A conditionally expressed third partner stabilizes or prevents the formation of a transcriptional activator in a three-hybrid system.
We describe a three-hybrid system that involves three polypeptides that allow or prevent the formation of the transcriptional activator. Beside the two-hybrid fusion proteins, the third partner is under the control of the Met25 promoter, which is positively regulated in medium lacking methionine. We document a situation where such a third partner promotes interaction between two proteins, one fused to a DNA-binding domain and the other fused to an activator domain. This is demonstrated for cdk7-MAT1 interaction stabilized by the presence of cyclin H; these three polypeptides are found either free or associated with the transcription/DNA repair factor TFIIH. We also document the capacity of our system to conditionally inhibit the interaction between two polypeptides that otherwise elicit a positive two-hybrid response. This is demonstrated for Ras-Raf interaction precluded by an excess of Raf. The presence of a methionine-regulated promoter provides an "on" or "off" switch for the formation of the transcriptional activator, thus also providing an excellent control to evaluate the activation or inhibition properties of the third partner.
Tirode
F
F
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM, 1 rue Laurent Fries, BP 163, 67404 Illkirch Cédex, Communauté Urbaine de Strasbourg, France.
Malaguti
C
C
Romero
F
F
Attar
R
R
Camonis
J
J
Egly
J M
JM
D016477:Artifacts; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D056748:Cyclin H; D018844:Cyclin-Dependent Kinases; D016213:Cyclins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D019473:Transcription Factors, TFII; D014170:Transformation, Genetic; D015003:Yeasts / Q000235:genetics; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D056748:Cyclin H; D016213:Cyclins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D051758:Transcription Factor TFIIH; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D018844:Cyclin-Dependent Kinases; D018631:ras Proteins; D000095043:Cyclin-Dependent Kinase-Activating Kinase