scholarly journals Mechanism of membrane-tethered mitochondrial protein synthesis

Science ◽  
2021 ◽  
Vol 371 (6531) ◽  
pp. 846-849
Author(s):  
Yuzuru Itoh ◽  
Juni Andréll ◽  
Austin Choi ◽  
Uwe Richter ◽  
Priyanka Maiti ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Conformational Changes ◽  
Large Subunit ◽  
Mitochondrial Protein ◽  
Mitochondrial Ribosomes ◽  
Helix Formation ◽  
Mitochondrial Inner Membrane ◽  
Cryo Electron Microscopy ◽  
Nascent Chain ◽  
Exit Tunnel

Mitochondrial ribosomes (mitoribosomes) are tethered to the mitochondrial inner membrane to facilitate the cotranslational membrane insertion of the synthesized proteins. We report cryo–electron microscopy structures of human mitoribosomes with nascent polypeptide, bound to the insertase oxidase assembly 1–like (OXA1L) through three distinct contact sites. OXA1L binding is correlated with a series of conformational changes in the mitoribosomal large subunit that catalyze the delivery of newly synthesized polypeptides. The mechanism relies on the folding of mL45 inside the exit tunnel, forming two specific constriction sites that would limit helix formation of the nascent chain. A gap is formed between the exit and the membrane, making the newly synthesized proteins accessible. Our data elucidate the basis by which mitoribosomes interact with the OXA1L insertase to couple protein synthesis and membrane delivery.

scholarly journals Cardiolipin is required for membrane docking of mitochondrial ribosomes and protein synthesis

2020 ◽  
Vol 133 (14) ◽  
pp. jcs240374 ◽  
Author(s):  
Richard G. Lee ◽  
Junjie Gao ◽  
Stefan J. Siira ◽  
Anne-Marie Shearwood ◽  
Judith A. Ermer ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Stress Responses ◽  
Protein Complexes ◽  
Mitochondrial Protein ◽  
Mitochondrial Morphology ◽  
Mitochondrial Translation ◽  
Inner Membrane ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Inner Membrane ◽  
Mitochondrial Ribosome

ABSTRACTThe mitochondrial inner membrane contains a unique phospholipid known as cardiolipin (CL), which stabilises the protein complexes embedded in the membrane and supports its overall structure. Recent evidence indicates that the mitochondrial ribosome may associate with the inner membrane to facilitate co-translational insertion of the hydrophobic oxidative phosphorylation (OXPHOS) proteins into the inner membrane. We generated three mutant knockout cell lines for the CL biosynthesis gene Crls1 to investigate the effects of CL loss on mitochondrial protein synthesis. Reduced CL levels caused altered mitochondrial morphology and transcriptome-wide changes that were accompanied by uncoordinated mitochondrial translation rates and impaired respiratory chain supercomplex formation. Aberrant protein synthesis was caused by impaired formation and distribution of mitochondrial ribosomes. Reduction or loss of CL resulted in divergent mitochondrial and endoplasmic reticulum stress responses. We show that CL is required to stabilise the interaction of the mitochondrial ribosome with the membrane via its association with OXA1 (also known as OXA1L) during active translation. This interaction facilitates insertion of newly synthesised mitochondrial proteins into the inner membrane and stabilises the respiratory supercomplexes.

scholarly journals Negamycin Binds to the Wall of the Nascent Chain Exit Tunnel of the 50S Ribosomal Subunit

2007 ◽  
Vol 51 (12) ◽  
pp. 4462-4465 ◽  
Author(s):  
Susan J. Schroeder ◽  
Gregor Blaha ◽  
Peter B. Moore
Keyword(s):  
Protein Synthesis ◽  
Small Molecule ◽  
Ribosomal Subunit ◽  
Small Molecule Inhibitor ◽  
Single Site ◽  
Haloarcula Marismortui ◽  
Molecule Inhibitor ◽  
Nascent Chain ◽  
Exit Tunnel ◽  
Inhibitor Of Protein Synthesis

