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1
Jovanovic, Bogdan, Lisa Schubert, Fabian Poetz y Georg Stoecklin. "Tagging of RPS9 as a tool for ribosome purification and identification of ribosome-associated proteins". Archives of Biological Sciences, n.º 00 (2020): 57. http://dx.doi.org/10.2298/abs20120557j.
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Ribosomes, the catalytic machinery required for protein synthesis, are comprised of 4 ribosomal RNAs and about 80 ribosomal proteins in mammals. Ribosomes further interact with numerous associated factors that regulate their biogenesis and function. As mutations of ribosomal proteins and ribosome associated proteins cause many diseases, it is important to develop tools by which ribosomes can be purified efficiently and with high specificity. Here, we designed a method to purify ribosomes from human cell lines by C-terminally tagging human RPS9, a protein of the small ribosomal subunit. The tag consists of a flag peptide and a streptavidin-binding peptide (SBP) separated by the tobacco etch virus (TEV) protease cleavage site. We demonstrate that RPS9-Flag-TEV-SBP (FTS) is efficiently incorporated into the ribosome without interfering with regular protein synthesis. Using HeLa-GFP-G3BP1 cells stably expressing RPS9-FTS or, as a negative control, mCherry-FTS, we show that complete ribosomes as well as numerous ribosome-associated proteins are efficiently and specifically purified following pull-down of RPS9-FTS using streptavidin beads. This tool will be helpful for the characterization of human ribosome heterogeneity, post-translational modifications of ribosomal proteins, and changes in ribosome-associated factors after exposing human cells to different stimuli and conditions.
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Akanuma, Genki. "Diverse relationships between metal ions and the ribosome". Bioscience, Biotechnology, and Biochemistry 85, n.º 7 (20 de abril de 2021): 1582–93. http://dx.doi.org/10.1093/bbb/zbab070.
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ABSTRACT The ribosome requires metal ions for structural stability and translational activity. These metal ions are important for stabilizing the secondary structure of ribosomal RNA, binding of ribosomal proteins to the ribosome, and for interaction of ribosomal subunits. In this review, various relationships between ribosomes and metal ions, especially Mg2+ and Zn2+, are presented. Mg2+ regulates gene expression by modulating the translational stability and synthesis of ribosomes, which in turn contribute to the cellular homeostasis of Mg2+. In addition, Mg2+ can partly complement the function of ribosomal proteins. Conversely, a reduction in the cellular concentration of Zn2+ induces replacement of ribosomal proteins, which mobilizes free-Zn2+ in the cell and represses translation activity. Evolutional relationships between these metal ions and the ribosome are also discussed.
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Sulima y Dinman. "The Expanding Riboverse". Cells 8, n.º 10 (5 de octubre de 2019): 1205. http://dx.doi.org/10.3390/cells8101205.
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Subverting the conventional concept of “the” ribosome, a wealth of information gleaned from recent studies is revealing a much more diverse and dynamic ribosomal reality than has traditionally been thought possible. A diverse array of researchers is collectively illuminating a universe of heterogeneous and adaptable ribosomes harboring differences in composition and regulatory capacity: These differences enable specialization. The expanding universe of ribosomes not only comprises an incredible richness in ribosomal specialization between species, but also within the same tissues and even cells. In this review, we discuss ribosomal heterogeneity and speculate how the emerging understanding of the ribosomal repertoire is impacting the biological sciences today. Targeting pathogen-specific and pathological “diseased” ribosomes promises to provide new treatment options for patients, and potential applications for “designer ribosomes” are within reach. Our deepening understanding of and ability to manipulate the ribosome are establishing both the technological and theoretical foundations for major advances for the 21st century and beyond.
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Guth-Metzler, Rebecca, Marcus S. Bray, Moran Frenkel-Pinter, Suttipong Suttapitugsakul, Claudia Montllor-Albalate, Jessica C. Bowman, Ronghu Wu et al. "Cutting in-line with iron: ribosomal function and non-oxidative RNA cleavage". Nucleic Acids Research 48, n.º 15 (14 de julio de 2020): 8663–74. http://dx.doi.org/10.1093/nar/gkaa586.
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Abstract Divalent metal cations are essential to the structure and function of the ribosome. Previous characterizations of the ribosome performed under standard laboratory conditions have implicated Mg2+ as a primary mediator of ribosomal structure and function. Possible contributions of Fe2+ as a ribosomal cofactor have been largely overlooked, despite the ribosome's early evolution in a high Fe2+ environment, and the continued use of Fe2+ by obligate anaerobes inhabiting high Fe2+ niches. Here, we show that (i) Fe2+ cleaves RNA by in-line cleavage, a non-oxidative mechanism that has not previously been shown experimentally for this metal, (ii) the first-order in-line rate constant with respect to divalent cations is >200 times greater with Fe2+ than with Mg2+, (iii) functional ribosomes are associated with Fe2+ after purification from cells grown under low O2 and high Fe2+ and (iv) a small fraction of Fe2+ that is associated with the ribosome is not exchangeable with surrounding divalent cations, presumably because those ions are tightly coordinated by rRNA and deeply buried in the ribosome. In total, these results expand the ancient role of iron in biochemistry and highlight a possible new mechanism of iron toxicity.
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Jiang, M., S. M. Sullivan, A. K. Walker, J. R. Strahler, P. C. Andrews y J. R. Maddock. "Identification of Novel Escherichia coli Ribosome-Associated Proteins Using Isobaric Tags and Multidimensional Protein Identification Techniques". Journal of Bacteriology 189, n.º 9 (2 de marzo de 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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Yang, Rui, Luis R. Cruz-Vera y Charles Yanofsky. "23S rRNA Nucleotides in the Peptidyl Transferase Center Are Essential for Tryptophanase Operon Induction". Journal of Bacteriology 191, n.º 11 (27 de marzo de 2009): 3445–50. http://dx.doi.org/10.1128/jb.00096-09.
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ABSTRACT Distinct features of the ribosomal peptide exit tunnel are known to be essential for recognition of specific amino acids of a nascent peptidyl-tRNA. Thus, a tryptophan residue at position 12 of the peptidyl-tRNA TnaC-tRNAPro leads to the creation of a free tryptophan binding site within the ribosome at which bound tryptophan inhibits normal ribosome functions. The ribosomal processes that are inhibited are hydrolysis of TnaC-tRNAPro by release factor 2 and peptidyl transfer of TnaC of TnaC-tRNAPro to puromycin. These events are normally performed in the ribosomal peptidyl transferase center. In the present study, changes of 23S rRNA nucleotides in the 2585 region of the peptidyl transferase center, G2583A and U2584C, were observed to reduce maximum induction of tna operon expression by tryptophan in vivo without affecting the concentration of tryptophan necessary to obtain 50% induction. The growth rate of strains with ribosomes with either of these changes was not altered appreciably. In vitro analyses with mutant ribosomes with these changes showed that tryptophan was not as efficient in protecting TnaC-tRNAPro from puromycin action as wild-type ribosomes. However, added tryptophan did prevent sparsomycin action as it normally does with wild-type ribosomes. These findings suggest that these two mutational changes act by reducing the ability of ribosome-bound tryptophan to inhibit peptidyl transferase activity rather than by reducing the ability of the ribosome to bind tryptophan. Thus, the present study identifies specific nucleotides within the ribosomal peptidyl transferase center that appear to be essential for effective tryptophan induction of tna operon expression.
