Starvation sensing by mycobacterial RelA/SpoT homologue through constitutive surveillance 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.

Fragile X mental retardation protein regulates translation by binding directly to the ribosome.

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.

Structural insights into unique features of the human mitochondrial ribosome recycling.

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.

Replacement of S14 Protein in Ribosomes of Zinc-Starved Mycobacteria Reduces Spectinamide Sensitivity.

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.

Ribosome hibernation: a new molecular framework for targeting nonreplicating persisters of mycobacteria.

Treatment of tuberculosis requires a multi-drug regimen administered for at least 6 months. The long-term chemotherapy is attributed in part to a minor subpopulation of nonreplicating Mycobacterium tuberculosis cells that exhibit phenotypic tolerance to antibiotics. The origins of these cells in infected hosts remain unclear. Here we discuss some recent evidence supporting the hypothesis that hibernation of ribosomes in M. tuberculosis, induced by zinc starvation, could be one of the primary mechanisms driving the development of nonreplicating persisters in hosts. We further analyse inconsistencies in previously reported studies to clarify the molecular principles underlying mycobacterial ribosome hibernation.

Zinc depletion induces ribosome hibernation in mycobacteria.

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.

Low dose mycophenolate mofetil versus cyclophosphamide in the induction therapy of lupus nephritis in Nepalese population: a randomized control trial.

BACKGROUND: The management of proliferative lupus nephritis (LN) comprises timely and coordinated immunosuppressive therapy. This study aimed to evaluate and compare the effectiveness and safety profile of low dose mycophenolate mofetil (MMF) and cyclophosphamide (CYC) in induction therapy of LN in Nepalese population. METHODS: We conducted a prospective, open-label, randomized trial over a period of one and half years. Forty-nine patients with class III to V lupus nephritis were enrolled, out of which 42 patients (21 in each group) could complete the study. CYC was given intravenously as a monthly pulse and MMF was administered orally in the tablet form in the maximum daily dose of 1.5 g in two divided doses. RESULTS: The mean age of the patients was 25.43 ± 10.17 years with female to male ratio of 7.3:1. Mean baseline serum creatinine was 1.58 ± 1.38 mg/dL and eGFR was 62.38 ± 26.76 ml/min/1.73m2. Mean 24-h urinary protein was 4.35 ± 3.71 g per 1.73 m2 body surface area. At 6 months, serum creatinine (mg/dL) decreased from 1.73 to 0.96 in CYC and from 1.24 to 0.91 in the MMF group with improvement in eGFR (ml/min/1.73 m2) from 60.33 to 88.52 in CYC and from 64.42 to 89.09 in MMF group. Twenty-four-hour urinary protein (gm/1.73m2) reduced from 4.47 to 0.94 in CYC and from 4.5 to 0.62 in the MMF group. Primary end point was achieved in higher percentage of patients with MMF than CYC (28.6% vs. 19%) while equal proportion of patients (67% in each group) achieved secondary end point in both groups. Number of non-responders was higher in CYC group than in the MMF group (14.3% vs. 4.8%). There was no difference in the rate of achievement of secondary end point in both CYC and MMF groups (3.16 vs. 3.05 months). The occurrence of adverse events was higher in the CYC than in MMF group (56 vs. 15 events). CONCLUSION: Present study has concluded that MMF, used in relatively lower dose, is equally effective in inducing remission with reduction of proteinuria and improvement of kidney function with lesser adverse events than CYC in the induction therapy of proliferative lupus nephritis. TRIAL REGISTRATION: Retrospectively registered to ClinicalTrials.gov PRS. NCT03200002 (Registered date: June 28, 2017).

Initial bridges between two ribosomal subunits are formed within 9.4 milliseconds, as studied by time-resolved cryo-EM.

Association of the two ribosomal subunits during the process of translation initiation is a crucial step of protein synthesis. The two subunits (30S and 50S) of the bacterial 70S ribosome are held together by 12 dynamic bridges involving RNA-RNA, RNA-protein, and protein-protein interactions. The process of bridge formation, such as whether all these bridges are formed simultaneously or in a sequential order, is poorly understood. To understand such processes, we have developed and implemented a class of microfluidic devices that mix two components to completion within 0.4 ms and spray the mixture in the form of microdroplets onto an electron microscopy grid, yielding a minimum reaction time of 9.4 ms before cryofixation. Using these devices, we have obtained cryo-EM data corresponding to reaction times of 9.4 and 43 ms and have determined 3D structures of ribosomal subunit association intermediates. Molecular analyses of the cryo-EM maps reveal that eight intersubunit bridges (bridges B1a, B1b, B2a, B2b, B3, B7a, B7b, and B8) form within 9.4 ms, whereas the remaining four bridges (bridges B2c, B4, B5, and B6) take longer than 43 ms to form, suggesting that bridges are formed in a stepwise fashion. Our approach can be used to characterize sequences of various dynamic functional events on complex macromolecular assemblies such as ribosomes.

