Table 3. List of Y2H activators of unknown function predicted to be TRs ORF YFL049W YJR070C YDR520C YGL066W YKR064W YCR082W Name YFL049W YJR070C YDR520C SGF73 YKR064W YCR082W Activation strength (in mM 3AT) Biological process (GO) 100 200 >200 50 200 200 Unknown Unknown Unknown Protein modification Unknown Unknown Nucleic Acids Research, 2006, Vol. 34, No. 3 961 Localization Nucleus Cytoplasm, Nucleus Cytoplasm, Nucleus Nucleus Cytoplasm, Nucleus Cytoplasm, Nucleus Interaction with transcription machinery Yes No Yes Yes No Yes Additional evidence Yes Yes Yes No Yes No lower CAI and show speciﬁc properties like enrichment of asparagine clusters. Protein domains Transcriptional ADs are still not well deﬁned structurally. This is also reﬂected in specialized domain databases such as SMART (8) or Interpro (45) which have hardly any deﬁned entries for transcriptional ADs. Therefore we have analyzed the Y2H activators for enrichment of known domains. Not unexpectedly, certain DBDs such as helix–loop–helix motifs or zinc ﬁngers are indeed signiﬁcantly overrepresented among the activators (Supplementary Table 5). Certain domains are also signiﬁcantly underrepresented such as the AAA domain. However, these domains are not further considered here. Binary protein–protein interactions Speciﬁc transcription activation is mediated by physical contact with the transcriptional machinery or other factors necessary for transcription, like chromatin remodeling pro- teins (4,5,16,18–20). Activators form a tightly connected protein interaction net- work (Figure 5A). Therefore, we wondered if the Y2H activ- ators had any known interactions with the transcription machinery or other proteins (Figure 5B). Signiﬁcantly more nuclear Y2H activators ( 36%) interac- ted with components of the transcription machinery compared with all nuclear proteins ( 23%). The same is true for known transcription regulators which show an even higher fraction of proteins interacting with the transcription machinery ( 46%). Taking bridging proteins into account (i.e. transcription proteins that can be reached over two protein interaction edges), the number of proteins interacting with transcription factors even increases to 57% (nuclear Y2H activators) and 64% (transcription regulators), respectively. All mentioned differences are highly signiﬁcant judged by Fisher’s exact test (P < 0.01). Remarkably, activation strength showed no effect on the number of proteins interacting with the transcription machinery. Protein complexes Because transcription is carried out by several large protein complexes such as RNA polymerase, TAFs, mediator and the like, we wondered with which of these complexes the Y2H activators interacted. Therefore we analyzed complexes from large scale MS studies (35,46,47) which had at least 50% of their proteins annotated as ‘transcription’-related for the presence of Y2H activators (Figure 5C). Of nuclear activators 19% interacted with transcription complexes compared with 8% of all nuclear proteins. An even higher fraction (27%) of known TRs interacted with complexes involved in transcrip- tion. Strikingly, Y2H activators in the nucleus that were not annotated as TRs showed a signiﬁcant higher number of interactions with the transcription machinery compared with other nuclear non-TR proteins. This might indicate that many of these proteins are also bona ﬁde TRs (see below). All these differences are highly signiﬁcant judged by Fisher’s exact test (P < 0.01). To reveal the nature of proteins interacting with trans- criptional activators we compiled the most frequent interactors of our Y2H activators. The top 15 interacting proteins of dif- ferent protein query sets are given in Supplementary Table 4. This analysis reveals that a high percentage of activators, as well as known TRs interact with components required for transcription. These were components of the RNA- polymerase II holoenzyme and proteins involved in chromo- some remodeling, like the SAGA complex. Nuclear proteins in general do not show this tendency with only one interactor (YDL014W) shared between Y2H activators and nuclear pro- teins in general (Supplementary Table 4). Interestingly, both nuclear activators and transcription regulators interacted with the metabolic enzyme alcohol dehydrogenase. With 21 and 19%, respectively, the fractions were much higher than in the set of nuclear proteins ( 10%, not shown). In summary, Y2H activators as well as known TRs tend to speciﬁcally interact with components of the transcription machinery even though such interactions have not been shown for the majority of either group. Y2H activators as bona fide transcriptional regulators Many of the Y2H activators described here may be bona ﬁde TRs even if they have not been annotated as such. We therefore may have identiﬁed Y2H activators of yet unknown function given their ability to efﬁciently activate transcrip- tion, their nuclear localization and their protein interaction pattern with other components of the transcription machinery. The following Y2H activators of yet unknown function are predicted to be physiological activators. YFL049W, a protein with no assigned function, was co-puriﬁed with Rtt102p, Snf2p and Snf5p indicating that it is a real Swi/Snf component (48). The yeast SWI/SNF com- plex is required for transcription of several yeast genes and has been shown to alter nucleosome structure in an ATP- dependent reaction. Transcription stimulation by SWI/SNF requires an AD with which it directly interacts. Strikingly, the acidic ADs of VP16, Gcn4, Swi5 and Hap4 interacted