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Sample records for 70-kda ribosomal protein

  1. A DOUBLE KNOCKOUT; A NOVEL APPROACH TO UNDERSTANDING STRESS-INDUCIBLE 70 KDA HEAT SHOCK PROTEINS (HSP70S) ON DEVELOPMENT AND REPRODUCTION

    EPA Science Inventory

    Heat and chemical toxicants which disrupt spermatogenesis and cause male infertility are thought to induce the expression of Hsp70-1 and 70-3, the major inducible heat shock proteins of the 70kDa family. Previous studies from several laboratories including our own have characteri...

  2. BALANCED PRODUCTION OF RIBOSOMAL PROTEINS

    PubMed Central

    Perry, Robert P.

    2017-01-01

    Eukaryotic ribosomes contain one molecule each of 79 different proteins. The genes encoding these proteins are usually at widely scattered loci and have distinctive promoters with certain common features. This minireview discusses the means by which cells manage to balance the production of ribosomal proteins so as to end up with equimolar quantities in the ribosome. Regulation at all levels of gene expression, from transcription to protein turnover, is considered. PMID:17689889

  3. Paradigms of ribosome synthesis: Lessons learned from ribosomal proteins

    PubMed Central

    Gamalinda, Michael; Woolford, John L

    2015-01-01

    The proteome in all cells is manufactured via the intricate process of translation by multimolecular factories called ribosomes. Nevertheless, these ribonucleoprotein particles, the largest of their kind, also have an elaborate assembly line of their own. Groundbreaking discoveries that bacterial ribosomal subunits can be self-assembled in vitro jumpstarted studies on how ribosomes are constructed. Until recently, ribosome assembly has been investigated almost entirely in vitro with bacterial small subunits under equilibrium conditions. In light of high-resolution ribosome structures and a more sophisticated toolkit, the past decade has been defined by a burst of kinetic studies in vitro and, importantly, also a shift to examining ribosome maturation in living cells, especially in eukaryotes. In this review, we summarize the principles governing ribosome assembly that emerged from studies focusing on ribosomal proteins and their interactions with rRNA. Understanding these paradigms has taken center stage, given the linkage between anomalous ribosome biogenesis and proliferative disorders. PMID:26779413

  4. Chloroplast ribosomes and protein synthesis.

    PubMed Central

    Harris, E H; Boynton, J E; Gillham, N W

    1994-01-01

    Consistent with their postulated origin from endosymbiotic cyanobacteria, chloroplasts of plants and algae have ribosomes whose component RNAs and proteins are strikingly similar to those of eubacteria. Comparison of the secondary structures of 16S rRNAs of chloroplasts and bacteria has been particularly useful in identifying highly conserved regions likely to have essential functions. Comparative analysis of ribosomal protein sequences may likewise prove valuable in determining their roles in protein synthesis. This review is concerned primarily with the RNAs and proteins that constitute the chloroplast ribosome, the genes that encode these components, and their expression. It begins with an overview of chloroplast genome structure in land plants and algae and then presents a brief comparison of chloroplast and prokaryotic protein-synthesizing systems and a more detailed analysis of chloroplast rRNAs and ribosomal proteins. A description of the synthesis and assembly of chloroplast ribosomes follows. The review concludes with discussion of whether chloroplast protein synthesis is essential for cell survival. PMID:7854253

  5. Neuron-Like Networks Between Ribosomal Proteins Within the Ribosome

    NASA Astrophysics Data System (ADS)

    Poirot, Olivier; Timsit, Youri

    2016-05-01

    From brain to the World Wide Web, information-processing networks share common scale invariant properties. Here, we reveal the existence of neural-like networks at a molecular scale within the ribosome. We show that with their extensions, ribosomal proteins form complex assortative interaction networks through which they communicate through tiny interfaces. The analysis of the crystal structures of 50S eubacterial particles reveals that most of these interfaces involve key phylogenetically conserved residues. The systematic observation of interactions between basic and aromatic amino acids at the interfaces and along the extension provides new structural insights that may contribute to decipher the molecular mechanisms of signal transmission within or between the ribosomal proteins. Similar to neurons interacting through “molecular synapses”, ribosomal proteins form a network that suggest an analogy with a simple molecular brain in which the “sensory-proteins” innervate the functional ribosomal sites, while the “inter-proteins” interconnect them into circuits suitable to process the information flow that circulates during protein synthesis. It is likely that these circuits have evolved to coordinate both the complex macromolecular motions and the binding of the multiple factors during translation. This opens new perspectives on nanoscale information transfer and processing.

  6. Import of ribosomal proteins into yeast mitochondria.

    PubMed

    Woellhaf, Michael W; Hansen, Katja G; Garth, Christoph; Herrmann, Johannes M

    2014-12-01

    Mitochondrial ribosomes of baker's yeast contain at least 78 protein subunits. All but one of these proteins are nuclear-encoded, synthesized on cytosolic ribosomes, and imported into the matrix for biogenesis. The import of matrix proteins typically relies on N-terminal mitochondrial targeting sequences that form positively charged amphipathic helices. Interestingly, the N-terminal regions of many ribosomal proteins do not closely match the characteristics of matrix targeting sequences, suggesting that the import processes of these proteins might deviate to some extent from the general import route. So far, the biogenesis of only two ribosomal proteins, Mrpl32 and Mrp10, was studied experimentally and indeed showed surprising differences to the import of other preproteins. In this review article we summarize the current knowledge on the transport of proteins into the mitochondrial matrix, and thereby specifically focus on proteins of the mitochondrial ribosome.

  7. Ribosome Inactivating Proteins from Rosaceae.

    PubMed

    Shang, Chenjing; Rougé, Pierre; Van Damme, Els J M

    2016-08-22

    Ribosome-inactivating proteins (RIPs) are widespread among higher plants of different taxonomic orders. In this study, we report on the RIP sequences found in the genome/transcriptome of several important Rosaceae species, including many economically important edible fruits such as apple, pear, peach, apricot, and strawberry. All RIP domains from Rosaceae share high sequence similarity with conserved residues in the catalytic site and the carbohydrate binding sites. The genomes of Malus domestica and Pyrus communis contain both type 1 and type 2 RIP sequences, whereas for Prunus mume, Prunus persica, Pyrus bretschneideri, and Pyrus communis a complex set of type 1 RIP sequences was retrieved. Heterologous expression and purification of the type 1 as well as the type 2 RIP from apple allowed to characterize the biological activity of the proteins. Both RIPs from Malus domestica can inhibit protein synthesis. Furthermore, molecular modelling suggests that RIPs from Rosaceae possess three-dimensional structures that are highly similar to the model proteins and can bind to RIP substrates. Screening of the recombinant type 2 RIP from apple on a glycan array revealed that this type 2 RIP interacts with terminal sialic acid residues. Our data suggest that the RIPs from Rosaceae are biologically active proteins.

  8. The Ribosome Modulates Nascent Protein Folding

    PubMed Central

    Kaiser, Christian M.; Goldman, Daniel H.; Chodera, John D.; Tinoco, Ignacio; Bustamante, Carlos

    2014-01-01

    Proteins are synthesized by the ribosome and generally must fold to become functionally active. Although it is commonly assumed that the ribosome affects the folding process, this idea has been extremely difficult to demonstrate. We have developed an experimental system to investigate the folding of single ribosome-bound stalled nascent polypeptides with optical tweezers. In T4 lysozyme, synthesized in a reconstituted in vitro translation system, the ribosome slows the formation of stable tertiary interactions and the attainment of the native state relative to the free protein. Incomplete T4 lysozyme polypeptides misfold and aggregate when free in solution, but they remain folding-competent near the ribosomal surface. Altogether, our results suggest that the ribosome not only decodes the genetic information and synthesizes polypeptides, but also promotes efficient de novo attainment of the native state. PMID:22194581

  9. Ribosome-associated protein quality control

    PubMed Central

    Brandman, Onn; Hegde, Ramanujan S

    2016-01-01

    Protein synthesis by the ribosome can fail for numerous reasons including faulty mRNA, insufficient availability of charged tRNAs and genetic errors. All organisms have evolved mechanisms to recognize stalled ribosomes and initiate pathways for recycling, quality control and stress signaling. Here we review the discovery and molecular dissection of the eukaryotic ribosome-associated quality-control pathway for degradation of nascent polypeptides arising from interrupted translation. PMID:26733220

  10. Differential Stoichiometry among Core Ribosomal Proteins

    PubMed Central

    Slavov, Nikolai; Semrau, Stefan; Airoldi, Edoardo; Budnik, Bogdan; van Oudenaarden, Alexander

    2015-01-01

    Summary Understanding the regulation and structure of ribosomes is essential to understanding protein synthesis and its dysregulation in disease. While ribosomes are believed to have a fixed stoichiometry among their core ribosomal proteins (RPs), some experiments suggest a more variable composition. Testing such variability requires direct and precise quantification of RPs. We used mass spectrometry to directly quantify RPs across monosomes and polysomes of mouse embryonic stem cells (ESC) and budding yeast. Our data show that the stoichiometry among core RPs in wild-type yeast cells and ESC depends both on the growth conditions and on the number of ribosomes bound per mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes. Together, our findings support the existence of ribosomes with distinct protein composition and physiological function. PMID:26565899

  11. The other lives of ribosomal proteins

    PubMed Central

    2010-01-01

    Despite the fact that ribosomal proteins are the constituents of an organelle that is present in every cell, they show a surprising level of regulation, and several of them have also been shown to have other extra-ribosomal functions, such in replication, transcription, splicing or even ageing. This review provides a comprehensive summary of these important aspects. PMID:20650820

  12. A new system for naming ribosomal proteins.

    PubMed

    Ban, Nenad; Beckmann, Roland; Cate, Jamie H D; Dinman, Jonathan D; Dragon, François; Ellis, Steven R; Lafontaine, Denis L J; Lindahl, Lasse; Liljas, Anders; Lipton, Jeffrey M; McAlear, Michael A; Moore, Peter B; Noller, Harry F; Ortega, Joaquin; Panse, Vikram Govind; Ramakrishnan, V; Spahn, Christian M T; Steitz, Thomas A; Tchorzewski, Marek; Tollervey, David; Warren, Alan J; Williamson, James R; Wilson, Daniel; Yonath, Ada; Yusupov, Marat

    2014-02-01

    A system for naming ribosomal proteins is described that the authors intend to use in the future. They urge others to adopt it. The objective is to eliminate the confusion caused by the assignment of identical names to ribosomal proteins from different species that are unrelated in structure and function. In the system proposed here, homologous ribosomal proteins are assigned the same name, regardless of species. It is designed so that new names are similar enough to old names to be easily recognized, but are written in a format that unambiguously identifies them as 'new system' names.

  13. Protein synthesis by ribosomes with tethered subunits.

    PubMed

    Orelle, Cédric; Carlson, Erik D; Szal, Teresa; Florin, Tanja; Jewett, Michael C; Mankin, Alexander S

    2015-08-06

    The ribosome is a ribonucleoprotein machine responsible for protein synthesis. In all kingdoms of life it is composed of two subunits, each built on its own ribosomal RNA (rRNA) scaffold. The independent but coordinated functions of the subunits, including their ability to associate at initiation, rotate during elongation, and dissociate after protein release, are an established model of protein synthesis. Furthermore, the bipartite nature of the ribosome is presumed to be essential for biogenesis, since dedicated assembly factors keep immature ribosomal subunits apart and prevent them from translation initiation. Free exchange of the subunits limits the development of specialized orthogonal genetic systems that could be evolved for novel functions without interfering with native translation. Here we show that ribosomes with tethered and thus inseparable subunits (termed Ribo-T) are capable of successfully carrying out protein synthesis. By engineering a hybrid rRNA composed of both small and large subunit rRNA sequences, we produced a functional ribosome in which the subunits are covalently linked into a single entity by short RNA linkers. Notably, Ribo-T was not only functional in vitro, but was also able to support the growth of Escherichia coli cells even in the absence of wild-type ribosomes. We used Ribo-T to create the first fully orthogonal ribosome-messenger RNA system, and demonstrate its evolvability by selecting otherwise dominantly lethal rRNA mutations in the peptidyl transferase centre that facilitate the translation of a problematic protein sequence. Ribo-T can be used for exploring poorly understood functions of the ribosome, enabling orthogonal genetic systems, and engineering ribosomes with new functions.

  14. Regulation of ribosomal protein synthesis in an Escherichia coli mutant missing ribosomal protein L1.

    PubMed Central

    Jinks-Robertson, S; Nomura, M

    1981-01-01

    In an Escherichia coli B strain missing ribosomal protein L1, the synthesis rate of L11 is 50% greater than that of other ribosomal proteins. This finding is in agreement with the previous conclusion that L1 regulates synthesis of itself and L11 and indicates that this regulation is important for maintaining the balanced synthesis of ribosomal proteins under physiological conditions. PMID:7009590

  15. Reconstitution of functional eukaryotic ribosomes from Dictyostelium discoideum ribosomal proteins and RNA.

    PubMed

    Mangiarotti, G; Chiaberge, S

    1997-08-08

    40 and 60 S ribosomal subunits have been reconstituted in vitro from purified ribosomal RNA and ribosomal proteins of Dictyostelium discoideum. The functionality of the reconstituted ribosomes was demonstrated in in vitro mRNA-directed protein synthesis. The reassembly proceeded well with immature precursors of ribosomal RNA but poorly if at all with mature cytoplasmic RNA species. Reassembly also required a preparation of small nuclear RNA(s), acting as morphopoietic factor(s).

  16. Functional Importance of Mobile Ribosomal Proteins.

    PubMed

    Chang, Kai-Chun; Wen, Jin-Der; Yang, Lee-Wei

    2015-01-01

    Although the dynamic motions and peptidyl transferase activity seem to be embedded in the rRNAs, the ribosome contains more than 50 ribosomal proteins (r-proteins), whose functions remain largely elusive. Also, the precise forms of some of these r-proteins, as being part of the ribosome, are not structurally solved due to their high flexibility, which hinders the efforts in their functional elucidation. Owing to recent advances in cryo-electron microscopy, single-molecule techniques, and theoretical modeling, much has been learned about the dynamics of these r-proteins. Surprisingly, allosteric regulations have been found in between spatially separated components as distant as those in the opposite sides of the ribosome. Here, we focus on the functional roles and intricate regulations of the mobile L1 and L12 stalks and L9 and S1 proteins. Conformational flexibility also enables versatile functions for r-proteins beyond translation. The arrangement of r-proteins may be under evolutionary pressure that fine-tunes mass distributions for optimal structural dynamics and catalytic activity of the ribosome.

  17. Mitochondrial ribosomal proteins (MRPs) of yeast.

    PubMed Central

    Graack, H R; Wittmann-Liebold, B

    1998-01-01

    Mitochondrial ribosomal proteins (MRPs) are the counterparts in that organelle of the cytoplasmic ribosomal proteins in the host. Although the MRPs fulfil similar functions in protein biosynthesis, they are distinct in number, features and primary structures from the latter. Most progress in the eludication of the properties of individual MRPs, and in the characterization of the corresponding genes, has been made in baker's yeast (Saccharomyces cerevisiae). To date, 50 different MRPs have been determined, although biochemical data and mutational analysis propose a total number which is substantially higher. Surprisingly, only a minority of the MRPs that have been characterized show significant sequence similarities to known ribosomal proteins from other sources, thus limiting the deduction of their functions by simple comparison of amino acid sequences. Further, individual MRPs have been characterized functionally by mutational studies, and the regulation of expression of MRP genes has been described. The interaction of the mitochondrial ribosomes with transcription factors specific for individual mitochondrial mRNAs, and the communication between mitochondria and the nucleus for the co-ordinated expression of ribosomal constituents, are other aspects of current MRP research. Although the mitochondrial translational system is still far from being described completely, the yeast MRP system serves as a model for other organisms, including that of humans. PMID:9445368

  18. Interrelationships between yeast ribosomal protein assembly events and transient ribosome biogenesis factors interactions in early pre-ribosomes.

    PubMed

    Jakob, Steffen; Ohmayer, Uli; Neueder, Andreas; Hierlmeier, Thomas; Perez-Fernandez, Jorge; Hochmuth, Eduard; Deutzmann, Rainer; Griesenbeck, Joachim; Tschochner, Herbert; Milkereit, Philipp

    2012-01-01

    Early steps of eukaryotic ribosome biogenesis require a large set of ribosome biogenesis factors which transiently interact with nascent rRNA precursors (pre-rRNA). Most likely, concomitant with that initial contacts between ribosomal proteins (r-proteins) and ribosome precursors (pre-ribosomes) are established which are converted into robust interactions between pre-rRNA and r-proteins during the course of ribosome maturation. Here we analysed the interrelationship between r-protein assembly events and the transient interactions of ribosome biogenesis factors with early pre-ribosomal intermediates termed 90S pre-ribosomes or small ribosomal subunit (SSU) processome in yeast cells. We observed that components of the SSU processome UTP-A and UTP-B sub-modules were recruited to early pre-ribosomes independently of all tested r-proteins. On the other hand, groups of SSU processome components were identified whose association with early pre-ribosomes was affected by specific r-protein assembly events in the head-platform interface of the SSU. One of these components, Noc4p, appeared to be itself required for robust incorporation of r-proteins into the SSU head domain. Altogether, the data reveal an emerging network of specific interrelationships between local r-protein assembly events and the functional interactions of SSU processome components with early pre-ribosomes. They point towards some of these components being transient primary pre-rRNA in vivo binders and towards a role for others in coordinating the assembly of major SSU domains.

  19. Ribosomal Protein Methyltransferases in the Yeast Saccharomyces cerevisiae: Roles in Ribosome Biogenesis and Translation

    PubMed Central

    Al-Hadid, Qais; White, Jonelle; Clarke, Steven

    2016-01-01

    A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed −1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation. PMID:26801560

  20. Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation.

    PubMed

    Al-Hadid, Qais; White, Jonelle; Clarke, Steven

    2016-02-12

    A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation.

  1. Hold on to your friends: Dedicated chaperones of ribosomal proteins: Dedicated chaperones mediate the safe transfer of ribosomal proteins to their site of pre-ribosome incorporation.

    PubMed

    Pillet, Benjamin; Mitterer, Valentin; Kressler, Dieter; Pertschy, Brigitte

    2017-01-01

    Eukaryotic ribosomes are assembled from their components, the ribosomal RNAs and ribosomal proteins, in a tremendously complex, multi-step process, which primarily takes place in the nuclear compartment. Therefore, most ribosomal proteins have to travel from the cytoplasm to their incorporation site on pre-ribosomes within the nucleus. However, due to their particular characteristics, such as a highly basic amino acid composition and the presence of unstructured extensions, ribosomal proteins are especially prone to aggregation and degradation in their unassembled state, hence specific mechanisms must operate to ensure their safe delivery. Recent studies have uncovered a group of proteins, termed dedicated chaperones, specialized in accompanying and guarding individual ribosomal proteins. In this essay, we review how these dedicated chaperones utilize different folds to interact with their ribosomal protein clients and how they ensure their soluble expression and interconnect their intracellular transport with their efficient assembly into pre-ribosomes.

  2. Insights into the Mechanism of Ribosomal Incorporation of Mammalian L13a Protein during Ribosome Biogenesis

    PubMed Central

    Das, Priyanka; Basu, Abhijit; Biswas, Aditi; Poddar, Darshana; Andrews, Joel; Barik, Sailen; Komar, Anton A.

    2013-01-01

    In contrast to prokaryotes, the precise mechanism of incorporation of ribosomal proteins into ribosomes in eukaryotes is not well understood. For the majority of eukaryotic ribosomal proteins, residues critical for rRNA binding, a key step in the hierarchical assembly of ribosomes, have not been well defined. In this study, we used the mammalian ribosomal protein L13a as a model to investigate the mechanism(s) underlying eukaryotic ribosomal protein incorporation into ribosomes. This work identified the arginine residue at position 68 of L13a as being essential for L13a binding to rRNA and incorporation into ribosomes. We also demonstrated that incorporation of L13a takes place during maturation of the 90S preribosome in the nucleolus, but that translocation of L13a into the nucleolus is not sufficient for its incorporation into ribosomes. Incorporation of L13a into the 90S preribosome was required for rRNA methylation within the 90S complex. However, mutations abolishing ribosomal incorporation of L13a did not affect its ability to be phosphorylated or its extraribosomal function in GAIT element-mediated translational silencing. These results provide new insights into the mechanism of ribosomal incorporation of L13a and will be useful in guiding future studies aimed at fully deciphering mammalian ribosome biogenesis. PMID:23689135

  3. Identification and characterization of a Dictyostelium discoideum ribosomal protein gene.

    PubMed Central

    Szymkowski, D E; Deering, R A

    1990-01-01

    We have identified a developmentally repressed large-subunit ribosomal protein gene of Dictyostelium discoideum based on sequence similarity to other ribosomal proteins. Protein rpl7 is homologous to large subunit ribosomal proteins from the rat and possibly to Mycoplasma capricolum and Escherichia coli, but is not similar to three sequenced ribosomal proteins in Dictyostelium. The rpl7 gene is present at one copy per genome, as are six other cloned Dictyostelium ribosomal proteins. Restriction fragment length polymorphisms exist for ribosomal protein genes rpl7, rp1024, and rp110 in strain HU182; most Dictyostelium ribosomal protein genes examined are linked no closer than 30-100 kb to each other in the genome. Dictyostelium ribosomal proteins are known to be developmentally regulated, and levels of rpl7 transcript gradually decrease during the 24-hour development cycle. This drop correlates with that of rp1024, indicating these and other ribosomal protein genes may be coordinately regulated. To determine the cellular location of the protein, we raised antibodies to an rpl7-derived branched synthetic peptide. These antibodies cross-reacted with one protein of the expected size in a ribosomal protein fraction of Dictyostelium, indicating that the product of gene rpl7 is localized in the ribosome. Images PMID:1975664

  4. Potential extra-ribosomal functions of ribosomal proteins in Saccharomyces cerevisiae.

    PubMed

    Lu, Hui; Zhu, Yi-Fei; Xiong, Juan; Wang, Rong; Jia, Zhengping

    2015-08-01

    Ribosomal proteins (RPs), are essential components of the ribosomes, the molecular machines that turn mRNA blueprints into proteins, as they serve to stabilize the structure of the rRNA, thus improving protein biosynthesis. In addition, growing evidence suggests that RPs can function in other cellular roles. In the present review, we summarize several potential extra-ribosomal functions of RPs in ribosomal biogenesis, transcription activity, translation process, DNA repair, replicative life span, adhesive growth, and morphological transformation in Saccharomyces cerevisiae. However, the future in-depth studies are needed to identify these novel secondary functions of RPs in S. cerevisiae.

  5. Proteomic characterization of archaeal ribosomes reveals the presence of novel archaeal-specific ribosomal proteins.

    PubMed

    Márquez, Viter; Fröhlich, Thomas; Armache, Jean-Paul; Sohmen, Daniel; Dönhöfer, Alexandra; Mikolajka, Aleksandra; Berninghausen, Otto; Thomm, Michael; Beckmann, Roland; Arnold, Georg J; Wilson, Daniel N

    2011-02-04

    Protein synthesis occurs in macromolecular particles called ribosomes. All ribosomes are composed of RNA and proteins. While the protein composition of bacterial and eukaryotic ribosomes has been well-characterized, a systematic analysis of archaeal ribosomes has been lacking. Here we report the first comprehensive two-dimensional PAGE and mass spectrometry analysis of archaeal ribosomes isolated from the thermophilic Pyrobaculum aerophilum and the thermoacidophilic Sulfolobus acidocaldarius Crenarchaeota. Our analysis identified all 66 ribosomal proteins (r-proteins) of the P. aerophilum small and large subunits, as well as all but two (62 of 64; 97%) r-proteins of the S. acidocaldarius small and large subunits that are predicted genomically. Some r-proteins were identified with one or two lysine methylations and N-terminal acetylations. In addition, we identify three hypothetical proteins that appear to be bona fide r-proteins of the S. acidocaldarius large subunit. Dissociation of r-proteins from the S. acidocaldarius large subunit indicates that the novel r-proteins establish tighter interactions with the large subunit than some integral r-proteins. Furthermore, cryo electron microscopy reconstructions of the S. acidocaldarius and P. aerophilum 50S subunits allow for a tentative localization of the binding site of the novel r-proteins. This study illustrates not only the potential diversity of the archaeal ribosomes but also the necessity to experimentally analyze the archaeal ribosomes to ascertain their protein composition. The discovery of novel archaeal r-proteins and factors may be the first step to understanding how archaeal ribosomes cope with extreme environmental conditions.

  6. Sequestration of Ribosome during Protein Aggregate Formation: Contribution of ribosomal RNA

    PubMed Central

    Pathak, Bani K.; Mondal, Surojit; Banerjee, Senjuti; Ghosh, Amar Nath; Barat, Chandana

    2017-01-01

    An understanding of the mechanisms underlying protein aggregation and cytotoxicity of the protein aggregates is crucial in the prevention of several diseases in humans. Ribosome, the cellular protein synthesis machine is capable of acting as a protein folding modulator. The peptidyltransferase center residing in the domain V of large ribosomal subunit 23S rRNA is the centre for the protein folding ability of the ribosome and is also the cellular target of several antiprion compounds. Our in vitro studies unexpectedly reveal that the partial unfolding or aggregation of lysozyme under reducing conditions in presence of the ribosome can induce aggregation of ribosomal components. Electrostatic interactions complemented by specific rRNA-protein interaction drive the ribosome-protein aggregation process. Under similar conditions the rRNA, especially the large subunit rRNA and in vitro transcribed RNA corresponding to domain V of 23S rRNA (bDV RNA) stimulates lysozyme aggregation leading to RNA-protein aggregate formation. Protein aggregation during the refolding of non-disulfide containing protein BCAII at high concentrations also induces ribosome aggregation. BCAII aggregation was also stimulated in presence of the large subunit rRNA. Our observations imply that the specific sequestration of the translation machine by aggregating proteins might contribute to their cytotoxicity. PMID:28169307

  7. Convergent evolution led ribosome inactivating proteins to interact with ribosomal stalk.

    PubMed

    Lapadula, Walter J; Sanchez-Puerta, M Virginia; Ayub, Maximiliano Juri

    2012-03-01

    Ribosome-inactivating proteins (RIPs) inhibit protein synthesis by depurinating an adenine on the sarcin-ricin loop (SRL) of the large subunit ribosomal RNA. Several RIPs interact with the C-terminal end of ribosomal stalk P proteins, and this interaction is required for their full activity. In contrast, the activity of Pokeweed Antiviral Protein is not affected by blocking this stalk component. Here, we provide evidence from phylogenetic analyses and sequence alignments suggesting that the interaction with the C-terminal end of P proteins evolved independently in different RIPs by convergent evolution.

  8. Ribosomal History Reveals Origins of Modern Protein Synthesis

    PubMed Central

    Harish, Ajith; Caetano-Anollés, Gustavo

    2012-01-01

    The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17) and the oldest substructure (the ribosomal ratchet) in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world. PMID:22427882

  9. Ribosome-Inactivating and Related Proteins

    PubMed Central

    Schrot, Joachim; Weng, Alexander; Melzig, Matthias F.

    2015-01-01

    Ribosome-inactivating proteins (RIPs) are toxins that act as N-glycosidases (EC 3.2.2.22). They are mainly produced by plants and classified as type 1 RIPs and type 2 RIPs. There are also RIPs and RIP related proteins that cannot be grouped into the classical type 1 and type 2 RIPs because of their different sizes, structures or functions. In addition, there is still not a uniform nomenclature or classification existing for RIPs. In this review, we give the current status of all known plant RIPs and we make a suggestion about how to unify those RIPs and RIP related proteins that cannot be classified as type 1 or type 2 RIPs. PMID:26008228

  10. On the Ribosomal Density that Maximizes Protein Translation Rate

    PubMed Central

    Zarai, Yoram; Margaliot, Michael; Tuller, Tamir

    2016-01-01

    During mRNA translation, several ribosomes attach to the same mRNA molecule simultaneously translating it into a protein. This pipelining increases the protein translation rate. A natural and important question is what ribosomal density maximizes the protein translation rate. Using mathematical models of ribosome flow along both a linear and a circular mRNA molecules we prove that typically the steady-state protein translation rate is maximized when the ribosomal density is one half of the maximal possible density. We discuss the implications of our results to endogenous genes under natural cellular conditions and also to synthetic biology. PMID:27861564

  11. Functions of Ribosomal Proteins in Assembly of Eukaryotic Ribosomes In Vivo

    PubMed Central

    2016-01-01

    The proteome of cells is synthesized by ribosomes, complex ribonucleoproteins that in eukaryotes contain 79–80 proteins and four ribosomal RNAs (rRNAs) more than 5,400 nucleotides long. How these molecules assemble together and how their assembly is regulated in concert with the growth and proliferation of cells remain important unanswered questions. Here, we review recently emerging principles to understand how eukaryotic ribosomal proteins drive ribosome assembly in vivo. Most ribosomal proteins assemble with rRNA cotranscriptionally; their association with nascent particles is strengthened as assembly proceeds. Each subunit is assembled hierarchically by sequential stabilization of their subdomains. The active sites of both subunits are constructed last, perhaps to prevent premature engagement of immature ribosomes with active subunits. Late-assembly intermediates undergo quality-control checks for proper function. Mutations in ribosomal proteins that affect mostly late steps lead to ribosomopathies, diseases that include a spectrum of cell type–specific disorders that often transition from hypoproliferative to hyperproliferative growth. PMID:25706898

  12. Functions of ribosomal proteins in assembly of eukaryotic ribosomes in vivo.

    PubMed

    de la Cruz, Jesús; Karbstein, Katrin; Woolford, John L

    2015-01-01

    The proteome of cells is synthesized by ribosomes, complex ribonucleoproteins that in eukaryotes contain 79-80 proteins and four ribosomal RNAs (rRNAs) more than 5,400 nucleotides long. How these molecules assemble together and how their assembly is regulated in concert with the growth and proliferation of cells remain important unanswered questions. Here, we review recently emerging principles to understand how eukaryotic ribosomal proteins drive ribosome assembly in vivo. Most ribosomal proteins assemble with rRNA cotranscriptionally; their association with nascent particles is strengthened as assembly proceeds. Each subunit is assembled hierarchically by sequential stabilization of their subdomains. The active sites of both subunits are constructed last, perhaps to prevent premature engagement of immature ribosomes with active subunits. Late-assembly intermediates undergo quality-control checks for proper function. Mutations in ribosomal proteins that affect mostly late steps lead to ribosomopathies, diseases that include a spectrum of cell type-specific disorders that often transition from hypoproliferative to hyperproliferative growth.

  13. Role of ribosomal protein mutations in tumor development (Review)

    PubMed Central

    GOUDARZI, KAVEH M.; LINDSTRÖM, MIKAEL S.

    2016-01-01

    Ribosomes are cellular machines essential for protein synthesis. The biogenesis of ribosomes is a highly complex and energy consuming process that initiates in the nucleolus. Recently, a series of studies applying whole-exome or whole-genome sequencing techniques have led to the discovery of ribosomal protein gene mutations in different cancer types. Mutations in ribosomal protein genes have for example been found in endometrial cancer (RPL22), T-cell acute lymphoblastic leukemia (RPL10, RPL5 and RPL11), chronic lymphocytic leukemia (RPS15), colorectal cancer (RPS20), and glioma (RPL5). Moreover, patients suffering from Diamond-Blackfan anemia, a bone marrow failure syndrome caused by mutant ribosomal proteins are also at higher risk for developing leukemia, or solid tumors. Different experimental models indicate potential mechanisms whereby ribosomal proteins may initiate cancer development. In particular, deregulation of the p53 tumor suppressor network and altered mRNA translation are mechanisms likely to be involved. We envisage that changes in expression and the occurrence of ribosomal protein gene mutations play important roles in cancer development. Ribosome biology constitutes a re-emerging vital area of basic and translational cancer research. PMID:26892688

  14. A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

    PubMed Central

    Schütz, Sabina; Fischer, Ute; Altvater, Martin; Nerurkar, Purnima; Peña, Cohue; Gerber, Michaela; Chang, Yiming; Caesar, Stefanie; Schubert, Olga T; Schlenstedt, Gabriel; Panse, Vikram G

    2014-01-01

    Within a single generation time a growing yeast cell imports ∼14 million ribosomal proteins (r-proteins) into the nucleus for ribosome production. After import, it is unclear how these intrinsically unstable and aggregation-prone proteins are targeted to the ribosome assembly site in the nucleolus. Here, we report the discovery of a conserved nuclear carrier Tsr2 that coordinates transfer of the r-protein eS26 to the earliest assembling pre-ribosome, the 90S. In vitro studies revealed that Tsr2 efficiently dissociates importin:eS26 complexes via an atypical RanGTP-independent mechanism that terminates the import process. Subsequently, Tsr2 binds the released eS26, shields it from proteolysis, and ensures its safe delivery to the 90S pre-ribosome. We anticipate similar carriers—termed here escortins—to securely connect the nuclear import machinery with pathways that deposit r-proteins onto developing pre-ribosomal particles. DOI: http://dx.doi.org/10.7554/eLife.03473.001 PMID:25144938

  15. Ribosome recycling: An essential process of protein synthesis.

    PubMed

    Kiel, Michael C; Kaji, Hideko; Kaji, Akira

    2007-01-01

    A preponderance of textbooks outlines cellular protein synthesis (translation) in three basic steps: initiation, elongation, and termination. However, researchers in the field of translation accept that a vital fourth step exists; this fourth step is called ribosome recycling. Ribosome recycling occurs after the nascent polypeptide has been released during the termination step. Despite the release of the polypeptide, ribosomes remain bound to the mRNA and tRNA. It is only during the fourth step of translation that ribosomes are ultimately released from the mRNA, split into subunits, and are free to bind new mRNA, thus the term "ribosome recycling." This step is essential to the viability of cells. In bacteria, it is catalyzed by two proteins, elongation factor G and ribosome recycling factor, a near perfect structural mimic of tRNA. Eukaryotic organelles such as mitochondria and chloroplasts possess ribosome recycling factor and elongation factor G homologues, but the nature of ribosome recycling in eukaryotic cytoplasm is still under investigation. In this review, the discovery of ribosome recycling and the basic mechanisms involved are discussed so that textbook writers and teachers can include this vital step, which is just as important as the three conventional steps, in sections dealing with protein synthesis.

  16. Prefabrication of a ribosomal protein subcomplex essential for eukaryotic ribosome formation

    PubMed Central

    Peña, Cohue; Schütz, Sabina; Fischer, Ute; Chang, Yiming; Panse, Vikram G

    2016-01-01

    Spatial clustering of ribosomal proteins (r-proteins) through tertiary interactions is a striking structural feature of the eukaryotic ribosome. However, the functional importance of these intricate inter-connections, and how they are established is currently unclear. Here, we reveal that a conserved ATPase, Fap7, organizes interactions between neighboring r-proteins uS11 and eS26 prior to their delivery to the earliest ribosome precursor, the 90S. In vitro, uS11 only when bound to Fap7 becomes competent to recruit eS26 through tertiary contacts found between these r-proteins on the mature ribosome. Subsequently, Fap7 ATPase activity unloads the uS11:eS26 subcomplex onto its rRNA binding site, and therefore ensures stoichiometric integration of these r-proteins into the 90S. Fap7-depletion in vivo renders uS11 susceptible to proteolysis, and precludes eS26 incorporation into the 90S. Thus, prefabrication of a native-like r-protein subcomplex drives efficient and accurate construction of the eukaryotic ribosome. DOI: http://dx.doi.org/10.7554/eLife.21755.001 PMID:27929371

  17. Cotranslational Protein Folding inside the Ribosome Exit Tunnel

    PubMed Central

    Nilsson, Ola B.; Hedman, Rickard; Marino, Jacopo; Wickles, Stephan; Bischoff, Lukas; Johansson, Magnus; Müller-Lucks, Annika; Trovato, Fabio; Puglisi, Joseph D.; O’Brien, Edward P.; Beckmann, Roland; von Heijne, Gunnar

    2015-01-01

    Summary At what point during translation do proteins fold? It is well established that proteins can fold cotranslationally outside the ribosome exit tunnel, whereas studies of folding inside the exit tunnel have so far detected only the formation of helical secondary structure and collapsed or partially structured folding intermediates. Here, using a combination of cotranslational nascent chain force measurements, inter-subunit fluorescence resonance energy transfer studies on single translating ribosomes, molecular dynamics simulations, and cryoelectron microscopy, we show that a small zinc-finger domain protein can fold deep inside the vestibule of the ribosome exit tunnel. Thus, for small protein domains, the ribosome itself can provide the kind of sheltered folding environment that chaperones provide for larger proteins. PMID:26321634

  18. Ribosomal Protein S14 Unties the MDM2-p53 Loop Upon Ribosomal Stress

    PubMed Central

    Zhou, Xiang; Hao, Qian; Liao, Jun-ming; Zhang, Qi; Lu, Hua

    2013-01-01

    The MDM2-p53 feedback loop is crucially important for restricting p53 level and activity during normal cell growth and proliferation, and is thus subjected to dynamic regulation in order for cells to activate p53 upon various stress signals. Several ribosomal proteins, such as RPL11, RPL5, RPL23, RPL26, or RPS7, have been shown to play a role in regulation of this feedback loop in response to ribosomal stress. Here, we identify another ribosomal protein S14, which is highly associated with 5q-syndrome, as a novel activator of p53 by inhibiting MDM2 activity. We found that RPS14, but not RPS19, binds to the central acidic domain of MDM2, like RPL5 and RPL23, and inhibits its E3 ubiquitin ligase activity toward p53. This RPS14-MDM2 binding was induced upon ribosomal stress caused by actinomycin D or mycophenolic acid. Overexpression of RPS14, but not RPS19, elevated p53 level and activity, leading to G1 or G2 arrest. Conversely, knockdown of RPS14 alleviated p53 induction by these two reagents. Interestingly, knockdown of either RPS14 or RPS19 caused a ribosomal stress that led to p53 activation, which was impaired by further knocking down the level of RPL11 or RPL5. Together, our results demonstrate that RPS14 and RPS19 play distinct roles in regulating the MDM2-p53 feedback loop in response to ribosomal stress. PMID:22391559

  19. Disassembly of yeast 80S ribosomes into subunits is a concerted action of ribosome-assisted folding of denatured protein.

    PubMed

    Chakraborty, Biprashekhar; Bhakta, Sayan; Sengupta, Jayati

    2016-01-22

    It has been shown by several groups that ribosome can assist folding of denatured protein in vitro and the process is conserved across the species. Domain V of large ribosomal rRNA which occupies the intersubunit side of the large subunit was identified as the key player responsible for chaperoning the folding process. Thus, it is conceivable that denatured protein needs to access the intersubunit space of the ribosome in order to get folded. In this study, we have investigated the mechanism of release of the protein from the eukaryotic ribosome following reactivation. We have observed significant splitting of yeast 80S ribosome when incubated with the denatured BCAII protein. Energy-free disassembly mechanism functions in low Mg(+2) ion concentration for prokaryotic ribosomes. Eukaryotic ribosomes do not show significant splitting even at low Mg(+2) ion concentration. In this respect, denatured protein-induced disassembly of eukaryotic ribosome without the involvement of any external energy source is intriguing. For prokaryotic ribosomes, it was reported that the denatured protein induces ribosome splitting into subunits in order to access domain V-rRNA. In contrast, our results suggest an alternative mechanism for eukaryotic ribosomal rRNA-mediated protein folding and subsequent separation of the subunits by which release of the activated-protein occurs.

  20. Comprehensive Analysis of Phosphorylated Proteins of E. coli Ribosomes

    PubMed Central

    Soung, George Y.; Miller, Jennifer L.; Koc, Hasan; Koc, Emine C.

    2009-01-01

    Phosphorylation of bacterial ribosomal proteins has been known for decades; however, there is still very limited information available on specific locations of the phosphorylation sites in ribosomal proteins and the role they might play in protein synthesis. In this study, we have mapped the specific phosphorylation sites in twenty-four E. coli ribosomal proteins by tandem mass spectrometry. Specific detection of phosphorylation was achieved by either phosphorylation specific visualization techniques, ProQ staining and antibodies for phospho-Ser, Thr, and Tyr, or by mass spectrometry equipped with a capability to detect addition and the loss of the phosphate moiety. Enrichment by immobilized metal affinity and/or strong cation exchange chromatography was used to improve the success of detection of the low abundance phosphopeptides. We found the small subunit (30S) proteins S3, S4, S5, S7, S11, S12, S13, S18, and S21 and the large subunit (50S) proteins L1, L2, L3, L5, L6, L7/L12, L13, L14, L16, L18, L19, L21, L22, L28, L31 to be phosphorylated at one or more residues. Potential roles for each specific site in ribosome function were deduced through careful evaluation of the given site of the phosphorylation in 3D-crystal structure models of ribosomes and the previous mutational studies of E. coli ribosomal proteins. PMID:19469554

  1. Structures of eukaryotic ribosomal stalk proteins and its complex with trichosanthin, and their implications in recruiting ribosome-inactivating proteins to the ribosomes.

    PubMed

    Choi, Andrew K H; Wong, Eddie C K; Lee, Ka-Ming; Wong, Kam-Bo

    2015-02-25

    Ribosome-inactivating proteins (RIP) are RNA N-glycosidases that inactivate ribosomes by specifically depurinating a conserved adenine residue at the α-sarcin/ricin loop of 28S rRNA. Recent studies have pointed to the involvement of the C-terminal domain of the eukaryotic stalk proteins in facilitating the toxic action of RIPs. This review highlights how structural studies of eukaryotic stalk proteins provide insights into the recruitment of RIPs to the ribosomes. Since the C-terminal domain of eukaryotic stalk proteins is involved in specific recognition of elongation factors and some eukaryote-specific RIPs (e.g., trichosanthin and ricin), we postulate that these RIPs may have evolved to hijack the translation-factor-recruiting function of ribosomal stalk in reaching their target site of rRNA.

  2. Cryo-EM structure of the spinach chloroplast ribosome reveals the location of plastid-specific ribosomal proteins and extensions.

    PubMed

    Graf, Michael; Arenz, Stefan; Huter, Paul; Dönhöfer, Alexandra; Nováček, Jiří; Wilson, Daniel N

    2016-12-15

    Ribosomes are the protein synthesizing machines of the cell. Recent advances in cryo-EM have led to the determination of structures from a variety of species, including bacterial 70S and eukaryotic 80S ribosomes as well as mitoribosomes from eukaryotic mitochondria, however, to date high resolution structures of plastid 70S ribosomes have been lacking. Here we present a cryo-EM structure of the spinach chloroplast 70S ribosome, with an average resolution of 5.4 Å for the small 30S subunit and 3.6 Å for the large 50S ribosomal subunit. The structure reveals the location of the plastid-specific ribosomal proteins (RPs) PSRP1, PSRP4, PSRP5 and PSRP6 as well as the numerous plastid-specific extensions of the RPs. We discover many features by which the plastid-specific extensions stabilize the ribosome via establishing additional interactions with surrounding ribosomal RNA and RPs. Moreover, we identify a large conglomerate of plastid-specific protein mass adjacent to the tunnel exit site that could facilitate interaction of the chloroplast ribosome with the thylakoid membrane and the protein-targeting machinery. Comparing the Escherichia coli 70S ribosome with that of the spinach chloroplast ribosome provides detailed insight into the co-evolution of RP and rRNA.

  3. ABC-F Proteins Mediate Antibiotic Resistance through Ribosomal Protection

    PubMed Central

    Sharkey, Liam K. R.; Edwards, Thomas A.

    2016-01-01

    ABSTRACT Members of the ABC-F subfamily of ATP-binding cassette proteins mediate resistance to a broad array of clinically important antibiotic classes that target the ribosome of Gram-positive pathogens. The mechanism by which these proteins act has been a subject of long-standing controversy, with two competing hypotheses each having gained considerable support: antibiotic efflux versus ribosomal protection. Here, we report on studies employing a combination of bacteriological and biochemical techniques to unravel the mechanism of resistance of these proteins, and provide several lines of evidence that together offer clear support to the ribosomal protection hypothesis. Of particular note, we show that addition of purified ABC-F proteins to an in vitro translation assay prompts dose-dependent rescue of translation, and demonstrate that such proteins are capable of displacing antibiotic from the ribosome in vitro. To our knowledge, these experiments constitute the first direct evidence that ABC-F proteins mediate antibiotic resistance through ribosomal protection. PMID:27006457

  4. The Ribosomal Protein Rpl22 Controls Ribosome Composition by Directly Repressing Expression of Its Own Paralog, Rpl22l1

    PubMed Central

    O'Leary, Monique N.; Schreiber, Katherine H.; Zhang, Yong; Duc, Anne-Cécile E.; Rao, Shuyun; Hale, J. Scott; Academia, Emmeline C.; Shah, Shreya R.; Morton, John F.; Holstein, Carly A.; Martin, Dan B.; Kaeberlein, Matt; Ladiges, Warren C.; Fink, Pamela J.; MacKay, Vivian L.; Wiest, David L.; Kennedy, Brian K.

    2013-01-01

    Most yeast ribosomal protein genes are duplicated and their characterization has led to hypotheses regarding the existence of specialized ribosomes with different subunit composition or specifically-tailored functions. In yeast, ribosomal protein genes are generally duplicated and evidence has emerged that paralogs might have specific roles. Unlike yeast, most mammalian ribosomal proteins are thought to be encoded by a single gene copy, raising the possibility that heterogenous populations of ribosomes are unique to yeast. Here, we examine the roles of the mammalian Rpl22, finding that Rpl22−/− mice have only subtle phenotypes with no significant translation defects. We find that in the Rpl22−/− mouse there is a compensatory increase in Rpl22-like1 (Rpl22l1) expression and incorporation into ribosomes. Consistent with the hypothesis that either ribosomal protein can support translation, knockdown of Rpl22l1 impairs growth of cells lacking Rpl22. Mechanistically, Rpl22 regulates Rpl22l1 directly by binding to an internal hairpin structure and repressing its expression. We propose that ribosome specificity may exist in mammals, providing evidence that one ribosomal protein can influence composition of the ribosome by regulating its own paralog. PMID:23990801

  5. Ribosome Dwell Times and the Protein Copy Number Distribution

    NASA Astrophysics Data System (ADS)

    Gorissen, Mieke; Vanderzande, Carlo

    2012-09-01

    Translation is the cellular process in which ribosomes make proteins from information encoded on messenger RNA (mRNA). We model translation with an exclusion process taking into account the experimentally determined, non-exponential, waiting time between steps of a ribosome. From numerical simulations using realistic parameter values, we determine the distribution P( E) of the number of proteins E produced by one mRNA. We find that for small E this distribution is not geometric. We present a simplified and analytically solvable model that relates P( E) to the distributions of the times to produce the first E proteins.

  6. Ribosomal Proteins Control or Bypass p53 during Nucleolar Stress

    PubMed Central

    Russo, Annapina; Russo, Giulia

    2017-01-01

    The nucleolus is the site of ribosome biogenesis, a complex process that requires the coordinate activity of all three RNA polymerases and hundreds of non-ribosomal factors that participate in the maturation of ribosomal RNA (rRNA) and assembly of small and large subunits. Nevertheless, emerging studies have highlighted the fundamental role of the nucleolus in sensing a variety of cellular stress stimuli that target ribosome biogenesis. This condition is known as nucleolar stress and triggers several response pathways to maintain cell homeostasis, either p53-dependent or p53-independent. The mouse double minute (MDM2)-p53 stress signaling pathways are activated by multiple signals and are among the most important regulators of cellular homeostasis. In this review, we will focus on the role of ribosomal proteins in p53-dependent and p53-independent response to nucleolar stress considering novel identified regulators of these pathways. We describe, in particular, the role of ribosomal protein uL3 (rpL3) in p53-independent nucleolar stress signaling pathways. PMID:28085118

  7. Ribosome-messenger recognition: mRNA target sites for ribosomal protein S1.

    PubMed Central

    Boni, I V; Isaeva, D M; Musychenko, M L; Tzareva, N V

    1991-01-01

    Ribosomal protein S1 is known to play an important role in translational initiation, being directly involved in recognition and binding of mRNAs by 30S ribosomal particles. Using a specially developed procedure based on efficient crosslinking of S1 to mRNA induced by UV irradiation, we have identified S1 binding sites on several phage RNAs in preinitiation complexes. Targets for S1 on Q beta and fr RNAs are localized upstream from the coat protein gene and contain oligo(U)-sequences. In the case of Q beta RNA, this S1 binding site overlaps the S-site for Q beta replicase and the site for S1 binding within a binary complex. It is reasonable that similar U-rich sequences represent S1 binding sites on bacterial mRNAs. To test this idea we have used E. coli ssb mRNA prepared in vitro with the T7 promoter/RNA polymerase system. By the methods of toeprinting, enzymatic footprinting, and UV crosslinking we have shown that binding of the ssb mRNA to 30S ribosomes is S1-dependent. The oligo(U)-sequence preceding the SD domain was found to be the target for S1. We propose that S1 binding sites, represented by pyrimidine-rich sequences upstream from the SD region, serve as determinants involved in recognition of mRNA by the ribosome. Images PMID:2011495

  8. Protein-guided RNA dynamics during early ribosome assembly.

    PubMed

    Kim, Hajin; Abeysirigunawarden, Sanjaya C; Chen, Ke; Mayerle, Megan; Ragunathan, Kaushik; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A

    2014-02-20

    The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the ribosomal RNA structure so that later proteins may join the complex is poorly understood. Here we use single-molecule fluorescence resonance energy transfer (FRET) to observe real-time encounters between Escherichia coli ribosomal protein S4 and the 16S 5' domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free-energy path to protein-RNA recognition. Three-colour FRET and molecular dynamics simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. These protein-guided dynamics offer an alternative explanation for induced fit in RNA-protein complexes.

  9. Protein-guided RNA dynamics during early ribosome assembly

    NASA Astrophysics Data System (ADS)

    Kim, Hajin; Abeysirigunawarden, Sanjaya C.; Chen, Ke; Mayerle, Megan; Ragunathan, Kaushik; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A.

    2014-02-01

    The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the ribosomal RNA structure so that later proteins may join the complex is poorly understood. Here we use single-molecule fluorescence resonance energy transfer (FRET) to observe real-time encounters between Escherichia coli ribosomal protein S4 and the 16S 5' domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free-energy path to protein-RNA recognition. Three-colour FRET and molecular dynamics simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. These protein-guided dynamics offer an alternative explanation for induced fit in RNA-protein complexes.

  10. Crosslinking of Ribosomal Proteins to RNA in Maize Ribosomes by UV-B and Its Effects on Translation1[w

    PubMed Central

    Casati, Paula; Walbot, Virginia

    2004-01-01

    Ultraviolet-B (UV-B) photons can cause substantial cellular damage in biomolecules, as is well established for DNA. Because RNA has the same absorption spectrum for UV as DNA, we have investigated damage to this cellular constituent. In maize (Zea mays) leaves, UV-B radiation damages ribosomes by crosslinking cytosolic ribosomal proteins S14, L23a, and L32, and chloroplast ribosomal protein L29 to RNA. Ribosomal damage accumulated during a day of UV-B exposure correlated with a progressive decrease in new protein production; however, de novo synthesis of some ribosomal proteins is increased after 6 h of UV-B exposure. After 16 h without UV-B, damaged ribosomes were eliminated and translation was restored to normal levels. Ribosomal protein S6 and an S6 kinase are phosphorylated during UV-B exposure; these modifications are associated with selective translation of some ribosomal proteins after ribosome damage in mammalian fibroblast cells and may be an adaptation in maize. Neither photosynthesis nor pigment levels were affected significantly by UV-B, demonstrating that the treatment applied is not lethal and that maize leaf physiology readily recovers. PMID:15466230

  11. Ribosome-inactivating proteins: from toxins to useful proteins.

    PubMed

    Stirpe, Fiorenzo

    2013-06-01

    Ribosome-inactivating proteins (RIPs) either single-chain (type 1) or two-chain (type 2) are frequent in plants, often in multiple forms. They are RNA N-glycosidases, have antiviral, antifungal and insecticidal activity. Their expression in plants is increased under stressful conditions. They are investigated for practical applications in medicine and in agriculture. In medicine, RIPs have been linked to, or fused with, appropriate antibodies or other carriers to form "immunotoxins" or other conjugates specifically toxic to the cells target of the carrier, with the aim of eliminating malignant or other undesired cells. In agriculture, it has been observed that an enhanced expression of RIPs confers to plants an increased resistance to viruses, fungi, insects, and also to drought and salinity.

