My times today.The two-hybrid system is a yeast-based genetic assay for detecting protein-protein interactions. In this method, genes encoding two hybrid proteins are constructed: one hybrid consists of the DNA-binding domain of a transcriptional activator fused to some protein “X”; the other consists of the transcriptional activation domain fused to some protein “Y”. Interaction between proteins X and Y reconstitutes the function of the transcriptional activator and leads to transcription of a reporter gene containing sites for the DNA-binding domain. This system has been used successfully to analyze interactions of nuclear, cytoplasmic, mitochondrial, and membrane-associated proteins from a variety of organisms. Applications of this system include the following: (i) testing for interaction between known proteins whose genes are available; (ii) delineating the domains of two proteins involved in an interaction; (iii) identifying specific residues of a protein important for interaction with another protein; and (iv) screening libraries of activation domain hybrids to identify proteins that bind to some protein of interest.
We describe genetic methods using yeast to analyze a DNA-binding protein to determine (i) the sequence of the DNA sites to which the protein binds and (ii) the location of the domain and specific amino acid residues in the protein responsible for DNA binding. These methods take advantage of the fact that a hybrid protein consisting of a particular DNA-binding domain and a transcriptional activation domain activates expression of a reporter gene that contains binding sites for the DNA-binding domain. We describe two applications of these methods. First, DNA fragments that contain binding sites for the DNA-binding protein of interest can be recovered from a library of fragments by their ability to mediate transcriptional activation of a reporter gene. If enough DNA fragments are identified, the consensus sequence of the DNA-binding site can usually be recognized. In addition, some of the DNA fragments may be derived from actual target genes regulated by the DNA-binding protein, and therefore these fragments might be used to identify such target genes. Second, a reporter gene whose expression inhibits cell growth and whose promoter contains binding sites for the DNA-binding protein can be used to select mutants defective in the DNA-binding domain. This procedure allows one to localize the DNA-binding domain within the protein and to identify amino acids important for DNA binding. The mutations that inactivate the DNA-binding domain are highly informative, since the method avoids the recovery of “uninteresting” mutations that simply destabilize the protein or prevent its synthesis.
Mammalian nuclear receptor function can be faithfully reconstituted in yeast, enabling a wide variety of genetic approaches to be taken toward defining the mechanisms of signal transduction and transcriptional regulation. This report describes vectors for the expression of mammalian receptors in yeast, reporter genes, yeast host strains, and simple assays that monitor receptor transcriptional activity. Methods for the generation of receptors with distinct defects in particular functions, such as DNA or hormone binding, that couple random mutagenesis with phenotypic screens are outlined as well. In addition, strategies for the identification of nonreceptor components whose gene products may act on receptors are discussed. The experimental advantages of yeast invite a detailed genetic analysis of mammalian nuclear receptor functions-hormone and DNA binding, nuclear localization, and interaction with nonreceptor factors-and should illuminate further the mechanisms of signal transduction and transcriptional regulation by this important class of regulatory molecules.