Christoph W. Müller

In eukaryotes, ribosome biogenesis initiates within the nucleolus, a hallmark multilayered compartment of the nucleus. Structures of pre-ribosomes have been characterized ex situ, but their assembly pathways within human nucleoli have not been described. Here, we used cryogenic correlative light and electron tomography to visualize molecular landscapes within HeLa cell nucleoli and obtained in-cell structural snapshots of both ribosomal subunit precursors, the small subunit processome and the pre-60S. These recapitulate major states resolved previously ex situ and reveal additional critical interaction partners, including the RNA exosome, rixosome, and nuclear export receptor CRM1-RanGTP. We further show how pre-ribosome assembly landscapes are altered upon RNA polymerase I inhibition. Our study combines molecular structures with cellular context to elucidate the spatiotemporal assembly pathway of the human ribosome.

Optimized sample preparation is essential for imaging biological macromolecules in their native state using single-particle cryo-electron microscopy (cryo-EM) or in situ cryo-electron tomography (cryo-ET). Here we present EasyGrid, a modular, automated platform designed to streamline and standardize cryo-EM/ET sample preparation. EasyGrid integrates in-line plasma treatment of the sample support, microfluidic dispensing, blot-less sample spreading, jet-based vitrification and grid quality control via light interferometry. We demonstrate its effectiveness by preparing grids for multiple purified macromolecular complexes and resolving their structures with cryo-EM. Additionally, EasyGrid achieves improved vitrification of large mammalian cells compared to conventional plunge-freezing. By enabling systematic and high-throughput optimization, EasyGrid provides a robust and time-saving solution for both structural and cellular cryo-EM applications.

Ribosome biogenesis is an essential multi-step process. In eukaryotes, it is orchestrated within the nucleolus, a hallmark multi-layered compartment of the nucleus. Structures of pre-ribosomes have been characterized in vitro , but their assembly pathways within human nucleoli have not been described. Here, we used cryogenic correlative light and electron tomography to visualize the molecular landscapes within HeLa cell nucleoli and obtained in-cell structural snapshots of both ribosomal subunit precursors, the SSU processome and the pre-60S. These recapitulate major states resolved previously in vitro while additionally revealing critical interaction partners, including the RNA exosome, rixosome, and nuclear export receptor CRM1-RanGTP. We further show how pre-ribosome assembly is altered upon RNA polymerase I inhibition. Our study combines molecular structures with cellular context to elucidate the spatiotemporal assembly pathways and regulation of human ribosome biogenesis.

Activation of virulence in Vibrio cholerae , the etiological agent of cholera disease, is mediated by two transmembrane one-component signal-transduction proteins, ToxR and TcpP, which are also transcription factors. Using cryo–electron microscopy, we have solved five structures of the ompU and toxT transcription activation complexes, including the RNA polymerase (RNAP) holoenzyme, promoter DNAs, transcribed RNA, and their corresponding transcription factors, ToxR or TcpP and ToxR-TcpP, respectively. Activation is achieved through the interaction of ToxR or TcpP with the α–C-terminal repeat domain of RNAP where a single residue of the activator, a phenylalanine, appears to be the most critical contact, as confirmed by mutagenesis. No interactions of the transcription factors were observed with other subunits of the RNAP, i.e., the σ subunit as it occurs in the structurally related PhoB family of two-component transcription factors. The structures, and their comparison with our previously solved DNA promoter–ToxR x-ray structures, unveil the molecular mechanism of cholera virulence gene activation.

Transfer-RNA genes (tDNAs) in budding yeast recruit varying amounts of Fpt1, a regulator of RNA polymerase III (RNAPIII) occupancy. Fpt1 occupancy resembles that of the general transcription factor TFIIIC but how Fpt1 is recruited to tDNAs remains unclear. Here we show that both TFIIIB and TFIIIC are required for Fpt1 binding under active as well as repressive RNAPIII conditions. Depletion of TFIIIB reduced Fpt1 occupancy without affecting TFIIIC. In contrast, TFIIIC depletion led to reduced Fpt1 and a gene-specific reduction in TFIIIB occupancy. Moreover, upon depletion of TFIIIC, Fpt1 and TFIIIB were lost to different extents. We identified the C-terminal intrinsically disordered region of Fpt1 as critical for increased Fpt1 binding under repressive conditions. Within this region, a short α-helix was predicted to interact with the N-terminal tetratricopeptide repeat array of the TFIIIC subunit τ131, a region also known to interact with TFIIIB. Deletion of this α-helix abrogated stress-induced Fpt1 recruitment to tDNAs, as did mutations in the predicted interaction surface of τ131, while having a milder effect on TFIIIB occupancy. Together, these findings uncovered a dual and dynamic mechanism of Fpt1 recruitment to tDNAs with independent contributions of TFIIIB and TFIIIC.

