Journal articles: 'Large ribosomal subunit'

1

Siibak, Triinu, Lauri Peil, Liqun Xiong, Alexander Mankin, Jaanus Remme, and Tanel Tenson. "Erythromycin- and Chloramphenicol-Induced Ribosomal Assembly Defects Are Secondary Effects of Protein Synthesis Inhibition." Antimicrobial Agents and Chemotherapy 53, no. 2 (November 24, 2008): 563–71. http://dx.doi.org/10.1128/aac.00870-08.

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ABSTRACT Several protein synthesis inhibitors are known to inhibit ribosome assembly. This may be a consequence of direct binding of the antibiotic to ribosome precursor particles, or it could result indirectly from loss of coordination in the production of ribosomal components due to the inhibition of protein synthesis. Here we demonstrate that erythromycin and chloramphenicol, inhibitors of the large ribosomal subunit, affect the assembly of both the large and small subunits. Expression of a small erythromycin resistance peptide acting in cis on mature ribosomes relieves the erythromycin-mediated assembly defect for both subunits. Erythromycin treatment of bacteria expressing a mixture of erythromycin-sensitive and -resistant ribosomes produced comparable effects on subunit assembly. These results argue in favor of the view that erythromycin and chloramphenicol affect the assembly of the large ribosomal subunit indirectly.

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Moraleva, Anastasia A., Alexander S. Deryabin, Yury P. Rubtsov, Maria P. Rubtsova, and Olga A. Dontsova. "Eukaryotic Ribosome Biogenesis: The 60S Subunit." Acta Naturae 14, no. 2 (July 21, 2022): 39–49. http://dx.doi.org/10.32607/actanaturae.11541.

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Ribosome biogenesis is consecutive coordinated maturation of ribosomal precursors in the nucleolus, nucleoplasm, and cytoplasm. The formation of mature ribosomal subunits involves hundreds of ribosomal biogenesis factors that ensure ribosomal RNA processing, tertiary structure, and interaction with ribosomal proteins. Although the main features and stages of ribosome biogenesis are conservative among different groups of eukaryotes, this process in human cells has become more complicated due to the larger size of the ribosomes and pre-ribosomes and intricate regulatory pathways affecting their assembly and function. Many of the factors involved in the biogenesis of human ribosomes have been identified using genome-wide screening based on RNA interference. A previous part of this review summarized recent data on the processing of the primary rRNA transcript and compared the maturation of the small 40S subunit in yeast and human cells. This part of the review focuses on the biogenesis of the large 60S subunit of eukaryotic ribosomes.

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Petrov, Anton S., Burak Gulen, Ashlyn M. Norris, Nicholas A. Kovacs, Chad R. Bernier, Kathryn A. Lanier, George E. Fox, et al. "History of the ribosome and the origin of translation." Proceedings of the National Academy of Sciences 112, no. 50 (November 30, 2015): 15396–401. http://dx.doi.org/10.1073/pnas.1509761112.

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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.

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Aoyama, Ryo, Keiko Masuda, Masaru Shimojo, Takashi Kanamori, Takuya Ueda, and Yoshihiro Shimizu. "In vitro reconstitution of the Escherichia coli 70S ribosome with a full set of recombinant ribosomal proteins." Journal of Biochemistry 171, no. 2 (November 8, 2021): 227–37. http://dx.doi.org/10.1093/jb/mvab121.

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Abstract Many studies of the reconstitution of the Escherichia coli small ribosomal subunit from its individual molecular parts have been reported, but contrastingly, similar studies of the large ribosomal subunit have not been well performed to date. Here, we describe protocols for preparing the 33 ribosomal proteins of the E. coli 50S subunit and demonstrate successful reconstitution of a functionally active 50S particle that can perform protein synthesis in vitro. We also successfully reconstituted both ribosomal subunits (30S and 50S) and 70S ribosomes using a full set of recombinant ribosomal proteins by integrating our developed method with the previously developed fully recombinant-based integrated synthesis, assembly and translation. The approach described here makes a major contribution to the field of ribosome engineering and could be fundamental to the future studies of ribosome assembly processes.

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Moy, Terence I., and Pamela A. Silver. "Requirements for the nuclear export of the small ribosomal subunit." Journal of Cell Science 115, no. 14 (July 15, 2002): 2985–95. http://dx.doi.org/10.1242/jcs.115.14.2985.

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Eukaryotic ribosome biogenesis requires multiple steps of nuclear transport because ribosomes are assembled in the nucleus while protein synthesis occurs in the cytoplasm. Using an in situ RNA localization assay in the yeast Saccharomyces cerevisiae, we determined that efficient nuclear export of the small ribosomal subunit requires Yrb2, a factor involved in Crm1-mediated export. Furthermore, in cells lacking YRB2, the stability and abundance of the small ribosomal subunit is decreased in comparison with the large ribosomal subunit. To identify additional factors affecting small subunit export, we performed a large-scale screen of temperature-sensitive mutants. We isolated new alleles of several nucleoporins and Ran-GTPase regulators. Together with further analysis of existing mutants,we show that nucleoporins previously shown to be defective in ribosomal assembly are also defective in export of the small ribosomal subunit.

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Jiang, Mengxi, Kaustuv Datta, Angela Walker, John Strahler, Pia Bagamasbad, Philip C. Andrews, and Janine R. Maddock. "The Escherichia coli GTPase CgtAE Is Involved in Late Steps of Large Ribosome Assembly." Journal of Bacteriology 188, no. 19 (October 1, 2006): 6757–70. http://dx.doi.org/10.1128/jb.00444-06.

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ABSTRACT The bacterial ribosome is an extremely complicated macromolecular complex the in vivo biogenesis of which is poorly understood. Although several bona fide assembly factors have been identified, their precise functions and temporal relationships are not clearly defined. Here we describe the involvement of an Escherichia coli GTPase, CgtAE, in late steps of large ribosomal subunit biogenesis. CgtAE belongs to the Obg/CgtA GTPase subfamily, whose highly conserved members are predominantly involved in ribosome function. Mutations in CgtAE cause both polysome and rRNA processing defects; small- and large-subunit precursor rRNAs accumulate in a cgtAE mutant. In this study we apply a new semiquantitative proteomic approach to show that CgtAE is required for optimal incorporation of certain late-assembly ribosomal proteins into the large ribosomal subunit. Moreover, we demonstrate the interaction with the 50S ribosomal subunits of specific nonribosomal proteins (including heretofore uncharacterized proteins) and define possible temporal relationships between these proteins and CgtAE. We also show that purified CgtAE associates with purified ribosomal particles in the GTP-bound form. Finally, CgtAE cofractionates with the mature 50S but not with intermediate particles accumulated in other large ribosome assembly mutants.

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Ling, Clarence, and Dmitri N. Ermolenko. "Initiation factor 2 stabilizes the ribosome in a semirotated conformation." Proceedings of the National Academy of Sciences 112, no. 52 (December 14, 2015): 15874–79. http://dx.doi.org/10.1073/pnas.1520337112.

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Intersubunit rotation and movement of the L1 stalk, a mobile domain of the large ribosomal subunit, have been shown to accompany the elongation cycle of translation. The initiation phase of protein synthesis is crucial for translational control of gene expression; however, in contrast to elongation, little is known about the conformational rearrangements of the ribosome during initiation. Bacterial initiation factors (IFs) 1, 2, and 3 mediate the binding of initiator tRNA and mRNA to the small ribosomal subunit to form the initiation complex, which subsequently associates with the large subunit by a poorly understood mechanism. Here, we use single-molecule FRET to monitor intersubunit rotation and the inward/outward movement of the L1 stalk of the large ribosomal subunit during the subunit-joining step of translation initiation. We show that, on subunit association, the ribosome adopts a distinct conformation in which the ribosomal subunits are in a semirotated orientation and the L1 stalk is positioned in a half-closed state. The formation of the semirotated intermediate requires the presence of an aminoacylated initiator, fMet-tRNAfMet, and IF2 in the GTP-bound state. GTP hydrolysis by IF2 induces opening of the L1 stalk and the transition to the nonrotated conformation of the ribosome. Our results suggest that positioning subunits in a semirotated orientation facilitates subunit association and support a model in which L1 stalk movement is coupled to intersubunit rotation and/or IF2 binding.

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8

Stern, Seth, and Prakash Purohit. "An oligonucleotide analog approach to the decoding region of 16S rRNA." Biochemistry and Cell Biology 73, no. 11-12 (December 1, 1995): 899–905. http://dx.doi.org/10.1139/o95-097.

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Despite the passage of about 30 years since the discovery of the translational activities of ribosomes and the outlining of the roles of the large and small subunits, the actual molecular basis for the mRNA decoding activities of the small subunit has remained essentially obscure. In this paper, we describe a new approach using oligonucleotide analogs of 16S ribosomal RNA, in which the small ribosomal subunit is effectively deconstructed into a smaller more experimentally tractable form. Specifically, we review the results of experiments using an oligonucleotide analog of the decoding region of 16S ribosomal RNA, suggesting that the decoding region is the functional core of the small subunit, that it contacts both mRNA codons and tRNA anticodons, and that it mediates and probably enhances codon–anticodon base pairing, that is, decoding.Key words: translation, ribosome, 30S, 16S, RNA, decoding, antibiotic.

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Levy, Michael, Reuven Falkovich, Shirley S. Daube, and Roy H. Bar-Ziv. "Autonomous synthesis and assembly of a ribosomal subunit on a chip." Science Advances 6, no. 16 (April 2020): eaaz6020. http://dx.doi.org/10.1126/sciadv.aaz6020.

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Ribosome biogenesis is an efficient and complex assembly process that has not been reconstructed outside a living cell so far, yet is the most critical step for establishing a self-replicating artificial cell. We recreated the biogenesis of Escherichia coli’s small ribosomal subunit by synthesizing and capturing all its ribosomal proteins and RNA on a chip. Surface confinement provided favorable conditions for autonomous stepwise assembly of new subunits, spatially segregated from original intact ribosomes. Our real-time fluorescence measurements revealed hierarchal assembly, cooperative interactions, unstable intermediates, and specific binding to large ribosomal subunits. Using only synthetic genes, our methodology is a crucial step toward creation of a self-replicating artificial cell and a general strategy for the mechanistic investigation of diverse multicomponent macromolecular machines.

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Bhattacharya, Arpita, Kerri B. McIntosh, Ian M. Willis, and Jonathan R. Warner. "Why Dom34 Stimulates Growth of Cells with Defects of 40S Ribosomal Subunit Biosynthesis." Molecular and Cellular Biology 30, no. 23 (September 27, 2010): 5562–71. http://dx.doi.org/10.1128/mcb.00618-10.

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ABSTRACT A set of genome-wide screens for proteins whose absence exacerbates growth defects due to pseudo-haploinsufficiency of ribosomal proteins in Saccharomyces cerevisiae identified Dom34 as being particularly important for cell growth when there is a deficit of 40S ribosomal subunits. In contrast, strains with a deficit of 60S ribosomal proteins were largely insensitive to the loss of Dom34. The slow growth of cells lacking Dom34 and haploinsufficient for a protein of the 40S subunit is caused by a severe shortage of 40S subunits available for translation initiation due to a combination of three effects: (i) the natural deficiency of 40S subunits due to defective synthesis, (ii) the sequestration of 40S subunits due to the large accumulation of free 60S subunits, and (iii) the accumulation of ribosomes “stuck” in a distinct 80S form, insensitive to the Mg2+ concentration, and at least temporarily unavailable for further translation. Our data suggest that these stuck ribosomes have neither mRNA nor tRNA. We postulate, based on our results and on previously published work, that the stuck ribosomes arise because of the lack of Dom34, which normally resolves a ribosome stalled due to insufficient tRNAs, to structural problems with its mRNA, or to a defect in the ribosome itself.

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11

Gregory, Brian, Nusrat Rahman, Ananth Bommakanti, Md Shamsuzzaman, Mamata Thapa, Alana Lescure, Janice M. Zengel, and Lasse Lindahl. "The small and large ribosomal subunits depend on each other for stability and accumulation." Life Science Alliance 2, no. 2 (March 5, 2019): e201800150. http://dx.doi.org/10.26508/lsa.201800150.

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The 1:1 balance between the numbers of large and small ribosomal subunits can be disturbed by mutations that inhibit the assembly of only one of the subunits. Here, we have investigated if the cell can counteract an imbalance of the number of the two subunits. We show that abrogating 60S assembly blocks 40S subunit accumulation. In contrast, cessation of the 40S pathways does not prevent 60S accumulation, but does, however, lead to fragmentation of the 25S rRNA in 60S subunits and formation of a 55S ribosomal particle derived from the 60S. We also present evidence suggesting that these events occur post assembly and discuss the possibility that the turnover of subunits is due to vulnerability of free subunits not paired with the other subunit to form 80S ribosomes.

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12

Gadal, Olivier, Daniela Strauß, Jacques Kessl, Bernard Trumpower, David Tollervey, and Ed Hurt. "Nuclear Export of 60S Ribosomal Subunits Depends on Xpo1p and Requires a Nuclear Export Sequence-Containing Factor, Nmd3p, That Associates with the Large Subunit Protein Rpl10p." Molecular and Cellular Biology 21, no. 10 (May 15, 2001): 3405–15. http://dx.doi.org/10.1128/mcb.21.10.3405-3415.2001.

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ABSTRACT Nuclear export of ribosomes requires a subset of nucleoporins and the Ran system, but specific transport factors have not been identified. Using a large subunit reporter (Rpl25p-eGFP), we have isolated several temperature-sensitive ribosomal export (rix) mutants. One of these corresponds to the ribosomal protein Rpl10p, which interacts directly with Nmd3p, a conserved and essential protein associated with 60S subunits. We find that thermosensitive nmd3 mutants are impaired in large subunit export. Strikingly, Nmd3p shuttles between the nucleus and cytoplasm and is exported by the nuclear export receptor Xpo1p. Moreover, we show that export of 60S subunits is Xpo1p dependent. We conclude that nuclear export of 60S subunits requires the nuclear export sequence-containing nonribosomal protein Nmd3p, which directly binds to the large subunit protein Rpl10p.

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Fan, Haitian, Joseph Hahm, Stephen Diggs, J. Jefferson P. Perry, and Gregor Blaha. "Structural and Functional Analysis of BipA, a Regulator of Virulence in Enteropathogenic Escherichia coli." Journal of Biological Chemistry 290, no. 34 (July 10, 2015): 20856–64. http://dx.doi.org/10.1074/jbc.m115.659136.

