958 Nucleic Acids Research, 2006, Vol. 34, No. 3 Figure 1. Activation strength in Y2H activators. (A) Number of activators showing the indicated activation strength in the LTH assay. They were divided into weak (LTHw), medium (LTHm) and strong (LTHs) activators as indicated. (B) Correlation of activation strength in LTH and bGal assays. The actually measured values are shown as dots, the median and two quartiles by an overlayed boxplot. Dotted lines indicate the median of bGal activity and LTH activity, respectively. For an explanation of datasets see Table 1. were termed LTHw, LTHm and LTHs, respectively. The bGal assay distribution was divided into strong activators (bGals) which activate above the median and weak activators that activate below the median (bGalW). A comparison of activation strength in both assays revealed an intermediate (Pearson’s correlation coefﬁcient of 0.58) but highly signiﬁcant correlation (P < 2.2e 16) of activation values (Figure 1B). Although both reporters are driven by the Gal1 promoter, the lacZ reporter is actually present in two copies with two different promoters (a GAL7 promoter in PJ69-4a and a GAL1 promoter in YULH). We have not tried to determine the contribution of each of the two lacZ genes and restricted the following analysis to the His3 data. In summary, a total of 72 weak, 179 medium and 75 strong transcriptional activators were identiﬁed in these experiments (Table 2 and Supplementary Table 1). Function First, we wondered if Y2H activators also act in vivo as tran- scriptional activators. Surprisingly, only 92 (or 20%) of the 451 activators had the GO attribute ‘transcription regulator activity’ (GO 0030528). Transcription activity was signiﬁc- antly correlated with activation strength: 27% of all strong activators are annotated as having ‘transcription regulatory activity’ (Figure 2A) whereas this annotation drops to 21 and 7% of the medium and weak activators, respectively. These numbers are signiﬁcantly higher than for the Y2H non-activators ( 3%). As an additional reference set we selected 138 proteins annotated to possess ‘transcriptional activator activity’ in the YPD database. Only 55 (or 12%) of the Y2H activators were annotated as transcriptional activ- ators in YPD. This annotation correlated with the activational strength: 19% of strong, 16% of medium and 4% of weak activators were annotated with this term (Supplementary Table 1). This suggests that many more proteins may act as transcriptional activators than currently known. Localization Transcription takes place in the nucleus. We wondered if Y2H activators were also localized in the nucleus and analyzed their localization using the large-scale localization study by Huh et al. (33). While 22% of the Y2H non-activators were localized to the nucleus, 41% of the activators were nuclear (Figure 2B). The activation strength was also correlated with nuclear localization: e.g. the percentage of nuclear localization grew from 35% for weak activators to almost 50% in strong activators. However, the percentage of strong activators in the nucleus was still lower than the value of 67% found for known transcriptional activators (GO annotation ‘transcription regulator activity’). Correlating GO function and localization revealed a sig- niﬁcant overrepresentation of known TRs among nuclear activators and among nuclear-cytoplasmic proteins, but not in the set of exclusively cytoplasmic proteins (Supplementary Figure 1). Strikingly, 47% of nuclear Y2H activators are known TRs. This number indicates that a large fraction of these activators may act as transcription factors in vivo although this activity has not been recognized previously. As a large fraction of activator proteins have no assigned func- tion ( 35%) this ﬁnding can contribute to the functional annotation of these proteins (see below) (Table 3). Abundance It has been known for a long time that transcriptional activ- ators are often expressed at low levels. This notion is also reﬂected in the lower concentration of known transcription regulators (Figure 2C). With an average copy number of 4000 proteins per cell the Y2H activators conﬁrmed this ﬁnding and even showed a trend towards lower abundance the stronger the activation was (Figure 2C). However, this trend was not signiﬁcant. General physicochemical properties of Y2H activators Although characteristics such as abundance and localization allow us to classify activators, they do not explain their behavior. In fact, it has been unclear which properties turn a protein into a transcriptional activator even though certain