The multi-protein Mediator complex, conserved throughout eukaryotic organisms, is critical for in vivo
assembly and stabilization of the preinitiation complex and conveys regulatory signals to the basal transcription machinery (Kornberg, 2005
; Malik and Roeder, 2000
; Naar et al., 2001
). Nearly 20 years after Mediator was first identified in the yeast Saccharomyces cerevisiae
(Flanagan et al., 1991
; Kelleher et al., 1990
) and despite its paramount importance in transcription, structural understanding of the complex is limited. A low resolution structure of yeast Mediator calculated from electron microscope (EM) images of Mediator particles preserved in stain (Dotson et al., 2000
) showed few features and revealed only the overall morphology of the complex. Nonetheless, comparison with the structure of the Mediator-RNA polymerase II (RNAPII) holoenzyme (Davis et al., 2002
) revealed large-scale changes in Mediator structure related to interaction of the complex with polymerase and brought up the question of how structural changes might contribute to the regulation mechanism (Chadick and Asturias, 2005
EM studies of human Mediator also detected changes in its structure resulting from interaction with activators (Taatjes et al., 2002
) and nuclear receptors (Taatjes et al., 2004
), again suggesting that structural rearrangements might be important for Mediator function. However, the low resolution of the Mediator reconstructions and the absence of structural information about the metazoan Mediator-RNAPII holoenzyme have prevented the detailed comparison of the Mediator and holoenzyme structures necessary to attain a better understanding of critical issues such as the significance of conformational changes in Mediator triggered by interaction with RNAPII and other components of the basal transcription machinery and the extent of conservation of structural and mechanistic properties of Mediator.
Statistical analysis of single Mediator particle images preserved in stain can result in a detailed description of conformational changes in the complex and pave the way for cryo-EM analysis in which single particle images are instantaneously frozen from physiologically relevant conditions and analyzed in the absence of staining or any constraints related to crystallization. Unfortunately, such studies have been hindered by lack of a robust purification protocol capable of producing sufficient amounts of soluble, homogeneous, and transcriptionally-active Mediator. We report here on a new affinity purification method that readily and reproducibly yields functional yeast Mediator. This has made possible conformational analysis of Mediator particles preserved in stain and design of a strategy to pursue refinement of a cryo-EM reconstruction of Mediator that shows its structure in detail. Comparison with a previous structure of the RNA polymerase II (RNAPII)-associated form of yeast Mediator and with the structure of human Mediator results in a precise identification of Mediator structural modules, a description of the motions these modules undergo to accommodate interaction with RNAPII, and demonstrates that the structure and rearrangement of Mediator that enables interaction with polymerase are conserved from yeast to humans.