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The translational GTPase BipA regulates the expression of virulence and pathogenicity factors in several eubacteria. BipA-dependent expression of virulence factors occurs under starvation conditions, such as encountered during infection of a host. Under these conditions, BipA associates with the small ribosomal subunit. BipA also has a second function to promote the efficiency of late steps in biogenesis of large ribosomal subunits at low temperatures, presumably while bound to the ribosome. During starvation, the cellular concentration of stress alarmone guanosine-3′, 5′-bis pyrophosphate (ppGpp) is increased. This increase allows ppGpp to bind to BipA and switch its binding specificity from ribosomes to small ribosomal subunits. A conformational change of BipA upon ppGpp binding could explain the ppGpp regulation of the binding specificity of BipA. Here, we present the structures of the full-length BipA from Escherichia coli in apo, GDP-, and ppGpp-bound forms. The crystal structure and small-angle x-ray scattering data of the protein with bound nucleotides, together with a thermodynamic analysis of the binding of GDP and of ppGpp to BipA, indicate that the ppGpp-bound form of BipA adopts the structure of the GDP form. This suggests furthermore, that the switch in binding preference only occurs when both ppGpp and the small ribosomal subunit are present. This molecular mechanism would allow BipA to interact with both the ribosome and the small ribosomal subunit during stress response.

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Pöll, Gisela, Michael Pilsl, Joachim Griesenbeck, Herbert Tschochner, and Philipp Milkereit. "Analysis of subunit folding contribution of three yeast large ribosomal subunit proteins required for stabilisation and processing of intermediate nuclear rRNA precursors." PLOS ONE 16, no. 11 (November 23, 2021): e0252497. http://dx.doi.org/10.1371/journal.pone.0252497.

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In yeast and human cells many of the ribosomal proteins (r-proteins) are required for the stabilisation and productive processing of rRNA precursors. Functional coupling of r-protein assembly with the stabilisation and maturation of subunit precursors potentially promotes the production of ribosomes with defined composition. To further decipher mechanisms of such an intrinsic quality control pathway we analysed here the contribution of three yeast large ribosomal subunit r-proteins rpL2 (uL2), rpL25 (uL23) and rpL34 (eL34) for intermediate nuclear subunit folding steps. Structure models obtained from single particle cryo-electron microscopy analyses provided evidence for specific and hierarchic effects on the stable positioning and remodelling of large ribosomal subunit domains. Based on these structural and previous biochemical data we discuss possible mechanisms of r-protein dependent hierarchic domain arrangement and the resulting impact on the stability of misassembled subunits.

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Bachand, François, Daniel H. Lackner, Jürg Bähler, and Pamela A. Silver. "Autoregulation of Ribosome Biosynthesis by a Translational Response in Fission Yeast." Molecular and Cellular Biology 26, no. 5 (March 1, 2006): 1731–42. http://dx.doi.org/10.1128/mcb.26.5.1731-1742.2006.

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ABSTRACT Maintaining the appropriate balance between the small and large ribosomal subunits is critical for translation and cell growth. We previously identified the 40S ribosomal protein S2 (rpS2) as a substrate of the protein arginine methyltransferase 3 (RMT3) and reported a misregulation of the 40S/60S ratio in rmt3 deletion mutants of Schizosaccharomyces pombe. For this study, using DNA microarrays, we have investigated the genome-wide biological response of rmt3-null cells to this ribosomal subunit imbalance. Whereas little change was observed at the transcriptional level, a number of genes showed significant alterations in their polysomal-to-monosomal ratios in rmt3Δ mutants. Importantly, nearly all of the 40S ribosomal protein-encoding mRNAs showed increased ribosome density in rmt3 disruptants. Sucrose gradient analysis also revealed that the ribosomal subunit imbalance detected in rmt3-null cells is due to a deficit in small-subunit levels and can be rescued by rpS2 overexpression. Our results indicate that rmt3-null fission yeast compensate for the reduced levels of small ribosomal subunits by increasing the ribosome density, and likely the translation efficiency, of 40S ribosomal protein-encoding mRNAs. Our findings support the existence of autoregulatory mechanisms that control ribosome biosynthesis and translation as an important layer of gene regulation.

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Schuwirth, Barbara S., Maria A. Borovinskaya, Cathy W. Hau, Wen Zhang, Antón Vila-Sanjurjo, James M. Holton, and Jamie H. Doudna Cate. "Structures of the Bacterial Ribosome at 3.5 Å Resolution." Science 310, no. 5749 (November 3, 2005): 827–34. http://dx.doi.org/10.1126/science.1117230.

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We describe two structures of the intact bacterial ribosome from Escherichia coli determined to a resolution of 3.5 angstroms by x-ray crystallography. These structures provide a detailed view of the interface between the small and large ribosomal subunits and the conformation of the peptidyl transferase center in the context of the intact ribosome. Differences between the two ribosomes reveal a high degree of flexibility between the head and the rest of the small subunit. Swiveling of the head of the small subunit observed in the present structures, coupled to the ratchet-like motion of the two subunits observed previously, suggests a mechanism for the final movements of messenger RNA (mRNA) and transfer RNAs (tRNAs) during translocation.

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Schaefer, Laura, William C. Uicker, Catherine Wicker-Planquart, Anne-Emmanuelle Foucher, Jean-Michel Jault, and Robert A. Britton. "Multiple GTPases Participate in the Assembly of the Large Ribosomal Subunit in Bacillus subtilis." Journal of Bacteriology 188, no. 23 (September 22, 2006): 8252–58. http://dx.doi.org/10.1128/jb.01213-06.

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ABSTRACT GTPases have been demonstrated to be necessary for the proper assembly of the ribosome in bacteria and eukaryotes. Here, we show that the essential GTPases YphC and YsxC are required for large ribosomal subunit biogenesis in Bacillus subtilis. Sucrose density gradient centrifugation of large ribosomal subunits isolated from YphC-depleted cells and YsxC-depleted cells indicates that they are similar to the 45S intermediate previously identified in RbgA-depleted cells. The sedimentation of the large-subunit intermediate isolated from YphC-depleted cells was identical to the intermediate found in RbgA-depleted cells, while the intermediate isolated from YsxC-depleted cells sedimented slightly slower than 45S, suggesting that it is a novel intermediate. Analysis of the protein composition of the large-subunit intermediates isolated from either YphC-depleted cells or YsxC-depleted cells indicated that L16 and L36 are missing. Purified YphC and YsxC are able to interact with the ribosome in vitro, supporting a direct role for these two proteins in the assembly of the 50S subunit. Our results indicate that, as has been demonstrated for Saccharomyces cerevisiae ribosome biogenesis, bacterial 50S ribosome assembly requires the function of multiple essential GTPases.

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Videira, A., M. L. Teles Grilo, S. Werner, and H. Bertrand. "Mitochondrial gene expression in a nuclear mutant of Neurospora deficient in large subunits of mitochondrial ribosomes." Genome 30, no. 5 (October 1, 1988): 802–7. http://dx.doi.org/10.1139/g88-129.

