Tra1p Is a Component of the Yeast Ada·Spt Transcriptional Regulatory Complexes*

The yeast Ada and TBP class of Spt proteins interact in multiple complexes that are required for transcriptional regulation. We have identified Tra1p as a component of these complexes through tandem mass spectrometry analysis of proteins that associate with Ngg1p/Ada3p. TRA1 is an essential gene and encodes a 3744-amino acid protein that is a member of a group of proteins including the catalytic subunit of DNA-dependent protein kinase, ATM and TRRAP, with carboxyl-terminal regions related to phosphatidylinositol 3-kinases. The interaction between Tra1p and Ada/Spt components was verified by the reciprocal coimmunoprecipitation of Ada2p and Tra1p from whole cell extracts in one or more complexes containing Spt7p. Tra1p cofractionated with Ngg1p and Spt7p through consecutive chromatography on Mono Q, DNA-cellulose, and Superose 6 columns. Binding of Tra1p to DNA-cellulose required Ada components. The association of Tra1p with two Ada·Spt complexes was suggested by its cofractionation with Ngg1p and Spt7p in two peaks on the Mono Q column. In the absence of Ada2p, the elution profile of Tra1p shifted to a distinct peak. Despite the similarity of Tra1p to a group of putative protein kinases, we have not detected protein kinase activity within immunoprecipitates of Tra1p or the Ada·Spt complexes.

The ADA genes were identified in Saccharomyces cerevisiae based on their requirement for the regulated activation and repression of transcription (1)(2)(3). In initial studies, Ada2p, Ngg1p/Ada3p, and Gcn5p/Ada4p were shown to function in a complex (4 -6). The identity of Gcn5p and its human homolog hGCN5 as histone acetyltransferases suggested that one role of the complex was to modulate nucleosome structure (reviewed in Ref. 7). Biochemical analyses of the Ada proteins demonstrated that they are found in at least four high molecular weight complexes, two with sizes of more than 2 MDa and others of 900 and 200 kDa (8 -10). Other proteins identified within these complexes include the product of ADA1 (1,11) and the products of the TBP class of SPT genes, SPT3, SPT7, SPT8, and SPT20/ADA5 (9,12,13). The latter group of proteins are found within at least one of the ADA-containing complexes, the SAGA complex (9). The association between the Spt and Ada proteins agrees with the functional link between the Ada proteins and TBP which was suggested by immunoprecipitation and affinity chromatography (8, 14 -16).
A detailed understanding of the mechanisms and regulation of the Ada and Spt protein-containing complexes (Ada⅐Spt complexes for simplicity) requires the identification of component proteins. We now identify Tra1p as a component of the Ada⅐Spt complexes. Tra1p is a member of a group of putative protein kinases, including the catalytic subunit of human DNA-dependent protein kinase (DNA-PK CS ) and ATM, that contain a carboxyl-terminal region related to phosphatidylinositol 3-kinases (PI3K 1 ; reviewed in Refs. [17][18][19][20]. In addition, Tra1p shows extensive sequence similarity throughout its entire length to the human protein TRRAP that associates with both c-Myc and E2F-1 (21).

DNA Constructs, Yeast Strains, and Genetic Analysis
Epitope-tagging of TRA1. A BstI1071-BglII fragment of YCp88-NGG1 (3), containing the DED1 promoter, Myc tag, and NotI restriction site, was cloned into HindII-BamHI sites of YCplac111. A 11,832-base pair FspI fragment from ATCC cosmid 70,897 was cloned into the SmaI site of this construct placing TRA1 downstream of the promoter. The 5Ј segment of TRA1 was synthesized by PCR, with a NotI site at the position of the translational start. Alleles expressing myc-NGG1 and HA-ADA2 have been described (5,8). Six histidine-tagged myc-NGG1 (HM-NGG1) was constructed by inserting a NotI fragment expressing 6-His into pDMYC-NGG1 (5).
