Two-hybrid selection assay to identify proteins interacting with polymerase II transcription factors and regulators.
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.
University of Zürich, Switzerland.
M
M
Petrascheck
F
F
Castagna
A
A
Barberis
D000818:Animals; D017930:Genes, Reporter; D051379:Mice; D011506:Proteins / Q000378:metabolism*; D012320:RNA Polymerase III / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000235:genetics*; D035561:Transcription Factor TFIIA; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000378:metabolism*; D019473:Transcription Factors, TFII / Q000378:metabolism*; D015003:Yeasts / Q000235:genetics
D011506:Proteins; D012342:RNA, Small Nuclear; D035561:Transcription Factor TFIIA; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII; C405486:U6 small nuclear RNA; D012320:RNA Polymerase III