70S Ribosome Research Articles

Nuclear 80S Ribosomes Present Across the Stages of Cell Cycle in Drosophila Melanogaster Cells

Nuclear translation has been a subject of controversy between scientists for over 5 decades. Despite the existence of evidence to the contrary, most biologists agree that translation exclusively takes place in the cytoplasm of eukaryotes. In recent years, more evidences are being presented that disprove this theory. Here we employed the Ribo-BiFC technique which can detect assembled, and potentially translating, ribosomes invivo and studied nuclear 80S assembly and translation at all the stages of cell cycle in Drosophila S2 cells. The results obtained suggest that 80S ribosomes are present in the nucleus particularly within the nucleolus across all the cell cycle stages in Drosophila S2 cells that were visualised. The signal observed is more apparent in S-phase. This investigation supports the many other previous findings that nuclear translation may occur in eukaryotic organisms.

Cryo-electron microscopy structure and translocation mechanism of the crenarchaeal ribosome.

Archaeal ribosomes have many domain-specific features; however, our understanding of these structures is limited. We present 10 cryo-electron microscopy (cryo-EM) structures of the archaeal ribosome from crenarchaeota Sulfolobus acidocaldarius (Sac) at 2.7-5.7 Å resolution. We observed unstable conformations of H68 and h44 of ribosomal RNA (rRNA) in the subunit structures, which may interfere with subunit association. These subunit structures provided models for 12 rRNA expansion segments and 3 novel r-proteins. Furthermore, the 50S-aRF1 complex structure showed the unique domain orientation of aRF1, possibly explaining P-site transfer RNA (tRNA) release after translation termination. Sac 70S complexes were captured in seven distinct steps of the tRNA translocation reaction, confirming conserved structural features during archaeal ribosome translocation. In aEF2-engaged 70S ribosome complexes, 3D classification of cryo-EM data based on 30S head domain identified two new translocation intermediates with 30S head domain tilted 5-6° enabling its disengagement from the translocated tRNA and its release post-translocation. Additionally, we observed conformational changes to aEF2 during ribosome binding and switching from three different states. Our structural and biochemical data provide new insights into archaeal translation and ribosome translocation.

Arabidopsis NOTCHLESS plays an important role in root and embryo development

ABSTRACT Ribosome biogenesis is a fundamental process in eukaryotic cells. NOTCHLESS (NLE) is involved in 60S ribosome biogenesis in yeast, but its role in Arabidopsis (A. thaliana) remains exclusive. Here, we found that Arabidopsis NLE (AtNLE) is highly conservative in phylogeny, which encoding a WD40-repeat protein. AtNLE is expressed in actively dividing tissues. AtNLE-GFP is localized in the nucleus. AtNLE physically interacts with the MIDAS domain of AtMDN1, a protein involved in the biogenesis of the 60S ribosomal subunit in Arabidopsis. The underexpressing mutant nle-2 shows short roots and reduced cell number in the root meristem. In addition, the null mutant nle-1 is embryo lethal, and defective embryos are arrested at the early globular stage. This work suggests that AtNLE interacts with AtMDN1, and AtNLE functions in root and embryo development.

Genotypic and phenotypic analysis of Elizabethkingia meningoseptica in bullfrog Rana catesbeiana isolated in Taiwan.

Elizabethkingia meningoseptica is a hazardous bacterium for agriculture production and human health. The present study identified E. meningoseptica from the bullfrog, human and reference strain BCRC 10677 by API 20NE, 50S ribosome protein L27 sequencing and pulse field gel electrophoresis to differentiate isolates of E. meningoseptica from aquatic animals and humans. All isolates from bullfrogs and humans were identified as E. meningoseptica by DNA sequencing with 98.8%-100% sequence identity. E. meningoseptica displayed significant genetic diversity when analysed using pulsed-field gel electrophoresis (PFGE). There were six distinct pulsotypes, including one pulsotype found in bullfrog isolates and five pulsotypes found in human isolates. However, E. meningoseptica from bullfrog exhibited one genotype only by PFGE. Overall, molecular epidemiological analysis of PFGE results indicated that the frog E. meningoseptica outbreaks in Taiwan were produced by genetically identical clones. The bullfrog isolates were not genetically related to other E. meningoseptica from human and reference isolates. This research provided the first comparisons of biochemical characteristics and genetic differences of E. meningoseptica from human and bullfrog isolates.

