![]() Received: JAccepted: FebruPublished: March 29, 2011Ĭopyright: © 2011 Bernecky et al. Petsko, Brandeis University, United States of America PLoS Biol 9(3):Īcademic Editor: Gregory A. These data in combination with the Mediator–pol II–TFIIF structure described here allow us to propose the structural organization of the entire 3.5 MDa human PIC.Ĭitation: Bernecky C, Grob P, Ebmeier CC, Nogales E, Taatjes DJ (2011) Molecular Architecture of the Human Mediator–RNA Polymerase II–TFIIF Assembly. The locations of TFIIH, TBP (a subunit within TFIID), TFIIA, TFIIB, TFIIE, and TFIIF relative to the pol II enzyme itself have been determined by previous studies. Additionally, we accurately dock the pol II crystal structure within the human Mediator–pol II–TFIIF cryo-EM map. We observe that TFIIF is required to stably orient the pol II enzyme within the Mediator–pol II assembly, indicating a novel structural role for TFIIF in transcription initiation. This study outlines a cryo-EM analysis of the Mediator–pol II assembly in the presence or absence of the dimeric TFIIF complex. Although the 1.2 MDa Mediator seems to have a major role in regulating assembly and function of the PIC, a structural understanding of the complex has yet to be established. Collectively, these factors are known as the Pre-Initiation Complex (PIC). Transcription initiation in humans is regulated by a macromolecular complex formed by the RNA polymerase II enzyme (pol II), Mediator, and the general transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. Finally, parallel structural analysis of Mediator–pol II complexes lacking TFIIF reveal that TFIIF plays a key role in stabilizing pol II orientation within the assembly. The data also reveal how pol II binding excludes Mediator–CDK8 subcomplex interactions and provide a structural basis for Mediator-dependent control of PIC assembly and function. Pol II surfaces required for interacting with TFIIB, TFIIE, and promoter DNA (i.e., the pol II cleft) are exposed within the Mediator–pol II–TFIIF structure RNA exit is unhindered along the RPB4/7 subunits upstream and downstream DNA is accessible for binding additional factors and no major structural re-organization is necessary to accommodate the large, multi-subunit TFIIH or TFIID complexes. Significantly, pol II orientation within the Mediator–pol II–TFIIF assembly can be reconciled with past studies that determined the location of other PIC components relative to pol II itself. The orientation of pol II within this assembly was determined by crystal structure docking and further validated with projection matching experiments, allowing the structural organization of the entire human PIC to be envisioned. We have assembled the complete, 1.9 MDa human Mediator–pol II–TFIIF complex from purified components and have characterized its structural organization using cryo-electron microscopy and single-particle reconstruction techniques. ![]() The structure of the human Mediator–pol II interface is not well-characterized, whereas attempts to structurally define the Mediator–pol II interaction in yeast have relied on incomplete assemblies of Mediator and/or pol II and have yielded inconsistent interpretations. ![]() At the heart of this assembly is the Mediator complex, which helps regulate PIC activity and interacts with the RNA polymerase II (pol II) enzyme. The macromolecular assembly required to initiate transcription of protein-coding genes, known as the Pre-Initiation Complex (PIC), consists of multiple protein complexes and is approximately 3.5 MDa in size. ![]()
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