To construct a disruption allele of TRA1 for genetic analysis, tailed PCR was performed using a 5Ј-oligonucleotide containing 60 base pairs corresponding to the sequence immediately upstream of TRA1 followed by sequence from the 5Ј end of HIS3. The 3Ј primer included 3Ј HIS3 sequence flanked by 60 base pairs corresponding to sequence immediately downstream of the TRA1 stop codon. HIS3-containing plasmid was used as template, and the resulting PCR fragment was used to transform the diploid strain YPH501 using lithium acetate (24,25). Transformants were plated onto histidine-deficient media, and colonies were obtained. DNA was isolated from transformants and subjected either to NheI digestion and Southern analysis with a HIS3 probe or used as a template in PCR reactions designed to amplify the unique junctional region formed between sequences upstream of TRA1 and the recombined HIS3 gene. Sporulation and tetrad dissection were performed using standard techniques (26).

Purification of HM-Ngg1p
Whole cell extract was prepared from 8-liter cultures of strains expressing HM-Ngg1p (CY979) or Myc-Ngg1p (CY1077) in Buffer E (50 mM HEPES (pH 7.6), 50 mM NaCl, 20 mM imidazole, 10% glycerol and 0.1% Nonidet P-40; see Ref. 8). Protease inhibitors (8) and 1.0 mM dithiothreitol were included in all solutions. 150 mg of protein (ϳ4 ml) was rotated with 1.5 ml of Sepharose CL-4B for 30 min at 4°C. Unbound protein was rotated with 2.5 ml of Ni 2ϩ -nitriloacetic acid-agarose (Ni 2ϩ -NTA; Qiagen) for 1 h. The mix was applied to a column, washed with 50 ml of Buffer E, then consecutively with 5 ml of Buffer E containing 50 and 350 mM imidazole. Fractions were concentrated (Centricon-30; Amicon) and separated on a 6% SDS gel. From the Coomassie-stained gel of the 350 mM imidazole fraction, gel slices corresponding to an ϳ400-kDa protein from the HM-NGG1 strain and its parallel position from the myc-NGG1 control were excised and washed with water for 20 min.

Protein Identification
Protein was eluted and in-gel trypsin digested by the method of Shevchenko et al. (27). Protein was identified by micro-column high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry and data base searching. A 100-by 200-m fused silica capillary (Ref. 28; Polymetrics, Inc.) was packed to a length of ϳ15 cm with 10 mm POROS 10 R2 reverse phase material (Perspective Biosystems). The fritted end of the column was inserted into the needle of the electrospray ion source and sample loaded by helium pressurization in a stainless steel bomb (29). Chromatography was performed with a dual syringe pump (Applied Biosystems). The mobile phase consisted of 0.5% acetic acid (solvent A) and 80:20 acetonitrile/ water containing 0.5% acetic acid (solvent B). A 100:1 precolumn split was used to deliver a flow rate of 1 to 1.5 ml/min. The high performance liquid chromatography pump was programmed to ramp solvent B from 0 to 60% in 30 min. Electrospray ionization was carried out at voltage of 4.6 kV. Tandem mass spectra were acquired automatically during the entire gradient run (30).
Tandem mass spectra were searched against an S. cerevisiae protein data base (Saccharomyces Genome Data base) using the SEQUEST program (31). Parameters for the SEQUEST program were set to locate potential sites of phosphorylation at serine, threonine, and tyrosine residues (32). Every sequence with high scores that matched a tandem mass spectrum was manually verified.

Fractionation of Tra1p, Ngg1p, Ada2p, and Spt7p
Ion Exchange Chromatography-32 mg (2 ml) of a mixture of whole cell extract containing Myc-Tra1p, Spt7p, and Myc-Ngg1p 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. The extract was applied to an FPLC Mono Q column (1.0 ml; Amersham Pharmacia Biotech) at a flow rate of 0.1 ml/min. After washing the column with 4 ml of the extraction buffer, protein was eluted with a 21-ml gradient of 20 -700 mM NaCl at a flow rate of 0.25 ml/ml. Protein from 45-l aliquots of 250-l fractions was separated by SDS-PAGE and analyzed by Western blotting with anti-Myc and anti-Spt7p antibody. Similarly, extracts prepared from an ada2 deletion strain (CY927) expressing Myc-Tra1p (myc-TRA1 ada2) or from a wild-type strain (TRA1 ADA2) were cofractionated on the Mono Q column and the peak fraction containing Myc-Tra1p and/or Spt7p used for subsequent analysis.