Structural insights into the mechanism of overcoming Erm-mediated resistance by macrolides acting together with hygromycin-A

The ever-growing rise of antibiotic resistance among bacterial pathogens is one of the top healthcare threats today. Although combination antibiotic therapies represent a potential approach to more efficiently combat infections caused by susceptible and drug-resistant bacteria, only a few known drug pairs exhibit synergy/cooperativity in killing bacteria. Here, we discover that well-known ribosomal antibiotics, hygromycin A (HygA) and macrolides, which target peptidyl transferase center and peptide exit tunnel, respectively, can act cooperatively against susceptible and drug-resistant bacteria. Remarkably, HygA slows down macrolide dissociation from the ribosome by 60-fold and enhances the otherwise weak antimicrobial activity of the newest-generation macrolide drugs known as ketolides against macrolide-resistant bacteria. By determining a set of high-resolution X-ray crystal structures of drug-sensitive wild-type and macrolide-resistant Erm-methylated 70S ribosomes in complex with three HygA-macrolide pairs, we provide a structural rationale for the binding cooperativity of these drugs and also uncover the molecular mechanism of overcoming Erm-type resistance by macrolides acting together with hygromycin A. Altogether our structural, biochemical, and microbiological findings lay the foundation for the subsequent development of synergistic antibiotic tandems with improved bactericidal properties against drug-resistant pathogens, including those expressing erm genes.

The structure and mechanism of action of a distinct class of dicistrovirus intergenic region IRESs.

Internal ribosomal entry sites (IRESs) engage with the eukaryotic translation apparatus to promote end-independent initiation. We identified a conserved class of ∼150 nt long intergenic region (IGR) IRESs in dicistrovirus genomes derived from members of the phyla Arthropoda, Bryozoa, Cnidaria, Echinodermata, Entoprocta, Mollusca and Porifera. These IRESs, exemplified by Wenling picorna-like virus 2, resemble the canonical cricket paralysis virus (CrPV) IGR IRES in comprising two nested pseudoknots (PKII/PKIII) and a 3'-terminal pseudoknot (PKI) that mimics a tRNA anticodon stem-loop base-paired to mRNA. However, they are ∼50 nt shorter than CrPV-like IRESs, and PKIII is an H-type pseudoknot that lacks the SLIV and SLV stem-loops that are primarily responsible for the affinity of CrPV-like IRESs for the 40S ribosomal subunit and that restrict initial binding of PKI to its aminoacyl (A) site. Wenling-class IRESs bound strongly to 80S ribosomes but only weakly to 40S subunits. Whereas CrPV-like IRESs must be translocated from the A site to the peptidyl (P) site by elongation factor 2 for elongation to commence, Wenling-class IRESs bound directly to the P site of 80S ribosomes, and decoding begins without a prior translocation step. A chimeric CrPV clone containing a Wenling-class IRES was infectious, confirming that the IRES functioned in cells.

Investigation of the Anti-Plasmodial Properties of N-2-Oxo Pyrrolidine Pentanamide-Based Sulfonamides: Experimental and Theoretical Approach

The study aimed to investigate novel pyrrolidine-pentanamide derivatives of sulfonamides as potential anti-malaria agents through a comprehensive approach involving experimental, theoretical, and in silico evaluations. The compounds were synthesized using standard methods and characterized using NMR, FT-IR, and HRMS techniques. The molecular properties of the compounds were investigated using density functional theory (DFT) at the B3LYP-D3 meta-functional/GenECP theoretical level. The calculated vibrational frequencies of the compounds showed good agreement with experimental results, validating the accuracy of the theoretical approach in capturing the behavior of different functional groups. Molecular docking simulations were performed to assess the binding affinity of the compounds to the amino acid residues binding sites of two target proteins: farnesyl pyrophosphate synthase (PDB ID: 1RQJ) and Plasmodium falciparum 80S ribosome (PDB ID: 3J7A). Substantially, the results of the docking simulations demonstrated that three compounds, namely 3-Methyl-2-(4-methylphenylsulfonamido)-N-(2-oxo-2-(pyrrolidin-1-yl)ethyl)pentanamide (MMOPEP), 3-Methyl-N-(2-oxo-2-(pyrrolidin-1-yl)ethyl)-2-(phenylsulfonamido)pentanamide (MOPEPP), and 3-Methyl-2-(4-nitrophenylsulfonamido)-N-(2-oxo-2-(pyrrolidin-1-yl)ethyl)pentanamide (MNOPEP), exhibited tight binding to the target proteins. These compounds showed favorable docking scores compared to standard drugs, indicating their potential as anti-malaria agents. Additionally, the high electrophilicity index recorded for the three compounds further supports their suitability as anti-malaria agents. The results collectively suggest that the pyrrolidine-pentanamide derivatives of sulfonamides have promising interactions with the target proteins, indicating their potential effectiveness against malaria. This study provides valuable insights into the molecular properties and binding affinities of the synthesized compounds, contributing to the development of new anti-malaria therapies.

