Non-homologous end joining
DNA double-strand breaks (DSBs) are a highly cytotoxic form of DNA damage and the incorrect repair of DSBs is linked to carcinogenesis. The conserved error-prone non-homologous end joining (NHEJ) pathway has a key role in determining the effects of DSB-inducing agents that are used to treat cancer as well as the generation of the diversity in antibodies and T cell receptors. Here we applied single-particle cryo-electron microscopy to visualize two key DNA–protein complexes that are formed by human NHEJ factors. The Ku70/80 heterodimer (Ku), the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), DNA ligase IV (LigIV), XRCC4 and XLF form a long-range synaptic complex, in which the DNA ends are held approximately 115 Å apart. Two DNA end-bound subcomplexes comprising Ku and DNA-PKcs are linked by interactions between the DNA-PKcs subunits and a scaffold comprising LigIV, XRCC4, XLF, XRCC4 and LigIV. The relative orientation of the DNA-PKcs molecules suggests a mechanism for autophosphorylation in trans, which leads to the dissociation of DNA-PKcs and the transition into the short-range synaptic complex. Within this complex, the Ku-bound DNA ends are aligned for processing and ligation by the XLF-anchored scaffold, and a single catalytic domain of LigIV is stably associated with a nick between the two Ku molecules, which suggests that the joining of both strands of a DSB involves both LigIV molecules.
Human Mediator’s role in RNA polymerase II transcription initiation
Eukaryotic transcription requires the assembly of a multi-subunit preinitiation complex (PIC) comprised of RNA polymerase II (Pol II) and the general transcription factors. The co-activator Mediator is recruited by transcription factors, facilitates the assembly of the PIC, and stimulates phosphorylation of the Pol II C-terminal domain (CTD) by the TFIIH subunit CDK7. Here, we present the cryo-electron microscopy structure of the human Mediator-bound PIC at sub-4 Å. Transcription factor binding sites within Mediator are primarily flexibly tethered to the tail module. CDK7 is stabilized by multiple contacts with Mediator. Two binding sites exist for the Pol II CTD, one between the head and middle modules of Mediator and the other in the active site of CDK7, providing structural evidence for Pol II CTD phosphorylation within the Mediator-bound PIC.
SWI/SNF chromatin remodeling complex
The chromatin remodeling complex SWI/SNF is highly conserved and plays critical roles in various cellular processes including transcription and DNA damage repair. It hydrolyzes ATP to remodel chromatin structure by sliding and evicting histone octamers, creating DNA regions that become accessible to other essential factors. We have determined the structure of SWI/SNF from the yeast S. cerevisiae bound to a nucleosome using cryo-electron microscopy (cryo-EM). In the structure, the Arp module is sandwiched between the ATPase and the rest of the complex, with the Snf2 HSA domain connecting all modules. The body contains an assembly scaffold composed of conserved subunits Snf12 (SMARCD/BAF60), Snf5 (SMARCB1/BAF47/ INI1) and an asymmetric dimer of Swi3 (SMARCC/BAF155/170). Another conserved subunit Swi1 (ARID1/BAF250) resides in the core of SWI/SNF, acting as a molecular hub. We also observed interactions between Snf5 and the histones at the acidic patch, which could serve as an anchor during active DNA translocation. Our structure allows us to map and rationalize a subset of cancer-related mutations in the human SWI/SNF complex and propose a model of how SWI/SNF recognizes and remodels the +1 nucleosome to generate nucleosome-depleted regions during gene activation.
Eukaryotic RNA polymerase III transcription
RNA polymerase III (Pol III) transcription initiation requires the action of the transcription factor IIIB (TFIIIB) and is highly regulated. Here, we determine the structures of Pol III pre-initiation complexes (PICs) using single particle cryo-electron microscopy (cryo-EM). We observe stable Pol III–TFIIIB complexes using nucleic acid scaffolds mimicking various functional states, in which TFIIIB tightly encircles the upstream promoter DNA. There is an intricate interaction between TFIIIB and Pol III, which stabilizes the winged-helix domains of the C34 subunit of Pol III over the active site cleft. The architecture of Pol III PIC more resembles that of the Pol II PIC than the Pol I PIC. In addition, we also obtain a 3D reconstruction of Pol III in complex with TFIIIB using the elongation complex (EC) scaffold, shedding light on the mechanism of facilitated recycling of Pol III prior to transcription re-initiation.
Eukaryotic RNA polymerase I transcription
Eukaryotic RNA synthesis is catalyzed by at least three classes of RNA Polymerases (Pol I-III). The large ribosomal RNA precursor (pre-rRNA) is transcribed by Pol I, accounting for up to 60% of total cellular RNA synthesis in Saccharomyces cerevisiae. Pre-Initiation Complex (PIC) formation is a key regulatory step in the control of gene transcription by eukaryotic RNA polymerases. We have determined the structure of Pol I transcription initiation complex at near-atomic resolution using single particle cryo-EM. The structure reveals the architecture of Core Factor binding to promoter DNA and that Pol I and promoter DNA are pre-conditioned in an elongation-competent form. We have also obtained three functional states of the Pol I Initial Transcribing Complex, which allows us to propose a molecular mechanism in which Pol I utilizes the intrinsic mobility of the DNA-bound Core Factor in the process of promoter opening. This model explains why TFIIH is not necessary for Pol I promoter opening, and implies that a similar mechanism could also be used by Pol III, the other TFIIH-independent RNA polymerase.
Eukaryotic RNA polymerase II transcription
Eukaryotic gene transcription is tightly controlled during the initiation stage, when RNA polymerase II (Pol II) and the general transcription factors (GTFs) (TFIID, TFIIA, TFIIB, TFIIE, TFIIF and TFIIH) assemble at the promoter into a PIC. The initial closed promoter complex (CC) must transition into an open complex (OC), where the melted single-stranded template DNA is inserted into the active site and Pol II locates the transcription start site (TSS). This transient OC is then converted into an initial transcribing complex (ITC), where the first phosphodiester bond forms and messenger RNA starts to be synthesized. Following abortive cycles of synthesis of short RNA products, Pol II eventually clears the promoter and a stable elongation complex (EC) forms. Our cryo-EM study, which provides nearly complete pseudo-atomic models of all the structural elements within a functional human PIC, has allowed us to define the structural transitions through the processes of DNA engagement by the CC, opening of the transcription bubble in the OC, and initiation of transcription in the ITC. The present structures constitute a comprehensive structural framework for past and future studies of the complex process of eukaryotic transcription initiation.