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

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.

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Structure of the full kinetoplastids mitoribosome and insight on its large subunit maturation

10.1101/2020.05.02.073890 ◽

2020 ◽

Author(s):

Heddy Soufari ◽

Florent Waltz ◽

Camila Parrot ◽

Stéphanie Durrieu ◽

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

AbstractKinetoplastids are unicellular eukaryotic parasites responsible for human pathologies such as Chagas disease, sleeping sickness or Leishmaniasis1. They possess a single large mitochondrion, essential for the parasite survival2. In kinetoplastids mitochondrion, most of the molecular machineries and gene expression processes have significantly diverged and specialized, with an extreme example being their mitochondrial ribosomes3. These large complexes are in charge of translating the few essential mRNAs encoded by mitochondrial genomes4,5. Structural studies performed in Trypanosoma brucei already highlighted the numerous peculiarities of these mitoribosomes and the maturation of their small subunit3,6. However, several important aspects mainly related to the large subunit remain elusive, such as the structure and maturation of its ribosomal RNA3. Here, we present a cryo-electron microscopy study of the protozoans Leishmania tarentolae and Trypanosoma cruzi mitoribosomes. For both species, we obtained the structure of their mature mitoribosomes, complete rRNA of the large subunit as well as previously unidentified ribosomal proteins. Most importantly, we introduce the structure of an LSU assembly intermediate in presence of 16 identified maturation factors. These maturation factors act both on the intersubunit and solvent sides of the LSU, where they refold and chemically modify the rRNA and prevent early translation before full maturation of the LSU.

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Cryo-EM structure of the RNA-rich plant mitochondrial ribosome

10.1101/777342 ◽

2019 ◽

Cited By ~ 2

Author(s):

Florent Waltz ◽

Heddy Soufari ◽

Anthony Bochler ◽

Philippe Giegé ◽

Yaser Hashem

Keyword(s):

Ribosomal Proteins ◽

Microscopy Study ◽

Electron Microscopy Study ◽

Pentatricopeptide Repeat ◽

Structural Investigations ◽

Mitochondrial Ribosomes ◽

Mitochondrial Ribosome ◽

Eukaryotic Species ◽

Specific Proteins ◽

Pentatricopeptide Repeat Proteins

The vast majority of eukaryotic cells contain mitochondria, essential powerhouses and metabolic hubs1. These organelles have a bacterial origin and were acquired during an early endosymbiosis event2. Mitochondria possess specialized gene expression systems composed of various molecular machines including the mitochondrial ribosomes (mitoribosomes). Mitoribosomes are in charge of translating the few essential mRNAs still encoded by mitochondrial genomes3. While chloroplast ribosomes strongly resemble those of bacteria4,5, mitoribosomes have diverged significantly during evolution and present strikingly different structures across eukaryotic species6–10. In contrast to animals and trypanosomatides, plants mitoribosomes have unusually expanded ribosomal RNAs and conserved the short 5S rRNA, which is usually missing in mitoribosomes11. We have previously characterized the composition of the plant mitoribosome6revealing a dozen plant-specific proteins, in addition to the common conserved mitoribosomal proteins. In spite of the tremendous recent advances in the field, plant mitoribosomes remained elusive to high-resolution structural investigations, and the plant-specific ribosomal features of unknown structures. Here, we present a cryo-electron microscopy study of the plant 78S mitoribosome from cauliflower at near-atomic resolution. We show that most of the plant-specific ribosomal proteins are pentatricopeptide repeat proteins (PPR) that deeply interact with the plant-specific rRNA expansion segments. These additional rRNA segments and proteins reshape the overall structure of the plant mitochondrial ribosome, and we discuss their involvement in the membrane association and mRNA recruitment prior to translation initiation. Finally, our structure unveils an rRNA-constructive phase of mitoribosome evolution across eukaryotes.

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Microtubule dynamics and microtubule caps: a time-resolved cryo-electron microscopy study

Trends in Cell Biology ◽

10.1016/0962-8924(91)90015-2 ◽

1991 ◽

Vol 1 (6) ◽

pp. 153

Author(s):

E.M. Mandelkow ◽

E. Mandelkow ◽

R.A. Milligan

Keyword(s):

Electron Microscopy ◽

Microscopy Study ◽

Electron Microscopy Study ◽

Microtubule Dynamics ◽

Time Resolved ◽

Cryo Electron Microscopy

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Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

10.1101/2021.03.17.435767 ◽

2021 ◽

Author(s):

Hauke S. Hillen ◽

Elena Lavdovskaia ◽

Franziska Nadler ◽

Elisa Hanitsch ◽

Andreas Linden ◽

...

Keyword(s):

Molecular Basis ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Large Subunit ◽

Structural Basis ◽

Catalytic Core ◽

Mitochondrial Ribosomes ◽

Mitochondrial Ribosome ◽

Translation Systems

Ribosome biogenesis is an essential process that requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. In particular, maturation of the peptidyl transferase center (PTC), the catalytic core of the ribosome, is mediated by universally conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial ribosomal large subunit (mtLSU) using a combination of endogenous complex purification, in vitro reconstitution and cryo-electron microscopy (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. Subsequent addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch by releasing MTERF4-NSUN4 and GTPBP5 accompanied by the progression to a near-mature PTC state. In addition, 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 define the molecular basis of dynamic GTPase-mediated PTC maturation during mitochondrial ribosome biogenesis and provide a framework for understanding step-wise progression of PTC folding as a critical quality control checkpoint in all translation systems.

