Design of a single plasmid-based modified yeast one-hybrid system for investigation of in vivo protein-protein and protein-DNA interactions.

We have developed a modified yeast one-hybrid system (MY1H) useful for in vivo investigation of protein-protein and protein-DNA interactions. Our single-plasmid expression system is capable of differential protein expression levels; in addition to a GAL4 activation domain (AD) fusion protein, a second protein can be coexpressed at either comparable or higher transcriptional levels from expression vectors pCETT or pCETF, respectively. This second protein can play a structural, modifying, or inhibitory role that restores or blocks reporter gene expression. Our MY1H was validated by use of the well-characterized DNA-binding protein p53 and its inhibitory partners, large T antigen (LTAg) and 53BP2. By coexpressing LTAg or 53BP2 at comparable or higher levels than the GAL4AD-p53 fusion in the MY1H, we show that DNA binding of p53 decreases by different, measurable extents dependent on the expression level of inhibitory partner. As with the traditional Y1H, our system could also be used to investigate proteins that provide coactivational or bridging functions and to identify novel protein- or DNA-binding partners through library screening. Our MY1H provides a system for investigation of simultaneous protein-protein and protein-DNA interactions, and thus is a useful addition to current methods for in vivo investigation of such interactions.