-
Proceedings of the National Academy of... Dec 2017Aminoglycosides are chemically diverse, broad-spectrum antibiotics that target functional centers within the bacterial ribosome to impact all four principle stages...
Aminoglycosides are chemically diverse, broad-spectrum antibiotics that target functional centers within the bacterial ribosome to impact all four principle stages (initiation, elongation, termination, and recycling) of the translation mechanism. The propensity of aminoglycosides to induce miscoding errors that suppress the termination of protein synthesis supports their potential as therapeutic interventions in human diseases associated with premature termination codons (PTCs). However, the sites of interaction of aminoglycosides with the eukaryotic ribosome and their modes of action in eukaryotic translation remain largely unexplored. Here, we use the combination of X-ray crystallography and single-molecule FRET analysis to reveal the interactions of distinct classes of aminoglycosides with the 80S eukaryotic ribosome. Crystal structures of the 80S ribosome in complex with paromomycin, geneticin (G418), gentamicin, and TC007, solved at 3.3- to 3.7-Å resolution, reveal multiple aminoglycoside-binding sites within the large and small subunits, wherein the 6'-hydroxyl substituent in ring I serves as a key determinant of binding to the canonical eukaryotic ribosomal decoding center. Multivalent binding interactions with the human ribosome are also evidenced through their capacity to affect large-scale conformational dynamics within the pretranslocation complex that contribute to multiple aspects of the translation mechanism. The distinct impacts of the aminoglycosides examined suggest that their chemical composition and distinct modes of interaction with the ribosome influence PTC read-through efficiency. These findings provide structural and functional insights into aminoglycoside-induced impacts on the eukaryotic ribosome and implicate pleiotropic mechanisms of action beyond decoding.
Topics: Aminoglycosides; Bacteria; Binding Sites; Eukaryota; Humans; Models, Molecular; Molecular Conformation; Protein Binding; Ribosome Subunits; Ribosomes
PubMed: 29208708
DOI: 10.1073/pnas.1715501114 -
Blood Apr 2010Ribosomopathies compose a collection of disorders in which genetic abnormalities cause impaired ribosome biogenesis and function, resulting in specific clinical... (Review)
Review
Ribosomopathies compose a collection of disorders in which genetic abnormalities cause impaired ribosome biogenesis and function, resulting in specific clinical phenotypes. Congenital mutations in RPS19 and other genes encoding ribosomal proteins cause Diamond-Blackfan anemia, a disorder characterized by hypoplastic, macrocytic anemia. Mutations in other genes required for normal ribosome biogenesis have been implicated in other rare congenital syndromes, Schwachman-Diamond syndrome, dyskeratosis congenita, cartilage hair hypoplasia, and Treacher Collins syndrome. In addition, the 5q- syndrome, a subtype of myelodysplastic syndrome, is caused by a somatically acquired deletion of chromosome 5q, which leads to haploinsufficiency of the ribosomal protein RPS14 and an erythroid phenotype highly similar to Diamond-Blackfan anemia. Acquired abnormalities in ribosome function have been implicated more broadly in human malignancies. The p53 pathway provides a surveillance mechanism for protein translation as well as genome integrity and is activated by defects in ribosome biogenesis; this pathway appears to be a critical mediator of many of the clinical features of ribosomopathies. Elucidation of the mechanisms whereby selective abnormalities in ribosome biogenesis cause specific clinical syndromes will hopefully lead to novel therapeutic strategies for these diseases.
Topics: Genetic Diseases, Inborn; Humans; Ribosomes; Syndrome
PubMed: 20194897
DOI: 10.1182/blood-2009-10-178129 -
RNA (New York, N.Y.) Feb 2003The recent structures of the ribosome and the ribosomal subunits only heighten the intrigue of trying to understand how the ribosome is assembled. Biochemical and... (Review)
Review
The recent structures of the ribosome and the ribosomal subunits only heighten the intrigue of trying to understand how the ribosome is assembled. Biochemical and mechanistic studies have mapped out the basic series of protein binding events that occur, but we do not yet have a clear picture of the RNA conformational changes that must accompany the protein binding. Recent studies point to roles of protein folding chaperones and RNA helicases as facilitators of ribosome assembly, but the basic process of assembly seems to be encoded in the RNA sequences and can occur for the most part spontaneously in vitro, and quite possibly in vivo as well.
