Structure of RNA polymerase II pre-initiation complex at 2.9 Å defines initial DNA opening.

Transcription initiation requires assembly of the RNA polymerase II (Pol II) pre-initiation complex (PIC) and opening of promoter DNA. Here, we present the long-sought high-resolution structure of the yeast PIC and define the mechanism of initial DNA opening. We trap the PIC in an intermediate state that contains half a turn of open DNA located 30-35 base pairs downstream of the TATA box. The initially opened DNA region is flanked and stabilized by the polymerase "clamp head loop" and the TFIIF "charged region" that both contribute to promoter-initiated transcription. TFIIE facilitates initiation by buttressing the clamp head loop and by regulating the TFIIH translocase. The initial DNA bubble is then extended in the upstream direction, leading to the open promoter complex and enabling start-site scanning and RNA synthesis. This unique mechanism of DNA opening may permit more intricate regulation than in the Pol I and Pol III systems.

Structures of mammalian RNA polymerase II pre-initiation complexes.

The initiation of transcription is a focal point for the regulation of gene activity during mammalian cell differentiation and development. To initiate transcription, RNA polymerase II (Pol II) assembles with general transcription factors into a pre-initiation complex (PIC) that opens promoter DNA. Previous work provided the molecular architecture of the yeast1-9 and human10,11 PIC and a topological model for DNA opening by the general transcription factor TFIIH12-14. Here we report the high-resolution cryo-electron microscopy structure of PIC comprising human general factors and Sus scrofa domesticus Pol II, which is 99.9% identical to human Pol II. We determine the structures of PIC with closed and opened promoter DNA at 2.5-2.8 Å resolution, and resolve the structure of TFIIH at 2.9-4.0 Å resolution. We capture the TFIIH translocase XPB in the pre- and post-translocation states, and show that XPB induces and propagates a DNA twist to initiate the opening of DNA approximately 30 base pairs downstream of the TATA box. We also provide evidence that DNA opening occurs in two steps and leads to the detachment of TFIIH from the core PIC, which may stop DNA twisting and enable RNA chain initiation.

Structure of the human Mediator-RNA polymerase II pre-initiation complex.

Mediator is a conserved coactivator complex that enables the regulated initiation of transcription at eukaryotic genes1-3. Mediator is recruited by transcriptional activators and binds the pre-initiation complex (PIC) to stimulate the phosphorylation of RNA polymerase II (Pol II) and promoter escape1-6. Here we prepare a recombinant version of human Mediator, reconstitute a 50-subunit Mediator-PIC complex and determine the structure of the complex by cryo-electron microscopy. The head module of Mediator contacts the stalk of Pol II and the general transcription factors TFIIB and TFIIE, resembling the Mediator-PIC interactions observed in the corresponding complex in yeast7-9. The metazoan subunits MED27-MED30 associate with exposed regions in MED14 and MED17 to form the proximal part of the Mediator tail module that binds activators. Mediator positions the flexibly linked cyclin-dependent kinase (CDK)-activating kinase of the general transcription factor TFIIH near the linker to the C-terminal repeat domain of Pol II. The Mediator shoulder domain holds the CDK-activating kinase subunit CDK7, whereas the hook domain contacts a CDK7 element that flanks the kinase active site. The shoulder and hook domains reside in the Mediator head and middle modules, respectively, which can move relative to each other and may induce an active conformation of the CDK7 kinase to allosterically stimulate phosphorylation of the C-terminal domain.

Promoter Distortion and Opening in the RNA Polymerase II Cleft.

Transcription initiation requires opening of promoter DNA in the RNA polymerase II (Pol II) pre-initiation complex (PIC), but it remains unclear how this is achieved. Here we report the cryo-electron microscopic (cryo-EM) structure of a yeast PIC that contains underwound, distorted promoter DNA in the closed Pol II cleft. The DNA duplex axis is offset at the upstream edge of the initially melted DNA region (IMR) where DNA opening begins. Unstable IMRs are found in a subset of yeast promoters that we show can still initiate transcription after depletion of the transcription factor (TF) IIH (TFIIH) translocase Ssl2 (XPB in human) from the nucleus in vivo. PIC-induced DNA distortions may thus prime the IMR for melting and may explain how unstable IMRs that are predicted in promoters of Pol I and Pol III can open spontaneously. These results suggest that DNA distortion in the polymerase cleft is a general mechanism that contributes to promoter opening.

Yeast PIC-Mediator structure with RNA polymerase II C-terminal domain.