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A new cytochrome a and b deficient nuclear mutant of Neurospora crassa, cyt-U-28, is defective in the assembly of large subunits of mitochondrial ribosomes. Nonetheless, this mutant overproduces apparently normal small subunits of mitochondrial ribosomes, even though it should be deficient for the S5 ribosomal protein required for assembly of the particles beyond the CAP30S stage. The mitochondria of cyt-U-28 indeed synthesize only small amounts of most mitochondrial polypeptides, including cytochrome oxidase subunits I, II, and III, contain very low amounts of the normal seven-polypeptide cytochrome oxidase complex, and, unlike the organelles from other cytochrome a deficient mutants, do not accumulate the nuclear-encoded cytochrome oxidase subunits 5 and 6. Nonetheless, the mutant markedly oversynthesizes a mitochondrial protein that comigrates with subunit 9 of the mitochondrial ATPase on S DS–polyacrylamide electrophoresis gels. The overproduction of this protein and the accumulation of mature small subunits of mitochondrial ribosomes indicate that the cyt-U-28 mutant preferentially expresses two mitochondrial genes, one coding for ATPase subunit 9, the other for the S5 ribosomal protein.Key words: Neurospora, mitochondria, ribosomes, protein synthesis.

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Oeffinger, Marlene. "Joining the interface: a site for Nmd3 association on 60S ribosome subunits." Journal of Cell Biology 189, no. 7 (June 28, 2010): 1071–73. http://dx.doi.org/10.1083/jcb.201006033.

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The adaptor protein Nmd3 is required for Crm1-dependent export of large ribosomal subunits from the nucleus. In this issue, Sengupta et al. (2010. J. Cell Biol. doi:10.1083/jcb.201001124) identify a binding site for yeast Nmd3 on 60S ribosomal subunits using cryoelectron microscopy and suggest a conformational model for its release in the cytoplasm. The study provides the first detailed structural description of a ribosome biogenesis factor in complex with the large subunit.

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Mandiyan, Valsan, and G. Ramananda Rao. "Separation of cytoplasmic ribosomal proteins of Microsporum canis." Canadian Journal of Microbiology 33, no. 4 (April 1, 1987): 339–43. http://dx.doi.org/10.1139/m87-058.

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The cytoplasmic ribosomal proteins of Microsporum canis were characterised in basic–acidic and basic–SDS two-dimensional polyacrylamide gel electrophoresis systems. The small subunit contained 28 proteins and the large subunit 38 proteins. The molecular weights of these proteins were in the range of 32 500 to 7600 and 48 000 to 11 000 in the small and large subunits, respectively. The 80S ribosomes showed 65 and 66 protein spots in basic–acidic and basic–SDS gel systems, respectively.

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Konikkat, Salini, and John L. Woolford,. "Principles of 60S ribosomal subunit assembly emerging from recent studies in yeast." Biochemical Journal 474, no. 2 (January 6, 2017): 195–214. http://dx.doi.org/10.1042/bcj20160516.

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Ribosome biogenesis requires the intertwined processes of folding, modification, and processing of ribosomal RNA, together with binding of ribosomal proteins. In eukaryotic cells, ribosome assembly begins in the nucleolus, continues in the nucleoplasm, and is not completed until after nascent particles are exported to the cytoplasm. The efficiency and fidelity of ribosome biogenesis are facilitated by >200 assembly factors and ∼76 different small nucleolar RNAs. The pathway is driven forward by numerous remodeling events to rearrange the ribonucleoprotein architecture of pre-ribosomes. Here, we describe principles of ribosome assembly that have emerged from recent studies of biogenesis of the large ribosomal subunit in the yeast Saccharomyces cerevisiae. We describe tools that have empowered investigations of ribosome biogenesis, and then summarize recent discoveries about each of the consecutive steps of subunit assembly.

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Jiang, M., S. M. Sullivan, A. K. Walker, J. R. Strahler, P. C. Andrews, and J. R. Maddock. "Identification of Novel Escherichia coli Ribosome-Associated Proteins Using Isobaric Tags and Multidimensional Protein Identification Techniques." Journal of Bacteriology 189, no. 9 (March 2, 2007): 3434–44. http://dx.doi.org/10.1128/jb.00090-07.

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ABSTRACT Biogenesis of the large ribosomal subunit requires the coordinate assembly of two rRNAs and 33 ribosomal proteins. In vivo, additional ribosome assembly factors, such as helicases, GTPases, pseudouridine synthetases, and methyltransferases, are also critical for ribosome assembly. To identify novel ribosome-associated proteins, we used a proteomic approach (isotope tagging for relative and absolute quantitation) that allows for semiquantitation of proteins from complex protein mixtures. Ribosomal subunits were separated by sucrose density centrifugation, and the relevant fractions were pooled and analyzed. The utility and reproducibility of the technique were validated via a double duplex labeling method. Next, we examined proteins from 30S, 50S, and translating ribosomes isolated at both 16°C and 37°C. We show that the use of isobaric tags to quantify proteins from these particles is an excellent predictor of the particles with which the proteins associate. Moreover, in addition to bona fide ribosomal proteins, additional proteins that comigrated with different ribosomal particles were detected, including both known ribosomal assembly factors and unknown proteins. The ribosome association of several of these proteins, as well as others predicted to be associated with ribosomes, was verified by immunoblotting. Curiously, deletion mutants for the majority of these ribosome-associated proteins had little effect on cell growth or on the polyribosome profiles.

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SZYMAŃSKI, Maciej, Mirosawa Z. BARCISZEWSKA, Volker A. ERDMANN, and Jan BARCISZEWSKI. "5 S rRNA: structure and interactions." Biochemical Journal 371, no. 3 (May 1, 2003): 641–51. http://dx.doi.org/10.1042/bj20020872.

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5S rRNA is an integral component of the large ribosomal subunit in all known organisms. Despite many years of intensive study, the function of 5S rRNA in the ribosome remains unknown. Advances in the analysis of ribosome structure that have revealed the crystal structures of large ribosomal subunits and of the complete ribosome from various organisms put the results of studies on 5S rRNA in a new perspective. This paper summarizes recently published data on the structure and function of 5S rRNA and its interactions in complexes with proteins, within and outside the ribosome.

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Maggi, Leonard B., Michael Kuchenruether, David Y. A. Dadey, Rachel M. Schwope, Silvia Grisendi, R. Reid Townsend, Pier Paolo Pandolfi, and Jason D. Weber. "Nucleophosmin Serves as a Rate-Limiting Nuclear Export Chaperone for the Mammalian Ribosome." Molecular and Cellular Biology 28, no. 23 (September 22, 2008): 7050–65. http://dx.doi.org/10.1128/mcb.01548-07.

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ABSTRACT Nucleophosmin (NPM) (B23) is an essential protein in mouse development and cell growth; however, it has been assigned numerous roles in very diverse cellular processes. Here, we present a unified mechanism for NPM's role in cell growth; NPM directs the nuclear export of both 40S and 60S ribosomal subunits. NPM interacts with rRNA and large and small ribosomal subunit proteins and also colocalizes with large and small ribosomal subunit proteins in the nucleolus, nucleus, and cytoplasm. The transduction of NPM shuttling-defective mutants or the loss of Npm1 inhibited the nuclear export of both the 40S and 60S ribosomal subunits, reduced the available pool of cytoplasmic polysomes, and diminished overall protein synthesis without affecting rRNA processing or ribosome assembly. While the inhibition of NPM shuttling can block cellular proliferation, the dramatic effects on ribosome export occur prior to cell cycle inhibition. Modest increases in NPM expression amplified the export of newly synthesized rRNAs, resulting in increased rates of protein synthesis and indicating that NPM is rate limiting in this pathway. These results support the idea that NPM-regulated ribosome export is a fundamental process in cell growth.

