960 Nucleic Acids Research, 2006, Vol. 34, No. 3 Figure 2. Function, localization and abundance of yeast activators. (A) Percentage of proteins annotated to have ‘TR activity’ (GO term, see also Supplementary Table 1). (B) Percentage of protein localized to the nucleus as analyzed by Huh et al. (33). (C) Abundance of proteins as analyzed by Ghaemmaghami et al. (34). physicochemical properties such as acidic stretches have been identiﬁed (10). We therefore revisited the inﬂuence of physicochemical properties on the propensity of a protein to activate transcription. Well-deﬁned general properties were analyzed ﬁrst and included the isoelectric point (Figure 3A), molecular mass (Figure 3B), overall hydrophobicity (GRAVY score) (Figure 3C) and aromaticity (Figure 3D). The most pro- nounced effect was found for the isoelectic point: the mean pI for activators was found to be 1.5 pH units below the value for non-activators. This effect was also observed for activators in the nucleus, although with 0.5 pH units the difference between nuclear localized activators and nuclear proteins in general was lower. Interestingly, known TRs had only a slightly reduced mean pI, which was even higher than for nuclear proteins in general. However, proteins annotated as ‘TRs’ may include non-activating regulators too. The inﬂu- ence of activation strength onto the mean pI was not signiﬁ- cant (judged by Student’s t-test). The mean molecular weight of activators was found to be 9 kDa higher than for non-activators. This tendency is also reﬂected by the higher mean molecular weight of known TRs. Activators in the nucleus were not signiﬁcantly larger than nuclear proteins in general but we found that the mean molecular weight increased with activation strength (Figure 3B). The overall hydrophobicity (GRAVY score) and the aromaticity showed a similar pattern. Both properties were reduced in activators as well as in known TRs. The effect was much smaller for the GRAVY score but highly signiﬁcant for nuclear localized proteins and activators (Students’t t-test P ¼ 2.4e 5). In addition, the CAI was analyzed (Figure 3E). As lower codon scores are associated with lower protein expression levels (34), the lower CAI for activators as well as for known TRs supports the previous ﬁnding of lower protein levels for these two protein sets (Figure 2C). As expected, known TRs generally showed similar properties as the Y2H activators except for the isoelectric point. Amino acid clusters It has been known for a long time that certain amino acids are overrepresented in transcriptional activators, in particular acidic and basic residues and certain other amino acids such as glutamine. We wondered if this is true for our activator set as well. As basic amino acid clusters are also acting as nuclear localization signals, we only took activators into account that are known to be localized to the nucleus and compared them with the remaining nuclear proteins. After correction for multiple testing using Holm’s procedure (41) 15 parameters involving certain amino acids remained highly signiﬁcant (Supplementary Table 2). These included the minimum pI in a 20 amino acid window, the overall percentage of several amino acids (Ala, Gly, Ser, Val, Lys, Asn, Gln) and amino acid clusters (Ser, Asp, Pro, Asn, Gln) and, in addition, the amino acids Lys and Arg and the net charge (amino acids [Lys + Arg] [Glu + Asp] ¼ KR ED). A detailed analysis of the amino acid clusters and the minimum pI is shown in Figure 4. A detailed analysis of the remaining properties is shown in Supplementary Figure 2. Taken together, the analysis of physicochemical properties showed that Y2H activators tend to possess a lower isoelectric point, have a lower hydrophobicity, higher molecular weight,