Structural organization of yeast and mammalian mediator complexes

Abstract

Structures of yeast Mediator complex, of a related complex from mouse cells and of thyroid hormone receptor-associated protein complex from human cells have been determined by three-dimensional reconstruction from electron micrographs of single particles. All three complexes show a division in two parts, a "head" domain and a combined "middle-tail" domain. The head domains of the three complexes appear most similar and interact most closely with RNA polymerase II. The middle-tail domains show the greatest structural divergence and, in the case of the tail domain, may not interact with polymerase at all. Consistent with this structural divergence, analysis of a yeast Mediator mutant localizes subunits that are not conserved between yeast and mammalian cells to the tail domain. Biochemically defined Rgr1 and Srb4 modules of yeast Mediator are then assigned to the middle and head domains.

Figure 1

Shown are 3-D structures of yeast Mediator (Left), murine Mediator (Center), and of the human TRAP complex (Right). The orientation of each complex in consecutive rows differs by 90°. (Top) Complexes in an orientation corresponding to the preferred orientation of the respective particles on the grid. (Bar = 100 Å.) The arrows near the head domain of the yeast Mediator indicate the “flaps” that must wrap around RNA polymerase II when the holoenzyme complex is formed. Cut-off levels for displaying the different maps were chosen to emphasize their overall domain organization. The resolution of the maps is 30–35 Å, as determined by the Fourier-ring correlation method (17), although a loss of resolution in the direction perpendicular to the plane of Top results from incomplete sampling of the structures in that direction.

Figure 2

Original (Upper), and contrast-enhanced (Lower) images of individual TRAP particles that appear partially extended, revealing middle- and tail-like domains that form the top portion of the TRAP structure shown in Fig. 1. The contrast-enhanced images were produced by applying a threshold to the corresponding original image and then partially obscuring the background around the particle.

Figure 3

Gray-scale projection map of a ΔSin4 mutant yeast holoenzyme (lacking subunits Sin4, Gal11, Med2, and Pgd1), calculated after alignment and averaging of ≈50 molecules preserved in uranyl acetate. (Bar = 200 Å.) An outline of the wild-type holoenzyme complex is shown for comparison (10). Mediator in the mutant holoenzyme appears wrapped around polymerase (pol II) in a extended conformation similar to that observed for the wild-type complex, but comparison of the structures reveals that the “tail” domain (labeled t in the wild-type holoenzyme outline) is missing, whereas the head (h) and middle (m) domains are present and interact with RNA polymerase as they do in the wild-type holoenzyme.

Figure 4

Outline of the wild-type yeast holoenzyme and proposed location of Mediator subunit modules identified by biochemical studies. The combined mass of the subunits assigned to each Mediator domain (shown in parenthesis along with the respective percentage of the total mass of the complex) appears consistent with the apparent relative size of the different domains.