title: Isolation of compensatory inhibitor domain mutants to novel activation domain variants using the split-ubiquitin screen.
abstract: The control of transcription factor function plays an important role in the development of many processes in eukaryotes, such as drug resistance in fungi and human tumours undergoing chemotherapy. Detailed molecular mapping of the interactions between transcription factors and their protein partners can give important information about their mechanisms of action and reveal potential therapeutic targets. We devised a genetic screening system for mapping the interaction site between the Saccharomyces cerevisiae transcription factor-inhibitor pair Gal4p and Gal80p. A novel Gal4p activation domain mutant, L868K, was produced, which prevented it interacting with Gal80p. The split-ubiquitin system was used with a mutant GAL80 library in order to screen for compensatory mutants in Gal80p which would restore binding with L868K. Five single amino acid residue compensatory mutations in Gal80p which restored the interaction with Gal4p(L868K) were isolated. These compensatory mutations were specific to L868K as they were unable to restore the interaction with two other Gal4p mutants that were incapable of interacting with Gal80p. Mutations within Gal80p that were capable of compensating for Gal4p (L868K) clustered inside a Gal80p surface cleft, supporting the idea that this area is important for Gal4p binding. Our data suggest a way to generate information about interaction sites that should be applicable to any transcription factor.
authors: Faculty of Life Sciences, University of Manchester, UK.
authors: Robert N
authors: RN
authors: Campbell
authors: Frederick
authors: F
authors: Westhorpe
authors: Richard J
authors: RJ
authors: Reece
mesh_terms: D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000379:methods*; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; C055002:GAL80 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D025801:Ubiquitin