  12. Structure of the Escherichia coli S10 ribosomal protein operon.

    PubMed Central

    Zurawski, G; Zurawski, S M

    1985-01-01

    The complete structure of the Escherichia coli S10 ribosomal protein operon is presented. Based on the DNA sequence, the deduced order of the 11 genes in the operon is rpsJ, rplC, rplD, rplW, rplB, rpsS, rplV, rpsC, rplP, rpmC, rpsQ. The estimated transcribed length of the operon is 5181 base pairs. Putative sequences involved in ribosome binding are discussed. The DNA sequence data corrects several errors in previously determined protein sequence data. PMID:3892488

  13. Inactivation of Ribosomal Protein Genes in Bacillus subtilis Reveals Importance of Each Ribosomal Protein for Cell Proliferation and Cell Differentiation

    PubMed Central

    Akanuma, Genki; Nanamiya, Hideaki; Natori, Yousuke; Yano, Koichi; Suzuki, Shota; Omata, Shuya; Ishizuka, Morio; Sekine, Yasuhiko

    2012-01-01

    Among the 57 genes that encode ribosomal proteins in the genome of Bacillus subtilis, a Gram-positive bacterium, 50 genes were targeted by systematic inactivation. Individual deletion mutants of 16 ribosomal proteins (L1, L9, L15, L22, L23, L28, L29, L32, L33.1, L33.2, L34, L35, L36, S6, S20, and S21) were obtained successfully. In conjunction with previous reports, 22 ribosomal proteins have been shown to be nonessential in B. subtilis, at least for cell proliferation. Although several mutants that harbored a deletion of a ribosomal protein gene did not show any significant differences in any of the phenotypes that were tested, various mutants showed a reduced growth rate and reduced levels of 70S ribosomes compared with the wild type. In addition, severe defects in the sporulation frequency of the ΔrplA (L1) mutant and the motility of the ΔrpsU (S21) mutant were observed. These data provide the first evidence in B. subtilis that L1 and S21 are required for the progression of cellular differentiation. PMID:23002217

  14. Structural basis for precursor protein-directed ribosomal peptide macrocyclization

    PubMed Central

    Li, Kunhua; Condurso, Heather L.; Li, Gengnan; Ding, Yousong; Bruner, Steven D.

    2016-01-01

    Macrocyclization is a common feature of natural product biosynthetic pathways including the diverse family of ribosomal peptides. Microviridins are architecturally complex cyanobacterial ribosomal peptides whose members target proteases with potent reversible inhibition. The product structure is constructed by three macrocyclizations catalyzed sequentially by two members of the ATP-grasp family, a unique strategy for ribosomal peptide macrocyclization. Here, we describe the detailed structural basis for the enzyme-catalyzed macrocyclizations in the microviridin J pathway of Microcystis aeruginosa. The macrocyclases, MdnC and MdnB, interact with a conserved α-helix of the precursor peptide using a novel precursor peptide recognition mechanism. The results provide insight into the unique protein/protein interactions key to the chemistry, suggest an origin of the natural combinatorial synthesis of microviridin peptides and provide a framework for future engineering efforts to generate designed compounds. PMID:27669417

  15. Structural basis for precursor protein-directed ribosomal peptide macrocyclization.

    PubMed

    Li, Kunhua; Condurso, Heather L; Li, Gengnan; Ding, Yousong; Bruner, Steven D

    2016-11-01

    Macrocyclization is a common feature of natural product biosynthetic pathways including the diverse family of ribosomal peptides. Microviridins are architecturally complex cyanobacterial ribosomal peptides that target proteases with potent reversible inhibition. The product structure is constructed via three macrocyclizations catalyzed sequentially by two members of the ATP-grasp family, a unique strategy for ribosomal peptide macrocyclization. Here we describe in detail the structural basis for the enzyme-catalyzed macrocyclizations in the microviridin J pathway of Microcystis aeruginosa. The macrocyclases MdnC and MdnB interact with a conserved α-helix of the precursor peptide using a novel precursor-peptide recognition mechanism. The results provide insight into the unique protein-protein interactions that are key to the chemistry, suggest an origin for the natural combinatorial synthesis of microviridin peptides, and provide a framework for future engineering efforts to generate designed compounds.

  16. Epigenetic engineering of ribosomal RNA genes enhances protein production.

    PubMed

    Santoro, Raffaella; Lienemann, Philipp; Fussenegger, Martin

    2009-08-14

    Selection of mammalian high-producer cell lines remains a major challenge for the biopharmaceutical manufacturing industry. Ribosomal RNA (rRNA) genes encode the major component of the ribosome but many rRNA gene copies are not transcribed due to epigenetic silencing by the nucleolar remodelling complex (NoRC) [6], which may limit the cell's full production capacity. Here we show that the knockdown of TIP5, a subunit of NoRC, decreases the number of silent rRNA genes, upregulates rRNA transcription, enhances ribosome synthesis and increases production of recombinant proteins. However, general enhancement of rRNA transcription rate did not stimulate protein synthesis. Our data demonstrates that the number of transcriptionally competent rRNA genes limits efficient ribosome synthesis. Epigenetic engineering of ribosomal RNA genes offers new possibilities for improving biopharmaceutical manufacturing and provides novel insights into the complex regulatory network which governs the translation machinery in normal cellular processes as well as in pathological conditions like cancer.

  17. ABC-F Proteins Mediate Antibiotic Resistance through Ribosomal Protection.

    PubMed

    Sharkey, Liam K R; Edwards, Thomas A; O'Neill, Alex J

    2016-03-22

    Members of the ABC-F subfamily of ATP-binding cassette proteins mediate resistance to a broad array of clinically important antibiotic classes that target the ribosome of Gram-positive pathogens. The mechanism by which these proteins act has been a subject of long-standing controversy, with two competing hypotheses each having gained considerable support: antibiotic efflux versus ribosomal protection. Here, we report on studies employing a combination of bacteriological and biochemical techniques to unravel the mechanism of resistance of these proteins, and provide several lines of evidence that together offer clear support to the ribosomal protection hypothesis. Of particular note, we show that addition of purified ABC-F proteins to anin vitrotranslation assay prompts dose-dependent rescue of translation, and demonstrate that such proteins are capable of displacing antibiotic from the ribosomein vitro To our knowledge, these experiments constitute the first direct evidence that ABC-F proteins mediate antibiotic resistance through ribosomal protection.IMPORTANCEAntimicrobial resistance ranks among the greatest threats currently facing human health. Elucidation of the mechanisms by which microorganisms resist the effect of antibiotics is central to understanding the biology of this phenomenon and has the potential to inform the development of new drugs capable of blocking or circumventing resistance. Members of the ABC-F family, which includelsa(A),msr(A),optr(A), andvga(A), collectively yield resistance to a broader range of clinically significant antibiotic classes than any other family of resistance determinants, although their mechanism of action has been controversial since their discovery 25 years ago. Here we present the first direct evidence that proteins of the ABC-F family act to protect the bacterial ribosome from antibiotic-mediated inhibition.

  18. An extra-ribosomal function of ribosomal protein L13a in macrophage resolves inflammation

    PubMed Central

    Poddar, Darshana; Basu, Abhijit; Baldwin, William; Kondratov, Roman V; Barik, Sailen; Mazumder, Barsanjit

    2013-01-01

    Inflammation is an obligatory attempt of the immune system to protect the host from infections. However, unregulated synthesis of pro-inflammatory products can have detrimental effects. Although mechanisms that lead to inflammation are well appreciated, those that restrain it are not adequately understood. Creating macrophage-specific L13a-knockout (KO) mice here we report that depletion of ribosomal protein L13a abrogates the endogenous translation control of several chemokines in macrophages. Upon LPS-induced endotoxemia these animals displayed symptoms of severe inflammation caused by widespread infiltration of macrophages in major organs causing tissue injury and reduced survival rates. Macrophages from these KO animals show unregulated expression of several chemokines e.g. CXCL13, CCL22, CCL8 and CCR3. These macrophages failed to show L13a-dependent RNA binding complex formation on target mRNAs. In addition, increased polyribosomal abundance of these mRNAs shows a defect in translation control in the macrophages. Thus, our studies provide the first evidence of an essential extra-ribosomal function of ribosomal protein L13a in resolving physiological inflammation in a mammalian host. PMID:23460747

  19. N(α)-Acetylation of yeast ribosomal proteins and its effect on protein synthesis.

    PubMed

    Kamita, Masahiro; Kimura, Yayoi; Ino, Yoko; Kamp, Roza M; Polevoda, Bogdan; Sherman, Fred; Hirano, Hisashi

    2011-04-01

    N(α)-Acetyltransferases (NATs) cause the N(α)-acetylation of the majority of eukaryotic proteins during their translation, although the functions of this modification have been largely unexplored. In yeast (Saccharomyces cerevisiae), four NATs have been identified: NatA, NatB, NatC, and NatD. In this study, the N(α)-acetylation status of ribosomal protein was analyzed using NAT mutants combined with two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). A total of 60 ribosomal proteins were identified, of which 17 were N(α)-acetylated by NatA, and two by NatB. The N(α)-acetylation of two of these, S17 and L23, by NatA was not previously observed. Furthermore, we tested the effect of ribosomal protein N(α)-acetylation on protein synthesis using the purified ribosomes from each NAT mutant. It was found that the protein synthesis activities of ribosomes from NatA and NatB mutants were decreased by 27% and 23%, respectively, as compared to that of the normal strain. Furthermore, we have shown that ribosomal protein N(α)-acetylation by NatA influences translational fidelity in the presence of paromomycin. These results suggest that ribosomal protein N(α)-acetylation is necessary to maintain the ribosome's protein synthesis function.

  20. Protein-guided RNA dynamics during early ribosome assembly

    PubMed Central

    Kim, Hajin; Abeysirigunawardena, Sanjaya C.; Chen, Ke; Mayerle, Megan; Ragunathan, Kaushik; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A.

    2014-01-01

    The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the rRNA structure so that later proteins may join the complex is poorly understood. Here we use single molecule fluorescence resonance energy transfer (smFRET) to observe real-time encounters between ribosomal protein S4 and the 16S 5′ domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free energy path to protein-RNA recognition. Three-color FRET and molecular dynamics (MD) simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. This protein-guided dynamics offers an alternative explanation for induced fit in RNA-protein complexes. PMID:24522531

  1. Ribosomal protein uS19 mutants reveal its role in coordinating ribosome structure and function

    PubMed Central

    Bowen, Alicia M; Musalgaonkar, Sharmishtha; Moomau, Christine A; Gulay, Suna P; Mirvis, Mary; Dinman, Jonathan D

    2015-01-01

    Prior studies identified allosteric information pathways connecting functional centers in the large ribosomal subunit to the decoding center in the small subunit through the B1a and B1b/c intersubunit bridges in yeast. In prokaryotes a single SSU protein, uS13, partners with H38 (the A-site finger) and uL5 to form the B1a and B1b/c bridges respectively. In eukaryotes, the SSU component was split into 2 separate proteins during the course of evolution. One, also known as uS13, participates in B1b/c bridge with uL5 in eukaryotes. The other, called uS19 is the SSU partner in the B1a bridge with H38. Here, polyalanine mutants of uS19 involved in the uS19/uS13 and the uS19/H38 interfaces were used to elucidate the important amino acid residues involved in these intersubunit communication pathways. Two key clusters of amino acids were identified: one located at the junction between uS19 and uS13, and a second that appears to interact with the distal tip of H38. Biochemical analyses reveal that these mutations shift the ribosomal rotational equilibrium toward the unrotated state, increasing ribosomal affinity for tRNAs in the P-site and for ternary complex in the A-site, and inhibit binding of the translocase, eEF2. These defects in turn affect specific aspects of translational fidelity. These findings suggest that uS19 plays a critical role as a conduit of information exchange between the large and small ribosomal subunits directly through the B1a, and indirectly through the B1b/c bridges. PMID:26824029

  2. Structural and functional analyses of a yeast mitochondrial ribosomal protein homologous to ribosomal protein S15 of Escherichia coli.

    PubMed Central

    Dang, H; Ellis, S R

    1990-01-01

    We have purified a small subunit mitochondrial ribosomal protein, MRPS28p, from the yeast, Saccharomyces cerevisiae. Sequence from the amino terminus of MRPS28p was used to design a degenerate oligonucleotide that was complementary to the MRPS28 gene. The MRPS28 gene was isolated and its sequence determined. The MRPS28 sequence encodes a 28 kDa protein that has a region of homology with ribosomal protein S15 of E. coli. This region spans the entire length of the E. coli protein, but as MRPS28p is larger, includes only the portion of the MRPS28p sequence from amino acids 150 to 238. Based on this homology, we predict that MRPS28p, like E. coli S15, interacts directly with small subunit rRNA and functions as an early protein in ribosome assembly. Cells carrying a disrupted chromosomal copy of MRPS28 are unable to respire and spontaneously lose portions of their mitochondrial genomes at a high frequency. These phenotypes are consistent with an essential role for MRPS28p in the assembly and/or function of the mitochondrial ribosome. Images PMID:2263452

  3. Structural basis for precursor protein-directed ribosomal peptide macrocyclization

    SciTech Connect

    Li, Kunhua; Condurso, Heather L.; Li, Gengnan; Ding, Yousong; Bruner, Steven D.

    2016-11-11

    Macrocyclization is a common feature of natural product biosynthetic pathways including the diverse family of ribosomal peptides. Microviridins are architecturally complex cyanobacterial ribosomal peptides that target proteases with potent reversible inhibition. The product structure is constructed via three macrocyclizations catalyzed sequentially by two members of the ATP-grasp family, a unique strategy for ribosomal peptide macrocyclization. Here we describe in detail the structural basis for the enzyme-catalyzed macrocyclizations in the microviridin J pathway of Microcystis aeruginosa. The macrocyclases MdnC and MdnB interact with a conserved α-helix of the precursor peptide using a novel precursor-peptide recognition mechanism. The results provide insight into the unique protein–protein interactions that are key to the chemistry, suggest an origin for the natural combinatorial synthesis of microviridin peptides, and provide a framework for future engineering efforts to generate designed compounds.

  4. Crystal Structures of the uL3 Mutant Ribosome: Illustration of the Importance of Ribosomal Proteins for Translation Efficiency

    PubMed Central

    Mailliot, Justine; de Loubresse, Nicolas Garreau; Yusupova, Gulnara; Meskauskas, Arturas; Dinman, Jonathan D.; Yusupov, Marat

    2017-01-01

    The ribosome has been described as a ribozyme in which ribosomal RNA is responsible for peptidyl-transferase reaction catalysis. The W255C mutation of the universally conserved ribosomal protein uL3 has diverse effects on ribosome function (e.g., increased affinities for transfer RNAs, decreased rates of peptidyl-transfer), and cells harboring this mutation are resistant to peptidyl-transferase inhibitors (e.g., anisomycin). These observations beg the question of how a single amino acid mutation may have such wide ranging consequences. Here, we report the structure of the vacant yeast uL3 W255C mutant ribosome by X-ray crystallography, showing a disruption of the A-site side of the peptidyl-transferase center (PTC). An additional X-ray crystallographic structure of the anisomycin-containing mutant ribosome shows that high concentrations of this inhibitor restore a “WT-like” configuration to this region of the PTC, providing insight into the resistance mechanism of the mutant. Globally, our data demonstrate that ribosomal protein uL3 is structurally essential to ensure an optimal and catalytically efficient organization of the PTC, highlighting the importance of proteins in the RNA-centered ribosome. PMID:26906928

  5. Crystal Structures of the uL3 Mutant Ribosome: Illustration of the Importance of Ribosomal Proteins for Translation Efficiency.

    PubMed

    Mailliot, Justine; Garreau de Loubresse, Nicolas; Yusupova, Gulnara; Meskauskas, Arturas; Dinman, Jonathan D; Yusupov, Marat

    2016-05-22

    The ribosome has been described as a ribozyme in which ribosomal RNA is responsible for peptidyl-transferase reaction catalysis. The W255C mutation of the universally conserved ribosomal protein uL3 has diverse effects on ribosome function (e.g., increased affinities for transfer RNAs, decreased rates of peptidyl-transfer), and cells harboring this mutation are resistant to peptidyl-transferase inhibitors (e.g., anisomycin). These observations beg the question of how a single amino acid mutation may have such wide ranging consequences. Here, we report the structure of the vacant yeast uL3 W255C mutant ribosome by X-ray crystallography, showing a disruption of the A-site side of the peptidyl-transferase center (PTC). An additional X-ray crystallographic structure of the anisomycin-containing mutant ribosome shows that high concentrations of this inhibitor restore a "WT-like" configuration to this region of the PTC, providing insight into the resistance mechanism of the mutant. Globally, our data demonstrate that ribosomal protein uL3 is structurally essential to ensure an optimal and catalytically efficient organization of the PTC, highlighting the importance of proteins in the RNA-centered ribosome.

  6. Ubiquitination of newly synthesized proteins at the ribosome.

    PubMed

    Wang, Feng; Canadeo, Larissa A; Huibregtse, Jon M

    2015-07-01

    Newly synthesized proteins can be misfolded or damaged because of errors during synthesis or environmental insults (e.g., heat shock), placing a significant burden on protein quality control systems. In addition, numerous human diseases are associated with a deficiency in eliminating aberrant proteins or accumulation of aggregated proteins. Understanding the mechanisms of protein quality control and disposal pathways for misfolded proteins is therefore crucial for therapeutic intervention in these diseases. Quality control processes function at many points in the life cycle of proteins, and a subset act at the actual site of protein synthesis, the ribosome. Here we summarize recent advances in the role of the ubiquitin proteasome system in protein quality control during the process of translation.

  7. Ubiquitination of Newly Synthesized Proteins at the Ribosome

    PubMed Central

    Wang, Feng; Canadeo, Larissa A.; Huibregtse, Jon M.

    2015-01-01

    Newly synthesized proteins can be misfolded or damaged because of errors during synthesis or environmental insults (e.g., heat shock), placing a significant burden on protein quality control systems. In addition, numerous human diseases are associated with a deficiency in eliminating aberrant proteins or accumulation of aggregated proteins. Understanding the mechanisms of protein quality control and disposal pathways for misfolded proteins is therefore crucial for therapeutic intervention in these diseases. Quality control processes function at many points in the life cycle of proteins, and a subset act at the actual site of protein synthesis, the ribosome. Here we summarize recent advances in the role of the ubiquitin proteasome system in protein quality control during the process of translation. PMID:25701549

  8. Protein folding: When ribosomes pick the structure

    NASA Astrophysics Data System (ADS)

    Sivertsson, Elin M.; Itzhaki, Laura S.

    2014-05-01

    Anfinsen's principle tells us that the folded structure of a protein is determined solely by its sequence. Now, it has been shown that the rate at which a polypeptide chain is synthesized in the cell can affect which of two alternative folded structures it adopts.

  9. Repressed synthesis of ribosomal proteins generates protein-specific cell cycle and morphological phenotypes.

    PubMed

    Thapa, Mamata; Bommakanti, Ananth; Shamsuzzaman, Md; Gregory, Brian; Samsel, Leigh; Zengel, Janice M; Lindahl, Lasse

    2013-12-01

    The biogenesis of ribosomes is coordinated with cell growth and proliferation. Distortion of the coordinated synthesis of ribosomal components affects not only ribosome formation, but also cell fate. However, the connection between ribosome biogenesis and cell fate is not well understood. To establish a model system for inquiries into these processes, we systematically analyzed cell cycle progression, cell morphology, and bud site selection after repression of 54 individual ribosomal protein (r-protein) genes in Saccharomyces cerevisiae. We found that repression of nine 60S r-protein genes results in arrest in the G2/M phase, whereas repression of nine other 60S and 22 40S r-protein genes causes arrest in the G1 phase. Furthermore, bud morphology changes after repression of some r-protein genes. For example, very elongated buds form after repression of seven 60S r-protein genes. These genes overlap with, but are not identical to, those causing the G2/M cell cycle phenotype. Finally, repression of most r-protein genes results in changed sites of bud formation. Strikingly, the r-proteins whose repression generates similar effects on cell cycle progression cluster in the ribosome physical structure, suggesting that different topological areas of the precursor and/or mature ribosome are mechanistically connected to separate aspects of the cell cycle.

  10. Expression Trend of Selected Ribosomal Protein Genes in Nasopharyngeal Carcinoma

    PubMed Central

    Ma, Xiang-Ru; Sim, Edmund Ui-Hang; Ling, Teck-Yee; Tiong, Thung-Sing; Subramaniam, Selva Kumar; Khoo, Alan Soo-Beng

    2012-01-01

    Background: Ribosomal proteins are traditionally associated with protein biosynthesis until recent studies that implicated their extraribosomal functions in human diseases and cancers. Our previous studies using GeneFishing™ DEG method and microarray revealed underexpression of three ribosomal protein genes, RPS26, RPS27, and RPL32 in cancer of the nasopharynx. Herein, we investigated the expression pattern and nucleotide sequence integrity of these genes in nasopharyngeal carcinoma to further delineate their involvement in tumourigenesis. The relationship of expression level with clinicopathologic factors was also statistically studied. Methods: Quantitative Polymerase Chain Reaction was performed on nasopharyngeal carcinoma and their paired normal tissues. Expression and sequence of these three genes were analysed. Results: All three ribosomal protein genes showed no significant difference in transcript expressions and no association could be established with clinicopathologic factors studied. No nucleotide aberrancy was detected in the coding regions of these genes. Conclusion: There is no early evidence to substantiate possible involvement of RPS26, RPS27, and RPL32 genes in NPC tumourigenesis. PMID:23613646

  11. RNA-protein distance patterns in ribosomes reveal the mechanism of translational attenuation.

    PubMed

    Yu, DongMei; Zhang, Chao; Qin, PeiWu; Cornish, Peter V; Xu, Dong

    2014-11-01

    Elucidating protein translational regulation is crucial for understanding cellular function and drug development. A key molecule in protein translation is ribosome, which is a super-molecular complex extensively studied for more than a half century. The structure and dynamics of ribosome complexes were resolved recently thanks to the development of X-ray crystallography, Cryo-EM, and single molecule biophysics. Current studies of the ribosome have shown multiple functional states, each with a unique conformation. In this study, we analyzed the RNA-protein distances of ribosome (2.5 MDa) complexes and compared these changes among different ribosome complexes. We found that the RNA-protein distance is significantly correlated with the ribosomal functional state. Thus, the analysis of RNA-protein binding distances at important functional sites can distinguish ribosomal functional states and help understand ribosome functions. In particular, the mechanism of translational attenuation by nascent peptides and antibiotics was revealed by the conformational changes of local functional sites.

  12. Structure determination of archaea-specific ribosomal protein L46a reveals a novel protein fold

    SciTech Connect

    Feng, Yingang; Song, Xiaxia; Lin, Jinzhong; Xuan, Jinsong; Cui, Qiu; Wang, Jinfeng

    2014-07-18

    Highlights: • The archaea-specific ribosomal protein L46a has no homology to known proteins. • Three dimensional structure and backbone dynamics of L46a were determined by NMR. • The structure of L46a represents a novel protein fold. • A potential rRNA-binding surface on L46a was identified. • The potential position of L46a on the ribosome was proposed. - Abstract: Three archaea-specific ribosomal proteins recently identified show no sequence homology with other known proteins. Here we determined the structure of L46a, the most conserved one among the three proteins, from Sulfolobus solfataricus P2 using NMR spectroscopy. The structure presents a twisted β-sheet formed by the N-terminal part and two helices at the C-terminus. The L46a structure has a positively charged surface which is conserved in the L46a protein family and is the potential rRNA-binding site. Searching homologous structures in Protein Data Bank revealed that the structure of L46a represents a novel protein fold. The backbone dynamics identified by NMR relaxation experiments reveal significant flexibility at the rRNA binding surface. The potential position of L46a on the ribosome was proposed by fitting the structure into a previous electron microscopy map of the ribosomal 50S subunit, which indicated that L46a contacts to domain I of 23S rRNA near a multifunctional ribosomal protein L7ae.

  13. The unfolded protein response triggers site-specific regulatory ubiquitylation of 40S ribosomal proteins

    PubMed Central

    Rising, Lisa; Mak, Raymond; Webb, Kristofor; Kaiser, Stephen E.; Zuzow, Nathan; Riviere, Paul; Yang, Bing; Fenech, Emma; Tang, Xin; Lindsay, Scott A.; Christianson, John C.; Hampton, Randolph Y.; Wasserman, Steven A.; Bennett, Eric J.

    2015-01-01

    Summary Insults to endoplasmic reticulum (ER) homeostasis activate the unfolded protein response (UPR), which elevates protein folding and degradation capacity and attenuates protein synthesis. While a role for ubiquitin in regulating the degradation of misfolded ER-resident proteins is well described, ubiquitin-dependent regulation of translational reprogramming during the UPR remains uncharacterized. Using global quantitative ubiquitin proteomics, we identify evolutionarily conserved, site-specific regulatory ubiquitylation of 40S ribosomal proteins. We demonstrate that these events occur on assembled cytoplasmic ribosomes and are stimulated by both UPR activation and translation inhibition. We further show that ER stress-stimulated regulatory 40S ribosomal ubiquitylation occurs on a timescale similar to eIF2α phosphorylation, is dependent upon PERK signaling, and is required for optimal cell survival during chronic UPR activation. In total, these results reveal regulatory 40S ribosomal ubiquitylation as a previously uncharacterized and important facet of eukaryotic translational control. PMID:26051182

  14. Dynamic evolution of mitochondrial ribosomal proteins in Holozoa.

    PubMed

    Scheel, Bettina M; Hausdorf, Bernhard

    2014-07-01

    We studied the highly dynamic evolution of mitochondrial ribosomal proteins (MRPs) in Holozoa. Most major clades within Holozoa are characterized by gains and/or losses of MRPs. The usefulness of gains of MRPs as rare genomic changes in phylogenetics is undermined by the high frequency of secondary losses. However, phylogenetic analyses of the MRP sequences provide evidence for the Acrosomata hypothesis, a sister group relationship between Ctenophora and Bilateria. An extensive restructuring of the mitochondrial genome and, as a consequence, of the mitochondrial ribosomes occurred in the ancestor of metazoans. The last MRP genes encoded in the mitochondrial genome were either moved to the nuclear genome or were lost. The strong decrease in size of the mitochondrial genome was probably caused by selection for rapid replication of mitochondrial DNA during oogenesis in the metazoan ancestor. A phylogenetic analysis of MRPL56 sequences provided evidence for a horizontal gene transfer of the corresponding MRP gene between metazoans and Dictyostelidae (Amoebozoa). The hypothesis that the requisition of additional MRPs compensated for a loss of rRNA segments in the mitochondrial ribosomes is corroborated by a significant negative correlation between the number of MRPs and length of the rRNA. Newly acquired MRPs evolved faster than bacterial MRPs and positions in eukaryote-specific MRPs were more strongly affected by coevolution than positions in prokaryotic MRPs in accordance with the necessity to fit these proteins into the pre-existing structure of the mitoribosome.

  15. Nuclear and nucleolar targeting of human ribosomal protein S6.

    PubMed Central

    Schmidt, C; Lipsius, E; Kruppa, J

    1995-01-01

    Chimeric proteins were constructed to define the nuclear localization signals (NLSs) of human ribosomal protein S6. The complete cDNA sequence, different cDNA fragments and oligonucleotides of the human ribosomal proteins S6, respectively, were joined to the 5' end of the entire LacZ gene of Escherichia coli by using recombinant techniques. The hybrid genes were transfected into L cells, transiently expressed, and the intracellular location of the fusion proteins was determined by their beta-galactosidase activity. Three NLSs were identified in the C-terminal half of the S6 protein. Deletion mutagenesis demonstrated that a single NLS is sufficient for targeting the corresponding S6-beta-galactosidase chimera into the nucleus. Removal of all three putative NLSs completely blocked the nuclear import of the resulting S6-beta-galactosidase fusion protein, which instead became evenly distributed in the cytoplasm. Chimeras containing deletion mutants of S6 with at least one single NLS or unmodified S6 accumulated in the nucleolus. Analysis of several constructs reveals the existence of a specific domain that is essential but not sufficient for nucleolar accumulation of S6. Images PMID:8590812

  16. A novel RNA-binding motif in omnipotent suppressors of translation termination, ribosomal proteins and a ribosome modification enzyme?

    PubMed

    Koonin, E V; Bork, P; Sander, C

    1994-06-11

    Using computer methods for database search, multiple alignment, protein sequence motif analysis and secondary structure prediction, a putative new RNA-binding motif was identified. The novel motif is conserved in yeast omnipotent translation termination suppressor SUP1, the related DOM34 protein and its pseudogene homologue; three groups of eukaryotic and archaeal ribosomal proteins, namely L30e, L7Ae/S6e and S12e; an uncharacterized Bacillus subtilis protein related to the L7A/S6e group; and Escherichia coli ribosomal protein modification enzyme RimK. We hypothesize that a new type of RNA-binding domain may be utilized to deliver additional activities to the ribosome.

  17. Ribosome profiling: a Hi-Def monitor for protein synthesis at the genome-wide scale

    PubMed Central

    Michel, Audrey M; Baranov, Pavel V

    2013-01-01

    Ribosome profiling or ribo-seq is a new technique that provides genome-wide information on protein synthesis (GWIPS) in vivo. It is based on the deep sequencing of ribosome protected mRNA fragments allowing the measurement of ribosome density along all RNA molecules present in the cell. At the same time, the high resolution of this technique allows detailed analysis of ribosome density on individual RNAs. Since its invention, the ribosome profiling technique has been utilized in a range of studies in both prokaryotic and eukaryotic organisms. Several studies have adapted and refined the original ribosome profiling protocol for studying specific aspects of translation. Ribosome profiling of initiating ribosomes has been used to map sites of translation initiation. These studies revealed the surprisingly complex organization of translation initiation sites in eukaryotes. Multiple initiation sites are responsible for the generation of N-terminally extended and truncated isoforms of known proteins as well as for the translation of numerous open reading frames (ORFs), upstream of protein coding ORFs. Ribosome profiling of elongating ribosomes has been used for measuring differential gene expression at the level of translation, the identification of novel protein coding genes and ribosome pausing. It has also provided data for developing quantitative models of translation. Although only a dozen or so ribosome profiling datasets have been published so far, they have already dramatically changed our understanding of translational control and have led to new hypotheses regarding the origin of protein coding genes. © 2013 John Wiley & Sons, Ltd. PMID:23696005

  18. A general procedure for the production of antibody reagents against eukaryotic ribosomal proteins.

    PubMed

    Dieci, Giorgio; Bottarelli, Lorena; Ottonello, Simone

    2005-08-01

    Despite recent progress in the structural and functional analysis of bacterial and archaeal ribosomes, the structure and biogenesis of eukaryotic ribosomes still awaits a detailed characterization. Ribosomal protein-specific antibodies would be valuable tools for such studies, but their production is commonly hindered by the poor expression and solubility of eukaryotic ribosomal proteins in E. coli. We report here an improved general procedure for the over-production of recombinant eukaryotic ribosomal proteins and for the generation of the corresponding polyclonal antibodies. The specificity and sensitivity of detection of the antibodies produced by this procedure are documented.

  19. Positive modulation of RNA polymerase III transcription by ribosomal proteins

    SciTech Connect

    Dieci, Giorgio; Carpentieri, Andrea; Amoresano, Angela; Ottonello, Simone

    2009-02-06

    A yeast nuclear fraction of unknown composition, named TFIIIE, was reported previously to enhance transcription of tRNA and 5S rRNA genes in vitro. We show that TFIIIE activity co-purifies with a specific subset of ribosomal proteins (RPs) which, as revealed by chromatin immunoprecipitation analysis, generally interact with tRNA and 5S rRNA genes, but not with a Pol II-specific promoter. Only Rpl6Ap and Rpl6Bp, among the tested RPs, were found associated to a TATA-containing tRNA{sup Ile}(TAT) gene. The RPL6A gene also emerged as a strong multicopy suppressor of a conditional mutation in the basal transcription factor TFIIIC, while RPL26A and RPL14A behaved as weak suppressors. The data delineate a novel extra-ribosomal role for one or a few RPs which, by influencing 5S rRNA and tRNA synthesis, could play a key role in the coordinate regulation of the different sub-pathways required for ribosome biogenesis and functionality.

  20. Two Dictyostelium ribosomal proteins act as RNases for specific classes of mRNAs.

    PubMed Central

    Mangiarotti, Giorgio

    2003-01-01

    Phosphorylation of ribosomal protein S6 leads to the stabilization of pre-spore specific mRNAs during development of Dictyostelium discoideum. The purification of S6 kinase has allowed the identification of protein S11 as the mRNase specific for pre-spore mRNAs. Methylation of ribosomal protein S31 leads to the destabilization of ribosomal protein mRNAs. The purification of S31 methyltransferase has allowed the identification of protein S29 as the mRNAse specific for ribosomal protein mRNAs. PMID:12392449

  1. Two Dictyostelium ribosomal proteins act as RNases for specific classes of mRNAs.

    PubMed

    Mangiarotti, Giorgio

    2003-03-01

    Phosphorylation of ribosomal protein S6 leads to the stabilization of pre-spore specific mRNAs during development of Dictyostelium discoideum. The purification of S6 kinase has allowed the identification of protein S11 as the mRNase specific for pre-spore mRNAs. Methylation of ribosomal protein S31 leads to the destabilization of ribosomal protein mRNAs. The purification of S31 methyltransferase has allowed the identification of protein S29 as the mRNAse specific for ribosomal protein mRNAs.

  2. Mosaic tetracycline resistance genes encoding ribosomal protection proteins.

    PubMed

    Warburton, Philip J; Amodeo, Nina; Roberts, Adam P

    2016-12-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria.

  3. Mosaic tetracycline resistance genes encoding ribosomal protection proteins

    PubMed Central

    Warburton, Philip J.; Amodeo, Nina; Roberts, Adam P.

    2016-01-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria. PMID:27494928

  4. Fluorescent protein tagging confirms the presence of ribosomal proteins at Drosophila polytene chromosomes

    PubMed Central

    Ramanathan, Preethi; Matina, Tina; Wen, Jikai

    2013-01-01

    Most ribosomal proteins (RPs) are stoichiometrically incorporated into ribosomal subunits and play essential roles in ribosome biogenesis and function. However, a number of RPs appear to have non-ribosomal functions, which involve direct association with pre-mRNA and transcription factors at transcription sites. The consensus is that the RPs found at these sites are off ribosomal subunits, but observation that different RPs are usually found together suggests that ribosomal or ribosomal-like subunits might be present. Notably, it has previously been reported that antibodies against 20 different RPs stain the same Pol II transcription sites in Drosophila polytene chromosomes. Some concerns, however, were raised about the specificity of the antibodies. To investigate further whether RPs are present at transcription sites in Drosophila, we have generated several transgenic flies expressing RPs (RpS2, RpS5a, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, and RpL36) tagged with either green or red fluorescent protein. Imaging of salivary gland cells showed that these proteins are, as expected, abundant in the cytoplasm as well as in the nucleolus. However, these RPs are also apparent in the nucleus in the region occupied by the chromosomes. Indeed, polytene chromosome immunostaining of a representative subset of tagged RPs confirms the association with transcribed loci. Furthermore, characterization of a strain expressing RpL41 functionally tagged at its native genomic locus with YFP, also showed apparent nuclear accumulation and chromosomal association, suggesting that such a nuclear localization pattern might be a shared feature of RPs and is biologically important. We anticipate that the transgenes described here should provide a useful research tool to visualize ribosomal subunits in Drosophila tissues and to study the non-ribosomal functions of RPs. PMID:23638349

  5. Large Ribosomal Protein 4 Increases Efficiency of Viral Recoding Sequences

    PubMed Central

    Green, Lisa; Houck-Loomis, Brian; Yueh, Andrew

    2012-01-01

    Expression of retroviral replication enzymes (Pol) requires a controlled translational recoding event to bypass the stop codon at the end of gag. This recoding event occurs either by direct suppression of termination via the insertion of an amino acid at the stop codon (readthrough) or by alteration of the mRNA reading frame (frameshift). Here we report the effects of a host protein, large ribosomal protein 4 (RPL4), on the efficiency of recoding. Using a dual luciferase reporter assay, we found that transfection of cells with a plasmid encoding RPL4 cDNA increases recoding efficiency in a dose-dependent manner, with a maximal enhancement of nearly twofold. Expression of RPL4 increases recoding of reporters containing retroviral readthrough and frameshift sequences, as well as the Sindbis virus leaky termination signal. RPL4-induced enhancement of recoding is cell line specific and appears to be specific to RPL4 among ribosomal proteins. Cotransfection of RPL4 cDNA with Moloney murine leukemia proviral DNA results in Gag processing defects and a reduction of viral particle formation, presumably caused by the RPL4-dependent alteration of the Gag-to-Gag-Pol ratio required for virion assembly and release. PMID:22718819

  6. Molecular evolution of the mammalian ribosomal protein gene, RPS14.

    PubMed

    Rhoads, D D; Roufa, D J

    1991-07-01

    Ribosomal protein S14 genes (RPS14) in eukaryotic species from protozoa to primates exhibit dramatically different intron-exon structures yet share homologous polypeptide-coding sequences. To recognize common features of RPS14 gene architectures in closely related mammalian species and to evaluate similarities in their noncoding DNA sequences, we isolated the intron-containing S14 locus from Chinese hamster ovary (CHO) cell DNA by using a PCR strategy and compared it with human RPS14. We found that rodent and primate S14 genes are composed of identical protein-coding exons interrupted by introns at four conserved DNA sites. However, the structures of corresponding CHO and human RPS14 introns differ significantly. Nonetheless, individual intron splice donor, splice acceptor, and upstream flanking motifs have been conserved within mammalian S14 homologues as well as within RPS14 gene fragments PCR amplified from other vertebrate genera (birds and bony fish). Our data indicate that noncoding, intronic DNA sequences within highly conserved, single-copy ribosomal protein genes are useful molecular landmarks for phylogenetic analysis of closely related vertebrate species.

  7. Interplay between trigger factor and other protein biogenesis factors on the ribosome

    NASA Astrophysics Data System (ADS)

    Bornemann, Thomas; Holtkamp, Wolf; Wintermeyer, Wolfgang

    2014-06-01

    Nascent proteins emerging from translating ribosomes in bacteria are screened by a number of ribosome-associated protein biogenesis factors, among them the chaperone trigger factor (TF), the signal recognition particle (SRP) that targets ribosomes synthesizing membrane proteins to the membrane and the modifying enzymes, peptide deformylase (PDF) and methionine aminopeptidase (MAP). Here, we examine the interplay between these factors both kinetically and at equilibrium. TF rapidly scans the ribosomes until it is stabilized on ribosomes presenting TF-specific nascent chains. SRP binding to those complexes is strongly impaired. Thus, TF in effect prevents SRP binding to the majority of ribosomes, except those presenting SRP-specific signal sequences, explaining how the small amount of SRP in the cell can be effective in membrane targeting. PDF and MAP do not interfere with TF or SRP binding to translating ribosomes, indicating that nascent-chain processing can take place before or in parallel with TF or SRP binding.

  8. Ribosomal proteins produced in excess are degraded by the ubiquitin–proteasome system

    PubMed Central

    Sung, Min-Kyung; Reitsma, Justin M.; Sweredoski, Michael J.; Hess, Sonja; Deshaies, Raymond J.

    2016-01-01

    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. PMID:27385339

  9. The extended loops of ribosomal proteins uL4 and uL22 of Escherichia coli contribute to ribosome assembly and protein translation

    PubMed Central

    Lawrence, Marlon G.; Shamsuzzaman, Md; Kondopaka, Maithri; Pascual, Clarence; Zengel, Janice M.; Lindahl, Lasse

    2016-01-01

    Nearly half of ribosomal proteins are composed of a domain on the ribosome surface and a loop or extension that penetrates into the organelle's RNA core. Our previous work showed that ribosomes lacking the loops of ribosomal proteins uL4 or uL22 are still capable of entering polysomes. However, in those experiments we could not address the formation of mutant ribosomes, because we used strains that also expressed wild-type uL4 and uL22. Here, we have focused on ribosome assembly and function in strains in which loop deletion mutant genes are the only sources of uL4 or uL22 protein. The uL4 and uL22 loop deletions have different effects, but both mutations result in accumulation of immature particles that do not accumulate in detectable amounts in wild-type strains. Thus, our results suggest that deleting the loops creates kinetic barriers in the normal assembly pathway, possibly resulting in assembly via alternate pathway(s). Furthermore, deletion of the uL4 loop results in cold-sensitive ribosome assembly and function. Finally, ribosomes carrying either of the loop-deleted proteins responded normally to the secM translation pausing peptide, but the uL4 mutant responded very inefficiently to the cmlAcrb pause peptide. PMID:27257065

  10. Structural insights into the interaction of the ribosomal P stalk protein P2 with a type II ribosome-inactivating protein ricin

    PubMed Central

    Fan, Xiaojiao; Zhu, Yuwei; Wang, Chongyuan; Niu, Liwen; Teng, Maikun; Li, Xu

    2016-01-01

    Ricin is a type II ribosome-inactivating protein (RIP) that depurinates A4324 at the sarcin-ricin loop of 28 S ribosomal RNA (rRNA), thus inactivating the ribosome by preventing elongation factors from binding to the GTPase activation centre. Recent studies have disclosed that the conserved C-terminal domain (CTD) of eukaryotic ribosomal P stalk proteins is involved in the process that RIPs target ribosome. However, the details of the molecular interaction between ricin and P stalk proteins remain unknown. Here, we report the structure of ricin-A chain (RTA) in a complex with the CTD of the human ribosomal protein P2. The structure shows that the Phe111, Leu113 and Phe114 residues of P2 insert into a hydrophobic pocket formed by the Tyr183, Arg235, Phe240 and Ile251 residues of RTA, while Asp115 of P2 forms hydrogen bonds with Arg235 of RTA. The key residues in RTA and P2 for complex formation were mutated, and their importance was determined by pull-down assays. The results from cell-free translation assays further confirmed that the interaction with P stalk proteins is essential for the inhibition of protein synthesis by RTA. Taken together, our results provide a structural basis that will improve our understanding of the process by which ricin targets the ribosome, which will benefit the development of effective small-molecule inhibitors for use as therapeutic agents. PMID:27886256

  11. Histone deacetylase 6 associates with ribosomes and regulates de novo protein translation during arsenite stress.

    PubMed

    Kappeler, Kyle V; Zhang, Jack; Dinh, Thai Nho; Strom, Joshua G; Chen, Qin M

    2012-05-01

    Histone deacetylase 6 (HDAC6) is known as a cytoplasmic enzyme that regulates cell migration, cell adhesion, and degradation of misfolded proteins by deacetylating substrates such as α-tubulin and Hsp90. When HaCaT keratinocytes were exposed to 1-200μM sodium arsenite, we observed perinuclear localization of HDAC6 within 30 min. Although the overall level of HDAC6 protein did not change, sodium arsenite caused an increase of HDAC6 in ribosomal fractions. Separation of ribosomal subunits versus intact ribosomes or polysomes indicated that HDAC6 was mainly detected in 40/43S fractions containing the small ribosomal subunit in untreated cells but was associated with 40/43S and 60/80S ribosomal fractions in arsenite-treated cells. Immunocytochemistry studies revealed that arsenite caused colocalization of HDAC6 with the ribosomal large and small subunit protein L36a and S6. Both L36a and S6 were detected in the immunocomplex of HDAC6 isolated from arsenite-treated cells. The observed physical interaction of HDAC6 with ribosomes pointed to a role of HDAC6 in stress-induced protein translation. Among arsenite stress-induced proteins, de novo Nrf2 protein translation was inhibited by Tubastatin A. These data demonstrate that HDAC6 was recruited to ribosomes, physically interacted with ribosomal proteins, and regulated de novo protein translation in keratinocytes responding to arsenite stress.

  12. Ribosomal protein gene mapping and human chromosomal disorders

    SciTech Connect

    Kenmochi, N.; Goodman, N.; Page, D.C.

    1994-09-01

    In Drosophila, the Minute phenotype (reduced body size, diminished viability and fertility, and short, thin bristles) results from heterozygous deficiencies (deletions) at any one of 50 loci scattered about the genome. A handful of these Minute loci have been molecularly characterized, and all have been found to encode ribosomal proteins. Thus, the Minute phenotype appears to result from reduced protein synthetic capacity in flies with one rather than two copies of a given ribosomal protein (rp) gene. We are pursuing the possibility that similar reductions in protein synthetic capacity--again resulting from rp gene deficiencies--might underlie phenotypes associated with certain chromosomal disorders in humans. We and our colleagues have reported findings consistent with a role for RPS4 deficiency in the etiology of certain features of Turner syndrome, a complex human disorder classically associated with an XO karyotype. We are intrigued by the possibility that deficiencies of other human rp genes might cause phenotypic abnormalities similar to those seen in Turner syndrome--just as deficiencies of any of a number of Drosophila rp genes cause the Minute phenotype. We must first learn the chromosomal map position of each of the estimated 83 human rp genes. The task of mapping the functional (intron-containing) rp genes is complicated by the existence of processed pseudogenes elsewhere in the genome. To date, we have assigned (or confirmed the previous assignment of) 38 rp genes to individual human chromosomes by PCR analysis of human-rodent somatic cell hybrids containing subsets of human chromosomes, with all but four chromosomes carrying at least one rp gene. We have also identified more than 100 large-insert human YAC (yeast artificial chromosome) clones that contain individual rp genes. Such screening of YAC libraries will result in precise positioning of the rp genes on the emerging physical map of the human genome.

  13. Are there proteins between the ribosomal subunits? Hot tritium bombardment experiments.

    PubMed

    Yusupov, M M; Spirin, A S

    1986-03-03

    The hot tritium bombardment technique [(1976) Dokl. Akad. Nauk SSSR 228, 1237-1238] was used for studying the surface localization of ribosomal proteins on Escherichia coli ribosomes. The degree of tritium labeling of proteins was considered as a measure of their exposure (surface localization). Proteins S1, S4, S7, S9 and/or S11, S12 and/or L20, S13, S18, S20, S21, L5, L6, L7/L12, L10, L11, L16, L17, L24, L26 and L27 were shown to be the most exposed on the ribosome surface. The sets of exposed ribosomal proteins on the surface of 70 S ribosomes, on the one hand, and the surfaces of 50 S and 30 S ribosomal subunits in the dissociated state, on the other, were compared. It was found that the dissociation of ribosomes into subunits did not result in exposure of additional ribosomal proteins. The conclusion was drawn that proteins are absent from the contacting surfaces of the ribosomal subunits.

  14. Ribosomal protein methylation in Escherichia coli: the gene prmA, encoding the ribosomal protein L11 methyltransferase, is dispensable.

    PubMed

    Vanet, A; Plumbridge, J A; Guérin, M F; Alix, J H

    1994-12-01

    The prmA gene, located at 72 min on the Escherichia coli chromosome, is the genetic determinant of ribosomal protein L11-methyltransferase activity. Mutations at this locus, prmA1 and prmA3, result in a severely undermethylated form of L11. No effect, other than the lack of methyl groups on L11, has been ascribed to these mutations. DNA sequence analysis of the mutant alleles prmA1 and prmA3 detected point mutations near the C-terminus of the protein and plasmids overproducing the wild-type and the two mutant proteins have been constructed. The wild-type PrmA protein could be crosslinked to its radiolabelled substrate, S-adenosyl-L-methionine (SAM), by u.v. irradiation indicating that it is the gene for the methyltransferase rather than a regulatory protein. One of the mutant proteins, PrmA3, was also weakly crosslinked to SAM. Both mutant enzymes when expressed from the overproducing plasmids were capable of catalysing the incorporation of 3H-labelled methyl groups from SAM to L11 in vitro. This confirmed the observation that the mutant proteins possess significant residual activity which could account for their lack of growth phenotype. However, a strain carrying an in vitro-constructed null mutation of the prmA gene, transferred to the E. coli chromosome by homologous recombination, was perfectly viable.

  15. Isolation and characterization of ribosome-inactivating proteins from Cucurbitaceae.

    PubMed

    Zhang, Daoning; Halaweish, Fathi T

    2007-03-01

    Due to their RNA-N-glycosidase activity, ribosome-inactivating proteins (RIPs) are attractive candidates as antitumor and antiviral agents in biomedical and agricultural research. We have isolated and characterized two such proteins, foetidissimin II and texanin, from two Cucurbitaceae species. Foetidissimin II, obtained from the roots of Cucurbita foetidissima, was identified as a type-2 RIP, with a molecular weight of 61 kDa, as estimated by gel electrophoresis. It is composed of two chains, a 29-kDa chain A, and a 32-kDa chain B. Texanin, isolated from the fruits of Cucurbita texana, is a type-I RIP, with a single chain of molecular weight 29.7 kDa, as estimated by MALDI-TOF-MS. Both proteins exhibit RNA-N-glycosidase activity, with aniline playing a critical role in rRNA cleavage. The IC50 value of foetidissimin II, determined by cell-free protein-synthesis inhibition, was 0.251 muM. In an in vitro cytotoxicity assay, foetidissimin II exhibited IC50 values of ca. 70 nM to both adenocarcinoma and erythroleukemia cells. Texanin exhibited a weaker anticancer activity against erythroleukemia cells, with an IC50 value of 95 microM, but no activity against adenocarcinoma cells. The N-terminal sequences of both proteins were compared with those of reported RIPs.

  16. Phosphorylation of ribosomal proteins induced by auxins in maize embryonic tissues. [Zea mays

    SciTech Connect

    Perez, L.; Aguilar, R.; Mendez, A.P.; de Jimenez, E.S.

    1990-11-01

    The effect of auxin on ribosomal protein phosphorylation of germinating maize (Zea mays) tissues was investigated. Two-dimensional gel electrophoresis and autoradiography of ({sup 32}P) ribosomal protein patterns for natural and synthetic auxin-treated tissues were performed. Both the rate of {sup 32}P incorporation and the electrophoretic patterns were dependent on {sup 32}P pulse length, suggesting that active protein phosphorylation-dephosphorylation occurred in small and large subunit proteins, in control as well as in auxin-treated tissues. The effect of ribosomal protein phosphorylation on in vitro translation was tested. Measurements of poly(U) translation rates as a function of ribosome concentration provided apparent K{sub m} values significantly different for auxin-treated and nontreated tissues. These findings suggest that auxin might exert some kind of translational control by regulating the phosphorylated status of ribosomal proteins.

  17. Mutations in ribosomal proteins S4 and S12 influence the higher order structure of 16 S ribosomal RNA.

    PubMed

    Allen, P N; Noller, H F

    1989-08-05

    We have studied the effects of protein mutations on the higher order structure of 16 S rRNA in Escherichia coli ribosomes, using a set of structure-sensitive chemical probes. Ten mutant strains were studied, which contained alterations in ribosomal proteins S4 and S12, including double mutants containing both altered S4 and S12. Two ribosomal ambiguity (ram) S4 mutant strains, four streptomycin resistant (SmR) S12 mutant strains, one streptomycin pseudodependent (SmP) S12 mutant strain, one streptomycin dependent (SmD) S12 mutant strain and two streptomycin independent (Sm1) double mutants (containing both-SmD and ram mutations) were probed and compared to an isogenic wild-type strain. In ribosomes from strains containing S4 ram mutations, nucleotides A8 and A26 become more reactive to dimethyl sulfate (DMS) at their N-1 positions. In ribosomes from strains bearing the SmD allele, A908, A909, A1413 and G1487 are significantly less reactive to chemical probes. These same effects are observed when the S4 and S12 mutations are present simultaneously in the double mutants. An interesting correlation is found between the reactivity of A908 and the miscoding potential of SmR, SmD, SmP and wild-type ribosomes; the reactivity of A908 increases as the translational error frequency of the ribosomes increases. In the case of ram ribosomes, the reactivity of A908 resembles that of wild-type, unless tRNA is bound, in which case it becomes hyper-reactive. Similarly, streptomycin has little effect on A908 in wild-type ribosomes unless tRNA is bound, in which case its reactivity increases to resemble that of ram ribosomes with bound tRNA. Finally, interaction of streptomycin with SmP and SmD ribosomes causes the reactivity of A908 to increase to near-wild-type levels. A simple model is proposed, in which the reactivity of A908 reflects the position of an equilibrium between two conformational states of the 30 S subunit, one of which is DMS-reactive, and the other DMS

  18. Transfer RNA-mediated regulation of ribosome dynamics during protein synthesis.

    PubMed

    Fei, Jingyi; Richard, Arianne C; Bronson, Jonathan E; Gonzalez, Ruben L

    2011-08-21

    Translocation of tRNAs through the ribosome during protein synthesis involves large-scale structural rearrangement of the ribosome and ribosome-bound tRNAs that is accompanied by extensive and dynamic remodeling of tRNA-ribosome interactions. How the rearrangement of individual tRNA-ribosome interactions influences tRNA movement during translocation, however, remains largely unknown. To address this question, we used single-molecule FRET to characterize the dynamics of ribosomal pretranslocation (PRE) complex analogs carrying either wild-type or systematically mutagenized tRNAs. Our data reveal how specific tRNA-ribosome interactions regulate the rate of PRE complex rearrangement into a critical, on-pathway translocation intermediate and how these interactions control the stability of the resulting configuration. Notably, our results suggest that the conformational flexibility of the tRNA molecule has a crucial role in directing the structural dynamics of the PRE complex during translocation.