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Lavdovskaia, Elena, Kärt Denks, Franziska Nadler, Emely Steube, Andreas Linden, Henning Urlaub, Marina V. Rodnina, and Ricarda Richter-Dennerlein. "Dual function of GTPBP6 in biogenesis and recycling of human mitochondrial ribosomes." Nucleic Acids Research 48, no. 22 (December 2, 2020): 12929–42. http://dx.doi.org/10.1093/nar/gkaa1132.

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Abstract Translation and ribosome biogenesis in mitochondria require auxiliary factors that ensure rapid and accurate synthesis of mitochondrial proteins. Defects in translation are associated with oxidative phosphorylation deficiency and cause severe human diseases, but the exact roles of mitochondrial translation-associated factors are not known. Here we identify the functions of GTPBP6, a homolog of the bacterial ribosome-recycling factor HflX, in human mitochondria. Similarly to HflX, GTPBP6 facilitates the dissociation of ribosomes in vitro and in vivo. In contrast to HflX, GTPBP6 is also required for the assembly of mitochondrial ribosomes. GTPBP6 ablation leads to accumulation of late assembly intermediate(s) of the large ribosomal subunit containing ribosome biogenesis factors MTERF4, NSUN4, MALSU1 and the GTPases GTPBP5, GTPBP7 and GTPBP10. Our data show that GTPBP6 has a dual function acting in ribosome recycling and biogenesis. These findings contribute to our understanding of large ribosomal subunit assembly as well as ribosome recycling pathway in mitochondria.

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Saurer, Martin, David J. F. Ramrath, Moritz Niemann, Salvatore Calderaro, Céline Prange, Simone Mattei, Alain Scaiola, et al. "Mitoribosomal small subunit biogenesis in trypanosomes involves an extensive assembly machinery." Science 365, no. 6458 (September 12, 2019): 1144–49. http://dx.doi.org/10.1126/science.aaw5570.

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Mitochondrial ribosomes (mitoribosomes) are large ribonucleoprotein complexes that synthesize proteins encoded by the mitochondrial genome. An extensive cellular machinery responsible for ribosome assembly has been described only for eukaryotic cytosolic ribosomes. Here we report that the assembly of the small mitoribosomal subunit in Trypanosoma brucei involves a large number of factors and proceeds through the formation of assembly intermediates, which we analyzed by using cryo–electron microscopy. One of them is a 4-megadalton complex, referred to as the small subunit assemblosome, in which we identified 34 factors that interact with immature ribosomal RNA (rRNA) and recognize its functionally important regions. The assembly proceeds through large-scale conformational changes in rRNA coupled with successive incorporation of mitoribosomal proteins, providing an example for the complexity of the ribosomal assembly process in mitochondria.

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Datta, Kaustuv, Jennifer L. Fuentes, and Janine R. Maddock. "The Yeast GTPase Mtg2p Is Required for Mitochondrial Translation and Partially Suppresses an rRNA Methyltransferase Mutant,mrm2." Molecular Biology of the Cell 16, no. 2 (February 2005): 954–63. http://dx.doi.org/10.1091/mbc.e04-07-0622.

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The assembly of ribosomes involves the coordinated processing and modification of rRNAs with the temporal association of ribosomal proteins. This process is regulated by assembly factors such as helicases, modifying enzymes, and GTPases. In contrast to the assembly of cytoplasmic ribosomes, there is a paucity of information concerning the role of assembly proteins in the biogenesis of mitochondrial ribosomes. In this study, we demonstrate that the Saccharomyces cerevisiae GTPase Mtg2p (Yhr168wp) is essential for mitochondrial ribosome function. Cells lacking MTG2 lose their mitochondrial DNA, giving rise to petite cells. In addition, cells expressing a temperature-sensitive mgt2-1 allele are defective in mitochondrial protein synthesis and contain lowered levels of mitochondrial ribosomal subunits. Significantly, elevated levels of Mtg2p partially suppress the thermosensitive loss of mitochondrial DNA in a 21S rRNA methyltransferase mutant, mrm2. We propose that Mtg2p is involved in mitochondrial ribosome biogenesis. Consistent with this role, we show that Mtg2p is peripherally localized to the mitochondrial inner membrane and associates with the 54S large ribosomal subunit in a salt-dependent manner.

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Ho, Jennifer Hei-Ngam, George Kallstrom, and Arlen W. Johnson. "Nmd3p Is a Crm1p-Dependent Adapter Protein for Nuclear Export of the Large Ribosomal Subunit." Journal of Cell Biology 151, no. 5 (November 27, 2000): 1057–66. http://dx.doi.org/10.1083/jcb.151.5.1057.

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In eukaryotic cells, nuclear export of nascent ribosomal subunits through the nuclear pore complex depends on the small GTPase Ran. However, neither the nuclear export signals (NESs) for the ribosomal subunits nor the receptor proteins, which recognize the NESs and mediate export of the subunits, have been identified. We showed previously that Nmd3p is an essential protein from yeast that is required for a late step in biogenesis of the large (60S) ribosomal subunit. Here, we show that Nmd3p shuttles and that deletion of the NES from Nmd3p leads to nuclear accumulation of the mutant protein, inhibition of the 60S subunit biogenesis, and inhibition of the nuclear export of 60S subunits. Moreover, the 60S subunits that accumulate in the nucleus can be coimmunoprecipitated with the NES-deficient Nmd3p. 60S subunit biogenesis and export of truncated Nmd3p were restored by the addition of an exogenous NES. To identify the export receptor for Nmd3p we show that Nmd3p shuttling and 60S export is blocked by the Crm1p-specific inhibitor leptomycin B. These results identify Crm1p as the receptor for Nmd3p export. Thus, export of the 60S subunit is mediated by the adapter protein Nmd3p in a Crm1p-dependent pathway.

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Liu, Zheng, Cristina Gutierrez-Vargas, Jia Wei, Robert A. Grassucci, Madhumitha Ramesh, Noel Espina, Ming Sun, et al. "Structure and assembly model for the Trypanosoma cruzi 60S ribosomal subunit." Proceedings of the National Academy of Sciences 113, no. 43 (October 10, 2016): 12174–79. http://dx.doi.org/10.1073/pnas.1614594113.

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Ribosomes of trypanosomatids, a family of protozoan parasites causing debilitating human diseases, possess multiply fragmented rRNAs that together are analogous to 28S rRNA, unusually large rRNA expansion segments, and r-protein variations compared with other eukaryotic ribosomes. To investigate the architecture of the trypanosomatid ribosomes, we determined the 2.5-Å structure of the Trypanosoma cruzi ribosome large subunit by single-particle cryo-EM. Examination of this structure and comparative analysis of the yeast ribosomal assembly pathway allowed us to develop a stepwise assembly model for the eight pieces of the large subunit rRNAs and a number of ancillary “glue” proteins. This model can be applied to the characterization of Trypanosoma brucei and Leishmania spp. ribosomes as well. Together with other details, our atomic-level structure may provide a foundation for structure-based design of antitrypanosome drugs.

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Pollutri, Daniela, and Marianna Penzo. "Ribosomal Protein L10: From Function to Dysfunction." Cells 9, no. 11 (November 19, 2020): 2503. http://dx.doi.org/10.3390/cells9112503.

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Eukaryotic cytoplasmic ribosomes are highly structured macromolecular complexes made up of four different ribosomal RNAs (rRNAs) and 80 ribosomal proteins (RPs), which play a central role in the decoding of genetic code for the synthesis of new proteins. Over the past 25 years, studies on yeast and human models have made it possible to identify RPL10 (ribosomal protein L10 gene), which is a constituent of the large subunit of the ribosome, as an important player in the final stages of ribosome biogenesis and in ribosome function. Here, we reviewed the literature to give an overview of the role of RPL10 in physiologic and pathologic processes, including inherited disease and cancer.

