The complete structure of the large subunit of the mammalian mitochondrial ribosome

Abstract

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...

Using mitoribosomal profiling to investigate human mitochondrial translation

“…This unprecedented incorporation of a tRNA rather than a 5S rRNA is also seen in other mammalian mitoribosomes but appears to be restricted to either mt-tRNA Phe or mt-tRNA Val 24, 27 . We recently published that in human cells with a mutated, unstable mt-tRNA Val there is a preferential incorporation of mt-tRNA Phe into the mitoribosome 27, 28 .…”

“…It is now known that the mitochondrially-encoded tRNA Val is incorporated as a structural component of the human mitoribosome 24– 26 . This unprecedented incorporation of a tRNA rather than a 5S rRNA is also seen in other mammalian mitoribosomes but appears to be restricted to either mt-tRNA Phe or mt-tRNA Val 24, 27 .…”

Biallelic Mutations in MRPS34 Lead to Instability of the Small Mitoribosomal Subunit and Leigh Syndrome

“…2,3 In humans, 30 mitochondrial ribosomal small subunit proteins (MRPSs) assemble with the 12S mt-rRNA to form the small 28S subunit, while 50 mitochondrial ribosomal large subunit proteins (MRPLs) assemble with the 16S mt-rRNA and mt-tRNA Val to form the large 39S subunit. [4][5][6] Molecular defects that impair different components of the mitochondrial translation machinery can cause combined OXPHOS deficiency. 7 Specifically, 7 of the 80 genes encoding mitochondrial ribosomal proteins have had pathogenic mutations reported, including autosomal-recessive mutations in MRPS7 (MIM: 611974), 8 MRPS16 (MIM: 609204), 9 MRPS22 (MIM: 605810), 10 MRPS23 (MIM: 611985), 11 MRPL3 (MIM: 607118), 12 MRPL12 (MIM: 602375), 13 and MRPL44 (MIM: 611849).…”

Structure of the E. coli ribosome–EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM

“…For initial model building, the cryo-EM density map was sharpened by applying a B factor of 2120 Å 2 , filtered to 3.1 Å resolution and masked using a pseudo bulk solvent envelope obtained by merging different versions of the cryo-EM map filtered at different frequencies 40 and Chimera 41 . Fourier transform of the masked cryo-EM density map to reciprocal space structure factors was performed using Crystallography and NMR System (CNS) 42,43 employing phase significance blurring scale factors derived from FSC values in a resolution-dependent manner 44 . In detail, a modified CNS input file was used for the assignment of FOMs (figure of merit) estimated based on equation (1) for the map obtained from the full data set and equation (2) for the maps obtained from two half sets:…”