Chromatography on DNA-Cellulose-The peak fraction containing Myc-Tra1p, Spt7p, and Myc-Ngg1p from the ϳ350 mM NaCl Mono Q fraction (Complex I) was dialyzed against DB buffer (5 mM Tris-HCl (pH 7.4), 50 mM NaCl, 5 mM MgCl 2 , 0.025% Nonidet P-40, 5% glycerol) and loaded onto a 0.5-ml DNA-cellulose column (Amersham Pharmacia Biotech) at a flow rate of 1.5 ml/h. After washing to remove unbound protein, bound protein was eluted with 0.6 ml of DNA-binding buffer containing 100, 300, and 500 mM NaCl and analyzed by Western blotting. The ϳ250 mM NaCl Mono Q fraction from the ada2 strain CY927 was handled similarly.
Gel Filtration Chromatography-The DNA-cellulose fraction containing Myc-Tra1p, Spt7p, and Myc-Ngg1p (300 mM NaCl fraction) was dialyzed against 40 mM Tris-HCl buffer containing 300 mM NaCl, 4 g/ml ethidium bromide, 10% glycerol, 0.08% Nonidet P-40, 0.2 mM EDTA, 0.1 mM dithiothreitol and loaded at a flow rate of 0.2 ml/min onto an FPLC Superose 6HR10/30 column (Amersham Pharmacia Biotech). Protein from 150-l aliquots of 500-l fractions was precipitated with 10% trichloroacetic acid, solubilized in SDS sample buffer, and separated on a 5.5% SDS-polyacrylamide gel. Similarly, 16 mg of whole cell extract was prepared from strains expressing Myc-Tra1p, Spt7p, and Myc-Ngg1p, filtered through 0.22-mm membrane and applied to the Superose 6 column in the presence of ethidium bromide. Alternate fractions were analyzed by Western blotting with anti-Myc and anti-Spt7p antibody.

Immunoprecipitation and Immunoblotting
Immunoprecipitation of HA-Ada2p and Myc-Tra1p from whole cell extracts was performed as described (8). Immunoprecipitation from the Mono Q fraction containing Ada⅐Spt Complex I was done after adjusting the buffer to 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1% Nonidet P-40, and 5% glycerol (IP buffer) in a volume of 1.5 ml. 150 g of protein was rotated for 20 min with 50 l of protein A-Sepharose. Unbound protein was rotated with 7 l of ascites fluid (12CA5) and 50 l of protein A-Sepharose beads for 3 h. Beads were washed four times with 1.5 ml of IP buffer and twice with 1.5 ml of buffer containing 25 mM HEPES (pH 7.5), 50 mM KCl, and 5% glycerol and then eluted in SDS loading buffer at 60°C for 5 min and separated by SDS-PAGE. To analyze for the association of Spt7p with Tra1p in the presence and absence of Ada2p, protein from the ϳ350 mM (50 g) and ϳ250 mM NaCl (70 g) Mono Q eluting fractions from strains expressing Myc-Tra1p (KY320, myc-TRA1 ADA2; CY927, Myc-TRA1 ada2) and untagged Tra1p (TRA1 ADA2) were incubated with 4 g of anti-Myc antibody and 15 l of protein A-Sepharose after dialysis in IP buffer. Bound protein was eluted from the beads and analyzed by Western blotting for Spt7p. Western blotting with polyclonal anti-Spt7p antibody and monoclonal anti-Myc and HA antibodies has been described (5,8,16).