Topics: Bacteria; In Vitro Techniques; RNA, Ribosomal; Ribosomes
PubMed: 12554857
DOI: 10.1261/rna.2164903 -
BMC Biology Jun 2019Ribosome biogenesis is a central process in every growing cell. In eukaryotes, it requires more than 250 non-ribosomal assembly factors, most of which are essential....
BACKGROUND
Ribosome biogenesis is a central process in every growing cell. In eukaryotes, it requires more than 250 non-ribosomal assembly factors, most of which are essential. Despite this large repertoire of potential targets, only very few chemical inhibitors of ribosome biogenesis are known so far. Such inhibitors are valuable tools to study this highly dynamic process and elucidate mechanistic details of individual maturation steps. Moreover, ribosome biogenesis is of particular importance for fast proliferating cells, suggesting its inhibition could be a valid strategy for treatment of tumors or infections.
RESULTS
We systematically screened ~ 1000 substances for inhibitory effects on ribosome biogenesis using a microscopy-based screen scoring ribosomal subunit export defects. We identified 128 compounds inhibiting maturation of either the small or the large ribosomal subunit or both. Northern blot analysis demonstrates that these inhibitors cause a broad spectrum of different rRNA processing defects.
CONCLUSIONS
Our findings show that the individual inhibitors affect a wide range of different maturation steps within the ribosome biogenesis pathway. Our results provide for the first time a comprehensive set of inhibitors to study ribosome biogenesis by chemical inhibition of individual maturation steps and establish the process as promising druggable pathway for chemical intervention.
Topics: Ribosomes; Saccharomyces cerevisiae
PubMed: 31182083
DOI: 10.1186/s12915-019-0664-2 -
Proceedings of the National Academy of... Aug 2020The ribosome translates the genetic code into proteins in all domains of life. Its size and complexity demand long-range interactions that regulate ribosome function....
The ribosome translates the genetic code into proteins in all domains of life. Its size and complexity demand long-range interactions that regulate ribosome function. These interactions are largely unknown. Here, we apply a global coevolution method, statistical coupling analysis (SCA), to identify coevolving residue networks (sectors) within the 23S ribosomal RNA (rRNA) of the large ribosomal subunit. As in proteins, SCA reveals a hierarchical organization of evolutionary constraints with near-independent groups of nucleotides forming physically contiguous networks within the three-dimensional structure. Using a quantitative, continuous-culture-with-deep-sequencing assay, we confirm that the top two SCA-predicted sectors contribute to ribosome function. These sectors map to distinct ribosome activities, and their origins trace to phylogenetic divergences across all domains of life. These findings provide a foundation to map ribosome allostery, explore ribosome biogenesis, and engineer ribosomes for new functions. Despite differences in chemical structure, protein and RNA enzymes appear to share a common internal logic of interaction and assembly.
Topics: Escherichia coli; Evolution, Molecular; Nucleic Acid Conformation; Phylogeny; RNA, Bacterial; RNA, Ribosomal, 23S; Ribosomes
PubMed: 32747536
DOI: 10.1073/pnas.1909634117 -
Trends in Biochemical Sciences Aug 2009The molecular mechanisms by which tRNA molecules enter and transit the ribosome during mRNA translation remains elusive. However, recent genetic, biochemical and... (Review)
Review
The molecular mechanisms by which tRNA molecules enter and transit the ribosome during mRNA translation remains elusive. However, recent genetic, biochemical and structural studies offer important new findings into the ordered sequence of events underpinning the translocation process that help place the molecular mechanism within reach. In particular, new structural and kinetic insights have been obtained regarding tRNA movements through 'hybrid state' configurations. These dynamic views reveal that the macromolecular ribosome particle, like many smaller proteins, has an intrinsic capacity to reversibly sample an ensemble of similarly stable native states. Such perspectives suggest that substrates, factors and environmental cues contribute to translation regulation by helping the dynamic system navigate through a highly complex and metastable energy landscape.
