Fig. 4:

Principle of the reverse two-hybrid system. (A) the "split-hybrid" system is based on the E. coli TN10-encoded tet-repressor (TetR)/operator system: interaction of the hybrid proteins X and Y activates the transcription of a TetR; the TetR protein then represses HIS3 expression, resulting in a no-growth phenotype on plates without histidine in the growth medium (53). Abrogation of the interaction, either by mutating Y or by introducing a dissociator (D), shuts down TetR expression, leads to HIS3 expression and results in growth on selective plates. (B) The reverse two-hybrid system takes advantage of counterselectable yeast reporter genes (51, 54-55). URA3 expression sensitizes yeast cells to 5-fluoroorotic acid (5-Foa), CYH2 expression to cycloheximide. Abrogation of the interaction enables the cells to grow in the presence of these drugs.