Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions.
Macromolecular interactions define many biological phenomena. Although genetic methods are available to identify novel protein-protein and DNA-protein interactions, no genetic system has thus far been described to identify molecules or mutations that dissociate known interactions. Herein, we describe genetic systems that detect such events in the yeast Saccharomyces cerevisiae. We have engineered yeast strains in which the interaction of two proteins expressed in the context of the two-hybrid system or the interaction between a DNA-binding protein and its binding site in the context of the one-hybrid system is deleterious to growth. Under these conditions, dissociation of the interaction provides a selective growth advantage, thereby facilitating detection. These methods referred to as the "reverse two-hybrid system" and "reverse one-hybrid system" facilitate the study of the structure-function relationships and regulation of protein-protein and DNA-protein interactions. They should also facilitate the selection of dissociator molecules that could be used as therapeutic agents.
Vidal
M
M
Massachusetts General Hospital Cancer Center, Charlestown 02129, USA. Vidal@Helix.MGH.Harvard.edu
Brachmann
R K
RK
Fattaey
A
A
Harlow
E
E
Boeke
J D
JD
D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D016384:Consensus Sequence; D017931:DNA Primers; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D020798:Two-Hybrid System Techniques
D017931:DNA Primers; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D011994:Recombinant Proteins; C097192:SPO13 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors