 2836 (Brown et al., 2000). Recruitment
of Tra1-SRR3413 to the PHO5 promoter in vivo was examined by chromatin immunoprecipitation using
the TAP-tagged derivatives of Tra1. As shown in Figure 5b, Tra1-SRR3413 was recruited as efficiently
to the PHO5 promoter as Tra1SB (compare lanes 2 and 3 of the top panel; input DNAs are shown in the
lower panel).
18
To determine if Tra1-SRR3413 alters acetylation of either histone H3 or histone H4, chromatin
immunoprecipitations were performed. We first compared the ratio of acetylated histone H4 (at lysine
8) to total histone H3 at the ADE1 and PHO5 promoters for the strain containing tra1-SRR3413 with
strains containing deletions of the NuA4 components Vid21/Eaf1 and Yng2. Figure 6a shows the
relative level of acetylated histone H4 as a percentage of that found in the wild-type strain. For both
ADE1 and PHO5 promoters, tra1-SRR3413 resulted in a decrease in relative acetylation to
approximately 60% of that found in the TRA1SB strain. This decrease was comparable to that found
upon deletion of yng2 and somewhat less than deletion of vid21/eaf1. For each mutant strain, a smaller
effect was observed for sequences at the 3’ end of the PHO5 gene. Acetylation of histone H3 (at
lysine 9) was then analyzed at PHO5, PHO84 and PGK1 promoters (Figure 6b); the latter being
SAGA-independent. tra1-SRR3413 resulted in a decrease in the ratio of acetylated histone H3 to total
histone H3 at PHO5 and PHO84 promoters that was approximately 35% of that found in the wild-type
tra1SB strain, approaching that seen in strains deleted for spt7 and ada2. At the PGK1 promoter, the
decrease in acetylation of histone H3 was less apparent (~70% of wild-type for tra1-SRR3413).
Although we can not exclude an indirect role of Tra1 on acetylation, the simplest interpretation of
these results is that the PI3K domain of Tra1 is required for optimal histone acetylation by both the
NuA4 and SAGA complexes.
Tra1-SRR3413 results in shortened telomere length. The similarity of Tra1 to many of the PI3K-
domain containing proteins involved in telomere biology (for example, Tel1 and Mec1) led us to test if
the tra1-SRR3413, tra1-P3408A and tra1-S3463A alleles affected telomere length. As an initial test we
assayed the effects of these alleles on the ability of strains to generate and stably maintain linearized
plasmids after transformation with circular plasmids containing inverted repeats of telomeric
sequences (Lundblad and Szostak, 1989). Strains containing tra1-SRR3413, tra1-P3408A and tra1-S3463A
were transformed with the LEU2 containing centromeric plasmid pVL106 (kindly supplied by Dr. V.
Lundblad), which contains URA3 flanked by inverted telomeric sequences. Cells were grown in
nonselective media and plated onto media containing 5-FOA and lacking leucine. 5-FOA
19
resistant/Leu+ colonies were counted and normalized to the number of total cells plated. As shown in
Figure 7a, tra1-SRR3413 resulted in a reduced ability to linearize and maintain pVL106. To directly
examine telomere length, Southern blotting was used to compare the terminal restriction fragments
(TRFs) of chromosomes in the tra1-SRR3413 and TRA1SB strains. CY1021 containing wild-type TRA1
on a URA3-containing plasmid was transformed with TRA1SB or tra1-SRR3413 on TRP1-centromeric
plasmids. The wild-type TRA1 allele was lost after selection on 5-FOA, establishing generation zero.
Strains were grown through multiple generations, genomic DNA isolated and TRF length determined
after digestion of the genomic DNA with XhoI. Cells were grown at both 30o and 34o. The latter
significantly reduces growth rate and from the linearization assay appeared to suppress the effects of
SRR3413 on telomere shortening (not shown). As shown in Figure 7b, at 10 generations of growth, the
TRFs were of approximately equal lengths in TRA1SB and tra1-SRR3413 strains. By 50 generations,
TRFs were appreciably shorter in the tra1-SRR3413 strain, with a further shortening apparent at 100
generations when cells were grown at 30o. The median difference was approximately 120 and 140 base
pairs at 50 and 100 generations, respectively. The effect of SRR3413 was not apparent when cells were
grown at 34o, perhaps the result of increasing the period available for telomere extension.
Genetic analysis. To examine the molecular basis for the tra1-SRR3413 phenotype, we initiated a
suppressor analysis. tra1-SRR3413 was introduced into yeast strain CY1021 by plasmid shuffling.
Approximately 2 x 106 cells were plated onto YPD containing 6% ethanol. Growth of two colonies
was observed after 5 days. To determine if these were intragenic, the tra1 expressing plasmid was
isolated from both strains and the phenotype re-examined after plasmid shuffling in yeast strain
CY1021. In both cases the plasmid enabled growth in YPD containing 6% ethanol. The 3’ SalI-SacI
fragment of each allele was sequenced and found to contain the SRR3413 mutation and a common
mutation converting H3530Y. This mutation was shown to be sufficient for suppression by recloning the
SalI-SacI fragment into TRA1SB and plasmid shuffling. H3530Y suppressed the growth defects
associated with SRR3413 on both 6% ethanol containing plates (Figure 8a) and 37o (not shown). Allele
specificity was examined by introducing H3530Y into wild-type and tra1-P3408A containing
20
backgrounds. H3530Y on its own had no effect on growth but did suppress the ethanol sensitivity due to
P3408A and therefore is a general suppressor of the ethanol-sensitive class of tra1 alleles. As shown in
Figure 8b, the effects of H3530Y are most likely due to suppression of transcriptional defects since this
mutation suppressed the slow induction of PHO5 seen in a tra1-SRR3413 strain.
To detect genes whose disruption in parallel with tra1-SRR3413 generates a synthetic slow growth
phenotype, we performed a systematic genetic array (SGA) analysis in which a strain containing tra1-
SRR3413 was crossed into the array representing the collection of ~4700 viable haploid deletion strains.
Candidate genetic interactions, scored as slow growing colonies through two independent screenings,
were examined individually after generation of double mutant strains by tetrad dissection. Growth for
these strains was examined on synthetic complete media at 34o. The twenty-three genes identified and
their functions are listed in supplemental Table 2. Consistent with the finding that tra1-SRR3413 resulted
in sensitivity to ethanol and Calcofluor white, ten of these appear involved in cell membrane and cell
wall processes. This number is greater if indirect roles, for example a contribution to
phosphatidylserine synthesis by SER1 and SER2, are considered. The other prevalent groups
represented were genes involved with transport and mitochondria function. A hierarchal cluster
analysis was performed with the tra1-SRR3413 interacting genes and the datasets of Tong et al. (2004)
and Pan et al. (2006). tra1-SRR3413 did not cluster closely with any of the strains used in these analyses.
In fact, no common synthetic growth defects were found amongst those listed in the Saccharomyces
Genome Database for NuA4 and SAGA components and those for tra1-SRR3413.
21
DISCUSSION
Tra1 is one of the largest yeast proteins and is essential for viability. As a component of both NuA4
and SAGA complexes, Tra1 interacts directly with DNA-binding transcriptional regulators recruiting
these HAT complexes to promoters. Based upon the similarity of this domain with that found in other
cellular regulators it is likely that the PI3K domain of the molecule has a significant function.
Indicative of a key role for the PI3K domain, eight targeted mutations within this region resulted in
loss of viability. Although we lack detailed structural information for the Tra1/TRRAP subfamily, the
alignment of these residues in comparison to the structure of porcine PI3K-γ is interesting. Residues
R3567 and PFR3621 are not unique to Tra1/TRRAP and are found in the catalytic-loop and activation-
loop, respectively. As is the case for the kinase members of the PI3K family (Dhand et al., 1994; Stack
and Emr, 1994), R3567 is required for function of Tra1. Similarly, PFR3621 is generally conserved within
the activation loop of the PI3K family. The activation-loop plays a critical role in substrate binding for
these molecules (Bondeva et al., 1998), suggesting a similar essential role for this region in Tra1.
