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. 2010 Mar;17(3):273-9.
doi: 10.1038/nsmb.1757. Epub 2010 Feb 14.

Mediator head module structure and functional interactions

Gang Cai  1 , Tsuyoshi Imasaki, Kentaro Yamada, Francesco Cardelli, Yuichiro Takagi, Francisco J Asturias
Affiliations

Mediator head module structure and functional interactions

Gang Cai et al. Nat Struct Mol Biol. 2010 Mar.

Abstract

We used single-particle electron microscopy to characterize the structure and subunit organization of the Mediator Head module that controls Mediator-RNA polymerase II (RNAPII) and Mediator-promoter interactions. The Head module adopts several conformations differing in the position of a movable jaw formed by the Med18-Med20 subcomplex. We also characterized, by structural, biochemical and genetic means, the interactions of the Head module with TATA-binding protein (TBP) and RNAPII subunits Rpb4 and Rpb7. TBP binds near the Med18-Med20 attachment point and stabilizes an open conformation of the Head module. Rpb4 and Rpb7 bind between the Head jaws, establishing contacts essential for yeast-cell viability. These results, and consideration of the structure of the Mediator-RNAPII holoenzyme, shed light on the stabilization of the pre-initiation complex by Mediator and suggest how Mediator might influence initiation by modulating polymerase conformation and interaction with promoter DNA.

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Conflict of interest statement

COMPETING INTERESTS STATEMENT

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Mediator and Head module structure. (a) A cryo-EM reconstruction of Mediator shows the overall structure of the complex at ~25-Å resolution. Previous biochemical, functional and structural analyses suggest a modular organization of Mediator. The Head, Middle/Arm and Tail structural modules have been identified by comparing structures of Mediator in different conformations. A portion of the structurally defined Head module (dashed in green) comprises density corresponding to subunits biochemically identified with the Middle module. (b) A micrograph showing single Head module particles preserved in uranyl acetate. Scale bar, 200 Å. (c) Three different conformations of the Head module were identified through reference-free alignment and classification of EM images. Head module particles were nearly evenly distributed among the three different conformations that differ in the position of a smaller corner-shaped domain on the bottom-right of the structure. The angle (α) between the larger and smaller portions of the Head module structure (see diagram) is <90° in the collapsed conformation, ~90° in the closed conformation and >90° in the open conformation. Information from images of tilted particles was used to obtain 3D reconstruction of the Head module in all three different conformations. Scale bar, 100 Å. (d) Different views of Head module volumes in the collapsed (left column), closed (middle column) and open (right column) conformations. Scale bar, 100 Å.
Figure 2
Figure 2
EM analysis of Head module and subcomplexes. (a) Comparison of class averages obtained from images of Head and Core subcomplexes (corresponding to approximately the same projection direction) and difference mapping establishes that the extended domain flexibly attached to the rest of the Head module corresponds to subunits Med18 and Med20, with Med18 directly connected to the rest of the Head module structure and Med20 forming the distal end of the mobile domain. The above-mentioned comparison also indicates that the Mini complex corresponds to the left portion of the Core complex structure. All class averages are also shown as contour plots to facilitate visual comparison. (b) Contour plots calculated from 2D class averages of the Head and Core complexes were color-coded to highlight the approximate boundaries between different sets of Head module subunits. Two views (marked 1 and 2) of the 3D Head module structure matching the two projections of the Core (also marked 1 and 2) used for difference mapping indicate that the two 2D maps arise from roughly perpendicular orientations of the Head and Core particles in the EM samples (the Med18–Med20 portion of the Head structure is shown as a green mesh in orientation 2). (c) The EM results can be used to derive a description of the overall organization of subunits in the Head module and its interface to the rest of the Mediator complex (see Supplementary Fig. 4 for docking of Med7–Med21 and Med31 into the Mediator cryo-EM structure).
Figure 3
Figure 3
Head–TBP interactions. (a) A GST pulldown assay was used to measure the interaction between TBP and the Head, Core and Mini complexes. GST fusion complexes (as indicated) were immobilized on a glutathione-agarose resin incubated in the presence (lanes 4, 6 and 8) or absence (lanes 3, 5 and 7) of TBP. Controls are shown in lanes 1 and 2. Values for relative TBP binding to the different complexes are represented by the bars below the immunoblot. (b) Class averages obtained after incubation of the Head module with (top) and without TBP (bottom). Interaction with TBP had no effect on the range of conformations adopted by the Head module. (c) TBP interaction influences the distribution of particles among the different conformations of the Head module. The order of groups in the histogram is derived from the order of class averages in b. (d) Class averages calculated from images of Core particles alone (middle) and after incubation with TBP (left), and the difference between them (right). (e) An approximate 3D model of the Head–TBP complex based on the Core + TBP 2D class average and the 3D structure of the Head module. The Core portion of the Head module is shown in red, density corresponding to subunits Med18 and Med20 is represented as a gray mesh, and TBP is shown as a purple surface calculated by low-pass filtering the X-ray structure of TBP.
Figure 4
Figure 4
Head module–Rpb4–Rpb7 interaction. (a) A GST pulldown assay was used to measure the interactions between Rpb4–Rpb7 and the Head, Core and Mini complexes. GST fusion complexes (as indicated) were immobilized on glutathione-agarose resin incubated in the presence (lanes 4, 6 and 8) or absence (lanes 3, 5 and 7) of recombinant 6×His–Rpb4–Rpb7. Controls are shown in lanes 1 and 2. Values for relative binding to the different complexes are represented by the bars below the immunoblot. (b) Comparison of class averages obtained after alignment of Head alone (left, 1,748 images) and Head–Rpb4–Rpb7 particles (right, 1,332 images) shows the presence of additional density in the region corresponding to the jaws of the Head module. A 3D reconstruction of the Head–Rpb4–Rpb7 complex (solid yellow surface, right panel top) shows density (semitransparent yellow surface, right panel bottom) matching the size and shape of a low-resolution model calculated from the Rpb4–Rpb7 X-ray structure (purple surface, right panel bottom). (c) Genetic interaction between Rpb4 and Mediator subunits in the Head (Med18 and Med20), Middle (Med1, Med9 and Med31) and Tail (Med16 and Med5) modules was tested by assessing the viability of the synthetic double mutant strains as described in Online Methods. The wild-type (WT) and mutant strains were grown in SC –Leu medium, spotted in five-fold dilutions onto SC –Ura-Leu and SC +5-FOA plates and incubated at 30 °C for 3 (SC –Ura-Leu plates) or 5 d (SC +5-FOA plates).
Figure 5
Figure 5
Interaction of the Head module with components of the mPIC and a possible mechanism for initiation regulation. (a) Positioning TBP (shown as a purple surface calculated by low-pass filtering the X-ray structure of TBP) in its approximate binding location to the Head module (adjacent to the Med8 subunit) places the transcription factor in a position matching that predicted by current models of the minimal preinitiation complex structure. This positioning suggests how interaction of RNAPII (shown in orange) with Mediator in the Mediator–RNAPII holoenzyme structure (shown in gray) might help stabilize the preinitiation complex. The magenta circle denotes the approximate position of the RNAPII active site. (b) Interaction of the Head module with the Rpb4–Rpb7 polymerase subunit complex (shown in ruby) documented in this study could be important for enabling Mediator and the general transcription factors to affect the conformation of the polymerase clamp domain (shown in blue), possibly facilitating opening (as indicated by the yellow arrow) of the RNA polymerase II active-site cleft (outlined in black) to allow access of double-stranded promoter DNA to the polymerase active site.
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