A modified yeast three-hybrid system enabling both positive and negative selections
Objective: To increase the reporter repertoire of the yeast three-hybrid system and introduce the option of negative selection. Results: Two new versions of the yeast three-hybrid system were made by modifying the MS2 coat RNA-binding protein and fusing it to the Gal4 DNA-binding protein. This allow...
| Main Authors: | , , |
|---|---|
| Format: | Journal Article |
| Language: | English |
| Published: |
SPRINGER
2018
|
| Subjects: | |
| Online Access: | http://purl.org/au-research/grants/arc/DP180101656 http://hdl.handle.net/20.500.11937/79273 |
| _version_ | 1848764028278538240 |
|---|---|
| author | Wallis, C.P. Filipovska, A. Rackham, Oliver |
| author_facet | Wallis, C.P. Filipovska, A. Rackham, Oliver |
| author_sort | Wallis, C.P. |
| building | Curtin Institutional Repository |
| collection | Online Access |
| description | Objective: To increase the reporter repertoire of the yeast three-hybrid system and introduce the option of negative selection. Results: Two new versions of the yeast three-hybrid system were made by modifying the MS2 coat RNA-binding protein and fusing it to the Gal4 DNA-binding protein. This allows the use of Gal4 inducible reporters to measure RNA–protein interactions. We introduced two mutations, V29I and N55K into the tandem MS2 dimer and an 11 amino acid deletion to increase RNA–protein affinity and inhibit capsid formation. Introduction of these constructs into the yeast strains MaV204K and PJ69-2A (which contain more reporters than the conventional yeast three-hybrid strains L40-coat and YBZ-1) allows RNA–protein binding interactions with a wide range of affinities to be detected using histidine auxotrophy, and negative selection with 5-fluoroorotic acid. Conclusion: This yeast three-hybrid system has advantages over previous versions as demonstrated by the increased dynamic range of detectable binding interactions using yeast survival assays and colony forming assays with multiple reporters using known RNA–protein interactions. |
| first_indexed | 2025-11-14T11:12:50Z |
| format | Journal Article |
| id | curtin-20.500.11937-79273 |
| institution | Curtin University Malaysia |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T11:12:50Z |
| publishDate | 2018 |
| publisher | SPRINGER |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | curtin-20.500.11937-792732022-10-27T07:27:14Z A modified yeast three-hybrid system enabling both positive and negative selections Wallis, C.P. Filipovska, A. Rackham, Oliver Science & Technology Life Sciences & Biomedicine Biotechnology & Applied Microbiology Genetic selection RNA-binding protein RNA-protein interactions Yeast three-hybrid system RNA-PROTEIN INTERACTIONS BINDING SPECIFICITY 2-HYBRID AFFINITY Objective: To increase the reporter repertoire of the yeast three-hybrid system and introduce the option of negative selection. Results: Two new versions of the yeast three-hybrid system were made by modifying the MS2 coat RNA-binding protein and fusing it to the Gal4 DNA-binding protein. This allows the use of Gal4 inducible reporters to measure RNA–protein interactions. We introduced two mutations, V29I and N55K into the tandem MS2 dimer and an 11 amino acid deletion to increase RNA–protein affinity and inhibit capsid formation. Introduction of these constructs into the yeast strains MaV204K and PJ69-2A (which contain more reporters than the conventional yeast three-hybrid strains L40-coat and YBZ-1) allows RNA–protein binding interactions with a wide range of affinities to be detected using histidine auxotrophy, and negative selection with 5-fluoroorotic acid. Conclusion: This yeast three-hybrid system has advantages over previous versions as demonstrated by the increased dynamic range of detectable binding interactions using yeast survival assays and colony forming assays with multiple reporters using known RNA–protein interactions. 2018 Journal Article http://hdl.handle.net/20.500.11937/79273 10.1007/s10529-018-2567-7 English http://purl.org/au-research/grants/arc/DP180101656 SPRINGER fulltext |
| spellingShingle | Science & Technology Life Sciences & Biomedicine Biotechnology & Applied Microbiology Genetic selection RNA-binding protein RNA-protein interactions Yeast three-hybrid system RNA-PROTEIN INTERACTIONS BINDING SPECIFICITY 2-HYBRID AFFINITY Wallis, C.P. Filipovska, A. Rackham, Oliver A modified yeast three-hybrid system enabling both positive and negative selections |
| title | A modified yeast three-hybrid system enabling both positive and negative selections |
| title_full | A modified yeast three-hybrid system enabling both positive and negative selections |
| title_fullStr | A modified yeast three-hybrid system enabling both positive and negative selections |
| title_full_unstemmed | A modified yeast three-hybrid system enabling both positive and negative selections |
| title_short | A modified yeast three-hybrid system enabling both positive and negative selections |
| title_sort | modified yeast three-hybrid system enabling both positive and negative selections |
| topic | Science & Technology Life Sciences & Biomedicine Biotechnology & Applied Microbiology Genetic selection RNA-binding protein RNA-protein interactions Yeast three-hybrid system RNA-PROTEIN INTERACTIONS BINDING SPECIFICITY 2-HYBRID AFFINITY |
| url | http://purl.org/au-research/grants/arc/DP180101656 http://hdl.handle.net/20.500.11937/79273 |