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Mandon, Elisabet C., Ying Jiang y Reid Gilmore. "Dual recognition of the ribosome and the signal recognition particle by the SRP receptor during protein targeting to the endoplasmic reticulum". Journal of Cell Biology 162, n.º 4 (11 de agosto de 2003): 575–85. http://dx.doi.org/10.1083/jcb.200303143.
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We have analyzed the interactions between the signal recognition particle (SRP), the SRP receptor (SR), and the ribosome using GTPase assays, biosensor experiments, and ribosome binding assays. Possible mechanisms that could contribute to an enhanced affinity between the SR and the SRP–ribosome nascent chain complex to promote protein translocation under physiological ionic strength conditions have been explored. Ribosomes or 60S large ribosomal subunits activate the GTPase cycle of SRP54 and SRα by providing a platform for assembly of the SRP–SR complex. Biosensor experiments revealed high-affinity, saturable binding of ribosomes or large ribosomal subunits to the SR. Remarkably, the SR has a 100-fold higher affinity for the ribosome than for SRP. Proteoliposomes that contain the SR bind nontranslating ribosomes with an affinity comparable to that shown by the Sec61 complex. An NH2-terminal 319-residue segment of SRα is necessary and sufficient for binding of SR to the ribosome. We propose that the ribosome–SR interaction accelerates targeting of the ribosome nascent chain complex to the RER, while the SRP–SR interaction is crucial for maintaining the fidelity of the targeting reaction.
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Kazibwe, Zakayo, Ang-Yu Liu, Gustavo C. MacIntosh y Diane C. Bassham. "The Ins and Outs of Autophagic Ribosome Turnover". Cells 8, n.º 12 (10 de diciembre de 2019): 1603. http://dx.doi.org/10.3390/cells8121603.
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Ribosomes are essential for protein synthesis in all organisms and their biogenesis and number are tightly controlled to maintain homeostasis in changing environmental conditions. While ribosome assembly and quality control mechanisms have been extensively studied, our understanding of ribosome degradation is limited. In yeast or animal cells, ribosomes are degraded after transfer into the vacuole or lysosome by ribophagy or nonselective autophagy, and ribosomal RNA can also be transferred directly across the lysosomal membrane by RNautophagy. In plants, ribosomal RNA is degraded by the vacuolar T2 ribonuclease RNS2 after transport by autophagy-related mechanisms, although it is unknown if a selective ribophagy pathway exists in plants. In this review, we describe mechanisms of turnover of ribosomal components in animals and yeast, and, then, discuss potential pathways for degradation of ribosomal RNA and protein within the vacuole in plants.
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Pecoraro, Annalisa, Martina Pagano, Giulia Russo y Annapina Russo. "Ribosome Biogenesis and Cancer: Overview on Ribosomal Proteins". International Journal of Molecular Sciences 22, n.º 11 (23 de mayo de 2021): 5496. http://dx.doi.org/10.3390/ijms22115496.
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Cytosolic ribosomes (cytoribosomes) are macromolecular ribonucleoprotein complexes that are assembled from ribosomal RNA and ribosomal proteins, which are essential for protein biosynthesis. Mitochondrial ribosomes (mitoribosomes) perform translation of the proteins essential for the oxidative phosphorylation system. The biogenesis of cytoribosomes and mitoribosomes includes ribosomal RNA processing, modification and binding to ribosomal proteins and is assisted by numerous biogenesis factors. This is a major energy-consuming process in the cell and, therefore, is highly coordinated and sensitive to several cellular stressors. In mitochondria, the regulation of mitoribosome biogenesis is essential for cellular respiration, a process linked to cell growth and proliferation. This review briefly overviews the key stages of cytosolic and mitochondrial ribosome biogenesis; summarizes the main steps of ribosome biogenesis alterations occurring during tumorigenesis, highlighting the changes in the expression level of cytosolic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs) in different types of tumors; focuses on the currently available information regarding the extra-ribosomal functions of CRPs and MRPs correlated to cancer; and discusses the role of CRPs and MRPs as biomarkers and/or molecular targets in cancer treatment.
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Ghulam, Mustafa Malik, Mathieu Catala y Sherif Abou Elela. "Differential expression of duplicated ribosomal protein genes modifies ribosome composition in response to stress". Nucleic Acids Research 48, n.º 4 (21 de diciembre de 2019): 1954–68. http://dx.doi.org/10.1093/nar/gkz1183.
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Abstract In Saccharomyces cerevisiae, most ribosomal proteins are synthesized from duplicated genes, increasing the potential for ribosome heterogeneity. However, the contribution of these duplicated genes to ribosome production and the mechanism determining their relative expression remain unclear. Here we demonstrate that in most cases, one of the two gene copies generate the bulk of the active ribosomes under normal growth conditions, while the other copy is favored only under stress. To understand the origin of these differences in paralog expression and their contribution to ribosome heterogeneity we used RNA polymerase II ChIP-Seq, RNA-seq, polyribosome association and peptide-based mass-spectrometry to compare their transcription potential, splicing, mRNA abundance, translation potential, protein abundance and incorporation into ribosomes. In normal conditions a post-transcriptional expression hierarchy of the duplicated ribosomal protein genes is the product of the efficient splicing, high stability and efficient translation of the major paralog mRNA. Exposure of the cell to stress modifies the expression ratio of the paralogs by repressing the expression of the major paralog and thus increasing the number of ribosomes carrying the minor paralog. Together the data indicate that duplicated ribosomal protein genes underlie a modular network permitting the modification of ribosome composition in response to changing growth conditions.
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Collins, Jason C., Homa Ghalei, Joanne R. Doherty, Haina Huang, Rebecca N. Culver y Katrin Karbstein. "Ribosome biogenesis factor Ltv1 chaperones the assembly of the small subunit head". Journal of Cell Biology 217, n.º 12 (22 de octubre de 2018): 4141–54. http://dx.doi.org/10.1083/jcb.201804163.
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The correct assembly of ribosomes from ribosomal RNAs (rRNAs) and ribosomal proteins (RPs) is critical, as indicated by the diseases caused by RP haploinsufficiency and loss of RP stoichiometry in cancer cells. Nevertheless, how assembly of each RP is ensured remains poorly understood. We use yeast genetics, biochemistry, and structure probing to show that the assembly factor Ltv1 facilitates the incorporation of Rps3, Rps10, and Asc1/RACK1 into the small ribosomal subunit head. Ribosomes from Ltv1-deficient yeast have substoichiometric amounts of Rps10 and Asc1 and show defects in translational fidelity and ribosome-mediated RNA quality control. These defects provide a growth advantage under some conditions but sensitize the cells to oxidative stress. Intriguingly, relative to glioma cell lines, breast cancer cells have reduced levels of LTV1 and produce ribosomes lacking RPS3, RPS10, and RACK1. These data describe a mechanism to ensure RP assembly and demonstrate how cancer cells circumvent this mechanism to generate diverse ribosome populations that can promote survival under stress.
