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Published on: August 27, 2021
Isolation of compensatory inhibitor domain mutants to novel activation domain variants using the split-ubiquitin
Robert N Campbell1, Frederick Westhorpe, Richard J Reece
1Faculty of Life Sciences, University of Manchester, UK.
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.
Insights
Researchers developed a genetic screening system to map interactions between transcription factors and inhibitors. This method identified specific mutations in Gal80p that restore binding to a Gal4p mutant, revealing key interaction sites.
Area of Science:
- Molecular biology
- Genetics
- Biochemistry
Background:
- Transcription factor (TF) regulation is crucial in eukaryotic processes like drug resistance.
- Understanding TF-protein interactions is key to elucidating mechanisms and identifying therapeutic targets.
Purpose of the Study:
- To develop a genetic screening system for mapping TF-inhibitor interaction sites.
- To identify compensatory mutations in the Gal80p inhibitor that restore binding to a specific Gal4p mutant.
Main Methods:
- Utilized a split-ubiquitin system with a mutant GAL80 library.
- Generated a novel Gal4p activation domain mutant (L868K) that lost Gal80p interaction.
- Screened for compensatory Gal80p mutations restoring Gal4p(L868K) binding.
Main Results:
- Isolated five single amino acid compensatory mutations in Gal80p that restored interaction with Gal4p(L868K).
- Demonstrated specificity of these mutations, as they did not restore interaction with other Gal4p mutants.
- Identified that compensating mutations clustered in a Gal80p surface cleft, indicating its importance for Gal4p binding.
Conclusions:
- The developed genetic screening system effectively maps TF-inhibitor interaction sites.
- The findings provide insights into the Gal4p-Gal80p interaction interface.
- This approach is potentially applicable to mapping interactions for any transcription factor.
