title: Transient-state kinetic analysis of transcriptional activator·DNA complexes interacting with a key coactivator.
abstract: 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.
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Amberlyn M
authors.initials: AM
authors.lastname: Wands
authors.forename: Ningkun
authors.initials: N
authors.lastname: Wang
authors.forename: Jenifer K
authors.initials: JK
authors.lastname: Lum
authors.forename: John
authors.initials: J
authors.lastname: Hsieh
authors.forename: Carol A
authors.initials: CA
authors.lastname: Fierke
authors.forename: Anna K
authors.initials: AK
authors.lastname: Mapp
mesh_terms: D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism; D015533:Transcriptional Activation / Q000502:physiology*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D004247:DNA