Topics: Binding Sites; Models, Molecular; Nucleic Acid Conformation; Protein Biosynthesis; RNA, Messenger; RNA, Transfer; Ribosomes; Thermodynamics
PubMed: 19647434
DOI: 10.1016/j.tibs.2009.04.004 -
Phytochemistry Oct 2022Ribosome inactivating proteins (RIPs) are rRNA N-glycosylases (EC 3.2.2.22) best known for hydrolyzing an adenine base from the conserved sarcin/ricin loop of ribosomal... (Review)
Review
Ribosome inactivating proteins (RIPs) are rRNA N-glycosylases (EC 3.2.2.22) best known for hydrolyzing an adenine base from the conserved sarcin/ricin loop of ribosomal RNA. Protein translation is inhibited by ribosome depurination; therefore, RIPs are generally considered toxic to cells. The expression of some RIPs is upregulated by biotic and abiotic stress, though the connection between RNA depurination and defense response is not well understood. Despite their prevalence in approximately one-third of flowering plant orders, our knowledge of RIPs stems primarily from biochemical analyses of individuals or genomics-scale analyses of small datasets from a limited number of species. Here, we performed an unbiased search for proteins with RIP domains and identified several-fold more RIPs than previously known - more than 800 from 120 species, many with novel associated domains and physicochemical characteristics. Based on protein domain configuration, we established 15 distinct groups, suggesting diverse functionality. Surprisingly, most of these RIPs lacked a signal peptide, indicating they may be localized to the nucleocytoplasm of cells, raising questions regarding their toxicity against conspecific ribosomes. Our phylogenetic analysis significantly extends previous models for RIP evolution in plants, predicting an original single-domain RIP that later evolved to acquire a signal peptide and different protein domains. We show that RIPs are distributed throughout 21 plant orders with many species maintaining genes for more than one RIP group. Our analyses provide the foundation for further characterization of these new RIP types, to understand how these enzymes function in plants.
Topics: Phylogeny; Plant Proteins; Protein Sorting Signals; RNA, Ribosomal; Ribosome Inactivating Proteins; Ribosomes
PubMed: 35934106
DOI: 10.1016/j.phytochem.2022.113337 -
FEBS Letters Mar 2002General principles of structure and function of the ribosome are surveyed, and the translating ribosome is regarded as a molecular conveying machine. Two coupled... (Review)
Review
General principles of structure and function of the ribosome are surveyed, and the translating ribosome is regarded as a molecular conveying machine. Two coupled conveying processes, the passing of compact tRNA globules and the drawing of linear mRNA chain through intraribosomal channel, are considered driven by discrete acts of translocation during translation. Instead of mechanical transmission mechanisms and power-stroke 'motors', thermal motion and chemically induced changes in affinities of ribosomal binding sites for their ligands (tRNAs, mRNA, elongation factors) are proposed to underlie all the directional movements within the ribosomal complex. The GTP-dependent catalysis of conformational transitions by elongation factors during translation is also discussed.
Topics: Animals; Biological Transport; Guanosine Triphosphate; Humans; Models, Molecular; Peptide Elongation Factors; Protein Biosynthesis; Protein Conformation; Ribosomes
PubMed: 11904172
DOI: 10.1016/s0014-5793(02)02309-8 -
MBio Feb 2017We postulate that the inhibition of growth and low rates of mortality of bacteria exposed to ribosome-binding antibiotics deemed bacteriostatic can be attributed almost...