For transcription initiation, RNA polymerase II (Pol II) forms a preinitiation complex (PIC) that associates with the general coactivator Mediator. Whereas atomic models of the human PIC-Mediator structure have been reported, structures for its yeast counterpart remain incomplete. Here, we present an atomic model for the yeast PIC with core Mediator, including the Mediator middle module that was previously poorly resolved and including subunit Med1 that was previously lacking. We observe three peptide regions containing eleven of the 26 heptapeptide repeats of the flexible C-terminal repeat domain (CTD) of Pol II. Two of these CTD regions bind between the Mediator head and middle modules and form defined CTD-Mediator interactions. CTD peptide 1 binds between the Med6 shoulder and Med31 knob domains, whereas CTD peptide 2 forms additional contacts with Med4. The third CTD region (peptide 3) binds in the Mediator cradle and associates with the Mediator hook. Comparisons with the human PIC-Mediator structure show that the central region in peptide 1 is similar and forms conserved contacts with Mediator, whereas peptides 2 and 3 exhibit distinct structures and Mediator interactions.

Mediator structure and function in transcription initiation.

Recent advances in cryo-electron microscopy have led to multiple structures of Mediator in complex with the RNA polymerase II (Pol II) transcription initiation machinery. As a result we now hold in hands near-complete structures of both yeast and human Mediator complexes and have a better understanding of their interactions with the Pol II pre-initiation complex (PIC). Herein, we provide a summary of recent achievements and discuss their implications for future studies of Mediator and its role in gene regulation.

A cell cycle-regulated Slx4-Dpb11 complex promotes the resolution of DNA repair intermediates linked to stalled replication.

A key function of the cellular DNA damage response is to facilitate the bypass of replication fork-stalling DNA lesions. Template switch reactions allow such a bypass and involve the formation of DNA joint molecules (JMs) between sister chromatids. These JMs need to be resolved before cell division; however, the regulation of this process is only poorly understood. Here, we identify a regulatory mechanism in yeast that critically controls JM resolution by the Mus81-Mms4 endonuclease. Central to this regulation is a conserved complex comprising the scaffold proteins Dpb11 and Slx4 that is under stringent control. Cell cycle-dependent phosphorylation of Slx4 by Cdk1 promotes the Dpb11-Slx4 interaction, while in mitosis, phosphorylation of Mms4 by Polo-like kinase Cdc5 promotes the additional association of Mus81-Mms4 with the complex, thereby promoting JM resolution. Finally, the DNA damage checkpoint counteracts Mus81-Mms4 binding to the Dpb11-Slx4 complex. Thus, Dpb11-Slx4 integrates several cellular inputs and participates in the temporal program for activation of the JM-resolving nuclease Mus81.

Structures of transcription preinitiation complex engaged with the +1 nucleosome.

The preinitiation complex (PIC) assembles on promoters of protein-coding genes to position RNA polymerase II (Pol II) for transcription initiation. Previous structural studies revealed the PIC on different promoters, but did not address how the PIC assembles within chromatin. In the yeast Saccharomyces cerevisiae, PIC assembly occurs adjacent to the +1 nucleosome that is located downstream of the core promoter. Here we present cryo-EM structures of the yeast PIC bound to promoter DNA and the +1 nucleosome located at three different positions. The general transcription factor TFIIH engages with the incoming downstream nucleosome and its translocase subunit Ssl2 (XPB in human TFIIH) drives the rotation of the +1 nucleosome leading to partial detachment of nucleosomal DNA and intimate interactions between TFIIH and the nucleosome. The structures provide insights into how transcription initiation can be influenced by the +1 nucleosome and may explain why the transcription start site is often located roughly 60 base pairs upstream of the dyad of the +1 nucleosome in yeast.

Structural basis of SNAPc-dependent snRNA transcription initiation by RNA polymerase II.

RNA polymerase II (Pol II) carries out transcription of both protein-coding and non-coding genes. Whereas Pol II initiation at protein-coding genes has been studied in detail, Pol II initiation at non-coding genes, such as small nuclear RNA (snRNA) genes, is less well understood at the structural level. Here, we study Pol II initiation at snRNA gene promoters and show that the snRNA-activating protein complex (SNAPc) enables DNA opening and transcription initiation independent of TFIIE and TFIIH in vitro. We then resolve cryo-EM structures of the SNAPc-containing Pol IIpre-initiation complex (PIC) assembled on U1 and U5 snRNA promoters. The core of SNAPc binds two turns of DNA and recognizes the snRNA promoter-specific proximal sequence element (PSE), located upstream of the TATA box-binding protein TBP. Two extensions of SNAPc, called wing-1 and wing-2, bind TFIIA and TFIIB, respectively, explaining how SNAPc directs Pol II to snRNA promoters. Comparison of structures of closed and open promoter complexes elucidates TFIIH-independent DNA opening. These results provide the structural basis of Pol II initiation at non-coding RNA gene promoters.

