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Single-particle cryogenic electron microscopy (cryo-EM) has become a standard technique for determining protein structures at atomic resolution 1 – 3 . However, cryo-EM studies of protein-free RNA are in their early days. The Tetrahymena thermophila group I self-splicing intron was the first ribozyme to be discovered and has been a prominent model system for the study of RNA catalysis and structure–function relationships 4 , but its full structure remains unknown. Here we report cryo-EM structures of the full-length Tetrahymena ribozyme in substrate-free and bound states at a resolution of 3.1 Å. Newly resolved peripheral regions form two coaxially stacked helices; these are interconnected by two kissing loop pseudoknots that wrap around the catalytic core and include two previously unforeseen (to our knowledge) tertiary interactions. The global architecture is nearly identical in both states; only the internal guide sequence and guanosine binding site undergo a large conformational change and a localized shift, respectively, upon binding of RNA substrates. These results provide a long-sought structural view of a paradigmatic RNA enzyme and signal a new era for the cryo-EM-based study of structure–function relationships in ribozymes. Cryo-electron microscopy has been used to determine the structure of the Tetrahymena ribozyme (a catalytic RNA) at sufficiently high resolution to model side chains and metal ions.

Telomerase adds telomeric repeats to chromosome ends using an internal RNA template and a specialized telomerase reverse transcriptase (TERT), thereby maintaining genome integrity. Little is known about the physical relationships among protein and RNA subunits within a biologically functional holoenzyme. Here we describe the architecture of Tetrahymena thermophila telomerase holoenzyme determined by electron microscopy. Six of the seven proteins and the TERT-binding regions of telomerase RNA (TER) have been localized by affinity labelling. Fitting with high-resolution structures reveals the organization of TERT, TER and p65 in the ribonucleoprotein (RNP) catalytic core. p50 has an unanticipated role as a hub between the RNP catalytic core, p75–p19–p45 subcomplex, and the DNA-binding Teb1. A complete in vitro holoenzyme reconstitution assigns function to these interactions in processive telomeric repeat synthesis. These studies provide the first view of the extensive network of subunit associations necessary for telomerase holoenzyme assembly and physiological function. The long-awaited structure of a telomerase holoenzyme, from Tetrahymena , has been obtained by electron microscopy; affinity labelling of subunits and modelling with NMR and crystal structures of various components allowed the identification of the catalytic core and subunit interactions, and the functional role of the subunits in telomerase processivity was enabled by performing the first reconstitution of the holoenzyme in vitro . Telomerase structure revealed The ends of linear chromosomes in eukaryotes are maintained by a ribonucleoprotein complex known as telomerase. Juli Feigon and colleagues have obtained a long-awaited structure of the ciliate ( Tetrahymena thermophila ) telomerase by electron microscopy. Modelling with crystal structures of various components allowed identification of the catalytic core and subunit interactions, and a complete in vitro reconstitution of the enzyme was performed. The structure provides unprecedented detail about the architecture of the ribonucleoprotein catalytic core and reveals the organization of holoenzyme subunits that confer processivity and bridge telomerase to telomeres.

Mitochondrial ATP synthases form functional homodimers to induce cristae curvature that is a universal property of mitochondria. To expand on the understanding of this fundamental phenomenon, we characterized the unique type III mitochondrial ATP synthase in its dimeric and tetrameric form. The cryo-EM structure of a ciliate ATP synthase dimer reveals an unusual U-shaped assembly of 81 proteins, including a substoichiometrically bound ATPTT2, 40 lipids, and co-factors NAD and CoQ. A single copy of subunit ATPTT2 functions as a membrane anchor for the dimeric inhibitor IF 1 . Type III specific linker proteins stably tie the ATP synthase monomers in parallel to each other. The intricate dimer architecture is scaffolded by an extended subunit- a that provides a template for both intra- and inter-dimer interactions. The latter results in the formation of tetramer assemblies, the membrane part of which we determined to 3.1 Å resolution. The structure of the type III ATP synthase tetramer and its associated lipids suggests that it is the intact unit propagating the membrane curvature. Mitochondrial ATP synthases are involved in shaping the mitochondrial cristae. The cryo-EM structure of type III ATP synthase reveals the architecture of the unusual, asymmetrical, U-shaped dimer and offers insights into the interaction with the natural inhibitor IF1 and membrane lipids. The structure of the enzyme tetramer suggests the mechanism of membrane curvature generation.

