SUMMARY The multisubunit Mediator comprising ~30 distinct proteins, plays an essential role in gene expression regulation by acting as a bridge between DNA binding transcription factors and the RNA polymerase II (RNAPII) transcription machinery. Efforts to uncover the Mediator mechanism have been hindered by a poor understanding of its structure, subunit organization, and conformational rearrangements. By overcoming biochemical and image analysis hurdles, we obtained accurate EM structures of yeast and human Mediators. Subunit localization experiments, docking of partial X-ray structures, and biochemical analyses resulted in comprehensive mapping of yeast Mediator subunits and a complete reinterpretation of our previous Mediator organization model. Large-scale Mediator rearrangements depend on changes at the interfaces between previously described Mediator modules, which appear to be facilitated by factors conducive to transcription initiation. Conservation across eukaryotes of Mediator structure, subunit organization, and RNA polymerase II interaction suggest conservation of fundamental aspects of the Mediator mechanism.

The CDK8 kinase module (CKM) is a conserved, dissociable Mediator subcomplex whose component subunits were genetically linked to the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and individually recognized as transcriptional repressors before Mediator was identified as a preeminent complex in eukaryotic transcription regulation. We used macromolecular electron microscopy and biochemistry to investigate the subunit organization, structure, and Mediator interaction of the Saccharomyces cerevisiae CKM. We found that interaction of the CKM with Mediator’s Middle module interferes with CTD-dependent RNAPII binding to a previously unknown Middle module CTD-binding site targeted early on in a multi-step holoenzyme formation process. Taken together, our results reveal the basis for CKM repression, clarify the origin of the connection between CKM subunits and the CTD, and suggest that a combination of competitive interactions and conformational changes that facilitate holoenzyme formation underlie the Mediator mechanism.

Natural proteins can be versatile building blocks for multimeric, self-assembling structures. Yet, creating protein-based assemblies with specific geometries and chemical properties remains challenging. Highly porous materials represent particularly interesting targets for designed assembly. Here we utilize a strategy of fusing two natural protein oligomers using a continuous alpha-helical linker to design a novel protein that self assembles into a 750 kDa, 225 Å diameter, cube-shaped cage with large openings into a 130 Å diameter inner cavity. A crystal structure of the cage showed atomic level agreement with the designed model, while electron microscopy, native mass spectrometry, and small angle x-ray scattering revealed alternate assembly forms in solution. These studies show that accurate design of large porous assemblies with specific shapes is feasible, while further specificity improvements will likely require limiting flexibility to select against alternative forms. These results provide a foundation for the design of advanced materials with applications in bionanotechnology, nanomedicine and material sciences.

The conserved Mediator co-activator complex has an essential role in the regulation of RNA polymerase II transcription in all eukaryotes. Understanding the structure and interactions of Mediator is crucial for determining how the complex influences transcription initiation and conveys regulatory information to the basal transcription machinery. Here we present a 4.4 Å resolution cryo-electron microscopy map of Schizosaccharomyces pombe Mediator in which conserved Mediator subunits are individually resolved. The essential Med14 subunit works as a central backbone that connects the Mediator head, middle and tail modules. Comparison with a 7.8 Å resolution cryo-electron microscopy map of a Mediator-RNA polymerase II holoenzyme reveals that changes in the structure of Med14 facilitate a large-scale Mediator rearrangement that is essential for holoenzyme formation. Our study suggests that access to different conformations and crosstalk between structural elements are essential for the Mediator regulation mechanism, and could explain the capacity of the complex to integrate multiple regulatory signals.

FOXO3a is a transcription factor of the FOXO family. The FOXO proteins participate in multiple signaling pathways, and their transcriptional activity is regulated by several post-translational mechanisms, including phosphorylation, acetylation and ubiquitination. Because these post-translational modification sites are located within the C-terminal basic region of the FOXO DNA-binding domain (FOXO-DBD), it is possible that these post-translational modifications could alter the DNA-binding characteristics. To understand how FOXO mediate transcriptional activity, we report here the 2.7 Å crystal structure of the DNA-binding domain of FOXO3a (FOXO3a-DBD) bound to a 13-bp DNA duplex containing a FOXO consensus binding sequence (GTAAACA). Based on a unique structural feature in the C-terminal region and results from biochemical and mutational studies, our studies may explain how FOXO-DBD C-terminal phosphorylation by protein kinase B (PKB) or acetylation by cAMP-response element binding protein (CBP) can attenuate the DNA-binding activity and thereby reduce transcriptional activity of FOXO proteins. In addition, we demonstrate that the methyl groups of specific thymine bases within the consensus sequence are important for FOXO3a-DBD recognition of the consensus binding site.