Mammalian mitochondrial ribosomes (mitoribosomes) synthesize mitochondrially encoded membrane proteins that are critical for mitochondrial function. Here we present the complete atomic structure of the porcine 55S mitoribosome at 3.8 angstrom resolution by cryo-electron microscopy and chemical cross-linking/mass spectrometry. The structure of the 28S subunit in the complex was resolved at 3.6 angstrom resolution by focused alignment, which allowed building of a detailed atomic structure including all of its 15 mitoribosomal-specific proteins. The structure reveals the intersubunit contacts in the 55S mitoribosome, the molecular architecture of the mitoribosomal messenger RNA (mRNA) binding channel and its interaction with transfer RNAs, and provides insight into the highly specialized mechanism of mRNA recruitment to the 28S subunit. Furthermore, the structure contributes to a mechanistic understanding of aminoglycoside ototoxicity.

Summary: Mitochondrial ribosomes (mitoribosomes) are extensively modified ribosomes of bacterial descent specialized for the synthesis and insertion of membrane proteins that are critical for energy conversion and ATP production inside mitochondria 1 . Mammalian mitoribosomes, which are composed of 39S and 28S subunits 2 , have diverged dramatically from the bacterial ribosomes from which they are derived, rendering them unique compared to bacterial, eukaryotic cytosolic, and fungal mitochondrial ribosomes [3][4][5] . We have previously determined the architecture of the porcine (Sus scrofa) 39S subunit at 4.9 Å resolution 6 , which is highly homologous to the human mitoribosomal large subunit. Here we present the complete atomic structure of the porcine 39S large mitoribosomal subunit determined in the context of a stalled translating mitoribosome at 3.4 Å resolution by cryo-electron microscopy and chemical crosslinking/mass spectrometry. The structure reveals the locations and the detailed folds of 50 mitoribosomal proteins,shows the highly conserved mitoribosomal peptidyl transferase active site in complex with its substrate tRNAs, and defines the path of the nascent chain in mammalian mitoribosomes along their idiosyncratic exit tunnel. Furthermore, we present evidence that a mitochondrial tRNA has become an integral component of the central protuberance of the 39S subunit where it architecturally substitutes for the absence of the 5S rRNA, a ubiquitous component of all cytoplasmic ribosomes.Main Text: Our previous analysis of the porcine 39S mitoribosomal large subunit at 4.9 Å resolution showed the overall fold of the mitoribosomal 16S rRNA as well as the localization of seven mitoribosomal-specific proteins 6 .However, proteins and protein extensions for which no homology models could be generated could not be modeled at this resolution. Additionally, due to the extensive differences between yeast and mammalian mitoribosomes, the recently reported high-resolution structure of the yeast mitoribosomal large subunit 5 is of limited use for understanding of the mammalian-specific aspects of mitoribosomal structure and function 4,7 .Cryo-EM data of porcine 55S mitoribosomes acquired on a movie modeenabled direct electron detector combined with movie frame realignment to compensate for beam-induced specimen motion 8 and maximum-likelihood based image classification and alignment 9 yielded a 3D-reconstruction of the 55S mitoribosome (Extended Data Fig. 1a, b) at 3.6 Å resolution (FSC = 0.143, "gold standard"). However, due to differences in local resolution (Extended Data Fig. 1c), the quality of the density in the 28S subunit part of the cryo-EM map (28S subunit resolution 4.1 Å) was of insufficient quality for reliable model building and refinement. Therefore, we focused the refinement on the 39S subunit, resulting in an improved 3D-reconstruction of the 39S subunit at 3.4 Å resolution (Extended Data Fig. 2), suitable for de-novo modelbuilding, structure refinement, and validation.We were able to build and ref...

Abstract:Mitochondrial ribosomes synthesize a number of highly hydrophobic proteins encoded on the genome of mitochondria, the organelles in eukaryotic cells that are responsible for energy conversion by oxidative phosphorylation. The ribosomes in mammalian mitochondria have undergone massive structural changes throughout their evolution, including rRNA shortening and acquisition of mitochondrial-specific ribosomal proteins. Here, we present the three-dimensional structure of the 39S large subunit of the porcine mitochondrial ribosome determined by cryo-electron microscopy at 4.9 Å resolution. The structure, combined with data from chemical crosslinking and mass spectrometry experiments, reveals the unique features of the 39S subunit at near atomic resolution and provides detailed insight into the architecture of the polypeptide exit site. This region of the mitochondrial ribosome has been dramatically remodeled, providing a specialized platform for the synthesis and membrane insertion of the highly hydrophobic protein components of the respiratory chain. 3Main Text: Mitochondrial ribosomes (mitoribosomes) are responsible for protein synthesis in mitochondria. These organelles of endosymbiotic origin 1 are required for energy conversion by aerobic respiration in eukaryotic cells.Mitoribosomes are more closely related to bacterial ribosomes than to eukaryotic cytosolic ribosomes 2 . However, the mammalian mitoribosome has been strongly altered by acquisition of mitochondrial-specific ribosomal proteins and protein extensions 2-5 , as well as the shortening of the mitochondrial ribosomal RNA (rRNA) 6 . The large 39S subunit of the mammalian mitoribosome catalyzes peptide bond formation during protein synthesis and harbors the nascent polypeptide exit tunnel. The structural evolution of the mammalian mitoribosome was accompanied by a strong functional specialization towards the synthesis of the highly hydrophobic mitochondrial inner membrane proteins 7 . The region around the polypeptide tunnel exit of the mitoribosome serves as a specialized platform for membrane insertion and assembly of these critical mitochondrially-encoded respiratory chain components [7][8][9][10][11] . Defects of the mitochondrial translation system are causally involved in a range of human diseases 12 .While cryo-electron microscopy (cryo-EM) reconstructions of the bovine mitoribosome at 13.5 Å 13 and 12.1 Å 14 resolution have visualized large structural differences compared to the bacterial ribosome, detailed molecular insight into the architecture and arrangement of unique protein and rRNA elements of the mammalian mitoribosome is currently lacking. We have used cryo-EM combined with chemical crosslinking followed by mass spectrometry (CX-MS) to determine the structure of the large subunit of the mammalian 4 mitoribosome, providing insight into its overall structure and into the molecular architecture of the polypeptide exit site in particular. Structure determinationTo obtain structural insights into the mammalian mitochondrial ribosome...