  19. Generation of chemically engineered ribosomes for atomic mutagenesis studies on protein biosynthesis.

    PubMed

    Erlacher, Matthias D; Chirkova, Anna; Voegele, Paul; Polacek, Norbert

    2011-05-01

    The protocol describes the site-specific chemical modification of 23S rRNA of Thermus aquaticus ribosomes. The centerpiece of this 'atomic mutagenesis' approach is the site-specific incorporation of non-natural nucleoside analogs into 23S rRNA in the context of the entire 70S ribosome. This technique exhaustively makes use of the available crystallographic structures of the ribosome for designing detailed biochemical experiments aiming at unraveling molecular insights of ribosomal functions. The generation of chemically engineered ribosomes carrying a particular non-natural 23S rRNA residue at the site of interest, a procedure that typically takes less than 2 d, allows the study of translation at the molecular level and goes far beyond the limits of standard mutagenesis approaches. This methodology, in combination with the presented tests for ribosomal functions adapted to chemically engineered ribosomes, allows unprecedented molecular insight into the mechanisms of protein biosynthesis.

  20. Autogenous Regulation of Splicing of the Transcript of a Yeast Ribosomal Protein Gene

    NASA Astrophysics Data System (ADS)

    Dabeva, Mariana D.; Post-Beittenmiller, Martha A.; Warner, Jonathan R.

    1986-08-01

    The gene for a yeast ribosomal protein, RPL32, contains a single intron. The product of this gene appears to participate in feedback control of the splicing of the intron from the transcript. This autogenous regulation of splicing provides a striking analogy to the autogenous regulation of translation of ribosomal proteins in Escherichia coli.

  1. Ribosomal protein S6 associates with alphavirus nonstructural protein 2 and mediates expression from alphavirus messages.

    PubMed

    Montgomery, Stephanie A; Berglund, Peter; Beard, Clayton W; Johnston, Robert E

    2006-08-01

    Although alphaviruses dramatically alter cellular function within hours of infection, interactions between alphaviruses and specific host cellular proteins are poorly understood. Although the alphavirus nonstructural protein 2 (nsP2) is an essential component of the viral replication complex, it also has critical auxiliary functions that determine the outcome of infection in the host. To gain a better understanding of nsP2 function, we sought to identify cellular proteins with which Venezuelan equine encephalitis virus nsP2 interacted. We demonstrate here that nsP2 associates with ribosomal protein S6 (RpS6) and that nsP2 is present in the ribosome-containing fractions of a polysome gradient, suggesting that nsP2 associates with RpS6 in the context of the whole ribosome. This result was noteworthy, since viral replicase proteins have seldom been described in direct association with components of the ribosome. The association of RpS6 with nsP2 was detected throughout the course of infection, and neither the synthesis of the viral structural proteins nor the presence of the other nonstructural proteins was required for RpS6 interaction with nsP2. nsP1 also was associated with RpS6, but other nonstructural proteins were not. RpS6 phosphorylation was dramatically diminished within hours after infection with alphaviruses. Furthermore, a reduction in the level of RpS6 protein expression led to diminished expression from alphavirus subgenomic messages, whereas no dramatic diminution in cellular translation was observed. Taken together, these data suggest that alphaviruses alter the ribosome during infection and that this alteration may contribute to differential translation of host and viral messages.

  2. A ribosome-inactivating protein in a Drosophila defensive symbiont

    PubMed Central

    Hamilton, Phineas T.; Peng, Fangni; Boulanger, Martin J.; Perlman, Steve J.

    2016-01-01

    Vertically transmitted symbionts that protect their hosts against parasites and pathogens are well known from insects, yet the underlying mechanisms of symbiont-mediated defense are largely unclear. A striking example of an ecologically important defensive symbiosis involves the woodland fly Drosophila neotestacea, which is protected by the bacterial endosymbiont Spiroplasma when parasitized by the nematode Howardula aoronymphium. The benefit of this defense strategy has led to the rapid spread of Spiroplasma throughout the range of D. neotestacea, although the molecular basis for this protection has been unresolved. Here, we show that Spiroplasma encodes a ribosome-inactivating protein (RIP) related to Shiga-like toxins from enterohemorrhagic Escherichia coli and that Howardula ribosomal RNA (rRNA) is depurinated during Spiroplasma-mediated protection of D. neotestacea. First, we show that recombinant Spiroplasma RIP catalyzes depurination of 28S rRNAs in a cell-free assay, as well as Howardula rRNA in vitro at the canonical RIP target site within the α-sarcin/ricin loop (SRL) of 28S rRNA. We then show that Howardula parasites in Spiroplasma-infected flies show a strong signal of rRNA depurination consistent with RIP-dependent modification and large decreases in the proportion of 28S rRNA intact at the α-sarcin/ricin loop. Notably, host 28S rRNA is largely unaffected, suggesting targeted specificity. Collectively, our study identifies a novel RIP in an insect defensive symbiont and suggests an underlying RIP-dependent mechanism in Spiroplasma-mediated defense. PMID:26712000

  3. Organization of proteins in mammalian mitochondrial ribosomes: accessibility to lactoperoxidase-catalyzed radioiodination

    SciTech Connect

    Denslow, N.D.; O'Brien, T.W.

    1984-08-10

    To assess the relative exposure of individual ribosomal proteins (r-proteins) in the large and small subunits of the bovine mitochondrial ribosome, double label iodination technique was used. Regions of r-proteins exposed in purified ribosomal subunits were labeled with /sup 131/I using the lactoperoxidase-catalyzed iodination system, and additional reactive groups available upon denaturing the r-proteins in urea were labeled with /sup 125/I using the chloramine-T mediated reaction. The ratio of /sup 131/I to /sup 125/I incorporated into individual proteins under these conditions is representative of the degree of exposure for each of the proteins in the subunits. In this manner, the r-proteins have been grouped into 3 classes depending on their degree of exposure: high exposure, intermediate exposure, and essentially buried. While both subunits have a few proteins in the highly exposed group, and a large number of proteins in the intermediate exposure group, only the large ribosomal subunit has an appreciable number of proteins which appear essentially buried. The more buried proteins may serve mainly structural roles, perhaps acting as assembly proteins, since many from this group bind to ribosomal RNA. The more superficially disposed proteins may comprise binding sites for macromolecules that interact with ribosomes during protein synthesis, as well as stabilizing the association of the large and small subribosomal particles.

  4. Phosphorylated proteins of the mammalian mitochondrial ribosome: implications in protein synthesis

    PubMed Central

    Miller, Jennifer L.; Cimen, Huseyin; Koc, Hasan; Koc, Emine C.

    2009-01-01

    Mitochondria, the powerhouse of eukaryotic cells, have their own translation machinery that is solely responsible for synthesis of 13 mitochondrially-encoded protein subunits of oxidative phosphorylation complexes. Phosphorylation is a well-known post-translational modification in regulation of many processes in mammalian mitochondria including oxidative phosphorylation. However, there is still very limited knowledge on phosphorylation of mitochondrial ribosomal proteins and their role(s) in ribosome function. In this study, we have identified the mitochondrial ribosomal proteins that are phosphorylated at serine, threonine or tyrosine residues. Twenty-four phosphorylated proteins were visualized by phosphorylation-specific techniques including in vitro radiolabeling, residue specific antibodies for phosphorylated residues, or ProQ phospho dye and identified by tandem mass spectrometry. Translation assays with isolated ribosomes that were phosphorylated in vitro by kinases PKA, PKCδ, or Abl Tyr showed up to 30% inhibition due to phosphorylation. Findings from this study should serve as the framework for future studies addressing the regulation mechanisms of mitochondrial translation machinery by phosphorylation and other post-translational modifications. PMID:19702336

  5. Eukaryote-specific extensions in ribosomal proteins of the small subunit: Structure and function

    PubMed Central

    Ghosh, Arnab; Komar, Anton A

    2015-01-01

    High-resolution structures of yeast ribosomes have improved our understanding of the architecture and organization of eukaryotic rRNA and proteins, as well as eukaryote-specific extensions present in some conserved ribosomal proteins. Despite this progress, assignment of specific functions to individual proteins and/or eukaryote-specific protein extensions remains challenging. It has been suggested that eukaryote-specific extensions of conserved proteins from the small ribosomal subunit may facilitate eukaryote-specific reactions in the initiation phase of protein synthesis. This review summarizes emerging data describing the structural and functional significance of eukaryote-specific extensions of conserved small ribosomal subunit proteins, particularly their possible roles in recruitment and spatial organization of eukaryote-specific initiation factors. PMID:26779416

  6. Analysis of Ribosome Inactivating Protein (RIP): A Bioinformatics Approach

    NASA Astrophysics Data System (ADS)

    Jothi, G. Edward Gnana; Majilla, G. Sahaya Jose; Subhashini, D.; Deivasigamani, B.

    2012-10-01

    In spite of the medical advances in recent years, the world is in need of different sources to encounter certain health issues.Ribosome Inactivating Proteins (RIPs) were found to be one among them. In order to get easy access about RIPs, there is a need to analyse RIPs towards constructing a database on RIPs. Also, multiple sequence alignment was done towards screening for homologues of significant RIPs from rare sources against RIPs from easily available sources in terms of similarity. Protein sequences were retrieved from SWISS-PROT and are further analysed using pair wise and multiple sequence alignment.Analysis shows that, 151 RIPs have been characterized to date. Amongst them, there are 87 type I, 37 type II, 1 type III and 25 unknown RIPs. The sequence length information of various RIPs about the availability of full or partial sequence was also found. The multiple sequence alignment of 37 type I RIP using the online server Multalin, indicates the presence of 20 conserved residues. Pairwise alignment and multiple sequence alignment of certain selected RIPs in two groups namely Group I and Group II were carried out and the consensus level was found to be 98%, 98% and 90% respectively.

  7. Ribosome reinitiation at leader peptides increases translation of bacterial proteins.

    PubMed

    Korolev, Semen A; Zverkov, Oleg A; Seliverstov, Alexandr V; Lyubetsky, Vassily A

    2016-04-16

    Short leader genes usually do not encode stable proteins, although their importance in expression control of bacterial genomes is widely accepted. Such genes are often involved in the control of attenuation regulation. However, the abundance of leader genes suggests that their role in bacteria is not limited to regulation. Specifically, we hypothesize that leader genes increase the expression of protein-coding (structural) genes via ribosome reinitiation at the leader peptide in the case of a short distance between the stop codon of the leader gene and the start codon of the structural gene. For instance, in Actinobacteria, the frequency of leader genes at a distance of 10-11 bp is about 70 % higher than the mean frequency within the 1 to 65 bp range; and it gradually decreases as the range grows longer. A pronounced peak of this frequency-distance relationship is also observed in Proteobacteria, Bacteroidetes, Spirochaetales, Acidobacteria, the Deinococcus-Thermus group, and Planctomycetes. In contrast, this peak falls to the distance of 15-16 bp and is not very pronounced in Firmicutes; and no such peak is observed in cyanobacteria and tenericutes. Generally, this peak is typical for many bacteria. Some leader genes located close to a structural gene probably play a regulatory role as well.

  8. Effects of ribosome-inactivating proteins on Escherichia coli and Agrobacterium tumefaciens translation systems.

    PubMed Central

    Girbés, T; Barbieri, L; Ferreras, M; Arias, F J; Rojo, M A; Iglesias, R; Alegre, C; Escarmis, C; Stirpe, F

    1993-01-01

    The effects of 30 type 1 and of 2 (ricin and volkensin) type 2 ribosome-inactivating proteins (RIPs) on Escherichia coli and Agrobacterium tumefaciens cell-free translation systems were compared with the effects on a rabbit reticulocyte translation system. The depurinating activity of RIPs on E. coli ribosomes was also evaluated. Only six type 1 RIPs inhibited endogenous mRNA-directed translational activity of E. coli lysates, with submicromolar 50% inhibitory concentrations. Four RIPs had similar activities on poly(U)-directed phenylalanine polymerization by E. coli ribosomes, and three RIPs inhibited poly(U)-directed polyphenylalanine synthesis by A. tumefaciens ribosomes, with submicromolar 50% inhibitory concentrations. Images PMID:8407849

  9. Ribosomal proteins as biomarkers for bacterial identification by mass spectrometry in the clinical microbiology laboratory

    PubMed Central

    Suarez, Stéphanie; Ferroni, Agnès; Lotz, Aurélie; Jolley, Keith A.; Guérin, Philippe; Leto, Julie; Dauphin, Brunhilde; Jamet, Anne; Maiden, Martin C.J.; Nassif, Xavier; Armengaud, Jean

    2014-01-01

    Whole-cell matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for identification of microorganisms that is increasingly used in microbiology laboratories. This identification is based on the comparison of the tested isolate mass spectrum with reference databases. Using Neisseria meningitidis as a model organism, we showed that in one of the available databases, the Andromas database, 10 of the 13 species-specific biomarkers correspond to ribosomal proteins. Remarkably, one biomarker, ribosomal protein L32, was subject to inter-strain variability. The analysis of the ribosomal protein patterns of 100 isolates for which whole genome sequences were available, confirmed the presence of inter-strain variability in the molecular weight of 29 ribosomal proteins, thus establishing a correlation between the sequence type (ST) and/or clonal complex (CC) of each strain and its ribosomal protein pattern. Since the molecular weight of three of the variable ribosomal proteins (L30, L31 and L32) was included in the spectral window observed by MALDI-TOF MS in clinical microbiology, i.e., 3640–12000 m/z, we were able by analyzing the molecular weight of these three ribosomal proteins to classify each strain in one of six subgroups, each of these subgroups corresponding to specific STs and/or CCs. Their detection by MALDI-TOF allows therefore a quick typing of N. meningitidis isolates. PMID:23916798

  10. Proteomic profiling of the mitochondrial ribosome identifies Atp25 as a composite mitochondrial precursor protein

    PubMed Central

    Woellhaf, Michael W.; Sommer, Frederik; Schroda, Michael; Herrmann, Johannes M.

    2016-01-01

    Whereas the structure and function of cytosolic ribosomes are well characterized, we only have a limited understanding of the mitochondrial translation apparatus. Using SILAC-based proteomic profiling, we identified 13 proteins that cofractionated with the mitochondrial ribosome, most of which play a role in translation or ribosomal biogenesis. One of these proteins is a homologue of the bacterial ribosome-silencing factor (Rsf). This protein is generated from the composite precursor protein Atp25 upon internal cleavage by the matrix processing peptidase MPP, and in this respect, it differs from all other characterized mitochondrial proteins of baker’s yeast. We observed that cytosolic expression of Rsf, but not of noncleaved Atp25 protein, is toxic. Our results suggest that eukaryotic cells face the challenge of avoiding negative interference from the biogenesis of their two distinct translation machineries. PMID:27582385

  11. Protein quality control at the ribosome: focus on RAC, NAC and RQC.

    PubMed

    Gamerdinger, Martin

    2016-10-15

    The biogenesis of new polypeptides by ribosomes and their subsequent correct folding and localization to the appropriate cellular compartments are essential key processes to maintain protein homoeostasis. These complex mechanisms are governed by a repertoire of protein biogenesis factors that directly bind to the ribosome and chaperone nascent polypeptide chains as soon as they emerge from the ribosomal tunnel exit. This nascent chain 'welcoming committee' regulates multiple co-translational processes including protein modifications, folding, targeting and degradation. Acting at the front of the protein production line, these ribosome-associated protein biogenesis factors lead the way in the cellular proteostasis network to ensure proteome integrity. In this article, I focus on three different systems in eukaryotes that are critical for the maintenance of protein homoeostasis by controlling the birth, life and death of nascent polypeptide chains.

  12. Proteomic profiling of the mitochondrial ribosome identifies Atp25 as a composite mitochondrial precursor protein.

    PubMed

    Woellhaf, Michael W; Sommer, Frederik; Schroda, Michael; Herrmann, Johannes M

    2016-10-15

    Whereas the structure and function of cytosolic ribosomes are well characterized, we only have a limited understanding of the mitochondrial translation apparatus. Using SILAC-based proteomic profiling, we identified 13 proteins that cofractionated with the mitochondrial ribosome, most of which play a role in translation or ribosomal biogenesis. One of these proteins is a homologue of the bacterial ribosome-silencing factor (Rsf). This protein is generated from the composite precursor protein Atp25 upon internal cleavage by the matrix processing peptidase MPP, and in this respect, it differs from all other characterized mitochondrial proteins of baker's yeast. We observed that cytosolic expression of Rsf, but not of noncleaved Atp25 protein, is toxic. Our results suggest that eukaryotic cells face the challenge of avoiding negative interference from the biogenesis of their two distinct translation machineries.

  13. The Arabidopsis TOR Kinase Specifically Regulates the Expression of Nuclear Genes Coding for Plastidic Ribosomal Proteins and the Phosphorylation of the Cytosolic Ribosomal Protein S6

    PubMed Central

    Dobrenel, Thomas; Mancera-Martínez, Eder; Forzani, Céline; Azzopardi, Marianne; Davanture, Marlène; Moreau, Manon; Schepetilnikov, Mikhail; Chicher, Johana; Langella, Olivier; Zivy, Michel; Robaglia, Christophe; Ryabova, Lyubov A.; Hanson, Johannes; Meyer, Christian

    2016-01-01

    Protein translation is an energy consuming process that has to be fine-tuned at both the cell and organism levels to match the availability of resources. The target of rapamycin kinase (TOR) is a key regulator of a large range of biological processes in response to environmental cues. In this study, we have investigated the effects of TOR inactivation on the expression and regulation of Arabidopsis ribosomal proteins at different levels of analysis, namely from transcriptomic to phosphoproteomic. TOR inactivation resulted in a coordinated down-regulation of the transcription and translation of nuclear-encoded mRNAs coding for plastidic ribosomal proteins, which could explain the chlorotic phenotype of the TOR silenced plants. We have identified in the 5′ untranslated regions (UTRs) of this set of genes a conserved sequence related to the 5′ terminal oligopyrimidine motif, which is known to confer translational regulation by the TOR kinase in other eukaryotes. Furthermore, the phosphoproteomic analysis of the ribosomal fraction following TOR inactivation revealed a lower phosphorylation of the conserved Ser240 residue in the C-terminal region of the 40S ribosomal protein S6 (RPS6). These results were confirmed by Western blot analysis using an antibody that specifically recognizes phosphorylated Ser240 in RPS6. Finally, this antibody was used to follow TOR activity in plants. Our results thus uncover a multi-level regulation of plant ribosomal genes and proteins by the TOR kinase. PMID:27877176

  14. The Arabidopsis TOR Kinase Specifically Regulates the Expression of Nuclear Genes Coding for Plastidic Ribosomal Proteins and the Phosphorylation of the Cytosolic Ribosomal Protein S6.

    PubMed

    Dobrenel, Thomas; Mancera-Martínez, Eder; Forzani, Céline; Azzopardi, Marianne; Davanture, Marlène; Moreau, Manon; Schepetilnikov, Mikhail; Chicher, Johana; Langella, Olivier; Zivy, Michel; Robaglia, Christophe; Ryabova, Lyubov A; Hanson, Johannes; Meyer, Christian

    2016-01-01

    Protein translation is an energy consuming process that has to be fine-tuned at both the cell and organism levels to match the availability of resources. The target of rapamycin kinase (TOR) is a key regulator of a large range of biological processes in response to environmental cues. In this study, we have investigated the effects of TOR inactivation on the expression and regulation of Arabidopsis ribosomal proteins at different levels of analysis, namely from transcriptomic to phosphoproteomic. TOR inactivation resulted in a coordinated down-regulation of the transcription and translation of nuclear-encoded mRNAs coding for plastidic ribosomal proteins, which could explain the chlorotic phenotype of the TOR silenced plants. We have identified in the 5' untranslated regions (UTRs) of this set of genes a conserved sequence related to the 5' terminal oligopyrimidine motif, which is known to confer translational regulation by the TOR kinase in other eukaryotes. Furthermore, the phosphoproteomic analysis of the ribosomal fraction following TOR inactivation revealed a lower phosphorylation of the conserved Ser240 residue in the C-terminal region of the 40S ribosomal protein S6 (RPS6). These results were confirmed by Western blot analysis using an antibody that specifically recognizes phosphorylated Ser240 in RPS6. Finally, this antibody was used to follow TOR activity in plants. Our results thus uncover a multi-level regulation of plant ribosomal genes and proteins by the TOR kinase.

  15. The RNA-binding protein Gemin5 binds directly to the ribosome and regulates global translation

    PubMed Central

    Francisco-Velilla, Rosario; Fernandez-Chamorro, Javier; Ramajo, Jorge; Martinez-Salas, Encarnación

    2016-01-01

    RNA-binding proteins (RBPs) play crucial roles in all organisms. The protein Gemin5 harbors two functional domains. The N-terminal domain binds to snRNAs targeting them for snRNPs assembly, while the C-terminal domain binds to IRES elements through a non-canonical RNA-binding site. Here we report a comprehensive view of the Gemin5 interactome; most partners copurified with the N-terminal domain via RNA bridges. Notably, Gemin5 sediments with the subcellular ribosome fraction, and His-Gemin5 binds to ribosome particles via its N-terminal domain. The interaction with the ribosome was lost in F381A and Y474A Gemin5 mutants, but not in W14A and Y15A. Moreover, the ribosomal proteins L3 and L4 bind directly with Gemin5, and conversely, Gemin5 mutants impairing the binding to the ribosome are defective in the interaction with L3 and L4. The overall polysome profile was affected by Gemin5 depletion or overexpression, concomitant to an increase or a decrease, respectively, of global protein synthesis. Gemin5, and G5-Nter as well, were detected on the polysome fractions. These results reveal the ribosome-binding capacity of the N-ter moiety, enabling Gemin5 to control global protein synthesis. Our study uncovers a crosstalk between this protein and the ribosome, and provides support for the view that Gemin5 may control translation elongation. PMID:27507887

  16. The Saccharomyces cerevisiae protein Stm1p facilitates ribosome preservation during quiescence

    SciTech Connect

    Van Dyke, Natalya; Chanchorn, Ekkawit; Van Dyke, Michael W.

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer Stm1p confers increased resistance to the macrolide starvation-mimic rapamycin. Black-Right-Pointing-Pointer Stm1p maintains 80S ribosome integrity during stationary phase-induced quiescence. Black-Right-Pointing-Pointer Stm1p facilitates polysome formation following quiescence exit. Black-Right-Pointing-Pointer Stm1p facilitates protein synthesis following quiescence exit. Black-Right-Pointing-Pointer Stm1p is a ribosome preservation factor under conditions of nutrient deprivation. -- Abstract: Once cells exhaust nutrients from their environment, they enter an alternative resting state known as quiescence, whereby proliferation ceases and essential nutrients are obtained through internal stores and through the catabolism of existing macromolecules and organelles. One example of this is ribophagy, the degradation of ribosomes through the process of autophagy. However, some ribosomes need to be preserved for an anticipated recovery from nutrient deprivation. We found that the ribosome-associated protein Stm1p greatly increases the quantity of 80S ribosomes present in quiescent yeast cells and that these ribosomes facilitate increased protein synthesis rates once nutrients are restored. These findings suggest that Stm1p can act as a ribosome preservation factor under conditions of nutrient deprivation and restoration.

  17. Crystal Structure of Ribosome-Inactivating Protein Ricin A Chain in Complex with the C-Terminal Peptide of the Ribosomal Stalk Protein P2

    PubMed Central

    Shi, Wei-Wei; Tang, Yun-Sang; Sze, See-Yuen; Zhu, Zhen-Ning; Wong, Kam-Bo; Shaw, Pang-Chui

    2016-01-01

    Ricin is a type 2 ribosome-inactivating protein (RIP), containing a catalytic A chain and a lectin-like B chain. It inhibits protein synthesis by depurinating the N-glycosidic bond at α-sarcin/ricin loop (SRL) of the 28S rRNA, which thereby prevents the binding of elongation factors to the GTPase activation center of the ribosome. Here, we present the 1.6 Å crystal structure of Ricin A chain (RTA) complexed to the C-terminal peptide of the ribosomal stalk protein P2, which plays a crucial role in specific recognition of elongation factors and recruitment of eukaryote-specific RIPs to the ribosomes. Our structure reveals that the C-terminal GFGLFD motif of P2 peptide is inserted into a hydrophobic pocket of RTA, while the interaction assays demonstrate the structurally untraced SDDDM motif of P2 peptide contributes to the interaction with RTA. This interaction mode of RTA and P protein is in contrast to that with trichosanthin (TCS), Shiga-toxin (Stx) and the active form of maize RIP (MOD), implying the flexibility of the P2 peptide-RIP interaction, for the latter to gain access to ribosome. PMID:27754366

  18. Revisiting the mechanism of macrolide-antibiotic resistance mediated by ribosomal protein L22

    PubMed Central

    Moore, Sean D.; Sauer, Robert T.

    2008-01-01

    Bacterial antibiotic resistance can occur by many mechanisms. An intriguing class of mutants is resistant to macrolide antibiotics even though these drugs still bind to their targets. For example, a 3-residue deletion (ΔMKR) in ribosomal protein L22 distorts a loop that forms a constriction in the ribosome exit tunnel, apparently allowing nascent-chain egress and translation in the presence of bound macrolides. Here, however, we demonstrate that ΔMKR and wild-type ribosomes show comparable macrolide sensitivity in vitro. In Escherichia coli, we find that this mutation reduces antibiotic occupancy of the target site on ribosomes in a manner largely dependent on the AcrAB-TolC efflux system. We propose a model for antibiotic resistance in which ΔMKR ribosomes alter the translation of specific proteins, possibly via changes in programmed stalling, and modify the cell envelope in a manner that lowers steady-state macrolide levels. PMID:19015512

  19. Revisiting the mechanism of macrolide-antibiotic resistance mediated by ribosomal protein L22.

    PubMed

    Moore, Sean D; Sauer, Robert T

    2008-11-25

    Bacterial antibiotic resistance can occur by many mechanisms. An intriguing class of mutants is resistant to macrolide antibiotics even though these drugs still bind to their targets. For example, a 3-residue deletion (DeltaMKR) in ribosomal protein L22 distorts a loop that forms a constriction in the ribosome exit tunnel, apparently allowing nascent-chain egress and translation in the presence of bound macrolides. Here, however, we demonstrate that DeltaMKR and wild-type ribosomes show comparable macrolide sensitivity in vitro. In Escherichia coli, we find that this mutation reduces antibiotic occupancy of the target site on ribosomes in a manner largely dependent on the AcrAB-TolC efflux system. We propose a model for antibiotic resistance in which DeltaMKR ribosomes alter the translation of specific proteins, possibly via changes in programmed stalling, and modify the cell envelope in a manner that lowers steady-state macrolide levels.

  20. Massively Convergent Evolution for Ribosomal Protein Gene Content in Plastid and Mitochondrial Genomes

    PubMed Central

    Maier, Uwe-G; Zauner, Stefan; Woehle, Christian; Bolte, Kathrin; Hempel, Franziska; Allen, John F.; Martin, William F.

    2013-01-01

    Plastid and mitochondrial genomes have undergone parallel evolution to encode the same functional set of genes. These encode conserved protein components of the electron transport chain in their respective bioenergetic membranes and genes for the ribosomes that express them. This highly convergent aspect of organelle genome evolution is partly explained by the redox regulation hypothesis, which predicts a separate plastid or mitochondrial location for genes encoding bioenergetic membrane proteins of either photosynthesis or respiration. Here we show that convergence in organelle genome evolution is far stronger than previously recognized, because the same set of genes for ribosomal proteins is independently retained by both plastid and mitochondrial genomes. A hitherto unrecognized selective pressure retains genes for the same ribosomal proteins in both organelles. On the Escherichia coli ribosome assembly map, the retained proteins are implicated in 30S and 50S ribosomal subunit assembly and initial rRNA binding. We suggest that ribosomal assembly imposes functional constraints that govern the retention of ribosomal protein coding genes in organelles. These constraints are subordinate to redox regulation for electron transport chain components, which anchor the ribosome to the organelle genome in the first place. As organelle genomes undergo reduction, the rRNAs also become smaller. Below size thresholds of approximately 1,300 nucleotides (16S rRNA) and 2,100 nucleotides (26S rRNA), all ribosomal protein coding genes are lost from organelles, while electron transport chain components remain organelle encoded as long as the organelles use redox chemistry to generate a proton motive force. PMID:24259312

  1. The ribosomal protein genes and Minute loci of Drosophila melanogaster

    PubMed Central

    Marygold, Steven J; Roote, John; Reuter, Gunter; Lambertsson, Andrew; Ashburner, Michael; Millburn, Gillian H; Harrison, Paul M; Yu, Zhan; Kenmochi, Naoya; Kaufman, Thomas C; Leevers, Sally J; Cook, Kevin R

    2007-01-01

    Background Mutations in genes encoding ribosomal proteins (RPs) have been shown to cause an array of cellular and developmental defects in a variety of organisms. In Drosophila melanogaster, disruption of RP genes can result in the 'Minute' syndrome of dominant, haploinsufficient phenotypes, which include prolonged development, short and thin bristles, and poor fertility and viability. While more than 50 Minute loci have been defined genetically, only 15 have so far been characterized molecularly and shown to correspond to RP genes. Results We combined bioinformatic and genetic approaches to conduct a systematic analysis of the relationship between RP genes and Minute loci. First, we identified 88 genes encoding 79 different cytoplasmic RPs (CRPs) and 75 genes encoding distinct mitochondrial RPs (MRPs). Interestingly, nine CRP genes are present as duplicates and, while all appear to be functional, one member of each gene pair has relatively limited expression. Next, we defined 65 discrete Minute loci by genetic criteria. Of these, 64 correspond to, or very likely correspond to, CRP genes; the single non-CRP-encoding Minute gene encodes a translation initiation factor subunit. Significantly, MRP genes and more than 20 CRP genes do not correspond to Minute loci. Conclusion This work answers a longstanding question about the molecular nature of Minute loci and suggests that Minute phenotypes arise from suboptimal protein synthesis resulting from reduced levels of cytoribosomes. Furthermore, by identifying the majority of haplolethal and haplosterile loci at the molecular level, our data will directly benefit efforts to attain complete deletion coverage of the D. melanogaster genome. PMID:17927810

  2. Studies on the Assembly Characteristics of Large Subunit Ribosomal Proteins in S. cerevisae

    PubMed Central

    Ohmayer, Uli; Gamalinda, Michael; Sauert, Martina; Ossowski, Julius; Pöll, Gisela; Linnemann, Jan; Hierlmeier, Thomas; Perez-Fernandez, Jorge; Kumcuoglu, Beril; Leger-Silvestre, Isabelle; Faubladier, Marlène; Griesenbeck, Joachim; Woolford, John; Tschochner, Herbert; Milkereit, Philipp

    2013-01-01

    During the assembly process of ribosomal subunits, their structural components, the ribosomal RNAs (rRNAs) and the ribosomal proteins (r-proteins) have to join together in a highly dynamic and defined manner to enable the efficient formation of functional ribosomes. In this work, the assembly of large ribosomal subunit (LSU) r-proteins from the eukaryote S. cerevisiae was systematically investigated. Groups of LSU r-proteins with specific assembly characteristics were detected by comparing the protein composition of affinity purified early, middle, late or mature LSU (precursor) particles by semi-quantitative mass spectrometry. The impact of yeast LSU r-proteins rpL25, rpL2, rpL43, and rpL21 on the composition of intermediate to late nuclear LSU precursors was analyzed in more detail. Effects of these proteins on the assembly states of other r-proteins and on the transient LSU precursor association of several ribosome biogenesis factors, including Nog2, Rsa4 and Nop53, are discussed. PMID:23874617

  3. Ribosomal protein alterations in thiostrepton- and Micrococcin-resistant mutants of Bacillus subtilis.

    PubMed

    Wienen, B; Ehrlich, R; Stöffler-Meilicke, M; Stöffler, G; Smith, I; Weiss, D; Vince, R; Pestka, S

    1979-08-25

    Ribosomal proteins of parental thiostrepton- and micrococcin-sensitive Bacillus subtilis cysA14 and thiostrepton-and micrococcin-resistant mutants were compared. Several electrophoretic and immunochemical techniques showed unambiguously that BS-L11 was not present on 50 S ribosomal subunits from the six thiostrepton-resistant mutants. Protein BS-L11 reappeared in all six revertants from thiostrepton resistance to thiostrepton sensitivity. No definitive protein alteration could be ascribed to the mutation from micrococcin sensitivity to resistance. It was also demonstrated that B. subtilis protein BS-L11 is homologous to Escherichia coli ribosomal protein L11. The finding that ribosomes from thiostrepton-resistant mutants do not contain protein L11 suggests that L11 not only is involved in binding of thiostrepton, but also, when mutationally altered, confers resistance to this antibiotic. Although the ribosomes of these strains do not contain protein L11, all thiostrepton-resistant mutants showed the same viability as the parental strain. Thus protein L11 cannot be obligatory for the structure and function of the ribosome.

  4. YaeJ is a novel ribosome-associated protein in Escherichia coli that can hydrolyze peptidyl-tRNA on stalled ribosomes.

    PubMed

    Handa, Yoshihiro; Inaho, Noriyuki; Nameki, Nobukazu

    2011-03-01

    In bacteria, ribosomes often become stalled and are released by a trans-translation process mediated by transfer-messenger RNA (tmRNA). In the absence of tmRNA, however, there is evidence that stalled ribosomes are released from non-stop mRNAs. Here, we show a novel ribosome rescue system mediated by a small basic protein, YaeJ, from Escherichia coli, which is similar in sequence and structure to the catalytic domain 3 of polypeptide chain release factor (RF). In vitro translation experiments using the E. coli-based reconstituted cell-free protein synthesis system revealed that YaeJ can hydrolyze peptidyl-tRNA on ribosomes stalled by both non-stop mRNAs and mRNAs containing rare codon clusters that extend downstream from the P-site and prevent Ala-tmRNA•SmpB from entering the empty A-site. In addition, YaeJ had no effect on translation of a normal mRNA with a stop codon. These results suggested a novel tmRNA-independent rescue system for stalled ribosomes in E. coli. YaeJ was almost exclusively found in the 70S ribosome and polysome fractions after sucrose density gradient sedimentation, but was virtually undetectable in soluble fractions. The C-terminal basic residue-rich extension was also found to be required for ribosome binding. These findings suggest that YaeJ functions as a ribosome-attached rescue device for stalled ribosomes.

  5. Phosphorylation of ribosomal protein S6 mediates compensatory renal hypertrophy.

    PubMed

    Xu, Jinxian; Chen, Jianchun; Dong, Zheng; Meyuhas, Oded; Chen, Jian-Kang

    2015-03-01

    The molecular mechanism underlying renal hypertrophy and progressive nephron damage remains poorly understood. Here we generated congenic ribosomal protein S6 (rpS6) knock-in mice expressing nonphosphorylatable rpS6 and found that uninephrectomy-induced renal hypertrophy was significantly blunted in these knock-in mice. Uninephrectomy-induced increases in cyclin D1 and decreases in cyclin E in the remaining kidney were attenuated in the knock-in mice compared with their wild-type littermates. Uninephrectomy induced rpS6 phosphorylation in the wild-type mice; however, no rpS6 phosphorylation was detected in uninephrectomized or sham-operated knock-in mice. Nonetheless, uninephrectomy stimulated comparable 4E-BP1 phosphorylation in both knock-in and wild-type mice, indicating that mTORC1 was still activated in the knock-in mice. Moreover, the mTORC1 inhibitor rapamycin prevented both rpS6 and 4E-BP1 phosphorylation, significantly blunted uninephrectomy-induced renal hypertrophy in wild-type mice, but did not prevent residual renal hypertrophy despite inhibiting 4E-BP1 phosphorylation in uninephrectomized knock-in mice. Thus, both genetic and pharmacological approaches unequivocally demonstrate that phosphorylated rpS6 is a downstream effector of the mTORC1-S6K1 signaling pathway mediating renal hypertrophy. Hence, rpS6 phosphorylation facilitates the increase in cyclin D1 and decrease in cyclin E1 that underlie the hypertrophic nature of uninephrectomy-induced kidney growth.

  6. Sequential domain assembly of ribosomal protein S3 drives 40S subunit maturation

    PubMed Central

    Mitterer, Valentin; Murat, Guillaume; Réty, Stéphane; Blaud, Magali; Delbos, Lila; Stanborough, Tamsyn; Bergler, Helmut; Leulliot, Nicolas; Kressler, Dieter; Pertschy, Brigitte

    2016-01-01

    Eukaryotic ribosomes assemble by association of ribosomal RNA with ribosomal proteins into nuclear precursor particles, which undergo a complex maturation pathway coordinated by non-ribosomal assembly factors. Here, we provide functional insights into how successive structural re-arrangements in ribosomal protein S3 promote maturation of the 40S ribosomal subunit. We show that S3 dimerizes and is imported into the nucleus with its N-domain in a rotated conformation and associated with the chaperone Yar1. Initial assembly of S3 with 40S precursors occurs via its C-domain, while the N-domain protrudes from the 40S surface. Yar1 is replaced by the assembly factor Ltv1, thereby fixing the S3 N-domain in the rotated orientation and preventing its 40S association. Finally, Ltv1 release, triggered by phosphorylation, and flipping of the S3 N-domain into its final position results in the stable integration of S3. Such a stepwise assembly may represent a new paradigm for the incorporation of ribosomal proteins. PMID:26831757

  7. Fragile X mental retardation protein regulates translation by binding directly to the ribosome.

    PubMed

    Chen, Eileen; Sharma, Manjuli R; Shi, Xinying; Agrawal, Rajendra K; Joseph, Simpson

    2014-05-08

    Fragile X syndrome (FXS) is the most common form of inherited mental retardation, and it is caused by loss of function of the fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein that is involved in the translational regulation of several neuronal mRNAs. However, the precise mechanism of translational inhibition by FMRP is unknown. Here, we show that FMRP inhibits translation by binding directly to the L5 protein on the 80S ribosome. Furthermore, cryoelectron microscopic reconstruction of the 80S ribosome⋅FMRP complex shows that FMRP binds within the intersubunit space of the ribosome such that it would preclude the binding of tRNA and translation elongation factors on the ribosome. These findings suggest that FMRP inhibits translation by blocking the essential components of the translational machinery from binding to the ribosome.

  8. Molecular basis for protection of ribosomal protein L4 from cellular degradation

    PubMed Central

    Huber, Ferdinand M.; Hoelz, André

    2017-01-01

    Eukaryotic ribosome biogenesis requires the nuclear import of ∼80 nascent ribosomal proteins and the elimination of excess amounts by the cellular degradation machinery. Assembly chaperones recognize nascent unassembled ribosomal proteins and transport them together with karyopherins to their nuclear destination. We report the crystal structure of ribosomal protein L4 (RpL4) bound to its dedicated assembly chaperone of L4 (Acl4), revealing extensive interactions sequestering 70 exposed residues of the extended RpL4 loop. The observed molecular recognition fundamentally differs from canonical promiscuous chaperone–substrate interactions. We demonstrate that the eukaryote-specific RpL4 extension harbours overlapping binding sites for Acl4 and the nuclear transport factor Kap104, facilitating its continuous protection from the cellular degradation machinery. Thus, Acl4 serves a dual function to facilitate nuclear import and simultaneously protect unassembled RpL4 from the cellular degradation machinery. PMID:28148929

  9. Nuclear import of dimerized ribosomal protein Rps3 in complex with its chaperone Yar1

    PubMed Central

    Mitterer, Valentin; Gantenbein, Nadine; Birner-Gruenberger, Ruth; Murat, Guillaume; Bergler, Helmut; Kressler, Dieter; Pertschy, Brigitte

    2016-01-01

    After their cytoplasmic synthesis, ribosomal proteins need to be transported into the nucleus, where they assemble with ribosomal RNA into pre-ribosomal particles. Due to their physicochemical properties, they need protection from aggregation on this path. Newly synthesized ribosomal protein Rps3 forms a dimer that is associated with one molecule of its specific chaperone Yar1. Here we report that redundant pathways contribute to the nuclear import of Rps3, with the classical importin α/β pathway (Kap60/Kap95 in yeast) constituting a main import route. The Kap60/Kap95 heterodimer mediates efficient nuclear import of Rps3 by recognition of an N-terminal monopartite nuclear localization signal (NLS). This Rps3-NLS is located directly adjacent to the Yar1-binding site and, upon binding of Kap60 to Rps3, Yar1 is displaced from the ribosomal protein in vitro. While Yar1 does not directly interact with Kap60 in vitro, affinity purifications of Yar1 and Rps3, however, revealed that Kap60 is present in the Rps3/Yar1 complex in vivo. Indeed we could reconstitute such a protein complex containing Rps3 and both Yar1 and Kap60 in vitro. Our data suggest that binding of Yar1 to one N-domain and binding of Kap60 to the second N-domain of dimerized Rps3 orchestrates import and protection of the ribosomal protein. PMID:27819319

  10. Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

    PubMed Central

    Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B.; Webb, Kristofor; Bennett, Eric J.; Vinterbo, Staal; Potter, Clinton S.; Carragher, Bridget; Joazeiro, Claudio A. P.

    2014-01-01

    All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes. PMID:25349383

  11. Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex.

    PubMed

    Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B; Webb, Kristofor; Bennett, Eric J; Vinterbo, Staal; Potter, Clinton S; Carragher, Bridget; Joazeiro, Claudio A P

    2014-11-11

    All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes.

  12. Fabrication and characteristics of MOSFET protein chip for detection of ribosomal protein.

    PubMed

    Park, Keun-Yong; Kim, Min-Suk; Choi, Sie-Young

    2005-04-15

    A metal oxide silicon field effect transistor (MOSFET) protein chip for the easy detection of protein was fabricated and its characteristics were investigated. Generally, the drain current of the MOSFET is varied by the gate potential. It is expected that the formation of an antibody-antigen complex on the gate of MOSFET would lead to a detectable change in the charge distribution and thus, directly modulate the drain current of MOSFET. As such, the drain current of the MOSFET protein chip can be varied by ribosomal proteins absorbed by the self-assembled monolayer (SAM) immobilized on the gate (Au) surface, as ribosomal protein has positive charge, and these current variations then used as the response of the protein chip. The gate of MOSFET protein chip is not directly biased by an external voltage source, so called open gate or floating gate MOSFET, but rather chemically modified by immobilized molecular receptors called self-assembled monolayer (SAM). In our experiments, the current variation in the proposed protein chip was about 8% with a protein concentration of 0.7 mM. As the protein concentration increased, the drain current also gradually increased. In addition, there were some drift of the drain current in the device. It is considered that these drift might be caused by the drift from the MOSFET itself or protein absorption procedures that are relied on the facile attachment of thiol (-S) ligands to the gate (Au) surface. We verified the formation of SAM on the gold surface and the absorption of protein through the surface plasmon resonance (SPR) measurement.

  13. Ribosomal analysis of rapid rates of protein synthesis in the Antarctic sea urchin Sterechinus neumayeri.

    PubMed

    Pace, Douglas A; Maxson, Robert; Manahan, Donal T

    2010-02-01

    Previous research has shown that developing stages of the Antarctic sea urchin Sterechinus neumayeri have high rates of protein synthesis that are comparable to those of similar species living in much warmer waters. Direct measurements of the biosynthetic capacities of isolated ribosomes have not been reported for marine organisms living in the extreme-cold environment of Antarctica. Such measurements are required for a mechanistic understanding of how the critical and highly complex processes involved in protein synthesis are regulated in animals living in the coldest marine environment on Earth (< -1 degrees C). We tested the hypothesis that high rates of protein synthesis in the cold are a direct result of high biosynthetic capacities of ribosomes engaged in protein synthesis. Our results show that the rate at which ribosomes manufacture proteins (i.e., the peptide elongation rate) at -1 degrees C is surprisingly similar to rates measured in other sea urchin species at temperatures that are over 15 degrees C warmer. Average peptide elongation rates for a range of developmental stages of the Antarctic sea urchin were 0.36 codons s(-1) (+/- 0.05, SE). On the basis of subcellular rate determinations of ribosomal activity, we calculated stage-specific rates of protein synthesis for blastulae and gastrulae to be 3.7 and 6.5 ng protein h(-1), respectively. These findings support the conclusion that the high rates of biosynthesis previously reported for the Antarctic sea urchin are an outcome of high ribosomal activities.

  14. In vitro expression of Escherichia coli ribosomal protein genes: autogenous inhibition of translation.

    PubMed Central

    Yates, J L; Arfsten, A E; Nomura, M

    1980-01-01

    Escherichia coli ribosomal protein L1 (0.5 micro M) was found to inhibit the synthesis of both proteins of the L11 operon, L11 and L1, but not the synthesis of other proteins directed by lambda rifd 18 DNA. Similarly, S4 (1 micro M) selectively inhibited the synthesis of three proteins of the alpha operon, S13, S11, and S4, directed by lambda spcI DNA or a restriction enzyme fragment obtained from this DNA. S8 (3.6 micro M) also showed preferential inhibitory effects on the synthesis of some proteins encoded in the spc operon, L24 and L5 (and probably S14 and S8), directed by lambda spcl DNA or a restriction enzyme fragment carrying the genes for these proteins. The inhibitory effect of L1 was observed only with L1 and not with other proteins examined, including S4 and S8. Similarly, the effect of S4 was not observed with L1 or S8, and that of S8 was not seen with L1 or S4. Inhibition was shown to take place at the level of translation rather than transcription. Thus, at least some ribosomal proteins (L1 S4, and S8) have the ability to cause selective translational inhibition of the synthesis of certain ribosomal proteins whose genes are in the same operon as their own. These results support the hypothesis that certain free ribosomal proteins not assembled into ribosomes act as "autogenous" feedback inhibitors to regulate the synthesis of ribosomal proteins. Images PMID:6445562

  15. SRY interacts with ribosomal proteins S7 and L13a in nuclear speckles.

    PubMed

    Sato, Youichi; Yano, Shojiro; Ewis, Ashraf A; Nakahori, Yutaka

    2011-05-01

    The SRY (sex-determining region on the Y chromosome) is essential for male development; however, the molecular mechanism by which the SRY induces testis development is still unclear. To elucidate the mechanism of testis development, we identified SRY-interacting proteins using a yeast two-hybrid system. We found two ribosomal proteins, RPS7 (ribosomal protein S7) and RPL13a (ribosomal protein L13a) that interact with the HMG (high-mobility group) box domain of SRY. Furthermore, we confirmed the intracellular distributions of RPS7, RPL13a and SRY and found that the three proteins were co-expressed in COS1 cells. SRY, RPS7 and RPL13a were co-localized in nuclear speckles. These findings suggest that SRY plays an important role in activities associated with nuclear speckles via an unknown mechanism.

  16. Listerin-dependent nascent protein ubiquitination relies on ribosome subunit dissociation.

    PubMed

    Shao, Sichen; von der Malsburg, Karina; Hegde, Ramanujan S

    2013-06-06

    Quality control of defective mRNAs relies on their translation to detect the lesion. Aberrant proteins are therefore an obligate byproduct of mRNA surveillance and must be degraded to avoid disrupting protein homeostasis. These defective translation products are thought to be ubiquitinated at the ribosome, but the mechanism of ubiquitin ligase selectivity for these ribosomes is not clear. Here, we in vitro reconstitute ubiquitination of nascent proteins produced from aberrant mRNAs. Stalled 80S ribosome-nascent chain complexes are dissociated by the ribosome recycling factors Hbs1/Pelota/ABCE1 to a unique 60S-nascent chain-tRNA complex. The ubiquitin ligase Listerin preferentially recognizes 60S-nascent chains and triggers efficient nascent chain ubiquitination. Interfering with Hbs1 function stabilizes 80S complexes, precludes efficient Listerin recruitment, and reduces nascent chain ubiquitination. Thus, ribosome recycling factors control Listerin localization, explaining how translation products of mRNA surveillance are efficiently ubiquitinated while sparing translating ribosomes.

  17. Dissecting the transcriptional phenotype of ribosomal protein deficiency: implications for Diamond-Blackfan Anemia

    PubMed Central

    Aspesi, Anna; Pavesi, Elisa; Robotti, Elisa; Crescitelli, Rossella; Boria, Ilenia; Avondo, Federica; Moniz, Hélène; Da Costa, Lydie; Mohandas, Narla; Roncaglia, Paola; Ramenghi, Ugo; Ronchi, Antonella; Gustincich, Stefano; Merlin, Simone; Marengo, Emilio; Ellis, Steven R.; Follenzi, Antonia; Santoro, Claudio; Dianzani, Irma

    2014-01-01

    Defects in genes encoding ribosomal proteins cause Diamond Blackfan Anemia (DBA), a red cell aplasia often associated with physical abnormalities. Other bone marrow failure syndromes have been attributed to defects in ribosomal components but the link between erythropoiesis and the ribosome remains to be fully defined. Several lines of evidence suggest that defects in ribosome synthesis lead to “ribosomal stress” with p53 activation and either cell cycle arrest or induction of apoptosis. Pathways independent of p53 have also been proposed to play a role in DBA pathogenesis. We took an unbiased approach to identify p53-independent pathways activated by defects in ribosome synthesis by analyzing global gene expression in various cellular models of DBA. Ranking-Principal Component Analysis (Ranking-PCA) was applied to the identified datasets to determine whether there are common sets of genes whose expression is altered in these different cellular models. We observed consistent changes in the expression of genes involved in cellular amino acid metabolic process, negative regulation of cell proliferation and cell redox homeostasis. These data indicate that cells respond to defects in ribosome synthesis by changing the level of expression of a limited subset of genes involved in critical cellular processes. Moreover, our data support a role for p53-independent pathways in the pathophysiology of DBA. PMID:24835311

  18. Transfer RNA-mediated regulation of ribosome dynamics during protein synthesis

    PubMed Central

    Fei, Jingyi; Richard, Arianne C.; Bronson, Jonathan E.; Gonzalez, & Ruben L.

    2011-01-01

    Translocation of transfer RNAs (tRNAs) through the ribosome during protein synthesis involves large-scale structural rearrangements of the ribosome and the ribosome-bound tRNAs that are accompanied by extensive and dynamic remodeling of tRNA-ribosome interactions. The contributions that rearranging individual tRNA-ribosome interactions make to directing tRNA movements during translocation, however, remain largely unknown. To address this question, we have used single-molecule fluorescence resonance energy transfer to characterize the dynamics of ribosomal pre-translocation (PRE) complex analogs carrying either wild-type or systematically mutagenized tRNAs. Our data reveal how specific tRNA-ribosome interactions regulate the rate with which the PRE complex rearranges into a critical, on-pathway translocation intermediate and how these interactions control the stability of the resulting configuration. More interestingly, our results suggest that the conformational flexibility of the tRNA molecule itself plays a crucial role in directing the structural dynamics of the PRE complex during translocation. PMID:21857664

  19. Dosage Sensitivity of RPL9 and Concerted Evolution of Ribosomal Protein Genes in Plants

    PubMed Central

    Devis, Deborah; Firth, Sue M.; Liang, Zhe; Byrne, Mary E.

    2015-01-01

    The ribosome in higher eukaryotes is a large macromolecular complex composed of four rRNAs and eighty different ribosomal proteins. In plants, each ribosomal protein is encoded by multiple genes. Duplicate genes within a family are often necessary to provide a threshold dose of a ribosomal protein but in some instances appear to have non-redundant functions. Here, we addressed whether divergent members of the RPL9 gene family are dosage sensitive or whether these genes have non-overlapping functions. The RPL9 family in Arabidopsis thaliana comprises two nearly identical members, RPL9B and RPL9C, and a more divergent member, RPL9D. Mutations in RPL9C and RPL9D genes lead to delayed growth early in development, and loss of both genes is embryo lethal, indicating that these are dosage-sensitive and redundant genes. Phylogenetic analysis of RPL9 as well as RPL4, RPL5, RPL27a, RPL36a, and RPS6 family genes in the Brassicaceae indicated that multicopy ribosomal protein genes have been largely retained following whole genome duplication. However, these gene families also show instances of tandem duplication, small scale deletion, and evidence of gene conversion. Furthermore, phylogenetic analysis of RPL9 genes in angiosperm species showed that genes within a species are more closely related to each other than to RPL9 genes in other species, suggesting ribosomal protein genes undergo convergent evolution. Our analysis indicates that ribosomal protein gene retention following whole genome duplication contributes to the number of genes in a family. However, small scale rearrangements influence copy number and likely drive concerted evolution of these dosage-sensitive genes. PMID:26734020

  20. Molecular cloning and characterization of a cDNA encoding the Paracoccidioides brasiliensis 135 ribosomal protein.

    PubMed

    Jesuino, Rosália S A; Pereira, Maristela; Felipe, M Sueli S; Azevedo, Maristella O; Soares, Célia M A

    2004-06-01

    A 630 bp cDNA encoding an L35 ribosomal protein of Paracoccidioides brasiliensis, designated as Pbl35, was cloned from a yeast expression library. Pbl35 encodes a polypeptide of 125 amino acids, with a predicted molecular mass of 14.5 kDa and a pI of 11.0. The deduced PbL35 shows significant conservation in respect to other described ribosomal L35 proteins from eukaryotes and prokaryotes. Motifs of ribosomal proteins are present in PbL35, including a bipartite nuclear localization signal (NLS) that could be related to the protein addressing to the nucleolus for the ribosomal assembly. The mRNA for PbL35, about 700 nucleotides in length, is expressed at a high level in P. brasiliensis. The PbL35 and the deduced amino acid sequence constitute the first description of a ribosomal protein in P. brasiliensis. The cDNA was deposited in GenBank under accession number AF416509.