Substrates for Tra1 are unknown. Potentially these could include phospholipids; however, we have
detected only very weak binding of recombinant Tra1-PI3K domain to PIP strips (Echelon
Biosciences; not shown).
Eight alleles with mutations in the PI3K domain supported viability and displayed phenotypes, which
fell into two groups. The first group, tra1-P3446, -DKL3491, -P3612 and -N3617 were slightly resistant to
overexpression of VP16, although to a much lesser extent than the ada genes, and were partially
sensitive to hydroxyurea, characteristic of defects in DNA replication. This later phenotype has not
been previously ascribed to SAGA or NuA4 components and may reflect a novel function of one or
both of these complexes or perhaps defects in expression of genes involved in this process. The second
group of alleles containing mutations of P3408, SRR3413, S3463 and to a lesser extent EER3416, was more
marked. These alleles resulted in temperature sensitive growth, reduced growth in low phosphate
media and in media containing 6% ethanol. The latter has also been noted for deletions of ada2 and
22
based on their reduced growth in the presence of Calcofluor white, this phenotype likely reflects
changes in cell wall properties (Takahashi et al., 2001) or the response to cell wall stress. Unlike
deletions of Ada components, the tra1-P3408A, -SRR3413 and -S3463A alleles did not result in resistance
to overexpression of VP16, nor is their temperature sensitive growth indicative of deletions of ada
genes in the KY320 strain background. tra1-P3408 and tra1-SRR3413, the alleles with the most
pronounced phenotypes, were partially sensitive to the microtubule destabilizing agent benomyl. This
is characteristic of deletions of NuA4 components, but again for the tra1 alleles the effect was not as
severe. The tra1-SRR3413 allele was the only allele showing sensitivity to MMS but dramatically less so
than for deletions of components of the NuA4 complex. Growth of tra1-P3408, SRR3413 and S3463 strains
was modestly reduced on media depleted of inositol, a characteristic of deletions of spt genes;
however, unlike deletion of spt7, the inositol auxotrophy of the tra1 alleles was not the result of
reduced expression of INO1. Cumulatively, these phenotypes suggest that Tra1 possesses functions
overlapping yet distinct from SAGA or NuA4 components.
The transcriptional effects of the tra1-P3408A, SRR3413 and S3463A alleles were compared to deletions of
spt7, gcn5 and yng2 using lacZ fusion reporters. The transcriptional change for the tra1 alleles was less
than that seen upon deletion of the NuA4 or SAGA components but most closely resembled that seen
upon deletion of gcn5. Microarray analyses confirmed the transcription defect arising from these
alleles and that they were acting by altering a common function. Cluster analysis versus the expression
profiles seen for deletions of SAGA and NuA4 components showed the greatest similarity though not
robust, to ada2. Comparison of the growth phenotypes of strains containing deletion of strains carrying
single and double mutations of ada2Δ0 and tra1-P3408A confirmed that the tra1 mutations were not
functioning exclusively through effects on the ADA genes (data not shown). This lack of similarity
suggests that the transcriptional effects of the tra1 alleles result from the integrative effect of
disturbing histone acetylation by both SAGA and NuA4 complexes and/or the possibility that the PI3K
domain of Tra1 has one or more roles distinct from other members of these complexes. The
mechanism by which the tra1 mutations affect histone acetylation is unclear; interestingly, in the three-
23
dimensional structure of the SAGA complex revealed by electron microscopy, Tra1 and Gcn5 are
positioned on opposite sides of a nucleosome-sized cleft (Wu et al., 2004), suggesting that Tra1 may
play a role in orienting nucleosomes for acetylation.
The finding of a generation dependent telomere shortening in the tra1-SRR3413 strain points to a role of
Tra1 in telomere elongation or maintenance. The shortened telomeres may be the indirect result of
Tra1 regulating expression of genes required for these processes. In this regard eleven genes whose
deletion results in shortened telomeres (Askree et al, 2004) have decreased expression of 1.5-fold or
greater in the tra1-SRR3413 strain. Most notable of these is EST3 with decreased expression of 2.4-fold.
Alternatively, Tra1 may play a direct role in telomere elongation or maintenance. We do not believe
that the Tra1 effect is mediated solely through one of SAGA or NuA4-dependent histone acetylation,
as component genes were not found in the genome wide screen of Askree et al. (2004) nor in our
independent analyses of Spt7, Gcn5 and Eaf7 (data not shown). Additional studies will be required to
determine if Tra1 is recruited to telomeres and the role of the PI3K-domain in telomere elongation.
The ethanol and Calcofluor white sensitivity of tra1-SRR3413 as well as its genetic interactions with
genes involved in cell wall and membrane processes is consistent with the involvement of SAGA in
the response to stress (Huisinga and Pugh, 2004). Tra1 may be required to trigger transcriptional
changes necessary when membrane processes are disturbed during cellular stress. Associations
between SAGA components and membrane processes have previously been suggested by the finding
that Gcn5 and Spt20 are required for the unfolded protein response (Welihinda et al., 1997, 2000).
Interestingly, deletion of a number of genes involved in vesicular trafficking, including vps9, vps15,
vps28, vps34, vps36, vps39, bro1 and sur4, result in both telomere shortening and ethanol sensitivity
similar to tra1-SRR3413. The other two gene deletions with both of these phenotypes are bem2 and sit4,
each of which is involved in cytoskeletal and cell wall organization. (Expression of these genes is not
reduced in the tra1-SRR3413 background.) Other studies connect NuA4 components with membrane
processes; most notably, vid21/eaf1 (Vacuolar Import Degradation) was identified in a screen for
24
defects in sorting of carboxypeptidase Y to the vacuole (Bonangelino et al., 2002), is sensitive to
ethanol (Fugita et al., 2006), and interacts with Vac8, which is required for several aspects of vacuolar
function (Tang et al., 2006).
Possible functions of the PI3K domain. Given the importance of PI3K domains in key signaling
proteins and the conservation of this domain in the Tra1/TRRAP family of molecules, it is reasonable
to consider possible activities of the PI3K domain. In this regard P3408, SRR3413 and S3463 map
proximally to what would be the cleft between N and C-terminal lobes of the PI3K-γ structure. P3408, in
fact, aligns with K833, which in PI3K-γ interacts with the α-phosphate of ATP and is covalently
modified by the kinase inhibitor wortmanin (Wymann et al., 1996). While clearly an approximation of
the Tra1 structure, S3463 may be located within the cleft at a position that putatively could be involved
with substrate interaction. Though speculative, it is attractive to propose that the putative cleft of Tra1
is a substrate-binding pocket, where the domain catalyzes the transfer of a modifying unit other than
phosphate moieties, transfers water as a hydrolase or perhaps has a role in the nuclear signaling by
inositol polyphosphates (for example; Steger et al., 2003).
ACKNOWLEDGEMENTS
We thank Drs.Brian Shilton, Catherine Bateman, David Litchfield and Greg Gloor for their comments
on the manuscript, Drs. Victoria Lundblad, Fred Winston, Kathy Gould, Joan Curcio, V. Zakian and
Raymund Wellinger for reagents, Shayna Oldegard, Dana Abrassart, Monica Piasecki, Courtney
Coschi and Kim Grant for assistance in preparation of DNA constructs and Dr. Joe Martens and
Melissa Bradford for assistance with the chromatin immunoprecipitations. This work was supported by
Canadian Institutes of Health Research grants to CJB. SMTH is a holder of a NSERC studentship. CH
was supported by an OGSST scholarship. AIM is a holder of a Western Graduate Research
Scholarship.
