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Science Advances Jun 2017Bypassing is a recoding event that leads to the translation of two distal open reading frames into a single polypeptide chain. We present the structure of a translating...
Bypassing is a recoding event that leads to the translation of two distal open reading frames into a single polypeptide chain. We present the structure of a translating ribosome stalled at the bypassing take-off site of of bacteriophage T4. The nascent peptide in the exit tunnel anchors the P-site peptidyl-tRNA to the ribosome and locks an inactive conformation of the peptidyl transferase center (PTC). The mRNA forms a short dynamic hairpin in the decoding site. The ribosomal subunits adopt a rolling conformation in which the rotation of the small subunit around its long axis causes the opening of the A-site region. Together, PTC conformation and mRNA structure safeguard against premature termination and read-through of the stop codon and reconfigure the ribosome to a state poised for take-off and sliding along the noncoding mRNA gap.
Topics: Models, Molecular; Peptides; Protein Biosynthesis; Protein Conformation; RNA, Messenger; Ribosome Subunits, Small, Bacterial; Ribosomes; Structure-Activity Relationship
PubMed: 28630923
DOI: 10.1126/sciadv.1700147 -
Current Opinion in Pediatrics Feb 2010Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by erythroid failure, congenital anomalies and predisposition to cancer.... (Review)
Review
PURPOSE OF REVIEW
Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by erythroid failure, congenital anomalies and predisposition to cancer. Recently, the notion of DBA as a disorder of ribosome biogenesis has been clarified. Correlations between molecular underpinnings and disease pathophysiology, while elusive, are beginning to emerge. Advances in these areas will be explored in this review.
RECENT FINDINGS
All known genes mutated in DBA encode ribosomal proteins associated with either the small (RPS) or large (RPL) subunit and in these cases ribosomal protein haploinsufficiency gives rise to the disease. The number of genes affected, their potential interactions with the environment and modifier genes, and the myriad of potential signaling pathways linking abortive ribosome synthesis to cell-cycle regulators may all contribute to disease heterogeneity. Genotype/phenotype relationships emerging over the past year promise to shed light on these complex interrelationships and their role in DBA pathophysiology.
SUMMARY
The nosology of DBA has recently expanded to include two distinct disease categories: a classical inherited bone marrow failure syndrome and a 'ribosomopathy'. The description of DBA as a ribosomopathy has provided a context for scientific inquiry analogous to the description of Fanconi anemia as a disorder of DNA repair.
Topics: Anemia, Diamond-Blackfan; Humans; Mutation; Proto-Oncogene Proteins c-mdm2; Ribosomal Proteins; Ribosome Subunits; Signal Transduction
PubMed: 19915471
DOI: 10.1097/MOP.0b013e328334573b -
Nucleic Acids Research Apr 2013The bacterial homologues of ObgH1 and Mtg1, ObgE and RbgA, respectively, have been suggested to be involved in the assembly of large ribosomal subunits. We sought to...
The bacterial homologues of ObgH1 and Mtg1, ObgE and RbgA, respectively, have been suggested to be involved in the assembly of large ribosomal subunits. We sought to elucidate the functions of ObgH1 and Mtg1 in ribosome biogenesis in human mitochondria. ObgH1 and Mtg1 are localized in mitochondria in association with the inner membrane, and are exposed on the matrix side. Mtg1 and ObgH1 specifically associate with the large subunit of the mitochondrial ribosome in GTP-dependent manner. The large ribosomal subunit stimulated the GTPase activity of Mtg1, whereas only the intrinsic GTPase activity was detectable with ObgH1. The knockdown of Mtg1 decreased the overall mitochondrial translation activity, and caused defects in the formation of respiratory complexes. On the other hand, the depletion of ObgH1 led to the specific activation of the translation of subunits of Complex V, and disrupted its proper formation. Our results suggested that Mtg1 and ObgH1 function with the large subunit of the mitochondrial ribosome, and are also involved in both the translation and assembly of respiratory complexes. The fine coordination of ribosome assembly, translation and respiratory complex formation in mammalian mitochondria is affirmed.