RESULTS
Association of Tra1p with Ngg1p-To search for components of the Ada⅐Spt complexes, we analyzed proteins associated with Ngg1p after affinity purification of Ngg1p from whole cell extracts. A derivative of NGG1 was constructed that encodes a protein with both six histidine and Myc-epitope tags (HM-Ngg1p). Extracts from a strain containing HM-NGG1 and as a control from a strain containing myc-NGG1 were loaded onto parallel Ni 2ϩ -NTA columns. HM-Ngg1p eluted from the Ni 2ϩ -NTA column with buffer containing 350 mM imidazole as compared with Myc-Ngg1p which eluted in 50 mM imidazole. 350 mM imidazole eluants from both strains were compared by SDS-PAGE, revealing potential HM-Ngg1p-associated proteins (not shown). One of these proteins had an apparent molecular mass of ϳ400 kDa and thus did not match previously described Ada components. With the purification of the Ada components obtained by chromatography on Ni 2ϩ -NTA and the resolution afforded by its migration on SDS-PAGE, the ϳ400-kDa protein was isolated and its identity determined by micro-column high performance liquid chromatography coupled to tandem mass spectrometry and data base searching.
The gene product Tra1p (YHR099W) was identified from 36 unique peptides that correspond to 15.2% of the mass of the protein. TRA1 encodes a protein of 3744 amino acids (433 kDa) with 20 -50% sequence similarity throughout its length to TRRAP, a human protein that associates with c-Myc and E2F-1 (21). The absence of Tra1p in the parallel gel slice from the control strain suggested that its association with HM-Ngg1p was specific. In addition, mass spectrometry revealed that the peptide AEQGDLDSPKEPQADELLDEFSK between residues of 165 and 187 of Tra1p contained one phosphoserine.
TRA1 Encodes an Essential Gene-The approaches used to study further Tra1p in the Ada⅐Spt complexes depended on whether TRA1 was essential for cellular viability. The entire 11.2-kb coding region of one TRA1 allele was replaced with HIS3 in the diploid strain YPH501. To ensure that a TRA1 allele was disrupted several isolated His ϩ colonies were analyzed by Southern blotting. When correctly inserted into the TRA1 locus, the HIS3 disruption cassette generates an NheI fragment of 6.3 kb. When NheI-restricted genomic DNA was probed for HIS3, the predicted 6.3-kb band was visible in several of the transformed strains (Fig. 1A, clones D1, D3, D4, and D5). This band is absent from wild-type strain YPH501 (lane 1) and from some of the His ϩ clones. The insertion of HIS3 into TRA1 was also confirmed by PCR using primers that span the TRA1-HIS3 junction (not shown). Three representative strains containing the wild-type TRA1 gene and the tra1::HIS3 allele were sporulated and the resulting tetrads dissected. Although all spores from wild-type YPH501 were viable, tetrads from TRA1/tra1::his3 strains segregated for growth with a 2:2 ratio (Fig. 1B), indicating that disruption of TRA1 is incompatible with growth.
Coimmunoprecipitation of Tra1p and Ada2p-To allow biochemical and immunological approaches to verify the association of Tra1p with the Ada and Spt proteins, Tra1p was Myctagged at its amino terminus and inserted into a centromeric plasmid (myc-TRA1-YCplac111). The ability of this allele to functionally replace the wild-type allele was shown in a plasmid shuffle experiment. CY1021 contains myc-TRA1 on a URA3 centromeric in a background in which chromosomal TRA1 is disrupted. Growth of cells in the absence of the plasmid copy of TRA1 was determined in the presence and absence of myc-TRA1-YCplac111 by plating the cells on 5-fluoroorotic acid (Fig. 2). Cells containing myc-TRA1-YCplac111 were able to lose the URA3 plasmid and thus grow on 5-fluoroorotic acid, in contrast to CY1021 without myc-TRA1-YCPlac111.