25
REFERENCES
1. Allard, S., R. T. Utley, J. Savard, A. Clarke, P. Grant, C. J. Brandl, L. Pillus, J. L. Workman
and J. Côté, 1999 NuA4, an essential transcription adaptor/histone H4 acetyltransferase complex
containing Esa1p and the ATM-related cofactor Tra1p. EMBO J. 18:5108–5119.
2. Alekseyenko, A.A., E. Larschan, E., W.R. Lai, P.J. Park and M.I. Kuroda, 2006 High-
resolution ChIP-chip analysis reveals that the Drosophila MSL complex selectively identifies active
genes on the male X chromosome. Genes Dev. 20: 848-857.
3. Ard, P. G., C. Chatterjee, S. Kunjibettu, L. R. Adside, L. E. Gralinski and S. B. McMahon,
2002 Transcriptional regulation of the mdm2 oncogene by p53 requires TRRAP acetyltransferase
complexes. Mol. Cell. Biol. 22:5650-5661.
4. Askree, S. H., T. Yehuda, S. Smolikov, R. Gurevich, J. Hawk, C. Coker, A. Krauskopf, M.
Kupiec and M. J. McEachern, 2004 A genome-wide screen for Saccharomyces cerevisiae deletion
mutants that affect telomere length. Proc. Natl. Acad. Sci. U S A. 101:9515-9516.
5. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K.
Struhl, 1998 Protocols in molecular biology. Greene/Wiley-Interscience, New York, N. Y.
6. Balasubramanian, R., Pray-Grant, M.G., Selleck, W., Grant, P.A. and S. Tan, 2001 Role of the
Ada2 and Ada3 transcriptional coactivators in histone acetylation. J. Biol. Chem. 277: 7989-7995.
7. Berger, S. L. 2002 Histone modifications in transcriptional regulation. Curr. Opin.Genet. Dev.
12:142-148.
8. Berger, S. L., B. Pina, N. Silverman, G. A. Marcus, J. Agapite, J. L. Regier, S. J. Triezenberg
and L. Guarente, 1992 Genetic isolation of ADA2: a potential transcriptional adaptor required for
function of certain acidic activation domains. Cell. 70:251-265.
26
9. Bhaumik, S.R., T. Raha, D.P. Aiello and M. R. Green, 2004 In vivo target of a transcriptional
activator revealed by fluorescence resonance energy transfer. Genes Dev. 18:333-343.
10. Bird, A. W., D. Y. Yu, M. G. Pray-Grant, Q. Qiu, K. E. Harmon, P. C. Megee, P. A. Grant, M.
M. Smith and M. F. Christman, 2002 Acetylation of histone H4 by Esa1 is required for DNA double-
strand break repair. Nature 419:411-415.
11. Bittner, C. B., D. T. Zeisig, B. B. Zeisig and R. K. Slany, 2004 Direct physical and functional
interaction of the NuA4 complex components Yaf9p and Swc4p. Eukaryot. Cell. 3:976-983.
12. Bonangelino, C.J., E.M. Chavez and J.S. Bonifacino, 2002 Genomic screen for vacuolar
protein sorting genes in Saccharomyces cerevisiae. Mol. Biol. Cell 13: 2486-2501.
13. Bondeva, T., L. Pirola, G. Bulgarelli-Leva, I. Rubio, R. Wetzker and M. P. Wymann, 1998.
Bifurcation of lipid and protein signals of PI3Kgamma to the protein kinases PKB and MAPK. Science
282:293-296.
14. Bosotti, R., A. Isacchi and E. L. Sonnhammer, 2000 FAT: a novel domain in PIK-related
kinases. Trends Biochem. Sci. 25:225-227.
15. Bouchard, C., O. Dittrich, A. Kiermaier, K. Dohmann, A. Menkel, M. Eilers and B. Lüscher.
2001 Regulation of cyclin D2 gene expression by the Myc/Max/Mad network: Myc-dependent TRRAP
recruitment and histone acetylation at the cyclin D2 promoter. Genes Dev. 15:2042-2047.
16. Brandl, C. J., A. M. Furlanetto, J. A. Martens and K. S. Hamilton, 1993 Characterization of
NGG1, a novel yeast gene required for glucose repression of GAL4p-regulated transcription. EMBO J.
12:5255-5265.
17. Brown, C. E., L. Howe, K. Sousa, S. C. Alley, M. J. Carrozza, S. Tan and J. L. Workman, 2001
Recruitment of HAT complexes by direct activator interactions with the ATM-related Tra1 subunit.
Science 292:2333-2337.
27
18. Brownell, J. E., J. Zhou, T. Ranalli, R. Kobayashi, D. G. Edmondson, S. Y. Roth and C. D.
Allis, 1996 Tetrahymena histone acetyltransferase A: a transcriptional co-activator linking gene
expression to histone acetylation. Cell 84:843-851.
19. Chen, W. and K. Struhl, 1988 Saturation mutagenesis of a yeast his3 "TATA element": genetic
evidence for a specific TATA-binding protein. Proc. Natl. Acad. Sci. 85: 2691-2695.
20. Choy, J. S. and S. J. Kron, 2002 NuA4 subunit Yng2 function in intra-S-phase DNA damage
response. Mol. Cell. Biol. 22:8215-8225.
21. Clarke, A. S., J. E. Lowell, S. J. Jacobson and L. Pillus, 1999 Esa1p is an essential histone
acetyltransferase required for cell cycle progression. Mol. Cell. Biol. 19:2515–2526.
22. Deleu, L., S. Shellard, K. Alevizopoulos, B. Amati and H. Land, 2001 Recruitment of TRRAP
required for oncogenic transformation by E1A.Oncogene 20:8270-8275.
23. Dhand, R., I. Hiles, G. Panayotou, S. Roche, M. J. Fry, I. Gout, N. F. Totty, O. Truong, P.
Vicendo, K. Yonezawa, M. Kasuga, S. A. Courtneidge and M. D. Waterfield, 1994 PI3-kinase is a dual
specificity enzyme: autoregulation by an intrinsic protein-serine kinase activity. EMBO J. 13:522-533.
24. Downs, J. A., S. Allard, O. Jobin-Robitaille, A. Javaheri, A. Auger, N. Bouchard, S. J. Kron, S.
P. Jackson and J. Côté, 2004 Binding of chromatin-modifying activities to phosphorylated histone
H2A at DNA damage sites. Mol. Cell. 16:9979-9990.
25. Eisen, M.B., P.T. Spellman, P.O. Brown and D. Botstein, 1998 Cluster analysis and display of
genome-wide expression patterns. Proc. Natl. Acad. Sci. 95: 14863-14868.
26. Eisenmann, D. M., K. M. Arndt, S. L. Ricupero, J. W. Rodney and F. Winston, 1992 SPT3
interacts with TFIID to allow normal transcription in Saccharomyces cerevisiae. Genes Dev. 6:1319-
1331.
28
27. Eisenmann, D. M., C. Chapon, S. M. Roberts, C. Dollard, and F. Winston, 1994 The
Saccharomyces cerevisiae SPT8 gene encodes a very acidic protein that is functionally related to SPT3
and TATA-binding protein. Genetics 137:647-657.
28. Fishburn J., N. Mohibullah and S. Hahn, 2005 Function of a eukaryotic transcription activator
during the transcription cycle. Mol. Cell. 18:369-378.
29. Fujita, K, A. Matsuyama, Y. Kobayashi and H. Iwahashi, 2006 The genome-wide screening of
yeast deletion mutants to identify the genes required for tolerance to ethanol and other alcohols FEMS
Yeast Res 6: 744-750.
30. Gansheroff, L. J., C. Dollard, P. Tan and F. Winston, 1995 The Saccharomyces cerevisiae
SPT7 gene encodes a very acidic protein important for transcription in vivo. Genetics 139:523-536.
31. Gietz, R.D. and A. Sugino, 1988, New yeast E. coli shuttle vectors constructed with in vitro
mutagenized yeast genes lacking six-base pair restriction sites. Gene 74:255-269.