Topics: Electron Transport; GTP Phosphohydrolases; HeLa Cells; Humans; Mitochondria; Mitochondrial Proteins; Monomeric GTP-Binding Proteins; Protein Biosynthesis; Ribosome Subunits, Large, Eukaryotic
PubMed: 23396448
DOI: 10.1093/nar/gkt079 -
Journal of Molecular Biology May 2019rRNA is the single most abundant polymer in most cells. Mammalian rRNAs are nearly twice as large as those of prokaryotes. Differences in rRNA size are due to expansion...
rRNA is the single most abundant polymer in most cells. Mammalian rRNAs are nearly twice as large as those of prokaryotes. Differences in rRNA size are due to expansion segments, which contain extended tentacles in metazoans. Here we show that the terminus of an rRNA tentacle of Homo sapiens contains 10 tandem G-tracts that form highly stable G-quadruplexes in vitro. We characterized rRNA of the H. sapiens large ribosomal subunit by computation, circular dichroism, UV melting, fluorescent probes, nuclease accessibility, electrophoretic mobility shifts, and blotting. We investigated Expansion Segment 7 (ES7), oligomers derived from ES7, intact 28S rRNA, 80S ribosomes, and polysomes. We used mass spectrometry to identify proteins that bind to rRNA G-quadruplexes in cell lysates. These proteins include helicases (DDX3, CNBP, DDX21, DDX17) and heterogeneous nuclear ribonucleoproteins. Finally, by multiple sequence alignments, we observe that G-quadruplex-forming sequences are a general feature of LSU rRNA of Chordata but not, as far as we can tell, of other species. Chordata ribosomes present polymorphic tentacles with the potential to switch between inter- and intramolecular G-quadruplexes. To our knowledge, G-quadruplexes have not been reported previously in ribosomes.
Topics: Animals; Base Sequence; Circular Dichroism; Electrophoretic Mobility Shift Assay; G-Quadruplexes; Humans; Nucleic Acid Conformation; RNA, Ribosomal; Ribosome Subunits, Large; Sequence Alignment
PubMed: 30885721
DOI: 10.1016/j.jmb.2019.03.010 -
Nature Dec 2018Orthogonal ribosomes are unnatural ribosomes that are directed towards orthogonal messenger RNAs in Escherichia coli, through an altered version of the 16S ribosomal RNA...
Orthogonal ribosomes are unnatural ribosomes that are directed towards orthogonal messenger RNAs in Escherichia coli, through an altered version of the 16S ribosomal RNA of the small subunit. Directed evolution of orthogonal ribosomes has provided access to new ribosomal function, and the evolved orthogonal ribosomes have enabled the encoding of multiple non-canonical amino acids into proteins. The original orthogonal ribosomes shared the pool of 23S ribosomal RNAs, contained in the large subunit, with endogenous ribosomes. Selectively directing a new 23S rRNA to an orthogonal mRNA, by controlling the association between the orthogonal 16S rRNAs and 23S rRNAs, would enable the evolution of new function in the large subunit. Previous work covalently linked orthogonal 16S rRNA and a circularly permuted 23S rRNA to create orthogonal ribosomes with low activity; however, the linked subunits in these ribosomes do not associate specifically with each other, and mediate translation by associating with endogenous subunits. Here we discover engineered orthogonal 'stapled' ribosomes (with subunits linked through an optimized RNA staple) with activities comparable to that of the parent orthogonal ribosome; they minimize association with endogenous subunits and mediate translation of orthogonal mRNAs through the association of stapled subunits. We evolve cells with genomically encoded stapled ribosomes as the sole ribosomes, which support cellular growth at similar rates to natural ribosomes. Moreover, we visualize the engineered stapled ribosome structure by cryo-electron microscopy at 3.0 Å, revealing how the staple links the subunits and controls their association. We demonstrate the utility of controlling subunit association by evolving orthogonal stapled ribosomes which efficiently polymerize a sequence of monomers that the natural ribosome is intrinsically unable to translate. Our work provides a foundation for evolving the rRNA of the entire orthogonal ribosome for the encoded cellular synthesis of non-canonical biological polymers.
Topics: Base Sequence; Cross-Linking Reagents; Cryoelectron Microscopy; Directed Molecular Evolution; Escherichia coli; Models, Molecular; Peptides; Protein Biosynthesis; RNA, Messenger; RNA, Ribosomal, 16S; RNA, Ribosomal, 23S; Ribosome Subunits; Ribosomes
PubMed: 30518861
DOI: 10.1038/s41586-018-0773-z -
Biochemistry. Biokhimiia Sep 2021"Would it be possible to analyze molecular mechanisms and structural organisation of polyribosome assemblies using cryo electron tomography?" - we asked through a... (Review)
Review
"Would it be possible to analyze molecular mechanisms and structural organisation of polyribosome assemblies using cryo electron tomography?" - we asked through a longstanding collaboration between my research group and that of Alexander S. Spirin. Indeed, it was: we found that double-row polyribosomes can have both circular and linear arrangements of their mRNA [Afonina, Z. A., et al. (2013) Biochemistry (Moscow)], we figured out how eukaryotic ribosomes assemble on an mRNA to form supramolecular left-handed helices [Myasnikov, A. G., et al. (2014) Nat. Commun.], that the circularization of polyribosomes is poly-A and cap-independent [Afonina, Z. A., et al. (2014) Nucleic Acids Res.], and that intermediary polyribosomes with open structures exist after a transition from a juvenile phase to strongly translating polysomes of medium size [Afonina, Z. A., et al. (2015) Nucleic Acids Res.] until they form densely packed helical structures with reduced activity. Our joint fruitful exchanges, hence, led to major advances in the field, which are reviewed here from a personal and historical perspective in memory of Alexander S. Spirin.
Topics: Cryoelectron Microscopy; Eukaryota; Nucleic Acid Conformation; Poly A; Polyribosomes; RNA Caps; RNA, Messenger; Ribosome Subunits
PubMed: 34565311
DOI: 10.1134/S0006297921090030 -
Nucleic Acids Research May 2023The chemical modification of ribosomal RNA and proteins is critical for ribosome assembly, for protein synthesis and may drive ribosome specialisation in development and...
The chemical modification of ribosomal RNA and proteins is critical for ribosome assembly, for protein synthesis and may drive ribosome specialisation in development and disease. However, the inability to accurately visualise these modifications has limited mechanistic understanding of the role of these modifications in ribosome function. Here we report the 2.15 Å resolution cryo-EM reconstruction of the human 40S ribosomal subunit. We directly visualise post-transcriptional modifications within the 18S rRNA and four post-translational modifications of ribosomal proteins. Additionally, we interpret the solvation shells in the core regions of the 40S ribosomal subunit and reveal how potassium and magnesium ions establish both universally conserved and eukaryote-specific coordination to promote the stabilisation and folding of key ribosomal elements. This work provides unprecedented structural details for the human 40S ribosomal subunit that will serve as an important reference for unravelling the functional role of ribosomal RNA modifications.
Topics: Humans; Ribosome Subunits, Small, Eukaryotic; Cryoelectron Microscopy; Ribosomal Proteins; Ribosomes; RNA, Ribosomal; RNA, Ribosomal, 18S
PubMed: 36951107
DOI: 10.1093/nar/gkad194 -
Molecular Microbiology Feb 2018Protein synthesis relies on several translational GTPases (trGTPases), related proteins that couple the hydrolysis of GTP to specific molecular events on the ribosome.... (Review)
Review
Protein synthesis relies on several translational GTPases (trGTPases), related proteins that couple the hydrolysis of GTP to specific molecular events on the ribosome. Most bacterial trGTPases, including IF2, EF-Tu, EF-G and RF3, play well-known roles in translation. The cellular functions of LepA (also termed EF4) and BipA (also termed TypA), conversely, have remained enigmatic. Recent studies provide compelling in vivo evidence that LepA and BipA function in biogenesis of the 30S and 50S subunit respectively. These findings have important implications for ribosome biogenesis in bacteria. Because the GTPase activity of each of these proteins depends on interactions with both ribosomal subunits, some portion of 30S and 50S assembly must occur in the context of the 70S ribosome. In this review, we introduce the trGTPases of bacteria, describe the new functional data on LepA and BipA, and discuss the how these findings shape our current view of ribosome biogenesis in bacteria.
