Two-Hybrid Analysis of RasRaf Interactions

The yeast two-hybrid system ( 1 ) is a genetic method that enables the experimentor to determine whether two proteins can form complexes within yeast cells. The method comprises expressing the proteins of interest as “hybrid” proteins, one fused to a DNA-binding domain and the other protein fused to a transcription-activating domain. If the fusion proteins interact, a reporter gene IS transcribed ( see Fig. 1 ).   Fig. 1.  The two-hybrid system. ( A ) The hybrid of the DNA-binding domain (BD) and protein X does not activate transcription if protein X does not contain an activation domain. ( B ) The hybrid of the activation domain (AD) and protein Y does not activate transcription because it does not localize to the DNA-binding site. ( C ) Interaction between proteins X and Y brings the activation domain into close proximity to the DNA-binding site and results in transcription of the reporter genes: LacZ and HIS3 .