Mediator structure and rearrangements required for holoenzyme formation

Abstract

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.

Interrogation of Mammalian Protein Complex Structure, Function, and Membership Using Genome-Scale Fitness Screens

“…We hypothesized that for protein complexes with a sufficient number of subunits and sufficiently distinct functional componentry, this modular structure may be reflected in the functional similarity networks. The Mediator complex is an evolutionarily conserved transcriptional activator composed of three stable modules – the Head, Middle and Tail (Figure 3A) – and one detachable, cell cycle specific module (Cyclin Kinase Module, not crystallized) (Nozawa et al, 2017; Tsai et al, 2017). Recent biochemical and genetic studies have demonstrated that the Head, Middle and Tail modules exhibit differential genomic targeting and have specialized roles in the context of Mediator complex global function (Jeronimo et al, 2016; Petrenko et al, 2016).…”

The cryo-electron microscopy structure of human transcription factor IIH

“…This density localizes near the region of attachment of the CAK subcomplex to the TFIIH core, as visualized in 2D class averages and 3D reconstructions of negatively stained TFIIH (Extended Data Fig. 8a–c and refs 6, 26), and reconstructions of TFIIH in the context of the assembled Pol II-PIC (see below and refs 5, 27). Previous data additionally show that MAT1 interacts with the XPD ARCH domain, and that the C259Y mutation in the ARCH domain impairs binding 28 .…”

Towards a mechanistic understanding of core promoter recognition from cryo-EM studies of human TFIID

“…Recent cryo-EM studies using budding or fission yeast proteins have visualized the interaction of a partial or full TBP-based PIC with the large Mediator complex involved in gene-specific activation of transcription [38–40]. Those studies have shed light on Mediator’s role in PIC stabilization and recruitment.…”

“…This model was generated by superposition of the common elements of three different cryo-EM structures – the human TFIID-TFIIA-promoter complex [25**], the human TBP-based PIC (including TBP, TFIIA, TFIIB, TFIIF, TFIIS, TFIIE, TFIIH, and Pol II) [32**], and the Mediator-Pol II complex from Schizosaccharomyces pombe [40**]. The resulting model represents an ~3 MDa transcription initiation supracomplex including TFIID, TFIIA, TFIIB, TFIIF, TFIIS, TFIIE, TFIIH, Pol II, and the Mediator coactivator complex.…”