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

Nucleic Acids Research ◽

10.1093/nar/gkz770 ◽

2019 ◽

Vol 47 (19) ◽

pp. 10414-10425 ◽

Cited By ~ 10

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.

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Temperature dependent intermediate structures during the main phase transition of dimyristoyl phosphatidylcholine vesicles a combined iodine laser-temperature jump and time resolved cryo-electron microscopy study

Biophysical Chemistry ◽

10.1016/0301-4622(95)00085-2 ◽

1996 ◽

Vol 58 (1-2) ◽

pp. 53-65 ◽

Cited By ~ 7

Author(s):

Rainer Groll ◽

Artur Böttcher ◽

Joachim Jäger ◽

Josef F. Holzwarth

Keyword(s):

Electron Microscopy ◽

Temperature Jump ◽

Microscopy Study ◽

Electron Microscopy Study ◽

Temperature Dependent ◽

Time Resolved ◽

Iodine Laser ◽

Cryo Electron Microscopy ◽

Dimyristoyl Phosphatidylcholine ◽

Laser Temperature Jump

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The proteins of Xenopus ovary ribosomes

Biochemical Journal ◽

10.1042/bj1251091 ◽

1971 ◽

Vol 125 (4) ◽

pp. 1091-1107 ◽

Cited By ~ 13

Author(s):

P J Ford

Keyword(s):

Potassium Chloride ◽

Ribosomal Proteins ◽

Large Subunit ◽

Buoyant Density ◽

Small Subunit ◽

Chemical Methods ◽

Ribosomal Subunits ◽

Molecular Weights ◽

Caesium Chloride ◽

Chloride Treatment

1. The preparation of ribosomes and ribosomal subunits from Xenopus ovary is described. 2. The yield of once-washed ribosomes (buoyant density in caesium chloride 1.601g·cm-3; 44% RNA, 56% protein by chemical methods) was 10.1mg/g wet wt. of tissue. 3. Buoyant density in caesium chloride and RNA/protein ratios by chemical methods have been determined for ribosome subunits produced by 1.0mm-EDTA or 0.5m-potassium chloride treatment and also for EDTA subunits extracted with 0.5m-, 1.0m- or 1.5m-potassium chloride, 4. Analysis of ribosomal protein on acrylamide gels at pH4.5 in 6m-urea reveals 24 and 26 bands from small and large EDTA subunits respectively. The actual numbers of proteins are greater than this, as many bands are obviously doublets. 5. Analysis of the proteins in the potassium chloride extract and particle fractions showed that some bands are completely and some partially extracted. Taking partial extraction as an indication of possible doublet bands it was found that there were 12 and 20 such bands in the small and large subunits respectively, making totals of 36 and 46 proteins. 6. From the measured protein contents and assuming weight-average molecular weights for the proteins of large and small subunits close to those observed for eukaryote ribosomal proteins it is possible to compute the total numbers of protein molecules per particle. It appears that too few protein bands have been identified on acrylamide gels to account for all the protein in the large subunit, but probably enough for the small subunit.

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

Nature Communications ◽

10.1038/s41467-020-18830-w ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 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.

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Ribosome Dimerization Protects the Small Subunit

Journal of Bacteriology ◽

10.1128/jb.00009-20 ◽

2020 ◽

Vol 202 (10) ◽

Cited By ~ 6

Author(s):

Heather A. Feaga ◽

Mykhailo Kopylov ◽

Jenny Kim Kim ◽

Marko Jovanovic ◽

Jonathan Dworkin

Keyword(s):

Ribosomal Proteins ◽

Selective Advantage ◽

Small Subunit ◽

Content Type ◽

Dimer Interface ◽

Cryo Electron Microscopy ◽

Single Protein ◽

Mechanistic Basis ◽

Favorable Conditions ◽

Insight Into

ABSTRACT When nutrients become scarce, bacteria can enter an extended state of quiescence. A major challenge of this state is how to preserve ribosomes for the return to favorable conditions. Here, we show that the ribosome dimerization protein hibernation-promoting factor (HPF) functions to protect essential ribosomal proteins. Ribosomes isolated from strains lacking HPF (Δhpf) or encoding a mutant allele of HPF that binds the ribosome but does not mediate dimerization were substantially depleted of the small subunit proteins S2 and S3. Strikingly, these proteins are located directly at the ribosome dimer interface. We used single-particle cryo-electron microscopy (cryo-EM) to further characterize these ribosomes and observed that a high percentage of ribosomes were missing S2, S3, or both. These data support a model in which the ribosome dimerization activity of HPF evolved to protect labile proteins that are essential for ribosome function. HPF is almost universally conserved in bacteria, and HPF deletions in diverse species exhibit decreased viability during starvation. Our data provide mechanistic insight into this phenotype and establish a mechanism for how HPF protects ribosomes during quiescence. IMPORTANCE The formation of ribosome dimers during periods of dormancy is widespread among bacteria. Dimerization is typically mediated by a single protein, hibernation-promoting factor (HPF). Bacteria lacking HPF exhibit strong defects in viability and pathogenesis and, in some species, extreme loss of rRNA. The mechanistic basis of these phenotypes has not been determined. Here, we report that HPF from the Gram-positive bacterium Bacillus subtilis preserves ribosomes by preventing the loss of essential ribosomal proteins at the dimer interface. This protection may explain phenotypes associated with the loss of HPF, since ribosome protection would aid survival during nutrient limitation and impart a strong selective advantage when the bacterial cell rapidly reinitiates growth in the presence of sufficient nutrients.

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