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Proceedings of the National Academy of... Aug 2020Alternative ribosome subunit proteins are prevalent in the genomes of diverse bacterial species, but their functional significance is controversial. Attempts to study...
Alternative ribosome subunit proteins are prevalent in the genomes of diverse bacterial species, but their functional significance is controversial. Attempts to study microbial ribosomal heterogeneity have mostly relied on comparing wild-type strains with mutants in which subunits have been deleted, but this approach does not allow direct comparison of alternate ribosome isoforms isolated from identical cellular contexts. Here, by simultaneously purifying canonical and alternative RpsR ribosomes from , we show that alternative ribosomes have distinct translational features compared with their canonical counterparts. Both alternative and canonical ribosomes actively take part in protein synthesis, although they translate a subset of genes with differential efficiency as measured by ribosome profiling. We also show that alternative ribosomes have a relative defect in initiation complex formation. Furthermore, a strain of in which the alternative ribosome protein operon is deleted grows poorly in iron-depleted medium, uncovering a role for alternative ribosomes in iron homeostasis. Our work confirms the distinct and nonredundant contribution of alternative bacterial ribosomes for adaptation to hostile environments.
Topics: Bacterial Proteins; Iron; Mycobacterium smegmatis; Peptide Chain Initiation, Translational; Protein Biosynthesis; Ribosomal Proteins; Ribosome Subunits; Ribosomes
PubMed: 32723820
DOI: 10.1073/pnas.2009607117 -
Cellular and Molecular Life Sciences :... Feb 2009The assembly of the protein synthesis machinery occurs during translation initiation. In bacteria, this process involves the binding of messenger RNA(mRNA) start site... (Review)
Review
The assembly of the protein synthesis machinery occurs during translation initiation. In bacteria, this process involves the binding of messenger RNA(mRNA) start site and fMet-tRNA(fMet) to the ribosome, which results in the formation of the first codon-anticodon interaction and sets the reading frame for the decoding of the mRNA. This interaction takes place in the peptidyl site of the 30S ribosomal subunit and is controlled by the initiation factors IF1, IF2 and IF3 to form the 30S initiation complex. The binding of the 50S subunit and the ejection of the IFs mark the irreversible transition to the elongation phase. Visualization of these ligands on the ribosome has been achieved by cryo-electron microscopy and X-ray crystallography studies, which has helped to understand the mechanism of translation initiation at the molecular level. Conformational changes associated with different functional states provide a dynamic view of the initiation process and of its regulation.
Topics: Bacteria; Models, Molecular; Multiprotein Complexes; Nucleic Acid Conformation; Peptide Chain Initiation, Translational; Peptide Initiation Factors; Protein Biosynthesis; Protein Conformation; RNA, Messenger; RNA, Transfer, Met; Ribosome Subunits, Small, Bacterial
PubMed: 19011758
DOI: 10.1007/s00018-008-8416-4 -
Molecular Cell Aug 2020Translating ribosomes that slow excessively incur collisions with trailing ribosomes. Persistent collisions are detected by ZNF598, a ubiquitin ligase that ubiquitinates...
Translating ribosomes that slow excessively incur collisions with trailing ribosomes. Persistent collisions are detected by ZNF598, a ubiquitin ligase that ubiquitinates sites on the ribosomal 40S subunit to initiate pathways of mRNA and protein quality control. The collided ribosome complex must be disassembled to initiate downstream quality control, but the mechanistic basis of disassembly is unclear. Here, we reconstitute the disassembly of a collided polysome in a mammalian cell-free system. The widely conserved ASC-1 complex (ASCC) containing the ASCC3 helicase disassembles the leading ribosome in an ATP-dependent reaction. Disassembly, but not ribosome association, requires 40S ubiquitination by ZNF598, but not GTP-dependent factors, including the Pelo-Hbs1L ribosome rescue complex. Trailing ribosomes can elongate once the roadblock has been removed and only become targets if they subsequently stall and incur collisions. These findings define the specific role of ASCC during ribosome-associated quality control and identify the molecular target of its activity.
Topics: Amino Acid Transport System y+; Animals; Carrier Proteins; Cell-Free System; DNA Helicases; GTP-Binding Proteins; HEK293 Cells; Humans; Multiprotein Complexes; Nuclear Proteins; Peptide Termination Factors; Polyribosomes; Protein Biosynthesis; Rabbits; Ribosome Subunits; Ribosomes; Ubiquitination
PubMed: 32579943
DOI: 10.1016/j.molcel.2020.06.006 -
Proceedings of the National Academy of... Feb 2022Emerging evidence reveals that ribosomes are not monolithic but dynamic machines with heterogeneous protein compositions that can reshape ribosomal translational...
Emerging evidence reveals that ribosomes are not monolithic but dynamic machines with heterogeneous protein compositions that can reshape ribosomal translational abilities and cellular adaptation to environmental changes. Duplications of ribosomal protein (RP) genes are ubiquitous among organisms and are believed to affect cell function through paralog-specific regulation (e.g., by generating heterogeneous ribosomes) and/or gene dose amplification. However, direct evaluations of their impacts on cell function remain elusive due to the highly heterogeneous cellular RP pool. Here, we engineered a yeast with homogeneous 40S RP paralog compositions, designated homo-40S, by deleting the entire set of alternative duplicated genes encoding yeast 40S RP paralogs. Homo-40S displayed mild growth defects along with high sensitivity to the translation inhibitor paromomycin and a significantly increased stop codon readthrough. Moreover, doubling of the remaining RP paralogous genes in homo-40S rescued these phenotypes markedly, although not fully, compared to the wild-type phenotype, indicating that the dose of 40S RP genes together with the heterogeneity of the contents was vital for maintaining normal translational functionalities and growth robustness. Additional experiments revealed that homo-40S improved paromomycin tolerance via acquisition of bypass mutations or evolved to be diploid to generate fast-growing derivatives, highlighting the mutational robustness of engineered yeast to accommodate environmental and genetic changes. In summary, our work demonstrated that duplicated RP paralogs impart robustness and phenotypic plasticity through both gene dose amplification and paralog-specific regulation, paving the way for the direct study of ribosome biology through monotypic ribosomes with a homogeneous composition of specific RP paralogs.
Topics: Genetic Engineering; Protein Processing, Post-Translational; Ribosomal Proteins; Ribosome Subunits, Small, Eukaryotic; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 35105807
DOI: 10.1073/pnas.2114445119 -
Proceedings of the National Academy of... Jul 2022RtcB is involved in transfer RNA (tRNA) splicing in archaeal and eukaryotic organisms. However, most RtcBs are found in bacteria, whose tRNAs have no introns. Because...
RtcB is involved in transfer RNA (tRNA) splicing in archaeal and eukaryotic organisms. However, most RtcBs are found in bacteria, whose tRNAs have no introns. Because tRNAs are the substrates of archaeal and eukaryotic RtcB, it is assumed that bacterial RtcBs are for repair of damaged tRNAs. Here, we show that a subset of bacterial RtcB, denoted RtcB2 herein, specifically repair ribosomal damage in the decoding center. To access the damage site for repair, however, the damaged 70S ribosome needs to be dismantled first, and this is accomplished by bacterial PrfH. Peptide-release assays revealed that PrfH is only active with the damaged 70S ribosome but not with the intact one. A 2.55-Å cryo-electron microscopy structure of PrfH in complex with the damaged 70S ribosome provides molecular insight into PrfH discriminating between the damaged and the intact ribosomes via specific recognition of the cleaved 3'-terminal nucleotide. RNA repair assays demonstrated that RtcB2 efficiently repairs the damaged 30S ribosomal subunit but not the damaged tRNAs. Cell-based assays showed that the RtcB2-PrfH pair reverse the damage inflicted by ribosome-specific ribotoxins in vivo. Thus, our combined biochemical, structural, and cell-based studies have uncovered a bacterial defense system specifically evolved to reverse the lethal ribosomal damage in the decoding center for cell survival.
Topics: Amino Acyl-tRNA Synthetases; Cryoelectron Microscopy; Escherichia coli Proteins; Protein Conformation; RNA Splicing; RNA, Transfer; Ribosome Subunits, Large, Bacterial
PubMed: 35858322
DOI: 10.1073/pnas.2202464119 -
Nucleic Acids Research Apr 2024During stress conditions such as heat shock and antibiotic exposure, ribosomes stall on messenger RNAs, leading to inhibition of protein synthesis. To remobilize...
During stress conditions such as heat shock and antibiotic exposure, ribosomes stall on messenger RNAs, leading to inhibition of protein synthesis. To remobilize ribosomes, bacteria use rescue factors such as HflXr, a homolog of the conserved housekeeping GTPase HflX that catalyzes the dissociation of translationally inactive ribosomes into individual subunits. Here we use time-resolved cryo-electron microscopy to elucidate the mechanism of ribosome recycling by Listeria monocytogenes HflXr. Within the 70S ribosome, HflXr displaces helix H69 of the 50S subunit and induces long-range movements of the platform domain of the 30S subunit, disrupting inter-subunit bridges B2b, B2c, B4, B7a and B7b. Our findings unveil a unique ribosome recycling strategy by HflXr which is distinct from that mediated by RRF and EF-G. The resemblance between HflXr and housekeeping HflX suggests that the alternative ribosome recycling mechanism reported here is universal in the prokaryotic kingdom.