Ngg1p and Ada2p are intimately associated in multiple complexes, some of which contain the TBP class of Spt proteins (8 -11). To verify the interaction of Tra1p with the ADA components, we determined if Tra1p coimmunoprecipitates with Ada2p. Whole cell extracts were prepared from strains expressing myc-TRA1 and HA-ADA2 independently and from a strain expressing both myc-TRA1 and HA-ADA2. HA-Ada2p and associated proteins were immunoprecipitated with anti-HA antibody, separated by SDS-PAGE, and Western blotted with anti-Myc antibody (Fig. 3A). In certain whole cell extracts Spt7p appeared as multiple bands (Fig. 3B, lanes 1 and 4). These may arise as a result of the modification of Spt7p by ubiquitination (16), although the reason for variability in the appearance of the bands is unclear.
Tra1p Cofractionates with ADA and SPT Proteins-To examine if Tra1p interacts with one or more of the Ada complexes, we compared the elution of Myc-Ngg1p, Spt7p, and Myc-Tra1p after fractionation by gel filtration and ion exchange chromatography. First, whole cell extract was chromatographed on a Superose 6 column, and equal volumes of alternate fractions were examined for Myc-Ngg1p, Spt7p, and Myc-Tra1p by Western blotting. As shown in the densitometric scanning profile in Fig. 4 and as described previously (8) To analyze further the association of Myc-Tra1p with the Ada⅐Spt complexes, whole cell extract was chromatographed on an FPLC Mono Q column. Protein was eluted from a Mono Q column in a linear gradient of 20 -700 mM NaCl with equal volumes of alternate fractions examined by Western blotting (Fig. 5A). Myc-Ngg1p was found in three peaks at fractions 34 (ϳ350 mM NaCl), 48 (ϳ450 mM NaCl), and 68 (ϳ550 mM NaCl). Ada2p peaks with Ngg1p in each of these fractions (not shown, see Ref. 8). Spt7p was present within the ϳ350 mM NaCl fraction (Ada⅐Spt Complex I) and the ϳ550 mM NaCl fraction (Ada⅐Spt Complex II) and perhaps coeluted with Ngg1p in the ϳ450 mM NaCl fraction. The presence of Spt7p in fractions centered at fraction 52 again suggests that Spt7p may exist independently of Ngg1p. Myc-Tra1p cofractionated with Myc-Ngg1p and Spt7p in both Ada⅐Spt Complex I and Complex II but was absent from the ϳ450 mM NaCl fraction.
As an additional step to demonstrate the association of Myc-Tra1p with the Ada/Spt proteins in Complex I, we immunoprecipitated HA-Ada2p from the ϳ350 mM NaCl Mono Q fraction and analyzed by Western blotting for Myc-Tra1p and Spt7p (Fig. 6). Both of these proteins specifically coimmunoprecipitated with HA-Ada2p from Complex I (lane 2) as they did from a whole cell extract (lane 3).
The amount of Myc-Tra1p in Complex II prohibited the same analyses done with Complex I. Therefore, to address whether coelution of Myc-Tra1p with the Ada⅐Spt components in this complex was indicative of their association, we analyzed whether removal of Ada2p altered the elution profile of Myc-Tra1p from gel filtration and ion exchange columns. As shown in Fig. 7, when extracts prepared from the ada2 deletion strain CY927 were chromatographed on a Mono Q column, Myc-Tra1p eluted in fractions centered at ϳ250 mM NaCl and was absent from both complexes (I and II) in which it was found when prepared from the wild-type ADA2 strain. This altered elution profile for Myc-Tra1p from the ada2 strain suggests that it is a component of both the Ada⅐Spt Complexes I and II.
Tra1p Associates with Spt7p in the Absence of Ada2p and with Ada2p in the Absence of Spt7p-To investigate the interdependence of the associations of Tra1p with Ada2p and Spt7p, Myc-Tra1p immunoprecipitates from yeast strain CY927 were analyzed for Spt7p by Western blotting (Fig. 8A). Immunoprecipitates from both the ϳ250 mM NaCl Mono Q fraction after chromatography of extract from CY927 (ada2; lane 5) and as a positive control from the ϳ350 mM fraction (Complex I) from KY320 (ADA2; lane 2) contained Spt7p. This association of Myc-Tra1p and Spt7p was specific since Spt7p was not found in any of the fractions which lacked Myc- Tra1p (lanes 1, 3, 4, and  6).
