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Cell Reports Feb 2022Eukaryotic ribosome biogenesis is facilitated and regulated by numerous ribosome biogenesis factors (RBFs). High-resolution cryoelectron microscopy (cryo-EM) maps have...
Eukaryotic ribosome biogenesis is facilitated and regulated by numerous ribosome biogenesis factors (RBFs). High-resolution cryoelectron microscopy (cryo-EM) maps have defined the molecular interactions of RBFs during maturation, but many transient and dynamic interactions, particularly during early assembly, remain uncharacterized. Using quantitative proteomics and crosslinking coupled to mass spectrometry (XL-MS) data from an extensive set of pre-ribosomal particles, we derive a comprehensive and time-resolved interaction map of RBF engagement during 60S maturation. We localize 22 previously unmapped RBFs to specific biogenesis intermediates and validate our results by mapping the catalytic activity of the methyltransferases Bmt2 and Rcm1 to their predicted nucleolar 60S intermediates. Our analysis reveals the interaction sites for the RBFs Noc2 and Ecm1 and elucidates the interaction map and timing of 60S engagement by the DEAD-box ATPases Dbp9 and Dbp10. Our data provide a powerful resource for future studies of 60S ribosome biogenesis.
Topics: Cell Nucleolus; Cryoelectron Microscopy; Models, Molecular; Ribosomal Proteins; Ribosome Subunits, Large, Eukaryotic; Ribosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 35139378
DOI: 10.1016/j.celrep.2022.110353 -
Proceedings of the National Academy of... Aug 2020Zinc starvation in mycobacteria leads to remodeling of ribosomes, in which multiple ribosomal (r-) proteins containing the zinc-binding CXXC motif are replaced by their...
Zinc starvation in mycobacteria leads to remodeling of ribosomes, in which multiple ribosomal (r-) proteins containing the zinc-binding CXXC motif are replaced by their motif-free paralogues, collectively called C- r-proteins. We previously reported that the 70S C- ribosome is exclusively targeted for hibernation by mycobacterial-specific protein Y (Mpy), which binds to the decoding center and stabilizes the ribosome in an inactive and drug-resistant state. In this study, we delineate the conditions for ribosome remodeling and hibernation and provide further insight into how zinc depletion induces Mpy recruitment to C- ribosomes. Specifically, we show that ribosome hibernation in a batch culture is induced at an approximately two-fold lower cellular zinc concentration than remodeling. We further identify a growth phase in which the C- ribosome remains active, while its hibernation is inhibited by the caseinolytic protease (Clp) system in a zinc-dependent manner. The Clp protease system destabilizes a zinc-bound form of Mpy recruitment factor (Mrf), which is stabilized upon further depletion of zinc, presumably in a zinc-free form. Stabilized Mrf binds to the 30S subunit and recruits Mpy to the ribosome. Replenishment of zinc to cells harboring hibernating ribosomes restores Mrf instability and dissociates Mpy from the ribosome. Finally, we demonstrate zinc-responsive binding of Mpy to ribosomes in (Mtb) and show Mpy-dependent antibiotic tolerance of Mtb in mouse lungs. Together, we propose that ribosome hibernation is a specific and conserved response to zinc depletion in both environmental and pathogenic mycobacteria.
Topics: Animals; Antibiotics, Antitubercular; Bacterial Proteins; Drug Tolerance; Endopeptidase Clp; Mice; Mycobacterium tuberculosis; Protein Processing, Post-Translational; Ribosomal Proteins; Ribosome Subunits; Ribosomes; Zinc
PubMed: 32723821
DOI: 10.1073/pnas.2013409117 -
Proceedings of the National Academy of... Dec 2015We present a molecular-level model for the origin and evolution of the translation system, using a 3D comparative method. In this model, the ribosome evolved by...
We present a molecular-level model for the origin and evolution of the translation system, using a 3D comparative method. In this model, the ribosome evolved by accretion, recursively adding expansion segments, iteratively growing, subsuming, and freezing the rRNA. Functions of expansion segments in the ancestral ribosome are assigned by correspondence with their functions in the extant ribosome. The model explains the evolution of the large ribosomal subunit, the small ribosomal subunit, tRNA, and mRNA. Prokaryotic ribosomes evolved in six phases, sequentially acquiring capabilities for RNA folding, catalysis, subunit association, correlated evolution, decoding, energy-driven translocation, and surface proteinization. Two additional phases exclusive to eukaryotes led to tentacle-like rRNA expansions. In this model, ribosomal proteinization was a driving force for the broad adoption of proteins in other biological processes. The exit tunnel was clearly a central theme of all phases of ribosomal evolution and was continuously extended and rigidified. In the primitive noncoding ribosome, proto-mRNA and the small ribosomal subunit acted as cofactors, positioning the activated ends of tRNAs within the peptidyl transferase center. This association linked the evolution of the large and small ribosomal subunits, proto-mRNA, and tRNA.