Critical for the regulation of eukaryotic gene transcription is the assembly and interplay of general transcription factors (GTFs) with RNA polymerases (RNAPs), leading to the formation of pre-initiation complexes (PICs) as a rate-limiting step in transcription activation. Compared with RNAPII PIC assembly involving many GTFs, activators, and co-activators, RNAPIII PIC assembly is less complex, involving mainly the four GTFs TFIIIA, TFIIIB, TFIIIC, and snRNA activating protein complex with only a few additional factors. The RNAPIII-specific GTF TFIIIC is present in type I and II promoters. One prominent area of investigation has been the dynamic interaction between TFIIIC and its promoter elements, the varying affinities of TFIIIC toward these elements, and the flexible linker within TFIIIC. Additionally, evidence suggests that TFIIIC may play a dual role, acting as an assembly factor that positions TFIIIB during PIC formation and as a barrier during RNAPIII-mediated transcription. By summarizing recent structural, biochemical, and genomic data, this review explores the mechanisms by which RNAPIII-specific GTFs, with a focus on TFIIIC, dynamically regulate RNAPIII transcription.

RNA polymerase III (Pol III) transcribes short, essential RNAs, including the U6 small nuclear RNA (snRNA). At U6 snRNA genes, Pol III is recruited by the snRNA Activating Protein Complex (SNAPc) and a Brf2-containing TFIIIB complex, forming a pre-initiation complex (PIC). Uniquely, SNAPc also recruits Pol II at the remaining splicesosomal snRNA genes (U1, 2, 4 and 5). The mechanism of SNAPc cross-polymerase engagement and the role of the SNAPC2 and SNAPC5 subunits remain poorly defined. Here, we present cryo-EM structures of the full-length SNAPc-containing Pol III PIC assembled on the U6 snRNA promoter in the open and melting states at 3.2–4.2 Å resolution. The structural comparison revealed differences with the Saccharomyces cerevisiae Pol III PIC and the basis of selective SNAPc engagement within Pol III and Pol II PICs. Additionally, crosslinking mass spectrometry localizes SNAPC2 and SNAPC5 near the promoter DNA, expanding upon existing descriptions of snRNA Pol III PIC structure.

Transcription of transfer RNA (tRNA) genes by RNA polymerase (Pol) III requires the general transcription factor IIIC (TFIIIC), which recognizes intragenic A-box and B-box DNA motifs of type II gene promoters. However, the underlying mechanism has remained elusive, in part due to missing structural information for A-box recognition. In this study, we use single-particle cryogenic electron microscopy (cryo-EM) and single-molecule fluorescence resonance energy transfer (smFRET) to reveal structural and real-time kinetic insights into how the 520-kDa yeast TFIIIC complex engages A-box and B-box DNA motifs in the context of a tRNA gene promoter. Cryo-EM structures of τA and τB subcomplexes bound to the A-box and B-box were obtained at 3.7 and 2.5 Å resolution, respectively, while cryo-EM single-particle mapping determined the specific distance and relative orientation of the τA and τB subcomplexes revealing a fully engaged state of TFIIIC. smFRET experiments show that overall recruitment and residence times of TFIIIC on a tRNA gene are primarily governed by B-box recognition, while footprinting experiments suggest a key role of τA and the A-box in TFIIIB and Pol III recruitment following TFIIIC recognition of type II promoters.

RNA polymerase III (Pol III) is specialized in the transcription of short, essential RNAs, including the U6 small nuclear RNAs (snRNAs). At U6 snRNA genes, Pol III is recruited by the snRNA Activating Protein Complex (SNAPc) forming, together with a Brf2-containing TFIIIB complex, a transcriptionally competent pre-initiation complex (PIC). Additionally, SNAPc is responsible for the recruitment of Pol II at the remaining snRNAs genes (U1, 2, 4 and 5), representing a unique example of a multi subunit transcription factor shared among different RNA Polymerases. The mechanism of SNAPc cross-polymerase engagement and the role of the SNAPC2 and SNAPC5 subunits in transcription remain poorly defined. Here, we present cryo-EM structures of the full-length SNAPc-containing Pol III PIC assembled on the U6 snRNA promoter in the open and melting states at 3.2-4.2Å resolution. Comparative structural analysis revealed unexpected differences with the yeast PIC and revealed the molecular basis of selective and structurally distinct SNAPc engagement within Pol III and Pol II PICs. Harnessing crosslinking mass spectrometry, we also localize the SNAPC2 and SNAPC5 subunits in proximity to the bound promoter DNA, expanding upon existing descriptions of snRNA Pol III PIC structure.

DNMT1 is an essential DNA methyltransferase that catalyzes the transfer of methyl groups to CpG islands in DNA and generates a prominent epigenetic mark. The catalytic activity of DNMT1 relies on its conformational plasticity and ability to change conformation from an auto-inhibited to an activated state. Here, we present four cryo-EM reconstructions of apo DNMT1 and DNTM1: non-productive DNA, DNTM1: H3Ub2-peptide, DNTM1: productive DNA complexes. Our structures demonstrate the flexibility of DNMT1’s N-terminal regulatory domains during the transition from an apo ‘auto-inhibited’ to a DNA-bound ‘non-productive’ and finally a DNA-bound ‘productive’ state of DNMT1. Furthermore, we address the regulation of DNMT1’s methyltransferase activity by a DNMT1-selective small-molecule inhibitor and ubiquitinated histone H3. We observe that DNMT1 binds DNA in a ‘non-productive’ state despite the presence of the inhibitor and present the cryo-EM reconstruction of full-length DNMT1 in complex with a di-ubiquitinated H3 peptide analogue. Taken together, our results provide structural insights into the reaction cycle of DNMT1.