32. Grant, P. A., L. Duggan, J. Côté, S. M. Roberts, J. E. Brownell, R. Candau, R. Ohba, T. Owen-
Hughes, C. D. Allis, F. Winston, S. L. Berger and J. L. Workman, 1997 Yeast Gcn5 functions in two
multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the
SAGA (Spt/Ada) complex. Genes Dev. 11:1640-1650.
33. Grant, P. A., D. Schieltz, M. G. Pray-Grant, D. J. Steger, J. C. Reese, J. R. Yates, 3rd. and J. L.
Workman. 1998a A subset of TAFIIs are integral components of the SAGA complex required for
nucleosome acetylation and transcriptional stimulation. Cell 94:45-53.
34. Grant, P. A., D. Schieltz, M. G. Pray-Grant, J. R. Yates 3rd. and J. L. Workman, 1998b The
ATM-related cofactor Tra1 is a component of the purified SAGA complex. Mol. Cell 2:863-867.
35. Green, M. R. 2005 Eukaryotic transcription activation: right on target. Mol. Cell. 18:399-402.
29
36. Han, M., U. J. Kim, P. Kayne and M. Grunstein, 1988 Depletion of histone H4 and
nucleosomes activates the PHO5 gene in Saccharomyces cerevisiae. EMBO J. 7:2221-2228.
37. Herceg, Z., W. Hulla, D. Gell, C. Cuenin, M. Lleonart, S. Jackson and Z. Q. Wang. 2001
Disruption of Trrap causes early embryonic lethality and defects in cell cycle progression. Nat. Genet.
29:206-211.
38. Hoffman, C. S. and F. Winston, 1987. A ten-minute DNA preparation from yeast efficiently
releases autonomous plasmids for transformation of Escherichia coli. Gene. 57:267-272.
39. Horiuchi, J., N. Silverman, B. Pina, G. A. Marcus and L. Guarente, 1997 ADA1, a novel
component of the ADA/GCN5 complex has broader effects than GCN5, ADA2, or ADA3. Mol. Cell.
Biol. 17:3220-3228.
40. Hughes, T.R., M.J. Marton, A.R. Jones, C.J. Roberts, R. Stoughton, C.D. Armour, H.A.
Bennett, E. Coffey, H. Dai, Y.D. He, M.J. Kidd, A.M. King, M.R. Meyer, D. Slade, P.Y. Lum, S.B.
Stepaniants, D.D. Shoemaker, D. Gachotte, K. Chakraburtty, J. Simon, M. Bard and S.H. Friend, 2000
Functional discovery via a compendium of expression profiles. Cell 102:109-126.
41. Huisinga, K. L. and B. F. Pugh, 2004 A genome-wide housekeeping role for TFIID and a
highly regulated stress-related role for SAGA in Saccharomyces cerevisiae. Mol Cell 13:573-585.
42. Iizuka, M. and M. M. Smith, 2003 Functional consequences of histone modifications. Curr.
Opin. Gen. Dev. 13:154-160.
43. Ingvarsdottir, K., N. J. Krogan, N. C. Emre, A. Wyce, N. J. Thompson, A. Emili, T. R. Hughes,
J. F. Greenblatt and S. L. Berger, 2005 H2B ubiquitin protease Ubp8 and Sgf11 constitute a discrete
functional module within the Saccharomyces cerevisiae SAGA complex. Mol Cell Biol. 25:1162-
1172.
30
44. Keith, C. T. and S. L. Schreiber, 1999 PIK-related kinases: DNA repair, recombination, and
cell cycle checkpoints. Science. 270:50–51.
45. Keogh, M. C., T. A. Mennella, C. Sawa, S. Berthelet, N. J. Krogan, A. Wolek, V. Podolny, L.
R. Carpenter, J. F. Greenblatt, K. Baetz and S. Buratowski, 2006 The Saccharomyces cerevisiae
histone H2A variant Htz1 is acetylated by NuA4. Genes Dev. 20:660-665.
46. Krogan, N. J., K. Baetz, M. C., Keogh, N. Datta, C. Sawa, T. C. Kwok, N. J. Thompson, M. G.
Davey, J. Pootoolal, T. R. Hughes, A. Emili, S. Buratowski, P. Hieter and J. F. Greenblatt. 2004
Regulation of chromosome stability by the histone H2A variant Htz1, the Swr1 chromatin remodeling
complex, and the histone acetyltransferase NuA4. Proc. Natl. Acad. Sci. USA. 101:13513-13518.
47. Kulesza, C. A., H. A. van Buskirk, M. D. Cole, J. C. Reese, M. M. Smith and D. A. Engel,
2002 Adenovirus E1A requires the yeast SAGA histone acetyltransferase complex and associates with
SAGA components Gcn5 and Tra1. Oncogene. 21:1411-1422.
48. Kuo, M. H., J. Zhou, P. Jambeck, M. E. Churchill and C. D. Allis, 1998 Histone
acetyltransferase activity of yeast Gcn5p is required for the activation of target genes in vivo. Genes
Dev. 12:627-639.
49. Kuo, M. H. and C. D. Allis, 1999 In vivo cross-linking and immunoprecipitation for studying
dynamic Protein:DNA associations in a chromatin environment. Methods 19:425-433.
50. Le Masson, I., D.Y. Yu, K. Jensen, A. Chevalier, R. Courbeyrette, Y. Boulard, M. M. Smith
and C. Mann, 2003 Yaf9, a novel NuA4 histone acetyltransferase subunit, is required for the cellular
response to spindle stress in yeast. Mol. Cell. Biol. 23: 6086–6102.
51. Lundblad, V. and J. W. Szostak, 1989 A mutant with a defect in telomere elongation leads to
senescence in yeast. Cell 57:633-643.
31
52. McMahon, SB, H. A. Van Buskirk, K. A. Dugan, T. D. Copeland and M. D. Cole, 1998 The
novel ATM-related protein TRRAP is an essential cofactor for the c-Myc and E2F oncoproteins. Cell.
94:363-374.
53. Mutiu, A. I. and C. J. Brandl, 2005. RNA isolation from yeast silica matrices. J Biomol Tech.
16:316-317.
54. Mutiu, A.I., S.M.T. Hoke, J. Genereaux, G. Liang and C.J. Brandl, 2007 The role of histone
ubiquitylation and deubiquitylation in gene expression as determined by the analysis of an HTB1K123R
Saccharomyces cerevisiae strain. Mol. Genet. Gen. (in press).
55. Nourani, A., W. Doyon, R. T. Utley, S. Allard, W.S. Lane and J. Côté, 2001 Role of an ING1
growth regulator in transcriptional activation and targeted histone acetylation by the NuA4 complex.
Mol. Cell. Biol. 21:7629-7640.
56. Nourani, A., R. T. Utley, S. Allard and J. Côté, 2004 Recruitment of the NuA4 complex poises
the PHO5 promoter for chromatin remodeling and activation. EMBO J. 23:2597-2607.
57. Pan, X., D.S. Yuan, X. Wang, J.S. Bader and J.D. Boeke, 2006 A DNA integrity network in the
yeast Saccharomyces cerevisiae. Cell 10: 1069-1081.
58. Park, J., S. Kunjibettu, S. B. McMahon and M. D. Cole, 2001 The ATM-related domain of
TRRAP is required for histone acetyltransferase recruitment and Myc-dependent oncogenesis. Genes
Dev. 15:1619-1624.
59. Reeves, W. M. and S. Hahn, 2005 Targets of the Gal4 transcription activator in functional
transcription complexes. Mol. Cell. Biol. 25:9092-9102.
60. Ricci, A.R., J. Genereaux and C.J. Brandl, 2002 Components of the SAGA histone
acetyltransferase complex are required for repressed transcription of ARG1 in rich medium. Mol. Cell.
Biol. 22: 4033-4042.
