RPAP2 regulates a transcription initiation checkpoint by inhibiting assembly of pre-initiation complex.

RNA polymerase II (Pol II)-mediated transcription in metazoans requires precise regulation. RNA Pol II-associated protein 2 (RPAP2) was previously identified to transport Pol II from cytoplasm to nucleus and dephosphorylates Pol II C-terminal domain (CTD). Here, we show that RPAP2 binds hypo-/hyper-phosphorylated Pol II with undetectable phosphatase activity. The structure of RPAP2-Pol II shows mutually exclusive assembly of RPAP2-Pol II and pre-initiation complex (PIC) due to three steric clashes. RPAP2 prevents and disrupts Pol II-TFIIF interaction and impairs in vitro transcription initiation, suggesting a function in inhibiting PIC assembly. Loss of RPAP2 in cells leads to global accumulation of TFIIF and Pol II at promoters, indicating a critical role of RPAP2 in inhibiting PIC assembly independent of its putative phosphatase activity. Our study indicates that RPAP2 functions as a gatekeeper to inhibit PIC assembly and transcription initiation and suggests a transcription checkpoint.

Structures of the human Mediator and Mediator-bound preinitiation complex.

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.

Structural insights into preinitiation complex assembly on core promoters.

Transcription factor IID (TFIID) recognizes core promoters and supports preinitiation complex (PIC) assembly for RNA polymerase II (Pol II)-mediated eukaryotic transcription. We determined the structures of human TFIID-based PIC in three stepwise assembly states and revealed two-track PIC assembly: stepwise promoter deposition to Pol II and extensive modular reorganization on track I (on TATA-TFIID-binding element promoters) versus direct promoter deposition on track II (on TATA-only and TATA-less promoters). The two tracks converge at an ~50-subunit holo PIC in identical conformation, whereby TFIID stabilizes PIC organization and supports loading of cyclin-dependent kinase (CDK)-activating kinase (CAK) onto Pol II and CAK-mediated phosphorylation of the Pol II carboxyl-terminal domain. Unexpectedly, TBP of TFIID similarly bends TATA box and TATA-less promoters in PIC. Our study provides structural visualization of stepwise PIC assembly on highly diversified promoters.

Identification of Integrator-PP2A complex (INTAC), an RNA polymerase II phosphatase.

The 14-subunit metazoan-specific Integrator contains an endonuclease that cleaves nascent RNA transcripts. Here, we identified a complex containing Integrator and protein phosphatase 2A core enzyme (PP2A-AC), termed INTAC. The 3.5-angstrom-resolution structure reveals that nine human Integrator subunits and PP2A-AC assemble into a cruciform-shaped central scaffold formed by the backbone and shoulder modules, with the phosphatase and endonuclease modules flanking the opposite sides. As a noncanonical PP2A holoenzyme, the INTAC complex dephosphorylates the carboxy-terminal repeat domain of RNA polymerase II at serine-2, -5, and -7 and thus regulates transcription. Our study extends the function of PP2A to transcriptional regulation and reveals how dual enzymatic activities-RNA cleavage and RNA polymerase II dephosphorylation-are structurally and functionally integrated into the INTAC complex.

Cryo-EM structure of SMG1-SMG8-SMG9 complex.

Nonsense-mediated mRNA decay (NMD) targets premature stop codon (PTC)-containing mRNAs for rapid degradation, and is essential for mammalian embryonic development, brain development and modulation of the stress response. The key event in NMD is the SMG1-mediated phosphorylation of an RNA helicase UPF1 and SMG1 kinase activity is inhibited by SMG8 and SMG9 in an unknown mechanism. Here, we determined the cryo-EM structures of human SMG1 at 3.6 Å resolution and the SMG1-SMG8-SMG9 complex at 3.4 Å resolution, respectively. SMG8 has a C-terminal kinase inhibitory domain (KID), which covers the catalytic pocket and inhibits the kinase activity of SMG1. Structural analyses suggest that GTP hydrolysis of SMG9 would lead to a dramatic conformational change of SMG8-SMG9 and the KID would move away from the inhibitory position to restore SMG1 kinase activity. Thus, our structural and biochemical analyses provide a mechanistic understanding of SMG1-SMG8-SMG9 complex assembly and the regulatory mechanism of SMG1 kinase activity.

Cryo-EM structure of human mTOR complex 2.

Mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) plays an essential role in regulating cell proliferation through phosphorylating AGC protein kinase family members, including AKT, PKC and SGK1. The functional core complex consists of mTOR, mLST8, and two mTORC2-specific components, Rictor and mSin1. Here we investigated the intermolecular interactions within mTORC2 complex and determined its cryo-electron microscopy structure at 4.9 Å resolution. The structure reveals a hollow rhombohedral fold with a 2-fold symmetry. The dimerized mTOR serves as a scaffold for the complex assembly. The N-terminal half of Rictor is composed of helical repeat clusters and binds to mTOR through multiple contacts. mSin1 is located close to the FRB domain and catalytic cavity of mTOR. Rictor and mSin1 together generate steric hindrance to inhibit binding of FKBP12-rapamycin to mTOR, revealing the mechanism for rapamycin insensitivity of mTORC2. The mTOR dimer in mTORC2 shows more compact conformation than that of mTORC1 (rapamycin sensitive), which might result from the interaction between mTOR and Rictor-mSin1. Structural comparison shows that binding of Rictor and Raptor (mTORC1-specific component) to mTOR is mutually exclusive. Our study provides a basis for understanding the assembly of mTORC2 and a framework to further characterize the regulatory mechanism of mTORC2 pathway.