The GTPase Nog1 couples polypeptide exit tunnel quality control with ribosomal stalk assembly

2018 ◽  
Author(s):  
Purnima Nerurkar ◽  
Ludovic Gillet ◽  
Cohue Pena ◽  
Olga T Schubert ◽  
Martin Altvater ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Folding ◽  
Atpase Activity ◽  
Large Subunit ◽  
Eukaryotic Ribosome ◽  
Release Factors ◽  
Assembly Factors ◽  
Ribosomal Stalk ◽  
Exit Tunnel ◽  
Ribosome Formation

Eukaryotic ribosome precursors acquire translation competence in the cytoplasm through stepwise release of bound assembly factors, and proofreading of their functional centers. In case of the large subunit precursor (pre-60S), these essential steps include eviction of placeholders Arx1 and Mrt4 that prevent premature loading of the protein-folding machinery at the polypeptide exit tunnel (PET), and the ribosomal stalk, respectively. Here, we reveal that sequential ATPase and GTPase activities license release factors Rei1 and Yvh1 recruitment to the pre-60S in order to trigger Arx1 and Mrt4 removal. Drg1-ATPase activity extracts the C-terminal tail of Nog1 from the PET, enabling Rei1 to probe PET integrity, and then catalyze Arx1 release. Subsequently, GTPase hydrolysis stimulates Nog1 removal from the pre-60S, permitting Yvh1 to mediate Mrt4 release, and initiate ribosomal stalk assembly. Thus, Nog1 couples quality control and assembly of spatially distant functional centers during ribosome formation.

scholarly journals The GTPase Nog1 co-ordinates the assembly, maturation and quality control of distant ribosomal functional centers

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Purnima Klingauf-Nerurkar ◽  
Ludovic C Gillet ◽  
Daniela Portugal-Calisto ◽  
Michaela Oborská-Oplová ◽  
Martin Jäger ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Folding ◽  
Atpase Activity ◽  
Ribosomal Protein ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Eukaryotic Ribosome ◽  
Ribosomal Stalk ◽  
Exit Tunnel ◽  
Ribosome Formation

Eukaryotic ribosome precursors acquire translation competence in the cytoplasm through stepwise release of bound assembly factors, and proofreading of their functional centers. In case of the pre-60S, these steps include removal of placeholders Rlp24, Arx1 and Mrt4 that prevent premature loading of the ribosomal protein eL24, the protein-folding machinery at the polypeptide exit tunnel (PET), and the ribosomal stalk, respectively. Here, we reveal that sequential ATPase and GTPase activities license release factors Rei1 and Yvh1 to trigger Arx1 and Mrt4 removal. Drg1-ATPase activity removes Rlp24 from the GTPase Nog1 on the pre-60S; consequently, the C-terminal tail of Nog1 is extracted from the PET. These events enable Rei1 to probe PET integrity and catalyze Arx1 release. Concomitantly, Nog1 eviction from the pre-60S permits peptidyl transferase center maturation, and allows Yvh1 to mediate Mrt4 release for stalk assembly. Thus, Nog1 co-ordinates the assembly, maturation and quality control of distant functional centers during ribosome formation.

scholarly journals Interconnected assembly factors regulate the biogenesis of mitoribosomal large subunit

2020 ◽  
Author(s):  
Victor Tobiasson ◽  
Ondřej Gahura ◽  
Shintaro Aibara ◽  
Rozbeh Baradaran ◽  
Alena Zíková ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Acyl Carrier Protein ◽  
Large Subunit ◽  
Structural Characterisation ◽  
Protein Mass ◽  
Assembly Factors ◽  
Mammalian Genomes ◽  
Exit Tunnel ◽  
Protein Components ◽  
Assembly Intermediate

AbstractMitoribosomes consist of ribosomal RNA and protein components, coordinated assembly of which is critical for function. We used mitoribosomes with reduced RNA and increased protein mass from Trypanosoma brucei, to provide insights into the biogenesis of mitoribosomal large subunit. Structural characterisation of a stable assembly intermediate revealed 22 assembly factors, some of which are also encoded in mammalian genomes. The assembly factors form a protein network that spans over 180 Å, shielding the ribosomal RNA surface. The entire central protuberance and L7/L12 stalk are not assembled, and require removal of the factors and remodeling of the mitoribosomal proteins to become functional. The conserved proteins GTPBP7 and mt-EngA are bound together at the subunit interface in proximity to the peptidyl transferase center. A mitochondrial acyl-carrier protein plays a role in docking the L1 stalk which needs to be repositioned during maturation. Additional enzymatically deactivated factors scaffold the assembly, while the exit tunnel is blocked. Together, the extensive network of the factors stabilizes the immature sites and connects the functionally important regions of the mitoribosomal large subunit.

