Structural studies of the mitochondrial ribosome and co-translational insertion of membrane proteins

The mitochondrion, a constituent of eukaryotic cells is important in the synthesis of ATP through a process called oxidative phosphorylation. The enzymes that make up the subunits of oxidative phosphorylation in humans and yeast are encoded by both nuclear DNA and in mitochondrial DNA. As such the mitochondrion has retained its own translational system to include mitochondrial ribosomes (mitoribosome) to translate these proteins along with other proteins which is species dependent. The mitoribosome is also involved in co-translational insertion of proteins destined for the inner mitochondrial membrane through binding of its translocon OXA1. My study has revealed the structure of the complete 75-component yeast Saccharomyces cerevisiae mitochondrial ribosome solved to 3.3 Å by single particle cryo-electron microscopy (cryo-EM). The previously unsolved small subunit of the mitoribosome was built de novo to include 34 proteins, of which 7 are specific to the yeast mitochondrial ribosome and the 15S ribosomal RNA. This mitochondrial ribosome has a distinct architecture compared to other mitochondrial ribosome and its ancestral bacterial ribosome. Elements specific to the yeast mitoribosome give it a distinct shape and architecture to include a putatively active enzyme on the periphery. An expanded messenger RNA exit channel has also been found harbouring a platform suitable for translational activator binding. In general this structure has provided insight into translation specialisation amongst species and its continued evolution. Multiple states of actively translating human mitoribosome have been elucidated using single particle cryo-EM. The human mitoribosome structures, stalled by different antibiotics, have been seen with A/A and P/P site tRNAs in situ as well as structures with an unidentified factor in the mitoribosomal factor site. In addition the human mitochondrial large subunit and stalled complete mitoribosome has been found at low resolution likely complexed to its translocon OXA1L following detergent solubilisation.Wellcome Trust Clinical PhD Fellowship (110301/Z/15/Z

Visualizing formation of the active site in the mitochondrial ribosome.

Funder: Agouron InstituteFunder: Louis-Jeantet FoundationRibosome assembly is an essential and conserved process that is regulated at each step by specific factors. Using cryo-electron microscopy (cryo-EM), we visualize the formation of the conserved peptidyl transferase center (PTC) of the human mitochondrial ribosome. The conserved GTPase GTPBP7 regulates the correct folding of 16S ribosomal RNA (rRNA) helices and ensures 2'-O-methylation of the PTC base U3039. GTPBP7 binds the RNA methyltransferase NSUN4 and MTERF4, which sequester H68-71 of the 16S rRNA and allow biogenesis factors to access the maturing PTC. Mutations that disrupt binding of their Caenorhabditis elegans orthologs to the large subunit potently activate mitochondrial stress and cause viability, development, and sterility defects. Next-generation RNA sequencing reveals widespread gene expression changes in these mutant animals that are indicative of mitochondrial stress response activation. We also answer the long-standing question of why NSUN4, but not its enzymatic activity, is indispensable for mitochondrial protein synthesis

Elongational stalling activates mitoribosome-associated quality control.

The human mitochondrial ribosome (mitoribosome) and associated proteins regulate the synthesis of 13 essential subunits of the oxidative phosphorylation complexes. We report the discovery of a mitoribosome-associated quality control pathway that responds to interruptions during elongation, and we present structures at 3.1- to 3.3-angstrom resolution of mitoribosomal large subunits trapped during ribosome rescue. Release factor homolog C12orf65 (mtRF-R) and RNA binding protein C6orf203 (MTRES1) eject the nascent chain and peptidyl transfer RNA (tRNA), respectively, from stalled ribosomes. Recruitment of mitoribosome biogenesis factors to these quality control intermediates suggests additional roles for these factors during mitoribosome rescue. We also report related cryo-electron microscopy structures (3.7 to 4.4 angstrom resolution) of elongating mitoribosomes bound to tRNAs, nascent polypeptides, the guanosine triphosphatase elongation factors mtEF-Tu and mtEF-G1, and the Oxa1L translocase