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 Analysis of translating mitoribosome reveals functional characteristics of translation in mitochondria of fungi

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuzuru Itoh ◽  
Andreas Naschberger ◽  
Narges Mortezaei ◽  
Johannes M. Herrmann ◽  
Alexey Amunts
Keyword(s):  
Large Subunit ◽  
Small Subunit ◽  
Translation Regulation ◽  
Structural Rearrangements ◽  
Nascent Polypeptide ◽  
Maturation Factor ◽  
Exit Tunnel ◽  
Assembly Intermediate ◽  
Insight Into ◽  
Inhibitory Factor 1

Abstract Mitoribosomes are specialized protein synthesis machineries in mitochondria. However, how mRNA binds to its dedicated channel, and tRNA moves as the mitoribosomal subunit rotate with respect to each other is not understood. We report models of the translating fungal mitoribosome with mRNA, tRNA and nascent polypeptide, as well as an assembly intermediate. Nicotinamide adenine dinucleotide (NAD) is found in the central protuberance of the large subunit, and the ATPase inhibitory factor 1 (IF1) in the small subunit. The models of the active mitoribosome explain how mRNA binds through a dedicated protein platform on the small subunit, tRNA is translocated with the help of the protein mL108, bridging it with L1 stalk on the large subunit, and nascent polypeptide paths through a newly shaped exit tunnel involving a series of structural rearrangements. An assembly intermediate is modeled with the maturation factor Atp25, providing insight into the biogenesis of the mitoribosomal large subunit and translation regulation.

scholarly journals Modular assembly of the nucleolar large subunit processome

2017 ◽  
Author(s):  
Zahra Assur Sanghai ◽  
Linamarie Miller ◽  
Kelly R. Molloy ◽  
Jonas Barandun ◽  
Mirjam Hunziker ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Molecular Mimicry ◽  
Large Subunit ◽  
Ribosome Assembly ◽  
Modular Assembly ◽  
Assembly Result ◽  
Concerted Activity ◽  
Folding Pathways ◽  
Exit Tunnel

Early co-transcriptional events of eukaryotic ribosome assembly result in the formation of the small and large subunit processomes. We have determined cryo-EM reconstructions of the nucleolar large subunit processome in different conformational states at resolutions up to 3.4 Ångstroms. These structures reveal how steric hindrance and molecular mimicry are used to prevent premature folding states and binding of later factors. This is accomplished by the concerted activity of 21 ribosome assembly factors that stabilize and remodel pre-ribosomal RNA and ribosomal proteins. Mutually exclusive conformations of these particles suggest that the formation of the polypeptide exit tunnel is achieved through different folding pathways during subsequent stages of ribosome assembly.

scholarly journals Analysis of translating mitoribosome reveals functional characteristics of translation in mitochondria of fungi

2020 ◽  
Author(s):  
Yuzuru Itoh ◽  
Andreas Naschberger ◽  
Narges Mortezaei ◽  
Johannes M. Herrmann ◽  
Alexey Amunts
Keyword(s):  
Nicotinamide Adenine Dinucleotide ◽  
Large Subunit ◽  
Small Subunit ◽  
Translation Regulation ◽  
Structural Rearrangements ◽  
Nascent Polypeptide ◽  
Maturation Factor ◽  
Exit Tunnel ◽  
Assembly Intermediate ◽  
Inhibitory Factor 1

AbstractMitoribosomes are specialized protein synthesis machineries in mitochondria. However, how mRNA binds to its dedicated channel, and tRNA moves as the mitoribosomal subunit rotate with respect to each other is not understood. We report models of the translating fungal mitoribosome with mRNA, tRNA and nascent polypeptide, as well as an assembly intermediate. Nicotinamide adenine dinucleotide (NAD) is found in the central protuberance of the large subunit, and the ATPase inhibitory factor 1 (IF1) in the small subunit. The models of the active mitoribosome explain how mRNA binds through a dedicated protein platform on the small subunit, tRNA is translocated with the help of the protein mL108, bridging it with L1 stalk on the large subunit, and nascent polypeptide paths through a newly shaped exit tunnel involving a series of structural rearrangements. An assembly intermediate is modeled with the maturation factor Atp25, providing insight in to the biogenesis of the mitoribosomal large subunit and translation regulation.

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.

Visualisation of E. coli ribosomal RNA in situ by electron spectroscopic imaging and image averaging

1992 ◽  
Vol 50 (1) ◽  
pp. 466-467
Author(s):  
Daniel Beniac ◽  
George Harauz
Keyword(s):  
Ribosomal Rna ◽  
Three Dimensional ◽  
Spectroscopic Imaging ◽  
Electron Spectroscopic Imaging ◽  
E Coli ◽  
Microscopical Technique ◽  
Quantitative Image ◽  
Dimensional Reconstruction ◽  
Image Averaging ◽  
Protein Components

The structures of E. coli ribosomes have been extensively probed by electron microscopy of negatively stained and frozen hydrated preparations. Coupled with quantitative image analysis and three dimensional reconstruction, such approaches are worthwhile in defining size, shape, and quaternary organisation. The important question of how the nucleic acid and protein components are arranged with respect to each other remains difficult to answer, however. A microscopical technique that has been proposed to answer this query is electron spectroscopic imaging (ESI), in which scattered electrons with energy losses characteristic of inner shell ionisations are used to form specific elemental maps. Here, we report the use of image sorting and averaging techniques to determine the extent to which a phosphorus map of isolated ribosomal subunits can define the ribosomal RNA (rRNA) distribution within them.

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.

Developmental expression of fibrillarin and U3 snRNA in Xenopus laevis

Development ◽  
1991 ◽  
Vol 112 (1) ◽  
pp. 317-326
Author(s):  
M. Caizergues-Ferrer ◽  
C. Mathieu ◽  
P. Mariottini ◽  
F. Amalric ◽  
F. Amaldi
Keyword(s):  
Ribosomal Rna ◽  
Rna Processing ◽  
Protein Detection ◽  
Developmental Expression ◽  
Oocyte Growth ◽  
Small Nuclear Ribonucleoprotein ◽  
Hybridization Probes ◽  
Ribosomal Rna Processing ◽  
Protein Components ◽  
Nuclear Ribonucleoprotein

Fibrillarin is one of the protein components that together with U3 snRNA constitute the U3 snRNP, a small nuclear ribonucleoprotein particle involved in ribosomal RNA processing in eucaryotic cells. Using an antifibrillarin antiserum for protein detection and a fibrillarin cDNA and a synthetic oligonucleotide complementary to U3 snRNA as hybridization probes, the expression of these two components has been studied during Xenopus development. Fibrillarin mRNA is accumulated early in oogenesis, like many other messengers, and translated during oocyte growth. Fibrillarin protein is thus progressively accumulated throughout oogenesis to be assembled with U3 snRNA and used for ribosome production in the amplified nucleoli. After fertilization, the amount of U3 snRNA decreases while the maternally accumulated fibrillarin mRNA is maintained and utilized to produce more protein. After the mid-blastula transition, stored fibrillarin is assembled with newly synthesized U3 snRNA and becomes localized in the prenucleolar bodies and reforming nucleoli.

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