We have analyzed Leishmania tarentolae mitochondrial ribonucleoprotein (RNP) complexes using the 9S small subunit (SSU) rRNA and the 12S large subunit (LSU) rRNA as markers, and have identified a 50S RNP particle as the putative mitochondrial monosome, a 40S particle as the putative LSU and a 30S particle as the putative SSU. These assignments are supported by morphological analysis by cryo-electron microscopy and proteomics analyses by mass spectrometry. The presence of additional rRNA-containing particles complicated the analysis and most likely was the basis for previous difficulties in identification of these ribosomes; thus, in addition to the monosomes and their subunits, there are abundant stable 45S particles (SSU(*)) containing only 9S rRNA, which may represent homodimers of the SSU or SSU associated with additional proteins, and variable minor amounts of 65S and 70S particles, which represent homodimers of the LSU and SSU(*), respectively. These additional rRNA particles might be due to the lengthy mitochondrial isolation and ribosome isolation procedures or may be present in vivo and play yet undetermined roles.

Ribosome binding factor A (RbfA) is a bacterial cold shock response protein, required for an efficient processing of the 5' end of the 16S ribosomal RNA (rRNA) during assembly of the small (30S) ribosomal subunit. Here we present a crystal structure of Thermus thermophilus (Tth) RbfA and a three-dimensional cryo-electron microscopic (EM) map of the Tth 30S*RbfA complex. RbfA binds to the 30S subunit in a position overlapping the binding sites of the A and P site tRNAs, and RbfA's functionally important C terminus extends toward the 5' end of the 16S rRNA. In the presence of RbfA, a portion of the 16S rRNA encompassing helix 44, which is known to be directly involved in mRNA decoding and tRNA binding, is displaced. These results shed light on the role played by RbfA during maturation of the 30S subunit, and also indicate how RbfA provides cells with a translational advantage under conditions of cold shock.

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-G1mt) 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-G1mt in mitochondrial tRNA (tRNAmt) translocation. In particular, the mito-specific C-terminal extension in EF-G1mt is directly involved in translocation of the acceptor arm of the A-site tRNAmt. 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.

Treatment of tuberculosis continues to be challenging due to the widespread latent form of the disease and the emergence of antibiotic-resistant strains of the pathogen, Mycobacterium tuberculosis . Bacterial ribosomes are a common and effective target for antibiotics. Several second line anti-tuberculosis drugs, e.g. kanamycin, amikacin, and capreomycin, target ribosomal RNA to inhibit protein synthesis. However, M. tuberculosis can acquire resistance to these drugs, emphasizing the need to identify new drug targets. Previous cryo-EM structures of the M. tuberculosis and M. smegmatis ribosomes identified two novel ribosomal proteins, bS22 and bL37, in the vicinity of two crucial drug-binding sites: the mRNA-decoding center on the small (30S), and the peptidyl-transferase center on the large (50S) ribosomal subunits, respectively. The functional significance of these two small proteins is unknown. In this study, we observe that an M. smegmatis strain lacking the bs22 gene shows enhanced susceptibility to kanamycin compared to the wild-type strain. Cryo-EM structures of the ribosomes lacking bS22 in the presence and absence of kanamycin suggest a direct role of bS22 in modulating the 16S rRNA kanamycin-binding site. Our structures suggest that amino-acid residue Lys-16 of bS22 interacts directly with the phosphate backbone of helix 44 of 16S rRNA to influence the micro-configuration of the kanamycin-binding pocket. Our analysis shows that similar interactions occur between eukaryotic homologues of bS22, and their corresponding rRNAs, pointing to a common mechanism of aminoglycoside resistance in higher organisms.

Zinc is an essential micronutrient for mycobacteria, and its depletion induces multiple adaptive changes in cellular physiology, the most remarkable of which are remodeling and hibernation of ribosomes. Ribosome remodeling, induced upon relatively moderate depletion of zinc, involves replacement of multiple ribosomal proteins containing the zinc-binding CXXC motif (called C+ r proteins) by their motif-free C- paralogs. Severe zinc depletion induces binding of mycobacterial protein Y (Mpy) to the 70S C- ribosome, thereby stabilizing the ribosome in an inactive state that is also resistant to kanamycin and streptomycin. Because the Mpy binding region on the ribosome is proximal to the binding pocket of spectinamides (Spa), the preclinical drug candidates for tuberculosis, we addressed the impact of remodeling and hibernation of ribosomes on Spa sensitivity. We report here that while Mpy binding has no significant effect on Spa sensitivity to the ribosome, replacement of S14C+ with its C- counterpart reduces the binding affinity of the drug by ∼2-fold, causing increased Spa tolerance in Mycobacterium smegmatis and Mycobacterium tuberculosis cells harboring the C- ribosome. The altered interaction between Spa and ribosomes likely results from new contact points for D67 and R83 residues of S14C- with U1138 and C1184 of 16S rRNA helix 34, respectively. Given that M. tuberculosis induces ribosome remodeling during progression from the acute to chronic phase of lung infection, our findings highlight new considerations in the development of Spa as effective drugs against tuberculosis.