  1. Ribosome profiling reveals pervasive translation outside of annotated protein-coding genes.

    PubMed

    Ingolia, Nicholas T; Brar, Gloria A; Stern-Ginossar, Noam; Harris, Michael S; Talhouarne, Gaëlle J S; Jackson, Sarah E; Wills, Mark R; Weissman, Jonathan S

    2014-09-11

    Ribosome profiling suggests that ribosomes occupy many regions of the transcriptome thought to be noncoding, including 5' UTRs and long noncoding RNAs (lncRNAs). Apparent ribosome footprints outside of protein-coding regions raise the possibility of artifacts unrelated to translation, particularly when they occupy multiple, overlapping open reading frames (ORFs). Here, we show hallmarks of translation in these footprints: copurification with the large ribosomal subunit, response to drugs targeting elongation, trinucleotide periodicity, and initiation at early AUGs. We develop a metric for distinguishing between 80S footprints and nonribosomal sources using footprint size distributions, which validates the vast majority of footprints outside of coding regions. We present evidence for polypeptide production beyond annotated genes, including the induction of immune responses following human cytomegalovirus (HCMV) infection. Translation is pervasive on cytosolic transcripts outside of conserved reading frames, and direct detection of this expanded universe of translated products enables efforts at understanding how cells manage and exploit its consequences.

  2. Peptide Chain Termination: Effect of Protein S on Ribosomal Binding of Release Factors

    PubMed Central

    Goldstein, J. L.; Caskey, C. T.

    1970-01-01

    The protein factor S, previously shown to stimulate polypeptide chain termination in bacterial extracts, has two effects upon the complex formed between ribosomes, release factor, and terminator (trinucleotide) codon: (1) in the absence of GTP or GDP, S stimulates formation of an [R·UAA·ribosome] intermediate, and (2) in the presence of GTP or GDP, S participates in dissociation of this intermediate. Factor S can stimulate fMet release from [fMet-tRNAf·AUG·ribosome] intermediates in either the presence or absence of GTP or GDP. A model is proposed which relates the in vitro effects of S ± GTP (or GDP) on fMet release to the effects of S ± GTP (or GDP) on the binding and dissociation of R factor from ribosomes. PMID:5289007

  3. Specific N-terminal cleavage of ribosomal protein L27 in Staphylococcus aureus and related bacteria

    PubMed Central

    Wall, Erin A.; Caufield, J. Harry; Lyons, Charles E.; Manning, Keith A.; Dokland, Terje; Christie, Gail E.

    2015-01-01

    Summary Ribosomal protein L27 is a component of the eubacterial large ribosomal subunit that has been shown to play a critical role in substrate stabilization during protein synthesis. This function is mediated by the L27 N-terminus, which protrudes into the peptidyl transferase center. In this report we demonstrate that L27 in Staphylococcus aureus and other Firmicutes is encoded with an N-terminal extension that is not present in most Gram-negative organisms, and is absent from mature ribosomes. We have identified a cysteine protease, conserved among bacteria containing the L27 N-terminal extension, which performs post-translational cleavage of L27. Ribosomal biology in eubacteria has largely been studied in the Gram negative bacterium Escherichia coli; our findings indicate that there are aspects of the basic biology of the ribosome in S. aureus and other related bacteria that differ substantially from that of the E. coli ribosome. This research lays the foundation for the development of new therapeutic approaches that target this novel pathway. PMID:25388641

  4. Mutual protection of ribosomal proteins L5 and L11 from degradation is essential for p53 activation upon ribosomal biogenesis stress

    PubMed Central

    Bursać, Sladana; Brdovčak, Maja Cokarić; Pfannkuchen, Martin; Orsolić, Ines; Golomb, Lior; Zhu, Yan; Katz, Chen; Daftuar, Lilyn; Grabušić, Kristina; Vukelić, Iva; Filić, Vedrana; Oren, Moshe; Prives, Carol; Volarević, Siniša

    2012-01-01

    Impairment of ribosomal biogenesis can activate the p53 protein independently of DNA damage. The ability of ribosomal proteins L5, L11, L23, L26, or S7 to bind Mdm2 and inhibit its ubiquitin ligase activity has been suggested as a critical step in p53 activation under these conditions. Here, we report that L5 and L11 are particularly important for this response. Whereas several other newly synthesized ribosomal proteins are degraded by proteasomes upon inhibition of Pol I activity by actinomycin D, L5 and L11 accumulate in the ribosome-free fraction where they bind to Mdm2. This selective accumulation of free L5 and L11 is due to their mutual protection from proteasomal degradation. Furthermore, the endogenous, newly synthesized L5 and L11 continue to be imported into nucleoli even after nucleolar disruption and colocalize with Mdm2, p53, and promyelocytic leukemia protein. This suggests that the disrupted nucleoli may provide a platform for L5- and L11-dependent p53 activation, implying a role for the nucleolus in p53 activation by ribosomal biogenesis stress. These findings may have important implications with respect to understanding the pathogenesis of diseases caused by impaired ribosome biogenesis. PMID:23169665

  5. Gene clusters for ribosomal proteins in the mitochondrial genome of a liverwort, Marchantia polymorpha.

    PubMed Central

    Takemura, M; Oda, K; Yamato, K; Ohta, E; Nakamura, Y; Nozato, N; Akashi, K; Ohyama, K

    1992-01-01

    We detected 16 genes for ribosomal proteins in the complete sequence of the mitochondrial DNA from a liverwort, Marchantia polymorpha. The genes formed two major clusters, rps12-rps7 and rps10-rpl2-rps19-rps3-rpl16-rpl5- rps14-rps8- rpl6-rps13-rps11-rps1, very similar in organization to Escherichia coli ribosomal protein operons (str and S10-spc-alpha operons, respectively). In contrast, rps2 and rps4 genes were located separately in the liverwort mitochondrial genome (the latter was part of the alpha operon in E. coli). Furthermore, several ribosomal proteins encoded by the liverwort mitochondrial genome differed substantially in size from their counterparts in E. coli and liverwort chloroplast. PMID:1620617

  6. Pokeweed Antiviral Protein, a Ribosome Inactivating Protein: Activity, Inhibition and Prospects

    PubMed Central

    Domashevskiy, Artem V.; Goss, Dixie J.

    2015-01-01

    Viruses employ an array of elaborate strategies to overcome plant defense mechanisms and must adapt to the requirements of the host translational systems. Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome inactivating protein (RIP) and is an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin (S/R) loop of large rRNA, arresting protein synthesis at the translocation step. PAP is thought to play an important role in the plant’s defense mechanism against foreign pathogens. This review focuses on the structure, function, and the relationship of PAP to other RIPs, discusses molecular aspects of PAP antiviral activity, the novel inhibition of this plant toxin by a virus counteraction—a peptide linked to the viral genome (VPg), and possible applications of RIP-conjugated immunotoxins in cancer therapeutics. PMID:25635465

  7. Reduced expression of the mouse ribosomal protein Rpl17 alters the diversity of mature ribosomes by enhancing production of shortened 5.8S rRNA

    PubMed Central

    Wang, Minshi; Parshin, Andrey V.; Shcherbik, Natalia; Pestov, Dimitri G.

    2015-01-01

    Processing of rRNA during ribosome assembly can proceed through alternative pathways but it is unclear whether this could affect the structure of the ribosome. Here, we demonstrate that shortage of a ribosomal protein can change pre-rRNA processing in a way that over time alters ribosome diversity in the cell. Reducing the amount of Rpl17 in mouse cells led to stalled 60S subunit maturation, causing degradation of most of the synthesized precursors. A fraction of pre-60S subunits, however, were able to complete maturation, but with a 5′-truncated 5.8S rRNA, which we named 5.8SC. The 5′ exoribonuclease Xrn2 is involved in the generation of both 5.8SC and the canonical long form of 5.8S rRNA. Ribosomes containing 5.8SC rRNA are present in various mouse and human cells and engage in translation. These findings uncover a previously undescribed form of mammalian 5.8S rRNA and demonstrate that perturbations in ribosome assembly can be a source of heterogeneity in mature ribosomes. PMID:25995445

  8. Reduced expression of the mouse ribosomal protein Rpl17 alters the diversity of mature ribosomes by enhancing production of shortened 5.8S rRNA.

    PubMed

    Wang, Minshi; Parshin, Andrey V; Shcherbik, Natalia; Pestov, Dimitri G

    2015-07-01

    Processing of rRNA during ribosome assembly can proceed through alternative pathways but it is unclear whether this could affect the structure of the ribosome. Here, we demonstrate that shortage of a ribosomal protein can change pre-rRNA processing in a way that over time alters ribosome diversity in the cell. Reducing the amount of Rpl17 in mouse cells led to stalled 60S subunit maturation, causing degradation of most of the synthesized precursors. A fraction of pre-60S subunits, however, were able to complete maturation, but with a 5'-truncated 5.8S rRNA, which we named 5.8SC. The 5' exoribonuclease Xrn2 is involved in the generation of both 5.8S(C) and the canonical long form of 5.8S rRNA. Ribosomes containing 5.8S(C) rRNA are present in various mouse and human cells and engage in translation. These findings uncover a previously undescribed form of mammalian 5.8S rRNA and demonstrate that perturbations in ribosome assembly can be a source of heterogeneity in mature ribosomes.

  9. Recent advances in the uses and applications of ribosome-inactivating proteins from plants.

    PubMed

    Girbés, T; Ferreras, J M; Iglesias, R; Citores, L; De Torre, C; Carbajales, M L; Jiménez, P; De Benito, F M; Muñoz, R

    1996-06-01

    Plant ribosome-inactivating proteins (RIPs) are inhibitors present in all parts of plants that irreversibly inactivate eukaryotic ribosomes, thus impairing protein synthesis. RIPs are enzymes with N-glycosidase activity on the large rRNA. Their powerful inhibitory activity has been made use of advantageously to construct conjugates with suitable carriers targeted to altered specific cells. RIPs may be used to inhibit replication of both animal and plant viruses. The introduction of genes coding for RIPs into the genome of plants leads to an increase in resistance towards fungal pathogens and viruses. RIPs are important tools for the treatment of cancer and AIDS and for the protection of crop production.

  10. E. coli metabolic protein aldehyde-alcohol dehydrogenase-E binds to the ribosome: a unique moonlighting action revealed

    PubMed Central

    Shasmal, Manidip; Dey, Sandip; Shaikh, Tanvir R.; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    It is becoming increasingly evident that a high degree of regulation is involved in the protein synthesis machinery entailing more interacting regulatory factors. A multitude of proteins have been identified recently which show regulatory function upon binding to the ribosome. Here, we identify tight association of a metabolic protein aldehyde-alcohol dehydrogenase E (AdhE) with the E. coli 70S ribosome isolated from cell extract under low salt wash conditions. Cryo-EM reconstruction of the ribosome sample allows us to localize its position on the head of the small subunit, near the mRNA entrance. Our study demonstrates substantial RNA unwinding activity of AdhE which can account for the ability of ribosome to translate through downstream of at least certain mRNA helices. Thus far, in E. coli, no ribosome-associated factor has been identified that shows downstream mRNA helicase activity. Additionally, the cryo-EM map reveals interaction of another extracellular protein, outer membrane protein C (OmpC), with the ribosome at the peripheral solvent side of the 50S subunit. Our result also provides important insight into plausible functional role of OmpC upon ribosome binding. Visualization of the ribosome purified directly from the cell lysate unveils for the first time interactions of additional regulatory proteins with the ribosome. PMID:26822933

  11. RNA editing in six mitochondrial ribosomal protein genes of Didymium iridis.

    PubMed

    Hendrickson, Peter G; Silliker, Margaret E

    2010-06-01

    Similarity searches with Didymium iridis mitochondrial genomic DNA identified six possible ribosomal protein-coding regions, however, each region contained stop codons that would need to be removed by RNA editing to produce functional transcripts. RT-PCR was used to amplify these regions from total RNA for cloning and sequencing. Six functional transcripts were verified for the following ribosomal protein genes: rpS12, rpS7, rpL2, rpS19, rpS3, and rpL16. The editing events observed, such as single C and U nucleotide insertions and a dinucleotide insertion, were consistent with previously observed editing patterns seen in D. iridis. Additionally, a new form of insertional editing, a single A insertion, was observed in a conserved region of the rpL16 gene. While the majority of codons created by editing specify hydrophobic amino acids, a greater proportion of the codons created in these hydrophilic ribosomal proteins called for positively charged amino acids in comparison to the previously characterized hydrophobic respiratory protein genes. This first report of edited soluble mitochondrial ribosomal proteins in myxomycetes expands upon the RNA editing patterns previously seen; there was: a greater proportion of created codons specifying positively charged amino acids, a shift in the codon position edited, and the insertion of single A nucleotides.

  12. Ribosomal Proteins and Human Diseases: Pathogenesis, Molecular Mechanisms, and Therapeutic Implications

    PubMed Central

    Wang, Wei; Nag, Subhasree; Zhang, Xu; Wang, Ming-Hai; Wang, Hui; Zhou, Jianwei; Zhang, Ruiwen

    2014-01-01

    Ribosomes are essential components of the protein synthesis machinery. The process of ribosome biogenesis is well organized and tightly regulated. Recent studies have shown that ribosomal proteins (RPs) have extraribosomal functions that are involved in cell proliferation, differentiation, apoptosis, DNA repair, and other cellular processes. The dysfunction of RPs has been linked to the development and progression of hematological, metabolic, and cardiovascular diseases and cancer. Perturbation of ribosome biogenesis results in ribosomal stress, which triggers activation of the p53 signaling pathway through RPs-MDM2 interactions, resulting in p53-dependent cell cycle arrest and apoptosis. RPs also regulate cellular functions through p53-independent mechanisms. We herein review the recent advances in several forefronts of RP research, including the understanding of their biological features and roles in regulating cellular functions, maintaining cell homeostasis, and their involvement in the pathogenesis of human diseases. We also highlight the translational potential of this research for the identification of molecular biomarkers, and in the discovery and development of novel treatments for human diseases. PMID:25164622

  13. Ribosome-associated pentatricopeptide repeat proteins function as translational activators in mitochondria of trypanosomes

    PubMed Central

    Aphasizheva, Inna; Maslov, Dmitri A.; Qian, Yu; Huang, Lan; Wang, Qi; Costello, Catherine E.; Aphasizhev, Ruslan

    2016-01-01

    Summary Mitochondrial ribosomes of Trypanosoma brucei are composed of 9S and 12S rRNAs, eubacterial-type ribosomal proteins, polypeptides lacking discernible motifs and approximately 20 pentatricopeptide repeat (PPR) RNA binding proteins. Several PPRs also populate the polyadenylation complex; among these, KPAF1 and KPAF2 function as general mRNA 3′ adenylation/uridylation factors. The A/U-tail enables mRNA binding to the small ribosomal subunit and is essential for translation. The presence of A/U-tail also correlates with requirement for translation of certain mRNAs in mammalian and insect parasite stages. Here, we inquired whether additional PPRs activate translation of individual mRNAs. Proteomic analysis identified KRIPP1 and KRIPP8 as components of the small ribosomal subunit in mammalian and insect forms, but also revealed their association with the polyadenylation complex in the latter. RNAi knockdowns demonstrated essential functions of KRIPP1 and KRIPP8 in the actively respiring insect stage, but not in the mammalian stage. In the KRIPP1 knockdown, A/U-tailed mRNA encoding cytochrome c oxidase subunit 1 declined concomitantly with the de novo synthesis of this subunit whereas polyadenylation and translation of cyb mRNA were unaffected. In contrast, the KRIPP8 knockdown inhibited A/U-tailing and translation of both CO1 and cyb mRNAs. Our findings indicate that ribosome-associated PPRs may selectively activate mRNAs for translation. PMID:26713541

  14. Recognition of Ribosomal Protein L11 by the Protein Trimethyltransferase PrmA

    SciTech Connect

    Demirci,H.; Gregory, S.; Dahlberg, A.; Jogl, G.

    2007-01-01

    Bacterial ribosomal protein L11 is post-translationally trimethylated at multiple residues by a single methyltransferase, PrmA. Here, we describe four structures of PrmA from the extreme thermophile Thermus thermophilus. Two apo-PrmA structures at 1.59 and 2.3 {angstrom} resolution and a third with bound cofactor S-adenosyl-L-methionine at 1.75 {angstrom} each exhibit distinct relative positions of the substrate recognition and catalytic domains, revealing how PrmA can position the L11 substrate for multiple, consecutive side-chain methylation reactions. The fourth structure, the PrmA-L11 enzyme-substrate complex at 2.4 {angstrom} resolution, illustrates the highly specific interaction of the N-terminal domain with its substrate and places Lys39 in the PrmA active site. The presence of a unique flexible loop in the cofactor-binding site suggests how exchange of AdoMet with the reaction product S-adenosyl-L-homocysteine can occur without necessitating the dissociation of PrmA from L11. Finally, the mode of interaction of PrmA with L11 explains its observed preference for L11 as substrate before its assembly into the 50S ribosomal subunit.

  15. NAD+-dependent Deacetylase SIRT3 Regulates Mitochondrial Protein Synthesis by Deacetylation of the Ribosomal Protein MRPL10*

    PubMed Central

    Yang, Yongjie; Cimen, Huseyin; Han, Min-Joon; Shi, Tong; Deng, Jian-Hong; Koc, Hasan; Palacios, Orsolya M.; Montier, Laura; Bai, Yidong; Tong, Qiang; Koc, Emine C.

    2010-01-01

    A member of the sirtuin family of NAD+-dependent deacetylases, SIRT3, is located in mammalian mitochondria and is important for regulation of mitochondrial metabolism, cell survival, and longevity. In this study, MRPL10 (mitochondrial ribosomal protein L10) was identified as the major acetylated protein in the mitochondrial ribosome. Ribosome-associated SIRT3 was found to be responsible for deacetylation of MRPL10 in an NAD+-dependent manner. We mapped the acetylated Lys residues by tandem mass spectrometry and determined the role of these residues in acetylation of MRPL10 by site-directed mutagenesis. Furthermore, we observed that the increased acetylation of MRPL10 led to an increase in translational activity of mitochondrial ribosomes in Sirt3−/− mice. In a similar manner, ectopic expression and knockdown of SIRT3 in C2C12 cells resulted in the suppression and enhancement of mitochondrial protein synthesis, respectively. Our findings constitute the first evidence for the regulation of mitochondrial protein synthesis by the reversible acetylation of the mitochondrial ribosome and characterize MRPL10 as a novel substrate of the NAD+-dependent deacetylase, SIRT3. PMID:20042612

  16. Synaptic Activation of Ribosomal Protein S6 Phosphorylation Occurs Locally in Activated Dendritic Domains

    ERIC Educational Resources Information Center

    Pirbhoy, Patricia Salgado; Farris, Shannon; Steward, Oswald

    2016-01-01

    Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylation of ribosomal protein S6 (rpS6) in postsynaptic neurons, but the functional significance of rpS6 phosphorylation is poorly understood. Here, we show that synaptic stimulation that induces perforant path LTP triggers phosphorylation of rpS6 (p-rpS6)…

  17. Tempo and Mode of Gene Duplication in Mammalian Ribosomal Protein Evolution

    PubMed Central

    Gajdosik, Matthew D.; Simon, Amanda; Nelson, Craig E.

    2014-01-01

    Gene duplication has been widely recognized as a major driver of evolutionary change and organismal complexity through the generation of multi-gene families. Therefore, understanding the forces that govern the evolution of gene families through the retention or loss of duplicated genes is fundamentally important in our efforts to study genome evolution. Previous work from our lab has shown that ribosomal protein (RP) genes constitute one of the largest classes of conserved duplicated genes in mammals. This result was surprising due to the fact that ribosomal protein genes evolve slowly and transcript levels are very tightly regulated. In our present study, we identified and characterized all RP duplicates in eight mammalian genomes in order to investigate the tempo and mode of ribosomal protein family evolution. We show that a sizable number of duplicates are transcriptionally active and are very highly conserved. Furthermore, we conclude that existing gene duplication models do not readily account for the preservation of a very large number of intact retroduplicated ribosomal protein (RT-RP) genes observed in mammalian genomes. We suggest that selection against dominant-negative mutations may underlie the unexpected retention and conservation of duplicated RP genes, and may shape the fate of newly duplicated genes, regardless of duplication mechanism. PMID:25369106

  18. Enhanced pest resistance of maize leaves expressing monocot crop plant derived ribosome inactivating protein and agglutinin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although many insect resistance genes have been identified, the number of studies examining their effects in combination using transgenic systems is limited. We introduced a construct into maize containing the coding sequence for maize ribosome inactivating protein (MRIP), wheat germ agglutinin (WGA...

  19. Protein Folding Activity of the Ribosome (PFAR) –– A Target for Antiprion Compounds

    PubMed Central

    Banerjee, Debapriya; Sanyal, Suparna

    2014-01-01

    Prion diseases are fatal neurodegenerative diseases affecting mammals. Prions are misfolded amyloid aggregates of the prion protein (PrP), which form when the alpha helical, soluble form of PrP converts to an aggregation-prone, beta sheet form. Thus, prions originate as protein folding problems. The discovery of yeast prion(s) and the development of a red-/white-colony based assay facilitated safe and high-throughput screening of antiprion compounds. With this assay three antiprion compounds; 6-aminophenanthridine (6AP), guanabenz acetate (GA), and imiquimod (IQ) have been identified. Biochemical and genetic studies reveal that these compounds target ribosomal RNA (rRNA) and inhibit specifically the protein folding activity of the ribosome (PFAR). The domain V of the 23S/25S/28S rRNA of the large ribosomal subunit constitutes the active site for PFAR. 6AP and GA inhibit PFAR by competition with the protein substrates for the common binding sites on the domain V rRNA. PFAR inhibition by these antiprion compounds opens up new possibilities for understanding prion formation, propagation and the role of the ribosome therein. In this review, we summarize and analyze the correlation between PFAR and prion processes using the antiprion compounds as tools. PMID:25341659

  20. Tempo and mode of gene duplication in mammalian ribosomal protein evolution.

    PubMed

    Dharia, Asav P; Obla, Ajay; Gajdosik, Matthew D; Simon, Amanda; Nelson, Craig E

    2014-01-01

    Gene duplication has been widely recognized as a major driver of evolutionary change and organismal complexity through the generation of multi-gene families. Therefore, understanding the forces that govern the evolution of gene families through the retention or loss of duplicated genes is fundamentally important in our efforts to study genome evolution. Previous work from our lab has shown that ribosomal protein (RP) genes constitute one of the largest classes of conserved duplicated genes in mammals. This result was surprising due to the fact that ribosomal protein genes evolve slowly and transcript levels are very tightly regulated. In our present study, we identified and characterized all RP duplicates in eight mammalian genomes in order to investigate the tempo and mode of ribosomal protein family evolution. We show that a sizable number of duplicates are transcriptionally active and are very highly conserved. Furthermore, we conclude that existing gene duplication models do not readily account for the preservation of a very large number of intact retroduplicated ribosomal protein (RT-RP) genes observed in mammalian genomes. We suggest that selection against dominant-negative mutations may underlie the unexpected retention and conservation of duplicated RP genes, and may shape the fate of newly duplicated genes, regardless of duplication mechanism.

  1. A conserved quality-control pathway that mediates degradation of unassembled ribosomal proteins

    PubMed Central

    Sung, Min-Kyung; Porras-Yakushi, Tanya R; Reitsma, Justin M; Huber, Ferdinand M; Sweredoski, Michael J; Hoelz, André; Hess, Sonja; Deshaies, Raymond J

    2016-01-01

    Overproduced yeast ribosomal protein (RP) Rpl26 fails to assemble into ribosomes and is degraded in the nucleus/nucleolus by a ubiquitin-proteasome system quality control pathway comprising the E2 enzymes Ubc4/Ubc5 and the ubiquitin ligase Tom1. tom1 cells show reduced ubiquitination of multiple RPs, exceptional accumulation of detergent-insoluble proteins including multiple RPs, and hypersensitivity to imbalances in production of RPs and rRNA, indicative of a profound perturbation to proteostasis. Tom1 directly ubiquitinates unassembled RPs primarily via residues that are concealed in mature ribosomes. Together, these data point to an important role for Tom1 in normal physiology and prompt us to refer to this pathway as ERISQ, for excess ribosomal protein quality control. A similar pathway, mediated by the Tom1 homolog Huwe1, restricts accumulation of overexpressed hRpl26 in human cells. We propose that ERISQ is a key element of the quality control machinery that sustains protein homeostasis and cellular fitness in eukaryotes. DOI: http://dx.doi.org/10.7554/eLife.19105.001 PMID:27552055

  2. A Possible Role of the Full-Length Nascent Protein in Post-Translational Ribosome Recycling

    PubMed Central

    Das, Debasis; Samanta, Dibyendu; Bhattacharya, Arpita; Basu, Arunima; Das, Anindita; Ghosh, Jaydip; Chakrabarti, Abhijit; Das Gupta, Chanchal

    2017-01-01

    Each cycle of translation initiation in bacterial cell requires free 50S and 30S ribosomal subunits originating from the post-translational dissociation of 70S ribosome from the previous cycle. Literature shows stable dissociation of 70S from model post-termination complexes by the concerted action of Ribosome Recycling Factor (RRF) and Elongation Factor G (EF-G) that interact with the rRNA bridge B2a/B2b joining 50S to 30S. In such experimental models, the role of full-length nascent protein was never considered seriously. We observed relatively slow release of full-length nascent protein from 50Sof post translation ribosome, and in that process, its toe prints on the rRNA in vivo and in in vitro translation with E.coli S30 extract. We reported earlier that a number of chemically unfolded proteins like bovine carbonic anhydrase (BCA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), lysozyme, ovalbumin etc., when added to free 70Sin lieu of the full length nascent proteins, also interact with identical RNA regions of the 23S rRNA. Interestingly the rRNA nucleotides that slow down release of the C-terminus of full-length unfolded protein were found in close proximity to the B2a/B2b bridge. It indicated a potentially important chemical reaction conserved throughout the evolution. Here we set out to probe that conserved role of unfolded protein conformation in splitting the free or post-termination 70S. How both the RRF-EFG dependent and the plausible nascent protein–EFG dependent ribosome recycling pathways might be relevant in bacteria is discussed here. PMID:28099529

  3. Inhibition of Protein Synthesis on the Ribosome by Tildipirosin Compared with Other Veterinary Macrolides

    PubMed Central

    Andersen, Niels Møller; Poehlsgaard, Jacob; Warrass, Ralf

    2012-01-01

    Tildipirosin is a 16-membered-ring macrolide developed to treat bacterial pathogens, including Mannheimia haemolytica and Pasteurella multocida, that cause respiratory tract infections in cattle and swine. Here we evaluated the efficacy of tildipirosin at inhibiting protein synthesis on the ribosome (50% inhibitory concentration [IC50], 0.23 ± 0.01 μM) and compared it with the established veterinary macrolides tylosin, tilmicosin, and tulathromycin. Mutation and methylation at key rRNA nucleotides revealed differences in the interactions of these macrolides within their common ribosomal binding site. PMID:22926570

  4. Small-Molecule Inhibitor Leads of Ribosome-Inactivating Proteins Developed Using the Doorstop Approach

    DTIC Science & Technology

    2011-03-01

    producing E. coli O157:H7 is an emerging bacterial pathogen responsible for outbreaks of foodborne disease with significant morbidity and mortality in the...ribosome. Nucleic Acids Res 37: 602–610. 29. Li XP, Chiou JC, Remacha M, Ballesta JP, Tumer NE (2009) A two-step binding model proposed for the...toxicity. Microb Pathog 43: 88–95. Inhibitors of Ribosome-Inactivating Proteins PLoS ONE | www.plosone.org 15 March 2011 | Volume 6 | Issue 3 | e17883

  5. Inhibition of protein synthesis on the ribosome by tildipirosin compared with other veterinary macrolides.

    PubMed

    Andersen, Niels Møller; Poehlsgaard, Jacob; Warrass, Ralf; Douthwaite, Stephen

    2012-11-01

    Tildipirosin is a 16-membered-ring macrolide developed to treat bacterial pathogens, including Mannheimia haemolytica and Pasteurella multocida, that cause respiratory tract infections in cattle and swine. Here we evaluated the efficacy of tildipirosin at inhibiting protein synthesis on the ribosome (50% inhibitory concentration [IC(50)], 0.23 ± 0.01 μM) and compared it with the established veterinary macrolides tylosin, tilmicosin, and tulathromycin. Mutation and methylation at key rRNA nucleotides revealed differences in the interactions of these macrolides within their common ribosomal binding site.

  6. Dietary protein intake and skeletal-muscle protein metabolism in rats. Studies with salt-washed ribosomes and transfer factors

    PubMed Central

    Alexis, S. D.; Basta, S.; Young, Vernon R.

    1972-01-01

    1. Aspects of skeletal muscle protein synthesis in vitro were studied in young rats given a low-protein diet for up to 10 days and during re-feeding with an adequate diet. 2. Partially purified muscle transfer factors (transferases I and II), crude and purified (NH4Cl-washed) ribosomes and a pH5 enzyme fraction were prepared for this purpose. 3. A marked decrease in the capacity of crude ribosomes to carry out cell-free polypeptide synthesis occurred within 4 days of feeding the low-protein diet. 4. The capacity of salt-washed ribosomes to promote amino acid polymerization, in the presence of added transfer factors and aminoacyl-tRNA, was only slightly decreased by the dietary treatment. 5. However, the capacity of salt-washed ribosomes to bind 14C-labelled aminoacyl-tRNA was decreased by feeding the low-protein diet. 6. The capacity of the pH5 enzyme fraction to promote amino acid incorporation in a complete cell-free system was decreased within 2 days of feeding the low-protein diet. There is no evidence that the change is associated with aminoacyl-tRNA synthetase or binding enzyme activities of the pH5 fractions. 7. These changes are discussed in relation to the diminished rate of protein synthesis in the intact muscle cell when rats are given a low-protein diet. PMID:4634827

  7. Fluctuations in protein synthesis from a single RNA template: Stochastic kinetics of ribosomes

    NASA Astrophysics Data System (ADS)

    Garai, Ashok; Chowdhury, Debashish; Ramakrishnan, T. V.

    2009-01-01

    Proteins are polymerized by cyclic machines called ribosomes, which use their messenger RNA (mRNA) track also as the corresponding template, and the process is called translation. We explore, in depth and detail, the stochastic nature of the translation. We compute various distributions associated with the translation process; one of them—namely, the dwell time distribution—has been measured in recent single-ribosome experiments. The form of the distribution, which fits best with our simulation data, is consistent with that extracted from the experimental data. For our computations, we use a model that captures both the mechanochemistry of each individual ribosome and their steric interactions. We also demonstrate the effects of the sequence inhomogeneities of real genes on the fluctuations and noise in translation. Finally, inspired by recent advances in the experimental techniques of manipulating single ribosomes, we make theoretical predictions on the force-velocity relation for individual ribosomes. In principle, all our predictions can be tested by carrying out in vitro experiments.

  8. A Rab escort protein integrates the secretion system with TOR signaling and ribosome biogenesis.

    PubMed

    Singh, Jaspal; Tyers, Mike

    2009-08-15

    The coupling of environmental conditions to cell growth and division is integral to cell fitness. In Saccharomyces cerevisiae, the transcription factor Sfp1 couples nutrient status to cell growth rate by controlling the expression of ribosome biogenesis (Ribi) and ribosomal protein (RP) genes. Sfp1 is localized to the nucleus in rich nutrients, but upon nutrient limitation or target of rapamycin (TOR) pathway inhibition by rapamycin, Sfp1 rapidly exits the nucleus, leading to repression of the Ribi/RP regulons. Through systematic cell-based screens we found that many components of the secretory system influence Sfp1 localization. Notably, the essential Rab escort protein Mrs6 exhibited a nutrient-sensitive interaction with Sfp1. Overexpression of Mrs6 prevented nuclear localization of Sfp1 in rich nutrients, whereas loss of Mrs6 resulted in nuclear Sfp1 localization in poor nutrients. These effects were specific to Sfp1 and independent of the protein kinase C (PKC) pathway, suggesting that Mrs6 lies in a distinct branch of TOR and ribosome biogenesis regulation. Rapamycin-resistant alleles of MRS6 were defective in the cytoplasmic retention of Sfp1, the control of cell size, and in the repression of the Ribi/RP regulons. The Sfp1-Mrs6 interaction is a nexus for growth regulation that links the secretory system and TOR-dependent nutrient signaling to ribosome biogenesis.

  9. Yeast ribosomal protein L10 helps coordinate tRNA movement through the large subunit

    PubMed Central

    Petrov, Alexey N.; Meskauskas, Arturas; Roshwalb, Sara C.; Dinman, Jonathan D.

    2008-01-01

    Yeast ribosomal protein L10 (E. coli L16) is located at the center of a topological nexus that connects many functional regions of the large subunit. This essential protein has previously been implicated in processes as diverse as ribosome biogenesis, translational fidelity and mRNA stability. Here, the inability to maintain the yeast Killer virus was used as a proxy for large subunit defects to identify a series of L10 mutants. These mapped to roughly four discrete regions of the protein. A detailed analysis of mutants located in the N-terminal ‘hook’ of L10, which inserts into the bulge of 25S rRNA helix 89, revealed strong effects on rRNA structure corresponding to the entire path taken by the tRNA 3′ end as it moves through the large subunit during the elongation cycle. The mutant-induced structural changes are wide-ranging, affecting ribosome biogenesis, elongation factor binding, drug resistance/hypersensitivity, translational fidelity and virus maintenance. The importance of L10 as a potential transducer of information through the ribosome, and of a possible role of its N-terminal domain in switching between the pre- and post-translocational states are discussed. PMID:18824477

  10. YqjD is an inner membrane protein associated with stationary-phase ribosomes in Escherichia coli.

    PubMed

    Yoshida, Hideji; Maki, Yasushi; Furuike, Shou; Sakai, Akiko; Ueta, Masami; Wada, Akira

    2012-08-01

    Here, we provide evidence that YqjD, a hypothetical protein of Escherichia coli, is an inner membrane and ribosome binding protein. This protein is expressed during the stationary growth phase, and expression is regulated by stress response sigma factor RpoS. YqjD possesses a transmembrane motif in the C-terminal region and associates with 70S and 100S ribosomes at the N-terminal region. Interestingly, E. coli possesses two paralogous proteins of YqjD, ElaB and YgaM, which are expressed and bind to ribosomes in a similar manner to YqjD. Overexpression of YqjD leads to inhibition of cell growth. It has been suggested that YqjD loses ribosomal activity and localizes ribosomes to the membrane during the stationary phase.

  11. Automated Design of Synthetic Ribosome Binding Sites to Precisely Control Protein Expression

    PubMed Central

    Salis, Howard M.; Mirsky, Ethan A.; Voigt, Christopher A.

    2009-01-01

    Microbial engineering often requires fine control over protein expression; for example, to connect genetic circuits 1-7 or control flux through a metabolic pathway 8-13. We have developed a predictive design method for synthetic ribosome binding sites that enables the rational control of a protein's production rate on a proportional scale. Experimental validation of over 100 predictions in Escherichia coli shows that the method is accurate to within a factor of 2.3 over a range of 100,000-fold. The design method also correctly predicts that reusing a ribosome binding site sequence in different genetic contexts can result in different protein expression levels. We demonstrate the method's utility by rationally optimizing a protein's expression level to connect a genetic sensor to a synthetic circuit. The proposed forward engineering approach will accelerate the construction and systematic optimization of large genetic systems. PMID:19801975

  12. Protein Folding Activity of the Ribosome is involved in Yeast Prion Propagation

    PubMed Central

    Blondel, Marc; Soubigou, Flavie; Evrard, Justine; Nguyen, Phu hai; Hasin, Naushaba; Chédin, Stéphane; Gillet, Reynald; Contesse, Marie-Astrid; Friocourt, Gaëlle; Stahl, Guillaume; Jones, Gary W.; Voisset, Cécile

    2016-01-01

    6AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of the large subunit of the ribosome. We therefore explored the link between PFAR and yeast prion [PSI+] using both PFAR-enriched mutants and site-directed methylation. We demonstrate that PFAR is involved in propagation and de novo formation of [PSI+]. PFAR and the yeast heat-shock protein Hsp104 partially compensate each other for [PSI+] propagation. Our data also provide insight into new functions for the ribosome in basal thermotolerance and heat-shocked protein refolding. PFAR is thus an evolutionarily conserved cell component implicated in the prion life cycle, and we propose that it could be a potential therapeutic target for human protein misfolding diseases. PMID:27633137

  13. Mitochondrial Ribosomal Protein L10 Associates with Cyclin B1/Cdk1 Activity and Mitochondrial Function

    PubMed Central

    Li, Hai-Bo; Wang, Ruo-Xi; Jiang, Hai-Bo; Zhang, En-dong; Tan, Jie-Qiong; Xu, Hui-Zhuo

    2016-01-01

    Mitochondrial ribosomal proteins are important for mitochondrial-encoded protein synthesis and mitochondrial function. In addition to their roles in mitoribosome assembly, several mitochondrial ribosome proteins are also implicated in cellular processes like cell cycle regulation, apoptosis, and mitochondrial homeostasis regulation. Here, we demonstrate that MRPL10 regulates cyclin B1/Cdk1 (cyclin-dependent kinase 1) activity and mitochondrial protein synthesis in mammalian cells. In Drosophila, inactivation of mRpL10 (the Drosophila ortholog of mammalian MRPL10) in eyes results in abnormal eye development. Furthermore, expression of human cyclin B1 suppresses eye phenotypes and mitochondrial abnormality of mRpL10 knockdown Drosophila. This study identified that the physiological regulatory pathway of MRPL10 and providing new insights into the role of MRPL10 in growth control and mitochondrial function. PMID:27726420

  14. The three-dimensional structure of the RNA-binding domain of ribosomal protein L2; a protein at the peptidyl transferase center of the ribosome.

    PubMed Central

    Nakagawa, A; Nakashima, T; Taniguchi, M; Hosaka, H; Kimura, M; Tanaka, I

    1999-01-01

    Ribosomal protein L2 is the largest protein component in the ribosome. It is located at or near the peptidyl transferase center and has been a prime candidate for the peptidyl transferase activity. It binds directly to 23S rRNA and plays a crucial role in its assembly. The three-dimensional structure of the RNA-binding domain of L2 from Bacillus stearothermophilus has been determined at 2.3 A resolution by X-ray crystallography using the selenomethionyl MAD method. The RNA-binding domain of L2 consists of two recurring motifs of approximately 70 residues each. The N-terminal domain (positions 60-130) is homologous to the OB-fold, and the C-terminal domain (positions 131-201) is homologous to the SH3-like barrel. Residues Arg86 and Arg155, which have been identified by mutation experiments to be involved in the 23S rRNA binding, are located at the gate of the interface region between the two domains. The molecular architecture suggests how this important protein has evolved from the ancient nucleic acid-binding proteins to create a 23S rRNA-binding domain in the very remote past. PMID:10075918

  15. Mutations of ribosomal protein S5 suppress a defect in late-30S ribosomal subunit biogenesis caused by lack of the RbfA biogenesis factor

    PubMed Central

    Nord, Stefan; Bhatt, Monika J.; Tükenmez, Hasan; Farabaugh, Philip J.; Wikström, P. Mikael

    2015-01-01

    The in vivo assembly of ribosomal subunits requires assistance by maturation proteins that are not part of mature ribosomes. One such protein, RbfA, associates with the 30S ribosomal subunits. Loss of RbfA causes cold sensitivity and defects of the 30S subunit biogenesis and its overexpression partially suppresses the dominant cold sensitivity caused by a C23U mutation in the central pseudoknot of 16S rRNA, a structure essential for ribosome function. We have isolated suppressor mutations that restore partially the growth of an RbfA-lacking strain. Most of the strongest suppressor mutations alter one out of three distinct positions in the carboxy-terminal domain of ribosomal protein S5 (S5) in direct contact with helix 1 and helix 2 of the central pseudoknot. Their effect is to increase the translational capacity of the RbfA-lacking strain as evidenced by an increase in polysomes in the suppressed strains. Overexpression of RimP, a protein factor that along with RbfA regulates formation of the ribosome's central pseudoknot, was lethal to the RbfA-lacking strain but not to a wild-type strain and this lethality was suppressed by the alterations in S5. The S5 mutants alter translational fidelity but these changes do not explain consistently their effect on the RbfA-lacking strain. Our genetic results support a role for the region of S5 modified in the suppressors in the formation of the central pseudoknot in 16S rRNA. PMID:26089326

  16. Increased phosphorylation of ribosomal protein S6 during meiotic maturation of Xenopus oocytes.

    PubMed Central

    Nielsen, P J; Thomas, G; Maller, J L

    1982-01-01

    A single ribosomal protein (Mr, 32,000) becomes phosphorylated during progesterone-induced in vitro maturation of Xenopus laevis oocytes. The protein is identified as 40S ribosomal protein S6. Phosphorylation of S6 is monitored by incorporation of 32Pi and by two-dimensional polyacrylamide gel electrophoresis. S6 is minimally phosphorylated in unstimulated oocytes. After progesterone treatment, phosphorylation of S6 precedes germinal vesicle breakdown (GVBD) and is maximal at the time when 50% of the oocytes have undergone GVBD. S6, when maximally phosphorylated, exists in derivatives that correspond to the most highly phosphorylated forms observed in other systems, and the increase in S6 phosphorylation occurs at approximately the same time as the increase in the overall protein synthesis rate reported to occur during oocyte maturation. S6 is also maximally phosphorylated in unfertilized eggs following maturation in vivo. Injection of a partially purified preparation of maturation-promoting factor into immature oocytes induces immediate phosphorylation of S6 and rapidly increases the rate of protein synthesis. Moreover, incubation of ribosomes with this factor and radiolabeled ATP results in labeling of S6. These findings suggest that S6 phosphorylation may be important in the control of protein synthesis during maturation and may also play a role in the mechanism of action of maturation-promoting factor. Images PMID:7045876

  17. Crystal structure of bacillus subtilis YdaF protein : a putative ribosomal N-acetyltransferase.

    SciTech Connect

    Brunzelle, J. S.; Wu, R.; Korolev, S. V.; Collart, F. R.; Joachimiak, A.; Anderson, W. F.; Biosciences Division; Northwestern Univ.; Saint Louis Univ. School of Medicine

    2004-12-01

    Comparative sequence analysis suggests that the ydaF gene encodes a protein (YdaF) that functions as an N-acetyltransferase, more specifically, a ribosomal N-acetyltransferase. Sequence analysis using basic local alignment search tool (BLAST) suggests that YdaF belongs to a large family of proteins (199 proteins found in 88 unique species of bacteria, archaea, and eukaryotes). YdaF also belongs to the COG1670, which includes the Escherichia coli RimL protein that is known to acetylate ribosomal protein L12. N-acetylation (NAT) has been found in all kingdoms. NAT enzymes catalyze the transfer of an acetyl group from acetyl-CoA (AcCoA) to a primary amino group. For example, NATs can acetylate the N-terminal {alpha}-amino group, the {epsilon}-amino group of lysine residues, aminoglycoside antibiotics, spermine/speridine, or arylalkylamines such as serotonin. The crystal structure of the alleged ribosomal NAT protein, YdaF, from Bacillus subtilis presented here was determined as a part of the Midwest Center for Structural Genomics. The structure maintains the conserved tertiary structure of other known NATs and a high sequence similarity in the presumed AcCoA binding pocket in spite of a very low overall level of sequence identity to other NATs of known structure.

  18. Euglena gracilis chloroplast ribosomal protein operon: a new chloroplast gene for ribosomal protein L5 and description of a novel organelle intron category designated group III.

    PubMed Central

    Christopher, D A; Hallick, R B

    1989-01-01

    We describe the structure (3840 bp) of a novel Euglena gracilis chloroplast ribosomal protein operon that encodes the five genes rpl16-rpl14-rpl5-rps8-rpl36. The gene organization resembles the spc and the 3'-end of the S10 ribosomal protein operons of E. coli. The rpl5 is a new chloroplast gene not previously reported for any chloroplast genome to date and also not described as a nuclear-encoded, chloroplast protein gene. The operon contains at least 7 introns. We present evidence from primer extension analysis of chloroplast RNA for the correct in vivo splicing of five of the introns. Two of the introns within the rps8 gene flank an 8 bp exon, the smallest exon yet characterized in a chloroplast gene. Three introns resemble the classical group II introns of organelle genomes. The remaining 4 introns appear to be unique to the Euglena chloroplast DNA. They are uniform in size (95-109 nt), share common features with each other and are distinct from both group I and group II introns. We designate this new intron category as 'group III'. Images PMID:2477800

  19. GATA1 and PU.1 Bind to Ribosomal Protein Genes in Erythroid Cells: Implications for Ribosomopathies

    PubMed Central

    Amanatiadou, Elsa P.; Papadopoulos, Giorgio L.; Strouboulis, John; Vizirianakis, Ioannis S.

    2015-01-01

    The clear connection between ribosome biogenesis dysfunction and specific hematopoiesis-related disorders prompted us to examine the role of critical lineage-specific transcription factors in the transcriptional regulation of ribosomal protein (RP) genes during terminal erythroid differentiation. By applying EMSA and ChIP methodologies in mouse erythroleukemia cells we show that GATA1 and PU.1 bind in vitro and in vivo the proximal promoter region of the RPS19 gene which is frequently mutated in Diamond-Blackfan Anemia. Moreover, ChIPseq data analysis also demonstrates that several RP genes are enriched as potential GATA1 and PU.1 gene targets in mouse and human erythroid cells, with GATA1 binding showing an association with higher ribosomal protein gene expression levels during terminal erythroid differentiation in human and mouse. Our results suggest that RP gene expression and hence balanced ribosome biosynthesis may be specifically and selectively regulated by lineage specific transcription factors during hematopoiesis, a finding which may be clinically relevant to ribosomopathies. PMID:26447946

  20. The ribosome as a missing link in prebiotic evolution II: Ribosomes encode ribosomal proteins that bind to common regions of their own mRNAs and rRNAs.

    PubMed

    Root-Bernstein, Robert; Root-Bernstein, Meredith

    2016-05-21

    We have proposed that the ribosome may represent a missing link between prebiotic chemistries and the first cells. One of the predictions that follows from this hypothesis, which we test here, is that ribosomal RNA (rRNA) must have encoded the proteins necessary for ribosomal function. In other words, the rRNA also functioned pre-biotically as mRNA. Since these ribosome-binding proteins (rb-proteins) must bind to the rRNA, but the rRNA also functioned as mRNA, it follows that rb-proteins should bind to their own mRNA as well. This hypothesis can be contrasted to a "null" hypothesis in which rb-proteins evolved independently of the rRNA sequences and therefore there should be no necessary similarity between the rRNA to which rb-proteins bind and the mRNA that encodes the rb-protein. Five types of evidence reported here support the plausibility of the hypothesis that the mRNA encoding rb-proteins evolved from rRNA: (1) the ubiquity of rb-protein binding to their own mRNAs and autogenous control of their own translation; (2) the higher-than-expected incidence of Arginine-rich modules associated with RNA binding that occurs in rRNA-encoded proteins; (3) the fact that rRNA-binding regions of rb-proteins are homologous to their mRNA binding regions; (4) the higher than expected incidence of rb-protein sequences encoded in rRNA that are of a high degree of homology to their mRNA as compared with a random selection of other proteins; and (5) rRNA in modern prokaryotes and eukaryotes encodes functional proteins. None of these results can be explained by the null hypothesis that assumes independent evolution of rRNA and the mRNAs encoding ribosomal proteins. Also noteworthy is that very few proteins bind their own mRNAs that are not associated with ribosome function. Further tests of the hypothesis are suggested: (1) experimental testing of whether rRNA-encoded proteins bind to rRNA at their coding sites; (2) whether tRNA synthetases, which are also known to bind to their

  1. Derivative of Extremophilic 50S Ribosomal Protein L35Ae as an Alternative Protein Scaffold

    PubMed Central

    Lomonosova, Anna V.; Ulitin, Andrei B.; Kazakov, Alexei S.; Mirzabekov, Tajib A.; Permyakov, Eugene A.

    2017-01-01

    Small antibody mimetics, or alternative binding proteins (ABPs), extend and complement antibody functionality with numerous applications in research, diagnostics and therapeutics. Given the superiority of ABPs, the last two decades have witnessed development of dozens of alternative protein scaffolds (APSs) for the design of ABPs. Proteins from extremophiles with their high structural stability are especially favorable for APS design. Here, a 10X mutant of the 50S ribosomal protein L35Ae from hyperthermophilic archaea Pyrococcus horikoshii has been probed as an APS. A phage display library of L35Ae 10X was generated by randomization of its three CDR-like loop regions (repertoire size of 2×108). Two L35Ae 10X variants specific to a model target, the hen egg-white lysozyme (HEL), were isolated from the resulting library using phage display. The affinity of these variants (L4 and L7) to HEL ranges from 0.10 μM to 1.6 μM, according to surface plasmon resonance data. While L4 has 1–2 orders of magnitude lower affinity to HEL homologue, bovine α-lactalbumin (BLA), L7 is equally specific to HEL and BLA. The reference L35Ae 10X is non-specific to both HEL and BLA. L4 and L7 are more resistant to denaturation by guanidine hydrochloride compared to the reference L35Ae 10X (mid-transition concentration is higher by 0.1–0.5 M). Chemical crosslinking experiments reveal an increased propensity of L4 and L7 to multimerization. Overall, the CDR-like loop regions of L35Ae 10X represent a proper interface for generation of functional ABPs. Hence, L35Ae is shown to extend the growing family of protein scaffolds dedicated to the design of novel binding proteins. PMID:28103321

  2. Error-prone and error-restrictive mutations affecting ribosomal protein S12.

    PubMed

    Agarwal, Deepali; Gregory, Steven T; O'Connor, Michael

    2011-07-01

    Ribosomal protein S12 plays a pivotal role in decoding functions on the ribosome. X-ray crystallographic analyses of ribosomal complexes have revealed that S12 is involved in the inspection of codon-anticodon pairings in the ribosomal A site, as well as in the succeeding domain rearrangements of the 30S subunit that are essential for accommodation of aminoacyl-tRNA. A role for S12 in tRNA selection is also well supported by classical genetic analyses; mutations affecting S12 are readily isolated in bacteria and organelles, since specific alterations in S12 confer resistance to the error-inducing antibiotic streptomycin, and the ribosomes from many such streptomycin-resistant S12 mutants display decreased levels of miscoding. However, substitutions that confer resistance to streptomycin likely represent a very distinct class of all possible S12 mutants. Until recently, the technical difficulties in generating random, unselectable mutations in essential genes in complex operons have generally precluded the analysis of other classes of S12 alterations. Using a recombineering approach, we have targeted the Escherichia coli rpsL gene, encoding S12, for random mutagenesis and screened the resulting mutants for effects on decoding fidelity. We have recovered over 40 different substitutions located throughout the S12 protein that alter the accuracy of translation without substantially affecting the sensitivity to streptomycin. Moreover, this collection includes mutants that promote miscoding, as well as those that restrict decoding errors. These results affirm the importance of S12 in decoding processes and indicate that alterations in this essential protein can have diverse effects on the accuracy of decoding.

  3. Eukaryotic Initiation Factor 6, an evolutionarily conserved regulator of ribosome biogenesis and protein translation

    SciTech Connect

    Guo, Jianjun; Jin, Zhaoqing; Yang, Xiaohan; Li, Jian-Feng; Chen, Jay

    2011-01-01

    We recently identified Receptor for Activated C Kinase 1 (RACK1) as one of the molecular links between abscisic acid (ABA) signaling and its regulation on protein translation. Moreover, we identified Eukaryotic Initiation Factor 6 (eIF6) as an interacting partner of RACK1. Because the interaction between RACK1 and eIF6 in mammalian cells is known to regulate the ribosome assembly step of protein translation initiation, it was hypothesized that the same process of protein translation in Arabidopsis is also regulated by RACK1 and eIF6. In this article, we analyzed the amino acid sequences of eIF6 in different species from different lineages and discovered some intriguing differences in protein phosphorylation sites that may contribute to its action in ribosome assembly and biogenesis. In addition, we discovered that, distinct from non-plant organisms in which eIF6 is encoded by a single gene, all sequenced plant genomes contain two or more copies of eIF6 genes. While one copy of plant eIF6 is expressed ubiquitously and might possess the conserved function in ribosome biogenesis and protein translation, the other copy seems to be only expressed in specific organs and therefore may have gained some new functions. We proposed some important studies that may help us better understand the function of eIF6 in plants.