Cryo-EM structure of mammalian RNA polymerase II in complex with human RPAP2.

Nuclear import of RNA polymerase II (Pol II) involves the conserved factor RPAP2. Here we report the cryo-electron microscopy (cryo-EM) structure of mammalian Pol II in complex with human RPAP2 at 2.8 Å resolution. The structure shows that RPAP2 binds between the jaw domains of the polymerase subunits RPB1 and RPB5. RPAP2 is incompatible with binding of downstream DNA during transcription and is displaced upon formation of a transcription pre-initiation complex.

An experimentally generated peptide database increases the sensitivity of XL-MS with complex samples.

Cross-linking mass spectrometry (XL-MS) is steadily expanding its range of applications from purified protein complexes to more complex samples like organelles and even entire cells. One main challenge using non-cleavable cross-linkers is the so-called n2 problem: With linearly increasing database size, the search space for the identification of two covalently linked peptides per spectrum increases quadratically. Here, we report an alternative search strategy that focuses on only those peptides, which were demonstrated to cross-link under the applied experimental conditions. The performance of a parallel XL-MS experiment using a thiol-cleavable cross-linker enabled the identification of peptides that carried a cleaved cross-link moiety after reduction and hence were involved in cross-linking reactions. Based on these identifications, a peptide database was generated and used for the database search of the actual cross-linking experiment with a non-cleavable cross-linker. This peptide-focused approach was tested on protein complexes with a reported structural model and obtained results corresponded well to a conventional database search. An application of the strategy on in vivo cross-linked Bacillus subtilis and Bacillus cereus cells revealed a five- to tenfold reduction in search time and led to significantly more identifications with the latter species than a search against the entire proteome. SIGNIFICANCE: Instead of considering all theoretically cross-linkable peptides in a proteome, identification and pre-filtering for a subset of cross-link peptide candidates allows for a dramatically decreased search space. Hence, there is less potential for the random accumulation of false positives ultimately leading to a higher sensitivity in the XL-MS experiment. Using the peptide-focused approach, a cross-linking database search can be conducted in a fraction of time while yielding a similar or higher number of identifications, thereby enabling the cross-linking analysis of samples of mammalian proteome complexity.

Architecture of the symmetric core of the nuclear pore.

The nuclear pore complex (NPC) controls the transport of macromolecules between the nucleus and cytoplasm, but its molecular architecture has thus far remained poorly defined. We biochemically reconstituted NPC core protomers and elucidated the underlying protein-protein interaction network. Flexible linker sequences, rather than interactions between the structured core scaffold nucleoporins, mediate the assembly of the inner ring complex and its attachment to the NPC coat. X-ray crystallographic analysis of these scaffold nucleoporins revealed the molecular details of their interactions with the flexible linker sequences and enabled construction of full-length atomic structures. By docking these structures into the cryoelectron tomographic reconstruction of the intact human NPC and validating their placement with our nucleoporin interactome, we built a composite structure of the NPC symmetric core that contains ~320,000 residues and accounts for ~56 megadaltons of the NPC's structured mass. Our approach provides a paradigm for the structure determination of similarly complex macromolecular assemblies.

Architecture of the fungal nuclear pore inner ring complex.

The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic transport. We present the reconstitution and interdisciplinary analyses of the ~425-kilodalton inner ring complex (IRC), which forms the central transport channel and diffusion barrier of the NPC, revealing its interaction network and equimolar stoichiometry. The Nsp1•Nup49•Nup57 channel nucleoporin heterotrimer (CNT) attaches to the IRC solely through the adaptor nucleoporin Nic96. The CNT•Nic96 structure reveals that Nic96 functions as an assembly sensor that recognizes the three-dimensional architecture of the CNT, thereby mediating the incorporation of a defined CNT state into the NPC. We propose that the IRC adopts a relatively rigid scaffold that recruits the CNT to primarily form the diffusion barrier of the NPC, rather than enabling channel dilation.