Figure 2

Structure of the Head + Middle modules and boundary of the Tail module. (A) Schematic view of obtaining the Tail-less core Mediator by deletion of the med16 gene. (B) SDS-PAGE analysis of the native Head + Middle modules. (C) A typical micrograph of the Head + Middle particles preserved under negative staining. Scale bar, 50 nm. (D) Representative 2D structures of the Head + Middle modules. The yellow and green arrows indicate the regions showing high degree of structural flexibility. Scale bar, 100 Å. (E) Different views of the 3D reconstructions of the Head + Middle modules in two different conformations. Scale bar, 100 Å. (F) Comparison of the structures of the core Mediator and the Head + Middle modules and difference mapping establishes that the dense domain at the base of the core Mediator corresponds to the Tail module. Contour plots calculated from 2D structures are color-coded to highlight the modular correspondence and the boundary of the Tail module. (G) Localization of the C terminus of the Med14 subunit revealed by Ni-NTA-Nanogold labeling. Single particles of nanogold-labeled core Mediator complex (middle), a corresponding class average (left), and a diagram representing the localization of the Med14 C terminus (right) are shown. (H) Fitting of the structure of the Head + Middle modules (semitransparent purple surface) into the core Mediator structure (semitransparent yellow mesh). Comparison of the structures of the core Mediator and the Head + Middle modules combined with the nanogold labeling of the Med14 subunit firmly establishes the boundary of the Tail module.