Structure of the Yeast Mitochondrial Large Ribosomal Subunit

Abstract

# These authors contributed equally to this work. AbstractMitochondria have specialized ribosomes that have diverged from their bacterial and cytoplasmic counterparts. We have solved the structure of the yeast mitoribosomal large subunit using singleparticle electron cryo-microscopy. The resolution of 3.2 Ångstroms enabled a nearly complete atomic model to be built de novo and refined, including 39 proteins, 13 of which are unique to mitochondria, as well as expansion segments of mitoribosomal RNA. The structure reveals a new exit tunnel path and architecture, unique elements of the E site and a putative membrane docking site.Mitochondria are organelles in eukaryotic cells that play a major role in metabolism, especially the synthesis of ATP. During evolution, mitochondria have lost or transferred most of their genes to the nuclei, significantly reducing the size of their genome (1). In yeast, all but one of the few remaining protein genes encode subunits of respiratory chain complexes, whose synthesis involves insertion into the inner mitochondrial membrane along with incorporation of prosthetic groups (2). For the translation of these genes, mitochondria maintain their own ribosomes (mitoribosomes) and translation system. The mitochondrial ribosomal RNA and several tRNAs are encoded by the mitochondrial genome, whereas all but one of its ribosomal proteins are nuclear encoded and imported from the cytoplasm. Mitoribosomes have diverged greatly from their counterparts in the cytosol of bacterial and eukaryotic cells and also exhibit high variability depending on species (Table S1) (3). Several genetic diseases map to mitoribosomes (4). In addition, the toxicity of many ribosomal antibiotics, in particular aminoglycosides, is thought to be due to their interaction with the mitoribosome (5).Mitochondrial translation in the yeast Saccharomyces cerevisiae (6) has been used as a model to study human mitochondrial diseases (7). The 74S yeast mitoribosome has an overall molecular weight of 3 MDa, or 30% higher than that of their bacterial counterpart. It consists of a 54S large subunit (1.9 MDa) and a 37S small subunit (1.1 MDa). Highresolution crystal structures have been solved for bacterial (8, 9) and eukaryotic ribosomes (10-12), as well as for an archaeal large subunit (13). In comparison, the only structures to † To whom correspondence should be addressed at scheres@mrc-lmb.cam.ac.uk or ramak@mrc-lmb.cam.ac.uk. Europe PMC Funders Group Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts date for mitoribosomes come from electron cryo-microscopy (cryo-EM) reconstructions at 12-14 Å (14-16).The very low yields of mitoribosomes from most tissues have impeded their crystallization. However, the advent of high-speed direct electron detectors (that allow accounting for particle movement during imaging) (17, 18), and improved algorithms for the classification and alignment of particles (19), allowed us to determine the structure of the large subunit of the yeast mitoribosome (hereafter referred t...

Trajectories of the ribosome as a Brownian nanomachine

“…This number is about an order of magnitude larger than previously achieved without timing information or templates. In combination with the recent availability of large cryo-EM datasets with near-atomic resolution (7,35), our approach promises the possibility to extract conformational information with unprecedented detail.…”

“…Powerful algorithms (4,5) have been used to sort cryo-EM snapshots into a small number of discrete classes, each presumed to represent an intermediate state (6). In some cases, however, snapshots of major ribosomal regions with large conformational flexibility have defied classification into discrete states altogether, even by the most advanced analytical methods (7). Single-molecule FRET experiments have yielded evidence for discrete conformational changes in single, freely equilibrating pretranslocational ribosomes, and provided ensemble averages for such changes, but have been unable to provide data for short-lived intermediates (8,9).…”

Crosslinking and Mass Spectrometry: An Integrated Technology to Understand the Structure and Function of Molecular Machines

“…Unfortunately, its low abundance has made the mitoribosome practically inaccessible to structure elucidation by crystallography. Thus, high-resolution cryo-EM was used in several recent projects to decipher the architecture of the mitoribosome of yeast and mammals [34,35,[77][78][79][80]. For three of these studies [34,35,77], XL-MS contributed restraints that were critical in localizing individual subunits within this massive complex ( Figure 3C).…”

RNA structure through multidimensional chemical mapping

“…In either case, these possibilities are, for the most part, untested. On one hand, structure determination methods that achieve high-resolution are growing in power and applicability, with recent improvements in cryo-electron microscopy achieving near-atomic-resolution models for RNA complexes extracted from living cells (Amunts et al 2014;Greber et al 2014;Hang et al 2015;Nguyen et al 2015). On the other hand, these methods, along with crystallography and nuclear magnetic resonance (NMR) approaches, continue to face challenges in RNAs that form non-compact states, form multiple structures, bind a heterogeneous complement of partners, or that have large unstructured regions.…”