Bacteria respond to zinc starvation by replacing ribosomal proteins that have the zinc-binding CXXC motif (C+) with their zinc-free (C-) paralogues. Consequences of this process beyond zinc homeostasis are unknown. Here, we show that the C- ribosome in Mycobacterium smegmatis is the exclusive target of a bacterial protein Y homolog, referred to as mycobacterial-specific protein Y (MPY), which binds to the decoding region of the 30S subunit, thereby inactivating the ribosome. MPY binding is dependent on another mycobacterial protein, MPY recruitment factor (MRF), which is induced on zinc depletion, and interacts with C- ribosomes. MPY binding confers structural stability to C- ribosomes, promoting survival of growth-arrested cells under zinc-limiting conditions. Binding of MPY also has direct influence on the dynamics of aminoglycoside-binding pockets of the C- ribosome to inhibit binding of these antibiotics. Together, our data suggest that zinc limitation leads to ribosome hibernation and aminoglycoside resistance in mycobacteria. Furthermore, our observation of the expression of the proteins of C- ribosomes in Mycobacterium tuberculosis in a mouse model of infection suggests that ribosome hibernation could be relevant in our understanding of persistence and drug tolerance of the pathogen encountered during chemotherapy of TB.

The spirochete bacterial pathogen Borrelia (Borreliella) burgdorferi (Bbu) affects more than 10% of the world population and causes Lyme disease in about half a million people in the US annually. Therapy for Lyme disease includes antibiotics that target the Bbu ribosome. Here we present the structure of the Bbu 70S ribosome obtained by single particle cryo-electron microscopy at 2.9 Å resolution, revealing a bound hibernation promotion factor protein and two genetically non-annotated ribosomal proteins bS22 and bL38. The ribosomal protein uL30 in Bbu has an N-terminal α-helical extension, partly resembling the mycobacterial bL37 protein, suggesting evolution of bL37 and a shorter uL30 from a longer uL30 protein. Its analogy to proteins uL30m and mL63 in mammalian mitochondrial ribosomes also suggests a plausible evolutionary pathway for expansion of protein content in mammalian mitochondrial ribosomes. Computational binding free energy predictions for antibiotics reflect subtle distinctions in antibiotic-binding sites in the Bbu ribosome. Discovery of these features in the Bbu ribosome may enable better ribosome-targeted antibiotic design for Lyme disease treatment.

Fragile X syndrome (FXS) is the most common form of inherited mental retardation, and it is caused by loss of function of the fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein that is involved in the translational regulation of several neuronal mRNAs. However, the precise mechanism of translational inhibition by FMRP is unknown. Here, we show that FMRP inhibits translation by binding directly to the L5 protein on the 80S ribosome. Furthermore, cryoelectron microscopic reconstruction of the 80S ribosome⋅FMRP complex shows that FMRP binds within the intersubunit space of the ribosome such that it would preclude the binding of tRNA and translation elongation factors on the ribosome. These findings suggest that FMRP inhibits translation by blocking the essential components of the translational machinery from binding to the ribosome.

Cryo-EM density maps showing the 70S ribosome of E. coli in two different functional states related by a ratchet-like motion were analyzed using real-space refinement. Comparison of the two resulting atomic models shows that the ribosome changes from a compact structure to a looser one, coupled with the rearrangement of many of the proteins. Furthermore, in contrast to the unchanged inter-subunit bridges formed wholly by RNA, the bridges involving proteins undergo large conformational changes following the ratchet-like motion, suggesting an important role of ribosomal proteins in facilitating the dynamics of translation.

The stringent response, which leads to persistence of nutrient-starved mycobacteria, is induced by activation of the RelA/SpoT homolog (Rsh) upon entry of a deacylated-tRNA in a translating ribosome. However, the mechanism by which Rsh identifies such ribosomes in vivo remains unclear. Here, we show that conditions inducing ribosome hibernation result in loss of intracellular Rsh in a Clp protease-dependent manner. This loss is also observed in nonstarved cells using mutations in Rsh that block its interaction with the ribosome, indicating that Rsh association with the ribosome is important for Rsh stability. The cryo-EM structure of the Rsh-bound 70S ribosome in a translation initiation complex reveals unknown interactions between the ACT domain of Rsh and components of the ribosomal L7/L12 stalk base, suggesting that the aminoacylation status of A-site tRNA is surveilled during the first cycle of elongation. Altogether, we propose a surveillance model of Rsh activation that originates from its constitutive interaction with the ribosomes entering the translation cycle.