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Möller-Hergt, Braulio Vargas, Andreas Carlström, Katharina Stephan, Axel Imhof, and Martin Ott. "The ribosome receptors Mrx15 and Mba1 jointly organize cotranslational insertion and protein biogenesis in mitochondria." Molecular Biology of the Cell 29, no. 20 (October 2018): 2386–96. http://dx.doi.org/10.1091/mbc.e18-04-0227.

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Mitochondrial gene expression in Saccharomyces cerevisiae is responsible for the production of highly hydrophobic subunits of the oxidative phosphorylation system. Membrane insertion occurs cotranslationally on membrane-bound mitochondrial ribosomes. Here, by employing a systematic mass spectrometry–based approach, we discovered the previously uncharacterized membrane protein Mrx15 that interacts via a soluble C-terminal domain with the large ribosomal subunit. Mrx15 contacts mitochondrial translation products during their synthesis and plays, together with the ribosome receptor Mba1, an overlapping role in cotranslational protein insertion. Taken together, our data reveal how these ribosome receptors organize membrane protein biogenesis in mitochondria.

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Eisinger, D. P., F. A. Dick, and B. L. Trumpower. "Qsr1p, a 60S ribosomal subunit protein, is required for joining of 40S and 60S subunits." Molecular and Cellular Biology 17, no. 9 (September 1997): 5136–45. http://dx.doi.org/10.1128/mcb.17.9.5136.

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QSR1 is a recently discovered, essential Saccharomyces cerevisiae gene, which encodes a 60S ribosomal subunit protein. Thirty-one unique temperature-sensitive alleles of QSR1 were generated by regional codon randomization within a conserved 20-amino-acid sequence of the QSR1-encoded protein. The temperature-sensitive mutants arrest as viable, large, unbudded cells 24 to 48 h after a shift to 37 degrees C. Polysome and ribosomal subunit analysis by velocity gradient centrifugation of lysates from temperature-sensitive qsr1 mutants and from cells in which Qsr1p was depleted by down regulation of an inducible promoter revealed the presence of half-mer polysomes and a large pool of free 60S subunits that lack Qsr1p. In vitro subunit-joining assays and analysis of a mutant conditional for the synthesis of Qsr1p demonstrate that 60S subunits devoid of Qsr1p are unable to join with 40S subunits whereas 60S subunits that contain either wild-type or mutant forms of the protein are capable of subunit joining. The defective 60S subunits result from a reduced association of mutant Qsr1p with 60S subunits. These results indicate that Qsr1p is required for ribosomal subunit joining.

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Boublik, M., and J. S. Wall. "Structure of rRNA in the ribosome." Proceedings, annual meeting, Electron Microscopy Society of America 50, no. 1 (August 1992): 462–63. http://dx.doi.org/10.1017/s042482010012271x.

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Ribosomes are complex subcellular organelles playing a central role in protein biosynthesis. They are composed of more than fifty different proteins and three or four ribonucleic acids (rRNA) unevenly distributed (with no symmetry) between the large and small ribosomal subunit. It has been well established that ribosomal proteins and rRNAs are both involved in formation of the internal architecture of the ribosome as well as its function in protein synthesis. Understanding the fundamental relationship between structure and function requires establishment of the 3-D structure of the ribosome and its components at a molecular level.

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Marshall, R. Andrew, Magdalena Dorywalska, and Joseph D. Puglisi. "Irreversible chemical steps control intersubunit dynamics during translation." Proceedings of the National Academy of Sciences 105, no. 40 (September 29, 2008): 15364–69. http://dx.doi.org/10.1073/pnas.0805299105.

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The ribosome, a two-subunit macromolecular machine, deciphers the genetic code and catalyzes peptide bond formation. Dynamic rotational movement between ribosomal subunits is likely required for efficient and accurate protein synthesis, but direct observation of intersubunit dynamics has been obscured by the repetitive, multistep nature of translation. Here, we report a collection of single-molecule fluorescence resonance energy transfer assays that reveal a ribosomal intersubunit conformational cycle in real time during initiation and the first round of elongation. After subunit joining and delivery of correct aminoacyl-tRNA to the ribosome, peptide bond formation results in a rapid conformational change, consistent with the counterclockwise rotation of the 30S subunit with respect to the 50S subunit implied by prior structural and biochemical studies. Subsequent binding of elongation factor G and GTP hydrolysis results in a clockwise rotation of the 30S subunit relative to the 50S subunit, preparing the ribosome for the next round of tRNA selection and peptide bond formation. The ribosome thus harnesses the free energy of irreversible peptidyl transfer and GTP hydrolysis to surmount activation barriers to large-scale conformational changes during translation. Intersubunit rotation is likely a requirement for the concerted movement of tRNA and mRNA substrates during translocation.

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Verma, J., and N. Agrawal. "Molecular characterization of Indian species of the genus Cornudiscoides Kulkarni, 1969 (Monogenoidea: Dactylogyridae)." Journal of Applied and Natural Science 13, no. 1 (January 31, 2021): 1–7. http://dx.doi.org/10.31018/jans.v13i1.2434.

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Molecular characterization and phylogenetic study based on partial sequences of 28S and 18S ribosomal DNA (rDNA) of sixteen Indian species of the genus Cornudiscoides (Monogenoidea: Dactylogyridae) were conducted to decode the genetic relationship between them and with other members of the family Dactylogyridae. Blastn searches disclosed the significant similarity among the species of the Cornudiscoides for large ribosomal subunits as well as for small ribosomal subunit showing genetic relatedness. The phylogenetic tree using neighbour-joining (NJ) and minimum evolution (ME) methods for 28S ribosomal subunit depicted that all Cornudiscoides species clustering in a single clade and forming sister clade with other members of the family Dactylogyridae and similar results were obtained from 18S ribosomal subunit. Thus, the present study demonstrated that both 28S and 18S ribosomal subunits are very helpful in discriminating Cornudiscoides species (intra or interspecific variation) and in the establishment of the evolutionary relationship among them.

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Umaer, Khan, Martin Ciganda, and Noreen Williams. "Ribosome Biogenesis in African Trypanosomes Requires Conserved and Trypanosome-Specific Factors." Eukaryotic Cell 13, no. 6 (April 4, 2014): 727–37. http://dx.doi.org/10.1128/ec.00307-13.

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ABSTRACTLarge ribosomal subunit protein L5 is responsible for the stability and trafficking of 5S rRNA to the site of eukaryotic ribosomal assembly. InTrypanosoma brucei, in addition to L5, trypanosome-specific proteins P34 and P37 also participate in this process. These two essential proteins form a novel preribosomal particle through interactions with both the ribosomal protein L5 and 5S rRNA. We have generated a procyclic L5 RNA interference cell line and found that L5 itself is a protein essential for trypanosome growth, despite the presence of other 5S rRNA binding proteins. Loss of L5 decreases the levels of all large-subunit rRNAs, 25/28S, 5.8S, and 5S rRNAs, but does not alter small-subunit 18S rRNA. Depletion of L5 specifically reduced the levels of the other large ribosomal proteins, L3 and L11, whereas the steady-state levels of the mRNA for these proteins were increased. L5-knockdown cells showed an increase in the 40S ribosomal subunit and a loss of the 60S ribosomal subunits, 80S monosomes, and polysomes. In addition, L5 was involved in the processing and maturation of precursor rRNAs. Analysis of polysomal fractions revealed that unprocessed rRNA intermediates accumulate in the ribosome when L5 is depleted. Although we previously found that the loss of P34 and P37 does not result in a change in the levels of L5, the loss of L5 resulted in an increase of P34 and P37 proteins, suggesting the presence of a compensatory feedback loop. This study demonstrates that ribosomal protein L5 has conserved functions, in addition to nonconserved trypanosome-specific features, which could be targeted for drug intervention.