  4. The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein.

    PubMed

    Barkan, Alice; Klipcan, Larik; Ostersetzer, Oren; Kawamura, Tetsuya; Asakura, Yukari; Watkins, Kenneth P

    2007-01-01

    The CRS1-YhbY domain (also called the CRM domain) is represented as a stand-alone protein in Archaea and Bacteria, and in a family of single- and multidomain proteins in plants. The function of this domain is unknown, but structural data and the presence of the domain in several proteins known to interact with RNA have led to the proposal that it binds RNA. Here we describe a phylogenetic analysis of the domain, its incorporation into diverse proteins in plants, and biochemical properties of a prokaryotic and eukaryotic representative of the domain family. We show that a bacterial member of the family, Escherichia coli YhbY, is associated with pre-50S ribosomal subunits, suggesting that YhbY functions in ribosome assembly. GFP fused to a single-domain CRM protein from maize localizes to the nucleolus, suggesting that an analogous activity may have been retained in plants. We show further that an isolated maize CRM domain has RNA binding activity in vitro, and that a small motif shared with KH RNA binding domains, a conserved "GxxG" loop, contributes to its RNA binding activity. These and other results suggest that the CRM domain evolved in the context of ribosome function prior to the divergence of Archaea and Bacteria, that this function has been maintained in extant prokaryotes, and that the domain was recruited to serve as an RNA binding module during the evolution of plant genomes.

  5. The antituberculosis antibiotic capreomycin inhibits protein synthesis by disrupting interaction between ribosomal proteins L12 and L10.

    PubMed

    Lin, Yuan; Li, Yan; Zhu, Ningyu; Han, Yanxing; Jiang, Wei; Wang, Yanchang; Si, Shuyi; Jiang, Jiandong

    2014-01-01

    Capreomycin is a second-line drug for multiple-drug-resistant tuberculosis (TB). However, with increased use in clinics, the therapeutic efficiency of capreomycin is decreasing. To better understand TB resistance to capreomycin, we have done research to identify the molecular target of capreomycin. Mycobacterium tuberculosis ribosomal proteins L12 and L10 interact with each other and constitute the stalk of the 50S ribosomal subunit, which recruits initiation and elongation factors during translation. Hence, the L12-L10 interaction is considered to be essential for ribosomal function and protein synthesis. Here we provide evidence showing that capreomycin inhibits the L12-L10 interaction by using an established L12-L10 interaction assay. Overexpression of L12 and/or L10 in M. smegmatis, a species close to M. tuberculosis, increases the MIC of capreomycin. Moreover, both elongation factor G-dependent GTPase activity and ribosome-mediated protein synthesis are inhibited by capreomycin. When protein synthesis was blocked with thiostrepton, however, the bactericidal activity of capreomycin was restrained. All of these results suggest that capreomycin seems to inhibit TB by interrupting the L12-L10 interaction. This finding might provide novel clues for anti-TB drug discovery.

  6. NAD(+)- dependent deacetylase SIRT3 regulates mitochondrial protein synthesis by deacetylation of the ribosomal protein MRPL10

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A member of the sirtuin family of NAD (+)-dependent deacetylases, SIRT3, is located in mammalian mitochondria and is important for regulation of mitochondrial metabolism, cell survival, and longevity. In this study, MRPL10 (mitochondrial ribosomal protein L10) was identified as the major acetylated ...

  7. The effect of ribosomal protein S1 from Escherichia coli and Micrococcus luteus on protein synthesis in vitro by E. coli and Bacillus subtilis.

    PubMed

    Farwell, M A; Roberts, M W; Rabinowitz, J C

    1992-11-01

    We have designed a set of nine plasmids containing the Bacillus pumilis cat gene with one of three Shine-Dalgarno (SD) sequences (weak, strong or stronger) and one of three initiation codons (AUG, GUG or UUG). These constructions have been used to determine the effect of ribosomal protein S1, SD and initiation codon sequences and Escherichia coli ribosomal protein S1 on translation in vitro by E. coli and B. subtilis ribosomes. Translation of these nine constructions was determined with three types of ribosomes: E. coli containing ribosomal protein S1, E. coli depleted of S1, and B. subtilis which is naturally free of S1. E. coli ribosomes were able to translate all nine transcripts with variable efficiencies. B. subtilis and S1-depleted E. coli ribosomes were similar to each other and differed from non-depleted E. coli ribosomes in that they required strong or stronger SD sequences and were unable to translate any of the weak transcripts. Addition of S1 from either E. coli or Micrococcus luteus, a Gram-positive bacterium, enabled S1-depleted E. coli ribosomes to translate mRNAs with weak SD sequences but had no effect on B. subtilis ribosomes. AUG was the preferred initiation codon for all ribosome types; however, B. subtilis ribosomes showed greater tolerance for the non-AUG codons than either type of E. coli ribosome. The presence of a strong or stronger SD sequence increased the efficiency by which E. coli ribosomes could utilize non-AUG codons.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Protein Synthesis with Ribosomes Selected for the Incorporation of β-Amino Acids.

    PubMed

    Maini, Rumit; Chowdhury, Sandipan Roy; Dedkova, Larisa M; Roy, Basab; Daskalova, Sasha M; Paul, Rakesh; Chen, Shengxi; Hecht, Sidney M

    2015-06-16

    In an earlier study, β³-puromycin was used for the selection of modified ribosomes, which were utilized for the incorporation of five different β-amino acids into Escherichia coli dihydrofolate reductase (DHFR). The selected ribosomes were able to incorporate structurally disparate β-amino acids into DHFR, in spite of the use of a single puromycin for the selection of the individual clones. In this study, we examine the extent to which the structure of the β³-puromycin employed for ribosome selection influences the regio- and stereochemical preferences of the modified ribosomes during protein synthesis; the mechanistic probe was a single suppressor tRNA(CUA) activated with each of four methyl-β-alanine isomers (1-4). The modified ribosomes were found to incorporate each of the four isomeric methyl-β-alanines into DHFR but exhibited a preference for incorporation of 3(S)-methyl-β-alanine (β-mAla; 4), i.e., the isomer having the same regio- and stereochemistry as the O-methylated β-tyrosine moiety of β³-puromycin. Also conducted were a selection of clones that are responsive to β²-puromycin and a demonstration of reversal of the regio- and stereochemical preferences of these clones during protein synthesis. These results were incorporated into a structural model of the modified regions of 23S rRNA, which included in silico prediction of a H-bonding network. Finally, it was demonstrated that incorporation of 3(S)-methyl-β-alanine (β-mAla; 4) into a short α-helical region of the nucleic acid binding domain of hnRNP LL significantly stabilized the helix without affecting its DNA binding properties.

  9. Crystal structure of a beta-finger domain of Prp8 reveals analogy to ribosomal proteins

    SciTech Connect

    Yang, K.; Heroux, A.; Zhang, L.; Zhao, R.

    2008-09-16

    Prp8 stands out among hundreds of splicing factors as a key regulator of spliceosome activation and a potential cofactor of the splicing reaction. We present here the crystal structure of a 274-residue domain (residues 1,822-2,095) near the C terminus of Saccharomyces cerevisiae Prp8. The most striking feature of this domain is a {beta}-hairpin finger protruding out of the protein (hence, this domain will be referred to as the {beta}-finger domain), resembling many globular ribosomal proteins with protruding extensions. Mutations throughout the {beta}-finger change the conformational equilibrium between the first and the second catalytic step. Mutations at the base of the {beta}-finger affect U4/U6 unwinding-mediated spliceosome activation. Prp8 may insert its {beta}-finger into the first-step complex (U2/U5/U6/pre-mRNA) or U4/U6.U5 tri-snRNP and stabilize these complexes. Mutations on the {beta}-finger likely alter these interactions, leading to the observed mutant phenotypes. Our results suggest a possible mechanism of how Prp8 regulates spliceosome activation. These results also demonstrate an analogy between a spliceosomal protein and ribosomal proteins that insert extensions into folded rRNAs and stabilize the ribosome.

  10. A protein inventory of human ribosome biogenesis reveals an essential function of exportin 5 in 60S subunit export.

    PubMed

    Wild, Thomas; Horvath, Peter; Wyler, Emanuel; Widmann, Barbara; Badertscher, Lukas; Zemp, Ivo; Kozak, Karol; Csucs, Gabor; Lund, Elsebet; Kutay, Ulrike

    2010-10-26

    The assembly of ribosomal subunits in eukaryotes is a complex, multistep process so far mostly studied in yeast. In S. cerevisiae, more than 200 factors including ribosomal proteins and trans-acting factors are required for the ordered assembly of 40S and 60S ribosomal subunits. To date, only few human homologs of these yeast ribosome synthesis factors have been characterized. Here, we used a systematic RNA interference (RNAi) approach to analyze the contribution of 464 candidate factors to ribosomal subunit biogenesis in human cells. The screen was based on visual readouts, using inducible, fluorescent ribosomal proteins as reporters. By performing computer-based image analysis utilizing supervised machine-learning techniques, we obtained evidence for a functional link of 153 human proteins to ribosome synthesis. Our data show that core features of ribosome assembly are conserved from yeast to human, but differences exist for instance with respect to 60S subunit export. Unexpectedly, our RNAi screen uncovered a requirement for the export receptor Exportin 5 (Exp5) in nuclear export of 60S subunits in human cells. We show that Exp5, like the known 60S exportin Crm1, binds to pre-60S particles in a RanGTP-dependent manner. Interference with either Exp5 or Crm1 function blocks 60S export in both human cells and frog oocytes, whereas 40S export is compromised only upon inhibition of Crm1. Thus, 60S subunit export is dependent on at least two RanGTP-binding exportins in vertebrate cells.

  11. Impact of P-Site tRNA and Antibiotics on Ribosome Mediated Protein Folding: Studies Using the Escherichia coli Ribosome

    PubMed Central

    Mondal, Surojit; Pathak, Bani Kumar; Ray, Sutapa; Barat, Chandana

    2014-01-01

    Background The ribosome, which acts as a platform for mRNA encoded polypeptide synthesis, is also capable of assisting in folding of polypeptide chains. The peptidyl transferase center (PTC) that catalyzes peptide bond formation resides in the domain V of the 23S rRNA of the bacterial ribosome. Proper positioning of the 3′ –CCA ends of the A- and P-site tRNAs via specific interactions with the nucleotides of the PTC are crucial for peptidyl transferase activity. This RNA domain is also the center for ribosomal chaperoning activity. The unfolded polypeptide chains interact with the specific nucleotides of the PTC and are released in a folding competent form. In vitro transcribed RNA corresponding to this domain (bDV RNA) also displays chaperoning activity. Results The present study explores the effects of tRNAs, antibiotics that are A- and P-site PTC substrate analogs (puromycin and blasticidin) and macrolide antibiotics (erythromycin and josamycin) on the chaperoning ability of the E. coli ribosome and bDV RNA. Our studies using mRNA programmed ribosomes show that a tRNA positioned at the P-site effectively inhibits the ribosome's chaperoning function. We also show that the antibiotic blasticidin (that mimics the interaction between 3′–CCA end of P/P-site tRNA with the PTC) is more effective in inhibiting ribosome and bDV RNA chaperoning ability than either puromycin or the macrolide antibiotics. Mutational studies of the bDV RNA could identify the nucleotides U2585 and G2252 (both of which interact with P-site tRNA) to be important for its chaperoning ability. Conclusion Both protein synthesis and their proper folding are crucial for maintenance of a functional cellular proteome. The PTC of the ribosome is attributed with both these abilities. The silencing of the chaperoning ability of the ribosome in the presence of P-site bound tRNA might be a way to segregate these two important functions. PMID:25000563

  12. Orsay virus utilizes ribosomal frameshifting to express a novel protein that is incorporated into virions

    SciTech Connect

    Jiang, Hongbing; Franz, Carl J.; Wu, Guang; Renshaw, Hilary; Zhao, Guoyan; Firth, Andrew E.; Wang, David

    2014-02-15

    Orsay virus is the first identified virus that is capable of naturally infecting Caenorhabditis elegans. Although it is most closely related to nodaviruses, Orsay virus differs from nodaviruses in its genome organization. In particular, the Orsay virus RNA2 segment encodes a putative novel protein of unknown function, termed delta, which is absent from all known nodaviruses. Here we present evidence that Orsay virus utilizes a ribosomal frameshifting strategy to express a novel fusion protein from the viral capsid (alpha) and delta ORFs. Moreover, the fusion protein was detected in purified virus fractions, demonstrating that it is most likely incorporated into Orsay virions. Furthermore, N-terminal sequencing of both the fusion protein and the capsid protein demonstrated that these proteins must be translated from a non-canonical initiation site. While the function of the alpha–delta fusion remains cryptic, these studies provide novel insights into the fundamental properties of this new clade of viruses. - Highlights: • Orsay virus encodes a novel fusion protein by a ribosomal frameshifting mechanism. • Orsay capsid and fusion protein is translated from a non-canonical initiation site. • The fusion protein is likely incorporated into Orsay virions.

  13. Contamination of ribosome inactivating proteins with ribonucleases, separated by affinity chromatography on red sepharose.

    PubMed

    Wang, H X; Ng, T B; Cheng, C H K; Fong, W P

    2003-05-01

    Three preparations of type 1 ribosome inactivating proteins (RIPs), namely, agrostin, saporin, and luffin, were subjected to affinity chromatography on Red Sepharose and eluted with a linear concentration gradient of NaCl in 10 mM Tris-HCl buffer (pH 7.4). The eluate was assayed for ability to inhibit translation in a cell-free rabbit reticulocyte lysate system which measures RIP activity, and for ability to hydrolyze yeast transfer RNA which measures RNase activity. It was found that, in all three RIP preparations, the peak of RIP activity, which coincided with the peak of absorbance at 280 nm, was eluted earlier than the peak of RNase activity. It appears that RNase is a possible contaminant of ribosome inactivating protein preparations and that this contamination can be minimized by using Red Sepharose.

  14. Co-evolution of Bacterial Ribosomal Protein S15 with Diverse mRNA Regulatory Structures.

    PubMed

    Slinger, Betty L; Newman, Hunter; Lee, Younghan; Pei, Shermin; Meyer, Michelle M

    2015-12-01

    RNA-protein interactions are critical in many biological processes, yet how such interactions affect the evolution of both partners is still unknown. RNA and protein structures are impacted very differently by mechanisms of genomic change. While most protein families are identifiable at the nucleotide level across large phylogenetic distances, RNA families display far less nucleotide similarity and are often only shared by closely related bacterial species. Ribosomal protein S15 has two RNA binding functions. First, it is a ribosomal protein responsible for organizing the rRNA during ribosome assembly. Second, in many bacterial species S15 also interacts with a structured portion of its own transcript to negatively regulate gene expression. While the first interaction is conserved in most bacteria, the second is not. Four distinct mRNA structures interact with S15 to enable regulation, each of which appears to be independently derived in different groups of bacteria. With the goal of understanding how protein-binding specificity may influence the evolution of such RNA regulatory structures, we examine whether examples of these mRNA structures are able to interact with, and regulate in response to, S15 homologs from organisms containing distinct mRNA structures. We find that despite their shared RNA binding function in the rRNA, S15 homologs have distinct RNA recognition profiles. We present a model to explain the specificity patterns observed, and support this model by with further mutagenesis. After analyzing the patterns of conservation for the S15 protein coding sequences, we also identified amino acid changes that alter the binding specificity of an S15 homolog. In this work we demonstrate that homologous RNA-binding proteins have different specificity profiles, and minor changes to amino acid sequences, or to RNA structural motifs, can have large impacts on RNA-protein recognition.

  15. Co-evolution of Bacterial Ribosomal Protein S15 with Diverse mRNA Regulatory Structures

    PubMed Central

    Slinger, Betty L.; Newman, Hunter; Lee, Younghan; Pei, Shermin; Meyer, Michelle M.

    2015-01-01

    RNA-protein interactions are critical in many biological processes, yet how such interactions affect the evolution of both partners is still unknown. RNA and protein structures are impacted very differently by mechanisms of genomic change. While most protein families are identifiable at the nucleotide level across large phylogenetic distances, RNA families display far less nucleotide similarity and are often only shared by closely related bacterial species. Ribosomal protein S15 has two RNA binding functions. First, it is a ribosomal protein responsible for organizing the rRNA during ribosome assembly. Second, in many bacterial species S15 also interacts with a structured portion of its own transcript to negatively regulate gene expression. While the first interaction is conserved in most bacteria, the second is not. Four distinct mRNA structures interact with S15 to enable regulation, each of which appears to be independently derived in different groups of bacteria. With the goal of understanding how protein-binding specificity may influence the evolution of such RNA regulatory structures, we examine whether examples of these mRNA structures are able to interact with, and regulate in response to, S15 homologs from organisms containing distinct mRNA structures. We find that despite their shared RNA binding function in the rRNA, S15 homologs have distinct RNA recognition profiles. We present a model to explain the specificity patterns observed, and support this model by with further mutagenesis. After analyzing the patterns of conservation for the S15 protein coding sequences, we also identified amino acid changes that alter the binding specificity of an S15 homolog. In this work we demonstrate that homologous RNA-binding proteins have different specificity profiles, and minor changes to amino acid sequences, or to RNA structural motifs, can have large impacts on RNA-protein recognition. PMID:26675164

  16. Pokeweed antiviral protein region Gly209-Lys225 is critical for RNA N-glycosidase activity of the prokaryotic ribosome.

    PubMed

    Nagasawa, Yoshimi; Fujii, Kazuyuki; Yoshikawa, Takafumi; Kobayashi, Yoshinori; Kondo, Toshiya

    2008-05-01

    Pokeweed antiviral protein (PAP) isolated from Phytolacca americana is a ribosome-inactivating protein (RIP) that has RNA N-glycosidase (RNG) activity towards both eukaryotic and prokaryotic ribosomes. In contrast, karasurin-A (KRN), a RIP from Trichosanthes kirilowii var. japonica, is active only on eukaryotic ribosomes. Stepwise selection of chimera proteins between PAP and KRN indicated that the C-terminal region of PAP (residues 209-225) was critical for RNG activity toward prokaryotic ribosomes. When the region of PAP (residues 209-225) was replaced with the corresponding region of KRN the PAP chimera protein, like KRN, was active only on eukaryotic ribosomes. Furthermore, insertion of the region of PAP (residues 209-225) into the KRN chimera protein resulted not only in the detectable RNG activity toward prokaryotic ribosome, but also activity toward the eukaryotic ribosomes as well that was seven-fold higher than for the original KRN. In this study, the possibility of genetic manipulation of the activity and substrate specificity of RIPs is demonstrated.

  17. The ribosomal protein L10/QM-like protein is a component of the NIK-mediated antiviral signaling

    SciTech Connect

    Rocha, Carolina S.; Santos, Anesia A.; Machado, Joao Paulo B.; Fontes, Elizabeth P.B.

    2008-10-25

    The NIK (NSP-interacting kinase)-mediated antiviral signaling pathway was identified as a virulence target of the begomovirus nuclear shuttle protein (NSP). Here, we further characterized this layer of plant innate defense by identifying the ribosomal protein L10 (rpL10), a QM-like protein, as a downstream effector of the antiviral signaling. Although both ribosomal proteins rpL10 and rpL18 were found to associate with NIK1 through yeast two-hybrid screening, the NIK receptors specifically phosphorylated rpL10 in vitro. Furthermore, loss of rpL10 function significantly increased susceptibility to begomovirus infection, recapitulating the phenotype of nik knockout lines. Our results genetically linked rpL10 to the NIK-mediated antiviral signaling.

  18. Maximizing protein translation rate in the non-homogeneous ribosome flow model: a convex optimization approach.

    PubMed

    Poker, Gilad; Zarai, Yoram; Margaliot, Michael; Tuller, Tamir

    2014-11-06

    Translation is an important stage in gene expression. During this stage, macro-molecules called ribosomes travel along the mRNA strand linking amino acids together in a specific order to create a functioning protein. An important question, related to many biomedical disciplines, is how to maximize protein production. Indeed, translation is known to be one of the most energy-consuming processes in the cell, and it is natural to assume that evolution shaped this process so that it maximizes the protein production rate. If this is indeed so then one can estimate various parameters of the translation machinery by solving an appropriate mathematical optimization problem. The same problem also arises in the context of synthetic biology, namely, re-engineer heterologous genes in order to maximize their translation rate in a host organism. We consider the problem of maximizing the protein production rate using a computational model for translation-elongation called the ribosome flow model (RFM). This model describes the flow of the ribosomes along an mRNA chain of length n using a set of n first-order nonlinear ordinary differential equations. It also includes n + 1 positive parameters: the ribosomal initiation rate into the mRNA chain, and n elongation rates along the chain sites. We show that the steady-state translation rate in the RFM is a strictly concave function of its parameters. This means that the problem of maximizing the translation rate under a suitable constraint always admits a unique solution, and that this solution can be determined using highly efficient algorithms for solving convex optimization problems even for large values of n. Furthermore, our analysis shows that the optimal translation rate can be computed based only on the optimal initiation rate and the elongation rate of the codons near the beginning of the ORF. We discuss some applications of the theoretical results to synthetic biology, molecular evolution, and functional genomics.

  19. Ribosome-Mediated Incorporation of Dipeptides and Dipeptide Analogues into Proteins in Vitro.

    PubMed

    Maini, Rumit; Dedkova, Larisa M; Paul, Rakesh; Madathil, Manikandadas M; Chowdhury, Sandipan Roy; Chen, Shengxi; Hecht, Sidney M

    2015-09-09

    Plasmids containing 23S rRNA randomized at positions 2057-2063 and 2502-2507 were introduced into Escherichia coli, affording a library of clones which produced modified ribosomes in addition to the pre-existing wild-type ribosomes. These clones were screened with a derivative of puromycin, a natural product which acts as an analogue of the 3'-end of aminoacyl-tRNA and terminates protein synthesis by accepting the growing polypeptide chain, thereby killing bacterial cells. The puromycin derivative in this study contained the dipeptide p-methoxyphenylalanylglycine, implying the ability of the modified ribosomes in clones sensitive to this puromycin analogue to recognize dipeptides. Several clones inhibited by the puromycin derivative were used to make S-30 preparations, and some of these were shown to support the incorporation of dipeptides into proteins. The four incorporated species included two dipeptides (Gly-Phe (2) and Phe-Gly (3)), as well as a thiolated dipeptide analogue (4) and a fluorescent oxazole (5) having amine and carboxyl groups approximately the same distance apart as in a normal dipeptide. A protein containing both thiolated dipeptide 4 and a 7-methoxycoumarin fluorophore was found to undergo fluorescence quenching. Introduction of the oxazole fluorophore 5 into dihydrofolate reductase or green fluorescent protein resulted in quite strong enhancement of its fluorescence emission, and the basis for this enhancement was studied. The aggregate results demonstrate the feasibility of incorporating dipeptides as a single ribosomal event, and illustrate the lack of recognition of the central peptide bond in the dipeptide, potentially enabling the incorporation of a broad variety of structural analogues.

  20. Antibacterial discovery in actinomycetes strains with mutations in RNA polymerase or ribosomal protein S12.

    PubMed

    Hosaka, Takeshi; Ohnishi-Kameyama, Mayumi; Muramatsu, Hideyuki; Murakami, Kana; Tsurumi, Yasuhisa; Kodani, Shinya; Yoshida, Mitsuru; Fujie, Akihiko; Ochi, Kozo

    2009-05-01

    We show that selection of drug-resistant bacterial mutants allows the discovery of antibacterial compounds. Mutant strains of a soil-isolated Streptomyces species that does not produce antibacterials synthesize a previously unknown class of antibacterial, which we name piperidamycin. Overall, 6% of non-Streptomyces actinomycetes species and 43% of Streptomyces species that do not produce antibacterials are activated to produce them. The antibacterial-producing mutants all carried mutations in RNA polymerase and/or the ribosomal protein S12.

  1. Maximizing Protein Translation Rate in the Ribosome Flow Model: The Homogeneous Case.

    PubMed

    Zarai, Yoram; Margaliot, Michael; Tuller, Tamir

    2014-01-01

    Gene translation is the process in which intracellular macro-molecules, called ribosomes, decode genetic information in the mRNA chain into the corresponding proteins. Gene translation includes several steps. During the elongation step, ribosomes move along the mRNA in a sequential manner and link amino-acids together in the corresponding order to produce the proteins. The homogeneous ribosome flow model (HRFM) is a deterministic computational model for translation-elongation under the assumption of constant elongation rates along the mRNA chain. The HRFM is described by a set of n first-order nonlinear ordinary differential equations, where n represents the number of sites along the mRNA chain. The HRFM also includes two positive parameters: ribosomal initiation rate and the (constant) elongation rate. In this paper, we show that the steady-state translation rate in the HRFM is a concave function of its parameters. This means that the problem of determining the parameter values that maximize the translation rate is relatively simple. Our results may contribute to a better understanding of the mechanisms and evolution of translation-elongation. We demonstrate this by using the theoretical results to estimate the initiation rate in M. musculus embryonic stem cell. The underlying assumption is that evolution optimized the translation mechanism. For the infinite-dimensional HRFM, we derive a closed-form solution to the problem of determining the initiation and transition rates that maximize the protein translation rate. We show that these expressions provide good approximations for the optimal values in the n-dimensional HRFM already for relatively small values of n. These results may have applications for synthetic biology where an important problem is to re-engineer genomic systems in order to maximize the protein production rate.

  2. Humoral autoimmune response to ribosomal P proteins in chronic Chagas heart disease.

    PubMed Central

    Levitus, G; Hontebeyrie-Joskowicz, M; Van Regenmortel, M H; Levin, M J

    1991-01-01

    The C terminal region of a Trypanosoma cruzi ribosomal P protein, encoded by the lambda gt11 JL5 recombinant, defined a major antigenic determinant in chronic Chagas heart disease. Immunopurified anti-JL5 antibodies were tested for anti-human ribosome reactivity by immunoblotting. They recognized the parasite ribosomal P proteins and clearly reacted with the corresponding human P proteins. The peptide R-13, that comprises the 13 C terminal residues of the JL5 recombinant and defines the specificity shared between chronic Chagas heart disease anti-JL5 antibodies and the systemic lupus erythematosus (SLE) anti-P antibodies, was used to study the specificity and the IgG subclass distribution of the anti-R-13 response by ELISA. The R-13 autoepitope is recognized mainly by sera from chagasic patients, but not by sera from malaria patients. Moreover, there was a significant correlation between anti-R-13 antibody levels and anti-T. cruzi antibody titres. The anti-R-13 response was mainly restricted to the IgG1 heavy chain isotype and correlated with the anti-T. cruzi isotype distribution. Images Fig. 1 Fig. 2 Fig. 3 PMID:1893622

  3. Role of a ribosome-associated E3 ubiquitin ligase in protein quality control.

    PubMed

    Bengtson, Mario H; Joazeiro, Claudio A P

    2010-09-23

    Messenger RNA lacking stop codons ('non-stop mRNA') can arise from errors in gene expression, and encode aberrant proteins whose accumulation could be deleterious to cellular function. In bacteria, these 'non-stop proteins' become co-translationally tagged with a peptide encoded by ssrA/tmRNA (transfer-messenger RNA), which signals their degradation by energy-dependent proteases. How eukaryotic cells eliminate non-stop proteins has remained unknown. Here we show that the Saccharomyces cerevisiae Ltn1 RING-domain-type E3 ubiquitin ligase acts in the quality control of non-stop proteins, in a process that is mechanistically distinct but conceptually analogous to that performed by ssrA: Ltn1 is predominantly associated with ribosomes, and it marks nascent non-stop proteins with ubiquitin to signal their proteasomal degradation. Ltn1-mediated ubiquitylation of non-stop proteins seems to be triggered by their stalling in ribosomes on translation through the poly(A) tail. The biological relevance of this process is underscored by the finding that loss of Ltn1 function confers sensitivity to stress caused by increased non-stop protein production. We speculate that defective protein quality control may underlie the neurodegenerative phenotype that results from mutation of the mouse Ltn1 homologue Listerin.

  4. Ribosomal Protein Mutations Result in Constitutive p53 Protein Degradation through Impairment of the AKT Pathway

    PubMed Central

    Hermkens, Dorien; Wlodarski, Marcin W.; Da Costa, Lydie; MacInnes, Alyson W.

    2015-01-01

    Mutations in ribosomal protein (RP) genes can result in the loss of erythrocyte progenitor cells and cause severe anemia. This is seen in patients with Diamond-Blackfan anemia (DBA), a pure red cell aplasia and bone marrow failure syndrome that is almost exclusively linked to RP gene haploinsufficiency. While the mechanisms underlying the cytopenia phenotype of patients with these mutations are not completely understood, it is believed that stabilization of the p53 tumor suppressor protein may induce apoptosis in the progenitor cells. In stark contrast, tumor cells from zebrafish with RP gene haploinsufficiency are unable to stabilize p53 even when exposed to acute DNA damage despite transcribing wild type p53 normally. In this work we demonstrate that p53 has a limited role in eliciting the anemia phenotype of zebrafish models of DBA. In fact, we find that RP-deficient embryos exhibit the same normal p53 transcription, absence of p53 protein, and impaired p53 response to DNA damage as RP haploinsufficient tumor cells. Recently we reported that RP mutations suppress activity of the AKT pathway, and we show here that this suppression results in proteasomal degradation of p53. By re-activating the AKT pathway or by inhibiting GSK-3, a downstream modifier that normally represses AKT signaling, we are able to restore the stabilization of p53. Our work indicates that the anemia phenotype of zebrafish models of DBA is dependent on factors other than p53, and may hold clinical significance for both DBA and the increasing number of cancers revealing spontaneous mutations in RP genes. PMID:26132763

  5. Defect in the formation of 70S ribosomes caused by lack of ribosomal protein L34 can be suppressed by magnesium.

    PubMed

    Akanuma, Genki; Kobayashi, Ako; Suzuki, Shota; Kawamura, Fujio; Shiwa, Yuh; Watanabe, Satoru; Yoshikawa, Hirofumi; Hanai, Ryo; Ishizuka, Morio

    2014-11-01

    To elucidate the biological functions of the ribosomal protein L34, which is encoded by the rpmH gene, the rpmH deletion mutant of Bacillus subtilis and two suppressor mutants were characterized. Although the ΔrpmH mutant exhibited a severe slow-growth phenotype, additional mutations in the yhdP or mgtE gene restored the growth rate of the ΔrpmH strain. Either the disruption of yhdP, which is thought to be involved in the efflux of Mg(2+), or overexpression of mgtE, which plays a major role in the import of Mg(2+), could suppress defects in both the formation of the 70S ribosome and growth caused by the absence of L34. Interestingly, the Mg(2+) content was lower in the ΔrpmH cells than in the wild type, and the Mg(2+) content in the ΔrpmH cells was restored by either the disruption of yhdP or overexpression of mgtE. In vitro experiments on subunit association demonstrated that 50S subunits that lacked L34 could form 70S ribosomes only at a high concentration of Mg(2+). These results showed that L34 is required for efficient 70S ribosome formation and that L34 function can be restored partially by Mg(2+). In addition, the Mg(2+) content was consistently lower in mutants that contained significantly reduced amounts of the 70S ribosome, such as the ΔrplA (L1) and ΔrplW (L23) strains and mutant strains with a reduced number of copies of the rrn operon. Thus, the results indicated that the cellular Mg(2+) content is influenced by the amount of 70S ribosomes.

  6. Post-transcriptional regulation of ribosomal protein genes during serum starvation in Entamoeba histolytica.

    PubMed

    Ahamad, Jamaluddin; Ojha, Sandeep; Srivastava, Ankita; Bhattacharya, Alok; Bhattacharya, Sudha

    2015-06-01

    Ribosome synthesis involves all three RNA polymerases which are co-ordinately regulated to produce equimolar amounts of rRNAs and ribosomal proteins (RPs). Unlike model organisms where transcription of rRNA and RP genes slows down during stress, in E. histolytica rDNA transcription continues but pre-rRNA processing slows down and unprocessed pre-rRNA accumulates during serum starvation. To investigate the regulation of RP genes under stress we measured transcription of six selected RP genes from the small- and large-ribosomal subunits (RPS6, RPS3, RPS19, RPL5, RPL26, RPL30) representing the early-, mid-, and late-stages of ribosomal assembly. Transcripts of these genes persisted in growth-stressed cells. Expression of luciferase reporter under the control of two RP genes (RPS19 and RPL30) was studied during serum starvation and upon serum replenishment. Although luciferase transcript levels remained unchanged during starvation, luciferase activity steadily declined to 7.8% and 15% of control cells, respectively. After serum replenishment the activity increased to normal levels, suggesting post-transcriptional regulation of these genes. Mutations in the sequence -2 to -9 upstream of AUG in the RPL30 gene resulted in the phenotype expected of post-transcriptional regulation. Transcription of luciferase reporter was unaffected in this mutant, and luciferase activity did not decline during serum starvation, showing that this sequence is required to repress translation of RPL30 mRNA, and mutations in this region relieve repression. Our data show that during serum starvation E. histolytica blocks ribosome biogenesis post-transcriptionally by inhibiting pre-rRNA processing on the one hand, and the translation of RP mRNAs on the other.

  7. Mechanistic insight into the ribosome biogenesis functions of the ancient protein KsgA

    PubMed Central

    Connolly, Keith; Rife, Jason P.; Culver, Gloria

    2009-01-01

    Summary While the general blueprint of ribosome biogenesis is evolutionarily conserved, most details have diverged considerably. A striking exception to this divergence is the universally conserved KsgA/Dim1p enzyme family, which modifies two adjacent adenosines in the terminal helix of small subunit ribosomal RNA (rRNA). While localization of KsgA on 30S subunits (SSUs) and genetic interaction data have suggested that KsgA acts as a ribosome biogenesis factor, mechanistic details and a rationale for its extreme conservation are still lacking. To begin to address these questions we have characterized the function of E. coli KsgA in vivo using both a ksgA deletion strain and a methyltransferase deficient form of this protein. Our data reveals cold sensitivity and altered ribosomal profiles are associated with a ΔksgA genotype in E. coli. Our work also indicates that loss of KsgA alters 16S rRNA processing. These findings allow KsgAs role in SSU biogenesis to be integrated into the network of other identified factors. Moreover, a methyltransferase-inactive form of KsgA, which we show to be deleterious to cell growth, profoundly impairs ribosome biogenesis prompting discussion of KsgA as a possible anti-microbial drug target. These unexpected data suggest that methylation is a second layer of function for KsgA and that its critical role is as a supervisor of biogenesis of SSUs in vivo. These new findings and this proposed regulatory role offer a mechanistic explanation for the extreme conservation of the KsgA/Dim1p enzyme family. PMID:18990185

  8. S18 family of mitochondrial ribosomal proteins: evolutionary history and Gly132 polymorphism in colon carcinoma.

    PubMed

    Mushtaq, Muhammad; Ali, Raja Hashim; Kashuba, Vladimir; Klein, George; Kashuba, Elena

    2016-08-23

    S18 family of mitochondrial ribosomal proteins (MRPS18, S18) consists of three members, S18-1 to -3. Earlier, we found that overexpression of S18-2 protein resulted in immortalization and eventual transformation of primary rat fibroblasts. The S18-1 and -3 have not exhibited such abilities. To understand the differences in protein properties, the evolutionary history of S18 family was analyzed. The S18-3, followed by S18-1 and S18-2 emerged as a result of ancient gene duplication in the root of eukaryotic species tree, followed by two metazoan-specific gene duplications. However, the most conserved metazoan S18 homolog is the S18-1; it shares the most sequence similarity with S18 proteins of bacteria and of other eukaryotic clades. Evolutionarily conserved residues of S18 proteins were analyzed in various cancers. S18-2 is mutated at a higher rate, compared with S18-1 and -3 proteins. Moreover, the evolutionarily conserved residue, Gly132 of S18-2, shows genetic polymorphism in colon adenocarcinomas that was confirmed by direct DNA sequencing.Concluding, S18 family represents the yet unexplored important mitochondrial ribosomal proteins.

  9. S18 family of mitochondrial ribosomal proteins: evolutionary history and Gly132 polymorphism in colon carcinoma

    PubMed Central

    Mushtaq, Muhammad; Ali, Raja Hashim; Kashuba, Vladimir; Klein, George; Kashuba, Elena

    2016-01-01

    S18 family of mitochondrial ribosomal proteins (MRPS18, S18) consists of three members, S18-1 to −3. Earlier, we found that overexpression of S18-2 protein resulted in immortalization and eventual transformation of primary rat fibroblasts. The S18-1 and −3 have not exhibited such abilities. To understand the differences in protein properties, the evolutionary history of S18 family was analyzed. The S18-3, followed by S18-1 and S18-2 emerged as a result of ancient gene duplication in the root of eukaryotic species tree, followed by two metazoan-specific gene duplications. However, the most conserved metazoan S18 homolog is the S18-1; it shares the most sequence similarity with S18 proteins of bacteria and of other eukaryotic clades. Evolutionarily conserved residues of S18 proteins were analyzed in various cancers. S18-2 is mutated at a higher rate, compared with S18-1 and −3 proteins. Moreover, the evolutionarily conserved residue, Gly132 of S18-2, shows genetic polymorphism in colon adenocarcinomas that was confirmed by direct DNA sequencing. Concluding, S18 family represents the yet unexplored important mitochondrial ribosomal proteins. PMID:27489352

  10. Complex RNA maturation pathway for a chloroplast ribosomal protein operon with an internal tRNA cistron.

    PubMed Central

    Christopher, D A; Hallick, R B

    1990-01-01

    We have studied the expression of a large chloroplast ribosomal protein operon from Euglena gracilis that resembles the Escherichia coli S10 and spc ribosomal protein operons. We present evidence that 11 ribosomal protein genes, a tRNA gene, and a new locus, orf214/orf302, are expressed as a single transcription unit. The primary transcript also contains at least 15 group II and group III introns. Gene-specific probes for each ribosomal protein gene, orf214/orf302, and for trnl hybridized to a common pre-mRNA of an estimated size of 8.3 kilobases. This is the RNA size predicted for a full-length transcript of the entire operon after splicing of all 15 introns. Polycistronic ribosomal protein mRNAs accumulated primarily as spliced hepta-, hexa-, penta-, tetra-, tri-, and dicistronic mRNAs, which were presumed to arise by stepwise processing of the 8.3-kilobase pre-mRNA. A novel finding was the cotranscription of the trnl gene as an internal cistron within the ribosomal protein operon. Several combined mRNA/tRNA molecules, such as the pentacistronic rpl5-rps8-rpl36-trnl-rps14, were characterized. The occurrence of the orf214/orf302 is a unique feature of the Euglena operon, distinguishing it from all chloroplast and prokaryotic ribosomal protein operons characterized to date. The orf214/orf302 are not similar to any known genes but are cotranscribed with the ribosomal protein loci and encode stable RNA species of 2.4, 1.8, and 1.4 kilobases. PMID:2136640

  11. Beta-methylthio-aspartic acid: identification of a novel posttranslational modification in ribosomal protein S12 from Escherichia coli.

    PubMed Central

    Kowalak, J. A.; Walsh, K. A.

    1996-01-01

    Utilizing microscale chemical derivatization reactions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we have identified a novel posttranslational modification of aspartic acid, beta-methylthio-aspartic acid. The modified residue is located at position 88 in ribosomal protein S12 from Escherichia coli, a phylogenetically conserved protein that has been implicated in maintaining translational accuracy of the ribosome. PMID:8844851

  12. The structure of SAV1646 from Staphylococcus aureus belonging to a new `ribosome-associated' subfamily of bacterial proteins.

    PubMed

    Chirgadze, Yuri N; Clarke, Teresa E; Romanov, Vladimir; Kisselman, Gera; Wu-Brown, Jean; Soloveychik, Maria; Chan, Tiffany S Y; Gordon, Roni D; Battaile, Kevin P; Pai, Emil F; Chirgadze, Nickolay Y

    2015-02-01

    The crystal structure of the SAV1646 protein from the pathogenic microorganism Staphylococcus aureus has been determined at 1.7 Å resolution. The 106-amino-acid protein forms a two-layer sandwich with α/β topology. The protein molecules associate as dimers in the crystal and in solution, with the monomers related by a pseudo-twofold rotation axis. A sequence-homology search identified the protein as a member of a new subfamily of yet uncharacterized bacterial `ribosome-associated' proteins with at least 13 members to date. A detailed analysis of the crystal protein structure along with the genomic structure of the operon containing the sav1646 gene allowed a tentative functional model of this protein to be proposed. The SAV1646 dimer is assumed to form a complex with ribosomal proteins L21 and L27 which could help to complete the assembly of the large subunit of the ribosome.

  13. Ribosomal protein biomarkers provide root nodule bacterial identification by MALDI-TOF MS.

    PubMed

    Ziegler, Dominik; Pothier, Joël F; Ardley, Julie; Fossou, Romain Kouakou; Pflüger, Valentin; de Meyer, Sofie; Vogel, Guido; Tonolla, Mauro; Howieson, John; Reeve, Wayne; Perret, Xavier

    2015-07-01

    Accurate identification of soil bacteria that form nitrogen-fixing associations with legume crops is challenging given the phylogenetic diversity of root nodule bacteria (RNB). The labor-intensive and time-consuming 16S ribosomal RNA (rRNA) sequencing and/or multilocus sequence analysis (MLSA) of conserved genes so far remain the favored molecular tools to characterize symbiotic bacteria. With the development of mass spectrometry (MS) as an alternative method to rapidly identify bacterial isolates, we recently showed that matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) can accurately characterize RNB found inside plant nodules or grown in cultures. Here, we report on the development of a MALDI-TOF RNB-specific spectral database built on whole cell MS fingerprints of 116 strains representing the major rhizobial genera. In addition to this RNB-specific module, which was successfully tested on unknown field isolates, a subset of 13 ribosomal proteins extracted from genome data was found to be sufficient for the reliable identification of nodule isolates to rhizobial species as shown in the putatively ascribed ribosomal protein masses (PARPM) database. These results reveal that data gathered from genome sequences can be used to expand spectral libraries to aid the accurate identification of bacterial species by MALDI-TOF MS.

  14. Versatile roles of Arabidopsis plastid ribosomal proteins in plant growth and development.

    PubMed

    Romani, Isidora; Tadini, Luca; Rossi, Fabio; Masiero, Simona; Pribil, Mathias; Jahns, Peter; Kater, Martin; Leister, Dario; Pesaresi, Paolo

    2012-12-01

    A lack of individual plastid ribosomal proteins (PRPs) can have diverse phenotypic effects in Arabidopsis thaliana, ranging from embryo lethality to compromised vitality, with the latter being associated with photosynthetic lesions and decreases in the expression of plastid proteins. In this study, reverse genetics was employed to study the function of eight PRPs, five of which (PRPS1, -S20, -L27, -L28 and -L35) have not been functionally characterised before. In the case of PRPS17, only leaky alleles or RNA interference lines had been analysed previously. PRPL1 and PRPL4 have been described as essential for embryo development, but their mutant phenotypes are analysed in detail here. We found that PRPS20, -L1, -L4, -L27 and -L35 are required for basal ribosome activity, which becomes crucial at the globular stage and during the transition from the globular to the heart stage of embryogenesis. Thus, lack of any of these PRPs leads to alterations in cell division patterns, and embryo development ceases prior to the heart stage. PRPL28 is essential at the latest stages of embryo-seedling development, during the greening process. PRPS1, -S17 and -L24 appear not to be required for basal ribosome activity and the organism can complete its entire life cycle in their absence. Interestingly, despite the prokaryotic origin of plastids, the significance of individual PRPs for plant development cannot be predicted from the relative phenotypic severity of the corresponding mutants in prokaryotic systems.

  15. Transient ribosomal attenuation coordinates protein synthesis and co-translational folding.

    PubMed

    Zhang, Gong; Hubalewska, Magdalena; Ignatova, Zoya

    2009-03-01

    Clustered codons that pair to low-abundance tRNA isoacceptors can form slow-translating regions in the mRNA and cause transient ribosomal arrest. We report that folding efficiency of the Escherichia coli multidomain protein SufI can be severely perturbed by alterations in ribosome-mediated translational attenuation. Such alterations were achieved by global acceleration of the translation rate with tRNA excess in vitro or by synonymous substitutions to codons with highly abundant tRNAs both in vitro and in vivo. Conversely, the global slow-down of the translation rate modulated by low temperature suppresses the deleterious effect of the altered translational attenuation pattern. We propose that local discontinuous translation temporally separates the translation of segments of the peptide chain and actively coordinates their co-translational folding.

  16. Sucralose activates an ERK1/2-ribosomal protein S6 signaling axis.

    PubMed

    Guerra, Marcy L; Kalwat, Michael A; McGlynn, Kathleen; Cobb, Melanie H

    2017-02-01

    The sweetener sucralose can signal through its GPCR receptor to induce insulin secretion from pancreatic β cells, but the downstream signaling pathways involved are not well-understood. Here we measure responses to sucralose, glucagon-like peptide 1, and amino acids in MIN6 β cells. Our data suggest a signaling axis, whereby sucralose induces calcium and cAMP, activation of ERK1/2, and site-specific phosphorylation of ribosomal protein S6. Interestingly, sucralose acted independently of mTORC1 or ribosomal S6 kinase (RSK). These results suggest that sweeteners like sucralose can influence β-cell responses to secretagogues like glucose through metabolic as well as GPCR-mediated pathways. Future investigation of novel sweet taste receptor signaling pathways in β cells will have implications for diabetes and other emergent fields involving these receptors.

  17. The eukaryote-specific N-terminal extension of ribosomal protein S31 contributes to the assembly and function of 40S ribosomal subunits

    PubMed Central

    Fernández-Pevida, Antonio; Martín-Villanueva, Sara; Murat, Guillaume; Lacombe, Thierry; Kressler, Dieter; de la Cruz, Jesús

    2016-01-01

    The archaea-/eukaryote-specific 40S-ribosomal-subunit protein S31 is expressed as an ubiquitin fusion protein in eukaryotes and consists of a conserved body and a eukaryote-specific N-terminal extension. In yeast, S31 is a practically essential protein, which is required for cytoplasmic 20S pre-rRNA maturation. Here, we have studied the role of the N-terminal extension of the yeast S31 protein. We show that deletion of this extension partially impairs cell growth and 40S subunit biogenesis and confers hypersensitivity to aminoglycoside antibiotics. Moreover, the extension harbours a nuclear localization signal that promotes active nuclear import of S31, which associates with pre-ribosomal particles in the nucleus. In the absence of the extension, truncated S31 inefficiently assembles into pre-40S particles and two subpopulations of mature small subunits, one lacking and another one containing truncated S31, can be identified. Plasmid-driven overexpression of truncated S31 partially suppresses the growth and ribosome biogenesis defects but, conversely, slightly enhances the hypersensitivity to aminoglycosides. Altogether, these results indicate that the N-terminal extension facilitates the assembly of S31 into pre-40S particles and contributes to the optimal translational activity of mature 40S subunits but has only a minor role in cytoplasmic cleavage of 20S pre-rRNA at site D. PMID:27422873

  18. Cloning and characterization of nuclear genes for two mitochondrial ribosomal proteins in Saccharomyces cerevisiae.

    PubMed Central

    Kitakawa, M; Grohmann, L; Graack, H R; Isono, K

    1990-01-01

    The genes for two large subunit proteins, YmL8 and YmL20, of the mitochondrial ribosome of Saccharomyces cerevisiae were cloned by hybridization with synthetic oligonucleotide mixtures corresponding to their N-terminal amino acid sequences. They were termed MRP-L8 and MRP-L20, respectively, and their nucleotide sequences were determined using a DNA sequencer. The MRP-L8 gene was found to encode a 26.8-kDa protein whose deduced amino acid sequence has a high degree of similarity to ribosomal protein L17 of Escherichia coli. The gene MRP-L20 was found to encode a 22.3-kDa protein with a presequence consisting of 18 amino acid residues. By Southern blot hybridization to the yeast chromosomes separated by field-inversion gel electrophoresis, the MRP-L8 and MRP-L20 genes were located on chromosomes X and XI, respectively. Gene disruption experiments indicate that their products, YmL8 and YmL20 proteins, are essential for the mitochondrial function and the absence of these proteins causes instability of the mitochondrial DNA. Images PMID:2183197

  19. Synthesis of ribosomal proteins in developing Dictyostelium discoideum cells is controlled by the methylation of proteins S24 and S31.

    PubMed

    Mangiarotti, Giorgio

    2002-01-01

    Ribosomal protein mRNAs left over from growth are selectively excluded from polyribosomes in the first half of Dictyostelium discoideum development. This is due to the fact that they are sequestered by a class of free 40S ribosomal subunits, characterized by possessing a methylated S24 protein. At the time of formation of tight cell aggregates, the methylated S24 is substituted by an unmethylated S24, while protein S31 of the same or other 40S subunits becomes methylated. This leads to a rapid degradation of the ribosomal protein mRNAs.

  20. Role of Protein L25 and Its Contact with Protein L16 in Maintaining the Active State of Escherichia coli Ribosomes in vivo.

    PubMed

    Anikaev, A Y; Isaev, A B; Korobeinikova, A V; Garber, M B; Gongadze, G M

    2016-01-01

    A ribosomal protein of the L25 family specifically binding to 5S rRNA is an evolutionary feature of bacteria. Structural studies showed that within the ribosome this protein contacts not only 5S rRNA, but also the C-terminal region of protein L16. Earlier we demonstrated that ribosomes from the ΔL25 strain of Escherichia coli have reduced functional activity. In the present work, it is established that the reason for this is a fraction of functionally inactive 50S ribosomal subunits. These subunits have a deficit of protein L16 and associate very weakly with 30S subunits. To study the role of the contact of these two proteins in the formation of the active ribosome, we created a number of E. coli strains containing protein L16 with changes in its C-terminal region. We found that some mutations (K133L or K127L/K133L) in this protein lead to a noticeable slowing of cell growth and decrease in the activity of their translational apparatus. As in the case of the ribosomes from the ΔL25 strain, the fraction of 50S subunits, which are deficient in protein L16, is present in the ribosomes of the mutant strains. All these data indicate that the contact with protein L25 is important for the retention of protein L16 within the E. coli ribosome in vivo. In the light of these findings, the role of the protein of the L25 family in maintaining the active state of the bacterial ribosome is discussed.

  1. Characterization and analysis of ribosomal proteins in two marine calanoid copepods

    NASA Astrophysics Data System (ADS)

    Yang, Feifei; Xu, Donghui; Zhuang, Yunyun; Huang, Yousong; Yi, Xiaoyan; Chen, Hongju; Liu, Guangxing; Zhang, Huan

    2016-11-01

    Copepods are among the most abundant and successful metazoans in the marine ecosystem. However, genomic resources related to fundamental cellular processes are still limited in this particular group of crustaceans. Ribosomal proteins are the building blocks of ribosomes, the primary site for protein synthesis. In this study, we characterized and analyzed the cDNAs of cytoplasmic ribosomal proteins (cRPs) of two calanoid copepods, Pseudodiaptomus poplesia and Acartia pacifica. We obtained 79 cRP cDNAs from P. poplesia and 67 from A. pacifica by cDNA library construction/sequencing and rapid amplification of cDNA ends. Analysis of the nucleic acid composition showed that the copepod cRP-encoding genes had higher GC content in the protein-coding regions (CDSs) than in the untranslated regions (UTRs), and single nucleotide repeats (>3 repeats) were common, with "A" repeats being the most frequent, especially in the CDSs. The 3'-UTRs of the cRP genes were significantly longer than the 5'-UTRs. Codon usage analysis showed that the third positions of the codons were dominated by C or G. The deduced amino acid sequences of the cRPs contained high proportions of positively charged residues and had high pI values. This is the first report of a complete set of cRP-encoding genes from copepods. Our results shed light on the characteristics of cRPs in copepods, and provide fundamental data for further studies of protein synthesis in copepods. The copepod cRP information revealed in this study indicates that additional comparisons and analysis should be performed on different taxonomic categories such as orders and families.