A similar experiment was performed to determine if Tra1p associates with Ada2p in the absence of Spt7p. The spt7 disruption strain FY1093 and its parent strain FY630 were transformed with plasmids expressing Myc-Tra1p and HA-Ada2p. Immunoprecipitates were analyzed for the presence of Myc-Tra1p by Western blotting. As shown in Fig. 8B, Myc-Tra1p, identified by its absence in control extracts lacking tagged protein (lane 3), was found within immunoprecipitates from both SPT7 wild-type (lane 1) and spt7 deletion (lane 2) strains. Tra1p thus independently associates with Ada2p and Spt7p.
Binding of Tra1p to DNA-Cellulose Requires ADA Proteins-The binding of Ada⅐Spt Complex I to DNA-cellulose was of particular note since it may reflect a function of the complex. To determine if Ada proteins are required for the binding of Myc-Tra1p to DNA-cellulose the ϳ250 mM NaCl, Myc-Tra1p-containing fraction after chromatography of a CY927 (ada2) extract on Mono Q was loaded onto DNA-cellulose. In contrast to Myc-Tra1p from a wild-type ADA2 extract, for the CY927 extract Myc-Tra1p eluted in the flow-through fraction ( Fig. 9; compare top two panels). To address whether factors in a wildtype extract could restore DNA binding of this Myc-Tra1p, Myc-Tra1p (ϳ250 mM NaCl Mono Q fraction) from the ada2 deletion strain was mixed with the ϳ350 mM NaCl Mono Q fraction from a wild-type strain lacking tagged Ada2p (TRA1 ADA2). As shown in the second to last panel, binding of approximately 25% of the Myc-Tra1p to DNA-cellulose was restored upon mixing with the wild-type Ada/Spt-containing frac- Protein was eluted with a 21-ml gradient of 20 -700 mM NaCl. Equal volumes of alternate fractions (0.25 ml) were separated on a 5.5% SDS gel to probe for Myc-Tra1p, Spt7p, and Myc-Ngg1p. Fractions are numbered as they were eluted. The two arrows indicate the peak fractions for elution of the three proteins (fraction 34, ϳ350 mM NaCl; fraction 68, ϳ550 mM NaCl). Unbound protein and fractions 1-28 and 74 -84 did not show immunoreactive bands that correspond to the three proteins. B, the Mono Q ϳ350 mM NaCl fraction containing Myc-Tra1p, Spt7p, and Myc-Ngg1p was dialyzed and chromatographed on a DNA-cellulose column. After washing with 50 mM NaCl buffer, bound protein was eluted with a step gradient of NaCl (100, 300, and 500 mM). The elution of Myc-Tra1p, Spt7p, and Myc-Ngg1p was followed by Western blotting. Load is 50 g of the sample applied to the column. FT is the flow-through of the DNA-cellulose column. C, the 300 mM NaCl fraction from the DNA-cellulose column was dialyzed against 40 mM Tris-HCl buffer (pH 7.7), containing 300 mM NaCl and 4 g/ml ethidium bromide and applied to an FPLC Superose 6 column at a flow rate of 0.3 ml/min. 50 fractions of 0.5 ml were collected, and the elution of Myc-Tra1p, Spt7p, and Myc-Ngg1p was followed by Western blotting of alternate fractions with anti-Myc and anti-Spt7p antibodies. Load represents 25 g of sample applied into the column. Void is the void volume of the Superose 6 column as determined by a high molecular mass DNA and is followed by the column fractions numbered as they were eluted. Other column fractions were shown to be negative for Myc-Tra1p, Spt7p, and Myc-Ngg1p. tion. The reconstitution of binding was due to Ada/Spt proteins because mixing with ϳ350 mM NaCl Mono Q fraction obtained from the ada2 strain did not restore binding of Tra1p to DNA-cellulose (lower panel). These results demonstrate that the binding of Myc-Tra1p to DNA-cellulose requires Ada/Spt components.