Cryo-EM structure of the small subunit of the mammalian mitochondrial ribosome.

The mammalian mitochondrial ribosomes (mitoribosomes) are responsible for synthesizing 13 membrane proteins that form essential components of the complexes involved in oxidative phosphorylation or ATP generation for the eukaryotic cell. The mammalian 55S mitoribosome contains significantly smaller rRNAs and a large mass of mitochondrial ribosomal proteins (MRPs), including large mito-specific amino acid extensions and insertions in MRPs that are homologous to bacterial ribosomal proteins and an additional 35 mito-specific MRPs. Here we present the cryo-EM structure analysis of the small (28S) subunit (SSU) of the 55S mitoribosome. We find that the mito-specific extensions in homologous MRPs generally are involved in inter-MRP contacts and in contacts with mito-specific MRPs, suggesting a stepwise evolution of the current architecture of the mitoribosome. Although most of the mito-specific MRPs and extensions of homologous MRPs are situated on the peripheral regions, they also contribute significantly to the formation of linings of the mRNA and tRNA paths, suggesting a tailor-made structural organization of the mito-SSU for the recruitment of mito-specific mRNAs, most of which do not possess a 5' leader sequence. In addition, docking of previously published coordinates of the large (39S) subunit (LSU) into the cryo-EM map of the 55S mitoribosome reveals that mito-specific MRPs of both the SSU and LSU are involved directly in the formation of six of the 15 intersubunit bridges.

Structural insights into initial and intermediate steps of the ribosome-recycling process.

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.

Insertion domain within mammalian mitochondrial translation initiation factor 2 serves the role of eubacterial initiation factor 1.

Mitochondria have their own translational machineries for the synthesis of thirteen polypeptide chains that are components of the complexes that participate in the process of oxidative phosphorylation (or ATP generation). Translation initiation in mammalian mitochondria requires two initiation factors, IF2(mt) and IF3(mt), instead of the three that are present in eubacteria. The mammalian IF2(mt) possesses a unique 37 amino acid insertion domain, which is known to be important for the formation of the translation initiation complex. We have obtained a three-dimensional cryoelectron microscopic map of the mammalian IF2(mt) in complex with initiator fMet-tRNA(iMet) and the eubacterial ribosome. We find that the 37 amino acid insertion domain interacts with the same binding site on the ribosome that would be occupied by the eubacterial initiation factor IF1, which is absent in mitochondria. Our finding suggests that the insertion domain of IF2(mt) mimics the function of eubacterial IF1, by blocking the ribosomal aminoacyl-tRNA binding site (A site) at the initiation step.

Drug resistance through ribosome splitting and rRNA disordering in mycobacteria.

HflX is known to rescue stalled ribosomes and is implicated in antibiotic resistance in several bacteria. Here we present several high-resolution cryo-EM structures of mycobacterial HflX in complex with the ribosome and its 50S subunit, with and without antibiotics. These structures reveal a distinct mechanism for HflX- mediated ribosome splitting and antibiotic resistance in mycobacteria. In addition to dissociating ribosome into two subunits, mycobacterial HflX mediates persistent disordering of multiple 23S rRNA helices to generate an inactive pool of 50S subunits. Mycobacterial HflX also acts as an anti-association factor by binding to pre-dissociated 50S subunits. A mycobacteria-specific insertion in HflX reaches further into the peptidyl transferase center. The position of this insertion overlaps with ribosome-bound macrolides or lincosamide class of antibiotics. The extended conformation of insertion seen in the absence of these antibiotics retracts and adjusts around the bound antibiotics instead of physically displacing them. It therefore likely imparts antibiotic resistance by sequestration of the antibiotic- bound inactive 50S subunits.

Evolution: Mitochondrial Ribosomes Across Species.

The ribosome is among the most complex and ancient cellular macromolecular assemblies that plays a central role in protein biosynthesis in all living cells. Its function of translation of genetic information encoded in messenger RNA into protein molecules also extends to subcellular compartments in eukaryotic cells such as apicoplasts, chloroplasts, and mitochondria. The origin of mitochondria is primarily attributed to an early endosymbiotic event between an alpha-proteobacterium and a primitive (archaeal) eukaryotic cell. The timeline of mitochondrial acquisition, the nature of the host, and their diversification have been studied in great detail and are continually being revised as more genomic and structural data emerge. Recent advancements in high-resolution cryo-EM structure determination have provided architectural details of mitochondrial ribosomes (mitoribosomes) from various species, revealing unprecedented diversifications among them. These structures provide novel insights into the evolution of mitoribosomal structure and function. Here, we present a brief overview of the existing mitoribosomal structures in the context of the eukaryotic evolution tree showing their diversification from their last common ancestor.

The small mycobacterial ribosomal protein, bS22, modulates aminoglycoside accessibility to its 16S rRNA helix-44 binding site.

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.

The structure of a hibernating ribosome in a Lyme disease pathogen.

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.

The structure of a hibernating ribosome in a Lyme disease pathogen.