  2. Kinetoplastid Specific RNA-Protein Interactions in Trypanosoma cruzi Ribosome Biogenesis.

    PubMed

    Umaer, Khan; Williams, Noreen

    2015-01-01

    RNA binding proteins (RBP) play essential roles in the highly conserved and coordinated process of ribosome biogenesis. Our laboratory has previously characterized two essential and abundant RBPs, P34 and P37, in Trypanosoma brucei which are required for several critical steps in ribosome biogenesis. The genes for these proteins have only been identified in kinetoplastid organisms but not in the host genome. We have identified a homolog of the TbP34 and TbP37 in a T. cruzi strain (termed TcP37/NRBD). Although the N-terminal APK-rich domain and RNA recognition motifs are conserved, the C-terminal region which contains putative nuclear and nucleolar localization signals in TbP34 and TbP37 is almost entirely missing from TcP37/NRBD. We have shown that TcP37/NRBD is expressed in T. cruzi epimastigotes at the level of mature mRNA and protein. Despite the loss of the C-terminal domain, TcP37/NRBD is present in the nucleus, including the nucleolus, and the cytoplasm. TcP37/NRBD interacts directly with Tc 5S rRNA, but does not associate with polyadenylated RNA. TcP37/NRBD also associates in vivo and in vitro with large ribosomal protein TcL5 and, unlike the case of T. brucei, this association is strongly enhanced by the presence of 5S rRNA, suggesting that the loss of the C-terminal domain of TcP37/NRBD may alter the interactions within the complex. These results indicate that the unique preribosomal complex comprised of L5, 5S rRNA, and the trypanosome-specific TcP37/NRBD or TbP34 and TbP37 is functionally conserved in trypanosomes despite the differences in the C-termini of the trypanosome-specific protein components.

  3. Rice Ribosomal Protein Large Subunit Genes and Their Spatio-temporal and Stress Regulation

    PubMed Central

    Moin, Mazahar; Bakshi, Achala; Saha, Anusree; Dutta, Mouboni; Madhav, Sheshu M.; Kirti, P. B.

    2016-01-01

    Ribosomal proteins (RPs) are well-known for their role in mediating protein synthesis and maintaining the stability of the ribosomal complex, which includes small and large subunits. In the present investigation, in a genome-wide survey, we predicted that the large subunit of rice ribosomes is encoded by at least 123 genes including individual gene copies, distributed throughout the 12 chromosomes. We selected 34 candidate genes, each having 2–3 identical copies, for a detailed characterization of their gene structures, protein properties, cis-regulatory elements and comprehensive expression analysis. RPL proteins appear to be involved in interactions with other RP and non-RP proteins and their encoded RNAs have a higher content of alpha-helices in their predicted secondary structures. The majority of RPs have binding sites for metal and non-metal ligands. Native expression profiling of 34 ribosomal protein large (RPL) subunit genes in tissues covering the major stages of rice growth shows that they are predominantly expressed in vegetative tissues and seedlings followed by meiotically active tissues like flowers. The putative promoter regions of these genes also carry cis-elements that respond specifically to stress and signaling molecules. All the 34 genes responded differentially to the abiotic stress treatments. Phytohormone and cold treatments induced significant up-regulation of several RPL genes, while heat and H2O2 treatments down-regulated a majority of them. Furthermore, infection with a bacterial pathogen, Xanthomonas oryzae, which causes leaf blight also induced the expression of 80% of the RPL genes in leaves. Although the expression of RPL genes was detected in all the tissues studied, they are highly responsive to stress and signaling molecules indicating that their encoded proteins appear to have roles in stress amelioration besides house-keeping. This shows that the RPL gene family is a valuable resource for manipulation of stress tolerance in

  4. The ribosomal subunit assembly line

    PubMed Central

    Dlakić, Mensur

    2005-01-01

    Recent proteomic studies in Saccharomyces cerevisiae have identified nearly 200 proteins, other than the structural ribosomal proteins, that participate in the assembly of ribosomal subunits and their transport from the nucleus. In a separate line of research, proteomic studies of mature plant ribosomes have revealed considerable variability in the protein composition of individual ribosomes. PMID:16207363

  5. Identification of an unconventional nuclear localization signal in human ribosomal protein S2

    SciTech Connect

    Antoine, M.; Reimers, K.; Wirz, W.; Gressner, A.M.; Mueller, R.; Kiefer, P. . E-Mail: pkiefer@ukaachen.de

    2005-09-16

    Ribosomal proteins must be imported into the nucleus after being synthesized in the cytoplasm. Since the rpS2 amino acid sequence does not contain a typical nuclear localization signal, we used deletion mutant analysis and rpS2-{beta}-galactosidase chimeric proteins to identify the nuclear targeting domains in rpS2. Nuclear rpS2 is strictly localized in the nucleoplasm and is not targeted to the nucleoli. Subcellular localization analysis of deletion mutants of rpS2-{beta}-galactosidase chimeras identified a central domain comprising 72 amino acids which is necessary and sufficient to target the chimeric {beta}-galactosidase to the nucleus. The nuclear targeting domain shares no significant similarity to already characterized nuclear localization signals in ribosomal proteins or other nuclear proteins. Although a Nup153 fragment containing the importin{beta} binding site fused to VP22 blocks nuclear import of rpS2-{beta}-galactosidase fusion proteins, nuclear uptake of rpS2 could be mediated by several import receptors since it binds to importin{alpha}/{beta} and transportin.

  6. Site-Specific Cleavage of Ribosomal RNA in Escherichia coli-Based Cell-Free Protein Synthesis Systems

    PubMed Central

    Failmezger, Jurek; Nitschel, Robert; Sánchez-Kopper, Andrés; Kraml, Michael; Siemann-Herzberg, Martin

    2016-01-01

    Cell-free protein synthesis, which mimics the biological protein production system, allows rapid expression of proteins without the need to maintain a viable cell. Nevertheless, cell-free protein expression relies on active in vivo translation machinery including ribosomes and translation factors. Here, we examined the integrity of the protein synthesis machinery, namely the functionality of ribosomes, during (i) the cell-free extract preparation and (ii) the performance of in vitro protein synthesis by analyzing crucial components involved in translation. Monitoring the 16S rRNA, 23S rRNA, elongation factors and ribosomal protein S1, we show that processing of a cell-free extract results in no substantial alteration of the translation machinery. Moreover, we reveal that the 16S rRNA is specifically cleaved at helix 44 during in vitro translation reactions, resulting in the removal of the anti-Shine-Dalgarno sequence. These defective ribosomes accumulate in the cell-free system. We demonstrate that the specific cleavage of the 16S rRNA is triggered by the decreased concentrations of Mg2+. In addition, we provide evidence that helix 44 of the 30S ribosomal subunit serves as a point-of-entry for ribosome degradation in Escherichia coli. Our results suggest that Mg2+ homeostasis is fundamental to preserving functional ribosomes in cell-free protein synthesis systems, which is of major importance for cell-free protein synthesis at preparative scale, in order to create highly efficient technical in vitro systems. PMID:27992588

  7. Unbiased Quantitative Models of Protein Translation Derived from Ribosome Profiling Data

    PubMed Central

    Gritsenko, Alexey A.; Hulsman, Marc; Reinders, Marcel J. T.; de Ridder, Dick

    2015-01-01

    Translation of RNA to protein is a core process for any living organism. While for some steps of this process the effect on protein production is understood, a holistic understanding of translation still remains elusive. In silico modelling is a promising approach for elucidating the process of protein synthesis. Although a number of computational models of the process have been proposed, their application is limited by the assumptions they make. Ribosome profiling (RP), a relatively new sequencing-based technique capable of recording snapshots of the locations of actively translating ribosomes, is a promising source of information for deriving unbiased data-driven translation models. However, quantitative analysis of RP data is challenging due to high measurement variance and the inability to discriminate between the number of ribosomes measured on a gene and their speed of translation. We propose a solution in the form of a novel multi-scale interpretation of RP data that allows for deriving models with translation dynamics extracted from the snapshots. We demonstrate the usefulness of this approach by simultaneously determining for the first time per-codon translation elongation and per-gene translation initiation rates of Saccharomyces cerevisiae from RP data for two versions of the Totally Asymmetric Exclusion Process (TASEP) model of translation. We do this in an unbiased fashion, by fitting the models using only RP data with a novel optimization scheme based on Monte Carlo simulation to keep the problem tractable. The fitted models match the data significantly better than existing models and their predictions show better agreement with several independent protein abundance datasets than existing models. Results additionally indicate that the tRNA pool adaptation hypothesis is incomplete, with evidence suggesting that tRNA post-transcriptional modifications and codon context may play a role in determining codon elongation rates. PMID:26275099

  8. Unbiased Quantitative Models of Protein Translation Derived from Ribosome Profiling Data.

    PubMed

    Gritsenko, Alexey A; Hulsman, Marc; Reinders, Marcel J T; de Ridder, Dick

    2015-08-01

    Translation of RNA to protein is a core process for any living organism. While for some steps of this process the effect on protein production is understood, a holistic understanding of translation still remains elusive. In silico modelling is a promising approach for elucidating the process of protein synthesis. Although a number of computational models of the process have been proposed, their application is limited by the assumptions they make. Ribosome profiling (RP), a relatively new sequencing-based technique capable of recording snapshots of the locations of actively translating ribosomes, is a promising source of information for deriving unbiased data-driven translation models. However, quantitative analysis of RP data is challenging due to high measurement variance and the inability to discriminate between the number of ribosomes measured on a gene and their speed of translation. We propose a solution in the form of a novel multi-scale interpretation of RP data that allows for deriving models with translation dynamics extracted from the snapshots. We demonstrate the usefulness of this approach by simultaneously determining for the first time per-codon translation elongation and per-gene translation initiation rates of Saccharomyces cerevisiae from RP data for two versions of the Totally Asymmetric Exclusion Process (TASEP) model of translation. We do this in an unbiased fashion, by fitting the models using only RP data with a novel optimization scheme based on Monte Carlo simulation to keep the problem tractable. The fitted models match the data significantly better than existing models and their predictions show better agreement with several independent protein abundance datasets than existing models. Results additionally indicate that the tRNA pool adaptation hypothesis is incomplete, with evidence suggesting that tRNA post-transcriptional modifications and codon context may play a role in determining codon elongation rates.

  9. Alopecia, Neurological Defects, and Endocrinopathy Syndrome Caused by Decreased Expression of RBM28, a Nucleolar Protein Associated with Ribosome Biogenesis

    PubMed Central

    Nousbeck, Janna; Spiegel, Ronen; Ishida-Yamamoto, Akemi; Indelman, Margarita; Shani-Adir, Ayelet; Adir, Noam; Lipkin, Ehud; Bercovici, Sivan; Geiger, Dan; van Steensel, Maurice A.; Steijlen, Peter M.; Bergman, Reuven; Bindereif, Albrecht; Choder, Mordechai; Shalev, Stavit; Sprecher, Eli

    2008-01-01

    Single-gene disorders offer unique opportunities to shed light upon fundamental physiological processes in humans. We investigated an autosomal-recessive phenotype characterized by alopecia, progressive neurological defects, and endocrinopathy (ANE syndrome). By using homozygosity mapping and candidate-gene analysis, we identified a loss-of-function mutation in RBM28, encoding a nucleolar protein. RBM28 yeast ortholog, Nop4p, was previously found to regulate ribosome biogenesis. Accordingly, electron microscopy revealed marked ribosome depletion and structural abnormalities of the rough endoplasmic reticulum in patient cells, ascribing ANE syndrome to the restricted group of inherited disorders associated with ribosomal dysfunction. PMID:18439547

  10. Ribosomal protein L3 bound to 23S precursor rRNA stimulates its maturation by Mini-III ribonuclease.

    PubMed

    Redko, Yulia; Condon, Ciarán

    2009-03-01

    Ribosomal RNAs (rRNAs) are processed from larger primary transcripts in every living system known. The maturation of 23S rRNA in Bacillus subtilis is catalysed by Mini-III, a member of the RNase III family of enzymes that lacks the characteristic double-stranded RNA binding domain of its relatives. We have previously shown that Mini-III processing of 23S precursor rRNA in assembled 50S ribosomal subunits is much more efficient than a substrate with no ribosomal proteins bound, suggesting that one or more large subunit proteins act as a cofactor for Mini-III cleavage. Here we show that this cofactor is ribosomal protein L3. Stimulation of the Mini-III cleavage reaction is through L3 binding to its normal site at the 3' end of 23S rRNA. We present indirect evidence that suggests that L3 acts at the level of substrate, rather than enzyme conformation. We also discuss the potential implication of using ribosomal protein cofactors in rRNA processing for ribosome quality control.

  11. Ribosomal Protein RPL27a Promotes Female Gametophyte Development in a Dose-Dependent Manner1[C][W][OPEN

    PubMed Central

    Zsögön, Agustin; Szakonyi, Dóra; Shi, Xiuling; Byrne, Mary E.

    2014-01-01

    Ribosomal protein mutations in Arabidopsis (Arabidopsis thaliana) result in a range of specific developmental phenotypes. Why ribosomal protein mutants have specific phenotypes is not fully known, but such defects potentially result from ribosome insufficiency, ribosome heterogeneity, or extraribosomal functions of ribosomal proteins. Here, we report that ovule development is sensitive to the level of Ribosomal Protein L27a (RPL27a) and is disrupted by mutations in the two paralogs RPL27aC and RPL27aB. Mutations in RPL27aC result in high levels of female sterility, whereas mutations in RPL27aB have a significant but lesser effect on fertility. Progressive reduction in RPL27a function results in increasing sterility, indicating a dose-dependent relationship between RPL27a and female fertility. RPL27a levels in both the sporophyte and gametophyte affect female gametogenesis, with different developmental outcomes determined by the dose of RPL27a. These results demonstrate that RPL27aC and RPL27aB act redundantly and reveal a function for RPL27a in coordinating complex interactions between sporophyte and gametophyte during ovule development. PMID:24872379

  12. Translation of CGA codon repeats in yeast involves quality control components and ribosomal protein L1.

    PubMed

    Letzring, Daniel P; Wolf, Andrew S; Brule, Christina E; Grayhack, Elizabeth J

    2013-09-01

    Translation of CGA codon repeats in the yeast Saccharomyces cerevisiae is inefficient, resulting in dose-dependent reduction in expression and in production of an mRNA cleavage product, indicative of a stalled ribosome. Here, we use genetics and translation inhibitors to understand how ribosomes respond to CGA repeats. We find that CGA codon repeats result in a truncated polypeptide that is targeted for degradation by Ltn1, an E3 ubiquitin ligase involved in nonstop decay, although deletion of LTN1 does not improve expression downstream from CGA repeats. Expression downstream from CGA codons at residue 318, but not at residue 4, is improved by deletion of either ASC1 or HEL2, previously implicated in inhibition of translation by polybasic sequences. Thus, translation of CGA repeats likely causes ribosomes to stall and exploits known quality control systems. Expression downstream from CGA repeats at amino acid 4 is improved by paromomycin, an aminoglycoside that relaxes decoding specificity. Paromomycin has no effect if native tRNA(Arg(ICG)) is highly expressed, consistent with the idea that failure to efficiently decode CGA codons might occur in part due to rejection of the cognate tRNA(Arg(ICG)). Furthermore, expression downstream from CGA repeats is improved by inactivation of RPL1B, one of two genes encoding the universally conserved ribosomal protein L1. The effects of rpl1b-Δ and of either paromomycin or tRNA(Arg(ICG)) on CGA decoding are additive, suggesting that the rpl1b-Δ mutant suppresses CGA inhibition by means other than increased acceptance of tRNA(Arg(ICG)). Thus, inefficient decoding of CGA likely involves at least two independent defects in translation.

  13. The host antimicrobial peptide Bac71-35 binds to bacterial ribosomal proteins and inhibits protein synthesis.

    PubMed

    Mardirossian, Mario; Grzela, Renata; Giglione, Carmela; Meinnel, Thierry; Gennaro, Renato; Mergaert, Peter; Scocchi, Marco

    2014-12-18

    Antimicrobial peptides (AMPs) are molecules from innate immunity with high potential as novel anti-infective agents. Most of them inactivate bacteria through pore formation or membrane barrier disruption, but others cross the membrane without damages and act inside the cells, affecting vital processes. However, little is known about their intracellular bacterial targets. Here we report that Bac71-35, a proline-rich AMP belonging to the cathelicidin family, can reach high concentrations (up to 340 μM) inside the E. coli cytoplasm. The peptide specifically and completely inhibits in vitro translation in the micromolar concentration range. Experiments of incorporation of radioactive precursors in macromolecules with E. coli cells confirmed that Bac71-35 affects specifically protein synthesis. Ribosome coprecipitation and crosslinking assays showed that the peptide interacts with ribosomes, binding to a limited subset of ribosomal proteins. Overall, these results indicate that the killing mechanism of Bac71-35 is based on a specific block of protein synthesis.

  14. Hepatitis C virus 3'X region interacts with human ribosomal proteins.

    PubMed

    Wood, J; Frederickson, R M; Fields, S; Patel, A H

    2001-02-01

    To identify proteins that can bind the 3' untranslated region (UTR) of hepatitis C virus (HCV) we screened human cDNA libraries using the Saccharomyces cerevisiae three-hybrid system. Screening with an RNA sequence derived from the 3'-terminal 98 nucleotides (3'X region) of an infectious clone of HCV (H77c) yielded clones of human ribosomal proteins L22, L3, S3, and mL3, a mitochondrial homologue of L3. We performed preliminary characterization of the binding between the 3'X region and these proteins by a three-hybrid mating assay using mutant 3'X sequences. We have further characterized the interaction between 3'X and L22, since this protein is known to be associated with two small Epstein-Barr virus (EBV)-encoded RNA species (EBERs) which are abundantly produced in cells latently infected with EBV. The EBERs, which have similar predicted secondary structure to the HCV 3'X, assemble into ribonucleoprotein particles that include L22 and La protein. To confirm that L22 binds HCV 3'X we performed in vitro binding assays using recombinant L22 (expressed as a glutathione S-transferase [GST] fusion protein) together with a 3'X riboprobe. The 3'X region binds to the GST-L22 fusion protein (but not to GST alone), and this interaction is subject to competition with unlabeled 3'X RNA. To establish the functional role played by L22 in internal ribosome entry site (IRES)-mediated translation of HCV sequences we performed translational analysis in HuH-7 cells using monocistronic and bicistronic reporter constructs. The relative amount of core-chloramphenicol acetyltransferase reporter protein translated under the control of the HCV IRES was stimulated in the presence of L22 and La when these proteins were supplied in trans.

  15. Reading the Evolution of Compartmentalization in the Ribosome Assembly Toolbox: The YRG Protein Family.

    PubMed

    Mier, Pablo; Pérez-Pulido, Antonio J; Reynaud, Emmanuel G; Andrade-Navarro, Miguel A

    2017-01-01

    Reconstructing the transition from a single compartment bacterium to a highly compartmentalized eukaryotic cell is one of the most studied problems of evolutionary cell biology. However, timing and details of the establishment of compartmentalization are unclear and difficult to assess. Here, we propose the use of molecular markers specific to cellular compartments to set up a framework to advance the understanding of this complex intracellular process. Specifically, we use a protein family related to ribosome biogenesis, YRG (YlqF related GTPases), whose evolution is linked to the establishment of cellular compartments, leveraging the current genomic data. We analyzed orthologous proteins of the YRG family in a set of 171 proteomes for a total of 370 proteins. We identified ten YRG protein subfamilies that can be associated to six subcellular compartments (nuclear bodies, nucleolus, nucleus, cytosol, mitochondria, and chloroplast), and which were found in archaeal, bacterial and eukaryotic proteomes. Our analysis reveals organism streamlining related events in specific taxonomic groups such as Fungi. We conclude that the YRG family could be used as a compartmentalization marker, which could help to trace the evolutionary path relating cellular compartments with ribosome biogenesis.

  16. Molecular cloning and characterization of the porcine ribosomal protein L21.

    PubMed

    Sun, Wu-Sheng; Chun, Ju-Lan; Kim, Dong-Hwan; Ahn, Jin-Seop; Kim, Min-Kyu; Hwang, In-Sul; Kwon, Dae-Jin; Hwang, Seong-Soo; Lee, Jeong-Woong

    2017-01-04

    Ribosomal protein L21 (RPL21) is a structural component of the 60S subunit of the eukaryotic ribosome. This protein plays an important role in protein synthesis and the occurrence of hereditary diseases. Pig is a common laboratory model, however, to the best of our knowledge, its RPL21 gene has not been cloned to date. In this study, we cloned and identified the full-length sequence of the pig RPL21 gene for the first time. Then we studied its expression pattern and function by overexpression or knockdown approach. As a result, we obtained a 604-bp segment that contains a 483-bp open reading frame encoding 160 amino acids. We found the pig RPL21 gene is located in the "+" strand of chromosome 11, which spans 2167 bp from 4199792 to 4201958. Pig RPL21 protein has nine strands and two helices in its secondary structure. Pig RPL21 is predominantly expressed in the ovary and lung compared to the kidney, small intestine and skin but expressed at lower levels in the heart and liver. Furthermore, we found RPL21 expression level is closely connected with cell proliferation and cell cycle arrest. These results are intended to provide valid information for the further study of pig RPL21.

  17. Reading the Evolution of Compartmentalization in the Ribosome Assembly Toolbox: The YRG Protein Family

    PubMed Central

    Pérez-Pulido, Antonio J.; Reynaud, Emmanuel G.; Andrade-Navarro, Miguel A.

    2017-01-01

    Reconstructing the transition from a single compartment bacterium to a highly compartmentalized eukaryotic cell is one of the most studied problems of evolutionary cell biology. However, timing and details of the establishment of compartmentalization are unclear and difficult to assess. Here, we propose the use of molecular markers specific to cellular compartments to set up a framework to advance the understanding of this complex intracellular process. Specifically, we use a protein family related to ribosome biogenesis, YRG (YlqF related GTPases), whose evolution is linked to the establishment of cellular compartments, leveraging the current genomic data. We analyzed orthologous proteins of the YRG family in a set of 171 proteomes for a total of 370 proteins. We identified ten YRG protein subfamilies that can be associated to six subcellular compartments (nuclear bodies, nucleolus, nucleus, cytosol, mitochondria, and chloroplast), and which were found in archaeal, bacterial and eukaryotic proteomes. Our analysis reveals organism streamlining related events in specific taxonomic groups such as Fungi. We conclude that the YRG family could be used as a compartmentalization marker, which could help to trace the evolutionary path relating cellular compartments with ribosome biogenesis. PMID:28072865

  18. Involvement of human ribosomal proteins in nucleolar structure and p53-dependent nucleolar stress

    PubMed Central

    Nicolas, Emilien; Parisot, Pascaline; Pinto-Monteiro, Celina; de Walque, Roxane; De Vleeschouwer, Christophe; Lafontaine, Denis L. J.

    2016-01-01

    The nucleolus is a potent disease biomarker and a target in cancer therapy. Ribosome biogenesis is initiated in the nucleolus where most ribosomal (r-) proteins assemble onto precursor rRNAs. Here we systematically investigate how depletion of each of the 80 human r-proteins affects nucleolar structure, pre-rRNA processing, mature rRNA accumulation and p53 steady-state level. We developed an image-processing programme for qualitative and quantitative discrimination of normal from altered nucleolar morphology. Remarkably, we find that uL5 (formerly RPL11) and uL18 (RPL5) are the strongest contributors to nucleolar integrity. Together with the 5S rRNA, they form the late-assembling central protuberance on mature 60S subunits, and act as an Hdm2 trap and p53 stabilizer. Other major contributors to p53 homeostasis are also strictly late-assembling large subunit r-proteins essential to nucleolar structure. The identification of the r-proteins that specifically contribute to maintaining nucleolar structure and p53 steady-state level provides insights into fundamental aspects of cell and cancer biology. PMID:27265389

  19. Structure and function of ribosomal protein S4 genes on the human and mouse sex chromosomes.

    PubMed

    Zinn, A R; Alagappan, R K; Brown, L G; Wool, I; Page, D C

    1994-04-01

    The human sex-linked genes RPS4X and RPS4Y encode distinct isoforms of ribosomal protein S4. Insufficient expression of S4 may play a role in the development of Turner syndrome, the complex human phenotype associated with monosomy X. In mice, the S4 protein is encoded by an X-linked gene, Rps4, and is identical to human S4X; there is no mouse Y homolog. We report here the organization of the human RPS4X and RPS4Y and mouse Rps4 genes. Each gene comprises seven exons; the positions of introns are conserved. The 5' flanking sequences of human RPS4X and mouse Rps4 are very similar, while RPS4Y diverges shortly upstream of the transcription start site. In chickens, S4 is encoded by a single gene that is not sex linked. The chicken protein differs from human S4X by four amino acid substitutions, all within a region encoded by a single exon. Three of the four substitutions are also present in human S4Y, suggesting that the chicken S4 gene may have arisen by recombination between S4X- and S4Y-like sequences. Using isoform-specific antisera, we determined that human S4X and S4Y are both present in translationally active ribosomes. S4Y is about 10 to 15% as abundant as S4X in ribosomes from normal male placental tissue and 46,XY cultured cells. In 49,XYYYY cells, S4Y is about half as abundant as S4X. In 49,XXXXY cells, S4Y is barely detectable. These results bear on the hypothesized role of S4 deficiency in Turner syndrome.

  20. Interaction of Bacillus thuringiensis vegetative insecticidal protein with ribosomal S2 protein triggers larvicidal activity in Spodoptera frugiperda.

    PubMed

    Singh, Gatikrushna; Sachdev, Bindiya; Sharma, Nathilal; Seth, Rakesh; Bhatnagar, Raj K

    2010-11-01

    Vegetative insecticidal protein (Vip3A) is synthesized as an extracellular insecticidal toxin by certain strains of Bacillus thuringiensis. Vip3A is active against several lepidopteran pests of crops. Polyphagous pest, Spodoptera frugiperda, and its cell line Sf21 are sensitive for lyses to Vip3A. Screening of cDNA library prepared from Sf21 cells through yeast two-hybrid system with Vip3A as bait identified ribosomal protein S2 as a toxicity-mediating interacting partner protein. The Vip3A-ribosomal-S2 protein interaction was validated by in vitro pulldown assays and by RNA interference-induced knockdown experiments. Knockdown of expression of S2 protein in Sf21 cells resulted in reduced toxicity of the Vip3A protein. These observations were further extended to adult fifth-instar larvae of Spodoptera litura. Knockdown of S2 expression by injecting corresponding double-stranded RNA resulted in reduced mortality of larvae to Vip3A toxin. Intracellular visualization of S2 protein and Vip3A through confocal microscopy revealed their interaction and localization in cytoplasm and surface of Sf21 cells.

  1. Mutations in the Drosophila gene encoding ribosomal protein S6 cause tissue overgrowth.

    PubMed Central

    Stewart, M J; Denell, R

    1993-01-01

    We have characterized two P-element-induced, lethal mutations in Drosophila melanogaster which affect the larval hemocytes, mediators of the insect immune response. Each mutant displays larval melanotic tumors characteristic of mutations affecting the insect cellular immune system, and the moribund animals develop grossly hypertrophied hematopoietic organs because of increased cell proliferation and extra rounds of endoreduplication in some hematopoietic cells. Surprisingly, these mutations are due to P element insertions in the 5' regulatory region of the Drosophila gene encoding ribosomal protein S6 and cause a reduction of S6 transcript abundance in mutant larvae. Images PMID:8384310

  2. Role of ribosomal protein S12 in peptide chain elongation: analysis of pleiotropic, streptomycin-resistant mutants of Escherichia coli.

    PubMed Central

    Zengel, J M; Young, R; Dennis, P P; Nomura, M

    1977-01-01

    Some of the spontaneous streptomycin-resistant mutants of Escherichia coli strain C600 exhibit pleiotropic effects in addition to the antibiotic resistance. These effects include decreased growth rates, reduced levels of certain enzymes, and poor support of bacteriophage growth. One of these mutants, strain SM3, was studied further. We have examined the question of whether the reduced growth rate of the mutant SM3 is related to the reduction in relative amounts of ribosomes or to the reduction in the efficiency of ribosomes in protein synthesis. Measurements of alpha, the differential synthesis rate of ribosomal protein, revealed that the protein synthesis effeciency of ribosomes from the mutant strain SM3 was reduced about twofold relative to that of the parent strain C600. Measurements of the induction lag for beta-galactosidase and of the synthesis time of several different molecular-weight classes of proteins indicated that the mutation resulted in a marked reduction in the peptide chain growth rate. This reduction in the chain growth rate probably accounted for most of the observed reduction in the growth rate of the mutant strain. These experimental results show that the strA gene product, the S12 protein of the 30S subunit, is involved in some aspect of protein chain elongation. Presumably this involvement occurs during the messenger ribonucleic acid-directed binding of transfer ribonucleic acid to the ribosome. PMID:321423

  3. Role of ribosomal protein S12 in peptide chain elongation: analysis of pleiotropic, streptomycin-resistant mutants of Escherichia coli.

    PubMed

    Zengel, J M; Young, R; Dennis, P P; Nomura, M

    1977-03-01

    Some of the spontaneous streptomycin-resistant mutants of Escherichia coli strain C600 exhibit pleiotropic effects in addition to the antibiotic resistance. These effects include decreased growth rates, reduced levels of certain enzymes, and poor support of bacteriophage growth. One of these mutants, strain SM3, was studied further. We have examined the question of whether the reduced growth rate of the mutant SM3 is related to the reduction in relative amounts of ribosomes or to the reduction in the efficiency of ribosomes in protein synthesis. Measurements of alpha, the differential synthesis rate of ribosomal protein, revealed that the protein synthesis effeciency of ribosomes from the mutant strain SM3 was reduced about twofold relative to that of the parent strain C600. Measurements of the induction lag for beta-galactosidase and of the synthesis time of several different molecular-weight classes of proteins indicated that the mutation resulted in a marked reduction in the peptide chain growth rate. This reduction in the chain growth rate probably accounted for most of the observed reduction in the growth rate of the mutant strain. These experimental results show that the strA gene product, the S12 protein of the 30S subunit, is involved in some aspect of protein chain elongation. Presumably this involvement occurs during the messenger ribonucleic acid-directed binding of transfer ribonucleic acid to the ribosome.

  4. Alpha-momorcharin: a ribosome-inactivating protein from Momordica charantia, possessing DNA cleavage properties.

    PubMed

    Wang, Shuzhen; Zheng, Yinzhen; Yan, Junjie; Zhu, Zhixuan; Wu, Zhihua; Ding, Yi

    2013-11-01

    Ribosome-inactivating proteins (RIPs) function to inhibit protein synthesis through the removal of specific adenine residues from eukaryotic ribosomal RNA and rending the 60S subunit unable to bind elongation factor 2. They have received much attention in biological and biomedical research due to their unique activities toward tumor cells, as well as the important roles in plant defense. Alpha-momorcharin (α-MC), a member of the type I family of RIPs, is rich in the seeds of Momordica charantia L. Previous studies demonstrated that α-MC is an effective antifungal and antibacterial protein. In this study, a detailed analysis of the DNase-like activity of α-MC was conducted. Results showed that the DNase-like activity toward plasmid DNA was time-dependent, temperature-related, and pH-stable. Moreover, a requirement for divalent metal ions in the catalytic domain of α-MC was confirmed. Additionally, Tyr(93) was found to be a critical residue for the DNase-like activity, while Tyr(134), Glu(183), Arg(186), and Trp(215) were activity-related residues. This study on the chemico-physical properties and mechanism of action of α-MC will improve its utilization in scientific research, as well as its potential industrial uses. These results may also assist in the characterization and elucidation of the DNase-like enzymatic properties of other RIPs.

  5. Characterization of silk gland ribosomes from a bivoltine caddisfly, Stenopsyche marmorata: translational suppression of a silk protein in cold conditions.

    PubMed

    Nomura, Takaomi; Ito, Miho; Kanamori, Mai; Shigeno, Yuta; Uchiumi, Toshio; Arai, Ryoichi; Tsukada, Masuhiro; Hirabayashi, Kimio; Ohkawa, Kousaku

    2016-01-08

    Larval Stenopsyche marmorata constructs food capture nets and fixed retreats underwater using self-produced proteinaceous silk fibers. In the Chikuma River (Nagano Prefecture, Japan) S. marmorata has a bivoltine life cycle; overwintering larvae grow slowly with reduced net spinning activity in winter. We recently reported constant transcript abundance of S. marmorata silk protein 1 (Smsp-1), a core S. marmorata silk fiber component, in all seasons, implying translational suppression in the silk gland during winter. Herein, we prepared and characterized silk gland ribosomes from seasonally collected S. marmorata larvae. Ribosomes from silk glands immediately frozen in liquid nitrogen (LN2) after dissection exhibited comparable translation elongation activity in spring, summer, and autumn. Conversely, silk glands obtained in winter did not contain active ribosomes and Smsp-1. Ribosomes from silk glands immersed in ice-cold physiological saline solution for approximately 4 h were translationally inactive, despite summer collection and Smsp-1 expression. The ribosomal inactivation occurs because of defects in the formation of 80S ribosomes, presumably due to splitting of 60S subunits containing 28S rRNA with central hidden break, in response to cold stress. These results suggest a novel-type ribosome-regulated translation control mechanism.

  6. Translational control of ribosomal protein synthesis during early Dictyostelium discoideum development.

    PubMed Central

    Steel, L F; Jacobson, A

    1987-01-01

    Throughout the developmental program of Dictyostelium discoideum there are substantial changes in the rates of both ribosome utilization and rRNA transcription and processing. We examined the regulation of ribosomal protein (r-protein) gene expression and found that, at the start of development, expression of these genes was drastically and specifically reduced by a block to translational initiation. An apparently separate event signals a sudden decrease in the relative amount of r-protein mRNA at about 10 h of development, a time when aggregated amoebae are forming tight cell-cell contacts. For the first 9 h of development, the relative amount of r-protein mRNA remained essentially unchanged and comparable to levels detected in growing cells. While the r-protein mRNAs were almost fully loaded on polysomes during vegetative growth, they were specifically excluded from polysomes at the start of development. The translational block was not the result of irreversible structural changes which inactivate the r-protein mRNAs since they remained translatable both in vitro, in wheat germ extracts, and in vivo, where they were recruited onto polysomes in the presence of the elongation inhibitor cycloheximide. In addition, precise measurements of poly(A) tail lengths on individual hybrid-selected mRNA species showed that there is no difference in the poly(A) tail length of r-protein mRNA isolated from growing cells and 1-h developing cells. Therefore, changes in translational efficiency cannot be attributed to cleavage of poly(A) tails. Images PMID:2882416

  7. Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR

    PubMed Central

    Lee, Sang-Won; Berger, Scott J.; Martinović, Suzana; Paša-Tolić, Ljiljana; Anderson, Gordon A.; Shen, Yufeng; Zhao, Rui; Smith, Richard D.

    2002-01-01

    Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry coupled with capillary reverse-phase liquid chromatography was used to characterize intact proteins from the large subunit of the yeast ribosome. High mass measurement accuracy, achieved by “mass locking” with an internal standard from a dual electrospray ionization source, allowed identification of ribosomal proteins. Analyses of the intact proteins revealed information on cotranslational and posttranslational modifications of the ribosomal proteins that included loss of the initiating methionine, acetylation, methylation, and proteolytic maturation. High-resolution separations permitted differentiation of protein isoforms having high structural similarity as well as proteins from their modified forms, facilitating unequivocal assignments. The study identified 42 of the 43 core large ribosomal subunit proteins and 58 (of 64 possible) core large subunit protein isoforms having unique masses in a single analysis. These results demonstrate the basis for the high-throughput analyses of complex mixtures of intact proteins, which we believe will be an important complement to other approaches for defining protein modifications and their changes resulting from physiological processes or environmental perturbations. PMID:11983894

  8. Ribosomal protein L4 interacts with viral protein VP3 and regulates the replication of infectious bursal disease virus.

    PubMed

    Chen, Yuming; Lu, Zhen; Zhang, Lizhou; Gao, Li; Wang, Nian; Gao, Xiang; Wang, Yongqiang; Li, Kai; Gao, Yulong; Cui, Hongyu; Gao, Honglei; Liu, Changjun; Zhang, Yanping; Qi, Xiaole; Wang, Xiaomei

    2016-01-04

    VP3 protein is a structural protein which plays important roles in the virus assembly and the inhibition of antiviral innate immunity of infectious bursal disease virus (IBDV). To explore the potential roles of VP3 in the interplay of IBDV with the host cell, an immunoprecipitation (IP)-coupled mass spectra (MS) screening was performed and the host cellular ribosomal protein L4 (RPL4) was identified as a putative interacting partner of VP3 protein. The interaction of RPL4 with VP3 was further confirmed by co-immunoprecipitation (co-IP) and their colocalization in DF1 cells were observed by confocal microscopy. In addition, knockdown of RPL4 in DF1 cells resulted in reductions of the viral protein pVP2 expression and the virus titers, which reveals a significant role of RPL4 in IBDV replication. Taken together, we indicated for the first time that ribosomal protein L4 (RPL4) was an interacting partner of VP3 and involved in the modulation of IBDV replication. The present study contributes to further understanding the pathogenic mechanism of IBDV.

  9. In vitro selection and evolution of functional proteins by using ribosome display

    PubMed Central

    Hanes, Jozef; Plückthun, Andreas

    1997-01-01

    We report here a system with which a correctly folded complete protein and its encoding mRNA both remain attached to the ribosome and can be enriched for the ligand-binding properties of the native protein. We have selected a single-chain fragment (scFv) of an antibody 108-fold by five cycles of transcription, translation, antigen-affinity selection, and PCR. The selected scFv fragments all mutated in vitro by acquiring up to four unrelated amino acid exchanges over the five generations, but they remained fully compatible with antigen binding. Libraries of native folded proteins can now be screened and made to evolve in a cell-free system without any transformation or constraints imposed by the host cell. PMID:9144168

  10. Appraisal of the Missing Proteins Based on the mRNAs Bound to Ribosomes.

    PubMed

    Xu, Shaohang; Zhou, Ruo; Ren, Zhe; Zhou, Baojin; Lin, Zhilong; Hou, Guixue; Deng, Yamei; Zi, Jin; Lin, Liang; Wang, Quanhui; Liu, Xin; Xu, Xun; Wen, Bo; Liu, Siqi

    2015-12-04

    Considering the technical limitations of mass spectrometry in protein identification, the mRNAs bound to ribosomes (RNC-mRNA) are assumed to reflect the mRNAs participating in the translational process. The RNC-mRNA data are reasoned to be useful for appraising the missing proteins. A set of the multiomics data including free-mRNAs, RNC-mRNAs, and proteomes was acquired from three liver cancer cell lines. On the basis of the missing proteins in neXtProt (release 2014-09-19), the bioinformatics analysis was carried out in three phases: (1) finding how many neXtProt missing proteins have or do not have RNA-seq and/or MS/MS evidence, (2) analyzing specific physicochemical and biological properties of the missing proteins that lack both RNA-seq and MS/MS evidence, and (3) analyzing the combined properties of these missing proteins. Total of 1501 missing proteins were found by neither RNC-mRNA nor MS/MS in the three liver cancer cell lines. For these missing proteins, some are expected higher hydrophobicity, unsuitable detection, or sensory functions as properties at the protein level, while some are predicted to have nonexpressing chromatin structures on the corresponding gene level. With further integrated analysis, we could attribute 93% of them (1391/1501) to these causal factors, which result in the expression products scarcely detected by RNA-seq or MS/MS.

  11. Isolation of Mitochondrial Ribosomes.

    PubMed

    Carroll, Adam J

    2017-01-01

    Translation of mitochondrial encoded mRNAs by mitochondrial ribosomes is thought to play a major role in regulating the expression of mitochondrial proteins. However, the structure and function of plant mitochondrial ribosomes remains poorly understood. To study mitochondrial ribosomes, it is necessary to separate them from plastidic and cytosolic ribosomes that are generally present at much higher concentrations. Here, a straight forward protocol for the preparation of fractions highly enriched in mitochondrial ribosomes from plant cells is described. The method begins with purification of mitochondria followed by mitochondrial lysis and ultracentrifugation of released ribosomes through sucrose cushions and gradients. Dark-grown Arabidopsis cells were used in this example because of the ease with which good yields of pure mitochondria can be obtained from them. However, the steps for isolation of ribosomes from mitochondria could be applied to mitochondria obtained from other sources. Proteomic analyses of resulting fractions have confirmed strong enrichment of mitochondrial ribosomal proteins.

  12. Yeast Asc1p and Mammalian RACK1 Are Functionally Orthologous Core 40S Ribosomal Proteins That Repress Gene Expression

    PubMed Central

    Gerbasi, Vincent R.; Weaver, Connie M.; Hill, Salisha; Friedman, David B.; Link, Andrew J.

    2004-01-01

    Translation of mRNA into protein is a fundamental step in eukaryotic gene expression requiring the large (60S) and small (40S) ribosome subunits and associated proteins. By modern proteomic approaches, we previously identified a novel 40S-associated protein named Asc1p in budding yeast and RACK1 in mammals. The goals of this study were to establish Asc1p or RACK1 as a core conserved eukaryotic ribosomal protein and to determine the role of Asc1p or RACK1 in translational control. We provide biochemical, evolutionary, genetic, and functional evidence showing that Asc1p or RACK1 is indeed a conserved core component of the eukaryotic ribosome. We also show that purified Asc1p-deficient ribosomes have increased translational activity compared to that of wild-type yeast ribosomes. Further, we demonstrate that asc1Δ null strains have increased levels of specific proteins in vivo and that this molecular phenotype is complemented by either Asc1p or RACK1. Our data suggest that one of Asc1p's or RACK1's functions is to repress gene expression. PMID:15340087

  13. Yeast Asc1p and mammalian RACK1 are functionally orthologous core 40S ribosomal proteins that repress gene expression.

    PubMed

    Gerbasi, Vincent R; Weaver, Connie M; Hill, Salisha; Friedman, David B; Link, Andrew J

    2004-09-01

    Translation of mRNA into protein is a fundamental step in eukaryotic gene expression requiring the large (60S) and small (40S) ribosome subunits and associated proteins. By modern proteomic approaches, we previously identified a novel 40S-associated protein named Asc1p in budding yeast and RACK1 in mammals. The goals of this study were to establish Asc1p or RACK1 as a core conserved eukaryotic ribosomal protein and to determine the role of Asc1p or RACK1 in translational control. We provide biochemical, evolutionary, genetic, and functional evidence showing that Asc1p or RACK1 is indeed a conserved core component of the eukaryotic ribosome. We also show that purified Asc1p-deficient ribosomes have increased translational activity compared to that of wild-type yeast ribosomes. Further, we demonstrate that asc1Delta null strains have increased levels of specific proteins in vivo and that this molecular phenotype is complemented by either Asc1p or RACK1. Our data suggest that one of Asc1p's or RACK1's functions is to repress gene expression.

  14. The yeast omnipotent suppressor SUP46 encodes a ribosomal protein which is a functional and structural homolog of the Escherichia coli S4 ram protein.

    PubMed

    Vincent, A; Liebman, S W

    1992-10-01

    The accurate synthesis of proteins is crucial to the existence of a cell. In yeast, several genes that affect the fidelity of translation have been identified (e.g., omnipotent suppressors, antisuppressors and allosuppressors). We have found that the dominant omnipotent suppressor SUP46 encodes the yeast ribosomal protein S13. S13 is encoded by two similar genes, but only the sup46 copy of the gene is able to fully complement the recessive phenotypes of SUP46 mutations. Both copies of the S13 genes contain introns. Unlike the introns of other duplicated ribosomal protein genes which are highly diverged, the duplicated S13 genes have two nearly identical DNA sequences of 25 and 31 bp in length within their introns. The SUP46 protein has significant homology to the S4 ribosomal protein in prokaryotic-type ribosomes. S4 is encoded by one of the ram (ribosomal ambiguity) genes in Escherichia coli which are the functional equivalent of omnipotent suppressors in yeast. Thus, SUP46 and S4 demonstrate functional as well as sequence conservation between prokaryotic and eukaryotic ribosomal proteins. SUP46 and S4 are most similar in their central amino acid sequences. Interestingly, the alterations resulting from the SUP46 mutations and the segment of the S4 protein involved in binding to the 16S rRNA are within this most conserved region.

  15. The Modular Adaptive Ribosome

    PubMed Central

    Yadav, Anupama; Radhakrishnan, Aparna; Panda, Anshuman; Singh, Amartya; Sinha, Himanshu; Bhanot, Gyan

    2016-01-01

    The ribosome is an ancient machine, performing the same function across organisms. Although functionally unitary, recent experiments suggest specialized roles for some ribosomal proteins. Our central thesis is that ribosomal proteins function in a modular fashion to decode genetic information in a context dependent manner. We show through large data analyses that although many ribosomal proteins are essential with consistent effect on growth in different conditions in yeast and similar expression across cell and tissue types in mice and humans, some ribosomal proteins are used in an environment specific manner. The latter set of variable ribosomal proteins further function in a coordinated manner forming modules, which are adapted to different environmental cues in different organisms. We show that these environment specific modules of ribosomal proteins in yeast have differential genetic interactions with other pathways and their 5’UTRs show differential signatures of selection in yeast strains, presumably to facilitate adaptation. Similarly, we show that in higher metazoans such as mice and humans, different modules of ribosomal proteins are expressed in different cell types and tissues. A clear example is nervous tissue that uses a ribosomal protein module distinct from the rest of the tissues in both mice and humans. Our results suggest a novel stratification of ribosomal proteins that could have played a role in adaptation, presumably to optimize translation for adaptation to diverse ecological niches and tissue microenvironments. PMID:27812193

  16. The Modular Adaptive Ribosome.

    PubMed

    Yadav, Anupama; Radhakrishnan, Aparna; Panda, Anshuman; Singh, Amartya; Sinha, Himanshu; Bhanot, Gyan

    2016-01-01

    The ribosome is an ancient machine, performing the same function across organisms. Although functionally unitary, recent experiments suggest specialized roles for some ribosomal proteins. Our central thesis is that ribosomal proteins function in a modular fashion to decode genetic information in a context dependent manner. We show through large data analyses that although many ribosomal proteins are essential with consistent effect on growth in different conditions in yeast and similar expression across cell and tissue types in mice and humans, some ribosomal proteins are used in an environment specific manner. The latter set of variable ribosomal proteins further function in a coordinated manner forming modules, which are adapted to different environmental cues in different organisms. We show that these environment specific modules of ribosomal proteins in yeast have differential genetic interactions with other pathways and their 5'UTRs show differential signatures of selection in yeast strains, presumably to facilitate adaptation. Similarly, we show that in higher metazoans such as mice and humans, different modules of ribosomal proteins are expressed in different cell types and tissues. A clear example is nervous tissue that uses a ribosomal protein module distinct from the rest of the tissues in both mice and humans. Our results suggest a novel stratification of ribosomal proteins that could have played a role in adaptation, presumably to optimize translation for adaptation to diverse ecological niches and tissue microenvironments.

  17. A novel insertion mutation in Streptomyces coelicolor ribosomal S12 protein results in paromomycin resistance and antibiotic overproduction.

    PubMed

    Wang, Guojun; Inaoka, Takashi; Okamoto, Susumu; Ochi, Kozo

    2009-03-01

    We identified a novel paromomycin resistance-associated mutation in rpsL, caused by the insertion of a glycine residue at position 92, in Streptomyces coelicolor ribosomal protein S12. This insertion mutation (GI92) resulted in a 20-fold increase in the paromomycin resistance level. In combination with another S12 mutation, K88E, the GI92 mutation markedly enhanced the production of the blue-colored polyketide antibiotic actinorhodin and the red-colored antibiotic undecylprodigiosin. The gene replacement experiments demonstrated that the K88E-GI92 double mutation in the rpsL gene was responsible for the marked enhancement of antibiotic production observed. Ribosomes with the K88E-GI92 double mutation were characterized by error restrictiveness (i.e., hyperaccuracy). Using a cell-free translation system, we found that mutant ribosomes harboring the K88E-GI92 double mutation but not ribosomes harboring the GI92 mutation alone displayed sixfold greater translation activity relative to that of the wild-type ribosomes at late growth phase. This resulted in the overproduction of actinorhodin, caused by the transcriptional activation of the pathway-specific regulatory gene actII-orf4, possibly due to the increased translation of transcripts encoding activators of actII-orf4. The mutant with the K88E-GI92 double mutation accumulated a high level of ribosome recycling factor at late stationary phase, underlying the high level of protein synthesis activity observed.

  18. Identification of Novel RNA-Protein Contact in Complex of Ribosomal Protein S7 and 3'-Terminal Fragment of 16S rRNA in E. coli.

    PubMed

    Golovin, A V; Khayrullina, G A; Kraal, B; Kopylov, Capital A Cyrillic М

    2012-10-01

    For prokaryotes in vitro, 16S rRNA and 20 ribosomal proteins are capable of hierarchical self- assembly yielding a 30S ribosomal subunit. The self-assembly is initiated by interactions between 16S rRNA and three key ribosomal proteins: S4, S8, and S7. These proteins also have a regulatory function in the translation of their polycistronic operons recognizing a specific region of mRNA. Therefore, studying the RNA-protein interactions within binary complexes is obligatory for understanding ribosome biogenesis. The non-conventional RNA-protein contact within the binary complex of recombinant ribosomal protein S7 and its 16S rRNA binding site (236 nucleotides) was identified. UV-induced RNA-protein cross-links revealed that S7 cross-links to nucleotide U1321 of 16S rRNA. The careful consideration of the published RNA- protein cross-links for protein S7 within the 30S subunit and their correlation with the X-ray data for the 30S subunit have been performed. The RNA - protein cross-link within the binary complex identified in this study is not the same as the previously found cross-links for a subunit both in a solution, and in acrystal. The structure of the binary RNA-protein complex formed at the initial steps of self-assembly of the small subunit appears to be rearranged during the formation of the final structure of the subunit.

  19. Mefloquine targets the Plasmodium falciparum 80S ribosome to inhibit protein synthesis.

    PubMed

    Wong, Wilson; Bai, Xiao-Chen; Sleebs, Brad E; Triglia, Tony; Brown, Alan; Thompson, Jennifer K; Jackson, Katherine E; Hanssen, Eric; Marapana, Danushka S; Fernandez, Israel S; Ralph, Stuart A; Cowman, Alan F; Scheres, Sjors H W; Baum, Jake

    2017-03-13

    Malaria control is heavily dependent on chemotherapeutic agents for disease prevention and drug treatment. Defining the mechanism of action for licensed drugs, for which no target is characterized, is critical to the development of their second-generation derivatives to improve drug potency towards inhibition of their molecular targets. Mefloquine is a widely used antimalarial without a known mode of action. Here, we demonstrate that mefloquine is a protein synthesis inhibitor. We solved a 3.2 Å cryo-electron microscopy structure of the Plasmodium falciparum 80S ribosome with the (+)-mefloquine enantiomer bound to the ribosome GTPase-associated centre. Mutagenesis of mefloquine-binding residues generates parasites with increased resistance, confirming the parasite-killing mechanism. Furthermore, structure-guided derivatives with an altered piperidine group, predicted to improve binding, show enhanced parasiticidal effect. These data reveal one possible mode of action for mefloquine and demonstrate the vast potential of cryo-electron microscopy to guide the development of mefloquine derivatives to inhibit parasite protein synthesis.

  20. Human recombinant antibodies against Trypanosoma cruzi ribosomal P2β protein.

    PubMed

    Grippo, Vanina; Niborski, Leticia L; Gomez, Karina A; Levin, Mariano J

    2011-05-01

    Patients with chronic Chagas' Heart Disease (cChHD) develop an antibody response that is suspected to be involved in the cardiac pathogenesis. The response against Trypanosoma cruzi ribosomal P proteins is of particular interest, as these antibodies can cross-react with host cardiac receptors causing electrophysiological alterations. To better understand the humoral anti-P response we constructed a single-chain variable fragment library derived from a cChHD patient. The variable heavy and light regions were amplified from bone-marrow RNA and subcloned into the vector pComb3X. The phage library was subsequently panned against T. cruzi ribosomal P2β protein (TcP2β). We obtained 3 different human recombinant antibodies that specifically reacted with TcP2β in ELISA and Western blots. Two of them reacted with the C-terminal region of TcP2β, peptide R13, as the recombinant autoanti-P antibodies from Systemic Lupus Erythematosus (SLE) patients. Interestingly, the third one was specific for TcP2β but did not recognize R13, confirming the specific nature of the anti-P response in Chagas disease. Neither sequence nor VH usage similarities between Chagas and SLE anti-P autoantibodies were observed. Herein, the first human mAbs against TcP2β have been obtained and characterized showing that the humoral anti-P response is directed against the parasite and does not include an autoimmune component.