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Siibak, Triinu, Lauri Peil, Liqun Xiong, Alexander Mankin, Jaanus Remme y Tanel Tenson. "Erythromycin- and Chloramphenicol-Induced Ribosomal Assembly Defects Are Secondary Effects of Protein Synthesis Inhibition". Antimicrobial Agents and Chemotherapy 53, n.º 2 (24 de noviembre de 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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Lavdovskaia, Elena, Kärt Denks, Franziska Nadler, Emely Steube, Andreas Linden, Henning Urlaub, Marina V. Rodnina y Ricarda Richter-Dennerlein. "Dual function of GTPBP6 in biogenesis and recycling of human mitochondrial ribosomes". Nucleic Acids Research 48, n.º 22 (2 de diciembre de 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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Meskauskas, Arturas, Alexey N. Petrov y Jonathan D. Dinman. "Identification of Functionally Important Amino Acids of Ribosomal Protein L3 by Saturation Mutagenesis". Molecular and Cellular Biology 25, n.º 24 (15 de diciembre de 2005): 10863–74. http://dx.doi.org/10.1128/mcb.25.24.10863-10874.2005.
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ABSTRACT There is accumulating evidence that many ribosomal proteins are involved in shaping rRNA into their functionally correct conformations through RNA-protein interactions. Moreover, although rRNA seems to play the central role in all aspects of ribosome function, ribosomal proteins may be involved in facilitating communication between different functional regions in ribosome, as well as between the ribosome and cellular factors. In an effort to more fully understand how ribosomal proteins may influence ribosome function, we undertook large-scale mutational analysis of ribosomal protein L3, a core protein of the large subunit that has been implicated in numerous ribosome-associated functions in the past. A total of 98 different rpl3 alleles were genetically characterized with regard to their effects on killer virus maintenance, programmed −1 ribosomal frameshifting, resistance/hypersensitivity to the translational inhibitor anisomycin and, in specific cases, the ability to enhance translation of a reporter mRNA lacking the 5′ 7mGppp cap structure and 3′ poly(A) tail. Biochemical studies reveal a correlation between an increased affinity for aminoacyl-tRNA and the extent of anisomycin resistance and a decreased peptidyltransferase activity and increased frameshifting efficiency. Immunoblot analyses reveal that the superkiller phenotype is not due to a defect in the ability of ribosomes to recruit the Ski-complex, suggesting that the defect lies in a reduced ability of mutant ribosomes to distinguish between cap+/poly(A)+ and cap−/poly(A)− mRNAs. The results of these analyses are discussed with regard to how protein-rRNA interactions may affect ribosome function.
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Sulima, Sergey, Kim Kampen y Kim De Keersmaecker. "Cancer Biogenesis in Ribosomopathies". Cells 8, n.º 3 (11 de marzo de 2019): 229. http://dx.doi.org/10.3390/cells8030229.
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Ribosomopathies are congenital diseases with defects in ribosome assembly and are characterized by elevated cancer risks. Additionally, somatic mutations in ribosomal proteins have recently been linked to a variety of cancers. Despite a clear correlation between ribosome defects and cancer, the molecular mechanisms by which these defects promote tumorigenesis are unclear. In this review, we focus on the emerging mechanisms that link ribosomal defects in ribosomopathies to cancer progression. This includes functional “onco-specialization” of mutant ribosomes, extra-ribosomal consequences of mutations in ribosomal proteins and ribosome assembly factors, and effects of ribosomal mutations on cellular stress and metabolism. We integrate some of these recent findings in a single model that can partially explain the paradoxical transition from hypo- to hyperproliferation phenotypes, as observed in ribosomopathies. Finally, we discuss the current and potential strategies, and the associated challenges for therapeutic intervention in ribosome-mutant diseases.
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Pollutri, Daniela y Marianna Penzo. "Ribosomal Protein L10: From Function to Dysfunction". Cells 9, n.º 11 (19 de noviembre de 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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Datta, Kaustuv, Jennifer L. Fuentes y 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, n.º 2 (febrero de 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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Herskovits, Anat A., Eyal Shimoni, Abraham Minsky y Eitan Bibi. "Accumulation of endoplasmic membranes and novel membrane-bound ribosome–signal recognition particle receptor complexes in Escherichia coli". Journal of Cell Biology 159, n.º 3 (4 de noviembre de 2002): 403–10. http://dx.doi.org/10.1083/jcb.200204144.
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In Escherichia coli, ribosomes must interact with translocons on the membrane for the proper integration of newly synthesized membrane proteins, cotranslationally. Previous in vivo studies indicated that unlike the E. coli signal recognition particle (SRP), the SRP receptor FtsY is required for membrane targeting of ribosomes. Accordingly, a putative SRP-independent, FtsY-mediated ribosomal targeting pathway has been suggested (Herskovits, A.A., E.S. Bochkareva, and E. Bibi. 2000. Mol. Microbiol. 38:927–939). However, the nature of the early contact of ribosomes with the membrane, and the involvement of FtsY in this interaction are unknown. Here we show that in cells depleted of the SRP protein, Ffh or the translocon component SecE, the ribosomal targeting pathway is blocked downstream and unprecedented, membrane-bound FtsY–ribosomal complexes are captured. Concurrently, under these conditions, novel, ribosome-loaded intracellular membrane structures are formed. We propose that in the absence of a functional SRP or translocon, ribosomes remain jammed at their primary membrane docking site, whereas FtsY-dependent ribosomal targeting to the membrane continues. The accumulation of FtsY-ribosome complexes induces the formation of intracellular membranes needed for their quantitative accommodation. Our results with E. coli, in conjunction with recent observations made with the yeast Saccharomyces cerevisiae, raise the possibility that the SRP receptor–mediated formation of intracellular membrane networks is governed by evolutionarily conserved principles.
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Konikkat, Salini y John L. Woolford,. "Principles of 60S ribosomal subunit assembly emerging from recent studies in yeast". Biochemical Journal 474, n.º 2 (6 de enero de 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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Hirokawa, Go, Hideko Kaji y Akira Kaji. "Inhibition of Antiassociation Activity of Translation Initiation Factor 3 by Paromomycin". Antimicrobial Agents and Chemotherapy 51, n.º 1 (6 de noviembre de 2006): 175–80. http://dx.doi.org/10.1128/aac.01096-06.
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ABSTRACT The effect of paromomycin on the interaction of ribosomal subunits was studied. Paromomycin inhibited the antiassociation activity of initiation factor 3 (IF3). Furthermore, ribosomal subunits were associated to form 70S ribosomes by paromomycin even in the presence of 1 mM Mg2+. Paromomycin did not inhibit the binding of IF3 to the 30S ribosomal subunits. On the other hand, IF3 bound to the 30S subunits was expelled by paromomycin-induced subunit association (70S formation). These results indicate that the stabilization of 70S ribosomes by paromomycin may in part be responsible for its inhibitory effects on translocation and ribosome recycling.