32
61. Rigaut, G., A. Shevchenko, B. Rutz, M. Wilm, M. Mann and B. Séraphin, 1999 A generic
protein purification method for protein complex characterization and proteome exploration. Nature
Biotech. 17:1030-1032.
62. Roberts, S. M. and F. Winston, 1997 Essential functional interactions of SAGA, a
Saccharomyces cerevisiae complex of Spt, Ada, and Gcn5 proteins, with the Snf/Swi and Srb/mediator
complexes. Genetics 147:451-465.
63. Robinson, M.D., J. Grigull, J., N. Mohammad and T.R. Hughes, 2002 FunSpec: a web-based
cluster interpreter for yeast. BMC Bioinformatics 3: 35.
64. Saleh, A., V. Lang, R. Cook and C. J. Brandl, 1997 Identification of native complexes
containing the yeast coactivator/repressor proteins NGG1/ADA3 and ADA2. J. Biol. Chem. 272:5571-
5578.
65. Saleh, A., D. Schieltz, N. Ting, S. B. McMahon, D. W. Litchfield, J. R. Yates 3rd., S. P. Lees-
Miller, M. D. Cole and C. J. Brandl, 1998 Tra1 is a component of the yeast Ada-Spt transcriptional
regulatory complexes. J. Biol. Chem. 273:26559-26565.
66. Sikorski, R.S. and J.D. Boeke, 1991 In vitro mutagenesis and plasmid shuffling: from cloned
gene to mutant yeast. Methods Enzymol. 194:302-329.
67. Smith, E. R., A. Eisen, W. Gu, M. Sattah, A. Pannuti, J. Zhou, R. G. Cook, J. C. Lucchesi and
C. D. Allis, 1998. ESA1 is a histone acetyltransferase that is essential for growth in yeast. Proc. Natl.
Acad. Sci. U S A. 95:3561-3565.
68. Stack, J. H. and S. D. Emr, 1994 Vps34p required for yeast vacuolar protein sorting is a
multiple specificity kinase that exhibits both protein kinase and phosphatidylinositol-specific PI-3-
kinase activities. J. Biol. Chem. 269:31552-31562.
33
69. Steger, D.J., E.S. Haswell, A.L. Miller, S.R. Wente and E.K. O’Shea, 2003 Regulation of
chromatin remodelling by inositol polyphosphates. Science 299: 114-116.
70. Sterner D. E., P. A. Grant, S. M. Roberts, L. J. Duggan, R. Belotesrkovskaya, L. A. Pacella, F.
Winston, J. L. Workman and S. L. Berger, 1999 Functional organization of the yeast SAGA complex:
distinct components involved in structural integrity, nucleosome acetylation, and TATA-Binding
protein interaction. Mol. Cell. Biol. 19:86-98.
71. Strahl, B. D. and C. D. Allis, 2000 The language of covalent histone modifications. Nature
403:41-45.
72. Takahashi, T., H. Shimoi and K. Ito, 2001 Identification of genes required for growth under
ethanol stress using transposon mutagenesis in Saccharomyces cerevisiae. Mol. Genet. Genomics
2656:1112-1119.
73. Tang, F., Y. Peng, J.J. Nau, E.J. Kauffman and L.S. Weisman, 2006 Vac8p, an armadillo repeat
protein, coordinates vacuole inheritance with multiple vacuolar processes. Traffic 7: 1368-1377.
74. Teng, S.C. and V. A. Zakian, 1999 Telomere-telomere recombination is an efficient bypass
pathway for telomere maintenance in Saccharomyces cerevisiae. Mol. Cell. Biol. 19:8083-8093.
75. Tong, A. H. and C. Boone, 2006 Synthetic genetic array analysis in Saccharomyces cerevisiae.
Methods Mol Biol. 313:171-192.
76. Tong, A.H. G. Lesage, G.D. Bader, H. Ding, H. Xu, X. Xin, J. Young, G.F. Berriz, R.L. Brost
M. Chang, Y. Chen, X. Cheng, G. Chua, H. Friesen, D.S. Goldberg, J. Haynes, C. Humphries, G. He,
N. Krogan, Z. Li, J.N. Levinson, H. Lu, P. Menard, C. Munyana, A.B. parsons, O. Ryan, R. Tonikian,
T. Roberts, A.M. Sdicu, J. Shapiro, B. Sheikh, B. Suter, S.L. Wong, L.V. Zhang, H. Zhu, C.G. Burg, S.
Munro, C. Sander, J. Rine, J. Greenblatt, M. Peter, A. Bretscher, G. Bell, F.P. Roth, G.W. Brown, B.
34
Andrews, H. Bussey and C. Boone, 2004 Global mapping of the yeast genetic interaction network.
Science 303: 808–813.
77. Vignali, M., D. J. Steger, K. E. Neely and J. L. Workman, 2000 Distribution of acetylated
histones resulting from Gal4-VP16 recruitment of SAGA and NuA4 complexes. EMBO J. 19:2629–
2640.
78. Walker, E. H., O. Perisic, C. Ried, L. Stephens and R. L. Williams, 1999 Structural insights
into phosphoinositide 3-kinase catalysis and signaling. Nature 402:313-320.
79. Wang, L., L. Liu and S. L. Berger, 1998 Critical residues for histone acetylation by Gcn5,
functioning in Ada and SAGA complexes, are also required for transcriptional function in vivo. Genes
Dev. 12:640-653.
80. Welihinda, A. A., W. Tirasophon, S. R Green and R. J. Kaufman, 1997 Gene induction in
response to unfolded protein in the endoplasmic reticulum is mediated through Ire1p kinase interaction
with a transcriptional coactivator complex containing Ada5p. Proc. Natl. Acad. Sci. USA. 94: 4289-
4294.
81. Welihinda, A. A., W. Tirasophon and R. J. Kaufman, 2000 The transcriptional co-activator
ADA5 is required for HAC1 mRNA processing in vivo. J. Biol. Chem. 275:3377-3381.
82. Winzeler, E. A. and R. W. Davis, 1997 Functional analysis of the yeast genome. Curr. Opin.
Genet. Dev. 7:771-776.
83. Wu, P. Y., C. Ruhlmann, F. Winston and P. Schultz, 2004 Molecular architecture of the S.
cerevisiae SAGA complex. Mol. Cell. 15:199-208.
84. Wymann, M. P., G. Bulgarelli-Leva, M. J. Zvelebil, L. Pirola, B. Vanhaesebroeck, M. D.
Waterfield and G. Panayotou, 1996 Wortmannin inactivates phosphoinositide 3-kinase by covalent
35
modification of Lys-802, a residue involved in the phosphate transfer reaction. Mol. Cell. Biol.
16:1722-1733.
36
FIGURE LEGENDS
Figure 1. Phenotypes of Tra1-SRR3413, P3408A and S3463A. A. Yeast strains containing the tra1 mutant
alleles and Tra1SB (parent molecule) expressed from plasmids or endogenous wild-type Tra1 (KY320)
were grown to saturation and ten-fold serial dilutions plated onto YP media containing 2% glucose and
grown at 30o and 37o, or at 30o in YPD containing 3% ethanol or 5 μg/ml Calcofluor white (lower
panels). Note that expression of Tra1SB on a plasmid results in slightly reduced growth as compared to
endogenous wild-type Tra1. B. Growth curves of Tra1 mutant strains after temperature shift to 37o.
The indicated tra1 mutant strains or the background wild-type strain (TRA1SB) were grown to
saturation and diluted into YPD media at 34o (time 0). Cells were grown for 4 hrs with the absorbance
at 600 nm determined on hourly intervals. One half of the culture volume was removed and shifted to
37o and absorbance determined for cultures at both temperatures. Note the scale differences for the
graphs. The arrow indicates 30 minutes after the temperature shift, the first time-point a reading at both
temperatures was made. C. Analyses of a strain containing an integrated version of the tra1-SRR3413
that lacks the BamHI site found in the plasmid copy. Yeast strains CY2437 (integrated wild-type
TRA1) and CY2438 (integrated tra1-SRR3413) were grown to saturation in YPD. Serial dilutions were
spotted onto YPD plates and grown at 30o and 37o, or at 30o in YPD containing 5 μg/ml Calcofluor
white.
