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. 2009 Apr 15;17(4):559-67.
doi: 10.1016/j.str.2009.01.016.

Mediator structural conservation and implications for the regulation mechanism

Gang Cai  1 , Tsuyoshi Imasaki, Yuichiro Takagi, Francisco J Asturias
Affiliations

Mediator structural conservation and implications for the regulation mechanism

Gang Cai et al. Structure. .

Abstract

Mediator, the multisubunit complex that plays an essential role in the regulation of transcription initiation in all eukaryotes, was isolated using an affinity purification protocol that yields pure material suitable for structural analysis. Conformational sorting of yeast Mediator single-particle images characterized the inherent flexibility of the complex and made possible calculation of a cryo-EM reconstruction. Comparison of free and RNA polymerase II (RNAPII) -associated yeast Mediator reconstructions demonstrates that intrinsic flexibility allows structural modules to reorganize and establish a complex network of contacts with RNAPII. We demonstrate that, despite very low sequence homology, the structures of human and yeast Mediators are surprisingly similar and the structural rearrangement that enables interaction of yeast Mediator with RNAPII parallels the structural rearrangement triggered by interaction of human Mediator with a nuclear receptor. This suggests that the topology and structural dynamics of Mediator constitute important elements of a conserved regulation mechanism.

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Figures

Figure 1
Figure 1. Purification and characterization of Yeast Mediator
(A) Silver stain SDS-PAGE analysis of purified Yeast Mediator. (B) Reconstituted transcription assay of Mediator fractions performed with purified proteins at 24 °C as described (Materials and Methods), with ~0.2 pmol of each affinity-purified Mediator as indicated, in the absence (lanes 1, 2, and 4) or presence (lanes 3 and 5) of recombinant Gcn4 protein. Transcripts from templates containing Gcn4-binding sites (pGCN4) or Gal4-binding sites (pJJ470) and G-less cassettes (G-) are indicated on the right. 32P-labeled fX/Hinf III DNA molecular weight markers (M) are shown in the left with sizes. Transcripts (~360 bp an ~ 260 bp) were separated in a 6% denaturing PAGE and revealed by autoradiography (top). The ~360 bp bands were quantified by Fluorescent image analysis (bottom). (C) EM images of Mediator particles preserved in stain (left) and in amorphous ice (right). High-contrast images in stain were used for evaluation of Mediator conformational variability. (D) Individual Mediator particles preserved in amorphous ice are indicated by the arrowheads. The bar represents ~200nm.
Figure 2
Figure 2. Yeast Mediator conformational variability and Cryo-EM reconstruction
(A) A selection of class averages obtained by statistical classification of images of Mediator particles preserved in stain showing variability in the position of the Head domain (labeled H in the top middle average) and the top of the Tail domain (labeled T in the top middle average). The number of images included in each class average is indicated by the number under each average. (B) Initial models for alignment of cryo-images by projection matching were generated by filtering a previous stain reconstruction of Mediator to ~10 nm and computationally varying the position of the Head domain (as indicated by the red outline). (C) Cryo-EM reconstruction of yeast Mediator calculated from images of ~9,000 particles carefully selected based on their conformational homogeneity. Curved black arrows in the Front view indicate the approximate magnitude and direction of variability in the position of the Head and middle portions of the structure. Apparent multiplicity of the top portion of the Tail domain results from significant mobility. Red stars identify a portion of the density that seems to correspond best to the shape and most common position of the tip of the Tail domain.
Figure 3
Figure 3. Structural modularity of yeast Mediator
