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Abstract
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. At the heart of this assembly are the Mediator complex, RNA polymerase II (pol II), and TFIIF. Structures of Mediator-pol II complexes have been examined in yeast, but these involved incomplete assemblies and have yielded conflicting results. No studies to date have examined human Mediator-pol II structure. Consequently, examination of the human Mediator-pol II structure will grant insight into the way these two multisubunit protein complexes – each of which is essential for expression of protein-coding genes – work together to initiate transcription.
We have assembled the 1.9 MDa human Mediator–pol II–TFIIF complex from purified components and have characterized its structural organization using cryo-electron microscopy. The orientation of pol II within this assembly was determined by crystal structure docking, allowing us to propose the structural organization of the entire human PIC. Pol II assembles at the head region of Mediator, leaving the leg domain completely exposed for interaction with other cofactors. Rearrangements have occurred in the domain to which the repressive CDK8 module binds, intimating a structural rationale for the observation that pol II binding and CDK8 module binding are mutually exclusive. The observed pol II orientation leaves all of the sites necessary for general transcription factor binding exposed, and so is compatible with all current data describing PIC assembly around the enzyme.
Parallel structural analysis of Mediator–pol II complexes lacking TFIIF reveal that TFIIF plays a key role in stabilizing the pol II orientation within the assembly. TFIIF associates tightly with pol II and is generally required for both transcriptional initiation and elongation. By elucidating the importance of TFIIF in stabilizing the Mediator-pol II structure, this work has defined a key structural role for TFIIF that likely contributes to its essential function. Altogether, the data reveal a potential mechanism for how pol II binding excludes repressive Mediator–CDK8 module interactions and provide a structural basis for Mediator-dependent control of PIC assembly and activity.