Figure 2. Expression and interactions of the ethanol sensitive Tra1 mutants. A. Crude extracts were
prepared from yeast strain KY320 (- ve) or strains expressing TAP-Flag3x-tagged versions of Tra1SB,
Tra1-S3463A, Tra1-P4308A and Tra1-SRR3413. 25 μg of extract was separated by SDS PAGE (5%) and
Western blotted with anti-Flag antibody. The upper panel is the Western blot of the ~450 kDa band
corresponding to Tra1; the lower panel is a portion of an equivalent gel stained with Coomassie
Brilliant Blue to verify protein concentrations. B. tra1-SRR3413 associates with Spt7 and Yng2. Top
panel: Crude extracts were prepared from strains KY320 expressing wild-type Tra1 (-ve), TAP-Flag-
Tra1SB or TAP-Flag-Tra1-SRR3413 and with Flag-Yng2. 150 μg of crude extract was separated by
37
electrophoresis on 5% SDS PAGE and visualized by Western blotting using anti-Flag antibody. Co-
purification: Extracts were subjected to tandem affinity purification. Equal volumes were separated by
electrophoresis on 5% and 8% SDS PAGE and Western blotted using anti-Flag antibody to detect Tra1
(upper panel) or Yng2 (middle panel). The presence of Spt7 (lower panel) was detected using anti-Spt7
antibody (kindly provided by Dr. Fred Winston). The SAGA-form and SLIK-form of Spt7 are marked
as SA and SL, respectively. C. Tra1-SRR3413 forms Ada2-containing complexes that elute from a
HiTrap Q Sepharose Fast Flow column with a similar profile to Tra1SB. Whole cell extract from strains
containing Tra1-SRR3413 or Tra1SB, each with HA-Ada2, were fractionated on a HiTrap Q Sepharose
Fast Flow column. Protein was eluted with a gradient of 0-1.0 M NaCl. Equal volumes of successive
fractions were separated on 8% SDS gels, Western blotted for HA-Ada2 and relative amounts
determined by densitometry. D. Tra1-SRR3413 forms Yng2-containing complexes that elute from a
HiTrap Q Sepharose Fast Flow column with a similar profile to Tra1SB. 25 mg of whole cell extract
from strains containing Tra1-SRR3413 or Tra1SB, each with Flag-Yng2, were fractionated on a HiTrap
Q Sepharose Fast Flow column as above. Equal volumes of successive fractions were separated on 8%
SDS gels and Western blotted with anti-Flag antibody.
Figure 3. Expression of β-galactosidase reporter constructs in tra1 mutant strains. A. Promoter
fragments from PHO5, INO1, HIS4 and ADH2 were cloned as his3-LACZ reporter fusions into the
LEU2 centromeric plasmid YCp87 and transformed into FY1093 (spt7Δ0), FY1370 (gcn5Δ0),
CY1514 (tra1-S3463A), CY1507 (tra1-P3408A) and CY1531 (tra1-SRR3413). β-galactosidase activity was
determined after growth in low phosphate media (PHO5), inositol depleted media (INO1), minimal
media (HIS4) or YP containing 3% ethanol plus 0.01% glucose (ADH2) at 30o. Activity was compared
to the appropriate wild-type strains, FY630 for spt7Δ, FY86 for gcn5Δ and CY1524 for the tra1 mutant
strains and are shown as percentages. Measurements were made in triplicate in two independent
experiments with the standard error indicated. B. Expression of INO1-lacZ was analyzed at 30 o and
35o for strains containing TRA1SB and tra1-SRR3413.
38
Figure 4. Hierarchical cluster analysis of tra1-SRR3413 microarray expression data. Microarrary
analyses were performed for yeast strain CY1531 containing tra1-SRR3413 as the sole copy of Tra1
grown in YPD at 30oC using the Agilent Yeast Oligo Array Kit. The experiment was performed in
duplicate with dye-reversal using TRA1SB (yeast strain CY1524) as the control. Comparisons were
made to the expression profiles of strains with deletions of NuA4 (Krogan et al., 2004) and SAGA
(Ingvarsdottir et al., 2005) components. Also included were profiles of strains with deletions of cka2,
ckb2, gcn4, hat2, isw1, mbp1, rpd3, rtg1, sap30, sin3, ste12 and yap3 which were identified as being
most similar to tra1-SRR3413 in an initial clustering analysis using a subset of the compendium data
(Hughes et al., 2000). Gene families are indicated to the right.
Figure 5. Effects of Tra1-SRR3413 on interaction with the Gal4 activation domain and recruitment to
the PHO5 promoter. A. Interaction of Tra1-SRR3413 with the Gal4 activation domain. Yeast extract
was prepared from CY1021 (lane 1, negative control), CY1962 containing HA-tagged Ada2 (Tra1SB;
lane 2) and CY1963 containing HA-tagged Ada2 (Tra1-SRR3413; lane 3). Purified TAP-Tra1SB was
applied to the Glutathione Sepharose bound with GST-Gal4AD (lane 4) or control Glutathione
Sepharose bound with GST (lane 5). Similarly TAP-Tra1-SRR3413 was applied to GST-Gal4 AD beads
(lane 6) or GST beads (lane 7). Bound protein was eluted with reduced glutathione and the eluant
separated on a 10% SDS-PAGE gel. Interaction with Gal4 AD was monitored by Western blotting for
HA-Ada2. B . Recruitment of TAP-Tra1 to the PHO5 promoter was determined by chromatin
immunoprecipitation using strains BY4741 (lane 1) and BY4741 containing TAP-Tra13413 (lane 2) or
TAP-Tra1-SRR3413 (lane 3). The presence of PHO5 promoter DNA was determined by PCR and
separation on 5% native polyacrylamide gels stained with ethidium bromide. The lower panel shows
PCR products from the input DNA used for analysis. A serial dilution of the sample for Tra1-SRR3413
is shown in lanes 3 through 5. Lane 6 is a no template control.
39
Figure 6. Histone acetylation in Tra1-SRR3413 containing strains. A. Acetylation of lysine 8 of histone
H4. Yeast strains CY1531 (tra1-SRR3413), CY1524 (TRA1SB), QY202 (yng2Δ0), QY204 (YNG2),
BY4916 (vid21Δ0) and BY4741 (VID21) were grown to A600=1.5 in YPD and ChIP analysis
performed using antibody directed against acetylated K8 of histone H4 and against histone H3. Levels
of immunoprecipitated ADE1 promoter, PHO5 promoter and the 3’ coding region of PHO5, were
determined after PCR and separation by electrophoresis and staining with ethidium bromide. Serial
dilutions were examined to ensure detection in a linear range. The histogram shows the ratio of
acetylated histone H4 to total histone H3 for each of the mutant strains as a percentage of that found in
the relevant wild-type strain for experiments performed in duplicate. B. Histone H3 acetylation. Yeast
strains CY1531 (tra1-SRR3413), CY1524 (TRA1SB), BY3281 (spt7Δ0), BY4282 (ada2Δ0) and BY4741
(wild-type) were grown in phosphate depleted media (PHO5) or YPD (PHO84 and PGK1) to an
A600=1.5. Equal amounts of chromatin were immunoprecipitated with anti acetylated (K9) histone H3
antibody or anti histone H3 antibody and levels of promoter determined after PCR. The ratio of
acetylated histone H3 to total histone H3 for each mutant is shown as a percentage of that found in the
relevant wild-type strain.
Figure 7. TRA1-SRR3413 results in a generation dependent reduction in telomere length. A. Plasmid
linearization assay. Yeast strains CY1524 (TRA1SB), CY1531 (tra1-SRR3413), and CY1507 (tra1-
P3408A) and CY1514 (tra1-S3463A) were transformed with pVL106 (kindly supplied by Dr. V.
