A CONDITIONALLY EXPRESSED 3RD PARTNER STABILIZES OR PREVENTS THE FORMATION OF A TRANSCRIPTIONAL ACTIVATOR IN A 3-HYBRID SYSTEM

We describe a three-hybrid system that involves three polypeptides tha t allow or prevent the formation of the transcriptional activator. Bes ide the two-hybrid fusion proteins, the third partner is under the con trol of the Met25 promoter, which is positively regulated in medium la cking methionine. We document a situation where such a third partner p romotes interaction between two proteins, one fused to a DNA-binding d omain and the other fused to an activator domain. This is demonstrated for cdk7-MAT1 interaction stabilized by the presence of cyclin H; the se three polypeptides are found either free or associated with the tra nscription/DNA repair factor TFIIH. We also document the capacity of o ur system to conditionally inhibit the interaction between two polypep tides that otherwise elicit a positive two-hybrid response. This is de monstrated for Ras-Raf interaction precluded by an excess of Raf. The presence of a methionine-regulated promoter provides an ''on'' or ''of f'' switch for the formation of the transcriptional activator, thus al so providing an excellent control to evaluate the activation or inhibi tion properties of the third partner.