Unique Architectural and Evolutionary Features of the Mammalian Mitochondrial and the Plant Chloroplast Ribosome Revealed by Cryo-Electron Microscopy

Abstract

Mitochondria and chloroplasts are cellular organelles with important functions in the energy metabolism of eukaryotic cells by hosting the protein complexes catalysing the oxidative phosphorylation and the photosynthetic reaction, respectively. They have independently evolved by the endosymbiosis of ancestral bacteria with eukaryotic progenitor cells. Essential membrane proteins of the oxidative phosphorylation and the photosynthesis machinery are still encoded on the residual organellar genomes and synthesized by the in-house translation apparatus, the mitochondrial and the chloroplast ribosome, respectively. Although sharing a common ancestor, mitoribosomes have a dramatically diverged composition and architecture in comparison to contemporary bacterial ribosomes. The high-resolution cryo-EM structures of the complete mammalian 55S mitoribosome and its 39S large and 28S small subunits presented in the first and second chapter of this thesis reveal their unique structural features and provide many important insights into the mechanism of mitochondrial protein biosynthesis. The mammalian 55S mitoribosome has a highly reduced rRNA, and the bacterial 5S rRNA is replaced by a tRNA molecule (CP tRNA) that was incorporated as a permanent part of the central protuberance. In addition, new ribosomal proteins, 28 in total, were acquired to the 55S mitoribosome providing structural stability of the reduced rRNA core and additional functionality, for example, in mRNA recruitment, intersubunit contacts, and membrane attachment. Mutations in mitoribosomal components can lead to severe human diseases that can now be understood on molecular level by inspecting the high-resolution structure of the mammalian 55S mitoribosome. In chloroplasts of plants and algae, the protein biosynthesis is coordinated with the photosynthetic activity defined by the day-night cycle and is mainly regulated at the level of translation. The high-resolution cryo-EM structure of the spinach chloroplast 70S ribosome presented in the third chapter of this thesis shows the plastid translation factor pY bound to the mRNA channel of the small subunit, where it blocks the translation initiation and stabilizes the ribosome in a non-rotated conformation during the dark phase of the day. Further, the structure reveals the plastid-specific ribosomal elements including five additional ribosomal proteins and extensions of ribosomal proteins with bacterial homologs that either play an important role in translation regulation or in structural stability. The rRNA molecules of the large subunit are fragmented to include an additional 4.5S rRNA and two strand breaks in the 23S rRNA, called hidden breaks. Taken together, the work in this thesis reveals the unique architecture of the mammalian 55S mitoribosome and the plant chloroplast 70S ribosome and provides novel insights into the mechanism of organellar protein biosynthesis and the evolution of organellar ribosomes.