Telomerase is unique among the reverse transcriptases in containing a noncoding RNA (known as telomerase RNA (TER)) that includes a short template that is used for the processive synthesis of G-rich telomeric DNA repeats at the 3′ ends of most eukaryotic chromosomes 1 . Telomerase maintains genomic integrity, and its activity or dysregulation are critical determinants of human longevity, stem cell renewal and cancer progression 2 , 3 . Previous cryo-electron microscopy structures have established the general architecture, protein components and stoichiometries of Tetrahymena and human telomerase, but our understandings of the details of DNA–protein and RNA–protein interactions and of the mechanisms and recruitment involved remain limited 4 – 6 . Here we report cryo-electron microscopy structures of active Tetrahymena telomerase with telomeric DNA at different steps of nucleotide addition. Interactions between telomerase reverse transcriptase (TERT), TER and DNA reveal the structural basis of the determination of the 5′ and 3′ template boundaries, handling of the template–DNA duplex and separation of the product strand during nucleotide addition. The structure and binding interface between TERT and telomerase protein p50 (a homologue of human TPP1 7 , 8 ) define conserved interactions that are required for telomerase activation and recruitment to telomeres. Telomerase La-related protein p65 remodels several regions of TER, bridging the 5′ and 3′ ends and the conserved pseudoknot to facilitate assembly of the TERT–TER catalytic core. Cryo-electron microscopy structures of Tetrahymena telomerase with telomeric DNA at several steps of nucleotide addition provide insights into the structural basis of telomere repeat synthesis.

Telomerase is a ribonucleoprotein complex that counteracts the shortening of chromosome ends due to incomplete replication. Telomerase contains a catalytic core of telomerase reverse transcriptase (TERT) and telomerase RNA (TER). However, what defines TERT and separates it from other reverse transcriptases remains a subject of debate. A recent cryoelectron microscopy map of Tetrahymena telomerase revealed the structure of a previously uncharacterized TERT domain (TRAP) with unanticipated interactions with the telomerase essential N-terminal (TEN) domain and roles in telomerase activity. Both TEN and TRAP are absent in the putative Tribolium TERT that has been used as a model for telomerase for over a decade. To investigate the conservation of TRAP and TEN across species, we performed multiple sequence alignments and statistical coupling analysis on all identified TERTs and find that TEN and TRAP have coevolved as telomerase-specific domains. Integrating the data from bioinformatic analysis and the structure of Tetrahymena telomerase, we built a pseudoatomic model of human telomerase catalytic core that accounts for almost all of the cryoelectron microscopy density in a published map, including TRAP in previously unassigned density as well as telomerase RNA domains essential for activity. This more complete model of the human telomerase catalytic core illustrates how domains of TER and TERT, including the TEN–TRAP complex, can interact in a conserved manner to regulate telomere synthesis.

Lipin/Pah phosphatidic acid phosphatases (PAPs) generate diacylglycerol to regulate triglyceride synthesis and cellular signaling. Inactivating mutations cause rhabdomyolysis, autoinflammatory disease, and aberrant fat storage. Disease-mutations cluster within the conserved N-Lip and C-Lip regions that are separated by 500-residues in humans. To understand how the N-Lip and C-Lip combine for PAP function, we determined crystal structures of Tetrahymena thermophila Pah2 ( Tt Pah2) that directly fuses the N-Lip and C-Lip. Tt Pah2 adopts a two-domain architecture where the N-Lip combines with part of the C-Lip to form an immunoglobulin-like domain and the remaining C-Lip forms a HAD-like catalytic domain. An N-Lip C-Lip fusion of mouse lipin-2 is catalytically active, which suggests mammalian lipins function with the same domain architecture as Tt Pah2. HDX-MS identifies an N-terminal amphipathic helix essential for membrane association. Disease-mutations disrupt catalysis or destabilize the protein fold. This illustrates mechanisms for lipin/Pah PAP function, membrane association, and lipin-related pathologies. Lipin/Pah phosphatidic acid phosphatases generate diacylglycerol to regulate triglyceride synthesis and cellular signaling. Here authors determine structures of Tetrahymena thermophila Pah2 and identify an N-terminal amphipathic helix essential for membrane association.