Topics: Bacteria; GTP Phosphohydrolases; Peptide Elongation Factors; Peptide Initiation Factors; Phylogeny; Protein Biosynthesis; Ribosome Subunits, Large, Bacterial; Ribosome Subunits, Small, Bacterial
PubMed: 29235176
DOI: 10.1111/mmi.13895 -
Proceedings of the National Academy of... Jan 2022Mitochondrial ribosomes (mitoribosomes) play a central role in synthesizing mitochondrial inner membrane proteins responsible for oxidative phosphorylation. Although...
Mitochondrial ribosomes (mitoribosomes) play a central role in synthesizing mitochondrial inner membrane proteins responsible for oxidative phosphorylation. Although mitoribosomes from different organisms exhibit considerable structural variations, recent insights into mitoribosome assembly suggest that mitoribosome maturation follows common principles and involves a number of conserved assembly factors. To investigate the steps involved in the assembly of the mitoribosomal small subunit (mt-SSU) we determined the cryoelectron microscopy structures of middle and late assembly intermediates of the mitochondrial small subunit (mt-SSU) at 3.6- and 3.7-Å resolution, respectively. We identified five additional assembly factors that together with the mitochondrial initiation factor 2 (mt-IF-2) specifically interact with functionally important regions of the rRNA, including the decoding center, thereby preventing premature mRNA or large subunit binding. Structural comparison of assembly intermediates with mature mt-SSU combined with RNAi experiments suggests a noncanonical role of mt-IF-2 and a stepwise assembly process, where modular exchange of ribosomal proteins and assembly factors together with mt-IF-2 ensure proper 9S rRNA folding and protein maturation during the final steps of assembly.
Topics: Cell Line; Cryoelectron Microscopy; Mitochondria; Mitochondrial Proteins; Mitochondrial Ribosomes; Models, Molecular; Oxidative Phosphorylation; RNA, Ribosomal; Ribosomal Proteins; Ribosome Subunits; Trypanosoma brucei brucei
PubMed: 35042777
DOI: 10.1073/pnas.2114710118 -
Protein & Cell Mar 2016The human Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease caused by mutations in a highly conserved ribosome assembly factor SBDS. The functional role...
The human Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease caused by mutations in a highly conserved ribosome assembly factor SBDS. The functional role of SBDS is to cooperate with another assembly factor, elongation factor 1-like (Efl1), to promote the release of eukaryotic initiation factor 6 (eIF6) from the late-stage cytoplasmic 60S precursors. In the present work, we characterized, both biochemically and structurally, the interaction between the 60S subunit and SBDS protein (Sdo1p) from yeast. Our data show that Sdo1p interacts tightly with the mature 60S subunit in vitro through its domain I and II, and is capable of bridging two 60S subunits to form a stable 2:2 dimer. Structural analysis indicates that Sdo1p bind to the ribosomal P-site, in the proximity of uL16 and uL5, and with direct contact to H69 and H38. The dynamic nature of Sdo1p on the 60S subunit, together with its strategic binding position, suggests a surveillance role of Sdo1p in monitoring the conformational maturation of the ribosomal P-site. Altogether, our data support a conformational signal-relay cascade during late-stage 60S maturation, involving uL16, Sdo1p, and Efl1p, which interrogates the functional P-site to control the departure of the anti-association factor eIF6.
Topics: Crystallography, X-Ray; GTP Phosphohydrolases; Humans; Protein Domains; Ribosome Subunits, Large, Eukaryotic; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 26850260
DOI: 10.1007/s13238-015-0242-5