Ribosomal Protein S12 Hastens Nucleation of Co-Transcriptional Ribosome Assembly.

Ribosomal subunits begin assembly during transcription of the ribosomal RNA (rRNA), when the rRNA begins to fold and associate with ribosomal proteins (RPs). In bacteria, the first steps of ribosome assembly depend upon recognition of the properly folded rRNA by primary assembly proteins such as S4, which nucleates assembly of the 16S 5' domain. Recent evidence, however, suggests that initial recognition by S4 is delayed due to variable folding of the rRNA during transcription. Here, using single-molecule colocalization co-transcriptional assembly (smCoCoA), we show that the late-binding RP S12 specifically promotes the association of S4 with the pre-16S rRNA during transcription, thereby accelerating nucleation of 30S ribosome assembly. Order of addition experiments suggest that S12 helps chaperone the rRNA during transcription, particularly near the S4 binding site. S12 interacts transiently with the rRNA during transcription and, consequently, a high concentration is required for its chaperone activity. These results support a model in which late-binding RPs moonlight as RNA chaperones during transcription in order to facilitate rapid assembly.

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.

G-protein-coupled receptor 141 mediates breast cancer proliferation and metastasis by regulating oncogenic mediators and the p-mTOR/p53 axis.

Breast cancer morbidity is surging towards the peak in females across the globe. An inherent property of cancer cells is enhanced cell proliferation rate and migration capability, leading to deregulated cell signaling cascades. G-protein-coupled receptors (GPCRs) have recently emerged as a hot-spot target in cancer research. We identify aberrant expression of G-protein-coupled receptor 141 (GPR141) in different breast cancer subtypes that correlate with poor prognosis. However, the molecular mechanism via which GPR141 advances breast cancer remains elusive. Increased GPR141 expression enhances the migratory behavior of breast cancer, driving oncogenic pathways both in vitro and in vivo through activation of epithelial to mesenchymal transition (EMT), oncogenic mediators and regulation of p-mTOR/p53 signaling. Our study unveils a molecular mechanism for p53 downregulation and activation of p-mTOR1 and its substrates in GPR141 overexpressed cells, accelerating breast tumorigenesis. We find that an E3 ubiquitin ligase, Cullin1, partly mediates p53 degradation via proteasomal pathway. Co-immunoprecipitation result shows that the phosphorylated form of 40S ribosome protein S6 (ps6., a p-mTOR1 substrate) forms a complex with Cullin1. These findings suggest an interplay between Cullin1 and p-mTOR1 in GPR141 overexpressed cells that downregulates p53 expression, thus inducing tumor growth. GPR141 silencing restores p53 expression and attenuates p-mTOR1 signaling events, thereby impeding proliferation and migration in breast cancer cells. Our findings describe the role of GPR141 in breast cancer proliferation, and metastasis, as well as in influencing the tumor microenvironment. Modulating GPR141 expression could pave the way for a better therapeutic approach to regulating breast cancer progression and metastasis.

Streptothricin F is a bactericidal antibiotic effective against highly drug-resistant gram-negative bacteria that interacts with the 30S subunit of the 70S ribosome.

