A complete PIC-Mediator structure
As a critical transcription coactivator, the multisubunit Mediator complex binds RNA polymerase II (Pol II), facilitates preinitiation complex (PIC) assembly, and stimulates transcription and phosphorylation of the Pol II C-terminal domain (CTD). However, how these critical transcriptional events are coordinated by Mediator is not fully understood. Chen et al. determined the structures of human Mediator and Mediator-bound PIC in distinct conformational states, the latter of which represents a complete PIC-Mediator complex assembled on the 14-subunit transcription factor IID (TFIID). The structures show that Mediator undergoes reorganization during PIC-Mediator assembly, sandwiches and facilitates phosphorylation of Pol II CTD, and works with TFIID to organize TFIIH in PIC for transcription initiation.
Science, abg0635, this issue p. eabg0635
The multisubunit Mediator binds RNA polymerase II (Pol II), transduces regulatory signals from transcription factors to Pol II, facilitates preinitiation complex (PIC) assembly, and stimulates cyclin-dependent kinase 7 (CDK7)–mediated Pol II C-terminal domain (CTD) phosphorylation. The 14-subunit transcription factor IID (TFIID) is globally required for almost all Pol II–mediated transcription initiation and cannot be replaced by TATA box–binding protein (TBP), a TFIID subunit. Previous structural studies focused on the TBP-based system. However, the mechanism by which Mediator is assembled into TFIID-based PIC and regulates Pol II CTD phosphorylation remains elusive.
We reconstituted and determined the cryo–electron microcopy structures of human 26-subunit Mediator and its complex with TFIID-based PIC (76 polypeptides, ~4.1 megadaltons). Structural analyses were performed using the structures of Mediator and PIC-Mediator in distinct conformations, as well as the previously reported TBP-based PIC-Mediator structure.
Mediator in the Tail-extended (MEDE) and Tail-bent (MEDB) conformations reveals similar separation of the Head and Middle modules. The structure of MEDE at 3.5-Å resolution reveals the mechanism of Mediator assembly. Binding of PIC induces concerted modular reorganization (Head-tilting and Middle-down) of Mediator through two connected molecular levers. The α-helix bundle HB1 of the Head and the Knob of the Middle form a Head-Middle sandwich, which stabilizes two CTD segments with the longer segment extending toward the CDK7 active site. The CTD-Mediator-CDK7 binding pattern suggests a CTD-gating mechanism, by which Mediator binds and brings Pol II CTD to CDK7 for efficient and persistent phosphorylation. Structures of PIC-Mediator in distinct conformations indicate that PIC’s architecture modulates Mediator organization. Mediator and TFIID together position TFIIH, stabilize xeroderma pigmentosum type B (XPB)–promoter–Pol II contacts, and may facilitate XPB-mediated promoter melting and DNA translocation toward Pol II. Structural comparison with TBP-based PIC-Mediator reveals considerable differences in Mediator conformation, CTD-Mediator interaction, and XPB stabilization, underscoring the critical role of TFIID in organizing PIC-Mediator.
Our study provides the structure of the human Mediator at near-atomic resolution as well as the structure of the complete PIC-Mediator holocomplex. These structures provide insights into PIC-Mediator assembly and Mediator-stimulated CTD phosphorylation. TFIID may confer considerable complexity and dynamics of PIC-Mediator organization to accommodate highly dynamic processes of transcription initiation. The structures also provide a framework for further studies of Mediator-stimulated transcription activation by transcription factors.
The 1.3-megadalton transcription factor IID (TFIID) is required for preinitiation complex (PIC) assembly and RNA polymerase II (Pol II)–mediated transcription initiation on almost all genes. The 26-subunit Mediator stimulates transcription and cyclin-dependent kinase 7 (CDK7)–mediated phosphorylation of the Pol II C-terminal domain (CTD). We determined the structures of human Mediator in the Tail module–extended (at near-atomic resolution) and Tail-bent conformations and structures of TFIID-based PIC-Mediator (76 polypeptides, ~4.1 megadaltons) in four distinct conformations. PIC-Mediator assembly induces concerted reorganization (Head-tilting and Middle-down) of Mediator and creates a Head-Middle sandwich, which stabilizes two CTD segments and brings CTD to CDK7 for phosphorylation; this suggests a CTD-gating mechanism favorable for phosphorylation. The TFIID-based PIC architecture modulates Mediator organization and TFIIH stabilization, underscoring the importance of TFIID in orchestrating PIC-Mediator assembly.