Ribosomal RNA (rRNA) plays key functional and architectural roles in ribosomes. Using electron microscopy, we determined the atomic structure of a highly divergent ribosome found in mitochondria of , a unicellular parasite that causes sleeping sickness in humans. The trypanosomal mitoribosome features the smallest rRNAs and contains more proteins than all known ribosomes. The structure shows how the proteins have taken over the role of architectural scaffold from the rRNA: They form an autonomous outer shell that surrounds the entire particle and stabilizes and positions the functionally important regions of the rRNA. Our results also reveal the "minimal" set of conserved rRNA and protein components shared by all ribosomes that help us define the most essential functional elements.

Chloroplasts are cellular organelles of plants and algae that are responsible for energy conversion and carbon fixation by the photosynthetic reaction. As a consequence of their endosymbiotic origin, they still contain their own genome and the machinery for protein biosynthesis. Here, we present the atomic structure of the chloroplast 70S ribosome prepared from spinach leaves and resolved by cryo‐EM at 3.4 Å resolution. The complete structure reveals the features of the 4.5S rRNA, which probably evolved by the fragmentation of the 23S rRNA, and all five plastid‐specific ribosomal proteins. These proteins, required for proper assembly and function of the chloroplast translation machinery, bind and stabilize rRNA including regions that only exist in the chloroplast ribosome. Furthermore, the structure reveals plastid‐specific extensions of ribosomal proteins that extensively remodel the mRNA entry and exit site on the small subunit as well as the polypeptide tunnel exit and the putative binding site of the signal recognition particle on the large subunit. The translation factor pY, involved in light‐ and temperature‐dependent control of protein synthesis, is bound to the mRNA channel of the small subunit and interacts with 16S rRNA nucleotides at the A‐site and P‐site, where it protects the decoding centre and inhibits translation by preventing tRNA binding. The small subunit is locked by pY in a non‐rotated state, in which the intersubunit bridges to the large subunit are stabilized.

Biomolecular mass spectrometry has matured strongly over the past decades and has now reached a stage where it can provide deep insights into the structure and composition of large cellular assemblies. Here, we describe a three-tiered hybrid mass spectrometry approach that enables the dissection of macromolecular complexes in order to complement structural studies. To demonstrate the capabilities of the approach, we investigate ribosomes, large ribonucleoprotein particles consisting of a multitude of protein and RNA subunits. We identify sites of sequence processing, protein post-translational modifications, and the assembly and stoichiometry of individual ribosomal proteins in four distinct ribosomal particles of bacterial, plant and human origin. Amongst others, we report extensive cysteine methylation in the zinc finger domain of the human S27 protein, the heptameric stoichiometry of the chloroplastic stalk complex, the heterogeneous composition of human 40S ribosomal subunits and their association to the CrPV, and HCV internal ribosome entry site RNAs.

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.

The mitochondrial ribosome, which translates all mitochondrial DNA (mtDNA)-encoded proteins, should be tightly regulated pre- and post-transcriptionally. Recently, we found RNA-DNA differences (RDDs) at human mitochondrial 16S (large) rRNA position 947 that were indicative of post-transcriptional modification. Here, we show that these 16S rRNA RDDs result from a 1-methyladenosine (m1A) modification introduced by TRMT61B, thus being the first vertebrate methyltransferase that modifies both tRNA and rRNAs. m1A947 is conserved in humans and all vertebrates having adenine at the corresponding mtDNA position (90% of vertebrates). However, this mtDNA base is a thymine in 10% of the vertebrates and a guanine in the 23S rRNA of 95% of bacteria, suggesting alternative evolutionary solutions. m1A, uridine, or guanine may stabilize the local structure of mitochondrial and bacterial ribosomes. Experimental assessment of genome-edited Escherichia coli showed that unmodified adenine caused impaired protein synthesis and growth. Our findings revealed a conserved mechanism of rRNA modification that has been selected instead of DNA mutations to enable proper mitochondrial ribosome function.