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Zalewski, Kazimierz. "The metabolism of aged seeds. The ribonucleolytic activity of rye grain embryos of different ages". Acta Societatis Botanicorum Poloniae 55, n.º 1 (2014): 45–52. http://dx.doi.org/10.5586/asbp.1986.006.
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Winter rye grain, harvested in various years and exhibiting clearly differing viability. was studied. Higher total ribonucleolytic activity was found in the post-ribosomal supernatant than in the ribosomes themselves. The ribonucleolytic activity of cytoplasmic ribosomes declined as the germination ability of the rye grain decreased. A similar relationship was observed in the post-ribosomal supernatant. Two additional, in comparison with fully viable grain, ribosome-associated RNase activities (pH 5.0 and 8.3) were found in the grain with the lowest (2%) viability.
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Gupta, Ankit, Priyanka Shah, Afreen Haider, Kirti Gupta, Mohammad Imran Siddiqi, Stuart A. Ralph y Saman Habib. "Reduced ribosomes of the apicoplast and mitochondrion of Plasmodium spp. and predicted interactions with antibiotics". Open Biology 4, n.º 5 (mayo de 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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Aquino, Gerald Ryan R., Nicolai Krogh, Philipp Hackert, Roman Martin, Jimena Davila Gallesio, Robert W. van Nues, Claudia Schneider et al. "RNA helicase-mediated regulation of snoRNP dynamics on pre-ribosomes and rRNA 2′-O-methylation". Nucleic Acids Research 49, n.º 7 (15 de marzo de 2021): 4066–84. http://dx.doi.org/10.1093/nar/gkab159.
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Abstract RNA helicases play important roles in diverse aspects of RNA metabolism through their functions in remodelling ribonucleoprotein complexes (RNPs), such as pre-ribosomes. Here, we show that the DEAD box helicase Dbp3 is required for efficient processing of the U18 and U24 intron-encoded snoRNAs and 2′-O-methylation of various sites within the 25S ribosomal RNA (rRNA) sequence. Furthermore, numerous box C/D snoRNPs accumulate on pre-ribosomes in the absence of Dbp3. Many snoRNAs guiding Dbp3-dependent rRNA modifications have overlapping pre-rRNA basepairing sites and therefore form mutually exclusive interactions with pre-ribosomes. Analysis of the distribution of these snoRNAs between pre-ribosome-associated and ‘free’ pools demonstrated that many are almost exclusively associated with pre-ribosomal complexes. Our data suggest that retention of such snoRNPs on pre-ribosomes when Dbp3 is lacking may impede rRNA 2′-O-methylation by reducing the recycling efficiency of snoRNPs and by inhibiting snoRNP access to proximal target sites. The observation of substoichiometric rRNA modification at adjacent sites suggests that the snoRNPs guiding such modifications likely interact stochastically rather than hierarchically with their pre-rRNA target sites. Together, our data provide new insights into the dynamics of snoRNPs on pre-ribosomal complexes and the remodelling events occurring during the early stages of ribosome assembly.
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Chen, Yu-Xiang, Zhi-yu Xu, Xueliang Ge, Jia-Yao Hong, Suparna Sanyal, Zhi John Lu y Babak Javid. "Selective translation by alternative bacterial ribosomes". Proceedings of the National Academy of Sciences 117, n.º 32 (28 de julio de 2020): 19487–96. http://dx.doi.org/10.1073/pnas.2009607117.
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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 fromMycobacterium smegmatis, 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 ofM. smegmatisin 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.
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Leclerc, Daniel y Léa Brakier-Gingras. "Study of the function of Escherichia coli ribosomal RNA through site-directed mutagenesis". Biochemistry and Cell Biology 68, n.º 1 (1 de enero de 1990): 169–79. http://dx.doi.org/10.1139/o90-023.
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Various approaches have been developed to study how mutations in Escherichia coli ribosomal RNA affect the function of the ribosome. Most of them are in vivo approaches, where mutations are introduced in a specialized plasmid harboring the ribosomal RNA genes. The mutated plasmids are then expressed in an appropriate host, where they can confer resistance to antibiotics whose target is the ribosome. Conditions can be used where the host ribosomal RNA genes or the host ribosomes are selectively inactivated, and the effect of the mutations on ribosome assembly and function can be studied. Another approach, which has been developed mainly with 16S ribosomal RNA, can be used entirely in vitro. In this approach, a plasmid has been constructed which contains the 16S ribosomal RNA gene under control of a T7 promoter. Mutations can be introduced in the 16S ribosomal RNA sequence and the mutated 16S ribosomal RNAs are produced by in vitro transcription. It is then possible to investigate how the mutations affect the assembly of the 16S ribosomal RNA into 30S subunits and the activity of the reconstituted 30S subunits in cell-free protein synthesis assays. Although these approaches are recent, they have already provided a large body of interesting information, relating specific RNA sequences to interactions with ribosomal proteins, to ribosome function, and to its response to antibiotics.Key words: ribosomal RNA, ribosome, site-directed mutagenesis, antibiotic resistance.
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Sung, Min-Kyung, Justin M. Reitsma, Michael J. Sweredoski, Sonja Hess y Raymond J. Deshaies. "Ribosomal proteins produced in excess are degraded by the ubiquitin–proteasome system". Molecular Biology of the Cell 27, n.º 17 (septiembre de 2016): 2642–52. http://dx.doi.org/10.1091/mbc.e16-05-0290.
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Ribosome assembly is an essential process that consumes prodigious quantities of cellular resources. Ribosomal proteins cannot be overproduced in Saccharomyces cerevisiae because the excess proteins are rapidly degraded. However, the responsible quality control (QC) mechanisms remain poorly characterized. Here we demonstrate that overexpression of multiple proteins of the small and large yeast ribosomal subunits is suppressed. Rpl26 overexpressed from a plasmid can be detected in the nucleolus and nucleoplasm, but it largely fails to assemble into ribosomes and is rapidly degraded. However, if the endogenous RPL26 loci are deleted, plasmid-encoded Rpl26 assembles into ribosomes and localizes to the cytosol. Chemical and genetic perturbation studies indicate that overexpressed ribosomal proteins are degraded by the ubiquitin–proteasome system and not by autophagy. Inhibition of the proteasome led to accumulation of multiple endogenous ribosomal proteins in insoluble aggregates, consistent with the operation of this QC mechanism in the absence of ribosomal protein overexpression. Our studies reveal that ribosomal proteins that fail to assemble into ribosomes are rapidly distinguished from their assembled counterparts and ubiquitinated and degraded within the nuclear compartment.
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Ghosh, Arnab y Natalia Shcherbik. "Cooperativity between the Ribosome-Associated Chaperone Ssb/RAC and the Ubiquitin Ligase Ltn1 in Ubiquitination of Nascent Polypeptides". International Journal of Molecular Sciences 21, n.º 18 (17 de septiembre de 2020): 6815. http://dx.doi.org/10.3390/ijms21186815.
