Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling - dataset2
Ontology highlight
ABSTRACT:
SUBMITTER: Hauke S. Hillen
PROVIDER: EMPIAR-11317 | biostudies-other |
REPOSITORIES: biostudies-other
Publications
Cryo-EM for Structure Determination of Mitochondrial Ribosome Samples.
Methods in molecular biology (Clifton, N.J.) 20230101
Single-particle cryoelectron microscopy (cryo-EM) allows structure determination of large macromolecular complexes from conformationally and compositionally heterogeneous mixtures of particles. This technique has been used to reveal the architecture of the mitochondrial ribosome and to visualize transient states that occur during the translation cycle or during mitoribosome biogenesis. Here, we outline an exemplary workflow for the analysis of single-particle cryo-EM data of human mitoribosome s ...[more]
Similar Datasets
Project description:Ribosome biogenesis requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. Particularly, maturation of the peptidyl transferase center (PTC) is mediated by conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial large ribosomal subunit (mtLSU) using endogenous complex purification, in vitro reconstitution and cryo-EM. Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch and progression to a near-mature PTC state. Additionally, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results provide a framework for understanding step-wise PTC folding as a critical conserved quality control checkpoint.
| S-EPMC8209004 | biostudies-literature
Project description:The bacterial ribosome is recycled into subunits by two conserved proteins, elongation factor G (EF-G) and the ribosome recycling factor (RRF). The molecular basis for ribosome recycling by RRF and EF-G remains unclear. Here, we report the crystal structure of a posttermination Thermus thermophilus 70S ribosome complexed with EF-G, RRF and two transfer RNAs at a resolution of 3.5 Å. The deacylated tRNA in the peptidyl (P) site moves into a previously unsuspected state of binding (peptidyl/recycling, p/R) that is analogous to that seen during initiation. The terminal end of the p/R-tRNA forms nonfavorable contacts with the 50S subunit while RRF wedges next to central inter-subunit bridges, illuminating the active roles of tRNA and RRF in dissociation of ribosomal subunits. The structure uncovers a missing snapshot of tRNA as it transits between the P and exit (E) sites, providing insights into the mechanisms of ribosome recycling and tRNA translocation.
| S-EPMC9150878 | biostudies-literature
Project description:Ribosome-driven protein biosynthesis is comprised of four phases: initiation, elongation, termination and recycling. In bacteria, ribosome recycling requires ribosome recycling factor and elongation factor G, and several structures of bacterial recycling complexes have been determined. In the eukaryotic and archaeal kingdoms, however, recycling involves the ABC-type ATPase ABCE1 and little is known about its structural basis. Here we present cryo-electron microscopy reconstructions of eukaryotic and archaeal ribosome recycling complexes containing ABCE1 and the termination factor paralogue Pelota. These structures reveal the overall binding mode of ABCE1 to be similar to canonical translation factors. Moreover, the iron-sulphur cluster domain of ABCE1 interacts with and stabilizes Pelota in a conformation that reaches towards the peptidyl transferase centre, thus explaining how ABCE1 may stimulate peptide-release activity of canonical termination factors. Using the mechanochemical properties of ABCE1, a conserved mechanism in archaea and eukaryotes is suggested that couples translation termination to recycling, and eventually to re-initiation.
| S-EPMC6878762 | biostudies-literature
Project description:Mammalian mitochondrial ribosomes (mitoribosomes) are responsible for synthesizing proteins that are essential for oxidative phosphorylation (ATP generation). Despite their common ancestry with bacteria, the composition and structure of the human mitoribosome and its translational factors are significantly different from those of their bacterial counterparts. The mammalian mitoribosome recycling factor (RRFmt) carries a mito-specific N terminus extension (NTE), which is necessary for the function of RRFmt Here we present a 3.9-Å resolution cryo-electron microscopic (cryo-EM) structure of the human 55S mitoribosome-RRFmt complex, which reveals α-helix and loop structures for the NTE that makes multiple mito-specific interactions with functionally critical regions of the mitoribosome. These include ribosomal RNA segments that constitute the peptidyl transferase center (PTC) and those that connect PTC with the GTPase-associated center and with mitoribosomal proteins L16 and L27. Our structure reveals the presence of a tRNA in the pe/E position and a rotation of the small mitoribosomal subunit on RRFmt binding. In addition, we observe an interaction between the pe/E tRNA and a mito-specific protein, mL64. These findings help understand the unique features of mitoribosome recycling.
| S-EPMC6486771 | biostudies-literature
Project description:Mitochondrial fission is a critical cellular event to maintain organelle function. This multistep process is initiated by the enhanced recruitment and oligomerization of dynamin-related protein 1 (Drp1) at the surface of mitochondria. As such, Drp1 is essential for inducing mitochondrial division in mammalian cells, and homologous proteins are found in all eukaryotes. As a member of the dynamin superfamily of proteins (DSPs), controlled Drp1 self-assembly into large helical polymers stimulates its GTPase activity to promote membrane constriction. Still, little is known about the mechanisms that regulate correct spatial and temporal assembly of the fission machinery. Here we present a cryo-EM structure of a full-length Drp1 dimer in an auto-inhibited state. This dimer reveals two key conformational rearrangements that must be unlocked through intramolecular rearrangements to achieve the assembly-competent state observed in previous structures. This structural insight provides understanding into the mechanism for regulated self-assembly of the mitochondrial fission machinery.