scholarly journals Prefabrication of a ribosomal protein subcomplex essential for eukaryotic ribosome formation

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Cohue Peña ◽  
Sabina Schütz ◽  
Ute Fischer ◽  
Yiming Chang ◽  
Vikram G Panse
Keyword(s):  
Atpase Activity ◽  
Structural Feature ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Spatial Clustering ◽  
Functional Importance ◽  
Eukaryotic Ribosome ◽  
Ribosome Formation

Spatial clustering of ribosomal proteins (r-proteins) through tertiary interactions is a striking structural feature of the eukaryotic ribosome. However, the functional importance of these intricate inter-connections, and how they are established is currently unclear. Here, we reveal that a conserved ATPase, Fap7, organizes interactions between neighboring r-proteins uS11 and eS26 prior to their delivery to the earliest ribosome precursor, the 90S. In vitro, uS11 only when bound to Fap7 becomes competent to recruit eS26 through tertiary contacts found between these r-proteins on the mature ribosome. Subsequently, Fap7 ATPase activity unloads the uS11:eS26 subcomplex onto its rRNA binding site, and therefore ensures stoichiometric integration of these r-proteins into the 90S. Fap7-depletion in vivo renders uS11 susceptible to proteolysis, and precludes eS26 incorporation into the 90S. Thus, prefabrication of a native-like r-protein subcomplex drives efficient and accurate construction of the eukaryotic ribosome.

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.

scholarly journals Context-specific action of macrolide antibiotics on the eukaryotic ribosome

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Maxim S. Svetlov ◽  
Timm O. Koller ◽  
Sezen Meydan ◽  
Vaishnavi Shankar ◽  
Dorota Klepacki ◽  
...  
Keyword(s):  
Amino Acid Sequences ◽  
Macrolide Antibiotics ◽  
Single Mutation ◽  
Sequence Motifs ◽  
Eukaryotic Translation ◽  
Eukaryotic Ribosome ◽  
Cellular Translation ◽  
Distinct Sequence ◽  
Exit Tunnel ◽  
Context Specific

AbstractMacrolide antibiotics bind in the nascent peptide exit tunnel of the bacterial ribosome and prevent polymerization of specific amino acid sequences, selectively inhibiting translation of a subset of proteins. Because preventing translation of individual proteins could be beneficial for the treatment of human diseases, we asked whether macrolides, if bound to the eukaryotic ribosome, would retain their context- and protein-specific action. By introducing a single mutation in rRNA, we rendered yeast Saccharomyces cerevisiae cells sensitive to macrolides. Cryo-EM structural analysis showed that the macrolide telithromycin binds in the tunnel of the engineered eukaryotic ribosome. Genome-wide analysis of cellular translation and biochemical studies demonstrated that the drug inhibits eukaryotic translation by preferentially stalling ribosomes at distinct sequence motifs. Context-specific action markedly depends on the macrolide structure. Eliminating macrolide-arrest motifs from a protein renders its translation macrolide-tolerant. Our data illuminate the prospects of adapting macrolides for protein-selective translation inhibition in eukaryotic cells.

scholarly journals A distinct assembly pathway of the human 39S late pre-mitoribosome

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jingdong Cheng ◽  
Otto Berninghausen ◽  
Roland Beckmann
Keyword(s):  
16S Rrna ◽  
Affinity Purification ◽  
Large Subunit ◽  
Assembly Pathway ◽  
Assembly Factors ◽  
Peptidyltransferase Center

AbstractAssembly of the mitoribosome is largely enigmatic and involves numerous assembly factors. Little is known about their function and the architectural transitions of the pre-ribosomal intermediates. Here, we solve cryo-EM structures of the human 39S large subunit pre-ribosomes, representing five distinct late states. Besides the MALSU1 complex used as bait for affinity purification, we identify several assembly factors, including the DDX28 helicase, MRM3, GTPBP10 and the NSUN4-mTERF4 complex, all of which keep the 16S rRNA in immature conformations. The late transitions mainly involve rRNA domains IV and V, which form the central protuberance, the intersubunit side and the peptidyltransferase center of the 39S subunit. Unexpectedly, we find deacylated tRNA in the ribosomal E-site, suggesting a role in 39S assembly. Taken together, our study provides an architectural inventory of the distinct late assembly phase of the human 39S mitoribosome.

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