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Eukaryotic cells have evolved multiple mechanisms to detect and eliminate aberrant polypeptides. Co-translational protein surveillance systems play an important role in these mechanisms. These systems include ribosome-associated protein quality control (RQC) that detects aberrant nascent chains stalled on ribosomes and promotes their ubiquitination and degradation by the proteasome, and ribosome-associated chaperone Ssb/RAC, which ensures correct nascent chain folding. Despite the known function of RQC and Ssb/ribosome-associated complex (RAC) in monitoring the quality of newly generated polypeptides, whether they cooperate during initial stages of protein synthesis remains unexplored. Here, we provide evidence that Ssb/RAC and the ubiquitin ligase Ltn1, the major component of RQC, display genetic and functional cooperativity. Overexpression of Ltn1 rescues growth suppression of the yeast strain-bearing deletions of SSB genes during proteotoxic stress. Moreover, Ssb/RAC promotes Ltn1-dependent ubiquitination of nascent chains associated with 80S ribosomal particles but not with translating ribosomes. Consistent with this finding, quantitative western blot analysis revealed lower levels of Ltn1 associated with 80S ribosomes and with free 60S ribosomal subunits in the absence of Ssb/RAC. We propose a mechanism in which Ssb/RAC facilitates recruitment of Ltn1 to ribosomes, likely by detecting aberrations in nascent chains and leading to their ubiquitination and degradation.
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Koplin, Ansgar, Steffen Preissler, Yulia Ilina, Miriam Koch, Annika Scior, Marc Erhardt y Elke Deuerling. "A dual function for chaperones SSB–RAC and the NAC nascent polypeptide–associated complex on ribosomes". Journal of Cell Biology 189, n.º 1 (5 de abril de 2010): 57–68. http://dx.doi.org/10.1083/jcb.200910074.
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The yeast Hsp70/40 system SSB–RAC (stress 70 B–ribosome-associated complex) binds to ribosomes and contacts nascent polypeptides to assist cotranslational folding. In this study, we demonstrate that nascent polypeptide–associated complex (NAC), another ribosome-tethered system, is functionally connected to SSB–RAC and the cytosolic Hsp70 network. Simultaneous deletions of genes encoding NAC and SSB caused conditional loss of cell viability under protein-folding stress conditions. Furthermore, NAC mutations revealed genetic interaction with a deletion of Sse1, a nucleotide exchange factor regulating the cytosolic Hsp70 network. Cells lacking SSB or Sse1 showed protein aggregation, which is enhanced by additional loss of NAC; however, these mutants differ in their potential client repertoire. Aggregation of ribosomal proteins and biogenesis factors accompanied by a pronounced deficiency in ribosomal particles and translating ribosomes only occurs in ssbΔ and nacΔssbΔ cells, suggesting that SSB and NAC control ribosome biogenesis. Thus, SSB–RAC and NAC assist protein folding and likewise have important functions for regulation of ribosome levels. These findings emphasize the concept that ribosome production is coordinated with the protein-folding capacity of ribosome-associated chaperones.
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Shcherbik, Natalia y Dimitri G. Pestov. "The Impact of Oxidative Stress on Ribosomes: From Injury to Regulation". Cells 8, n.º 11 (2 de noviembre de 2019): 1379. http://dx.doi.org/10.3390/cells8111379.
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The ribosome is a complex ribonucleoprotein-based molecular machine that orchestrates protein synthesis in the cell. Both ribosomal RNA and ribosomal proteins can be chemically modified by reactive oxygen species, which may alter the ribosome′s functions or cause a complete loss of functionality. The oxidative damage that ribosomes accumulate during their lifespan in a cell may lead to reduced or faulty translation and contribute to various pathologies. However, remarkably little is known about the biological consequences of oxidative damage to the ribosome. Here, we provide a concise summary of the known types of changes induced by reactive oxygen species in rRNA and ribosomal proteins and discuss the existing experimental evidence of how these modifications may affect ribosome dynamics and function. We emphasize the special role that redox-active transition metals, such as iron, play in ribosome homeostasis and stability. We also discuss the hypothesis that redox-mediated ribosome modifications may contribute to adaptive cellular responses to stress.
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Sleiman, Sophie y Francois Dragon. "Recent Advances on the Structure and Function of RNA Acetyltransferase Kre33/NAT10". Cells 8, n.º 9 (5 de septiembre de 2019): 1035. http://dx.doi.org/10.3390/cells8091035.
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Ribosome biogenesis is one of the most energy demanding processes in the cell. In eukaryotes, the main steps of this process occur in the nucleolus and include pre-ribosomal RNA (pre-rRNA) processing, post-transcriptional modifications, and assembly of many non-ribosomal factors and ribosomal proteins in order to form mature and functional ribosomes. In yeast and humans, the nucleolar RNA acetyltransferase Kre33/NAT10 participates in different maturation events, such as acetylation and processing of 18S rRNA, and assembly of the 40S ribosomal subunit. Here, we review the structural and functional features of Kre33/NAT10 RNA acetyltransferase, and we underscore the importance of this enzyme in ribosome biogenesis, as well as in acetylation of non-ribosomal targets. We also report on the role of human NAT10 in Hutchinson–Gilford progeria syndrome.
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Maiväli, Ülo, Anton Paier y Tanel Tenson. "When stable RNA becomes unstable: the degradation of ribosomes in bacteria and beyond". Biological Chemistry 394, n.º 7 (1 de julio de 2013): 845–55. http://dx.doi.org/10.1515/hsz-2013-0133.
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Abstract This review takes a comparative look at the various scenarios where ribosomes are degraded in bacteria and eukaryotes with emphasis on studies involving Escherichia coli and Saccharomyces cerevisiae. While the molecular mechanisms of degradation in bacteria and yeast appear somewhat different, we argue that the underlying causes of ribosome degradation are remarkably similar. In both model organisms during ribosomal assembly, partially formed pre-ribosomal particles can be degraded by at least two different sequentially-acting quality control pathways and fully assembled but functionally faulty ribosomes can be degraded in a separate quality control pathway. In addition, ribosomes that are both structurally- and functionally-sound can be degraded as an adaptive measure to stress.
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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, n.º 50 (30 de noviembre de 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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Dalla Venezia, Nicole, Anne Vincent, Virginie Marcel, Frédéric Catez y Jean-Jacques Diaz. "Emerging Role of Eukaryote Ribosomes in Translational Control". International Journal of Molecular Sciences 20, n.º 5 (11 de marzo de 2019): 1226. http://dx.doi.org/10.3390/ijms20051226.
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Translation is one of the final steps that regulate gene expression. The ribosome is the effector of translation through to its role in mRNA decoding and protein synthesis. Many mechanisms have been extensively described accounting for translational regulation. However it emerged only recently that ribosomes themselves could contribute to this regulation. Indeed, though it is well-known that the translational efficiency of the cell is linked to ribosome abundance, studies recently demonstrated that the composition of the ribosome could alter translation of specific mRNAs. Evidences suggest that according to the status, environment, development, or pathological conditions, cells produce different populations of ribosomes which differ in their ribosomal protein and/or RNA composition. Those observations gave rise to the concept of “specialized ribosomes”, which proposes that a unique ribosome composition determines the translational activity of this ribosome. The current review will present how technological advances have participated in the emergence of this concept, and to which extent the literature sustains this concept today.