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. We determined the structure of the Bbu 70S ribosome by single particle cryo-electron microscopy (cryo-EM) at a resolution of 2.9 Å, revealing its distinctive features. In contrast to a previous study suggesting that the single hibernation promoting factor protein present in Bbu (bbHPF) may not bind to its ribosome, our structure reveals a clear density for bbHPF bound to the decoding center of the small ribosomal 30S subunit. The 30S subunit has a non-annotated ribosomal protein, bS22, that has been found only in mycobacteria and Bacteroidetes so far. The protein bL38, recently discovered in Bacteroidetes, is also present in the Bbu large 50S ribosomal subunit. The protein bL37, previously seen only in mycobacterial ribosomes, is replaced by an N-terminal α-helical extension of uL30, suggesting that the two bacterial ribosomal proteins uL30 and bL37 may have evolved from one longer uL30 protein. The longer uL30 protein interacts with both the 23S rRNA and the 5S rRNA, is near the peptidyl transferase center (PTC), and could impart greater stability to this region. 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 energies are predicted for antibiotics, bound to the decoding center or PTC and are in clinical use for Lyme disease, that account for subtle distinctions in antibiotic-binding regions in the Bbu ribosome structure. Besides revealing unanticipated structural and compositional features for the Bbu ribosome, our study thus provides groundwork to enable ribosome-targeted antibiotic design for more effective treatment of Lyme disease.

Structure of a mitochondrial ribosome with minimal RNA.

The Leishmania tarentolae mitochondrial ribosome (Lmr) is a minimal ribosomal RNA (rRNA)-containing ribosome. We have obtained a cryo-EM map of the Lmr. The map reveals several features that have not been seen in previously-determined structures of eubacterial or eukaryotic (cytoplasmic or organellar) ribosomes to our knowledge. Comparisons of the Lmr map with X-ray crystallographic and cryo-EM maps of the eubacterial ribosomes and a cryo-EM map of the mammalian mitochondrial ribosome show that (i) the overall structure of the Lmr is considerably more porous, (ii) the topology of the intersubunit space is significantly different, with fewer intersubunit bridges, but more tunnels, and (iii) several of the functionally-important rRNA regions, including the alpha-sarcin-ricin loop, have different relative positions within the structure. Furthermore, the major portions of the mRNA channel, the tRNA passage, and the nascent polypeptide exit tunnel contain Lmr-specific proteins, suggesting that the mechanisms for mRNA recruitment, tRNA interaction, and exiting of the nascent polypeptide in Lmr must differ markedly from the mechanisms deduced for ribosomes in other organisms. Our study identifies certain structural features that are characteristic solely of mitochondrial ribosomes and other features that are characteristic of both mitochondrial and chloroplast ribosomes (i.e., organellar ribosomes).

PSRP1 is not a ribosomal protein, but a ribosome-binding factor that is recycled by the ribosome-recycling factor (RRF) and elongation factor G (EF-G).

Plastid-specific ribosomal proteins (PSRPs) have been proposed to play roles in the light-dependent regulation of chloroplast translation. Here we demonstrate that PSRP1 is not a bona fide ribosomal protein, but rather a functional homologue of the Escherichia coli cold-shock protein pY. Three-dimensional Cryo-electron microscopic (Cryo-EM) reconstructions reveal that, like pY, PSRP1 binds within the intersubunit space of the 70S ribosome, at a site overlapping the positions of mRNA and A- and P-site tRNAs. PSRP1 induces conformational changes within ribosomal components that comprise several intersubunit bridges, including bridge B2a, thereby stabilizes the ribosome against dissociation. We find that the presence of PSRP1/pY lowers the binding of tRNA to the ribosome. Furthermore, similarly to tRNAs, PSRP1/pY is recycled from the ribosome by the concerted action of the ribosome-recycling factor (RRF) and elongation factor G (EF-G). These results suggest a novel function for EF-G and RRF in the post-stress return of PSRP1/pY-inactivated ribosomes to the actively translating pool.

Purification of Hibernating and Active C- Ribosomes from Zinc-Starved Mycobacteria.

Zinc starvation in Mycobacterium smegmatis and Mycobacterium tuberculosis induces ribosome remodeling and hibernation. Remodeling involves replacement of C+ ribosomal (r-) proteins containing the zinc-binding CXXC motif with their C- paralogues without the motif. Hibernation is characterized by binding of mycobacterial-specific protein Y (Mpy) to 70S C- ribosomes, stabilizing the ribosome in an inactive state that is also resistant to kanamycin and streptomycin. We observed that ribosome remodeling and hibernation occur at two different concentrations of cellular zinc. Here, we describe the methods to purify hibernating and active forms of C- ribosomes from zinc-starved mycobacteria, along with purification of C+ ribosomes from zinc-rich mycobacterial cells. In vitro analysis of these distinct types of ribosomes will facilitate screening of small molecule inhibitors of ribosome hibernation for improved therapeutics against mycobacterial infections.