Lundblad). Leu+ Ura+ transformants were grown sequentially in media lacking uracil then YPD,
washed, and spotted onto plates containing 5-FOA and lacking leucine. The number of 5-FOA resistant
Leu+ colonies were counted after 5 days of growth at 30o and normalized to the total number of cells
plated. The graph indicates the percentage of 5-FOA resistant Leu+ colonies in each of the tra1 mutant
strains as compared to that observed for TRA1SB for 5 experiments each performed in duplicate. B.
Southern blot of genomic DNA from TRA1SB (wt) and tra1-SRR3413 (SRR) containing strains cut with
XhoI and hybridized to a 32P-labeled TG1-3/C1-3A DNA probe. The terminal restriction fragment (TRF)
40
is derived from a subset of yeast telomeres that have a Y’ telomere element. The Y’ element has a
conserved XhoI site that is located proximal to the terminal telomeric repeat tract. The larger DNA
fragments are derived from either telomeres lacking the Y’ element or subtelomeric DNA sequences
containing TG1-3 tracks. Cells were serially passaged for the indicated number of generations. After 10
generations the average TRF length for the two strains (~1110 basepairs) did not differ significantly
(left-hand panel) at either growth temperature. After 50 and 100 generations at 30˚ (right-hand panel)
the average TRF length for the tra1-SRR3413 strain was reduced by ~125 and 140 basepairs,
respectively.
Figure 8. H3530Y suppresses the ethanol sensitivity and transcription defects due to tra1-SRR3413. A.
Yeast strains containing the TRA1 alleles indicated in the legend to the right were plated onto YPD or
YPD containing 6% ethanol and grown at 30o. B. Delay in the induction of PHO5 resulting from
SRR3413 is suppressed by alteration of H3530Y. Yeast strains containing Tra1SB, Tra1-SRR3413, and Tra1-
SRR3413-H3530Y were transformed with PHO5-lacZ, grown to saturation in YPD, washed 4-times in
water, and diluted 1:12 into low phosphate media. Samples were taken at the indicated time points and
β-galactosidase activity determined.
Tra1SB
Tra1WT
Tra1WT
S3463A
Tra1SB
P3408A
P3408A
S3463A
SRR3413
SRR3413
YPD 30o YPD 37o
YPD EtOH YPD CW
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 2 4 6 8 10 12
Ab
sorb
ance
(60
0 n
M)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 2 4 6 8 10 12
Ab
sorb
ance
(60
0 n
M)
0
0.5
1
1.5
2
2.5
0 2 4 6 8 10 12
Ab
sorb
ance
(60
0 n
M)
34C37C
BTra1SB
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 2 4 6 8 10 12
Ab
sorb
ance
(60
0 n
M)
P3408A
S3463A
SRR3413
Time (hr)
A
Figure 1
YPD 30o
YPD 37o
YPD CW
CSRR3413
SRR3413
SRR3413
Tra1WT
Tra1WT
Tra1WT
0
50
100
150
200
250
20 30 40 50 60 70
P 3408
A
SRR 34
13
S 3463
A
TRA
1 SB
-ve
A
Figure 2
TAP
CrudeextractsTra1
Tra1
SBSR
R 3413
-ve
SASL
Tra1
Spt7
Yng2
B0
50
100
150
200
250
300
350
10 20 30 40 50
FractionD
ensi
tom
etry
units
Ada2 elution profile
Yng2 elution profile
C
Fraction
FractionD
ensi
tom
etry
Uni
tsD
ensi
tom
etry
Uni
tsD
Tra1SB
SRR3413
Tra1SB
SRR3413Tra1
Cont
0
100
200
300
400
500
600
700
Rel
ativ
e IN
O1
exp
ress
ion 30
35
0
20
40
60
80
100
120
140
PHO5 INO1 HIS4 ADH2
Per
cen
t W
T e
xpre
ssio
nSRR3413P3408S3463yng2gcn5spt7
Tra1-SB Tra1-SRR
A B
o
o
Figure 3
gcn4
∆
cka2
∆
ckb2
∆
rtg1
∆
isw
1∆
hat2
∆
eaf5
∆
yaf9
∆
vid2
1∆yn
g2∆
ynl1
36w
∆
sgf1
1∆
ubp8
∆
yap3
∆
ste1
2∆
mbp
1∆
rpd3
∆
sap3
0∆
sin3
∆
ada2
∆
tra1
-SR
Rsp
t3
∆
spt8
∆
purine base metabolism (GO:0006144)
cell wall (GO:0005618)
carbohydrate catabolism (GO:0016052)
amino acid metabolism (GO:0006520)
phosphate metabolism (GO:0006796)
cell cycle (GO:0007049)
ion transporter (GO:0015075)
protein metabolism (GO:0019538)
fold repression fold induction1 ≥42≥4 323
Figure 4
B
Figure 5
Tra1
SBSR
R
cont
rol
HA-Ada2
Tra1SB SRR3413
1 2 3 4 5 6 7inputs bound
A
Tra1 wt SB SRR3413
TAP - + +
1 2 3 4 5 6
IP
Input
Column Matrix
Gal4AD Gal4AD
-GST -GSTGST GST
0
10
20
30
40
50
60
70
80
90
100
PHO84 PHO5 PGK1
Ac-
H3/
H3
(% m
uta
nt/
wt) tra1-SRR
spt7ada2
Figure 6
0
10
20
30
40
50
60
70
80
90
100
ADE1 PHO5
Ac-
H4/
H3
(% m
uta
nt/
wt) tra1-SRR
yng2vid21/eaf1
PHO5 (3’-coding)
A B
0
20
40
60
80
100
120
140
Tra1SB SRR3413 P3408A S3463A
Per
cent
5-F
OA
res
ista
nt L
eu+
A
Tra1SB SRR3413 P3408 S3463
Figure 7
StrainGenerations
Temp ˚C 30 34 30 34
wt SRR
10 10
TRF
1 2 3 4
1.2
1.9
4.3
Size (kb)
M
wt SRR wt SRR50 50 100 100
30 34 30 34 30 34 30 34
1 2 3 4 5 6 7 8
TRF1.2
1.9
0.7
4.3
B
H3530Y
P3408
SRRSRRH3530Y
P3408H3530YTra1SB
YPD
6%EtOH
A B
Figure 8
SRR3413
SRR3413H3530Y
Tra1SB
Time (hr)P
HO
5-Lac
ZE
xpre
ssio
n
Table 1. Strains used in this study Strain Genotype TRA1 plasmid Reference KY320 MATa ura3-52 ade2-101 trp1-∆1 lys2-
801 his3-∆200 leu2::PET56 Chen and Struhl
(1988) CY1021 Isogenic to KY320 but tra1::Tn10LUK myc-TRA1 Saleh et al., 1998 CY1962 Isogenic to CY1021 TAP- TRA1SB This work CY1963 Isogenic to CY1021 TAP-tra1-
SRR3413 This work
CY1524 Isogenic to CY1021 Flag3- TRA1SB This work CY1507 Isogenic to CY1021 Flag3-tra1-
P3408A This work
CY1531 Isogenic to CY1021 Flag3-tra1-SRR3413
This work
CY1514 Isogenic to CY1021 Flag3-tra1-S3463A
This work
FY630 MATα his4-917 lys2-173R2 leu2∆1 ura3-52 trp1∆63
Gansheroff et al. (1995)
FY1093 Isogenic to FY630 but spt7::LEU2 CY1381 Isogenic to FY1093 but
leu2::HIS3 This work
FY86 MATα his3∆200 leu2∆1 ura3-52 Roberts and Winston (1997)
FY1370 isogenic to FY86 but gcn5::HIS3 Y5565 MATα can1∆::MFA1pr-HIS3
mfα1∆::MFα1pr-LEU2 lyp1∆ ura3∆ his3∆ met15∆
Tong and Boone (2006)
BY4741 MATa his∆0 leu2∆0 met1∆0 ura3∆0 Winzeler and Davis (1997)
BY3281 Isogenic to BY4741 except spt7::Kanr BY4196 Isogenic to BY4741 except vid21::Kanr BY4282 Isogenic to BY4741 except ada2::Kanr QY204 MAT his3∆200 trp1∆ 63 ura3-52
leu2∆1 lys2-128δ Nourani et al.