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Boublik, M. y J. S. Wall. "Structure of rRNA in the ribosome". Proceedings, annual meeting, Electron Microscopy Society of America 50, n.º 1 (agosto de 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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Masłowski, P., M. Komoszyński y A. Śliwak. "Changes in chloroplastic and cytoplasmic ribosomal protein after GA3-treatment of Zea mays leaves". Acta Agrobotanica 27, n.º 1 (2015): 81–84. http://dx.doi.org/10.5586/aa.1974.007.
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Protein was isolated from chioroplastic and cytoplasmic ribosomes of 14-day-old maize leaves subjected to the action of gibberellic acid. The proteins were separated electrophoretically on polyacrylamide gel. Fourteen fractions of ribosomal protein were obtained exhibiting wide electrophoretic differences. Qualitative differences were found between the chloroplastic and cytoplasmic ribosomes. Gibberellic acid caused the appearance of an additional protein Traction in cytoplasmic ribosomes. It did not, however, affect the qualitative composition of ribosome proteins from chloroplasts.
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Bhattacharya, Arpita, Kerri B. McIntosh, Ian M. Willis y Jonathan R. Warner. "Why Dom34 Stimulates Growth of Cells with Defects of 40S Ribosomal Subunit Biosynthesis". Molecular and Cellular Biology 30, n.º 23 (27 de septiembre de 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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Coyle, Scott M., Wendy V. Gilbert y Jennifer A. Doudna. "Direct Link between RACK1 Function and Localization at the Ribosome In Vivo". Molecular and Cellular Biology 29, n.º 6 (29 de diciembre de 2008): 1626–34. http://dx.doi.org/10.1128/mcb.01718-08.
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ABSTRACT The receptor for activated C-kinase (RACK1), a conserved protein implicated in numerous signaling pathways, is a stoichiometric component of eukaryotic ribosomes located on the head of the 40S ribosomal subunit. To test the hypothesis that ribosome association is central to the function of RACK1 in vivo, we determined the 2.1-Å crystal structure of RACK1 from Saccharomyces cerevisiae (Asc1p) and used it to design eight mutant versions of RACK1 to assess roles in ribosome binding and in vivo function. Conserved charged amino acids on one side of the β-propeller structure were found to confer most of the 40S subunit binding affinity, whereas an adjacent conserved and structured loop had little effect on RACK1-ribosome association. Yeast mutations that confer moderate to strong defects in ribosome binding mimic some phenotypes of a RACK1 deletion strain, including increased sensitivity to drugs affecting cell wall biosynthesis and translation elongation. Furthermore, disruption of RACK1's position at the 40S ribosomal subunit results in the failure of the mRNA binding protein Scp160 to associate with actively translating ribosomes. These results provide the first direct evidence that RACK1 functions from the ribosome, implying a physical link between the eukaryotic ribosome and cell signaling pathways in vivo.
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Andreeva, Irena, Riccardo Belardinelli y Marina V. Rodnina. "Translation initiation in bacterial polysomes through ribosome loading on a standby site on a highly translated mRNA". Proceedings of the National Academy of Sciences 115, n.º 17 (9 de abril de 2018): 4411–16. http://dx.doi.org/10.1073/pnas.1718029115.
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During translation, consecutive ribosomes load on an mRNA and form a polysome. The first ribosome binds to a single-stranded mRNA region and moves toward the start codon, unwinding potential mRNA structures on the way. In contrast, the following ribosomes can dock at the start codon only when the first ribosome has vacated the initiation site. Here we show that loading of the second ribosome on a natural 38-nt-long 5′ untranslated region oflppmRNA, which codes for the outer membrane lipoprotein fromEscherichia coli, takes place before the leading ribosome has moved away from the start codon. The rapid formation of this standby complex depends on the presence of ribosomal proteins S1/S2 in the leading ribosome. The early recruitment of the second ribosome to the standby site before translation by the leading ribosome and the tight coupling between translation elongation by the first ribosome and the accommodation of the second ribosome can contribute to high translational efficiency of thelppmRNA.
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Campos, Rafael K., H. R. Sagara Wijeratne, Premal Shah, Mariano A. Garcia-Blanco y Shelton S. Bradrick. "Ribosomal stalk proteins RPLP1 and RPLP2 promote biogenesis of flaviviral and cellular multi-pass transmembrane proteins". Nucleic Acids Research 48, n.º 17 (5 de septiembre de 2020): 9872–85. http://dx.doi.org/10.1093/nar/gkaa717.
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Abstract The ribosomal stalk proteins, RPLP1 and RPLP2 (RPLP1/2), which form the ancient ribosomal stalk, were discovered decades ago but their functions remain mysterious. We had previously shown that RPLP1/2 are exquisitely required for replication of dengue virus (DENV) and other mosquito-borne flaviviruses. Here, we show that RPLP1/2 function to relieve ribosome pausing within the DENV envelope coding sequence, leading to enhanced protein stability. We evaluated viral and cellular translation in RPLP1/2-depleted cells using ribosome profiling and found that ribosomes pause in the sequence coding for the N-terminus of the envelope protein, immediately downstream of sequences encoding two adjacent transmembrane domains (TMDs). We also find that RPLP1/2 depletion impacts a ribosome density for a small subset of cellular mRNAs. Importantly, the polarity of ribosomes on mRNAs encoding multiple TMDs was disproportionately affected by RPLP1/2 knockdown, implying a role for RPLP1/2 in multi-pass transmembrane protein biogenesis. These analyses of viral and host RNAs converge to implicate RPLP1/2 as functionally important for ribosomes to elongate through ORFs encoding multiple TMDs. We suggest that the effect of RPLP1/2 at TMD associated pauses is mediated by improving the efficiency of co-translational folding and subsequent protein stability.
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Laughrea, Michael y Eric Higgins. "ΔLys120, a mutation which destabilizes the ribosome-binding domain of ribosomal protein L7/L12". Biochemistry and Cell Biology 68, n.º 5 (1 de mayo de 1990): 832–38. http://dx.doi.org/10.1139/o90-123.
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Five-residue-long deletions centered on A1a63, A1a75, and Glu118 of ribosomal protein L7/L12 gave low mutant yields (5% or less) when the mutant genes were cloned in phage M13mp18 and controlled by the L10 promotor. Deletions of Glu118–Lys120 or Lys120 (the COOH-terminus of L7/L12) gave higher mutant yields, up to 50% with L7/L12ΔLys120. L7/L12ΔLys120 was not preferentially found in the S100 and not preferentially removed by LiCl washing, but was preferentially extracted from 70S ribosomes in the presence of 28–35% ethanol in 0.25–0.5 M NH4Cl. It follows that ΔLys120 destabilizes the ribosome-binding domain of ribosomal protein L7/L12 in an ethanol-containing solvent, which raises the question whether Lys120 is part of the ribosome-binding domain of L7/L12 during some step of protein synthesis or whether it is essential to preserve the conformation of the physiological ribosome-binding domain under structurally stressful conditions.Key words: ribosome, ribosomal protein L7/L12, site-directed mutagenesis.