DISCUSSION
Tra1p Is a Component of Two Ada⅐Spt Complexes-Our identification of Tra1p within the Ada⅐Spt complexes was based on its association with ADA components after affinity purification on Ni 2ϩ -NTA. Several lines of evidence confirm that Tra1p associates with the Ada/Spt proteins. The association of Tra1p and Ada/Spt components was shown by their reciprocal coimmunoprecipitation from whole cell extracts. As well as their copurification on a Ni 2ϩ -NTA column, Tra1p, Ngg1p, and Spt7p remain associated through an approximately 1200-fold purification over consecutive chromatography on FPLC Mono Q, DNA-cellulose, and Superose 6 columns. Furthermore, chromatography on the FPLC Mono Q column revealed that all of the Tra1p coeluted with Ngg1p and Spt7p in two distinct peaks. The validity of this cofractionation as a measure of association was also verified by the finding that Tra1p coimmunoprecipitated with Ada2p in the ϳ350 mM NaCl Mono Q fraction (Complex I) and the demonstration that the elution profile of Tra1p on Mono Q and DNA-cellulose was altered when performed with an extract from a strain lacking Ada2p. Association of Tra1p with the Ada/Spt proteins was also supported by the ability of partially purified Ada/Spt proteins to reconstitute binding of Tra1p to DNA-cellulose.
We have previously identified biochemically four complexes that contain the ADA proteins as follows: two with sizes of approximately 2 MDa and single complexes of approximately 900 and 200 kDa (8). The appearance of Tra1p with Ngg1p in the 2-MDa peak on Superose 6 and in two peaks on a Mono Q column suggests that it is found within both of the 2-MDa complexes. Since the Tra1p-containing complexes also contained Spt7p, one of these probably represents the previously described 1.8-MDa SAGA complex (9); the second thus identifies an additional Ada⅐Spt complex that has also been observed by Grant and Workman. 2 Tra1p is likely a central molecule in the formation or stability of these complexes since the interaction of Ada2p with Tra1p was independent of Spt7p and similarly the interaction of Spt7p with Tra1p was independent of Ada2p.
The fractionation of the Ada and Spt proteins on Superose 6 and Mono Q also identified a possible Spt7p-containing complex that lacked the Ada proteins and Tra1p. This complex has an approximate size of 1.5 MDa based upon its elution from Superose 6. The existence of Ada-independent Spt7p agrees with the broader range of phenotypes seen in spt7 disruptions as compared with those seen for the adas (10,15).
Similarity of Tra1p to PI3K-related Molecules-Tra1p is a member of a group of molecules with carboxyl-terminal sequences similar to PI3Ks (33). Although related to the PI3Ks, these molecules can be distinguished from the PI3Ks by having a common region directly at their carboxyl terminus, their large size, and in some cases an ability to phosphorylate proteins (19,34). Similarly, 150 g of the ϳ250 mM NaCl Mono Q fraction containing Myc-Tra1p from CY929 (ada2) was chromatographed, individually (Q 250 mM NaCl myc-Tra1 ada2; second panel), and after mixing with 150 g of the Mono Q fraction 12 from KY320 (Q 250 mM NaCl myc-Tra1 ada2 ϩ Q 350 mM NaCl Tra1 ADA2; third panel), or after mixing with 150 g of the ϳ350 mM NaCl fraction from the same ada2 strain (Q 250 mM NaCl myc-Tra1 ada2 ϩ Q 350 mM NaCl myc-Tra1 ada2; bottom panel). The elution of Myc-Tra1p was assayed by Western blotting. The first lane (load) is 25 g of the protein applied to the column, followed by the flow-through (FT; 50 mM NaCl), 100, 300, and 500 mM NaCl fractions. Different amounts of protein were used for ADA2 and ada2 strains to equalize the amount of Myc-Tra1p. be identified with a BLAST search throughout the molecules. Of the PI3K-like molecules, two seem to be most closely related to Tra1p as follows: the product of the S. pombe gene C1F5.11C and the mammalian protein TRRAP (21). Both share 20 -50% homology with Tra1p along the entire length of the molecules.
