Transcriptional activation is the basis of the Y2H system (26
). However, some bait proteins activate transcription without requiring an interacting protein that bears a separate AD. In fact, this property prevents the study of many TRs by means of the two-hybrid system. Nevertheless, we realized that this observation can extend our knowledge about the properties of transcriptional ADs.
In the course of this study we took previously identified (but mostly unpublished) activators from several Y2H screens (26
) and measured their activation strength. In addition, we looked for properties distinguishing this set from non-activator proteins. Although many well-characterized TRs and nuclear proteins are highly overrepresented in the set of Y2H activators, we identified many which have not been associated with transcription previously. Irrespective of their physiological role, these activators must be able to interact with and recruit the transcriptional machinery.
Which features or sequences mediate the recruitment of the general transcriptional machinery to the activator? Although there is no single feature several rather general properties of transcription activators have been defined previously. These included the protein sequence composition as in acidic activators (25
) or more specific features such as defined interactions between activators and the basal transcription machinery [e.g. (21
Acidic activators, i.e. activators with stretches of acidic amino acids, were the first class to be identified in yeast and the ones studied most extensively (10
). Their importance was emphasized by screens for random activating fragments of the E.coli
genome or activating peptides, both of which mainly identified acidic activators (56
). The finding was confirmed in this study by the lower isoelectric point of activators (), more acidic stretches () and by the increased clustering of aspartate ().
Other previously defined activator classes were also mirrored by this study: glutamine-rich activators (, Supplementary Figure 2) (11
), proline-rich activators () (12
) and serine-rich activators () (57
). Elevated asparagine levels and cluster values ( and Supplementary Figure 2) of activators may indicate a role of an additional amino acid in transcription activation, possibly similar to the closely related amino-acid glutamine in glutamine-rich activators.
It is generally accepted that ADs mediate their function by specific interactions with the basal transcription machinery (5
) or additional factors involved in transcription, like chromosome remodeling complexes (58
). Transcription activators analyzed in this study form a highly interconnected protein interaction network involving transcription related proteins (). Protein interactions with transcription-related proteins are highly overrepresented, even in the set of activators which have not been described as TRs ().
A significant fraction of nuclear Y2H activators as well as known TRs interact with the basal transcription machinery (like RNA polymerase II) and chromosome remodeling complexes (like the SAGA complex) (Supplementary Table 4). These interactions possibly explain their activation properties. Strikingly, the top ranking interaction partner of nuclear activators not known to regulate transcription was also RNA polymerase II. This indicates that genuine transcription regulators are contained in this set and form the basis for annotating them as TRs (see Results). However, for a large fraction of the Y2H activators no interactions are known and thus their mode of action remains unclear.
A surprising finding is the frequent interaction of activators with alcohol dehydrogenase. This enzyme is found with ~10% of nuclear proteins (data not shown) but with ~20% in both the nuclear activator and known transcription regulator dataset. Thus, alcohol dehydrogenase might be more a specific component of activation complexes than an unspecific contamination. Such moonlighting functions (59
) have been found in other components of various transcription factor complexes but also among other proteins such as actin which acts both as a cytoskeletal protein and a transcription factor (60
Screening of protein fragments fused to the DBD of Gal4 has been used before to identify transcriptional activators. Ruden et al
) screened fragments of the E.coli
genome and mainly identified acidic stretches that activate transcription in yeast. Wiesner et al
) established a screening system based on a GFP reporter gene in a murine cell line and identified human transcription factors using a cDNA library. In contrast to this study we could make use of extensive large-scale studies which not only identified a set of transcription activators (26
), but also localized proteins (33
), measured protein concentrations (34
) and protein interactions (26
). The measurement of the activation strength let us focus on sets of proteins highly enriched in known TRs. Combining several data sources, for six uncharacterized yeast proteins we could identify additional evidence for their genuine role in transcriptional regulation.
We are aware that we may have missed a number of activators: of 138 proteins annotated with the GO-term ‘transcriptional activator activity’ in the YPD database 55 (40%) were detected as activators in this study (Supplementary Table 1), suggesting a false negative rate of ~60%. This discrepancy may be explained by the fact that some TRs are annotated as activators although they do not act as such in our assay. Other proteins may require specific promoters, cofactors or conditions to exhibit activating activity. For example, several activators are only active when yeast is grown in glucose-free media. Interestingly, Gal4 itself becomes a very weak activator when fused to another Gal4–DBD, i.e. the DBD appears to inhibit the activating properties of some proteins. Thus, our studies need to be extended in order to get a more complete picture of all transcriptional activators in yeast, e.g. by using different conditions or fusing the DBD C-terminally.
Although we have identified a large number of transcriptional activators in yeast and semi-quantitatively measured their activation strength, we have not measured activation in a truly quantitative way. New reporter strains with luciferase or some other enzyme need to be used in the future for more precise measurements.
In addition, we have not determined the actual ADs. Given the poor definition of ADs, it remains an important challenge to map these domains by fragmenting the proteins described here. As ADs do not appear to be well-defined structural domains it appears to be likely that they do not require a defined 3D structure. Instead, short linear peptides may contain the activation activity proper.
Once the ADs have been mapped, their interactions with the transcriptional machinery have to be identified. Unfortunately, the classical two-hybrid system cannot be used for this purpose. Thus either classical biochemistry or alternative systems have to be used such as the split-Ubiquitin system (61
Finally, activators can differ in their ability to activate transcription dependent on the promoters to which they are bound (62
). Hence all activators need to be tested in different promoter contexts and their transcriptional activity quantified.
Once their interactions have been mapped on a proteome-wide scale and their promoter context evaluated, it may be possible to generalize these findings and predict the transcriptional activity of every protein, including their activation strength. This would be a major step towards quantitative and thus systems biology.