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Abstract: Aphthovirus is a genus of the family Picornaviridae which includes Foot-and- mouth-disease virus (FMDV) and Equine rhinitis A virus (ERAV). FMDV is a highly contagious pathogen infecting cloven-hoofed animals and is hence economically important. FMDV replication takes place in the cytoplasm and induces massive rearrangement of the host cell membranes to facilitate virus replication. Rearranged membranes form structures providing the site of viral genome replication known as the replication organelle (RO). The understanding of the RO, viral proteins and site of virus assembly is not well established. This project applies various microscopy approaches to investigate details of aphthovirus replication in cells. FMDV 3A protein is known to play a key role in viral replication machinery. We generated recombinant viruses of FMDV with various tags fused to this protein, subsequently allowing 3A to be detected in confocal microscopy. We developed a split-GFP system to study the dynamics of 3A protein in vitro. We showed that 3A signals appeared contiguous to the Golgi membrane signals suggesting that it potentially serve as a main source of membrane associated with viral replication. This approach was taken with the aim of facilitating the development of a correlative light electron microscopy (CLEM) system to unravel the localisation of virus proteins and their link to RO in cells. ERAV was used as a surrogate model to study FMDV replication in a lower containment laboratory using cryo-electron vi tomography (cryo-ET). Virus particles were observed associated with membrane structures with single membrane vesicles being more predominant than double membrane vesicles in infected cells. By sub-tomogram averaging, we reconstructed 3-dimensional (3D) models of intracellular ERAV full and empty particles which were compared with structures obtained for the virus purified from tissue culture and crystallized. Additional density was identified in the ERAV empty particles potentially corresponding to RNA contact sites inside the capsid.

Open Access

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Abstract: Approximately 25% of eukaryotic genes code for integral membrane proteins that are assembled at the endoplasmic reticulum. An abundant and widely conserved multi-protein complex termed EMC has been implicated in membrane protein biogenesis, but its mechanism of action is poorly understood. Here, we define the composition and architecture of human EMC using biochemical assays, crystallography of individual subunits, site-specific photocrosslinking, and cryo-EM reconstruction. Our results suggest that EMC's cytosolic domain contains a large, moderately hydrophobic vestibule that can bind a substrate's transmembrane domain (TMD). The cytosolic vestibule leads into a lumenally-sealed, lipid-exposed intramembrane groove large enough to accommodate a single substrate TMD. A gap between the cytosolic vestibule and intramembrane groove provides a potential path for substrate egress from EMC. These findings suggest how EMC facilitates energy-independent membrane insertion of TMDs, explain why only short lumenal domains are translocated by EMC, and constrain models of EMC's proposed chaperone function.

Open Access

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Abstract: Transcription termination by RNA polymerase II (RNA Pol II) is linked to RNA 3′ end processing by the cleavage and polyadenylation factor (CPF or CPSF). CPF contains endonuclease, poly(A) polymerase, and protein phosphatase activities, which cleave and polyadenylate pre-mRNAs and dephosphorylate RNA Pol II to control transcription. Exactly how the RNA 3′ end processing machinery is coupled to transcription remains unclear. Here, we combine in vitro reconstitution, structural studies, and genome-wide analyses to show that yeast CPF physically and functionally interacts with RNA Pol II. Surprisingly, CPF-mediated dephosphorylation promotes the formation of an RNA Pol II stalk-to-stalk homodimer in vitro. This dimer is compatible with transcription but not with the binding of transcription elongation factors. Disruption of the dimerization interface in cells causes transcription defects, including altered RNA Pol II abundance on protein-coding genes, tRNA genes, and intergenic regions. We hypothesize that RNA Pol II dimerization may provide a mechanistic basis for the allosteric model of transcription termination.

Open Access

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Abstract: The poly-ADP-ribosyltransferase tankyrase (TNKS, TNKS2) controls a wide range of disease-relevant cellular processes, including WNT–β-catenin signalling, telomere length maintenance, Hippo signalling, DNA damage repair and glucose homeostasis1,2. This has incentivized the development of tankyrase inhibitors. Notwithstanding, our knowledge of the mechanisms that control tankyrase activity has remained limited. Both catalytic and non-catalytic functions of tankyrase depend on its filamentous polymerization3,4,5. Here we report the cryo-electron microscopy reconstruction of a filament formed by a minimal active unit of tankyrase, comprising the polymerizing sterile alpha motif (SAM) domain and its adjacent catalytic domain. The SAM domain forms a novel antiparallel double helix, positioning the protruding catalytic domains for recurring head-to-head and tail-to-tail interactions. The head interactions are highly conserved among tankyrases and induce an allosteric switch in the active site within the catalytic domain to promote catalysis. Although the tail interactions have a limited effect on catalysis, they are essential to tankyrase function in WNT–β-catenin signalling. This work reveals a novel SAM domain polymerization mode, illustrates how supramolecular assembly controls catalytic and non-catalytic functions, provides important structural insights into the regulation of a non-DNA-dependent poly-ADP-ribosyltransferase and will guide future efforts to modulate tankyrase and decipher its contribution to disease mechanisms.

Open Access

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Abstract: The structure of the dimeric ATP synthase from bovine mitochondria determined in three rotational states by electron cryo-microscopy provides evidence that the proton uptake from the mitochondrial matrix via the proton inlet half channel proceeds via a Grotthus mechanism, and a similar mechanism may operate in the exit half channel. The structure has given information about the architecture and mechanical constitution and properties of the peripheral stalk, part of the membrane extrinsic region of the stator, and how the action of the peripheral stalk damps the side-to-side rocking motions that occur in the enzyme complex during the catalytic cycle. It also describes wedge structures in the membrane domains of each monomer, where the skeleton of each wedge is provided by three α-helices in the membrane domains of the b-subunit to which the supernumerary subunits e, f, and g and the membrane domain of subunit A6L are bound. Protein voids in the wedge are filled by three specifically bound cardiolipin molecules and two other phospholipids. The external surfaces of the wedges link the monomeric complexes together into the dimeric structures and provide a pivot to allow the monomer–monomer interfaces to change during catalysis and to accommodate other changes not related directly to catalysis in the monomer–monomer interface that occur in mitochondrial cristae. The structure of the bovine dimer also demonstrates that the structures of dimeric ATP synthases in a tetrameric porcine enzyme have been seriously misinterpreted in the membrane domains.