Although closely related to the PI3K family members, Tra1p lacks the DFG sequence found in many protein kinases (35). It does contain the DXXXXN kinase motif but in a flanking sequence context different from other family members. BLAST and FASTA searches identify other sequence relationships that could relate to function. The PI3K region shows similarity to the transcriptional repressor RGM1p (27% identity over a 136amino acid overlap; see Ref. 36) and SIR4p (24% identity over a 244-amino acid overlap; see Refs. 37 and 38). Central regions of the protein are also similar to the transcriptional regulator TEC1p (39) and to MTR1p which is involved in nuclear protein import (40).
As a group the PI3K-related molecules are involved in many key cellular processes including DNA repair, meiotic recombination, V(D)J recombination, cell-cycle regulation, DNA-damage recognition, and transcription (17,18,20). Interestingly, the human molecule most likely the homologue of Tra1p (TRRAP) has recently been shown to associate with both c-Myc and E2F-1 in vivo (21). During the mammalian cell cycle, the Myc and E2F families of transcription factors are major regulators of G 1 /S phase progression (41,42). Furthermore, both c-Myc and E2F-1 interact with TRRAP through their transactivation domains, in keeping with the presence of TRA1p in a multi-protein complex implicated in transcriptional regulation (21).
Tra1p Does Not Possess Protein Kinase Activity-The essential nature of TRA1 has not allowed direct gene disruption experiments to determine its role in the Ada⅐Spt complexes. Many models reflect the possibility, based on sequence similarities, that Tra1p is a protein kinase and, in turn, the identity of substrates. The recent finding by Barlev et al. (43) that Ku70, which as a heterodimer with Ku80 regulates the DNA binding of DNA-PK cs (44,45), associates with hGCN5, would support this possibility. In the case of DNA-PK cs , hGCN5 is a target in vitro and phosphorylation correlates with decreased histone acetyltransferase activity (43).
To test if Tra1p is a protein kinase, we have examined kinase activity in immunoprecipitates of both Myc-Tra1p and HA-Ada2p, from partially purified Ada⅐Spt complexes. Under separate conditions in which DNA-PK cs , casein kinase II, or the cyclin-dependent kinase p34 cdc2 were active, we were unable to detect phosphorylation of ␤-casein, RPA, histone H1, myelin basic protein, or the amino terminus of p53 at a level above that found in control immunoprecipitates (not shown). Assays were performed ϩ/Ϫ DNA as well as ϩ/Ϫ Ku with no significant activity detected. In addition, under these same conditions we did not observe phosphorylation of endogenous components of the complex, including Gcn5p or Tra1p. A general loss in the integrity of the immunoprecipitated complexes was unlikely because they retained histone acetyltransferase activity (not shown, Ref. 16). Furthermore, it is unlikely that Tra1p is inhibited by components of the ADA complex because immunoprecipitates of Tra1p isolated from the ada2 deletion strain also lacked kinase activity. Clearly, we cannot at present exclude the possibility that Tra1p has unique substrates or assay conditions; however, the lack of kinase activity is consistent with the absence of the normally conserved DFG sequence in the kinase motif.
The sequence similarity of Tra1p to several key cellular regulators, its appearance in at least two of the Ada⅐Spt complexes, and even its large size suggests that Tra1p plays a key role in the structure, function, or regulation of the Ada⅐Spt complexes. Since all the detectable Tra1p cofractionated with Myc-Ngg1p, it appears to function principally through its association with the Ada/Spt proteins. The fact that unlike other Ada⅐Spt complex proteins, Tra1p is essential, does predict that it has a broader range of function(s) than the other components.