Poly-ADP-ribosylation is a post-translational modification that regulates processes involved in genome stability. Breakdown of the poly(ADP-ribose) (PAR) polymer is catalysed by poly(ADP-ribose) glycohydrolase (PARG), whose endo -glycohydrolase activity generates PAR fragments. Here we present the crystal structure of PARG incorporating the PAR substrate. The two terminal ADP-ribose units of the polymeric substrate are bound in exo -mode. Biochemical and modelling studies reveal that PARG acts predominantly as an exo -glycohydrolase. This preference is linked to Phe902 (human numbering), which is responsible for low-affinity binding of the substrate in endo -mode. Our data reveal the mechanism of poly-ADP-ribosylation reversal, with ADP-ribose as the dominant product, and suggest that the release of apoptotic PAR fragments occurs at unusual PAR/PARG ratios. Poly-ADP-ribosylation is a post-translational modification that is countered by poly(ADP-ribose) glycohydrolases (PARGs). In this study, the authors present the crystal structure of poly(ADP-ribose) glycohydrolase (PARGs) in complex with a poly(ADP-ribose) substrate, and reveal that poly(ADP-ribose) glycohydrolase (PARGs) enzymes act predominantly as exo - rather than as endo -glycohydrolases.

Glutathione-S-transferase enzymes (GSTs) are essential components of the phase II detoxification system and protect organisms from oxidative stress induced by xenobiotics and harmful toxins such as 1-chloro-2,4-dinitrobenzene (CDNB). In Tetrahymena thermophila, the TtGSTm34 gene was previously reported to be one of the most responsive GST genes to CDNB treatment (LD50 = 0.079 mM). This study aimed to determine the kinetic features of recombinantly expressed and purified TtGSTm34 with CDNB and glutathione (GSH). TtGSTm34-8xHis was recombinantly produced in T. thermophila as a 25-kDa protein after the cloning of the 660-bp full-length ORF of TtGSTm34 into the pIGF-1 vector. A three-dimensional model of the TtGSTm34 protein constructed by the AlphaFold and PyMOL programs confirmed that it has structurally conserved and folded GST domains. The recombinant production of TtGSTm34-8xHis was confirmed by SDS‒PAGE and Western blot analysis. A dual-affinity chromatography strategy helped to purify TtGSTm34-8xHis approximately 3166-fold. The purified recombinant TtGSTm34-8xHis exhibited significantly high enzyme activity with CDNB (190 µmol/min/mg) as substrate. Enzyme kinetic analysis revealed Km values of 0.68 mM with GSH and 0.40 mM with CDNB as substrates, confirming its expected high affinity for CDNB. The optimum pH and temperature were determined to be 7.0 and 25 °C, respectively. Ethacrynic acid inhibited fully TtGSTm34-8xHis enzyme activity. These results imply that TtGSTm34 of T. thermophila plays a major role in the detoxification of xenobiotics, such as CDNB, as a first line of defense in aquatic protists against oxidative damage.

Polyglutamylation of tubulin has been implicated in several functions of microtubules, but the identification of the responsible enzyme(s) has been challenging. We found that the neuronal tubulin polyglutamylase is a protein complex containing a tubulin tyrosine ligase-like (TTLL) protein, TTLL1. TTLL1 is a member of a large family of proteins with a TTL homology domain, whose members could catalyze ligations of diverse amino acids to tubulins or other substrates. In the model protist Tetrahymena thermophila, two conserved types of polyglutamylases were characterized that differ in substrate preference and subcellular localization.

Based on observations of markers for DNA lesions, such as phosphorylated histone H2AX (γH2AX) and open DNA ends, it has been suggested that post-meiotic DNA double-strand breaks (PM-DSBs) enable chromatin remodeling during animal spermiogenesis. However, the existence of PM-DSBs is unconfirmed, and the mechanism responsible for their formation is unclear. Here, we report the first direct observation of programmed PM-DSBs via the electrophoretic separation of DSB-generated DNA fragments in the ciliate Tetrahymena thermophila. These PM-DSBs are accompanied by switching from a heterochromatic to euchromatic chromatin structure in the haploid pronucleus. Both a topoisomerase II paralog with exclusive pronuclear expression and Spo11 are prerequisites for PM-DSB induction. Reduced PM-DSB induction blocks euchromatin formation, characterized by histone H3K56 acetylation, leading to a failure in gametic nuclei production. We propose that PM-DSBs are responsible for histone replacement during the reprogramming of generative to undifferentiated progeny nuclei.