Several lines of evidence suggest that the prototypical amphipathic transcriptional activators Gal4, Gcn4, and VP16 interact with the key coactivator Med15 (Gal11) during transcription initiation despite little sequence homology. Recent cross-linking data further reveal that at least two of the activators utilize the same binding surface within Med15 for transcriptional activation. To determine whether these three activators use a shared binding mechanism for Med15 recruitment, we characterized the thermodynamics and kinetics of Med15·activator·DNA complex formation by fluorescence titration and stopped-flow techniques. Combination of each activator·DNA complex with Med15 produced biphasic time courses. This is consistent with a minimum two-step binding mechanism composed of a bimolecular association step limited by diffusion, followed by a conformational change in the Med15·activator·DNA complex. Furthermore, the equilibrium constant for the conformational change (K(2)) correlates with the ability of an activator to stimulate transcription. VP16, the most potent of the activators, has the largest K(2) value, whereas Gcn4, the least potent, has the smallest value. This correlation is consistent with a model in which transcriptional activation is regulated at least in part by the rearrangement of the Med15·activator·DNA ternary complex. These results are the first detailed kinetic characterization of the transcriptional activation machinery and provide a framework for the future design of potent transcriptional activators.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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Evidence ID | Analyze ID | File | Description |
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