ABSTRACT Negamycin, a small-molecule inhibitor of protein synthesis, binds the Haloarcula marismortui 50S ribosomal subunit at a single site formed by highly conserved RNA nucleotides near the cytosolic end of the nascent chain exit tunnel. The mechanism of antibiotic action and the function of this unexplored tunnel region remain intriguingly elusive.

scholarly journals Structural consequences of the interaction of RbgA with a 50S ribosomal subunit assembly intermediate

2019 ◽  
Vol 47 (19) ◽  
pp. 10414-10425 ◽  
Author(s):  
Amal Seffouh ◽  
Nikhil Jain ◽  
Dushyant Jahagirdar ◽  
Kaustuv Basu ◽  
Aida Razi ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Catalytic Residue ◽  
Gtp Hydrolysis ◽  
Subunit Assembly ◽  
Cryo Electron Microscopy ◽  
Assembly Factor ◽  
Assembly Intermediate ◽  
Immature Particle

Abstract Bacteria harbor a number GTPases that function in the assembly of the ribosome and are essential for growth. RbgA is one of these GTPases and is required for the assembly of the 50S subunit in most bacteria. Homologs of this protein are also implicated in the assembly of the large subunit of the mitochondrial and eukaryotic ribosome. We present here the cryo-electron microscopy structure of RbgA bound to a Bacillus subtilis 50S subunit assembly intermediate (45SRbgA particle) that accumulates in cells upon RbgA depletion. Binding of RbgA at the P site of the immature particle stabilizes functionally important rRNA helices in the A and P-sites, prior to the completion of the maturation process of the subunit. The structure also reveals the location of the highly conserved N-terminal end of RbgA containing the catalytic residue Histidine 9. The derived model supports a mechanism of GTP hydrolysis, and it shows that upon interaction of RbgA with the 45SRbgA particle, Histidine 9 positions itself near the nucleotide potentially acting as the catalytic residue with minimal rearrangements. This structure represents the first visualization of the conformational changes induced by an assembly factor in a bacterial subunit intermediate.

scholarly journals The ribosome and its role in protein folding: looking through a magnifying glass

2017 ◽  
Vol 73 (6) ◽  
pp. 509-521 ◽  
Author(s):  
Abid Javed ◽  
John Christodoulou ◽  
Lisa D. Cabrita ◽  
Elena V. Orlova
Keyword(s):  
Protein Folding ◽  
Structural Biology ◽  
Polypeptide Chain ◽  
Structure And Function ◽  
Cellular Activity ◽  
Cryo Electron Microscopy ◽  
Nascent Chain ◽  
Exit Tunnel ◽  
Dynamics Simulations ◽  
And Function

Protein folding, a process that underpins cellular activity, begins co-translationally on the ribosome. During translation, a newly synthesized polypeptide chain enters the ribosomal exit tunnel and actively interacts with the ribosome elements – the r-proteins and rRNA that line the tunnel – prior to emerging into the cellular milieu. While understanding of the structure and function of the ribosome has advanced significantly, little is known about the process of folding of the emerging nascent chain (NC). Advances in cryo-electron microscopy are enabling visualization of NCs within the exit tunnel, allowing early glimpses of the interplay between the NC and the ribosome. Once it has emerged from the exit tunnel into the cytosol, the NC (still attached to its parent ribosome) can acquire a range of conformations, which can be characterized by NMR spectroscopy. Using experimental restraints within molecular-dynamics simulations, the ensemble of NC structures can be described. In order to delineate the process of co-translational protein folding, a hybrid structural biology approach is foreseeable, potentially offering a complete atomic description of protein folding as it occurs on the ribosome.

