EGOC inhibits TOROID polymerization by structurally activating TORC1.

Target of rapamycin complex 1 (TORC1) is a protein kinase controlling cell homeostasis and growth in response to nutrients and stresses. In Saccharomyces cerevisiae, glucose depletion triggers a redistribution of TORC1 from a dispersed localization over the vacuole surface into a large, inactive condensate called TOROID (TORC1 organized in inhibited domains). However, the mechanisms governing this transition have been unclear. Here, we show that acute depletion and repletion of EGO complex (EGOC) activity is sufficient to control TOROID distribution, independently of other nutrient-signaling pathways. The 3.9-Å-resolution structure of TORC1 from TOROID cryo-EM data together with interrogation of key interactions in vivo provide structural insights into TORC1-TORC1' and TORC1-EGOC interaction interfaces. These data support a model in which glucose-dependent activation of EGOC triggers binding to TORC1 at an interface required for TOROID assembly, preventing TORC1 polymerization and promoting release of active TORC1.

Cryo-EM structure of the SEA complex.

The SEA complex (SEAC) is a growth regulator that acts as a GTPase-activating protein (GAP) towards Gtr1, a Rag GTPase that relays nutrient status to the Target of Rapamycin Complex 1 (TORC1) in yeast1. Functionally, the SEAC has been divided into two subcomplexes: SEACIT, which has GAP activity and inhibits TORC1, and SEACAT, which regulates SEACIT2. This system is conserved in mammals: the GATOR complex, consisting of GATOR1 (SEACIT) and GATOR2 (SEACAT), transmits amino acid3 and glucose4 signals to mTORC1. Despite its importance, the structure of SEAC/GATOR, and thus molecular understanding of its function, is lacking. Here, we solve the cryo-EM structure of the native eight-subunit SEAC. The SEAC has a modular structure in which a COPII-like cage corresponding to SEACAT binds two flexible wings, which correspond to SEACIT. The wings are tethered to the core via Sea3, which forms part of both modules. The GAP mechanism of GATOR1 is conserved in SEACIT, and GAP activity is unaffected by SEACAT in vitro. In vivo, the wings are essential for recruitment of the SEAC to the vacuole, primarily via the EGO complex. Our results indicate that rather than being a direct inhibitor of SEACIT, SEACAT acts as a scaffold for the binding of TORC1 regulators.

The connecting cilium inner scaffold provides a structural foundation that protects against retinal degeneration.

Inherited retinal degeneration due to loss of photoreceptor cells is a leading cause of human blindness. These cells possess a photosensitive outer segment linked to the cell body through the connecting cilium (CC). While structural defects of the CC have been associated with retinal degeneration, its nanoscale molecular composition, assembly, and function are barely known. Here, using expansion microscopy and electron microscopy, we reveal the molecular architecture of the CC and demonstrate that microtubules are linked together by a CC inner scaffold containing POC5, CENTRIN, and FAM161A. Dissecting CC inner scaffold assembly during photoreceptor development in mouse revealed that it acts as a structural zipper, progressively bridging microtubule doublets and straightening the CC. Furthermore, we show that Fam161a disruption in mouse leads to specific CC inner scaffold loss and triggers microtubule doublet spreading, prior to outer segment collapse and photoreceptor degeneration, suggesting a molecular mechanism for a subtype of retinitis pigmentosa.

Cryo-EM snapshots of a native lysate provide structural insights into a metabolon-embedded transacetylase reaction.

Found across all kingdoms of life, 2-keto acid dehydrogenase complexes possess prominent metabolic roles and form major regulatory sites. Although their component structures are known, their higher-order organization is highly heterogeneous, not only across species or tissues but also even within a single cell. Here, we report a cryo-EM structure of the fully active Chaetomium thermophilum pyruvate dehydrogenase complex (PDHc) core scaffold at 3.85 Å resolution (FSC = 0.143) from native cell extracts. By combining cryo-EM with macromolecular docking and molecular dynamics simulations, we resolve all PDHc core scaffold interfaces and dissect the residing transacetylase reaction. Electrostatics attract the lipoyl domain to the transacetylase active site and stabilize the coenzyme A, while apolar interactions position the lipoate in its binding cleft. Our results have direct implications on the structural determinants of the transacetylase reaction and the role of flexible regions in the context of the overall 10 MDa PDHc metabolon architecture.

Structural basis of human separase regulation by securin and CDK1-cyclin B1.