| S-EPMC10864337 | biostudies-literature
Project description:Efficient adaptation to environmental changes is pivotal for all bacterial cells. Almost all bacterial species depend on the conserved stringent response system to prompt timely transcriptional and metabolic responses according to stress conditions and nutrient depletion. The stringent response relies on the stress-dependent synthesis of the second messenger nucleotides and alarmones (p)ppGpp, which pleiotropically target and reprogram processes that consume cellular resources, such as ribosome biogenesis. Here we show that (p)ppGpp acts on the ribosome biogenesis GTPase A (RbgA) of Gram-positive bacteria. Using X-ray crystallography, hydrogen-deuterium exchange MS (HDX-MS) and kinetic analysis, we demonstrate that the alarmones (p)ppGpp bind to RbgA in a manner similar to that of binding by GDP and GTP and thereby act as competitive inhibitors. Our structural analysis of Staphylococcus aureus RbgA bound to ppGpp and pppGpp at 1.8 and 1.65 Å resolution, respectively, suggested that the alarmones (p)ppGpp prevent the active GTPase conformation of RbgA by sterically blocking the association of its G2 motif via their 3'-pyrophosphate moieties. Taken together, our structural and biochemical characterization of RbgA in the context of the alarmone-mediated stringent response reveals how (p)ppGpp affects the function of RbgA and reprograms this GTPase to arrest the ribosomal large subunit.
| S-EPMC6314131 | biostudies-literature
Project description:Eukaryotic ribosome biogenesis begins with the co-transcriptional assembly of the 90S pre-ribosome. The 'U three protein' (UTP) complexes and snoRNP particles arrange around the nascent pre-ribosomal RNA chaperoning its folding and further maturation. The earliest event in this hierarchical process is the binding of the UTP-A complex to the 5'-end of the pre-ribosomal RNA (5'-ETS). This oligomeric complex predominantly consists of β-propeller and α-solenoidal proteins. Here we present the structure of the Utp4 subunit from the thermophilic fungus Chaetomium thermophilum at 2.15 Å resolution and analyze its function by UV RNA-crosslinking (CRAC) and in context of a recent cryo-EM structure of the 90S pre-ribosome. Utp4 consists of two orthogonal and highly basic β-propellers that perfectly fit the EM-data. The Utp4 structure highlights an unusual Velcro-closure of its C-terminal β-propeller as relevant for protein integrity and potentially Utp8 recognition in the context of the pre-ribosome. We provide a first model of the 5'-ETS RNA from the internally hidden 5'-end up to the region that hybridizes to the 3'-hinge sequence of U3 snoRNA and validate a specific Utp4/5'-ETS interaction by CRAC analysis.
| S-EPMC5456268 | biostudies-literature
Project description:Free L-tryptophan (L-Trp) stalls ribosomes engaged in the synthesis of TnaC, a leader peptide controlling the expression of the Escherichia coli tryptophanase operon. Despite extensive characterization, the molecular mechanism underlying the recognition and response to L-Trp by the TnaC-ribosome complex remains unknown. Here, we use a combined biochemical and structural approach to characterize a TnaC variant (R23F) with greatly enhanced sensitivity for L-Trp. We show that the TnaC-ribosome complex captures a single L-Trp molecule to undergo termination arrest and that nascent TnaC prevents the catalytic GGQ loop of release factor 2 from adopting an active conformation at the peptidyl transferase center. Importantly, the L-Trp binding site is not altered by the R23F mutation, suggesting that the relative rates of L-Trp binding and peptidyl-tRNA cleavage determine the tryptophan sensitivity of each variant. Thus, our study reveals a strategy whereby a nascent peptide assists the ribosome in detecting a small metabolite.
| S-EPMC8429421 | biostudies-literature
Project description:At the end of translation in bacteria, ribosome recycling factor (RRF) is used together with elongation factor G to recycle the 30S and 50S ribosomal subunits for the next round of translation. In x-ray crystal structures of RRF with the Escherichia coli 70S ribosome, RRF binds to the large ribosomal subunit in the cleft that contains the peptidyl transferase center. Upon binding of either E. coli or Thermus thermophilus RRF to the E. coli ribosome, the tip of ribosomal RNA helix 69 in the large subunit moves away from the small subunit toward RRF by 8 A, thereby disrupting a key contact between the small and large ribosomal subunits termed bridge B2a. In the ribosome crystals, the ability of RRF to destabilize bridge B2a is influenced by crystal packing forces. Movement of helix 69 involves an ordered-to-disordered transition upon binding of RRF to the ribosome. The disruption of bridge B2a upon RRF binding to the ribosome seen in the present structures reveals one of the key roles that RRF plays in ribosome recycling, the dissociation of 70S ribosomes into subunits. The structures also reveal contacts between domain II of RRF and protein S12 in the 30S subunit that may also play a role in ribosome recycling.
| S-EPMC2712656 | biostudies-literature
Project description:The universally conserved process of protein biosynthesis is crucial for maintaining cellular homoeostasis and in eukaryotes, mitochondrial translation is essential for aerobic energy production. Mitochondrial ribosomes (mitoribosomes) are highly specialized to synthesize 13 core subunits of the oxidative phosphorylation (OXPHOS) complexes. Although the mitochondrial translation machinery traces its origin from a bacterial ancestor, it has acquired substantial differences within this endosymbiotic environment. The cycle of mitoribosome function proceeds through the conserved canonical steps of initiation, elongation, termination and mitoribosome recycling. However, when mitoribosomes operate in the context of limited translation factors or on aberrant mRNAs, they can become stalled and activation of rescue mechanisms is required. This review summarizes recent advances in the understanding of protein biosynthesis in mitochondria, focusing especially on the mechanistic and physiological details of translation termination, and mitoribosome recycling and rescue.
| S-EPMC8786322 | biostudies-literature