Differential Sensitivity to 5-fluoro-orotic Acid as a Screen for Bait RNA Independent False Positives in a Yeast Three-Hybrid System

Abstract

The yeast three-hybrid system presents a valuable tool for detecting and analyzing RNA-protein interactions in vivo. A major drawback of the use of such a transcriptional reporter-based assay in a library screen is the frequent occurrence of false-positive results due to bait RNA-independent activation of the reporter gene. To minimize the isolation of false positives in three-hybrid library screens, we incorporated a rapid and simple procedure based on differential sensitivity to 5-fluro-orotic acid. The technique effectively eliminates bait RNA-independent false positives and thus greatly enhances the efficiency of the yeast three hybrid system.