In early mitosis, the duplicated chromosomes are held together by the ring-shaped cohesin complex1. Separation of chromosomes during anaphase is triggered by separase-a large cysteine endopeptidase that cleaves the cohesin subunit SCC1 (also known as RAD212-4). Separase is activated by degradation of its inhibitors, securin5 and cyclin B6, but the molecular mechanisms of separase regulation are not clear. Here we used cryogenic electron microscopy to determine the structures of human separase in complex with either securin or CDK1-cyclin B1-CKS1. In both complexes, separase is inhibited by pseudosubstrate motifs that block substrate binding at the catalytic site and at nearby docking sites. As in Caenorhabditis elegans7 and yeast8, human securin contains its own pseudosubstrate motifs. By contrast, CDK1-cyclin B1 inhibits separase by deploying pseudosubstrate motifs from intrinsically disordered loops in separase itself. One autoinhibitory loop is oriented by CDK1-cyclin B1 to block the catalytic sites of both separase and CDK19,10. Another autoinhibitory loop blocks substrate docking in a cleft adjacent to the separase catalytic site. A third separase loop contains a phosphoserine6 that promotes complex assembly by binding to a conserved phosphate-binding pocket in cyclin B1. Our study reveals the diverse array of mechanisms by which securin and CDK1-cyclin B1 bind and inhibit separase, providing the molecular basis for the robust control of chromosome segregation.

The Hsp70-Hsp90 co-chaperone Hop/Stip1 shifts the proteostatic balance from folding towards degradation.

Hop/Stip1/Sti1 is thought to be essential as a co-chaperone to facilitate substrate transfer between the Hsp70 and Hsp90 molecular chaperones. Despite this proposed key function for protein folding and maturation, it is not essential in a number of eukaryotes and bacteria lack an ortholog. We set out to identify and to characterize its eukaryote-specific function. Human cell lines and the budding yeast with deletions of the Hop/Sti1 gene display reduced proteasome activity due to inefficient capping of the core particle with regulatory particles. Unexpectedly, knock-out cells are more proficient at preventing protein aggregation and at promoting protein refolding. Without the restraint by Hop, a more efficient folding activity of the prokaryote-like Hsp70-Hsp90 complex, which can also be demonstrated in vitro, compensates for the proteasomal defect and ensures the proteostatic equilibrium. Thus, cells may act on the level and/or activity of Hop to shift the proteostatic balance between folding and degradation.

Architecture of the centriole cartwheel-containing region revealed by cryo-electron tomography.

Centrioles are evolutionarily conserved barrels of microtubule triplets that form the core of the centrosome and the base of the cilium. While the crucial role of the proximal region in centriole biogenesis has been well documented, its native architecture and evolutionary conservation remain relatively unexplored. Here, using cryo-electron tomography of centrioles from four evolutionarily distant species, we report on the architectural diversity of the centriole's proximal cartwheel-bearing region. Our work reveals that the cartwheel central hub is constructed from a stack of paired rings with cartwheel inner densities inside. In both Paramecium and Chlamydomonas, the repeating structural unit of the cartwheel has a periodicity of 25 nm and consists of three ring pairs, with 6 radial spokes emanating and merging into a single bundle that connects to the microtubule triplet via the D2-rod and the pinhead. Finally, we identified that the cartwheel is indirectly connected to the A-C linker through the triplet base structure extending from the pinhead. Together, our work provides unprecedented evolutionary insights into the architecture of the centriole proximal region, which underlies centriole biogenesis.

Molecular insight into RNA polymerase I promoter recognition and promoter melting.

RNA polymerase I (Pol I) assembles with core factor (CF) and Rrn3 on the rDNA core promoter for transcription initiation. Here, we report cryo-EM structures of closed, intermediate and open Pol I initiation complexes from 2.7 to 3.7 Å resolution to visualize Pol I promoter melting and to structurally and biochemically characterize the recognition mechanism of Pol I promoter DNA. In the closed complex, double-stranded DNA runs outside the DNA-binding cleft. Rotation of CF and upstream DNA with respect to Pol I and Rrn3 results in the spontaneous loading and opening of the promoter followed by cleft closure and positioning of the Pol I A49 tandem winged helix domain (tWH) onto DNA. Conformational rearrangement of A49 tWH leads to a clash with Rrn3 to initiate complex disassembly and promoter escape. Comprehensive insight into the Pol I transcription initiation cycle allows comparisons with promoter opening by Pol II and Pol III.

The cryo-EM structure of a 12-subunit variant of RNA polymerase I reveals dissociation of the A49-A34.5 heterodimer and rearrangement of subunit A12.2.

RNA polymerase (Pol) I is a 14-subunit enzyme that solely transcribes pre-ribosomal RNA. Cryo-electron microscopy (EM) structures of Pol I initiation and elongation complexes have given first insights into the molecular mechanisms of Pol I transcription. Here, we present cryo-EM structures of yeast Pol I elongation complexes (ECs) bound to the nucleotide analog GMPCPP at 3.2 to 3.4 Å resolution that provide additional insight into the functional interplay between the Pol I-specific transcription-like factors A49-A34.5 and A12.2. Strikingly, most of the nucleotide-bound ECs lack the A49-A34.5 heterodimer and adopt a Pol II-like conformation, in which the A12.2 C-terminal domain is bound in a previously unobserved position at the A135 surface. Our structural and biochemical data suggest a mechanism where reversible binding of the A49-A34.5 heterodimer could contribute to the regulation of Pol I transcription initiation and elongation.