Mitoribosomal small subunit biogenesis in trypanosomes involves an extensive assembly machinery

Science ◽  
2019 ◽  
Vol 365 (6458) ◽  
pp. 1144-1149 ◽  
Author(s):  
Martin Saurer ◽  
David J. F. Ramrath ◽  
Moritz Niemann ◽  
Salvatore Calderaro ◽  
Céline Prange ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Conformational Changes ◽  
Large Scale ◽  
Small Subunit ◽  
Ribosomal Assembly ◽  
Mitochondrial Ribosomes ◽  
Ribonucleoprotein Complexes ◽  
Cryo Electron Microscopy ◽  
Number Of Factors ◽  
Cellular Machinery

Mitochondrial ribosomes (mitoribosomes) are large ribonucleoprotein complexes that synthesize proteins encoded by the mitochondrial genome. An extensive cellular machinery responsible for ribosome assembly has been described only for eukaryotic cytosolic ribosomes. Here we report that the assembly of the small mitoribosomal subunit in Trypanosoma brucei involves a large number of factors and proceeds through the formation of assembly intermediates, which we analyzed by using cryo–electron microscopy. One of them is a 4-megadalton complex, referred to as the small subunit assemblosome, in which we identified 34 factors that interact with immature ribosomal RNA (rRNA) and recognize its functionally important regions. The assembly proceeds through large-scale conformational changes in rRNA coupled with successive incorporation of mitoribosomal proteins, providing an example for the complexity of the ribosomal assembly process in mitochondria.

scholarly journals Visualising nascent chain dynamics at the ribosome exit tunnel by cryo-electron microscopy

2019 ◽  
Author(s):  
Abid Javed ◽  
Tomasz Wlodarski ◽  
Anaïs. M.E. Cassaignau ◽  
Lisa D. Cabrita ◽  
John Christodoulou ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Active Component ◽  
Chain Dynamics ◽  
Cryo Electron Microscopy ◽  
Chain Complexes ◽  
Nascent Chain ◽  
Immunoglobulin Domain ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel

Ribosomes maintain a healthy cellular proteome by synthesising proteins. The nascent chain (NC), emerges into the cellular milieu via the ribosomal exit tunnel, which is an active component that regulates the NC passage. How the NC dynamics at the exit tunnel affect NC folding remains to be an important question, the answer on which has strong implications to medicine. Here, we report high-resolution cryo-EM maps of ribosome nascent-chain complexes (RNCs) displaying distinct steps during biosynthesis. These RNC structures reveal a range of pathways adopted by the NC. The most pronounced diversity in the NC trajectories were found in the vestibule region. Rearrangements of several ribosomal components further suggest that these elements may actively monitor the emerging NC during translation. The ribosome-NC contacts within the vestibule define these NC pathways and modulate position of a folded immunoglobulin domain outside the ribosome.

scholarly journals Structure of the mature kinetoplastids mitoribosome and insights into its large subunit biogenesis

2020 ◽  
Vol 117 (47) ◽  
pp. 29851-29861 ◽  
Author(s):  
Heddy Soufari ◽  
Florent Waltz ◽  
Camila Parrot ◽  
Stéphanie Durrieu-Gaillard ◽  
Anthony Bochler ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Large Subunit ◽  
Microscopy Study ◽  
Small Subunit ◽  
Electron Microscopy Study ◽  
Mitochondrial Ribosomes ◽  
Cryo Electron Microscopy ◽  
Parasite Survival ◽  
Assembly Intermediate ◽  
Early Translation

Kinetoplastids are unicellular eukaryotic parasites responsible for such human pathologies as Chagas disease, sleeping sickness, and leishmaniasis. They have a single large mitochondrion, essential for the parasite survival. In kinetoplastid mitochondria, most of the molecular machineries and gene expression processes have significantly diverged and specialized, with an extreme example being their mitochondrial ribosomes. These large complexes are in charge of translating the few essential mRNAs encoded by mitochondrial genomes. Structural studies performed inTrypanosoma bruceialready highlighted the numerous peculiarities of these mitoribosomes and the maturation of their small subunit. However, several important aspects mainly related to the large subunit (LSU) remain elusive, such as the structure and maturation of its ribosomal RNA. Here we present a cryo-electron microscopy study of the protozoansLeishmania tarentolaeandTrypanosoma cruzimitoribosomes. For both species, we obtained the structure of their mature mitoribosomes, complete rRNA of the LSU, as well as previously unidentified ribosomal proteins. In addition, we introduce the structure of an LSU assembly intermediate in the presence of 16 identified maturation factors. These maturation factors act on both the intersubunit and the solvent sides of the LSU, where they refold and chemically modify the rRNA and prevent early translation before full maturation of the LSU.