The streptothricin natural product mixture (also known as nourseothricin) was discovered in the early 1940s, generating intense initial interest because of excellent gram-negative activity. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin F (S-F, 1 lysine) and streptothricin D (S-D, 3 lysines), purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE) and Acinetobacter baumannii. For CRE, the MIC50 and MIC90 for S-F and S-D were 2 and 4 μM, and 0.25 and 0.5 μM, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes in in vitro translation assays. In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1-expressing Klebsiella pneumoniae Nevada strain with minimal or no toxicity. Cryo-EM characterization of S-F bound to the A. baumannii 70S ribosome defines extensive hydrogen bonding of the S-F steptolidine moiety, as a guanine mimetic, to the 16S rRNA C1054 nucleobase (Escherichia coli numbering) in helix 34, and the carbamoylated gulosamine moiety of S-F with A1196, explaining the high-level resistance conferred by corresponding mutations at the residues identified in single rrn operon E. coli. Structural analysis suggests that S-F probes the A-decoding site, which potentially may account for its miscoding activity. Based on unique and promising activity, we suggest that the streptothricin scaffold deserves further preclinical exploration as a potential therapeutic for drug-resistant, gram-negative pathogens.

Molecular basis for recognition and deubiquitination of 40S ribosomes by Otu2

In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not4 and deubiquitinated by Otu2 upon ribosomal subunit recycling. Despite its importance for translation efficiency the exact role and structural basis for this translational reset is poorly understood. Here, structural analysis by cryo-electron microscopy of native and reconstituted Otu2-bound ribosomal complexes reveals that Otu2 engages 40S subunits mainly between ribosome recycling and initiation stages. Otu2 binds to several sites on the intersubunit surface of the 40S that are not occupied by any other 40S-binding factors. This binding mode explains the discrimination against 80S ribosomes via the largely helical N-terminal domain of Otu2 as well as the specificity for mono-ubiquitinated eS7 on 40S. Collectively, this study reveals mechanistic insights into the Otu2-driven deubiquitination steps for translational reset during ribosome recycling/(re)initiation.

Structure of the actively translating plant 80S ribosome at 2.2 Å resolution

In plant cells, translation occurs in three compartments: the cytosol, the plastids and the mitochondria. While the structures of the (prokaryotic-type) ribosomes in plastids and mitochondria are well characterized, high-resolution structures of the eukaryotic 80S ribosomes in the cytosol have been lacking. Here the structure of translating tobacco (Nicotiana tabacum) 80S ribosomes was solved by cryo-electron microscopy with a global resolution of 2.2 Å. The ribosome structure includes two tRNAs, decoded mRNA and the nascent peptide chain, thus providing insights into the molecular underpinnings of the cytosolic translation process in plants. The map displays conserved and plant-specific rRNA modifications and the positions of numerous ionic cofactors, and it uncovers the role of monovalent ions in the decoding centre. The model of the plant 80S ribosome enables broad phylogenetic comparisons that reveal commonalities and differences in the ribosomes of plants and those of other eukaryotes, thus putting our knowledge about eukaryotic translation on a firmer footing.

The ASC-1 complex promotes translation initiation by scanning ribosomes.

Translation initiates when the eIF4F complex binds the 5' mRNA cap, followed by 5' untranslated region scanning for the start codon by scanning ribosomes. Here, we demonstrate that the ASC-1 complex (ASCC), which was previously shown to promote the dissociation of colliding 80S ribosomes, associates with scanning ribosomes to regulate translation initiation. Selective translation complex profiling (TCP-seq) analysis revealed that ASCC3, a helicase domain-containing subunit of ASCC, localizes predominantly to the 5' untranslated region of mRNAs. Ribo-seq, TCP-seq, and luciferase reporter analyses showed that ASCC3 knockdown impairs 43S preinitiation complex loading and scanning dynamics, thereby reducing translation efficiency. Whereas eIF4A, an RNA helicase in the eIF4F complex, is important for global translation, ASCC was found to regulate the scanning process for a specific subset of transcripts. Our results have thus revealed that ASCC is required not only for dissociation of colliding 80S ribosomes but also for efficient translation initiation by scanning ribosomes at a subset of transcripts.

Structural inventory of cotranslational protein folding by the eukaryotic RAC complex

The challenge of nascent chain folding at the ribosome is met by the conserved ribosome-associated complex (RAC), which forms a chaperone triad with the Hsp70 protein Ssb in fungi, and consists of the non-canonical Hsp70 Ssz1 and the J domain protein Zuotin (Zuo1). Here we determine cryo-EM structures of Chaetomium thermophilum RAC bound to 80S ribosomes. RAC adopts two distinct conformations accommodating continuous ribosomal rotation by a flexible lever arm. It is held together by a tight interaction between the Ssz1 substrate-binding domain and the Zuo1 N terminus, and additional contacts between the Ssz1 nucleotide-binding domain and Zuo1 J- and Zuo1 homology domains, which form a rigid unit. The Zuo1 HPD motif conserved in J-proteins is masked in a non-canonical interaction by the Ssz1 nucleotide-binding domain, and allows the positioning of Ssb for activation by Zuo1. Overall, we provide the basis for understanding how RAC cooperates with Ssb in a dynamic nascent chain interaction and protein folding.