UNLABELLED
We postulate that the inhibition of growth and low rates of mortality of bacteria exposed to ribosome-binding antibiotics deemed bacteriostatic can be attributed almost uniquely to these drugs reducing the number of ribosomes contributing to protein synthesis, i.e., the number of effective ribosomes. We tested this hypothesis with Escherichia coli K-12 MG1655 and constructs that had been deleted for 1 to 6 of the 7 rRNA (rrn) operons. In the absence of antibiotics, constructs with fewer rrn operons have lower maximum growth rates and longer lag phases than those with more ribosomal operons. In the presence of the ribosome-binding "bacteriostatic" antibiotics tetracycline, chloramphenicol, and azithromycin, E. coli strains with 1 and 2 rrn operons are killed at a substantially higher rate than those with more rrn operons. This increase in the susceptibility of E. coli with fewer rrn operons to killing by ribosome-targeting bacteriostatic antibiotics is not reflected in their greater sensitivity to killing by the bactericidal antibiotic ciprofloxacin, which does not target ribosomes, but also to killing by gentamicin, which does. Finally, when such strains are exposed to these ribosome-targeting bacteriostatic antibiotics, the time before these bacteria start to grow again when the drugs are removed, referred to as the post-antibiotic effect (PAE), is markedly greater for constructs with fewer rrn operons than for those with more rrn operons. We interpret the results of these other experiments reported here as support for the hypothesis that the reduction in the effective number of ribosomes due to binding to these structures provides a sufficient explanation for the action of bacteriostatic antibiotics that target these structures.
IMPORTANCE
Chemotherapeutic agents, including antibiotics, have been used for more than a century; nevertheless, there are still major gaps in our understanding of how these drugs operate which limit future advances in antibacterial chemotherapy. Although the molecular mechanisms by which antibiotics bind to their target structures are largely known, fundamental questions about how these drugs actually kill and/or inhibit the replication of bacteria remain unanswered and subjects of controversy. We postulate that for the broad class of ribosome-binding bacteriostatic antibiotics, their reducing the number of active (functional) ribosomes per cell provides a sufficient explanation for the abatement of replication and the low rate of decline in densities of viable cells of bacteria exposed to these drugs. Using E. coli K-12 constructs with deletions of from one to six of the seven ribosome-RNA operons and the ribosome-binding bacteriostatic antibiotics tetracycline, chloramphenicol, and azithromycin, we tested this hypothesis. The results of our experiments are consistent with this "numbers game" hypothesis.
Topics: Anti-Bacterial Agents; Escherichia coli K12; Microbial Viability; Protein Biosynthesis; Ribosomes
PubMed: 28174311
DOI: 10.1128/mBio.02253-16 -
Proceedings of the National Academy of... Feb 2022Researchers commonly anneal metals, alloys, and semiconductors to repair defects and improve microstructures via recrystallization. Theoretical studies indicate that...
Researchers commonly anneal metals, alloys, and semiconductors to repair defects and improve microstructures via recrystallization. Theoretical studies indicate that simulated annealing on biological macromolecules helps predict the final structures with minimum free energy. Experimental validation of this homogenizing effect and further exploration of its applications are fascinating scientific questions that remain elusive. Here, we chose the apo-state 70 ribosome from as a model, wherein the 30 subunit undergoes a thermally driven intersubunit rotation and exhibits substantial structural flexibility as well as distinct free energy. We experimentally demonstrate that annealing at a fast cooling rate enhances the 70 ribosome homogeneity and improves local resolution on the 30 subunit. After annealing, the 70 ribosome is in a nonrotated state with respect to corresponding intermediate structures in unannealed or heated ribosomes. Manifold-based analysis further indicates that the annealed 70 ribosome takes a narrow conformational distribution and exhibits a minimum-energy state in the free-energy landscape. Our experimental results offer a facile yet robust approach to enhance protein stability, which is ideal for high-resolution cryogenic electron microscopy. Beyond structure determination, annealing shows great potential for synchronizing proteins on a single-molecule level and can be extended to study protein folding and explore conformational and energy landscapes.
Topics: Cryoelectron Microscopy; Escherichia coli; Escherichia coli Proteins; Models, Molecular; Protein Conformation; RNA, Ribosomal; Ribosomal Proteins; Ribosomes
PubMed: 35177473
DOI: 10.1073/pnas.2111231119