scholarly journals Visualising formation of the ribosomal active site in mitochondria

2021 ◽  
Author(s):  
Viswanathan Chandrasekaran ◽  
Nirupa Desai ◽  
Nicholas O. Burton ◽  
Hanting Yang ◽  
Jon Price ◽  
...  
Keyword(s):  
Stress Responses ◽  
Large Subunit ◽  
Mitochondrial Protein ◽  
Mitochondrial Stress ◽  
Cryo Electron Microscopy ◽  
Complex Process ◽  
Mitochondrial Ribosome ◽  
Peptidyl Transferase Centre

SummaryRibosome assembly is an essential and complex process that is regulated at each step by specific biogenesis factors. Using cryo-electron microscopy, we identify and order major steps in the formation of the highly conserved peptidyl transferase centre (PTC) and tRNA binding sites in the large subunit of the human mitochondrial ribosome (mitoribosome). The conserved GTPase GTPBP7 regulates the folding and incorporation of core 16S ribosomal RNA (rRNA) helices and the ribosomal protein bL36m, and ensures that the PTC base U3039 has been 2′-O-methylated. Additionally, GTPBP7 binds the RNA methyltransferase NSUN4 and MTERF4, which facilitate earlier steps by sequestering H68-71 of the 16S rRNA and allowing biogenesis factors to access the maturing PTC. Consistent with the central role of NSUN4•MTERF4 and GTPBP7 during mitoribosome biogenesis, in vivo mutagenesis designed to disrupt binding of their Caenorhabditis elegans orthologs to the large subunit potently activates mitochondrial stress responses and results in severely reduced viability, developmental delays and sterility. Next-generation RNA sequencing reveals widespread gene expression changes in these mutant animals that are indicative of mitochondrial stress response activation. We also answer the long-standing question of why NSUN4 but not its enzymatic activity, is indispensable for mitochondrial protein synthesis in metazoans.

scholarly journals Human GTPBP5 (MTG2) fuels mitoribosome large subunit maturation by facilitating 16S rRNA methylation

2020 ◽  
Vol 48 (14) ◽  
pp. 7924-7943 ◽  
Author(s):  
Priyanka Maiti ◽  
Hana Antonicka ◽  
Anne-Claude Gingras ◽  
Eric A Shoubridge ◽  
Antoni Barrientos
Keyword(s):  
Quality Control ◽  
16S Rrna ◽  
Large Subunit ◽  
Mitochondrial Protein ◽  
Small Gtpases ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Ribosomes ◽  
Family Protein ◽  
Assembly Factors ◽  
Subunit Joining

Abstract Biogenesis of mammalian mitochondrial ribosomes (mitoribosomes) involves several conserved small GTPases. Here, we report that the Obg family protein GTPBP5 or MTG2 is a mitochondrial protein whose absence in a TALEN-induced HEK293T knockout (KO) cell line leads to severely decreased levels of the 55S monosome and attenuated mitochondrial protein synthesis. We show that a fraction of GTPBP5 co-sediments with the large mitoribosome subunit (mtLSU), and crosslinks specifically with the 16S rRNA, and several mtLSU proteins and assembly factors. Notably, the latter group includes MTERF4, involved in monosome assembly, and MRM2, the methyltransferase that catalyzes the modification of the 16S mt-rRNA A-loop U1369 residue. The GTPBP5 interaction with MRM2 was also detected using the proximity-dependent biotinylation (BioID) assay. In GTPBP5-KO mitochondria, the mtLSU lacks bL36m, accumulates an excess of the assembly factors MTG1, GTPBP10, MALSU1 and MTERF4, and contains hypomethylated 16S rRNA. We propose that GTPBP5 primarily fuels proper mtLSU maturation by securing efficient methylation of two 16S rRNA residues, and ultimately serves to coordinate subunit joining through the release of late-stage mtLSU assembly factors. In this way, GTPBP5 provides an ultimate quality control checkpoint function during mtLSU assembly that minimizes premature subunit joining to ensure the assembly of the mature 55S monosome.

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