The cryo-EM resolution revolution and transcription complexes.

Direct electron detector technology combined with improved imaging processing procedures has dramatically increased the resolution that can be obtained by single-particle cryo-electron microscopy and cryo-electron tomography. These developments-often referred to as the `resolution revolution' in cryo-EM-have had a profound impact on the structural biology of transcription as they allow the determination of atomic or near-atomic resolution structures of very large, flexible and often transient transcription complexes that in many cases had resisted crystal structure determination for decades. In this review, we will discuss recent advances and breakthroughs in the structural biology of transcription complexes enabled by the revolution in cryo-electron microscopy with particular focus on eukaryotic RNA polymerases and their pre-initiation complexes, but also chromatin remodelers and epigenetic regulators.

Structural insights into transcription initiation by yeast RNA polymerase I.

In eukaryotic cells, RNA polymerase I (Pol I) synthesizes precursor ribosomal RNA (pre-rRNA) that is subsequently processed into mature rRNA. To initiate transcription, Pol I requires the assembly of a multi-subunit pre-initiation complex (PIC) at the ribosomal RNA promoter. In yeast, the minimal PIC includes Pol I, the transcription factor Rrn3, and Core Factor (CF) composed of subunits Rrn6, Rrn7, and Rrn11. Here, we present the cryo-EM structure of the 18-subunit yeast Pol I PIC bound to a transcription scaffold. The cryo-EM map reveals an unexpected arrangement of the DNA and CF subunits relative to Pol I. The upstream DNA is positioned differently than in any previous structures of the Pol II PIC. Furthermore, the TFIIB-related subunit Rrn7 also occupies a different location compared to the Pol II PIC although it uses similar interfaces as TFIIB to contact DNA. Our results show that although general features of eukaryotic transcription initiation are conserved, Pol I and Pol II use them differently in their respective transcription initiation complexes.

Molecular Structures of Transcribing RNA Polymerase I.

RNA polymerase I (Pol I) is a 14-subunit enzyme that solely synthesizes pre-ribosomal RNA. Recently, the crystal structure of apo Pol I gave unprecedented insight into its molecular architecture. Here, we present three cryo-EM structures of elongating Pol I, two at 4.0 Å and one at 4.6 Å resolution, and a Pol I open complex at 3.8 Å resolution. Two modules in Pol I mediate the narrowing of the DNA-binding cleft by closing the clamp domain. The DNA is bound by the clamp head and by the protrusion domain, allowing visualization of the upstream and downstream DNA duplexes in one of the elongation complexes. During formation of the Pol I elongation complex, the bridge helix progressively folds, while the A12.2 C-terminal domain is displaced from the active site. Our results reveal the conformational changes associated with elongation complex formation and provide additional insight into the Pol I transcription cycle.

Specialization versus conservation: How Pol I and Pol III use the conserved architecture of the pre-initiation complex for specialized transcription.

Here, we discuss the overall architecture of the RNA polymerase I (Pol I) and III (Pol III) core enzymes and their associated general transcription factors in the context of models of the Pol I and Pol III pre-initiation complexes, thereby highlighting potential functional adaptations of the Pol I and Pol III enzymes to their respective transcription tasks. Several new insights demonstrate the great degree of specialization of each of the eukaryotic RNA polymerases that is only beginning to be revealed as the structural and functional characterization of all eukaryotic RNA polymerases and their pre-initiation complexes progresses.

The role of Cdc42 and Gic1 in the regulation of septin filament formation and dissociation.

Septins are guanine nucleotide-binding proteins that polymerize into filamentous and higher-order structures. Cdc42 and its effector Gic1 are involved in septin recruitment, ring formation and dissociation. The regulatory mechanisms behind these processes are not well understood. Here, we have used electron microscopy and cryo electron tomography to elucidate the structural basis of the Gic1-septin and Gic1-Cdc42-septin interaction. We show that Gic1 acts as a scaffolding protein for septin filaments forming long and flexible filament cables. Cdc42 in its GTP-form binds to Gic1, which ultimately leads to the dissociation of Gic1 from the filament cables. Surprisingly, Cdc42-GDP is not inactive, but in the absence of Gic1 directly interacts with septin filaments resulting in their disassembly. We suggest that this unanticipated dual function of Cdc42 is crucial for the cell cycle. Based on our results we propose a novel regulatory mechanism for septin filament formation and dissociation. DOI: http://dx.doi.org/10.7554/eLife.01085.001.