GENETIC-CHARACTERIZATION OF A MAMMALIAN PROTEIN-PROTEIN INTERACTION DOMAIN BY USING A YEAST REVERSE 2-HYBRID SYSTEM

Many biological processes rely upon protein-protein interactions, Henc e, detailed analysis of these interactions is critical for their under standing. Due to the complexities involved, genetic approaches are oft en needed, In yeast and phage, genetic characterizations of protein co mplexes are possible. However, in multicellular organisms, such charac terizations are limited by the lack of powerful selection systems, Her ein we describe genetic selections that allow single amino acid change s that disrupt protein-protein interactions to be selected from large libraries of randomly generated mutant alleles, The strategy, based on a yeast reverse two-hybrid system, involves a first-step negative sel ection for mutations that affect interaction, followed by a second-ste p positive selection for a subset of these mutations that maintain exp ression of full-length protein (two-step selection). We have selected such mutations in the transcription factor E2F1 that affect its abilit y to heterodimerize with DP1. The mutations obtained identified a puta tive helix in the marked box, a region conserved among E2F family memb ers, as an important determinant for interaction. This two-step select ion procedure can be used to characterize any interaction domain that can be tested in the two-hybrid system.