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The RNA polymerase III-based two-hybrid system has been developed to detect interactions between proteins such as RNA polymerase II transcription factors and regulators that cannot be studied by the original RNA polymerase II two-hybrid system. This novel method appears to be most useful for a refined analysis of already known protein-protein interactions. However, the application of this system in library screenings has been impaired by the lack of a suitable assay for the selection of the activated pol III reporter gene in yeast. Here, we describe a novel selection assay for the pol III-based two-hybrid system that makes it readily usable for screening expression libraries to search for interacting partners. Our system utilizes a temperature-sensitive (ts) U6 snRNA, which is synthesized by RNA polymerase III from a mutated SNR6 gene in yeast. In this ts strain, interactions between hybrid proteins activate an artificial pol III reporter construct (UASG-SNR6), which controls expression of wild-type U6 snRNA. This wild-type U6 snRNA can suppress the ts phenotype and allow growth at the nonpermissive temperature of 37 degrees C, thus providing a positive selection system for interacting proteins.

Two-Hybrid Selection Assay to Identify Proteins Interacting with Polymerase II Transcription Factors and Regulators