RNA polymerase II holoenzyme ͉ transcriptional regulation ͉ TRAP ͉ electron microscopy A Mediator complex with Ϸ20 polypeptide components is involved in regulation of RNA polymerase II transcription in the yeast Saccharomyces cerevisiae (1, 2). A number of complexes containing subunits homologous to those of the yeast Mediator have been identified in higher organisms. These related complexes include a murine Mediator (3), human thyroid hormone receptor-associated protein (TRAP) complex, a coactivator associated with the thyroid hormone nuclear receptor, and other human complexes (4-9). Mediator and related complexes interact with RNA polymerase II to form holoenzymes and confer on the polymerase both enhanced activity in basal transcription and responsiveness to transcriptional activators. Evidence of direct Mediator-activator interaction has come from the physical isolation of TRAP as a complex with thyroid hormone receptor from hormone-induced but not uninduced cells. Other human Mediators have been isolated as complexes with activators or isolated by activator-affinity chromatography.Initial structural analysis of yeast, murine Mediators, and holoenzymes was performed by averaging a small number (Ͻ100) of electron-microscope images of the complexes viewed in a single orientation in projection. Despite rather limited sequence homology, the yeast and murine complexes appeared remarkably alike in size, shape, and conformational changes associated with Mediator-polymerase interaction (10). Here we present three-dimensional (3-D) structures of yeast and murine Mediators and of human TRAP complex, obtained by averaging hundreds of electron-microscope images from views in random orientations. Beyond confirming the overall similarity of yeast and murine Mediators and extending this result to TRAP, the structures reveal details of surface and internal organization, disclosing further similarities and also notable differences. Materials and MethodsSample Preparation and Data Collection. Yeast Mediator purified from commercial yeast as described (11), mouse Mediator purified as described (3), and human TRAP complex immunopurified from HeLa cells as described (12) were diluted to a concentration of 15-25 g͞ml and applied to specimen grids covered with a thin amorphous carbon film. Because of their relatively low abundance, the amounts of murine Mediator and of the human TRAP complex available for our structural studies were not sufficient for the preparation of unstained specimens; thus, individual particles were imaged in negative stain. The carbon film substrate was glow discharged before preparation of the samples to facilitate adhesion of the molecules and aid staining by making the film surface more hydrophilic. The particles were negatively stained by using a 1% solution of uranyl acetate. After staining, a second, prestained carbon layer was placed over the specimen to ensure complete coverage of the particles by the stain. Samples were examined by using a Philips CM120 electron microscope, equipped with a LaB 6 f...