SignificanceMycobacteria as well as other bacteria remodel their ribosomes in response to zinc depletion by replacing zinc-binding ribosomal proteins with zinc-free paralogues, releasing zinc for other metabolic processes. In this study, we show that the remodeled ribosome acquires a structurally stable but functionally inactive and aminoglycoside-resistant state in zinc-starved Mycobacterium smegmatis. Conversely, M. smegmatis cells that are growth arrested in zinc-rich conditions have unstable ribosomes and reduced survival. We further provide evidence for ribosome remodeling in Mycobacterium tuberculosis in host tissues, suggesting that ribosome hibernation occurs during TB infections. Our findings could offer insights into mechanisms of persistence and antibiotic tolerance of mycobacterial infections.

During protein synthesis, elongation of the polypeptide chain by each amino acid is followed by a translocation step in which mRNA and transfer RNA (tRNA) are advanced by one codon. This crucial step is catalyzed by elongation factor G (EF-G), a guanosine triphosphatase (GTPase), and accompanied by a rotation between the two ribosomal subunits. A mutant of EF-G, H91A, renders the factor impaired in guanosine triphosphate (GTP) hydrolysis and thereby stabilizes it on the ribosome. We use cryogenic electron microscopy (cryo-EM) at near-atomic resolution to investigate two complexes formed by EF-G H91A in its GTP state with the ribosome, distinguished by the presence or absence of the intersubunit rotation. Comparison of these two structures argues in favor of a direct role of the conserved histidine in the switch II loop of EF-G in GTPase activation, and explains why GTP hydrolysis cannot proceed with EF-G bound to the unrotated form of the ribosome.

Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF‐G) on the ribosome in a post‐translocational state. It is used clinically against Gram‐positive bacteria such as pathogenic strains of Staphylococcus aureus, but no structural information has been available for EF‐G from these species. We have solved the apo crystal structure of EF‐G from S. aureus to 1.9 Å resolution. This structure shows a dramatically different overall conformation from previous structures of EF‐G, although the individual domains are highly similar. Between the different structures of free or ribosome‐bound EF‐G, domains III–V move relative to domains I–II, resulting in a displacement of the tip of domain IV relative to domain G. In S. aureus EF‐G, this displacement is about 25 Å relative to structures of Thermus thermophilus EF‐G in a direction perpendicular to that in previous observations. Part of the switch I region (residues 46–56) is ordered in a helix, and has a distinct conformation as compared with structures of EF‐Tu in the GDP and GTP states. Also, the switch II region shows a new conformation, which, as in other structures of free EF‐G, is incompatible with FA binding. We have analysed and discussed all known fusA‐based fusidic acid resistance mutations in the light of the new structure of EF‐G from S. aureus, and a recent structure of T. thermophilus EF‐G in complex with the 70S ribosome with fusidic acid [Gao YG et al. (2009) Science326, 694–699]. The mutations can be classified as affecting FA binding, EF‐G–ribosome interactions, EF‐G conformation, and EF‐G stability.

Termination of messenger RNA translation in Bacteria and Archaea is initiated by release factors (RFs) 1 or 2 recognizing a stop codon in the ribosomal A site and releasing the peptide from the P-site transfer RNA. After release, RF-dissociation is facilitated by the G-protein RF3. Structures of ribosomal complexes with RF1 or RF2 alone or with RF3 alone—RF3 bound to a non-hydrolyzable GTP-analog—have been reported. Here, we present the cryo-EM structure of a post-termination ribosome containing both apo-RF3 and RF1. The conformation of RF3 is distinct from those of free RF3•GDP and ribosome-bound RF3•GDP(C/N)P. Furthermore, the conformation of RF1 differs from those observed in RF3-lacking ribosomal complexes. Our study provides structural keys to the mechanism of guanine nucleotide exchange on RF3 and to an L12-mediated ribosomal recruitment of RF3. In conjunction with previous observations, our data provide the foundation to structurally characterize the complete action cycle of the G-protein RF3.DOI: http://dx.doi.org/10.7554/eLife.00411.001

The mammalian mitochondrial ribosome (mitoribosome) and its associated translational factors have evolved to accommodate greater participation of proteins in mitochondrial translation. Here we present the 2.68-3.96 Å cryo-EM structures of the human 55S mitoribosome in complex with the human mitochondrial elongation factor G1 (EF-G1 mt) in three distinct conformational states, including an intermediate state and a post-translocational state. These structures reveal the role of several mitochondria-specific (mito-specific) mitoribosomal proteins (MRPs) and a mito-specific segment of EF-G1 mt in mitochondrial tRNA (tRNA mt) translocation. In particular, the mito-specific C-terminal extension in EF-G1 mt is directly involved in translocation of the acceptor arm of the A-site tRNA mt. In addition to the ratchet-like and independent head-swiveling motions exhibited by the small mitoribosomal subunit, we discover significant conformational changes in MRP mL45 at the nascent polypeptide-exit site within the large mitoribosomal subunit that could be critical for tethering of the elongating mitoribosome onto the inner-mitochondrial membrane.

Mammalian mitochondrial ribosomes (mitoribosomes) are responsible for synthesizing proteins that are essential for oxidative phosphorylation (ATP generation). Despite their common ancestry with bacteria, the composition and structure of the human mitoribosome and its translational factors are significantly different from those of their bacterial counterparts. The mammalian mitoribosome recycling factor (RRFmt) carries a mito-specific N terminus extension (NTE), which is necessary for the function of RRFmt. Here we present a 3.9-Å resolution cryo-electron microscopic (cryo-EM) structure of the human 55S mitoribosome-RRFmtcomplex, which reveals α-helix and loop structures for the NTE that makes multiple mito-specific interactions with functionally critical regions of the mitoribosome. These include ribosomal RNA segments that constitute the peptidyl transferase center (PTC) and those that connect PTC with the GTPase-associated center and with mitoribosomal proteins L16 and L27. Our structure reveals the presence of a tRNA in the pe/E position and a rotation of the small mitoribosomal subunit on RRFmtbinding. In addition, we observe an interaction between the pe/E tRNA and a mito-specific protein, mL64. These findings help understand the unique features of mitoribosome recycling.

SummaryThe human mitochondrial translational initiation factor 3 (IF3mt) carries mitochondrial-specific amino acid extensions at both its N and C termini (N- and C-terminal extensions [NTE and CTE, respectively]), when compared with its eubacterial counterpart. Here we present 3.3- to 3.5-Å-resolution cryoelectron microscopic structures of the mammalian 28S mitoribosomal subunit in complex with human IF3mt. Unique contacts observed between the 28S subunit and N-terminal domain of IF3mt explain its unusually high affinity for the 28S subunit, whereas the position of the mito-specific NTE suggests NTE's role in binding of initiator tRNA to the 28S subunit. The location of the C-terminal domain (CTD) clarifies its anti-association activity, whereas the orientation of the mito-specific CTE provides a mechanistic explanation for its role in destabilizing initiator tRNA in the absence of mRNA. Furthermore, our structure hints at a possible role of the CTD in recruiting leaderless mRNAs for translation initiation. Our findings highlight unique features of IF3mt in mitochondrial translation initiation.

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