Topics: Biocatalysis; Escherichia coli; Evolution, Molecular; Models, Molecular; Nucleic Acid Conformation; Protein Biosynthesis; RNA, Messenger; RNA, Ribosomal; RNA, Transfer; Ribosome Subunits; Ribosomes
PubMed: 26621738
DOI: 10.1073/pnas.1509761112 -
Nucleic Acids Research Jan 2018Human translation initiation relies on the combined activities of numerous ribosome-associated eukaryotic initiation factors (eIFs). The largest factor, eIF3, is an...
Human translation initiation relies on the combined activities of numerous ribosome-associated eukaryotic initiation factors (eIFs). The largest factor, eIF3, is an ∼800 kDa multiprotein complex that orchestrates a network of interactions with the small 40S ribosomal subunit, other eIFs, and mRNA, while participating in nearly every step of initiation. How these interactions take place during the time course of translation initiation remains unclear. Here, we describe a method for the expression and affinity purification of a fluorescently-tagged eIF3 from human cells. The tagged eIF3 dodecamer is structurally intact, functions in cell-based assays, and interacts with the HCV IRES mRNA and the 40S-IRES complex in vitro. By tracking the binding of single eIF3 molecules to the HCV IRES RNA with a zero-mode waveguides-based instrument, we show that eIF3 samples both wild-type IRES and an IRES that lacks the eIF3-binding region, and that the high-affinity eIF3-IRES interaction is largely determined by slow dissociation kinetics. The application of single-molecule methods to more complex systems involving eIF3 may unveil dynamics underlying mRNA selection and ribosome loading during human translation initiation.
Topics: Eukaryotic Initiation Factor-3; Fluorescent Dyes; Hepacivirus; Humans; Internal Ribosome Entry Sites; Protein Biosynthesis; RNA, Messenger; RNA, Viral; Reproducibility of Results; Ribosome Subunits, Small, Eukaryotic; Single Molecule Imaging; Spectrum Analysis
PubMed: 29136179
DOI: 10.1093/nar/gkx1050 -
International Journal of Molecular... Dec 2023Ribosome is a major part of the protein synthesis machinery, and analysis of its structure is of paramount importance. However, the structure of ribosomes from only a...
Ribosome is a major part of the protein synthesis machinery, and analysis of its structure is of paramount importance. However, the structure of ribosomes from only a limited number of organisms has been resolved to date; it especially concerns plant ribosomes and ribosomal subunits. Here, we report a high-resolution cryo-electron microscopy reconstruction of the small subunit of the (common wheat) cytoplasmic ribosome. A detailed atomic model was built that includes the majority of the rRNA and some of the protein modifications. The analysis of the obtained data revealed structural peculiarities of the 40S subunit in the monocot plant ribosome. We applied the 3D Flexible Refinement approach to analyze the internal mobility of the 40S subunit and succeeded in decomposing it into four major motions, describing rotations of the head domain and a shift in the massive rRNA expansion segment. It was shown that these motions are almost uncorrelated and that the 40S subunit is flexible enough to spontaneously adopt any conformation it takes as a part of a translating ribosome or ribosomal complex. Here, we introduce the first high-resolution structure of an isolated plant 40S subunit and the first quantitative analysis of the flexibility of small ribosomal subunits, hoping that it will help in studying various aspects of ribosome functioning.
Topics: Ribosome Subunits, Small, Eukaryotic; Cryoelectron Microscopy; Ribosomes; RNA, Ribosomal; Protein Biosynthesis; Ribosomal Proteins
PubMed: 38139282
DOI: 10.3390/ijms242417453 -
Biochimie Jul 2015The accuracy of start codon selection is determined by the translation initiation process. In prokaryotes the initiation step on most mRNAs relies on recruitment of the... (Review)
Review
The accuracy of start codon selection is determined by the translation initiation process. In prokaryotes the initiation step on most mRNAs relies on recruitment of the small ribosomal subunit onto the initiation codon by base pairing between the mRNA and the 16S rRNA. Eukaryotes have evolved a complex molecular machinery involving at least 11 initiation factors, and mRNAs do not directly recruit the small ribosomal subunit. Instead the initiation complex is recruited to the 5' end of the mRNA through a complex protein network including eIF4E that interacts with the 5' cap structure and poly-A binding protein that interacts with the 3'end. However, some viral and cellular mRNAs are able to escape this pathway by internal recruitment of one or several components of the translation machinery. Here we review those eukaryotic mRNAs that have been reported to directly recruit the 40S ribosomal subunit internally. In the well characterized cases of viral IRESes, a specific RNA structure is involved in this process, and in addition to recruitment of the ribosome, the mRNA also manipulates the ribosome structure to stimulate the first translocation step. We also review recently described IRES/ribosome interactions in cases where the molecular mechanism leading to translation initiation has yet to be described. Finally we evaluate the possibility that mRNA may recruit the 40S ribosomal subunit through base pairing with the 18S rRNA.
Topics: Animals; Binding Sites; Humans; Protein Biosynthesis; RNA Transport; RNA, Messenger; RNA, Transfer; Ribosome Subunits, Large, Eukaryotic; Ribosome Subunits, Small, Eukaryotic
PubMed: 25530261
DOI: 10.1016/j.biochi.2014.12.008 -
Nature Structural & Molecular Biology Jan 2020The bacterial ribosome is recycled into subunits by two conserved proteins, elongation factor G (EF-G) and the ribosome recycling factor (RRF). The molecular basis for...