The ribosome-recycling factor (RRF) and elongation factor-G (EF-G) disassemble the 70S post-termination complex (PoTC) into mRNA, tRNA, and two ribosomal subunits. We have determined cryo-electron microscopic structures of the PoTC·RRF complex, with and without EF-G. We find that domain II of RRF initially interacts with universally conserved residues of the 23S rRNA helices 43 and 95, and protein L11 within the 50S ribosomal subunit. Upon EF-G binding, both RRF and tRNA are driven towards the tRNA-exit (E) site, with a large rotational movement of domain II of RRF towards the 30S ribosomal subunit. During this intermediate step of the recycling process, domain II of RRF and domain IV of EF-G adopt hitherto unknown conformations. Furthermore, binding of EF-G to the PoTC·RRF complex reverts the ribosome from ratcheted to unratcheted state. These results suggest that (i) the ribosomal intersubunit reorganizations upon RRF binding and subsequent EF-G binding could be instrumental in destabilizing the PoTC and (ii) the modes of action of EF-G during tRNA translocation and ribosome-recycling steps are markedly different.

After the termination step of translation, the posttermination complex (PoTC), composed of the ribosome, mRNA, and a deacylated tRNA, is processed by the concerted action of the ribosome-recycling factor (RRF), elongation factor G (EF-G), and GTP to prepare the ribosome for a fresh round of protein synthesis. However, the sequential steps of dissociation of the ribosomal subunits, and release of mRNA and deacylated tRNA from the PoTC, are unclear. Using three-dimensional cryo-electron microscopy, in conjunction with undecagold-labeled RRF, we show that RRF is capable of spontaneously moving from its initial binding site on the 70S Escherichia coli ribosome to a site exclusively on the large 50S ribosomal subunit. This movement leads to disruption of crucial intersubunit bridges and thereby to the dissociation of the two ribosomal subunits, the central event in ribosome recycling. Results of this study allow us to propose a model of ribosome recycling.

A novel type of ribonucleoprotein (RNP) complex has been described from the kinetoplast-mitochondria of Leishmania tarentolae. The complex, termed the 45S SSU*, contains the 9S small subunit rRNA but does not contain the 12S large subunit rRNA. This complex is the most stable and abundant mitochondrial RNP complex present in Leishmania. As shown by tandem mass spectrometry, the complex contains at least 39 polypeptides with a combined molecular mass of almost 2.1 MDa. These components include several homologs of small subunit ribosomal proteins (S5, S6, S8, S9, S11, S15, S16, S17, S18, MRPS29); however, most of the polypeptides present are unique. Only a few of them show recognizable motifs, such as protein-protein (coiled-coil, Rhodanese) or protein-RNA (pentatricopeptide repeat) interaction domains. A cryo-electron microscopy examination of the 45S SSU* fraction reveals that 27% of particles represent SSU homodimers arranged in a head-to-tail orientation, while the majority of particles are clearly different and show an asymmetric bilobed morphology. Multiple classes of two-dimensional averages were derived for the asymmetrical particles, probably reflecting random orientations of the particles and difficulties in correlating these views with the known projections of ribosomal complexes. One class of the two-dimensional averages shows a SSU moiety attached to a protein mass or masses in a monosome-like appearance. The combined mass spectrometry and electron microscopy data thus indicate that the majority 45S SSU* particles represents a heterodimeric complex in which the SSU of the Leishmania mitochondrial ribosome is associated with an additional protein mass. The biological role of these particles is not known.

Ribosomes are recycled for a new round of translation initiation by dissociation of ribosomal subunits, messenger RNA and transfer RNA from their translational post-termination complex. Here we present cryo-EM structures of the human 55S mitochondrial ribosome (mitoribosome) and the mitoribosomal large 39S subunit in complex with mitoribosome recycling factor (RRFmt) and a recycling-specific homolog of elongation factor G (EF-G2mt). These structures clarify an unusual role of a mitochondria-specific segment of RRFmt, identify the structural distinctions that confer functional specificity to EF-G2mt, and show that the deacylated tRNA remains with the dissociated 39S subunit, suggesting a distinct sequence of events in mitoribosome recycling. Furthermore, biochemical and structural analyses reveal that the molecular mechanism of antibiotic fusidic acid resistance for EF-G2mt is markedly different from that of mitochondrial elongation factor EF-G1mt, suggesting that the two human EF-Gmts have evolved diversely to negate the effect of a bacterial antibiotic.

Proteins involved in mammalian mitochondrial translation, when compared to analogous bacterial proteins, frequently have additional sequence regions whose structural or functional roles are not always clear. For example, an additional short insert sequence in the bovine mitochondrial initiation factor 2 (IF2(mt)) seems sufficient to fulfill the added role of eubacterial initiation factor IF1. Prior to our recent cryo-EM study that showed IF2(mt) to structurally occupy both the IF1 and IF2 binding sites, the spatial separation of these sites, and the short length of the insert sequence, posed ambiguity in whether it could perform the role of IF1 through occupation of the IF1 binding site on the ribosome.

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-RRFmt complex, 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 RRFmt binding. 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.

The 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.