956 Nucleic Acids Research, 2006, Vol. 34, No. 3 Only a few proteins have been identiﬁed as speciﬁc interaction partners of transcriptional activators, including TATA-box binding protein, TFIIB, TFIIH (4,16–20) and several others [reviewed in (21)]. Although previous studies have attempted to screen for transcriptional activators [e.g. (22)], we do not know of such a systematic screen in yeast. Incidentally, we approached the problem indirectly by doing systematic yeast-two-hybrid (Y2H) screens (23) which are based on a split transcription regulator, e.g. Gal4. In these screens all yeast open reading frames (ORFs) have been fused to the DBD of yeast Gal4 and tested for interaction with every other yeast protein. Unfortu- nately, in many cases no interactions could be detected because the Gal4–DBD fusion protein was activating tran- scription without requiring a second fusion protein, that is, the Gal4–AD fusion, also known as prey. Gal4–DBD fusion proteins that activate transcription of a reporter gene without requirement of a prey protein are called ‘auto-activators’ (or activators for short) and thereby possess properties of an AD. Our genome-wide two-hybrid screen thus provided data about activation properties of nearly all yeast proteins. Up to now this information has not been used for the understanding of transcription in yeast. However, similar assays have been used previously by Wiesner et al. (24) and Ma and Ptashne (25) for screening human and Escherichia coli proteins/peptides for their transcriptional activation properties. By design such screens do not necessar- ily identify physiological activators as the fusion partners are artiﬁcially targeted to the promoter of a single, arbitrarily selected reporter gene. Bearing these caveats in mind, we not only selected Y2H auto-activators but also measured their activation strength. In a second step we analyzed these auto-activators for their domain content and other sequence properties. We found that auto-activators possess speciﬁc physicochemical properties, like certain amino acid clusters, and can often be found in known complexes of the transcription machinery. Many of the activators identiﬁed in this study are known physiological activators. However, many do not appear to be localized to the nucleus under standard laboratory conditions and thus may not act as bona ﬁde transcription factors. Nevertheless, we believe that even such cases can shed light on the mechanisms of transcriptional activation similar to heterologous proteins such as VP16 which helped to uncover many mechanistic details of transcriptional activation. MATERIALS AND METHODS Selection of transcriptional activators Transcriptional activators were identiﬁed in two genome-wide Y2H studies of yeast proteins (26,27). The ‘Ito screen’ resulted in 392 auto-activators which were identiﬁed by testing essen- tially all yeast proteins for transcription activation when fused to the DBD of Gal4 as full-length ORFs (27). The ‘Uetz set’ contains 68 auto-activators and was identiﬁed in two-hybrid screens of 600 yeast proteins (26). For analysis of transcription activator properties activators identiﬁed by Ito et al. were selected from the Uetz bait library (26) and combined with the Uetz activators to form a set of 451 proteins. These two independent selection steps ensured correct identiﬁcation of activators. For further quality control, we sequenced 48 samples from the Ito collection and checked another 29 from the Uetz lab by colony PCR for correct insert size. Among these 77 clones only 1 (out of the 29 mentioned) did not match the expected identity and was thus excluded from further analysis. The activators of the ‘Ito set’ were then divided into activation strength groups (see below). However, 79 strains did not grow well enough to be quantiﬁed, although they were reported to be activators. Their activation strength has been annotated as ‘not available’ (NA) in Supplementary Table 1. Yeast strains The activators were selected from the bait proteins described by Uetz et al. (26) and Hazbun et al. (28), and consisted of full-length ORFs fused to the Gal4–DBD in the CEN4 plasmid pOBD2 (29). These DBD-activator gene constructs were expressed in the strain YULH (MATa, ura3-52, trp1, lys2, his3, leu2, gal4D, gal80D, GAL1-URA3, GAL1-lacZ). This ‘activator strain’ was re-arrayed onto 96-well plates and mated with yeast cells of the opposite mating type [namely PJ69-4a: MAT a, trp1-901, leu2–3, ura3–52, his3–200, gal4D, gal80D, GAL2-ADE2, LYS::GAL1-HIS3, met2::GAL7- lacZ (26,30)] carrying an empty prey vector pLP-GADT7 (Clontech). This mating was necessary in order to introduce the HIS3 reporter gene of the PJ69-4 a strain. The handling of yeast colonies was done by automated robotic procedures employing a Biomek 2000 robotic work- station (Beckman Coulter). Measurements of transcriptional activation strength The activation strength was measured by two different assays which measure the expression of two reporter genes: His3, which encodes imidazoleglycerol-phosphate dehydratase and catalyzes the sixth step in histidine biosynthesis (LTH assay) and beta-galactosidase (bGal assay). His3 gene expression was measured (LTH assay) by grow- ing yeast cells on selective media lacking leucine (L), trypto- phane (T) and histidine (H) with the former two corresponding to markers on the two vectors pOBD2 and pLP-GADT7. Activity of the HIS3 reporter was quantiﬁed by increasing amounts of 3-aminotriazole (3-AT), a competitive inhibitor of His3. The lowest concentration of 3-AT that inhibited growth was considered as the activation strength of the gene construct (29). This assay was done in quadruplicate in order to ensure reproducibility (for detailed results see Supplementary Table 1). The bGal assay was performed in 96-well plates using ONPG as a substrate (31). Brieﬂy, diploid yeast cells were grown overnight at 30 C in 100 ml selective media (leucine and tryptophane deﬁcient), the media was replaced by 50 ml Z-buffer [1.6% (w/v) Na2HPO4, 0.55% (w/v) NaH2PO4, 0.075% (w/v) KCl and 0.025% (w/v) MgSO4, pH 7] and the cells lysed by two freeze-thaw cycles. For normalization of the cell density OD600 was measured using a microplate reader (Elx808, Bio-Tek Instruments, VT). The assay was started by adding 50 ml of 1.5 mg/ml ONPG in Z-buffer and the initial OD405i was measured immediately. The reaction was incubated at 37 C and OD405 measured at different time