The bacterial ribosome is recycled into subunits by two conserved proteins, elongation factor G (EF-G) and the ribosome recycling factor (RRF). The molecular basis for ribosome recycling by RRF and EF-G remains unclear. Here, we report the crystal structure of a posttermination Thermus thermophilus 70S ribosome complexed with EF-G, RRF and two transfer RNAs at a resolution of 3.5 Å. The deacylated tRNA in the peptidyl (P) site moves into a previously unsuspected state of binding (peptidyl/recycling, p/R) that is analogous to that seen during initiation. The terminal end of the p/R-tRNA forms nonfavorable contacts with the 50S subunit while RRF wedges next to central inter-subunit bridges, illuminating the active roles of tRNA and RRF in dissociation of ribosomal subunits. The structure uncovers a missing snapshot of tRNA as it transits between the P and exit (E) sites, providing insights into the mechanisms of ribosome recycling and tRNA translocation.
Topics: Bacterial Proteins; Crystallography, X-Ray; Models, Molecular; Peptide Elongation Factor G; Protein Conformation; RNA, Transfer; Ribosomal Proteins; Ribosome Subunits, Large, Bacterial; Ribosomes; Thermus thermophilus
PubMed: 31873307
DOI: 10.1038/s41594-019-0350-7 -
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 -
Plant Physiology Aug 2018Chloroplast ribosomes, which originated from cyanobacteria, comprise a large subunit (50S) and a small subunit (30S) containing ribosomal RNAs (rRNAs) and various...
Chloroplast ribosomes, which originated from cyanobacteria, comprise a large subunit (50S) and a small subunit (30S) containing ribosomal RNAs (rRNAs) and various ribosomal proteins. Genes for many chloroplast ribosomal proteins, as well as proteins with auxiliary roles in ribosome biogenesis or functioning, reside in the nucleus. Here, we identified Arabidopsis () CHLOROPLAST RIBOSOME ASSOCIATED (CRASS), a member of the latter class of proteins, based on the tight coexpression of its mRNA with transcripts for nucleus-encoded chloroplast ribosomal proteins. CRASS was acquired during the evolution of embryophytes and is localized to the chloroplast stroma. Loss of CRASS results in minor defects in development, photosynthetic efficiency, and chloroplast translation activity under controlled growth conditions, but these phenotypes are greatly exacerbated under stress conditions induced by the translational inhibitors lincomycin and chloramphenicol or by cold treatment. The CRASS protein comigrates with chloroplast ribosomal particles and coimmunoprecipitates with the 16S rRNA and several chloroplast ribosomal proteins, particularly the plastid ribosomal proteins of the 30S subunit (PRPS1 and PRPS5). The association of CRASS with PRPS1 and PRPS5 is independent of rRNA and is not detectable in yeast two-hybrid experiments, implying that either CRASS interacts indirectly with PRPS1 and PRPS5 via another component of the small ribosomal subunit or that it recognizes structural features of the multiprotein/rRNA particle. CRASS plays a role in the biogenesis and/or stability of the chloroplast ribosome that becomes critical under certain stressful conditions when ribosomal activity is compromised.
Topics: Arabidopsis; Arabidopsis Proteins; Carrier Proteins; Chloroplasts; Cold-Shock Response; Embryophyta; Gene Expression Regulation, Plant; Immunoprecipitation; Plants, Genetically Modified; Protein Biosynthesis; RNA, Ribosomal, 16S; Ribosome Subunits, Small
PubMed: 29914890
DOI: 10.1104/pp.18.00602 -
Nature Communications Feb 2019Ribo-T is an engineered ribosome whose small and large subunits are tethered together by linking 16S rRNA and 23S rRNA in a single molecule. Although Ribo-T can support...
Ribo-T is an engineered ribosome whose small and large subunits are tethered together by linking 16S rRNA and 23S rRNA in a single molecule. Although Ribo-T can support cell proliferation in the absence of wild type ribosomes, Ribo-T cells grow slower than those with wild type ribosomes. Here, we show that cell growth defect is likely explained primarily by slow Ribo-T assembly rather than its imperfect functionality. Ribo-T maturation is stalled at a late assembly stage. Several post-transcriptional rRNA modifications and some ribosomal proteins are underrepresented in the accumulated assembly intermediates and rRNA ends are incompletely trimmed. Ribosome profiling of Ribo-T cells shows no defects in translation elongation but reveals somewhat higher occupancy by Ribo-T of the start codons and to a lesser extent stop codons, suggesting that subunit tethering mildly affects the initiation and termination stages of translation. Understanding limitations of Ribo-T system offers ways for its future development.
Topics: Codon, Initiator; Escherichia coli; Protein Biosynthesis; RNA Processing, Post-Transcriptional; RNA, Bacterial; RNA, Ribosomal, 16S; RNA, Ribosomal, 23S; Ribosomal Proteins; Ribosome Subunits
PubMed: 30804338
DOI: 10.1038/s41467-019-08892-w