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Fuchs, Gabriele, Alexey N. Petrov, Caleb D. Marceau, Lauren M. Popov, Jin Chen, Seán E. O’Leary, Richard Wang, Jan E. Carette, Peter Sarnow, and Joseph D. Puglisi. "Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site." Proceedings of the National Academy of Sciences 112, no. 2 (December 16, 2014): 319–25. http://dx.doi.org/10.1073/pnas.1421328111.

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Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.

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Gupta, Ankit, Priyanka Shah, Afreen Haider, Kirti Gupta, Mohammad Imran Siddiqi, Stuart A. Ralph, and Saman Habib. "Reduced ribosomes of the apicoplast and mitochondrion of Plasmodium spp. and predicted interactions with antibiotics." Open Biology 4, no. 5 (May 2014): 140045. http://dx.doi.org/10.1098/rsob.140045.

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Apicomplexan protists such as Plasmodium and Toxoplasma contain a mitochondrion and a relic plastid (apicoplast) that are sites of protein translation. Although there is emerging interest in the partitioning and function of translation factors that participate in apicoplast and mitochondrial peptide synthesis, the composition of organellar ribosomes remains to be elucidated. We carried out an analysis of the complement of core ribosomal protein subunits that are encoded by either the parasite organellar or nuclear genomes, accompanied by a survey of ribosome assembly factors for the apicoplast and mitochondrion. A cross-species comparison with other apicomplexan, algal and diatom species revealed compositional differences in apicomplexan organelle ribosomes and identified considerable reduction and divergence with ribosomes of bacteria or characterized organelle ribosomes from other organisms. We assembled structural models of sections of Plasmodium falciparum organellar ribosomes and predicted interactions with translation inhibitory antibiotics. Differences in predicted drug–ribosome interactions with some of the modelled structures suggested specificity of inhibition between the apicoplast and mitochondrion. Our results indicate that Plasmodium and Toxoplasma organellar ribosomes have a unique composition, resulting from the loss of several large and small subunit proteins accompanied by significant sequence and size divergences in parasite orthologues of ribosomal proteins.

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Horne, Z., and J. Hesketh. "Immunological localization of ribosomes in striated rat muscle. Evidence for myofibrillar association and ontological changes in the subsarcolemmal:myofibrillar distribution." Biochemical Journal 268, no. 1 (May 15, 1990): 231–36. http://dx.doi.org/10.1042/bj2680231.

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Ribosome distribution in skeletal-muscle fibres was investigated immunohistochemically by using polyclonal antibodies raised against large-ribosomal-subunit proteins isolated from rat liver. Immunoblot analysis showed the antibodies to recognize five major proteins of the large subunit; these were identified as L4, L6, L7, L15 and L17 by two-dimensional electrophoresis. Immunohistochemistry of frozen rat skeletal-muscle sections showed staining of both the subsarcolemmal and intermyofibrillar cytoplasm. A distinct banding pattern was observed, and when peroxidase and phase-contrast images of the same field were compared by image analysis the anti-ribosome staining was found to correspond to the A-bands. These results suggest that a proportion of muscle ribosomes are present in the myofibrillar cytoplasm in a regular fashion, possibly associated with myosin. Densitometric analysis of the peroxidase immunostaining showed that the ratio of myofibrillar to sub-sarcolemmal ribosomal material was lower in muscle from 51-day-old rats compared with those from 14-day-old animals.

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Sengupta, Jayati, Cyril Bussiere, Jesper Pallesen, Matthew West, Arlen W. Johnson, and Joachim Frank. "Characterization of the nuclear export adaptor protein Nmd3 in association with the 60S ribosomal subunit." Journal of Cell Biology 189, no. 7 (June 28, 2010): 1079–86. http://dx.doi.org/10.1083/jcb.201001124.

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The nucleocytoplasmic shuttling protein Nmd3 is an adaptor for export of the 60S ribosomal subunit from the nucleus. Nmd3 binds to nascent 60S subunits in the nucleus and recruits the export receptor Crm1 to facilitate passage through the nuclear pore complex. In this study, we present a cryoelectron microscopy (cryo-EM) reconstruction of the 60S subunit in complex with Nmd3 from Saccharomyces cerevisiae. The density corresponding to Nmd3 is directly visible in the cryo-EM map and is attached to the regions around helices 38, 69, and 95 of the 25S ribosomal RNA (rRNA), the helix 95 region being adjacent to the protein Rpl10. We identify the intersubunit side of the large subunit as the binding site for Nmd3. rRNA protection experiments corroborate the structural data. Furthermore, Nmd3 binding to 60S subunits is blocked in 80S ribosomes, which is consistent with the assigned binding site on the subunit joining face. This cryo-EM map is a first step toward a molecular understanding of the functional role and release mechanism of Nmd3.

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41

Garaeva, Natalia, Aydar Bikmullin, Evelina Klochkova, Shamil Validov, Marat Yusupov, and Konstantin Usachev. "Abstract P-31: Assembly of the Complex of the 30S Ribosomal Subunit and the Ribosome Maturation Factor P from Staphylococcus aureus for Structural Studies by Cryo-Electron Microscopy." International Journal of Biomedicine 11, Suppl_1 (June 1, 2021): S25. http://dx.doi.org/10.21103/ijbm.11.suppl_1.p31.

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Background: Staphylococcus aureus (S. aureus) is one of the main human pathogens causing numerous nosocomial soft tissue infections and is among the best-known causes of bacterial infections. The bacterial 70S ribosome consists of two subunits, designated the 30S (small) and 50S (large) subunits. The small subunit (30S) consists of 16S ribosomal RNA (rRNA), from which the assembly of 30S begins, and 21 ribosomal proteins (r-proteins). The ribosome maturation factor P (RimP protein) binds to the free 30S subunit. Strains lacking RimP accumulate immature 16S rRNA, and fewer polysomes and an increased amount of unassociated 30S and 50S subunits compared to wild-type strains are observed in the ribosomal profile. Structural studies of the 30S subunit complex and the ribosome maturation factor RimP will make it possible in the future to develop an antibiotic that slows down or completely stops the translation of Staphylococcus aureus, which will complicate the synthesis and isolation of its pathogenic factors. Here we present the protocol of the in vitro reconstruction of S. aureus 30S ribosome subunit in a complex with RimP for further structural studies by cryo-electron microscopy. Methods: Recombinant RimP protein from S. aureus was expressed in E. coli and purified by Ni-NTA chromatography and size exclusion chromatography. Reconstitution of the 30S–RimP complex was performed by mixing RimP protein with 30S ribosome. Unbound RimP protein was removed by Amicon Ultra Concentration (Merk KGaA, Darmstadt, Germany) with a cut-off limit of 100 kDa. The presence of RimP protein in the resulting 30S-RimP complex was confirmed by SDS-PAGE, and the quality of the final sample was analyzed by the negative staining EM. Results: Finally, by in vitro reconstruction, the 30S-RimP complex from S. aureus was obtained for further structural studies by cryo-electron microscopy.