(A) Boundaries of Head (red), Middle (green), Tail (orange), and Arm (purple) modules were defined based on independent mobility of these portions of the structure as semi-rigid modules identified by comparison with the conformation of Mediator in the Mediator-RNAPII holoenzyme structure (Davis et al., 2002). Some of the density (enclosed by black ellipse) assigned to the Head module forms an important connection between the Head and the rest of Mediator complex and most likely corresponds to Middle module subunits that are closely associated to the Head module and move along with it. The black arrows indicate module movement triggered by formation of the Mediator-RNAPII holoenzyme. The positions of the Middle and Tail modules change very significantly as they rotate and slide past one another (at an interface indicated by the dashed black lines), while the Arm module appears to rotate and move closer to the Middle and Tail modules. (B) Fitting of Mediator modules into the Mediator portion of a Mediator-RNAPII holoenzyme structure (semi-transparent gray surface). The Head module was fitted first and other modules were subsequently fitted as required to maximize overlap between the modules and Mediator density in the holoenzyme reconstruction. Further small rearrangement of specific features is suggested by the similarity between portions of the free and RNAPII-associated Mediator structures. For example, two small features (outlined in black) in the Tail module (third row from the top) match nearby features (outlined in red) in the Mediator portions of the holoenzyme structure. Apparent extra density at the top of the Tail module results from high mobility of that portion of the structure that results in apparent multiplicity of the feature. The Arm module seems to undergo significant conformational changes and could only be partially accommodated into the Mediator portions of the holoenzyme structure. Two fragments (outlined in black) of the Arm (bottom row) match nearby portions (outlined in red) of Mediator density in the holoenzyme structure but the Arm was not segmented to avoid over-fitting. (C) Mediator modules repositioned by comparison with the structure of the Mediator portion of the holoenzyme structure are shown along with the cryo-EM reconstruction of free Mediator (gray surface) to show the magnitude of the rearrangements undergone by Mediator modules upon interaction with RNAPII.
Figure 4
Figure 4. Arrangement of Mediator modules in the Mediator-RNAPII holoenzyme
(A) Fitting of Mediator modules into the Mediator portion of a Mediator-RNAPII holoenzyme reconstruction (semi-transparent gray surface) shows the way the modules are positioned when Mediator interacts with RNAPII in an arrangement that in its general characteristics must be equivalent to the one that Mediator would adopt as part of a complete preinitiation complex. (B) Two views of a Mediator-RNAPII complex model based on consideration of the free- and RNAPII-associated forms of Mediator and a projection map of the Mediator-RNAPII complex (inset) that reveals contacts between the Head module and RNAPII subunits Rpb4/Rpb7. The interaction of RNAPII with Mediator is very extensive, as the latter surrounds the entire back face of polymerase (top) where the general transcription factors TBP and TFIIB presumably bind. Transcription factor TFIIF is also partially located in the back face of RNAPII and around polymerase subunits Rpb4/Rpb7, likely explaining a TFIIF requirement for stable interaction of Mediator (and the Head module in particular) with RNAPII. All of these contacts are consistent with reported stabilization of the preinitiation complex by Mediator. The interaction between the Mediator Head module and Rpb4/Rpb7 (bottom) could facilitate changes in Rpb4/Rpb7 position that could affect the conformation of the RNAPII clamp domain and therefore indirectly modulate interaction of the enzyme with promoter DNA.
Figure 5
Figure 5. Structural similarity between yeast and human Mediators
The structural similarity between yeast and human Mediators is evidenced by comparison of their 3D structures. Different views of the structure of thyroid hormone receptor (TR)-bound human Mediator (Taatjes et al., 2004) are remarkably similar to corresponding views of the yeast Mediator cryo-EM reconstructions. Moreover, the main change in human Mediator structure induced by TR binding (Taatjes et al., 2004) is the same as the main change required for interaction of yeast Mediator with RNAPII, namely, a swinging of the Middle domain from a central position (indicated by a black line) to a lateral position (indicated by a red line) towards the back of the Head module. This rearrangement opens a space that in the yeast Mediator-RNAPII holoenzyme accommodates RNAPII and allows the enzyme to interact with different Mediator modules. This suggests that structural conservation might be related to conservation of basic aspects of the mechanism of transcription regulation from yeast to human cells.
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Comment in

  • Mediator comes out from the shadows.
    Conaway RC, Conaway JW. Conaway RC, et al. Structure. 2009 Apr 15;17(4):485-6. doi: 10.1016/j.str.2009.03.003. Structure. 2009. PMID: 19368880 No abstract available.

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