Colony color assay coupled with 5FOA negative selection greatly improves yeast three-hybrid library screening efficiency

The recently developed yeast three-hybrid system is a powerful tool for ana lyzing RNA-protein interactions in vivo. However, large numbers of false po sitives are frequently met due to bait RNA-independent activation of the re porter gene in the library screening using this system. In this report, we coupled the colony color assay with the 5FOA (5-fluoroorotic acid) negative selection in the library screening, and found that this coupled method eff ectively eliminated bait RNA-independent false positives and hence greatly improved library screening efficiency. We used this method successfully in isolation of cDNA of an RNA-binding protein that might play important roles in certain cellular process. This improvement will facilitate the use of t he yeast three-hybrid system in analyzing RNA-protein interaction.