  1. Importance of Host Cell Arginine Uptake in Francisella Phagosomal Escape and Ribosomal Protein Amounts*

    PubMed Central

    Ramond, Elodie; Gesbert, Gael; Guerrera, Ida Chiara; Chhuon, Cerina; Dupuis, Marion; Rigard, Mélanie; Henry, Thomas; Barel, Monique; Charbit, Alain

    2015-01-01

    Upon entry into mammalian host cells, the pathogenic bacterium Francisella must import host cell arginine to multiply actively in the host cytoplasm. We identified and functionally characterized an arginine transporter (hereafter designated ArgP) whose inactivation considerably delayed bacterial phagosomal escape and intracellular multiplication. Intramacrophagic growth of the ΔargP mutant was fully restored upon supplementation of the growth medium with excess arginine, in both F. tularensis subsp. novicida and F. tularensis subsp. holarctica LVS, demonstrating the importance of arginine acquisition in these two subspecies. High-resolution mass spectrometry revealed that arginine limitation reduced the amount of most of the ribosomal proteins in the ΔargP mutant. In response to stresses such as nutritional limitation, repression of ribosomal protein synthesis has been observed in all kingdoms of life. Arginine availability may thus contribute to the sensing of the intracellular stage of the pathogen and to trigger phagosomal egress. All MS data have been deposited in the ProteomeXchange database with identifier PXD001584 (http://proteomecentral.proteomexchange.org/dataset/PXD001584). PMID:25616868

  2. Arabidopsis ribosomal proteins control vacuole trafficking and developmental programs through the regulation of lipid metabolism

    DOE PAGES

    Li, Ruixi; Sun, Ruobai; Hicks, Glenn R.; ...

    2014-12-22

    The vacuole is the most prominent compartment in plant cells and is important for ion and protein storage. In our effort to search for key regulators in the plant vacuole sorting pathway, ribosomal large subunit 4 (rpl4d) was identified as a translational mutant defective in both vacuole trafficking and normal development. Polysome profiling of the rpl4d mutant showed reduction in polysome-bound mRNA compared with wild-type, but no significant change in the general mRNA distribution pattern. Ribsomal profiling data indicated that genes in the lipid metabolism pathways were translationally down-regulated in the rpl4d mutant. Live imaging studies by Nile red stainingmore » suggested that both polar and nonpolar lipid accumulation was reduced in meristem tissues of rpl4d mutants. Pharmacological evidence showed that sterol and sphingolipid biosynthetic inhibitors can phenocopy the defects of the rpl4d mutant, including an altered vacuole trafficking pattern. Genetic evidence from lipid biosynthetic mutants indicates that alteration in the metabolism of either sterol or sphingolipid biosynthesis resulted in vacuole trafficking defects, similar to the rpl4d mutant. Tissue-specific complementation with key enzymes from lipid biosynthesis pathways can partially rescue both vacuole trafficking and auxin-related developmental defects in the rpl4d mutant. These results indicate that lipid metabolism modulates auxin-mediated tissue differentiation and endomembrane trafficking pathways downstream of ribosomal protein function.« less

  3. Bactobolin Resistance Is Conferred by Mutations in the L2 Ribosomal Protein

    PubMed Central

    Chandler, Josephine R.; Truong, Thao T.; Silva, Patricia M.; Seyedsayamdost, Mohammad R.; Carr, Gavin; Radey, Matthew; Jacobs, Michael A.; Sims, Elizabeth H.; Clardy, Jon; Greenberg, E. Peter

    2012-01-01

    ABSTRACT Burkholderia thailandensis produces a family of polyketide-peptide molecules called bactobolins, some of which are potent antibiotics. We found that growth of B. thailandensis at 30°C versus that at 37°C resulted in increased production of bactobolins. We purified the three most abundant bactobolins and determined their activities against a battery of bacteria and mouse fibroblasts. Two of the three compounds showed strong activities against both bacteria and fibroblasts. The third analog was much less potent in both assays. These results suggested that the target of bactobolins might be conserved across bacteria and mammalian cells. To learn about the mechanism of bactobolin activity, we isolated four spontaneous bactobolin-resistant Bacillus subtilis mutants. We used genomic sequencing technology to show that each of the four resistant variants had mutations in rplB, which codes for the 50S ribosome-associated L2 protein. Ectopic expression of a mutant rplB gene in wild-type B. subtilis conferred bactobolin resistance. Finally, the L2 mutations did not confer resistance to other antibiotics known to interfere with ribosome function. Our data indicate that bactobolins target the L2 protein or a nearby site and that this is not the target of other antibiotics. We presume that the mammalian target of bactobolins involves the eukaryotic homolog of L2 (L8e). PMID:23249812

  4. High temperature, differentiation, and endoplasmic reticulum stress decrease but epigenetic and antioxidative agents increase Aspergillus ribosomal protein gene expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genome-wide gene expression assays using next-generation sequencing techniques have allowed the identification of transcriptomes in many species. Transcript abundance of ribosomal protein (RP) genes can serve as a proxy for the capacity of general transcription and synthesis of cellular proteins tha...

  5. Plastid ribosomal protein S5 plays a critical role in photosynthesis, plant development, and cold stress tolerance in arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plastid ribosomal proteins (RPs) are essential components for protein synthesis machinery and exert diverse roles in plant growth and development. Mutations in plastid RPs lead to a range of developmental phenotypes in plants. However, how they regulate these processes is not fully understood and th...

  6. Ricin A chain can be chemically cross-linked to the mammalian ribosomal proteins L9 and L10e.

    PubMed

    Vater, C A; Bartle, L M; Leszyk, J D; Lambert, J M; Goldmacher, V S

    1995-05-26

    Indirect immunofluorescence studies revealed that when fixed, permeabilized cultured human cells were incubated with ricin A chain, the toxin molecule localized in a staining pattern indicative of binding to the endoplasmic reticulum and to nucleoli. Chemical cross-linking experiments were performed to identify the cellular components that mediated the binding of ricin A chain. Conjugates were formed between 125I-labeled ricin A chain and two proteins present in preparations of total cell membranes and in samples of purified mammalian ribosomes. Specificity of the ricin A chain-ribosome interaction was demonstrated by inhibition of formation of the complexes by excess unlabeled ricin A chain, but not by excess unlabeled gelonin, another ribosome-inactivating protein. Complexes of ricin A chain cross-linked to the ribosomal proteins were purified and subjected to proteolytic digestion with trypsin. Amino acid sequencing of internal tryptic peptides enabled identification of the ricin A chain-binding proteins as L9 and L10e of the mammalian large ribosomal subunit.

  7. Promoter architecture and transcriptional regulation of Abf1-dependent ribosomal protein genes in Saccharomyces cerevisiae

    PubMed Central

    Fermi, Beatrice; Bosio, Maria Cristina; Dieci, Giorgio

    2016-01-01

    In Saccharomyces cerevisiae, ribosomal protein gene (RPG) promoters display binding sites for either Rap1 or Abf1 transcription factors. Unlike Rap1-associated promoters, the small cohort of Abf1-dependent RPGs (Abf1-RPGs) has not been extensively investigated. We show that RPL3, RPL4B, RPP1A, RPS22B and RPS28A/B share a common promoter architecture, with an Abf1 site upstream of a conserved element matching the sequence recognized by Fhl1, a transcription factor which together with Ifh1 orchestrates Rap1-associated RPG regulation. Abf1 and Fhl1 promoter association was confirmed by ChIP and/or gel retardation assays. Mutational analysis revealed a more severe requirement of Abf1 than Fhl1 binding sites for RPG transcription. In the case of RPS22B an unusual Tbf1 binding site promoted both RPS22B and intron-hosted SNR44 expression. Abf1-RPG down-regulation upon TOR pathway inhibition was much attenuated at defective mutant promoters unable to bind Abf1. TORC1 inactivation caused the expected reduction of Ifh1 occupancy at RPS22B and RPL3 promoters, but unexpectedly it entailed largely increased Abf1 association with Abf1-RPG promoters. We present evidence that Abf1 recruitment upon nutritional stress, also observed for representative ribosome biogenesis genes, favours RPG transcriptional rescue upon nutrient replenishment, thus pointing to nutrient-regulated Abf1 dynamics at promoters as a novel mechanism in ribosome biogenesis control. PMID:27016735

  8. Ribosomal Stalk Protein Silencing Partially Corrects the ΔF508-CFTR Functional Expression Defect

    PubMed Central

    Veit, Guido; Oliver, Kathryn; Apaja, Pirjo M.; Perdomo, Doranda; Bidaud-Meynard, Aurélien; Guo, Jingyu; Icyuz, Mert; Sorscher, Eric J.; Hartman, John L.; Lukacs, Gergely L.

    2016-01-01

    The most common cystic fibrosis (CF) causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del), results in functional expression defect of the CF transmembrane conductance regulator (CFTR) at the apical plasma membrane (PM) of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER). Deletion of phenylalanine 670 (ΔF670) in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter) of Saccharomyces cerevisiae phenocopies the ΔF508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic) analysis of the Yor1-ΔF670 biogenesis identified several modifier genes of mRNA processing and translation, which conferred oligomycin resistance to yeast. Silencing of orthologues of these candidate genes enhanced the ΔF508-CFTR functional expression at the apical PM in human CF bronchial epithelia. Although knockdown of RPL12, a component of the ribosomal stalk, attenuated the translational elongation rate, it increased the folding efficiency as well as the conformational stability of the ΔF508-CFTR, manifesting in 3-fold augmented PM density and function of the mutant. Combination of RPL12 knockdown with the corrector drug, VX-809 (lumacaftor) restored the mutant function to ~50% of the wild-type channel in primary CFTRΔF508/ΔF508 human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ΔF508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ΔF508-CFTR folding defect. PMID:27168400

  9. Expression of human serum albumin--L7/L12 (Brucella abortus ribosomal protein) fusion protein in Saccharomyces cerevisiae.

    PubMed

    Pakzad, Iraj; Rezaee, Abbas; Emaneini, Mohammad; Hosseini, Ahmad Zavaran; Tabbaraee, Bahman; Taherikalani, Morovat

    2009-01-01

    Brucella abortus is a facultative intracellular gram-negative bacterial pathogen that causes abortion in pregnant cattle and undulant fever in humans. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of subunit vaccines against brucellosis. It has already been expressed in several bacteria and has been used as DNA vaccine. In order to construct yeast expressing vector for the tHSA-L7/L12 fusion protein, the l7/l12 ribosomal gene was amplified by PCR. The expression plasmid pYtHSA-L7/L12 was constructed by inserting the L7/L12 gene into the pYHSA5 shuttle vector (containing inulinase signal sequence, HSA gene and Gal10 promoter). The recombinant vector was transformed into S. cerevisiae and was then induced by galactose. The secreted recombinant fusion protein was detected in supernatant by SDS-PAGE and confirmed by western blot analysis using anti-HSA and anti-L7/L12 antibodies. Fusion protein was purified by affinity chromatography and its amount was approximately 500 microg/liter.

  10. Cleavage of nicotinamide adenine dinucleotide by the ribosome-inactivating protein from Momordica charantia.

    PubMed

    Vinkovic, M; Dunn, G; Wood, G E; Husain, J; Wood, S P; Gill, R

    2015-09-01

    The interaction of momordin, a type 1 ribosome-inactivating protein from Momordica charantia, with NADP(+) and NADPH has been investigated by X-ray diffraction analysis of complexes generated by co-crystallization and crystal soaking. It is known that the proteins of this family readily cleave the adenine-ribose bond of adenosine and related nucleotides in the crystal, leaving the product, adenine, bound to the enzyme active site. Surprisingly, the nicotinamide-ribose bond of oxidized NADP(+) is cleaved, leaving nicotinamide bound in the active site in the same position but in a slightly different orientation to that of the five-membered ring of adenine. No binding or cleavage of NADPH was observed at pH 7.4 in these experiments. These observations are in accord with current views of the enzyme mechanism and may contribute to ongoing searches for effective inhibitors.

  11. Mapping of the RNA recognition site of Escherichia coli ribosomal protein S7.

    PubMed Central

    Robert, F; Gagnon, M; Sans, D; Michnick, S; Brakier-Gingras, L

    2000-01-01

    Bacterial ribosomal protein S7 initiates the folding of the 3' major domain of 16S ribosomal RNA by binding to its lower half. The X-ray structure of protein S7 from thermophilic bacteria was recently solved and found to be a modular structure, consisting of an alpha-helical domain with a beta-ribbon extension. To gain further insights into its interaction with rRNA, we cloned the S7 gene from Escherichia coli K12 into a pET expression vector and introduced 4 deletions and 12 amino acid substitutions in the protein sequence. The binding of each mutant to the lower half of the 3' major domain of 16S rRNA was assessed by filtration on nitrocellulose membranes. Deletion of the N-terminal 17 residues or deletion of the B hairpins (residues 72-89) severely decreased S7 affinity for the rRNA. Truncation of the C-terminal portion (residues 138-178), which includes part of the terminal alpha-helix, significantly affected S7 binding, whereas a shorter truncation (residues 148-178) only marginally influenced its binding. Severe effects were also observed with several strategic point mutations located throughout the protein, including Q8A and F17G in the N-terminal region, and K35Q, G54S, K113Q, and M115G in loops connecting the alpha-helices. Our results are consistent with the occurrence of several sites of contact between S7 and the 16S rRNA, in line with its role in the folding of the 3' major domain. PMID:11105763

  12. Giant panda ribosomal protein S14: cDNA, genomic sequence cloning, sequence analysis, and overexpression.

    PubMed

    Wu, G-F; Hou, Y-L; Hou, W-R; Song, Y; Zhang, T

    2010-10-13

    RPS14 is a component of the 40S ribosomal subunit encoded by the RPS14 gene and is required for its maturation. The cDNA and the genomic sequence of RPS14 were cloned successfully from the giant panda (Ailuropoda melanoleuca) using RT-PCR technology and touchdown-PCR, respectively; they were both sequenced and analyzed. The length of the cloned cDNA fragment was 492 bp; it contained an open-reading frame of 456 bp, encoding 151 amino acids. The length of the genomic sequence is 3421 bp; it contains four exons and three introns. Alignment analysis indicates that the nucleotide sequence shares a high degree of homology with those of Homo sapiens, Bos taurus, Mus musculus, Rattus norvegicus, Gallus gallus, Xenopus laevis, and Danio rerio (93.64, 83.37, 92.54, 91.89, 87.28, 84.21, and 84.87%, respectively). Comparison of the deduced amino acid sequences of the giant panda with those of these other species revealed that the RPS14 of giant panda is highly homologous with those of B. taurus, R. norvegicus and D. rerio (85.99, 99.34 and 99.34%, respectively), and is 100% identical with the others. This degree of conservation of RPS14 suggests evolutionary selection. Topology prediction shows that there are two N-glycosylation sites, three protein kinase C phosphorylation sites, two casein kinase II phosphorylation sites, four N-myristoylation sites, two amidation sites, and one ribosomal protein S11 signature in the RPS14 protein of the giant panda. The RPS14 gene can be readily expressed in Escherichia coli. When it was fused with the N-terminally His-tagged protein, it gave rise to accumulation of an expected 22-kDa polypeptide, in good agreement with the predicted molecular weight. The expression product obtained can be purified for studies of its function.

  13. Ribosomal Protein S6 Interacts with the Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus ▿

    PubMed Central

    Chen, Wuguo; Dittmer, Dirk P.

    2011-01-01

    The latency-associated nuclear antigen (LANA) is central to the maintenance of Kaposi's sarcoma-associated herpesvirus (KSHV) and to the survival of KSHV-carrying tumor cells. In an effort to identify interaction partners of LANA, we purified authentic high-molecular-weight complexes of LANA by conventional chromatography followed by immunoprecipitation from the BC-3 cell line. This is the first analysis of LANA-interacting partners that is not based on forced ectopic expression of LANA. Subsequent tandem mass spectrometry (MS/MS) analysis identified many of the known LANA-interacting proteins. We confirmed LANA's interactions with histones. Three classes of proteins survived our stringent four-step purification procedure (size, heparin, anion, and immunoaffinity chromatography): two heat shock proteins (Hsp70 and Hsp96 precursor), signal recognition particle 72 (SRP72), and 10 different ribosomal proteins. These proteins are likely involved in structural interactions within LANA high-molecular-weight complexes. Here, we show that ribosomal protein S6 (RPS6) interacts with LANA. This interaction is mediated by the N-terminal domain of LANA and does not require DNA or RNA. Depletion of RPS6 from primary effusion lymphoma (PEL) cells dramatically decreases the half-life of full-length LANA. The fact that RPS6 has a well-established nuclear function beyond its role in ribosome assembly suggests that RPS6 (and by extension other ribosomal proteins) contributes to the extraordinary stability of LANA. PMID:21734034

  14. Eukaryote-specific motif of ribosomal protein S15 neighbors A site codon during elongation and termination of translation.

    PubMed

    Khairulina, Julia; Graifer, Dmitri; Bulygin, Konstantin; Ven'yaminova, Aliya; Frolova, Ludmila; Karpova, Galina

    2010-07-01

    The eukaryotic ribosomal protein S15 is a key component of the decoding site in contrast to its prokaryotic counterpart, S19p, which is located away from the mRNA binding track on the ribosome. Here, we determined the oligopeptide of S15 neighboring the A site mRNA codon on the human 80S ribosome with the use of mRNA analogues bearing perfluorophenyl azide-modified nucleotides in the sense or stop codon targeted to the 80S ribosomal A site. The protein was cross-linked to mRNA analogues in specific ribosomal complexes that were obtained in the presence of eRF1 in the experiments with mRNAs bearing stop codon. Digestion of modified S15 with various specific proteolytic agents followed by identification of the resulting modified oligopeptides showed that cross-link was in C-terminal fragment in positions 131-145, most probably, in decapeptide 131-PGIGATHSSR-140. The position of cross-linking site on the S15 protein did not depend on the nature of the A site-bound codon (sense or stop codon) and on the presence of polypeptide chain release factor eRF1 in the ribosomal complexes with mRNA analogues bearing a stop codon. The results indicate an involvement of the mentioned decapeptide in the formation of the ribosomal decoding site during elongation and termination of translation. Alignment of amino acid sequences of eukaryotic S15 and its prokaryotic counterpart, S19p from eubacteria and archaea, revealed that decapeptide PGIGATHSSR in positions 131-140 is strongly conserved in eukaryotes and has minor variations in archaea but has no homology with any sequence in C-terminal part of eubacterial S19p, which suggests involvement of the decapeptide in the translation process in a eukaryote-specific manner.

  15. Ribosomal vaccines. I. Immunogenicity of ribosomal fractions isolated from Salmonella typhimurium and Yersinia pestis.

    PubMed

    Johnson, W

    1972-06-01

    The immunogenicity of ribosomes and ribosomal subfractions isolated from Yersina pestis and Salmonella typhimurium has been studied. Ribosomes and ribosomal protein isolated from S. typhimurium protected mice against lethal challenge. Ribosomal ribonucleic acid isolated by phenol extraction failed to induce any significant level of protection in mice. None of the ribosomes or ribosomal subfractions isolated from Y. pestis were effective in inducing immunity to lethal challenge. These results suggest that the immunogen of the ribosomal vaccine is protein.

  16. RPLP1, a crucial ribosomal protein for embryonic development of the nervous system.

    PubMed

    Perucho, Laura; Artero-Castro, Ana; Guerrero, Sergi; Ramón y Cajal, Santiago; LLeonart, Matilde E; Wang, Zhao-Qi

    2014-01-01

    Ribosomal proteins are pivotal to development and tissue homeostasis. RP Large P1 (Rplp1) overexpression is associated with tumorigenesis. However, the physiological function of Rplp1 in mammalian development remains unknown. In this study, we disrupted Rplp1 in the mouse germline and central nervous system (Rplp1CNSΔ). Rplp1 heterozygosity caused body size reductions, male infertility, systemic abnormalities in various tissues and a high frequency of early postnatal death. Rplp1CNSΔ newborn mice exhibited perinatal lethality and brain atrophy with size reductions of the neocortex, midbrain and ganglionic eminence. The Rplp1 knockout neocortex exhibited progenitor cell proliferation arrest and apoptosis due to the dysregulation of key cell cycle and apoptosis regulators (cyclin A, cyclin E, p21CIP1, p27KIP1, p53). Similarly, Rplp1 deletion in pMEFs led to proliferation arrest and premature senescence. Importantly, Rplp1 deletion in primary mouse embryonic fibroblasts did not alter global protein synthesis, but did change the expression patterns of specific protein subsets involved in protein folding and the unfolded protein response, cell death, protein transport and signal transduction, among others. Altogether, we demonstrated that the translation "fine-tuning" exerted by Rplp1 is essential for embryonic and brain development and for proper cell proliferation.

  17. Cloning and characterization of a gene from Rhizobium melilotii 2011 coding for ribosomal protein S1.

    PubMed Central

    Schnier, J; Thamm, S; Lurz, R; Hussain, A; Faist, G; Dobrinski, B

    1988-01-01

    A 7 kb chromosomal DNA fragment from R. melilotii was cloned, which complemented temperature-sensitivity of an E. coli amber mutant in rpsA, the gene for ribosomal protein S1 (ES1). From complementation and maxicell analysis a 58 kd protein was identified as the homolog of protein S1 (RS1). DNA sequence analysis of the R. melilotii rpsA gene identified a protein of 568 amino acids, which showed 47% identical amino acid homology to protein S1 from E. coli. The RS1 protein lacked the two Cys residues which had been reported to play an important role for the function of ES1. Two repeats containing Shine-Dalgarno sequences were identified upstream of the structural gene. Binding studies with RNA polymerase from E. coli and Pseudomonas putida located one RNA-polymerase binding site close to the RS1 gene and another one several hundred basepairs upstream. One possible promoter was also identified by DNA sequence comparison with the corresponding E. coli promoter. Images PMID:3368316

  18. Cloning and sequence analysis of the Chlamydia trachomatis spc ribosomal protein gene cluster.

    PubMed Central

    Kaul, R; Gray, G J; Koehncke, N R; Gu, L J

    1992-01-01

    We identified and sequenced a segment of Chlamydia trachomatis chromosomal DNA that shows homology to the Escherichia coli spc and distal region of the S10 ribosomal protein (r-protein) operons. Its sequence revealed a high degree of nucleotide and operon context conservation with the E. coli r-protein genes. The C. trachomatis spec operon contains the r-protein genes for L14, L24, L5, S8, L6, L18, S5, L15, and Sec Y along with the genes for r-proteins L16, L29, and S17 of the S10 operon. The two operons are separated by a 16-bp intragenic region which contains no transcription signals. However, a putative promoter for the transcription of the spc operon was found 162 nucleotides upstream of the CtrL14e start site; it revealed significant homology to the E. coli consensus promoter sequences. Interestingly, our results indicate the absence of any structure resembling an EcoS8 regulatory target site on C. trachomatis spc mRNA in spite of significant amino acid identity between E. coli and C. trachomatis r-proteins. Also, the intrinsic aminoglycoside resistance in C. trachomatis is unlikely to be mediated by CtrL6e since E. coli expressing CtrL6e remained susceptible to gentamicin (MIC less than 0.5 micrograms/ml). Images PMID:1735714

  19. Ribosomal Protein Mutations Induce Autophagy through S6 Kinase Inhibition of the Insulin Pathway

    PubMed Central

    Pereboom, Tamara C.; Goos, Yvonne J.; Seinen, Cor W.; van Oirschot, Brigitte A.; van Dooren, Rowie; Gastou, Marc; Giles, Rachel H.; van Solinge, Wouter; Kuijpers, Taco W.; Gazda, Hanna T.; Bierings, Marc B.; Da Costa, Lydie; MacInnes, Alyson W.

    2014-01-01

    Mutations affecting the ribosome lead to several diseases known as ribosomopathies, with phenotypes that include growth defects, cytopenia, and bone marrow failure. Diamond-Blackfan anemia (DBA), for example, is a pure red cell aplasia linked to the mutation of ribosomal protein (RP) genes. Here we show the knock-down of the DBA-linked RPS19 gene induces the cellular self-digestion process of autophagy, a pathway critical for proper hematopoiesis. We also observe an increase of autophagy in cells derived from DBA patients, in CD34+ erythrocyte progenitor cells with RPS19 knock down, in the red blood cells of zebrafish embryos with RP-deficiency, and in cells from patients with Shwachman-Diamond syndrome (SDS). The loss of RPs in all these models results in a marked increase in S6 kinase phosphorylation that we find is triggered by an increase in reactive oxygen species (ROS). We show that this increase in S6 kinase phosphorylation inhibits the insulin pathway and AKT phosphorylation activity through a mechanism reminiscent of insulin resistance. While stimulating RP-deficient cells with insulin reduces autophagy, antioxidant treatment reduces S6 kinase phosphorylation, autophagy, and stabilization of the p53 tumor suppressor. Our data suggest that RP loss promotes the aberrant activation of both S6 kinase and p53 by increasing intracellular ROS levels. The deregulation of these signaling pathways is likely playing a major role in the pathophysiology of ribosomopathies. PMID:24875531

  20. Human ribosomal protein L9 is a Bax suppressor that promotes cell survival in yeast.

    PubMed

    Eid, Rawan; Sheibani, Sara; Gharib, Nada; Lapointe, Jason F; Horowitz, Avital; Vali, Hojatollah; Mandato, Craig A; Greenwood, Michael T

    2014-05-01

    The identification of a human ribosomal protein L9 (hRPL9) cDNA as a sequence capable of suppressing the lethal effects of heterologously expressed murine Bax in yeast led us to investigate its antiapoptotic potential. Using growth and viability assays, we show that yeast cells heterologously expressing hRPL9 are resistant to the growth inhibitory and lethal effects of exogenously supplied copper, indicating that it has pro-survival properties. To explore potential mechanisms, we used yeast mutants defective in all three types of programmed cell death (apoptosis, necrosis, and autophagy). The ability to retain pro-survival function in all the mutants suggests that hRPL9 may regulate a common pro-death process. In contrast, the yeast RPL9 orthologues, RPL9A and RPL9B, have opposite effects when overexpressed in yeast. In effect, instead of showing resistance to stress, RPL9A and RPL9B overexpressing cells show reduced cell growth. Further analysis indicates that the effects of overexpressed RPL9A and RPL9B are not in themselves lethal, instead, they serve to increase cell doubling time. Thus, yeast RPL9s are more representative of RPs whose extra-ribosomal function is similar to that of tumor suppressors. Taken together, our results demonstrate that RPL9 represents a species- and sequence-specific regulator of cell growth and survival.

  1. Revising the Taxonomic Distribution, Origin and Evolution of Ribosome Inactivating Protein Genes

    PubMed Central

    Lapadula, Walter J.; Sánchez Puerta, María Virginia; Juri Ayub, Maximiliano

    2013-01-01

    Ribosome inactivating proteins are enzymes that depurinate a specific adenine residue in the alpha-sarcin-ricin loop of the large ribosomal RNA, being ricin and Shiga toxins the most renowned examples. They are widely distributed in plants and their presence has also been confirmed in a few bacterial species. According to this taxonomic distribution, the current model about the origin and evolution of RIP genes postulates that an ancestral RIP domain was originated in flowering plants, and later acquired by some bacteria via horizontal gene transfer. Here, we unequivocally detected the presence of RIP genes in fungi and metazoa. These findings, along with sequence and phylogenetic analyses, led us to propose an alternative, more parsimonious, hypothesis about the origin and evolutionary history of the RIP domain, where several paralogous RIP genes were already present before the three domains of life evolved. This model is in agreement with the current idea of the Last Universal Common Ancestor (LUCA) as a complex, genetically redundant organism. Differential loss of paralogous genes in descendants of LUCA, rather than multiple horizontal gene transfer events, could account for the complex pattern of RIP genes across extant species, as it has been observed for other genes. PMID:24039805

  2. Molecular dissection of the pseudoknot governing the translational regulation of Escherichia coli ribosomal protein S15.

    PubMed Central

    Philippe, C; Bénard, L; Portier, C; Westhof, E; Ehresmann, B; Ehresmann, C

    1995-01-01

    The ribosomal protein S15 controls its own translation by binding to a mRNA region overlapping the ribosome binding site. That region of the mRNA can fold in two mutually exclusive conformations that are in dynamic equilibrium: a structure with two hairpins and a pseudoknot. A mutational analysis provided evidence for the existence and requirement of the pseudoknot for translational control in vivo and S15 recognition in vitro. In this study, we used chemical probing to analyze the structural consequences of mutations and their effect on the stem-loop/pseudoknot equilibrium. Interactions between S15 and the pseudoknot structure were further investigated by footprinting experiments. These data, combined with computer modelling and the previously published data on S15 binding and in vivo control, provide important clues on pseudoknot formation and S15 recognition. An unexpected result is that the relevant control element, here the pseudoknot form, can exist in a variety of topologically equivalent structures recognizable and shapable by S15. S15 sits on the deep groove of the co-axial stack and makes contacts with both stems, shielding the bridging adenine. The only specific sequence determinants are found in the helix common to the pseudoknot and the hairpin structures. Images PMID:7532857

  3. Fibroblast growth factor 3, a protein with a dual subcellular fate, is interacting with human ribosomal protein S2

    SciTech Connect

    Antoine, Marianne; Reimers, Kerstin; Wirz, Werner; Gressner, Axel M.; Mueller, Robert; Kiefer, Paul . E-mail: pkiefer@ukaachen.de

    2005-12-16

    The secreted isoform of fibroblast growth factor 3 (FGF3) induces a mitogenic cell response, while the nuclear form inhibits cell proliferation. Recently, we identified a nucleolar FGF3-binding protein which is implicated in processing of pre-rRNA as a possible target of nuclear FGF3 signalling. Here, we report a second candidate protein identified by a yeast two-hybrid screen for nuclear FGF3 action, ribosomal protein S2, rpS2. Recombinant rpS2 binds to in vitro translated FGF3 and to nuclear FGF3 extracted from transfected COS-1 cells. Characterization of the FGF3 binding domain of rpS2 showed that both the Arg-Gly-rich N-terminal region and a short carboxyl-terminal sequence of rpS2 are necessary for FGF3 binding. Mapping the S2 binding domains of FGF3 revealed that these domains are important for both NoBP and rpS2 interaction. Transient co-expression of rpS2 and nuclear FGF3 resulted in a reduced nucleolar localization of the FGF. These findings suggest that the nuclear form of FGF3 inhibits cell proliferation by interfering with ribosomal biogenesis.

  4. Ribosomal protein insufficiency and the minute syndrome in Drosophila: a dose-response relationship.

    PubMed Central

    Saebøe-Larssen, S; Lyamouri, M; Merriam, J; Oksvold, M P; Lambertsson, A

    1998-01-01

    Minutes comprise > 50 phenotypically similar mutations scattered throughout the genome of Drosophila, many of which are identified as mutations in ribosomal protein (rp) genes. Common traits of the Minute phenotype are short and thin bristles, slow development, and recessive lethality. By mobilizing a P element inserted in the 5' UTR of M(3)95A, the gene encoding ribosomal protein S3 (RPS3), we have generated two homozygous viable heteroalleles that are partial revertants with respect to the Minute phenotype. Molecular characterization revealed both alleles to be imprecise excisions, leaving 40 and 110 bp, respectively, at the P-element insertion site. The weaker allele (40 bp insert) is associated with a approximately 15% decrease in RPS3 mRNA abundance and displays a moderate Minute phenotype. In the stronger allele (110 bp insert) RPS3 mRNA levels are reduced by approximately 60%, resulting in an extreme Minute phenotype that includes many morphological abnormalities as well as sterility in both males and females due to disruption of early gametogenesis. The results show that there is a correlation between reduced RPS3 mRNA levels and the severity of the Minute phenotype, in which faulty differentiation of somatic tissues and arrest of gametogenesis represent the extreme case. That heteroalleles in M(3)95A can mimic the phenotypic variations that exist between different Minute/rp-gene mutations strongly suggests that all phenotypes primarily are caused by reductions in maximum protein synthesis rates, but that the sensitivity for reduced levels of the individual rp-gene products is different. PMID:9539436

  5. Elderberries: a source of ribosome-inactivating proteins with lectin activity.

    PubMed

    Tejero, Jesús; Jiménez, Pilar; Quinto, Emiliano J; Cordoba-Diaz, Damián; Garrosa, Manuel; Cordoba-Diaz, Manuel; Gayoso, Manuel J; Girbés, Tomás

    2015-01-30

    Sambucus (Adoxaceae) species have been used for both food and medicine purposes. Among these, Sambucus nigra L. (black elder), Sambucus ebulus L. (dwarf elder), and Sambucus sieboldiana L. are the most relevant species studied. Their use has been somewhat restricted due to the presence of bioactive proteins or/and low molecular weight compounds whose ingestion could trigger deleterious effects. Over the last few years, the chemical and pharmacological characteristics of Sambucus species have been investigated. Among the proteins present in Sambucus species both type 1, and type 2 ribosome-inactivating proteins (RIPs), and hololectins have been reported. The biological role played by these proteins remains unknown, although they are conjectured to be involved in defending plants against insect predators and viruses. These proteins might have an important impact on the nutritional characteristics and food safety of elderberries. Type 2 RIPs are able to interact with gut cells of insects and mammals triggering a number of specific and mostly unknown cell signals in the gut mucosa that could significantly affect animal physiology. In this paper, we describe all known RIPs that have been isolated to date from Sambucus species, and comment on their antiviral and entomotoxic effects, as well as their potential uses.

  6. La Autoantigen Induces Ribosome Binding Protein 1 (RRBP1) Expression through Internal Ribosome Entry Site (IRES)-Mediated Translation during Cellular Stress Condition.

    PubMed

    Gao, Wenqing; Li, Qi; Zhu, Ruiyu; Jin, Jian

    2016-07-20

    The function of ribosome binding protein 1 (RRBP1) is regulating the transportation and secretion of some intracellular proteins in mammalian cells. Transcription of RRBP1 is induced by various cytokines. However, few studies focused on the process of RRPB1 mRNA translation. The RRBP1 mRNA has a long 5' untranslated region that potentially formed a stable secondary structure. In this study, we show that the 5' UTR of RRBP1 mRNA contains an internal ribosome entry site (IRES). Moreover, the RRBP1 expression is induced by chemotherapeutic drug paclitaxel or adriamycin in human hepatocellular carcinoma cells and accompanied with the increased expression of La autoantigen (La), which binds to RRBP1 IRES element and facilitates translation initiation. Interestingly, we found IRES-mediated RRBP1 translation is also activated during serum-starvation condition which can induce cytoplasmic localization of La. After mapping the entire RRBP1 5' UTR, we determine the core IRES activity is located between nt-237 and -58. Furthermore, two apical GARR loops within the functional RRBP1 IRES elements may be important for La binding. These results strongly suggest an important role for IRES-dependent translation of RRBP1 mRNA in hepatocellular carcinoma cells during cellular stress conditions.

  7. Late-assembly of human ribosomal protein S20 in the cytoplasm is essential for the functioning of the small subunit ribosome

    SciTech Connect

    Tai, Lin-Ru; Chou, Chang-Wei; Wu, Jing-Ying; Kirby, Ralph; Lin, Alan

    2013-11-15

    Using immuno-fluorescent probing and Western blotting analysis, we reveal the exclusive cytoplasm nature of the small subunit ribosomal protein S20. To illustrate the importance of the cellular compartmentation of S20 to the function of small subunit 40S, we created a nuclear resident S20{sub NLS} mutant gene and examined polysome profile of cells that had been transfected with the S20{sub NLS} gene. As a result, we observed the formation of recombinant 40S carried S20{sub NLS} but this recombinant 40S was never found in the polysome, suggesting such a recombinant 40S was translation incompetent. Moreover, by the tactic of the energy depletion and restoration, we were able to restrain the nuclear-resided S20{sub NLS} in the cytoplasm. Yet, along a progressive energy restoration, we observed the presence of recombinant 40S subunits carrying the S20{sub NLS} in the polysome. This proves that S20 needs to be cytoplasmic in order to make a functional 40S subunit. Furthermore, it also implies that the assembly order of ribosomal protein in eukaryote is orderly regulated. - Highlights: • The step of S20 assembled on 40S is happened in the cytoplasm. • A small subunit assembled with a nuclear S20{sub NLS} is translational incompetence. • Using energy depletion and recovery to manipulate the cellular compartment of S20{sub NLS}. • Cytoplasm-retained S20{sub NLS} is crucial for creating a functional small subunit.

  8. Loss of Ribosomal Protein L11 Blocks Stress Activation of the Bacillus subtilis Transcription Factor ςB

    PubMed Central

    Zhang, Shuyu; Scott, Janelle M.; Haldenwang, W. G.

    2001-01-01

    ςB, the general stress response sigma factor of Bacillus subtilis, is activated when the cell's energy levels decline or the bacterium is exposed to environmental stress (e.g., heat shock, ethanol). Physical stress activates ςB through a collection of regulatory kinases and phosphatases (the Rsb proteins) which catalyze the release of ςB from an anti-ςB factor inhibitor. The means by which diverse stresses communicate with the Rsb proteins is unknown; however, a role for the ribosome in this process was suggested when several of the upstream members of the ςB stress activation cascade (RsbR, -S, and -T) were found to cofractionate with ribosomes in crude B. subtilis extracts. We now present evidence for the involvement of a ribosome-mediated process in the stress activation of ςB. B. subtilis strains resistant to the antibiotic thiostrepton, due to the loss of ribosomal protein L11 (RplK), were found to be blocked in the stress activation of ςB. Neither the energy-responsive activation of ςB nor stress-dependent chaperone gene induction (a ςB-independent stress response) was inhibited by the loss of L11. The Rsb proteins required for stress activation of ςB are shown to be active in the RplK− strain but fail to be triggered by stress. The data demonstrate that the B. subtilis ribosomes provide an essential input for the stress activation of ςB and suggest that the ribosomes may themselves be the sensors for stress in this system. PMID:11244072

  9. Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila, Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria.

    PubMed

    Cammarano, P; Mazzei, F; Londei, P; Teichner, A; de Rosa, M; Gambacorta, A

    1983-08-02

    Ribosomal subunits of Caldariella acidophila (max.growth temp., 90 degrees C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70 degrees C) and Escherichia coli (max. growth temp., 47 degrees C) with respect to (a) bihelical content of rRNA; (b) G . C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. Subunits of C. acidophilia ribosomes (Tm = 90-93 degrees C) exhibit considerable thermal tolerance over their B. acidocaldarius (Tm = 77 degrees C) and E. coli counterparts (Tm = 72 degrees C). Based on the "melting' hyperchromicities of the intact ribosomal subunits a 51-55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67-70%, appears to be involved in hydrogen bonding in the naked rRNA species. The G . C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63-64% G . C, compared to 58.5% G . C for B. acidocaldarius and 55% G . C for E. coli. The increment of ribosome Tm values with increasing thermophily is greater than the increase in Tm for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release r

  10. Ribosomal Protein S6 Phosphorylation in the Nervous System: From Regulation to Function

    PubMed Central

    Biever, Anne; Valjent, Emmanuel; Puighermanal, Emma

    2015-01-01

    Since the discovery of the phosphorylation of the 40S ribosomal protein S6 (rpS6) about four decades ago, much effort has been made to uncover the molecular mechanisms underlying the regulation of this post-translational modification. In the field of neuroscience, rpS6 phosphorylation is commonly used as a readout of the mammalian target of rapamycin complex 1 signaling activation or as a marker for neuronal activity. Nevertheless, its biological role in neurons still remains puzzling. Here we review the pharmacological and physiological stimuli regulating this modification in the nervous system as well as the pathways that transduce these signals into rpS6 phosphorylation. Altered rpS6 phosphorylation observed in various genetic and pathophysiological mouse models is also discussed. Finally, we examine the current state of knowledge on the physiological role of this post-translational modification and highlight the questions that remain to be addressed. PMID:26733799

  11. Induction of apoptosis by ribosome inactivating proteins: importance of N-glycosidase activity.

    PubMed

    Das, Mrinal Kumar; Sharma, Radhey Shyam; Mishra, Vandana

    2012-03-01

    Apoptotic cell death is a fundamental process in the development and physiological homeostasis of multicellular organisms. It is associated with control of cell numbers in tissues and organs during development, with cell turnover, and with response to infection. Molecules that trigger this process in continuously proliferating cancer cells can be used as chemotherapeutic agents. Ribosome inactivating proteins (RIPs) that inhibit translation in a cell by depurinating (N-glycosidase activity) the 28S rRNA are known to serve as apoptosis inducers. However, the role of depurination activity of the RIPs in apoptosis induction is still controversial. Presently, there are three different hypotheses which propose that depurination is: (1) essential, (2) essential but not the sole factor, or (3) not essential for apoptosis induction. This article reviews various experimental outcomes on the importance of N-glycosidase activity of RIPs in the induction of apoptosis.

  12. Molecular cloning and analysis of the CRY1 gene: a yeast ribosomal protein gene.

    PubMed Central

    Larkin, J C; Woolford, J L

    1983-01-01

    Using cloned DNA from the vicinity of the yeast mating type locus (MAT) as a probe, the wild type allele of the cryptopleurine resistance gene CRY1 has been isolated by the technique of chromosome walking and has been shown to be identical to the gene for ribosomal protein 59. A recessive cryR1 allele has also been cloned, using the integration excision method. The genetic distance from MAT to CRY1 is 2.2 cM, while the physical distance is 21 kb, giving a ratio of about 10 kb/cM for this interval. The phenotypic expression of both plasmid borne alleles of the gene can be detected in vivo. The use of this gene as a hybridization probe to examine RNA processing defects in the rna 2, rna 3, rna 4, rna 8, and rna 11 mutants is also discussed. Images PMID:6338478

  13. Ubiquitous Autofragmentation of Fluorescent Proteins Creates Abundant Defective Ribosomal Products (DRiPs) for Immunosurveillance*

    PubMed Central

    Wei, Jiajie; Gibbs, James S.; Hickman, Heather D.; Cush, Stephanie S.; Bennink, Jack R.; Yewdell, Jonathan W.

    2015-01-01

    Green fluorescent protein (GFP) and other fluorescent proteins are essential tools for biological research. When fused to peptides or proteins as a reporter, GFP enables localization and quantitation of gene products in otherwise unmanipulated live cells or organisms. We previously reported that a sizable fraction of nascent GFP is post-translationally converted into a 20-kDa Triton X-100-insoluble proteasome substrate (Qian, S. B., Princiotta, M. F., Bennink, J. R., and Yewdell, J. W. (2006) J. Biol. Chem. 281, 392–400; Dolan, B. P., Li, L., Veltri, C. A., Ireland, C. M., Bennink, J. R., and Yewdell, J. W. (2011) J. Immunol. 186, 2065–2072). Here, we show that a similarly sized fragment is generated by all GFP and red fluorescent protein family members we examined. We demonstrate that fragmentation is a by-product of GFP chromophore rearrangement. A non-rearranging GFP mutant fails to fragment and generates diminished levels of Kb-SIINFEKL complexes when SIINFEKL is genetically fused to either the C- or N-terminal domains of GFP fusion proteins. Instructively, another fragmenting GFP mutant that cannot create the functional chromophore but still generates fragments also demonstrates diminished Kb-SIINFEKL generation. However, the mutant and wild-type fragments differ fundamentally in that wild-type fragments are rapidly liberated from the intact molecule and degraded quickly, accounting for increased Kb-SIINFEKL generation. In the fragmenting mutant, the fragments are generated slowly and remain associated, likely in a native conformation based on their original structural description (Barondeau, D. P., Kassmann, C. J., Tainer, J. A., and Getzoff, E. D. (2006) J. Am. Chem. Soc. 128, 4685–4693). The wild-type GFP fragments represent the first biochemically defined natural defective ribosomal products to contribute peptides for immunosurveillance, enabling quantitation of peptide generation efficiency from this source of defective ribosomal products. More

  14. Expression of a truncated form of ribosomal protein L3 confers resistance to pokeweed antiviral protein and the Fusarium mycotoxin deoxynivalenol.

    PubMed

    Di, Rong; Tumer, Nilgun E

    2005-08-01

    The contamination of important agricultural products such as wheat, barley, or maize with the trichothecene mycotoxin deoxynivalenol (DON) due to infection with Fusarium species is a worldwide problem. Trichothecenes inhibit protein synthesis by targeting ribosomal protein L3. Pokeweed antiviral protein (PAP), a ribosome-inactivating protein binds to L3 to depurinate the alpha-sarcin/loop of the large rRNA. Plants transformed with the wild-type PAP show lesions and express very low levels of PAP because PAP autoregulates its expression by destabilizing its own mRNA. We show here that transgenic tobacco plants expressing both the wild-type PAP and a truncated form of yeast L3 (L3delta) are phenotypically normal. PAP mRNA and protein levels are very high in these plants, indicating that L3delta suppresses the autoregulation of PAP mRNA expression. Ribosomes are not depurinated in the transgenic plants expressing PAP and L3delta, even though PAP is associated with ribosomes. The expression of the endogenous tobacco ribosomal protein L3 is up-regulated in these plants and they are resistant to the Fusarium mycotoxin DON. These results demonstrate that expression of an N-terminal fragment of yeast L3 leads to trans-dominant resistance to PAP and the trichothecene mycotoxin DON, providing evidence that both toxins target L3 by a common mechanism.

  15. Synaptic activation of ribosomal protein S6 phosphorylation occurs locally in activated dendritic domains.

    PubMed

    Pirbhoy, Patricia Salgado; Farris, Shannon; Steward, Oswald

    2016-06-01

    Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylation of ribosomal protein S6 (rpS6) in postsynaptic neurons, but the functional significance of rpS6 phosphorylation is poorly understood. Here, we show that synaptic stimulation that induces perforant path LTP triggers phosphorylation of rpS6 (p-rpS6) locally near active synapses. Using antibodies specific for phosphorylation at different sites (ser235/236 versus ser240/244), we show that strong synaptic activation led to dramatic increases in immunostaining throughout postsynaptic neurons with selectively higher staining for p-ser235/236 in the activated dendritic lamina. Following LTP induction, phosphorylation at ser235/236 was detectable by 5 min, peaked at 30 min, and was maintained for hours. Phosphorylation at both sites was completely blocked by local infusion of the NMDA receptor antagonist, APV. Despite robust induction of p-rpS6 following high frequency stimulation, assessment of protein synthesis by autoradiography revealed no detectable increases. Exploration of a novel environment led to increases in the number of p-rpS6-positive neurons throughout the forebrain in a pattern reminiscent of immediate early gene induction and many individual neurons that were p-rpS6-positive coexpressed Arc protein. Our results constrain hypotheses about the possible role of rpS6 phosphorylation in regulating postsynaptic protein synthesis during induction of synaptic plasticity.

  16. Insulin receptors mediate growth effects in cultured fetal neurons. II. Activation of a protein kinase that phosphorylates ribosomal protein S6

    SciTech Connect

    Heidenreich, K.A.; Toledo, S.P. )

    1989-09-01

    As an initial attempt to identify early steps in insulin action that may be involved in the growth responses of neurons to insulin, we investigated whether insulin receptor activation increases the phosphorylation of ribosomal protein S6 in cultured fetal neurons and whether activation of a protein kinase is involved in this process. When neurons were incubated for 2 h with 32Pi, the addition of insulin (100 ng/ml) for the final 30 min increased the incorporation of 32Pi into a 32K microsomal protein. The incorporation of 32Pi into the majority of other neuronal proteins was unaltered by the 30-min exposure to insulin. Cytosolic extracts from insulin-treated neurons incubated in the presence of exogenous rat liver 40S ribosomes and (gamma-32P)ATP displayed a 3- to 8-fold increase in the phosphorylation of ribosomal protein S6 compared to extracts from untreated cells. Inclusion of cycloheximide during exposure of the neurons to insulin did not inhibit the increased cytosolic kinase activity. Activation of S6 kinase activity by insulin was dose dependent (seen at insulin concentration as low as 0.1 ng/ml) and reached a maximum after 20 min of incubation. Addition of phosphatidylserine, diolein, and Ca2+ to the in vitro kinase reaction had no effect on the phosphorylation of ribosomal protein S6. Likewise, treatment of neurons with (Bu)2cAMP did not alter the phosphorylation of ribosomal protein S6 by neuronal cytosolic extracts. We conclude that insulin activates a cytosolic protein kinase that phosphorylates ribosomal S6 in neurons and is distinct from protein kinase-C and cAMP-dependent protein kinase. Stimulation of this kinase may play a role in insulin signal transduction in neurons.

  17. Chagas disease: a homology model for the three-dimensional structure of the Trypanosoma cruzi ribosomal P0 antigenic protein.

    PubMed

    Gomez Barroso, Juan Arturo; Aguilar, Carlos Fernando

    2014-09-01

    Ribosomal P proteins form a "stalk" complex in the large subunit of the ribosomes. In Trypanosoma cruzi, the etiological agent of Chagas disease, the complex is formed by five P protein members: TcP0, TcP1α, TcP1β, TcP2α and TcP2β. The TcP0 protein has 34 kDa, and TcP1 and TcP2 proteins have 10 kDa. The structure of T. cruzi P0 and the stalk complex TcP0-TcP1α-TcP1β-TcP2α-TcP2β have not been solved to date. In this work, we constructed a three-dimensional molecular model for TcP0 using homology modeling as implemented in the MODELLER 9v12 software. The model was constructed using different templates: the X-ray structures of the protein P0 from Pirococcus horikoshii, a segment from the Danio renio Ca(+2)/K(+) channel and the C-terminal peptide (C13) from T. cruzi ribosomal P2 protein; the Cryo-EM structure of Triticum aestivum P0 protein and the NMR structure of Homo sapiens P1 ribosomal protein. TcP0 has a 200-residue-long N-terminal, which is an α/β globular stable domain, and a flexible C-terminal, 120-residue-long domain. The molecular surface electrostatic potential and hydrophobic surface were calculated. The surface properties are important for the C-terminal's antigenic properties. They are also responsible for P0-specific binding to RNA26S and the binding to the P1-P2 proteins. We explored and identified protein interactions that may be involved in conformational stability. The structure proposed in this work represents a first structural report for the TcP0 protein.

  18. Reduced Dosage of Genes Encoding Ribosomal Protein S18 Suppresses a Mitochondrial Initiation Codon Mutation in Saccharomyces Cerevisiae

    PubMed Central

    Folley, L. S.; Fox, T. D.

    1994-01-01

    A yeast mitochondrial translation initiation codon mutation affecting the gene for cytochrome oxidase subunit III (COX3) was partially suppressed by a spontaneous nuclear mutation. The suppressor mutation also caused cold-sensitive fermentative growth on glucose medium. Suppression and cold sensitivity resulted from inactivation of the gene product of RPS18A, one of two unlinked genes that code the essential cytoplasmic small subunit ribosomal protein termed S18 in yeast. The two S18 genes differ only by 21 silent substitutions in their exons; both are interrupted by a single intron after the 15th codon. Yeast S18 is homologous to the human S11 (70% identical) and the Escherichia coli S17 (35% identical) ribosomal proteins. This highly conserved family of ribosomal proteins has been implicated in maintenance of translational accuracy and is essential for assembly of the small ribosomal subunit. Characterization of the original rps18a-1 missense mutant and rps18aΔ and rps18bΔ null mutants revealed that levels of suppression, cold sensitivity and paromomycin sensitivity all varied directly with a limitation of small ribosomal subunits. The rps18a-1 mutant was most affected, followed by rps18aΔ then rps18bΔ. Mitochondrial mutations that decreased COX3 expression without altering the initiation codon were not suppressed. This allele specificity implicates mitochondrial translation in the mechanism of suppression. We could not detect an epitope-tagged variant of S18 in mitochondria. Thus, it appears that suppression of the mitochondrial translation initiation defect is caused indirectly by reduced levels of cytoplasmic small ribosomal subunits, leading to changes in either cytoplasmic translational accuracy or the relative levels of cytoplasmic translation products. PMID:8070651

  19. Mutation of the rice ASL2 gene encoding plastid ribosomal protein L21 causes chloroplast developmental defects and seedling death.

    PubMed

    Lin, D; Jiang, Q; Zheng, K; Chen, S; Zhou, H; Gong, X; Xu, J; Teng, S; Dong, Y

    2015-05-01

    The plastid ribosome proteins (PRPs) play important roles in plastid protein biosynthesis, chloroplast differentiation and early chloroplast development. However, the specialised functions of individual protein components of the chloroplast ribosome in rice (Oryza sativa) remain unresolved. In this paper, we identified a novel rice PRP mutant named asl2 (Albino seedling lethality 2) exhibiting an albino, seedling death phenotype. In asl2 mutants, the alteration of leaf colour was associated with chlorophyll (Chl) content and abnormal chloroplast development. Through map-based cloning and complementation, the mutated ASL2 gene was isolated and found to encode the chloroplast 50S ribosome protein L21 (RPL21c), a component of the chloroplast ribosome large subunit, which was localised in chloroplasts. ASL2 was expressed at a higher level in the plumule and leaves, implying its tissue-specific expression. Additionally, the expression of ASL2 was regulated by light. The transcript levels of the majority of genes for Chl biosynthesis, photosynthesis and chloroplast development were strongly affected in asl2 mutants. Collectively, the absence of functional ASL2 caused chloroplast developmental defects and seedling death. This report establishes the important role of RPL21c in chloroplast development in rice.

