Isolation and Functional Characterization of the TRAP/Mediator Complex

This chapter describes the methods developed for isolating functionally active preparations of the TRAP/Mediator complex and for testing them in cell-free assay systems. The TRAP/Mediator complex was initially isolated from HeLa cells stably expressing FLAG-tagged thyroid hormone receptor (TRα). The propagation of this cell-line in the presence of the cognate ligand (thyroid hormone), but not in its absence, allowed the isolation (by subsequent affinity chromatography utilizing the FLAG tag) of a multiprotein complex (TRAP) associated with TRα. It is easier to isolate the human TRAP/Mediator by affinity purification from extracts of HeLa cells that stably express a FLAG-tagged version of an integral TRAP/Mediator subunit. The experimental systems presented in the chapter provide important insights into some of the mechanisms by which nuclear receptors activate their target genes. They establish the dominant role of the TRAP/Mediator coactivator, especially as it pertains to transcription from DNA templates. As the emphasis in the transcription field shifts to the study of distinct genes, it is likely that more natural templates–that is, chromatin templates carrying their physiological arrangements of regulatory elements (e.g., promoters andenhancers) will increasingly be used in in vitro experiments. The availability of pure TRAP/Mediator, together with the assays utilizing purified factors (to which new cofactors can be added) and with unfractionated extracts (from which cofactors can be selectively removed and resupplied), should provide a foundation for this next phase of investigation.