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Farrar, Jason E., Michelle Nater, Emi Caywood, Michael A. McDevitt, Jeanne Kowalski, Clifford M. Takemoto, C. Conover Talbot, et al. "Abnormalities of the large ribosomal subunit protein, Rpl35a, in Diamond-Blackfan anemia." Blood 112, no. 5 (September 1, 2008): 1582–92. http://dx.doi.org/10.1182/blood-2008-02-140012.

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Abstract Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by anemia, congenital abnormalities, and cancer predisposition. Small ribosomal subunit genes RPS19, RPS24, and RPS17 are mutated in approximately one-third of patients. We used a candidate gene strategy combining high-resolution genomic mapping and gene expression microarray in the analysis of 2 DBA patients with chromosome 3q deletions to identify RPL35A as a potential DBA gene. Sequence analysis of a cohort of DBA probands confirmed involvement RPL35A in DBA. shRNA inhibition shows that Rpl35a is essential for maturation of 28S and 5.8S rRNAs, 60S subunit biogenesis, normal proliferation, and cell survival. Analysis of pre-rRNA processing in primary DBA lymphoblastoid cell lines demonstrated similar alterations of large ribosomal subunit rRNA in both RPL35A-mutated and some RPL35A wild-type patients, suggesting additional large ribosomal subunit gene defects are likely present in some cases of DBA. These data demonstrate that alterations of large ribosomal subunit proteins cause DBA and support the hypothesis that DBA is primarily the result of altered ribosomal function. The results also establish that haploinsufficiency of large ribosomal subunit proteins contributes to bone marrow failure and potentially cancer predisposition.

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Li, Wenzhu, Jing Zhang, Wenpeng Cheng, Yuze Li, Jinwen Feng, Jun Qin, and Xiangwei He. "Differential Paralog-Specific Expression of Multiple Small Subunit Proteins Cause Variations in Rpl42/eL42 Incorporation in Ribosome in Fission Yeast." Cells 11, no. 15 (August 2, 2022): 2381. http://dx.doi.org/10.3390/cells11152381.

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Ribosomes within a cell are commonly viewed as biochemically homogenous RNA–protein super-complexes performing identical functions of protein synthesis. However, recent evidence suggests that ribosomes may be a more dynamic macromolecular complex with specialized roles. Here, we present extensive genetic and molecular evidence in the fission yeast S. pombe that the paralogous genes for many ribosomal proteins (RPs) are functionally different, despite that they encode the same ribosomal component, often with only subtle differences in the sequences. Focusing on the rps8 paralog gene deletions rps801d and rps802d, we showed that the mutant cells differ in the level of Rpl42p in actively translating ribosomes and that their phenotypic differences reside in the Rpl42p level variation instead of the subtle protein sequence difference between Rps801p and Rps802p. Additional 40S ribosomal protein paralog pairs also exhibit similar phenotypic differences via differential Rpl42p levels in actively translating ribosomes. Together, our work identifies variations in the Rpl42p level as a potential form of ribosome heterogeneity in biochemical compositions and suggests a possible connection between large and small subunits during ribosome biogenesis that may cause such heterogeneity. Additionally, it illustrates the complexity of the underlying mechanisms for the genetic specificity of ribosome paralogs.

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JOHNSON, A. W., J. H. N. HO, G. KALLSTROM, C. TROTTA, E. LUND, L. KAHAN, J. DAHLBERG, and J. HEDGES. "Nuclear Export of the Large Ribosomal Subunit." Cold Spring Harbor Symposia on Quantitative Biology 66 (January 1, 2001): 599–606. http://dx.doi.org/10.1101/sqb.2001.66.599.

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Harauz, G., H. Zuzan, P. T. Kim, and J. A. Holbrook. "SECRet of the Eukaryotic Large Ribosomal Subunit." Biochemical and Biophysical Research Communications 207, no. 2 (February 1995): 848–51. http://dx.doi.org/10.1006/bbrc.1995.1263.

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De Rijk, P. "The European Large Subunit Ribosomal RNA database." Nucleic Acids Research 28, no. 1 (January 1, 2000): 177–78. http://dx.doi.org/10.1093/nar/28.1.177.

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Wuyts, J. "The European Large Subunit Ribosomal RNA Database." Nucleic Acids Research 29, no. 1 (January 1, 2001): 175–77. http://dx.doi.org/10.1093/nar/29.1.175.

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De Rijk, Peter, Yves Van de Peer, Ilse Van den Broeck, and Rupert De Wachter. "Evolution according to large ribosomal subunit RNA." Journal of Molecular Evolution 41, no. 3 (September 1995): 366–75. http://dx.doi.org/10.1007/bf01215184.

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Nikolaeva, Daria D., Mikhail S. Gelfand, and Sofya K. Garushyants. "Simplification of Ribosomes in Bacteria with Tiny Genomes." Molecular Biology and Evolution 38, no. 1 (July 18, 2020): 58–66. http://dx.doi.org/10.1093/molbev/msaa184.

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Abstract The ribosome is an essential cellular machine performing protein biosynthesis. Its structure and composition are highly conserved in all species. However, some bacteria have been reported to have an incomplete set of ribosomal proteins. We have analyzed ribosomal protein composition in 214 small bacterial genomes (<1 Mb) and found that although the ribosome composition is fairly stable, some ribosomal proteins may be absent, especially in bacteria with dramatically reduced genomes. The protein composition of the large subunit is less conserved than that of the small subunit. We have identified the set of frequently lost ribosomal proteins and demonstrated that they tend to be positioned on the ribosome surface and have fewer contacts to other ribosome components. Moreover, some proteins are lost in an evolutionary correlated manner. The reduction of ribosomal RNA is also common, with deletions mostly occurring in free loops. Finally, the loss of the anti-Shine–Dalgarno sequence is associated with the loss of a higher number of ribosomal proteins.

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Lin, Bin, Desiree A. Thayer, and Janine R. Maddock. "The Caulobacter crescentus CgtAC Protein Cosediments with the Free 50S Ribosomal Subunit." Journal of Bacteriology 186, no. 2 (January 15, 2004): 481–89. http://dx.doi.org/10.1128/jb.186.2.481-489.2004.

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ABSTRACT The Obg family of GTPases is widely conserved and predicted to play an as-yet-unknown role in translation. Recent reports provide circumstantial evidence that both eukaryotic and prokaryotic Obg proteins are associated with the large ribosomal subunit. Here we provide direct evidence that the Caulobacter crescentus CgtAC protein is associated with the free large (50S) ribosomal subunit but not with 70S monosomes or with translating ribosomes. In contrast to the Bacillus subtilis and Escherichia coli proteins, CgtAC does not fractionate in a large complex by gel filtration, indicating a moderately weak association with the 50S subunit. Moreover, binding of CgtAC to the 50S particle is sensitive to salt concentration and buffer composition but not guanine nucleotide occupancy of CgtAC. Assays of epitope-tagged wild-type and mutant variants of CgtAC indicate that the C terminus of CgtAC is critical for 50S association. Interestingly, the addition of a C-terminal epitope tag also affected the ability of various cgtAC alleles to function in vivo. Depletion of CgtAC led to perturbations in the polysome profile, raising the possibility that CgtAC is involved in ribosome assembly or stability.

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Journal articles on the topic 'Large ribosomal subunit'

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