Initiation of translation on nedicistrovirus and related intergenic region IRESs by their factor-independent binding to the P site of 80S ribosomes.

Initiation of translation on many viral mRNAs occurs by noncanonical mechanisms that involve 5' end-independent binding of ribosomes to an internal ribosome entry site (IRES). The ∼190-nt-long intergenic region (IGR) IRES of dicistroviruses such as cricket paralysis virus (CrPV) initiates translation without Met-tRNAi Met or initiation factors. Advances in metagenomics have revealed numerous dicistrovirus-like genomes with shorter, structurally distinct IGRs, such as nedicistrovirus (NediV) and Antarctic picorna-like virus 1 (APLV1). Like canonical IGR IRESs, the ∼165-nt-long NediV-like IGRs comprise three domains, but they lack key canonical motifs, including L1.1a/L1.1b loops (which bind to the L1 stalk of the ribosomal 60S subunit) and the apex of stem-loop V (SLV) (which binds to the head of the 40S subunit). Domain 2 consists of a compact, highly conserved pseudoknot (PKIII) that contains a UACUA loop motif and a protruding CrPV-like stem--loop SLIV. In vitro reconstitution experiments showed that NediV-like IRESs initiate translation from a non-AUG codon and form elongation-competent 80S ribosomal complexes in the absence of initiation factors and Met-tRNAi Met Unlike canonical IGR IRESs, NediV-like IRESs bind directly to the peptidyl (P) site of ribosomes leaving the aminoacyl (A) site accessible for decoding. The related structures of NediV-like IRESs and their common mechanism of action indicate that they exemplify a distinct class of IGR IRES.

Abstract 3906: The mRNA translational plasticity of small cell lung carcinoma is associated with its phenotypic transdifferentiation

Abstract Small cell lung carcinoma(SCLC), classified as a recalcitrant cancer type, accounts for 13-15% of all lung cancers with a median overall survival less than 10 months in patients with extensive stage SCLC. SCLC was once considered as molecularly homogeneous due to nearly universal loss of TP53 and RB1, whereas subsets of MYC-driven, non-neuroendocrine (non-NE) SCLC variants, possess chemoresistance, suggesting its high plasticity and intertumoral heterogeneity related to rapid refractory to first-line chemotherapies. Current pathognomonic classification stratifies SCLC into two NE subtypes (ASCL1+, NEUROD1+) and two non-NE subtypes (POU2F3+ and triple negative), associated with different expression patterns of MYC family proteins. MYC is a master regulator of mRNA translation, whether translational regulation actively tunes SCLC plasticity and the associated chemosensitivity remains an enigma. Here, we delineate the translation landscape of NE and non-NE SCLC cells by using paired human NE SCLC cell line and constitutive or induced non-NE SCLC cells. As expected, non-NE SCLC cells are more resistant to the clinically relevant first-line chemotherapy than NE SCLC cells. With polysome profiling in combination with polysome RNA-seq, we showed differential expression at both transcription- and translation-level. However, transcription-level changes minimally overlapped with the translational changes between NE and non-NE SCLC cells, with a global reduction of translation activity observed in non-NE SCLC cells. Through analyzing the polysome profiles, we observed a striking accumulation of 60S monosomes and diminishment of 80S ribosomes derived from non-NE SCLC cells compared to those from NE SCLC cells. This unique translational pattern in non-NE SCLC cells was correlated with an increased expression of eIF6, a ‘sitting on the bench’ member of eukaryotic initiation factor (eIF) family. The eIF6 couples 60S monosome maturation and recycling, enrolling a quintessential role in functional 80S ribosome formation. Positive correlation between eIF6 expression and non-NE SCLC tumors was observed in 81 tumor samples derived from patients with limited stage SCLC and CCLE cell consortium, in which c-MYC expression was also positively correlated with eIF6 level. By using both ultracentrifugation and fraction analysis from polysome profiling, we found that less association of eIF6 with 60S may mitigate 80S ribosome assembly in non-NE SCLC cells. In addition, in vivo xenograft models showed that eIF6 expression was higher in residual tumors upon chemotherapy. Taken together, these data revealed an eIF6-dependent translational regulation in non-NE transdifferentiation of SCLC subtypes, suggesting an underappreciated function of translational regulation of SCLC plasticity and the potential therapeutic value of eIF6 for overcoming SCLC resistance to chemotherapy. Citation Format: Haoning Peng, Mengyao Wang, Lu Li, Lunxu Liu, Shensi Shen. The mRNA translational plasticity of small cell lung carcinoma is associated with its phenotypic transdifferentiation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3906.