Topics: Cryoelectron Microscopy; Bacterial Proteins; Ribosomes; Listeria monocytogenes; Ribosomal Proteins; Protein Biosynthesis; Models, Molecular; Ribosome Subunits, Large, Bacterial; Peptide Elongation Factor G
PubMed: 38407413
DOI: 10.1093/nar/gkae128 -
Advances in Biological Regulation Jan 2018Mutations that target the ubiquitous process of ribosome assembly paradoxically cause diverse tissue-specific disorders (ribosomopathies) that are often associated with... (Review)
Review
Mutations that target the ubiquitous process of ribosome assembly paradoxically cause diverse tissue-specific disorders (ribosomopathies) that are often associated with an increased risk of cancer. Ribosomes are the essential macromolecular machines that read the genetic code in all cells in all kingdoms of life. Following pre-assembly in the nucleus, precursors of the large 60S and small 40S ribosomal subunits are exported to the cytoplasm where the final steps in maturation are completed. Here, I review the recent insights into the conserved mechanisms of ribosome assembly that have come from functional characterisation of the genes mutated in human ribosomopathies. In particular, recent advances in cryo-electron microscopy, coupled with genetic, biochemical and prior structural data, have revealed that the SBDS protein that is deficient in the inherited leukaemia predisposition disorder Shwachman-Diamond syndrome couples the final step in cytoplasmic 60S ribosomal subunit maturation to a quality control assessment of the structural and functional integrity of the nascent particle. Thus, study of this fascinating disorder is providing remarkable insights into how the large ribosomal subunit is functionally activated in the cytoplasm to enter the actively translating pool of ribosomes.
Topics: Bone Marrow Diseases; Cryoelectron Microscopy; Exocrine Pancreatic Insufficiency; Humans; Lipomatosis; Mutation; Proteins; Ribosome Subunits, Large, Eukaryotic; Ribosome Subunits, Small, Eukaryotic; Shwachman-Diamond Syndrome
PubMed: 28942353
DOI: 10.1016/j.jbior.2017.09.002 -
Nature Communications Aug 2023Assembly of ribosomes in bacteria is highly efficient, taking ~2-3 min, but this makes the abundance of assembly intermediates very low, which is a challenge for...
Assembly of ribosomes in bacteria is highly efficient, taking ~2-3 min, but this makes the abundance of assembly intermediates very low, which is a challenge for mechanistic understanding. Genetic perturbations of the assembly process create bottlenecks where intermediates accumulate, facilitating structural characterization. We use cryo-electron microscopy, with iterative subclassification to identify intermediates in the assembly of the 50S ribosomal subunit from E. coli. The analysis of the ensemble of intermediates that spans the entire biogenesis pathway for the 50 S subunit was facilitated by a dimensionality reduction and cluster picking approach using PCA-UMAP-HDBSCAN. The identity of the cooperative folding units in the RNA with associated proteins is revealed, and the hierarchy of these units reveals a complete assembly map for all RNA and protein components. The assembly generally proceeds co-transcriptionally, with some flexibility in the landscape to ensure efficiency for this central cellular process under a variety of growth conditions.
Topics: Cryoelectron Microscopy; Escherichia coli; Ribosome Subunits, Large, Bacterial; Bacteria; RNA
PubMed: 37633970
DOI: 10.1038/s41467-023-40859-w -
RNA (New York, N.Y.) Sep 2021Many antibiotics that bind to the ribosome inhibit translation by blocking the movement of tRNAs and mRNA or interfering with ribosome dynamics, which impairs the...
Many antibiotics that bind to the ribosome inhibit translation by blocking the movement of tRNAs and mRNA or interfering with ribosome dynamics, which impairs the formation of essential translocation intermediates. Here we show how translocation inhibitors viomycin (Vio), neomycin (Neo), paromomycin (Par), kanamycin (Kan), spectinomycin (Spc), hygromycin B (HygB), and streptomycin (Str, an antibiotic that does not inhibit tRNA movement), affect principal motions of the small ribosomal subunits (SSU) during EF-G-promoted translocation. Using ensemble kinetics, we studied the SSU body domain rotation and SSU head domain swiveling in real time. We show that although antibiotics binding to the ribosome can favor a particular ribosome conformation in the absence of EF-G, their kinetic effect on the EF-G-induced transition to the rotated/swiveled state of the SSU is moderate. The antibiotics mostly inhibit backward movements of the SSU body and/or the head domains. Vio, Spc, and high concentrations of Neo completely inhibit the backward movements of the SSU body and head domain. Kan, Par, HygB, and low concentrations of Neo slow down both movements, but their sequence and coordination are retained. Finally, Str has very little effect on the backward rotation of the SSU body domain, but retards the SSU head movement. The data underscore the importance of ribosome dynamics for tRNA-mRNA translocation and provide new insights into the mechanism of antibiotic action.
Topics: Anti-Bacterial Agents; Biological Transport; Cinnamates; Escherichia coli; Hygromycin B; Kanamycin; Kinetics; Neomycin; Paromomycin; Peptide Elongation Factor G; Protein Biosynthesis; RNA, Messenger; RNA, Transfer; Ribosome Subunits; Spectinomycin; Streptomycin; Viomycin
PubMed: 34117118
DOI: 10.1261/rna.078758.121 -
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