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Tamm, Tiina, Ivan Kisly y Jaanus Remme. "Functional Interactions of Ribosomal Intersubunit Bridges in Saccharomyces cerevisiae". Genetics 213, n.º 4 (24 de octubre de 2019): 1329–39. http://dx.doi.org/10.1534/genetics.119.302777.
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Ribosomes of Archaea and Eukarya share higher homology with each other than with bacterial ribosomes. For example, there is a set of 35 r-proteins that are specific only for archaeal and eukaryotic ribosomes. Three of these proteins—eL19, eL24, and eL41—participate in interactions between ribosomal subunits. The eukaryote-specific extensions of r-proteins eL19 and eL24 form two intersubunit bridges eB12 and eB13, which are present only in eukaryotic ribosomes. The third r-protein, eL41, forms bridge eB14. Notably, eL41 is found in all eukaryotes but only in some Archaea. It has been shown that bridges eB12 and eB13 are needed for efficient translation, while r-protein eL41 plays a minor role in ribosome function. Here, the functional interactions between intersubunit bridges were studied using budding yeast strains lacking different combinations of the abovementioned bridges/proteins. The growth phenotypes, levels of in vivo translation, ribosome–polysome profiles, and in vitro association of ribosomal subunits were analyzed. The results show a genetic interaction between r-protein eL41 and the eB12 bridge-forming region of eL19, and between r-proteins eL41 and eL24. It was possible to construct viable yeast strains with Archaea-like ribosomes lacking two or three eukaryote-specific bridges. These strains display slow growth and a poor translation phenotype. In addition, bridges eB12 and eB13 appear to cooperate during ribosome subunit association. These results indicate that nonessential structural elements of r-proteins become highly important in the context of disturbed subunit interactions. Therefore, eukaryote-specific bridges may contribute to the evolutionary success of eukaryotic translation machinery.
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Gaviraghi, Vivori y Tonon. "How Cancer Exploits Ribosomal RNA Biogenesis: A Journey beyond the Boundaries of rRNA Transcription". Cells 8, n.º 9 (17 de septiembre de 2019): 1098. http://dx.doi.org/10.3390/cells8091098.
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The generation of new ribosomes is a coordinated process essential to sustain cell growth. As such, it is tightly regulated according to cell needs. As cancer cells require intense protein translation to ensure their enhanced growth rate, they exploit various mechanisms to boost ribosome biogenesis. In this review, we will summarize how oncogenes and tumor suppressors modulate the biosynthesis of the RNA component of ribosomes, starting from the description of well-characterized pathways that converge on ribosomal RNA transcription while including novel insights that reveal unexpected regulatory networks hacked by cancer cells to unleash ribosome production.
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Bayas, Camille A., Jiarui Wang, Marissa K. Lee, Jared M. Schrader, Lucy Shapiro y W. E. Moerner. "Spatial organization and dynamics of RNase E and ribosomes inCaulobacter crescentus". Proceedings of the National Academy of Sciences 115, n.º 16 (2 de abril de 2018): E3712—E3721. http://dx.doi.org/10.1073/pnas.1721648115.
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We report the dynamic spatial organization ofCaulobacter crescentusRNase E (RNA degradosome) and ribosomal protein L1 (ribosome) using 3D single-particle tracking and superresolution microscopy. RNase E formed clusters along the central axis of the cell, while weak clusters of ribosomal protein L1 were deployed throughout the cytoplasm. These results contrast with RNase E and ribosome distribution inEscherichia coli, where RNase E colocalizes with the cytoplasmic membrane and ribosomes accumulate in polar nucleoid-free zones. For both RNase E and ribosomes inCaulobacter, we observed a decrease in confinement and clustering upon transcription inhibition and subsequent depletion of nascent RNA, suggesting that RNA substrate availability for processing, degradation, and translation facilitates confinement and clustering. Importantly, RNase E cluster positions correlated with the subcellular location of chromosomal loci of two highly transcribed rRNA genes, suggesting that RNase E’s function in rRNA processing occurs at the site of rRNA synthesis. Thus, components of the RNA degradosome and ribosome assembly are spatiotemporally organized inCaulobacter, with chromosomal readout serving as the template for this organization.
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Albanèse, Véronique, Stefanie Reissmann y Judith Frydman. "A ribosome-anchored chaperone network that facilitates eukaryotic ribosome biogenesis". Journal of Cell Biology 189, n.º 1 (5 de abril de 2010): 69–81. http://dx.doi.org/10.1083/jcb.201001054.
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Molecular chaperones assist cellular protein folding as well as oligomeric complex assembly. In eukaryotic cells, several chaperones termed chaperones linked to protein synthesis (CLIPS) are transcriptionally and physically linked to ribosomes and are implicated in protein biosynthesis. In this study, we show that a CLIPS network comprising two ribosome-anchored J-proteins, Jjj1 and Zuo1, function together with their partner Hsp70 proteins to mediate the biogenesis of ribosomes themselves. Jjj1 and Zuo1 have overlapping but distinct functions in this complex process involving the coordinated assembly and remodeling of dozens of proteins on the ribosomal RNA (rRNA). Both Jjj1 and Zuo1 associate with nuclear 60S ribosomal biogenesis intermediates and play an important role in nuclear rRNA processing, leading to mature 25S rRNA. In addition, Zuo1, acting together with its Hsp70 partner, SSB (stress 70 B), also participates in maturation of the 35S rRNA. Our results demonstrate that, in addition to their known cytoplasmic roles in de novo protein folding, some ribosome-anchored CLIPS chaperones play a critical role in nuclear steps of ribosome biogenesis.
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Baßler, Jochen y Ed Hurt. "Eukaryotic Ribosome Assembly". Annual Review of Biochemistry 88, n.º 1 (20 de junio de 2019): 281–306. http://dx.doi.org/10.1146/annurev-biochem-013118-110817.
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Ribosomes, which synthesize the proteins of a cell, comprise ribosomal RNA and ribosomal proteins, which coassemble hierarchically during a process termed ribosome biogenesis. Historically, biochemical and molecular biology approaches have revealed how preribosomal particles form and mature in consecutive steps, starting in the nucleolus and terminating after nuclear export into the cytoplasm. However, only recently, due to the revolution in cryo–electron microscopy, could pseudoatomic structures of different preribosomal particles be obtained. Together with in vitro maturation assays, these findings shed light on how nascent ribosomes progress stepwise along a dynamic biogenesis pathway. Preribosomes assemble gradually, chaperoned by a myriad of assembly factors and small nucleolar RNAs, before they reach maturity and enter translation. This information will lead to a better understanding of how ribosome synthesis is linked to other cellular pathways in humans and how it can cause diseases, including cancer, if disturbed.