Structural Insights into the Distortion of the Ribosomal Small Subunit at Different Magnesium Concentrations

Magnesium ions are abundant and play indispensable functions in the ribosome. A decrease in Mg2+ concentration causes 70S ribosome dissociation and subsequent unfolding. Structural distortion at low Mg2+ concentrations has been observed in an immature pre50S, while the structural changes in mature subunits have not yet been studied. Here, we purified the 30S subunits of E. coli cells under various Mg2+ concentrations and analyzed their structural distortion by cryo-electron microscopy. Upon systematically interrogating the structural heterogeneity within the 1 mM Mg2+ dataset, we observed 30S particles with different levels of structural distortion in the decoding center, h17, and the 30S head. Our model showed that, when the Mg2+ concentration decreases, the decoding center distorts, starting from h44 and followed by the shifting of h18 and h27, as well as the dissociation of ribosomal protein S12. Mg2+ deficiency also eliminates the interactions between h17, h10, h15, and S16, resulting in the movement of h17 towards the tip of h6. More flexible structures were observed in the 30S head and platform, showing high variability in these regions. In summary, the structures resolved here showed several prominent distortion events in the decoding center and h17. The requirement for Mg2+ in ribosomes suggests that the conformational changes reported here are likely shared due to a lack of cellular Mg2+ in all domains of life.

Sarecycline inhibits protein translation in Cutibacterium acnes 70S ribosome using a two-site mechanism.

Acne vulgaris is a chronic disfiguring skin disease affecting ∼1 billion people worldwide, often having persistent negative effects on physical and mental health. The Gram-positive anaerobe, Cutibacterium acnes is implicated in acne pathogenesis and is, therefore, a main target for antibiotic-based acne therapy. We determined a 2.8-Å resolution structure of the 70S ribosome of Cutibacterium acnes by cryogenic electron microscopy and discovered that sarecycline, a narrow-spectrum antibiotic against Cutibacterium acnes, may inhibit two active sites of this bacterium's ribosome in contrast to the one site detected previously on the model ribosome of Thermus thermophilus. Apart from the canonical binding site at the mRNA decoding center, the second binding site for sarecycline exists at the nascent peptide exit tunnel, reminiscent of the macrolides class of antibiotics. The structure also revealed Cutibacterium acnes-specific features of the ribosomal RNA and proteins. Unlike the ribosome of the Gram-negative bacterium Escherichia coli, Cutibacterium acnes ribosome has two additional proteins, bS22 and bL37, which are also present in the ribosomes of Mycobacterium smegmatis and Mycobacterium tuberculosis. We show that bS22 and bL37 have antimicrobial properties and may be involved in maintaining the healthy homeostasis of the human skin microbiome.

Near-physiological in vitro assembly of 50S ribosomes involves parallel pathways.

Understanding the assembly principles of biological macromolecular complexes remains a significant challenge, due to the complexity of the systems and the difficulties in developing experimental approaches. As a ribonucleoprotein complex, the ribosome serves as a model system for the profiling of macromolecular complex assembly. In this work, we report an ensemble of large ribosomal subunit intermediate structures that accumulate during synthesis in a near-physiological and co-transcriptional in vitro reconstitution system. Thirteen pre-50S intermediate maps covering the entire assembly process were resolved using cryo-EM single-particle analysis and heterogeneous subclassification. Segmentation of the set of density maps reveals that the 50S ribosome intermediates assemble based on fourteen cooperative assembly blocks, including the smallest assembly core reported to date, which is composed of a 600-nucleotide-long folded rRNA and three ribosomal proteins. The cooperative blocks assemble onto the assembly core following defined dependencies, revealing the parallel pathways at both early and late assembly stages of the 50S subunit.