1. INTRODUCTION Recent studies have brought up the existence of several molecular complexes either stable, like the RNA polymerase II holoenzyme, transcription factor TFIID and mediators, or transient as could be observed in the various steps of the transcription process e.g. initiation, elongation and termination. These observations point out the fact that regulation of different cellular mechanisms is orchestrated by interactions between molecular species either to modify proteins or to position one of them within a complex that then will be functional. Several techniques such as affinity precipitation, glycerol gradient sedimentation and the yeast two-hybrid system are currently used to study protein-protein interactions (1). These various methods, usually investigating the connection between two partners, keep in account only the strong (and stable) interactions and neglect the weaker ones. Considering the complexes studied so far, it is suspected and sometimes shown that interactions often occur between more than two proteins, e.g. to stabilize the complex. In an effort to understand the various biological mechanisms-and in our case gene expression regulation-, we were interested in developing the yeast three-(or tri-)hybrid system. The three-hybrid system, as illustrated in figure 1, is based on the reconstitution of a transcriptional activator complex either to search for or to study a protein that interacts with two others and to acquire information about ternary complex assembly (2). This technique detects direct or mediated interactions between two fusion proteins that contain either a DNA binding domain (DBD; the DBD-X protein) or an activation domain (AD; the AD-Y protein). In some cases, when these two hybrid proteins interact weakly or not at all, a third partner (the protein Z) is necessary to promote (to induce) the formation of the transcriptional activator allowing the transcription of the reporter genes. Thus, specific and stable protein-protein interactions between X, Y and Z lead to the activation of the reporter genes that are integrated in the yeast genome. The HIS3 reporter gene contains a specific DNA sequence which can be recognized by the DBD of the transcriptional activator. Activation of the HIS3 gene permits the endogenous synthesis of histidine allowing the yeast to grow on histidine-lacking media. Activation of the LacZ gene, another reporter gene containing the same DNA binding element, will lead to the synthesis of the β-galactosidase (β-Gal) that catalyses the transformation of either X-Gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside) or ONPG (o-nitrophenyl-β-D-galactopyranoside) into a detectable blue or yellow product, respectively. There are several options to reconstitute the transcriptional activator: (i) the third partner Z can act as a bridging factor (Fig.1A) by interacting with both the DBD-X and AD-Y, thus allowing the DNA-binding protein X to target the basal transcription machinery; (ii) In case of weak interactions, Z may function as a stabilising factor (Fig.1B) that strengthens the interaction between X and Y; (iii) In some other cases, reconstitution of the transcriptional activator requires some post-transcriptional modifications of one of the two partners, in order to allow interaction between X and Y. The third partner Z will then be a regulating factor (Fig.1C), an enzyme that will not necessarily be part of the reconstituted transcriptional activator. When DBD-X and AD-Y are sufficient to reconstitute a stable transcriptional activator, the three-hybrid system can also be used for the search of inhibitors. In this case, the Z partner can act as an inhibitor (Fig.1D) by interacting with, or enzymatically modifying, one of the two main partners, thus preventing their interactions. This will result in the inhibition of the expression of the reporter genes followed by growth restriction on histidine-lacking media as well as repression of the β-Gal activity.