(2001) QY202 Isogenic to QY204 except yng2::Kanr
Table 2. Growth of strains containing mutations throughout the length of Tra1. Mutation1 Reason for analysis Viability2
30o 37o DSP171 Potential phosphorylation Yes Yes EFS184 Potential phosphorylation Yes Yes KEL345 Conserved3 Yes Yes VLL837 LXXLL sequence Yes Yes VRL867 Conserved Yes Yes KKK1686 Found in chromatin associated Sth1 Yes Yes GLW2677 Conserved in yeast4 Yes Yes GYH2939 Conserved in yeast Yes Yes TLP2972 Conserved in yeast Yes Yes HFQ3168 Conserved in FAT domain5 Yes Yes EER3416 Conserved Yes Reduced PFR3621 Conserved in PI3K domain No No 1 Alleles were constructed by placing a NotI linker encoding a triple alanine tag in place of the indicated three amino acids. The number refers to the amino acid position of the first of the three amino acids. 2 Growth on YPD media at 30o or 37o 3 Conserved indicates identity in S. pombe and human Tra1 molecules. 4 Sequence similarity or identity in yeast homologs 5 See Bosotti et al., 2000
Table 3. Phenotypes of tra1 mutations. Mutation1 30o 2 37 o 3 Phos4 Ino5 EtOH6 VP167 Ben8 K3571 ++++ ++++ ++++ ++++ ++++ - ++++ P3655 ++++ ++++ ++++ ++++ +++ - ++++ P3408 +++ + + +++ + - ++ SRR3413 ++ - + ++ - - + EER3416 ++++ ++ ++++ ++++ ++++ - ++++ S3463 ++++ + ++ +++ + - +++ F3443 + - - - - - - P3446 ++++ ++++ ++++ ++++ +++ +/- ++++ DKL3491 ++++ ++++ ++++ ++++ ++++ +/- ++++ P3612 ++++ ++++ ++++ ++++ +++ +/- ++++ N3617 ++++ ++++ ++++ ++++ +++ +/- ++++ 1 The indicated mutations were engineered into TRA1SB which contains a BamHI restriction site that results in N3580A and a silent SalI restrictions site that converts nucleotides A9714G and T9717G. All amino acid changes were to alanine. 2 Alleles were transformed into CY1021 and tested for viability by growth on mimimal plates containing 5-FOA. Growth at 30o was determined on YPD. Comparisons were made to a strain containing TRA1SB 3 Growth at 37 o on YPD plates 4 Growth on YPD plates deficient for phosphate 5 Growth on complete minimal media lacking inositol 6 Growth on YPD plates containing 6% ethanol 7 Strains were transformed with padhGV16-gal4-VP16 (Berger et al., 1992) and growth examined on minimal media lacking leucine. 8 Growth on synthetic complete media containing 20 µg/ml benomyl
Table 4: Gene expression profile in tra1-P3408A, -SRR3413, and -S3463A tra1-P3408A
GENE1 Fold2 tra1-SRR3413
GENE Fold tra1-S3463A
GENE Fold 1 FET3 3.2 TKL2 4.5 YGL262W 6.0 2 PCL1 3.2 YHR033W 3.9 AFT2 3.9 3 ATF2 3.2 SCW10 3.5 COS12 3.5 4 RNR1 3.1 YLR327C 3.4 SCW10 3.0 5 SCW10 3.1 YER053C 3.4 FET3 3.0 6 HXT4 2.9 PDR12 3.3 RNR1 3.0 7 SVS1 2.8 YLR312C 3.1 PDR12 2.9 8 YKL097W-A 2.7 SVS1 3.1 PCL1 2.8 9 PDR12 2.7 MNN1 3.1 PUS2 2.7 10 DBP2 2.6 RNR1 3.0 KAP122 2.7 11 YHR033W 2.6 ATF2 3.0 SVS1 2.7 12 RMD6 2.5 PCL1 3.0 RRN3 2.7 13 MNN1 2.5 RRN3 2.9 MNN1 2.7 14 NCE4 2.5 MND1 2.8 HXT4 2.6 15 RRN3 2.4 ECM11 2.8 DBP2 2.6 16 APG10 2.4 KCC4 2.7 AGP10 2.5 17 YER053C 2.4 TRA1 2.7 YHR033W 2.5 18 HO 2.4 APG14 2.7 HO 2.5 19 RPL7B 2.3 PUS2 2.7 TRA1 2.4 20 PLB3 2.3 INO1 2.6 YPL207W 2.3 21 RNA14 2.3 FET3 2.6 FDH2 2.3 22 MIR1 2.3 BDF2 2.6 DHR2 2.3 23 YLR312C 2.3 HRP1 2.6 YER053C 2.3 24 MEP3 2.3 SIT1 2.6 MDV1 2.3 25 PUS2 2.3 ATP7 2.5 TOP3 2.3 TOTAL#3 85 152 97 1 IMD2 -14.7 IMD2 -19.3 IMD2 -18.3 2 ADE17 -14.2 ADE17 -13.5 SPL2 -10.1 3 SPL2 -10.9 SPL2 -11.4 YBR285W -7.9 4 HIS4 -6.9 AAD4 -11.3 ADE17 -7.2 5 YBR285W -6.9 YLR053C -8.7 AAD4 -6.3 6 AAD4 -6.8 YBR285W -8.5 HIS4 -5.5 7 ADE1 -6.5 ADE1 -8.5 YAL061W -5.2 8 LAP4 -6.3 PHO84 -7.9 TSA2 -5.2 9 VPS73 -6.1 HIS4 -7.4 YMR090W -5.1 10 KHS1 -5.8 VPS73 -7.3 ZTA1 -5.1 11 YAL061W -5.8 GTT2 -7.2 EMI2 -5.0 12 SNZ1 -5.8 YMR090W -7.2 SNZ1 -4.9 13 YLR053C -5.5 YAL061W -6.5 YBR047W -4.9 14 SHM2 -5.2 TSA2 -6.5 YLR460C -4.8 15 PH05 -5.1 BNA1 -6.1 YLR053C -4.7 16 ZTA1 -5.1 LYS20 -6.1 YBR147W -4.6 17 PHO84 -5.0 ADE5,7 -6.1 YPS73 -4.5 18 TSA2 -5.0 LAP4 -5.8 YJL038C -4.3 19 EMI2 -4.9 IMD1 -5.8 SNO1 -4.3 20 IMD1 -4.8 ZTA1 -5.8 YMR118C -4.2 21 ADE5,7 -4.7 LYS9 -5.7 YKL107W -4.2 22 YJL038C -4.7 LYS7 -5.5 LAP4 -4.2 23 YBR147W -4.7 GCV2 -5.4 ECM12 -4.2 24 YNL194C -4.6 YPS6 -5.3 PHO84 -4.1 25 GCV2 -4.6 YKL107W -5.3 IMD1 -4.1 TOTAL#3 258 294 261
1 Genes in bold are found either a minimum of 2-fold elevated or 4-fold decreased in one of the other strains. Those italicized are 3-fold decreased in one of the other strains. Dubious ORFs are not listed. HIS4 was down regulated in the gene array data more so than in the β-galactosidase assays, likely reflecting different levels of Tra1 contribution in different media. 2 Values are the fold-change in expression relative to that in CY1524. Cells were grown in YPD media to an A600 nm ~1.5. Values are the average of two experiments with dye reversal. 3 Total number of genes affected 2-fold or greater.