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Bonincontro, Adalberto, Alessio De Francesco y Gianfranco Risuleo. "Dielectric Properties of Ribosomal Core Particles Lacking a Select Population of Proteins". Zeitschrift für Naturforschung C 54, n.º 7-8 (1 de agosto de 1999): 569–72. http://dx.doi.org/10.1515/znc-1999-7-817.
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Abstract In this communication we present a comparative investigation of the dielectric properties of native E. coli 70S and ribosomal cores obtained by LiCl treatment. Previous data obtained in our laboratory showed that ribosomes exhibit two different dielectric dispersions. We show that elimination of some select proteins modifies only the first one and therefore the overall dielectric properties of the ribosome result altered. Ribosomal RNA and proteins remaining in the core particle are mainly responsible for the second dielectric dispersion. Our experimental approach allows an estimation of the size of RNA traits exposed to solvent both in native ribosomes and in core particles where a higher portion of rRNA interacts with the external environment. Furthermore our results are consistent with the idea that proteins remaining after high salt treatment are necessary and sufficient for the maintenance of the basic structural properties of the ribosome.
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Slimane, Sophie Nait, Virginie Marcel, Tanguy Fenouil, Frédéric Catez, Jean-Christophe Saurin, Philippe Bouvet, Jean-Jacques Diaz y Hichem C. Mertani. "Ribosome Biogenesis Alterations in Colorectal Cancer". Cells 9, n.º 11 (27 de octubre de 2020): 2361. http://dx.doi.org/10.3390/cells9112361.
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Many studies have focused on understanding the regulation and functions of aberrant protein synthesis in colorectal cancer (CRC), leaving the ribosome, its main effector, relatively underappreciated in CRC. The production of functional ribosomes is initiated in the nucleolus, requires coordinated ribosomal RNA (rRNA) processing and ribosomal protein (RP) assembly, and is frequently hyperactivated to support the needs in protein synthesis essential to withstand unremitting cancer cell growth. This elevated ribosome production in cancer cells includes a strong alteration of ribosome biogenesis homeostasis that represents one of the hallmarks of cancer cells. None of the ribosome production steps escape this cancer-specific dysregulation. This review summarizes the early and late steps of ribosome biogenesis dysregulations described in CRC cell lines, intestinal organoids, CRC stem cells and mouse models, and their possible clinical implications. We highlight how this cancer-related ribosome biogenesis, both at quantitative and qualitative levels, can lead to the synthesis of ribosomes favoring the translation of mRNAs encoding hyperproliferative and survival factors. We also discuss whether cancer-related ribosome biogenesis is a mere consequence of cancer progression or is a causal factor in CRC, and how altered ribosome biogenesis pathways can represent effective targets to kill CRC cells. The association between exacerbated CRC cell growth and alteration of specific steps of ribosome biogenesis is highlighted as a key driver of tumorigenesis, providing promising perspectives for the implementation of predictive biomarkers and the development of new therapeutic drugs.
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Bogdanov, Alexey A., Olga A. Dontsova, Svetlana S. Dokudovskaya y Inna N. Lavrik. "Structure and function of 5S rRNA in the ribosome". Biochemistry and Cell Biology 73, n.º 11-12 (1 de diciembre de 1995): 869–76. http://dx.doi.org/10.1139/o95-094.
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5S rRNA is a small RNA molecule that is a component of a ribosome from almost all living organisms. In this review, we discuss the biogenesis of 5S rRNA and its properties as an independent structural domain of a ribosome as well as the current concepts concerning the higher order structure of 5S rRNA in free state and in its complexes with ribosomal proteins and its folding in the ribosome. Special attention is paid to recent experimental approaches that have been useful in 5S rRNA studies. Our own data on topography of 5S rRNA in the ribosomes are discussed in detail. The hypothesis describing the possible functional role of 5S rRNA for ribosome functioning is discussed.Key words: 5S rRNA, ribosomes, 23S rRNA, site-directed chemical cross-linking, RNA folding.
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Amirbeigiarab, Susan, Parnian Kiani, Ana Velazquez Sanchez, Christoph Krisp, Andriy Kazantsev, Lars Fester, Hartmut Schlüter y Zoya Ignatova. "Invariable stoichiometry of ribosomal proteins in mouse brain tissues with aging". Proceedings of the National Academy of Sciences 116, n.º 45 (21 de octubre de 2019): 22567–72. http://dx.doi.org/10.1073/pnas.1912060116.
Texto completoResumen
Across phyla, the ribosomes—the central molecular machines for translation of genetic information—exhibit an overall preserved architecture and a conserved functional core. The natural heterogeneity of the ribosome periodically phases a debate on their functional specialization and the tissue-specific variations of the ribosomal protein (RP) pool. Using sensitive differential proteomics, we performed a thorough quantitative inventory of the protein composition of ribosomes from 3 different mouse brain tissues, i.e., hippocampus, cortex, and cerebellum, across various ages, i.e., juvenile, adult, and middle-aged mouse groups. In all 3 brain tissues, in both monosomal and polysomal ribosome fractions, we detected an invariant set of 72 of 79 core RPs, RACK1 and 2 of the 8 RP paralogs, the stoichiometry of which remained constant across different ages. The amount of a few RPs punctually varied in either one tissue or one age group, but these fluctuations were within the tight bounds of the measurement noise. Further comparison with the ribosomes from a high-metabolic-rate organ, e.g., the liver, revealed protein composition identical to that of the ribosomes from the 3 brain tissues. Together, our data show an invariant protein composition of ribosomes from 4 tissues across different ages of mice and support the idea that functional heterogeneity may arise from factors other than simply ribosomal protein stoichiometry.
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McMullin, T. W. y R. L. Hallberg. "Effect of heat shock on ribosome structure: appearance of a new ribosome-associated protein." Molecular and Cellular Biology 6, n.º 7 (julio de 1986): 2527–35. http://dx.doi.org/10.1128/mcb.6.7.2527.
Texto completoResumen
After a nonlethal but heat shock protein-inducing hyperthermic treatment, ribosomes isolated from Tetrahymena thermophila contained an additional 22-kilodalton protein (p22). When maximally ribosome associated, this protein was found to be on the small subunit in a 1:1 stoichiometric ratio with other ribosomal proteins. Using an antiserum directed against the purified 22-kilodalton protein, we found that non-heat-shocked and heat-shocked cells contain identical amounts of this protein, the only difference being that in the stressed cells p22 is entirely ribosome bound, whereas in the unstressed cells p22 has little or no detectable ribosome association. Because the two-dimensional electrophoretic properties of p22 showed no alterations after heat shock, this change in state of ribosome-p22 interaction does not appear to be caused by a chemical modification of p22. When not strongly ribosome associated, p22 is not found free in the cytoplasm. During that time in heat shock when p22 is first becoming ribosome associated, it is found preferentially on polysomal ribosomes. Subsequently, all ribosomes, whether polysome bound or not, obtain a bound p22. The functional significance of this association is discussed.