  20. A Ribosomal Protein AgRPS3aE from Halophilic Aspergillus glaucus Confers Salt Tolerance in Heterologous Organisms

    PubMed Central

    Liang, Xilong; Liu, Yiling; Xie, Lixia; Liu, Xiaodan; Wei, Yi; Zhou, Xiaoyang; Zhang, Shihong

    2015-01-01

    High salt in soils is one of the abiotic stresses that significantly reduces crop yield, although saline lands are considered potential resources arable for agriculture. Currently, genetic engineering for enhancing salt tolerance is being tested as an efficient and viable strategy for crop improvement. We previously characterized a large subunit of the ribosomal protein RPL44, which is involved in osmotic stress in the extremely halophilic fungus Aspergillus glaucus. Here, we screened another ribosomal protein (AgRPS3aE) that also produced high-salt tolerance in yeast. Bioinformatics analysis indicated that AgRPS3aE encodes a 29.2 kDa small subunit of a ribosomal protein belonging to the RPS3Ae family in eukaryotes. To further confirm its protective function against salinity, we expressed AgRPS3aE in three heterologous systems, the filamentous fungus Magnaporthe oryzae and two model plants Arabidopsis and tobacco. Overexpression of AgRPS3aE in all tested transformants significantly alleviated stress symptoms compared with controls, suggesting that AgRPS3aE functions not only in fungi but also in plants. Considering that ribosomal proteins are housekeeping components in organisms from prokaryotes to eukaryotes, we propose that AgRPS3aE is one of the optimal genes for improving high-salt tolerance in crops. PMID:25642759

  1. Protein synthesis assessed by ribosome analysis in human papillary muscle in relation to oxidative capacity: a comparison with skeletal muscle.

    PubMed

    Wernerman, J; Sylvén, C; von der Decken, A; Jansson, E; Böök, K; Vinnars, E

    1988-08-01

    Protein synthesis as assessed by the concentration and size distribution of ribosomes was determined together with citrate synthase activity in papillary muscles obtained at open heart surgery from patients with mitral valve disease. The results were compared with corresponding data from the quadriceps femoris muscle of patients undergoing cholecystectomy. Citrate synthase activity was six times higher in papillary muscle than in skeletal muscle. The total ribosome concentration per mg DNA was similar in the two types of muscle. Compared with skeletal muscle, in papillary muscle polyribosomes constituted a higher proportion of the ribosomes (p less than 0.001), and there was a tendency towards larger polyribosome aggregates. It is proposed that the high concentration of polyribosomes in papillary muscle is related to the high oxidative capacity of that tissue.

  2. Ribosomal protein S6 phosphorylation is controlled by TOR and modulated by PKA in Candida albicans.

    PubMed

    Chowdhury, Tahmeena; Köhler, Julia R

    2015-10-01

    TOR and PKA signaling pathways control eukaryotic cell growth and proliferation. TOR activity in model fungi, such as Saccharomyces cerevisiae, responds principally to nutrients, e.g., nitrogen and phosphate sources, which are incorporated into the growing cell mass; PKA signaling responds to the availability of the cells' major energy source, glucose. In the fungal commensal and pathogen, Candida albicans, little is known of how these pathways interact. Here, the signal from phosphorylated ribosomal protein S6 (P-S6) was defined as a surrogate marker for TOR-dependent anabolic activity in C. albicans. Nutritional, pharmacologic and genetic modulation of TOR activity elicited corresponding changes in P-S6 levels. The P-S6 signal corresponded to translational activity of a GFP reporter protein. Contributions of four PKA pathway components to anabolic activation were then examined. In high glucose concentrations, only Tpk2 was required to upregulate P-S6 to physiologic levels, whereas all four tested components were required to downregulate P-S6 in low glucose. TOR was epistatic to PKA components with respect to P-S6. In many host niches inhabited by C. albicans, glucose is scarce, with protein being available as a nitrogen source. We speculate that PKA may modulate TOR-dependent cell growth to a rate sustainable by available energy sources, when monomers of anabolic processes, such as amino acids, are abundant.

  3. Ribosomal protein s15 phosphorylation mediates LRRK2 neurodegeneration in Parkinson's disease

    PubMed Central

    Martin, Ian; Kim, Jungwoo Wren; Lee, Byoung Dae; Kang, Ho Chul; Xu, Jin-Chong; Jia, Hao; Stankowski, Jeannette; Kim, Min-Sik; Zhong, Jun; Kumar, Manoj; Andrabi, Shaida A.; Xiong, Yulan; Dickson, Dennis W.; Wszolek, Zbigniew K.; Pandey, Akhilesh; Dawson, Ted M.; Dawson, Valina L.

    2014-01-01

    Summary Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial and sporadic Parkinson's disease (PD). Elevated LRRK2 kinase activity and neurodegeneration are linked, but the phosphosubstrate that connects LRRK2 kinase activity to neurodegeneration is not known. Here, we show that ribosomal protein s15 is a key pathogenic LRRK2 substrate in Drosophila and human neuron PD models. Phospho-deficient s15 carrying a threonine 136 to alanine substitution rescues dopamine neuron degeneration and age-related locomotor deficits in G2019S LRRK2 transgenic Drosophila and substantially reduces G2019S LRRK2-mediated neurite loss and cell death in human dopamine and cortical neurons. Remarkably, pathogenic LRRK2 stimulates both cap-dependent and cap-independent mRNA translation, and induces a bulk increase in protein synthesis in Drosophila, which can be prevented by phospho-deficient T136A s15. These results reveal a novel mechanism of PD pathogenesis linked to elevated LRRK2 kinase activity and aberrant protein synthesis in vivo. PMID:24725412

  4. Archaeal ribosomal stalk protein interacts with translation factors in a nucleotide-independent manner via its conserved C terminus

    PubMed Central

    Nomura, Naoko; Honda, Takayoshi; Baba, Kentaro; Naganuma, Takao; Tanzawa, Takehito; Arisaka, Fumio; Noda, Masanori; Uchiyama, Susumu; Tanaka, Isao; Yao, Min; Uchiumi, Toshio

    2012-01-01

    Protein synthesis on the ribosome requires translational GTPase factors to bind to the ribosome in the GTP-bound form, take individual actions that are coupled with GTP hydrolysis, and dissociate, usually in the GDP-bound form. The multiple copies of the flexible ribosomal stalk protein play an important role in these processes. Using biochemical approaches and the stalk protein from a hyperthermophilic archaeon, Pyrococcus horikoshii, we here provide evidence that the conserved C terminus of the stalk protein aP1 binds directly to domain I of the elongation factor aEF-2, irrespective of whether aEF-2 is bound to GTP or GDP. Site-directed mutagenesis revealed that four hydrophobic amino acids at the C terminus of aP1, Leu-100, 103, 106, and Phe-107, are crucial for the direct binding. P1 was also found to bind to the initiation factor aIF5B, as well as aEF-1α, but not aIF2γ, via its C terminus. Moreover, analytical ultracentrifugation and gel mobility shift analyses showed that a heptameric complex of aP1 and aP0, aP0(aP1)2(aP1)2(aP1)2, can bind multiple aEF-2 molecules simultaneously, which suggests that individual copies of the stalk protein are accessible to the factor. The functional significance of the C terminus of the stalk protein was also shown using the eukaryotic proteins P1/P2 and P0. It is likely that the conserved C terminus of the stalk proteins of archaea and eukaryotes can bind to translation factors both before and after GTP hydrolysis. This consistent binding ability of the stalk protein may contribute to maintaining high concentrations of translation factors around the ribosome, thus promoting translational efficiency. PMID:22355137

  5. Crystal structure of Gib2, a signal-transducing protein scaffold associated with ribosomes in Cryptococcus neoformans.

    PubMed

    Ero, Rya; Dimitrova, Valya Tenusheva; Chen, Yun; Bu, Wenting; Feng, Shu; Liu, Tongbao; Wang, Ping; Xue, Chaoyang; Tan, Suet Mien; Gao, Yong-Gui

    2015-03-03

    The atypical Gβ-like/RACK1 Gib2 protein promotes cAMP signalling that plays a central role in regulating the virulence of Cryptococcus neoformans. Gib2 contains a seven-bladed β transducin structure and is emerging as a scaffold protein interconnecting signalling pathways through interactions with various protein partners. Here, we present the crystal structure of Gib2 at a 2.2-Å resolution. The structure allows us to analyse the association between Gib2 and the ribosome, as well as to identify the Gib2 amino acid residues involved in ribosome binding. Our studies not only suggest that Gib2 has a role in protein translation but also present Gib2 as a physical link at the crossroads of various regulatory pathways important for the growth and virulence of C. neoformans.

  6. Altered chlorplast ribosomal proteins associated with erythromycin-resistant mutants in two genetic systems of Chlamydomonas reinhardi.

    PubMed

    Mets, L; Bogorad, L

    1972-12-01

    The phenotype of several erythromycin-resistant mutants of Chlamydomonas reinhardi was further characterized in terms of the electrophoretic properties of their chloroplast ribosomal proteins. In mutant ery-M2d a single protein of the large (52 S) subunit has altered properties, which probably result from a change in its primary sequence. This mutation is inherited in a Meudelian manner. In mutant ery-U1a, which is inherited in a uniparental manner, a different single protein of the 52 S subunit is altered. This change might result from a change in either the primary sequence of the protein or in some form of secondary modification. These results indicate that these two distinct genetic systems must cooperate in the production of chloroplast ribosomes.

  7. Crystal structure of Gib2, a signal-transducing protein scaffold associated with ribosomes in Cryptococcus neoformans

    NASA Astrophysics Data System (ADS)

    Ero, Rya; Dimitrova, Valya Tenusheva; Chen, Yun; Bu, Wenting; Feng, Shu; Liu, Tongbao; Wang, Ping; Xue, Chaoyang; Tan, Suet Mien; Gao, Yong-Gui

    2015-03-01

    The atypical Gβ-like/RACK1 Gib2 protein promotes cAMP signalling that plays a central role in regulating the virulence of Cryptococcus neoformans. Gib2 contains a seven-bladed β transducin structure and is emerging as a scaffold protein interconnecting signalling pathways through interactions with various protein partners. Here, we present the crystal structure of Gib2 at a 2.2-Å resolution. The structure allows us to analyse the association between Gib2 and the ribosome, as well as to identify the Gib2 amino acid residues involved in ribosome binding. Our studies not only suggest that Gib2 has a role in protein translation but also present Gib2 as a physical link at the crossroads of various regulatory pathways important for the growth and virulence of C. neoformans.

  8. Matching the crystallographic structure of ribosomal protein S7 to a three-dimensional model of the 16S ribosomal RNA.

    PubMed Central

    Tanaka, I; Nakagawa, A; Hosaka, H; Wakatsuki, S; Mueller, F; Brimacombe, R

    1998-01-01

    Two recently published but independently derived structures, namely the X-ray crystallographic structure of ribosomal protein S7 and the "binding pocket" for this protein in a three-dimensional model of the 16S rRNA, have been correlated with one another. The known rRNA-protein interactions for S7 include a minimum binding site, a number of footprint sites, and two RNA-protein crosslink sites on the 16S rRNA, all of which form a compact group in the published 16S rRNA model (despite the fact that these interactions were not used as primary modeling constraints in building that model). The amino acids in protein S7 that are involved in the two crosslinks to 16S rRNA have also been determined in previous studies, and here we have used these sites to orient the crystallographic structure of S7 relative to its rRNA binding pocket. Some minor alterations were made to the rRNA model to improve the fit. In the resulting structure, the principal positively charged surface of the protein is in contact with the 16S rRNA, and all of the RNA-protein interaction data are satisfied. The quality of the fit gives added confidence as to the validity of the 16S rRNA model. Protein S7 is furthermore known to be crosslinked both to P site-bound tRNA and to mRNA at positions upstream of the P site codon; the matched S7-16S rRNA structure makes a prediction as to the location of this crosslink site within the protein molecule. PMID:9582096

  9. Knock-out of the plastid ribosomal protein L11 in Arabidopsis: effects on mRNA translation and photosynthesis.

    PubMed

    Pesaresi, P; Varotto, C; Meurer, J; Jahns, P; Salamini, F; Leister, D

    2001-08-01

    The prpl11-1 mutant of Arabidopsis thaliana was identified among a collection of T-DNA tagged lines on the basis of a decrease in the effective quantum yield of photosystem II. The mutation responsible was localized to Prpl11, a single-copy nuclear gene that encodes PRPL11, a component of the large subunit of the plastid ribosome. The amino acid sequence of Arabidopsis PRPL11 is very similar to those of L11 proteins from spinach and prokaryotes. In the prpl11-1 mutant, photosensitivity and chlorophyll fluorescence parameters are significantly altered owing to changes in the levels of thylakoid protein complexes and stromal proteins. The abundance of most plastome transcripts examined, such as those of genes coding for the photosystem II core complex and RbcL, is not decreased. Plastid ribosomal RNA accumulates in wild-type amounts, and the assembly of plastid polysomes on the transcripts of the rbcL, psbA and psbE genes remains mainly unchanged in mutant plants, indicating that lack of PRPL11 affects neither the abundance of plastid ribosomes nor their assembly into polysomes. However, in vivo translation assays demonstrate that the rate of translation of the large subunit of Rubisco (RbcL) is significantly reduced in prpl11-1 plastids. Our data suggest a major role for PRPL11 in plastid ribosome activity per se, consistent with its location near the GTPase-binding centre of the chloroplast 50S ribosomal subunit. Additional effects of the mutation, including the pale green colour of the leaves and a drastic reduction in growth rate under greenhouse conditions, are compatible with reduced levels of protein synthesis in plastids.

  10. Selection of IgE-binding aptameric green fluorescent protein (Ap-GFP) by the ribosome display (RD) platform

    SciTech Connect

    Chen, S.-S. Yang Yongmin; Barankiewicz, Teresa J.

    2008-09-26

    GFP-C{kappa} fusion protein was previously shown selectable on ribosome display platform with solid phase antibodies against GFP determinant [Y.-M. Yang, T.J. Barankiewicz, M. He, M. Taussig, S.-S. Chen, Selection of antigenic markers on a GFP-C{kappa} fusion scaffold with high sensitivity by eukaryotic ribosome display, Biochem. Biophys. Res. Commun. 359 (2007) 251-257]. Herein, we show that members of aptameric peptide library constructed within the site 6 and site 8/9 loops of GFP of the ribosome display construct are selectable upon binding to the solid phase IgE antigen. An input of 1.0 {mu}g of the dual site aptameric GFP library exhibiting a diversity of 7.5 x 10{sup 11} was transcribed, translated and incubated with solid phase IgE. RT-PCR products were amplified from mRNA of the aptamer-ribosome-mRNA (ARM) complex captured on the solid phase IgE. Clones of aptameric GFP were prepared from RT-PCR product of ARM complex following repetitive selection. Recombinant aptameric GFP proteins from the selected clones bind IgE coated on the 96-well plate, and the binding was abrogated by incubation with soluble human IgE but not human IgG. Selected aptameric GFP proteins also exhibit binding to three different sources of human IgE (IgE PS, BED, and JW8) but not irrelevant proteins. These observations indicate that appropriately selected aptameric GFP on a solid phase ligand by ribosome display may serve as an affinity reagent for blocking reactivity of a biological ligand.

  11. Identifying Neisseria Species by Use of the 50S Ribosomal Protein L6 (rplF) Gene

    PubMed Central

    Bennett, Julia S.; Watkins, Eleanor R.; Jolley, Keith A.; Harrison, Odile B.

    2014-01-01

    The comparison of 16S rRNA gene sequences is widely used to differentiate bacteria; however, this gene can lack resolution among closely related but distinct members of the same genus. This is a problem in clinical situations in those genera, such as Neisseria, where some species are associated with disease while others are not. Here, we identified and validated an alternative genetic target common to all Neisseria species which can be readily sequenced to provide an assay that rapidly and accurately discriminates among members of the genus. Ribosomal multilocus sequence typing (rMLST) using ribosomal protein genes has been shown to unambiguously identify these bacteria. The PubMLST Neisseria database (http://pubmlst.org/neisseria/) was queried to extract the 53 ribosomal protein gene sequences from 44 genomes from diverse species. Phylogenies reconstructed from these genes were examined, and a single 413-bp fragment of the 50S ribosomal protein L6 (rplF) gene was identified which produced a phylogeny that was congruent with the phylogeny reconstructed from concatenated ribosomal protein genes. Primers that enabled the amplification and direct sequencing of the rplF gene fragment were designed to validate the assay in vitro and in silico. Allele sequences were defined for the gene fragment, associated with particular species names, and stored on the PubMLST Neisseria database, providing a curated electronic resource. This approach provides an alternative to 16S rRNA gene sequencing, which can be readily replicated for other organisms for which more resolution is required, and it has potential applications in high-resolution metagenomic studies. PMID:24523465

  12. Ribosomal protein L3 functions as a ‘rocker switch’ to aid in coordinating of large subunit-associated functions in eukaryotes and Archaea

    PubMed Central

    Meskauskas, Arturas

    2008-01-01

    Although ribosomal RNAs (rRNAs) comprise the bulk of the ribosome and carry out its main functions, ribosomal proteins also appear to play important structural and functional roles. Many ribosomal proteins contain long, nonglobular domains that extend deep into the rRNA cores. In eukaryotes and Archaea, ribosomal protein L3 contains two such extended domains tethered to a common globular hub, thus providing an excellent model to address basic questions relating to ribosomal protein structure/function relationships. Previous work in our laboratory identified the central ‘W-finger’ extension of yeast L3 in helping to coordinate ribosomal functions. New studies on the ‘N-terminal’ extension in yeast suggest that it works with the W-finger to coordinate opening and closing of the corridor through which the 3′ end of aa-tRNA moves during the process of accommodation. Additionally, the effect of one of the L3 N-terminal extension mutants on the interaction between C75 of the aa-tRNA and G2921 (Escherichia coli G2553) of 25S rRNA provides the first evidence of the effect of a ribosomal protein on aa-tRNA positioning and peptidyltransfer, possibly through the induced fit model. A model is presented describing how all three domains of L3 may function together as a ‘rocker switch’ to coordinate the stepwise processes of translation elongation. PMID:18832371

  13. Sequence, overproduction and purification of Vibrio proteolyticus ribosomal protein L18 for in vitro and in vivo studies

    NASA Technical Reports Server (NTRS)

    Setterquist, R. A.; Smith, G. K.; Oakley, T. H.; Lee, Y. H.; Fox, G. E.

    1996-01-01

    A strategy suggested by comparative genomic studies was used to amplify the entire Vibrio proteolyticus (Vp) gene for ribosomal protein L18. Vp L18 and its flanking regions were sequenced and compared with the deduced amino acid (aa) sequences of other known L18 proteins. A 26-aa residue segment at the carboxy terminus contains many strongly conserved residues and may be critical for the L18 interaction with 5S rRNA. This approach should allow rapid characterization of L18 from large numbers of bacteria. Both Vp L18 and Escherichia coli (Ec) L18 were overproduced and purified using a T7 expression vector which fuses an N-terminal peptide segment (His-tag) containing 6 histidine residues to the recombinant protein. The purified fusion proteins, Vp His::L18 and Ec His::L18, were both found to bind to either the Vp 5S or Ec 5S rRNAs in vitro. Vp His::L18 protein was also shown to incorporate into Ec ribosomes in vivo. This His-tag strategy likely will have general applicability for the study of ribosomal proteins in vitro and in vivo.

  14. Structure of the JmjC domain-containing protein NO66 complexed with ribosomal protein Rpl8

    SciTech Connect

    Wang, Chengliang; Zhang, Qiongdi; Hang, Tianrong; Tao, Yue; Ma, Xukai; Wu, Minhao; Zhang, Xuan Zang, Jianye

    2015-08-28

    The structure of the complex of NO66 and Rpl8 was solved in the native state and NO66 recognizes the consensus motif NHXH . Tetramerization is required for efficient substrate binding and catalysis by NO66. The JmjC domain-containing proteins belong to a large family of oxygenases possessing distinct substrate specificities which are involved in the regulation of different biological processes, such as gene transcription, RNA processing and translation. Nucleolar protein 66 (NO66) is a JmjC domain-containing protein which has been reported to be a histone demethylase and a ribosome protein 8 (Rpl8) hydroxylase. The present biochemical study confirmed the hydroxylase activity of NO66 and showed that oligomerization is required for NO66 to efficiently catalyze the hydroxylation of Rpl8. The structures of NO66{sup 176–C} complexed with Rpl8{sup 204–224} in a tetrameric form and of the mutant protein M2 in a dimeric form were solved. Based on the results of structural and biochemical analyses, the consensus sequence motif NHXH recognized by NO66 was confirmed. Several potential substrates of NO66 were found by a BLAST search according to the consensus sequence motif. When binding to substrate, the relative positions of each subunit in the NO66 tetramer shift. Oligomerization may facilitate the motion of each subunit in the NO66 tetramer and affect the catalytic activity.

  15. The Ribosome: The Cell's Protein-Synthesizing Machine and How Antibiotics Disrupt It

    SciTech Connect

    Venki Ramakrishnan

    2009-10-08

    Determining the structure of the ribosome has made it possible for Ramakrishnan and his colleagues to image antibiotics bound to the ribosome, leading to a better understanding of their action, which could help in the development of novel drugs. In his ta

  16. The Ribosome: The Cell's Protein-Synthesizing Machine and How Antibiotics Disrupt It

    ScienceCinema

    Venki Ramakrishnan

    2016-07-12

    Determining the structure of the ribosome has made it possible for Ramakrishnan and his colleagues to image antibiotics bound to the ribosome, leading to a better understanding of their action, which could help in the development of novel drugs. In his ta

  17. Molecular scanning: combining random mutagenesis, ribosome display, and bioinformatic analysis for protein engineering.

    PubMed

    Darmanin-Sheehan, Alfredo; Finlay, William James Jonathan; Cunningham, Orla; Fennell, Brian Joseph

    2012-01-01

    Protein engineering techniques can facilitate the direct de-convolution of specific domains, regions, and particular amino acids that contribute to protein function. Many tools are available to aid this enterprise and herein we describe one such tool, a technique we term "Molecular Scanning" (MS). MS is analogous to previously described alanine scanning in that it samples potentially functional sequence space, but differs in that it uses Error-Prone polymerase chain reaction to randomly introduce all amino acids across the sequence space, as opposed to simply introducing alanine at each desired position. We commonly use MS in conjunction with ribosome-display, selecting for specific character traits (e.g., improved affinity) which allows us to sample functionally relevant diversity on a reasonably large scale. This approach is amenable to a variety of different mutational techniques and display technologies as dictated by user requirements or needs. In this chapter we present a general outline of the process as we have previously successfully applied it.

  18. Transition state analogues in structures of ricin and saporin ribosome-inactivating proteins

    SciTech Connect

    Ho, Meng-Chiao; Sturm, Matthew B.; Almo, Steven C.; Schramm, Vern L.

    2010-01-12

    Ricin A-chain (RTA) and saporin-L1 (SAP) catalyze adenosine depurination of 28S rRNA to inhibit protein synthesis and cause cell death. We present the crystal structures of RTA and SAP in complex with transition state analogue inhibitors. These tight-binding inhibitors mimic the sarcin-ricin recognition loop of 28S rRNA and the dissociative ribocation transition state established for RTA catalysis. RTA and SAP share unique purine-binding geometry with quadruple {pi}-stacking interactions between adjacent adenine and guanine bases and 2 conserved tyrosines. An arginine at one end of the {pi}-stack provides cationic polarization and enhanced leaving group ability to the susceptible adenine. Common features of these ribosome-inactivating proteins include adenine leaving group activation, a remarkable lack of ribocation stabilization, and conserved glutamates as general bases for activation of the H{sub 2}O nucleophile. Catalytic forces originate primarily from leaving group activation evident in both RTA and SAP in complex with transition state analogues.

  19. Multiple-Site Trimethylation of Ribosomal Protein L11 by the PrmA Methyltransferase

    SciTech Connect

    Demirci,H.; Gregory, S.; Dahlberg, A.; Jogl, G.

    2008-01-01

    Ribosomal protein L11 is a universally conserved component of the large subunit, and plays a significant role during initiation, elongation, and termination of protein synthesis. In Escherichia coli, the lysine methyltransferase PrmA trimethylates the N-terminal a-amino group and the -amino groups of Lys3 and Lys39. Here, we report four PrmA-L11 complex structures in different orientations with respect to the PrmA active site. Two structures capture the L11 N-terminal a-amino group in the active site in a trimethylated postcatalytic state and in a dimethylated state with bound S-adenosyl-L-homocysteine. Two other structures show L11 in a catalytic orientation to modify Lys39 and in a noncatalytic orientation. The comparison of complex structures in different orientations with a minimal substrate recognition complex shows that the binding mode remains conserved in all L11 orientations, and that substrate orientation is brought about by the unusual interdomain flexibility of PrmA.

  20. Multiple-Site Trimethylation of Ribosomal Protein L11 by the PrmA Methyltransferase

    PubMed Central

    Demirci, Hasan; Gregory, Steven T.; Dahlberg, Albert E.; Jogl, Gerwald

    2008-01-01

    SUMMARY Ribosomal protein L11 is a universally conserved component of the large subunit, and plays a significant role during initiation, elongation, and termination of protein synthesis. In Escherichia coli, the lysine methyltransferase PrmA trimethylates the N-terminal α-amino group and the ε-amino groups of Lys3 and Lys39. Here, we report four PrmA-L11 complex structures in different orientations with respect to the PrmA active site. Two structures capture the L11 N-terminal α-amino group in the active site in a trimethylated postcatalytic state and in a dimethylated state with bound S-adenosyl-L-homocysteine. Two other structures show L11 in a catalytic orientation to modify Lys39 and in a noncatalytic orientation. The comparison of complex structures in different orientations with a minimal substrate recognition complex shows that the binding mode remains conserved in all L11 orientations, and that substrate orientation is brought about by the unusual interdomain flexibility of PrmA. PMID:18611379

  1. The structure and evolution of the ribosomal proteins encoded in the spc operon of the archaeon (Crenarchaeota) Sulfolobus acidocaldarius.

    PubMed

    Yang, D; Kusser, I; Köpke, A K; Koop, B F; Matheson, A T

    1999-07-01

    The genes for nine ribosomal proteins, L24, L5, S14, S8, L6, L18, S5, L30, and L15, have been isolated and sequenced from the spc operon in the archaeon (Crenarchaeota) Sulfolobus acidocaldarius, and the putative amino acid sequence of the proteins coded by these genes has been determined. In addition, three other genes in the spc operon, coding for ribosomal proteins S4E, L32E, and L19E (equivalent to rat ribosomal proteins S4, L32, and L19), were sequenced and the structure of the putative proteins was determined. The order of the ribosomal protein genes in the spc operon of the Crenarchaeota kingdom of Archaea is identical to that present in the Euryarchaeota kingdom of Archaea and also identical to that found in bacteria, except for the genes for r-proteins S4E, L32E, and L19E, which are absent in bacteria. Although AUG is the initiation codon in most of the spc genes, GUG (val) and UUG (leu) are also used as initiation codons in S. acidocaldarius. Over 70% of the codons in the Sulfolobus spc operon have A or U in the third position, reflecting the low GC content of Sulfolobus DNA. Phylogenetic analysis indicated that the archaeal r-proteins are a sister group of their eucaryotic counterparts but did not resolve the question of whether the Archaea is monophyletic, as suggested by the L6P, L15P, and L18P trees, or the question of whether the Crenarchaeota is separate from the Euryarchaeota and closer to the Eucarya, as suggested by the S8P, S5P, and L24P trees. In the case of the three Sulfolobus r-proteins that do not have a counterpart in the bacterial ribosome (S4E, L32E, and L19E), the archaeal r-proteins showed substantial identity to their eucaryotic equivalents, but in all cases the archaeal proteins formed a separate group from the eucaryotic proteins.

  2. The Dedicated Chaperone Acl4 Escorts Ribosomal Protein Rpl4 to Its Nuclear Pre-60S Assembly Site

    PubMed Central

    Pillet, Benjamin; García-Gómez, Juan J.; Pausch, Patrick; Falquet, Laurent; Bange, Gert; de la Cruz, Jesús; Kressler, Dieter

    2015-01-01

    Ribosomes are the highly complex macromolecular assemblies dedicated to the synthesis of all cellular proteins from mRNA templates. The main principles underlying the making of ribosomes are conserved across eukaryotic organisms and this process has been studied in most detail in the yeast Saccharomyces cerevisiae. Yeast ribosomes are composed of four ribosomal RNAs (rRNAs) and 79 ribosomal proteins (r-proteins). Most r-proteins need to be transported from the cytoplasm to the nucleus where they get incorporated into the evolving pre-ribosomal particles. Due to the high abundance and difficult physicochemical properties of r-proteins, their correct folding and fail-safe targeting to the assembly site depends largely on general, as well as highly specialized, chaperone and transport systems. Many r-proteins contain universally conserved or eukaryote-specific internal loops and/or terminal extensions, which were shown to mediate their nuclear targeting and association with dedicated chaperones in a growing number of cases. The 60S r-protein Rpl4 is particularly interesting since it harbours a conserved long internal loop and a prominent C-terminal eukaryote-specific extension. Here we show that both the long internal loop and the C-terminal eukaryote-specific extension are strictly required for the functionality of Rpl4. While Rpl4 contains at least five distinct nuclear localization signals (NLS), the C-terminal part of the long internal loop associates with a specific binding partner, termed Acl4. Absence of Acl4 confers a severe slow-growth phenotype and a deficiency in the production of 60S subunits. Genetic and biochemical evidence indicates that Acl4 can be considered as a dedicated chaperone of Rpl4. Notably, Acl4 localizes to both the cytoplasm and nucleus and it has the capacity to capture nascent Rpl4 in a co-translational manner. Taken together, our findings indicate that the dedicated chaperone Acl4 accompanies Rpl4 from the cytoplasm to its pre-60S

  3. The Dedicated Chaperone Acl4 Escorts Ribosomal Protein Rpl4 to Its Nuclear Pre-60S Assembly Site.

    PubMed

    Pillet, Benjamin; García-Gómez, Juan J; Pausch, Patrick; Falquet, Laurent; Bange, Gert; de la Cruz, Jesús; Kressler, Dieter

    2015-10-01

    Ribosomes are the highly complex macromolecular assemblies dedicated to the synthesis of all cellular proteins from mRNA templates. The main principles underlying the making of ribosomes are conserved across eukaryotic organisms and this process has been studied in most detail in the yeast Saccharomyces cerevisiae. Yeast ribosomes are composed of four ribosomal RNAs (rRNAs) and 79 ribosomal proteins (r-proteins). Most r-proteins need to be transported from the cytoplasm to the nucleus where they get incorporated into the evolving pre-ribosomal particles. Due to the high abundance and difficult physicochemical properties of r-proteins, their correct folding and fail-safe targeting to the assembly site depends largely on general, as well as highly specialized, chaperone and transport systems. Many r-proteins contain universally conserved or eukaryote-specific internal loops and/or terminal extensions, which were shown to mediate their nuclear targeting and association with dedicated chaperones in a growing number of cases. The 60S r-protein Rpl4 is particularly interesting since it harbours a conserved long internal loop and a prominent C-terminal eukaryote-specific extension. Here we show that both the long internal loop and the C-terminal eukaryote-specific extension are strictly required for the functionality of Rpl4. While Rpl4 contains at least five distinct nuclear localization signals (NLS), the C-terminal part of the long internal loop associates with a specific binding partner, termed Acl4. Absence of Acl4 confers a severe slow-growth phenotype and a deficiency in the production of 60S subunits. Genetic and biochemical evidence indicates that Acl4 can be considered as a dedicated chaperone of Rpl4. Notably, Acl4 localizes to both the cytoplasm and nucleus and it has the capacity to capture nascent Rpl4 in a co-translational manner. Taken together, our findings indicate that the dedicated chaperone Acl4 accompanies Rpl4 from the cytoplasm to its pre-60S

  4. Thermodynamics and kinetics of protein folding on the ribosome: Alteration in energy landscapes, denatured state, and transition state ensembles

    NASA Astrophysics Data System (ADS)

    O'Brien, Edward; Vendruscolo, Michele; Dobson, Christopher

    2010-03-01

    In vitro experiments examining cotranslational folding utilize ribosome-nascent chain complexes (RNCs) in which the nascent chain is stalled at different points of its biosynthesis on the ribosome. We investigate the thermodynamics, kinetics, and structural properties of RNCs containing five different globular and repeat proteins stalled at ten different nascent chain lengths using coarse grained replica exchange simulations. We find that when the proteins are stalled near the ribosome exit tunnel opening they exhibit altered folding coopserativity, quantified by the van't Hoff enthalpy criterion; a significantly altered denatured state ensemble, in terms of Rg and shape parameters (Rg tensor); and the appearance of partially folded intermediates during cotranslation, evidenced by the appearance of a third basin in the free energy profile. These trends are due in part to excluded volume (crowding) interactions between the ribosome and nascent chain. We perform in silico temperature-jump experiments on the RNCs and examine nascent chain folding kinetics and structural changes in the transition state ensemble at various stall lengths.

  5. Ricin uses arginine 235 as an anchor residue to bind to P-proteins of the ribosomal stalk

    PubMed Central

    Zhou, Yijun; Li, Xiao-Ping; Chen, Brian Y.; Tumer, Nilgun E.

    2017-01-01

    Ricin toxin A chain (RTA) binds to stalk P-proteins to reach the α–sarcin/ricin loop (SRL) where it cleaves a conserved adenine. Arginine residues at the RTA/RTB interface are involved in this interaction. To investigate the individual contribution of each arginine, we generated single, double and triple arginine mutations in RTA. The R235A mutation reduced toxicity and depurination activity more than any other single arginine mutation in yeast. Further reduction in toxicity, depurination activity and ribosome binding was observed when R235A was combined with a mutation in a nearby arginine. RTA interacts with the ribosome via a two-step process, which involves slow and fast interactions. Single arginine mutations eliminated the fast interactions with the ribosome, indicating that they increase the binding rate of RTA. Arginine residues form a positively charged patch to bind to negatively charged residues at the C-termini of P-proteins. When electrostatic interactions conferred by the arginines are lost, hydrophobic interactions are also abolished, suggesting that the hydrophobic interactions alone are insufficient to allow binding. We propose that Arg235 serves as an anchor residue and cooperates with nearby arginines and the hydrophobic interactions to provide the binding specificity and strength in ribosome targeting of RTA. PMID:28230053

  6. The ribosomal protein Asc1/RACK1 is required for efficient translation of short mRNAs

    PubMed Central

    Thompson, Mary K; Rojas-Duran, Maria F; Gangaramani, Paritosh; Gilbert, Wendy V

    2016-01-01

    Translation is a core cellular process carried out by a highly conserved macromolecular machine, the ribosome. There has been remarkable evolutionary adaptation of this machine through the addition of eukaryote-specific ribosomal proteins whose individual effects on ribosome function are largely unknown. Here we show that eukaryote-specific Asc1/RACK1 is required for efficient translation of mRNAs with short open reading frames that show greater than average translational efficiency in diverse eukaryotes. ASC1 mutants in S. cerevisiae display compromised translation of specific functional groups, including cytoplasmic and mitochondrial ribosomal proteins, and display cellular phenotypes consistent with their gene-specific translation defects. Asc1-sensitive mRNAs are preferentially associated with the translational ‘closed loop’ complex comprised of eIF4E, eIF4G, and Pab1, and depletion of eIF4G mimics the translational defects of ASC1 mutants. Together our results reveal a role for Asc1/RACK1 in a length-dependent initiation mechanism optimized for efficient translation of genes with important housekeeping functions. DOI: http://dx.doi.org/10.7554/eLife.11154.001 PMID:27117520

  7. Preservation of Gene Duplication Increases the Regulatory Spectrum of Ribosomal Protein Genes and Enhances Growth under Stress.

    PubMed

    Parenteau, Julie; Lavoie, Mathieu; Catala, Mathieu; Malik-Ghulam, Mustafa; Gagnon, Jules; Abou Elela, Sherif

    2015-12-22

    In baker's yeast, the majority of ribosomal protein genes (RPGs) are duplicated, and it was recently proposed that such duplications are preserved via the functional specialization of the duplicated genes. However, the origin and nature of duplicated RPGs' (dRPGs) functional specificity remain unclear. In this study, we show that differences in dRPG functions are generated by variations in the modality of gene expression and, to a lesser extent, by protein sequence. Analysis of the sequence and expression patterns of non-intron-containing RPGs indicates that each dRPG is controlled by specific regulatory sequences modulating its expression levels in response to changing growth conditions. Homogenization of dRPG sequences reduces cell tolerance to growth under stress without changing the number of expressed genes. Together, the data reveal a model where duplicated genes provide a means for modulating the expression of ribosomal proteins in response to stress.

  8. Pineapple translation factor SUI1 and ribosomal protein L36 promoters drive constitutive transgene expression patterns in Arabidopsis thaliana.

    PubMed

    Koia, Jonni; Moyle, Richard; Hendry, Caroline; Lim, Lionel; Botella, José Ramón

    2013-03-01

    The availability of a variety of promoter sequences is necessary for the genetic engineering of plants, in basic research studies and for the development of transgenic crops. In this study, the promoter and 5' untranslated regions of the evolutionally conserved protein translation factor SUI1 gene and ribosomal protein L36 gene were isolated from pineapple and sequenced. Each promoter was translationally fused to the GUS reporter gene and transformed into the heterologous plant system Arabidopsis thaliana. Both the pineapple SUI1 and L36 promoters drove GUS expression in all tissues of Arabidopsis at levels comparable to the CaMV35S promoter. Transient assays determined that the pineapple SUI1 promoter also drove GUS expression in a variety of climacteric and non-climacteric fruit species. Thus the pineapple SUI1 and L36 promoters demonstrate the potential for using translation factor and ribosomal protein genes as a source of promoter sequences that can drive constitutive transgene expression patterns.

  9. p73 expression is regulated by ribosomal protein RPL26 through mRNA translation and protein stability

    PubMed Central

    Yan, Wensheng; Chen, Xinbin

    2016-01-01

    p73, a p53 family tumor suppressor, is regulated by multiple mechanisms, including transcription and mRNA and protein stability. However, whether p73 expression is regulated via mRNA translation has not been explored. To test this, we examined whether ribosomal protein 26 (RPL26) plays a role in p73 expression. Here, we showed that p73 expression is controlled by RPL26 via protein stability and mRNA translation. To examine whether MDM2 mediates RPL26 to regulate p73 protein stability, we generated multiple MDM2-knockout cell lines by CRISPR-cas9. We found that in the absence of MDM2, the half-life of p73 protein is markedly increased. Interestingly, we also found that RPL26 is still capable of regulating p73 expression, albeit to a lesser extent, in MDM2-KO cells compared to that in isogenic control cells, suggesting that RPL26 regulates p73 expression via multiple mechanisms. Indeed, we found that RPL26 is necessary for efficient assembly of polysomes on p73 mRNA and de novo synthesis of p73 protein. Consistently, we found that RPL26 directly binds to p73 3′ untranslated region (3′UTR) and that RPL26 is necessary for efficient expression of an eGFP reporter that carries p73 3′UTR. We also found that RPL26 interacts with cap-binding protein eIF4E and enhances the association of eIF4E with p73 mRNA, leading to increased p73 mRNA translation. Finally, we showed that knockdown of RPL26 promotes, whereas ectopic expression of RPL26 inhibits, cell growth in a TAp73-dependent manner. Together, our data indicate that RPL26 regulates p73 expression via two distinct mechanisms: protein stability and mRNA translation. PMID:27825141

  10. Ribosome maturation in E. coli.

    PubMed

    Silengo, L; Altruda, F; Dotto, G P; Lacquaniti, F; Perlo, C; Turco, E; Mangiarotti, G

    1977-01-01

    In vivo and in vitro experiments have shown that processing of ribosomal RNA is a late event in ribosome biogenesis. The precursor form of RNA is probably necessary to speed up the assembly of ribomal proteins. Newly formed ribosomal particles which have already entered polyribosomes differ from mature ribosomes not only in their RNA content but also in their susceptibility to unfolding in low Mg concentration and to RNase attack. Final maturation of new ribosomes is probably dependent on their functioning in protein synthesis. Thus only those ribosomes which have proven to be functional may be converted into stable cellular structures.

  11. Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

    NASA Astrophysics Data System (ADS)

    Kallehauge, Thomas Beuchert; Li, Shangzhong; Pedersen, Lasse Ebdrup; Ha, Tae Kwang; Ley, Daniel; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup; Lee, Gyun Min; Lewis, Nathan E.

    2017-01-01

    Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production.

  12. Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

    PubMed Central

    Kallehauge, Thomas Beuchert; Li, Shangzhong; Pedersen, Lasse Ebdrup; Ha, Tae Kwang; Ley, Daniel; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup; Lee, Gyun Min; Lewis, Nathan E.

    2017-01-01

    Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production. PMID:28091612

  13. Skeletal muscle plasticity induced by seasonal acclimatization involves IGF1 signaling: implications in ribosomal biogenesis and protein synthesis.

    PubMed

    Fuentes, Eduardo N; Zuloaga, Rodrigo; Valdes, Juan Antonio; Molina, Alfredo; Alvarez, Marco

    2014-10-01

    One of the most fundamental biological processes in living organisms that are affected by environmental fluctuations is growth. In fish, skeletal muscle accounts for the largest proportion of body mass, and the growth of this tissue is mainly controlled by the insulin-like growth factor (IGF) system. By using the carp (Cyprinus carpio), a fish that inhabits extreme conditions during winter and summer, we assessed the skeletal muscle plasticity induced by seasonal acclimatization and the relation of IGF signaling with protein synthesis and ribosomal biogenesis. The expression of igf1 in muscle decreased during winter in comparison with summer, whereas the expression for both paralogues of igf2 did not change significantly between seasons. The expression of igf1 receptor a (igf1ra), but not of igf1rb, was down-regulated in muscle during the winter as compared to the summer. A decrease in protein contents and protein phosphorylation for IGF signaling molecules in muscle was observed in winter-acclimatized carp. This was related with a decreased expression in muscle for markers of myogenesis (myoblast determination factor (myod), myogenic factor 5 (myf5), and myogenin (myog)); protein synthesis (myosin heavy chain (mhc) and myosin light chain (mlc3 and mlc1b)); and ribosomal biogenesis (pre-rRNA and ribosomal proteins). IGF signaling, and key markers of ribosomal biogenesis, protein synthesis, and myogenesis were affected by seasonal acclimatization, with differential regulation in gene expression and signaling pathway activation observed in muscle between both seasons. This suggests that these molecules are responsible for the muscle plasticity induced by seasonal acclimatization in carp.

  14. Lagenin, a novel ribosome-inactivating protein with ribonucleolytic activity from bottle gourd (Lagenaria siceraria) seeds.

    PubMed

    Wang, H X; Ng, T B

    2000-10-13

    The seeds of Lagenaria siceraria (Family Cucurbitaceae) were extracted with water and the extract was lyophilized. The lyophilized extract was chromatographed on a DEAE-cellulose column in 10 mM Tris-HCl buffer (pH 7.2). The unadsorbed fraction was applied to an Affi-gel Blue gel column previously equilibrated with the same buffer. After removal of unadsorbed materials, the adsorbed proteins were eluted with 1.5 M NaCl in the Tris-HCl buffer. After dialysis the adsorbed fraction was loaded on a CM-Sepharose CL-6B column which had been equilibrated with and was eluted with the same buffer. After elution of unadsorbed proteins, the column was eluted with a gradient of 0-1 M NaCl in 10 mM Tris-HCl buffer (pH 7.2). The fraction eluting at about 0.55 M NaCl, which represented pure ribosome inactivating protein (RIP), inhibited cell-free translation in a rabbit reticulocyte system with an IC50 of 0.21 nM and exerted ribonuclease activity on yeast tRNA with an activity of 45 U/mg. The RIP was designated lagenin. It possessed a molecular weight of 20 kDa, smaller than the range of 26-32 kDa reported for other RIPs. The N-terminal sequence of lagenin exhibited a lesser extent of similarity to those of other Cucurbitaceae RIPs, characterized by a deletion of the first three amino acid residues and a replacement of the 4th (Phe), 17th (Phe), 18th (Ile) and 22nd (Arg) residues which are invariant in other RIPs.

  15. Expression profile of ribosomal protein L10a throughout gonadal development in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Makkapan, Walaiporn; Yoshizaki, Goro; Tashiro, Masami; Chotigeat, Wilaiwan

    2014-08-01

    Ribosomal protein L10a (RpL10A) has been previously established as a stimulator during the early stages of ovarian development in both the banana prawn and the fruit fly. In order to develop a greater understanding of the role of this protein in vertebrates, the present study aimed to characterize the expression profile of rpl10a during gonadal development in fish. It was determined that the expression of rpl10a within genital ridges increased during embryonic development. Although rpl10a expression was observed in both gonadal somatic cells and primordial germ cells, higher levels of both transcript and protein expression were detected in somatic cells. rpl10a transcripts were observed in all of the adult tissues examined. Cellular level expression of rpl10a was subsequently characterized across various maturational stages using in situ hybridization and immunohistochemistry of both testes and ovaries. Analysis of tissue derived from the testis showed high levels of rpl10a expression within spermatogonia and the Sertoli cells attached to them. In ovarian tissue, rpl10a was strongly expressed in chromatin-nucleolus-stage and peri-nucleolus-stage oocytes. The relationship between rpl10a expression and regulation of gonadal development was confirmed using real-time PCR, which was performed in order to analyze rpl10a expression in testicular and ovarian tissues subsequent to incubation with salmon pituitary extract and various sex steroids for 24 h. Among them, 11-ketotestosterone at 100 ng/mL effectively up-regulated expression of rpl10a in testicular tissues, while 17β-estradiol down-regulated rpl10a expression in ovarian tissues. These results suggested that rpl10a played a role in the regulation of gonadal development in fish.

  16. RibAlign: a software tool and database for eubacterial phylogeny based on concatenated ribosomal protein subunits

    PubMed Central

    Teeling, Hanno; Gloeckner, Frank Oliver

    2006-01-01

    Background Until today, analysis of 16S ribosomal RNA (rRNA) sequences has been the de-facto gold standard for the assessment of phylogenetic relationships among prokaryotes. However, the branching order of the individual phlya is not well-resolved in 16S rRNA-based trees. In search of an improvement, new phylogenetic methods have been developed alongside with the growing availability of complete genome sequences. Unfortunately, only a few genes in prokaryotic genomes qualify as universal phylogenetic markers and almost all of them have a lower information content than the 16S rRNA gene. Therefore, emphasis has been placed on methods that are based on multiple genes or even entire genomes. The concatenation of ribosomal protein sequences is one method which has been ascribed an improved resolution. Since there is neither a comprehensive database for ribosomal protein sequences nor a tool that assists in sequence retrieval and generation of respective input files for phylogenetic reconstruction programs, RibAlign has been developed to fill this gap. Results RibAlign serves two purposes: First, it provides a fast and scalable database that has been specifically adapted to eubacterial ribosomal protein sequences and second, it provides sophisticated import and export capabilities. This includes semi-automatic extraction of ribosomal protein sequences from whole-genome GenBank and FASTA files as well as exporting aligned, concatenated and filtered sequence files that can directly be used in conjunction with the PHYLIP and MrBayes phylogenetic reconstruction programs. Conclusion Up to now, phylogeny based on concatenated ribosomal protein sequences is hampered by the limited set of sequenced genomes and high computational requirements. However, hundreds of full and draft genome sequencing projects are on the way, and advances in cluster-computing and algorithms make phylogenetic reconstructions feasible even with large alignments of concatenated marker genes. Rib

  17. Identification, characterization and structure analysis of a type I ribosome-inactivating protein from Sapium sebiferum (Euphorbiaceae)

    SciTech Connect

    Wu, Ying; Mao, Yingji; Jin, Shan; Hou, Jinyan; Du, Hua; Yang, Minglei; Wu, Lifang

    2015-08-07

    Ribosome-inactivating proteins (RIPs) are N-glycosidases (EC3.2.2.22) that universally inactivate the ribosome, thereby inhibiting protein biosynthesis. In this study, a novel type I RIPs named SEBIN was identified in Sapium sebiferum. Nuclear acid depurine experiment showed that SEBIN had rRNA N-Glycosidase activity. Further experiment indicated that SEBIN significantly inhibited Caenorhabditis elegans development as well as resulted in worm cell apoptosis. This is the first report to evaluate RIPs toxicity using C. elegans. We proposed that SEBIN may impaire C. elegans reproduction in a DNA-damage manner besides traditional protein synthesis inhibition approach. The predicted 3D structure was modeled using threading and ab initio modeling, and the r-RNA binding residue of SEBIN was identified through the protein-ligand docking approach. It showed the amino acid residues, Glu195, Asn81, Ala82, Tyr83, Glu164, Ser163, Ile159 and Arg167, played critical roles in catalytic process. Our results provided the theoretical foundation of structure–function relationships between enzymatic properties, toxicity and structural characterization of SEBIN. - Graphical abstract: Superposition of main chains of ricin (cyan) and SEBIN (brown), and adenine binding site residues of SEBIN. - Highlights: • A Ribosome-inactivating proteins gene (SEBIN) was isolated from Sapium sebiferum. • SEBIN had DNase activity besides widely reported ribosome inactivation via N-glycosidases activity. • SEBIN significantly inhibited Caenorhabditis elegans development in vivo. • SEBIN may impaire C. elegans reproduction in a DNA-damage manner with the aid of mutant strains hus-1 and clk-2. • The possible active sites between SEBIN and the adenine of rRNA were predicted.

  18. The mushroom ribosome-inactivating protein lyophyllin exerts deleterious effects on mouse embryonic development in vitro.

    PubMed

    Chan, W Y; Ng, T B; Lam, Joyce S Y; Wong, Jack H; Chu, K T; Ngai, P H K; Lam, S K; Wang, H X

    2010-01-01

    Earlier investigations disclose that some plant ribosome-inactivating proteins (RIPs) adversely affect mouse embryonic development. In the present study, a mushroom RIP, namely lyophyllin from Lyophyllum shimeji, was isolated, partially sequenced, and its translation inhibitory activity determined. Its teratogenicity was studied by using a technique entailing microinjection and postimplantation whole-embryo culture. It was found that embryonic abnormalities during the period of organogenesis from E8.5 to E9.5 were induced by lyophyllin at a concentration as low as 50 microg/ml, and when the lyophyllin concentration was raised, the number of abnormal embryos increased, the final somite number decreased, and the abnormalities increased in severity. The affected embryonic structures included the cranial neural tube, forelimb buds, branchial arches, and body axis, while optic and otic placodes were more resistant. Lyophyllin at a concentration higher than 500 microg/ml also induced forebrain blisters within the cranial mesenchyme. When the abnormal embryos were examined histologically, an increase of cell death was found to be associated with abnormal structures, indicating that cell death may be one of the underlying causes of teratogenicity of the mushroom RIP. This constitutes the first report on the teratogenicity of a mushroom RIP.

  19. Dinoflagellate phylogeny revisited: Using ribosomal proteins to resolve deep branching dinoflagellate clades

    PubMed Central

    Bachvaroff, Tsvetan R.; Gornik, Sebastian G.; Concepcion, Gregory T.; Waller, Ross F.; Mendez, Gregory S.; Lippmeier, J. Casey; Delwiche, Charles F.

    2014-01-01

    The alveolates are composed of three major lineages, the ciliates, dinoflagellates, and apicomplexans. Together these ‘protist’ taxa play key roles in primary production and ecology, as well as in illness of humans and other animals. The interface between the dinoflagellate and apicomplexan clades has been an area of recent discovery, blurring the distinction between these two clades. Moreover, phylogenetic analysis has yet to determine the position of basal dinoflagellate clades hence the deepest branches of the dinoflagellate tree currently remain unresolved. Large-scale mRNA sequencing was applied to 11 species of dinoflagellates, including strains of the syndinean genera Hematodinium and Amoebophrya, parasites of crustaceans and dinoflagellates, respectively, to optimize and update the dinoflagellate tree. From the transcriptome-scale data a total of 73 ribosomal protein-coding genes were selected for phylogeny. After individual gene orthology assessment, the genes were concatenated into a >15,000 amino acid alignment with 76 taxa from dinoflagellates, apicomplexans, ciliates, and the outgroup heterokonts. Overall the tree was well resolved and supported, when the data was subsampled with gblocks or constraint trees were tested with the approximately unbiased test. The deepest branches of the dinoflagellate tree can now be resolved with strong support, and provides a clearer view of the evolution of the distinctive traits of dinoflagellates. PMID:24135237

  20. Critical Diamond-Blackfan anemia due to ribosomal protein S19 missense mutation.

    PubMed

    Ozono, Shuichi; Mitsuo, Miho; Noguchi, Maiko; Nakagawa, Shin-Ichiro; Ueda, Koichiro; Inada, Hiroko; Ohga, Shouichi; Ito, Etsuro

    2016-09-01

    Diamond-Blackfan anemia (DBA) is a rare congenital disorder characterized by pure erythrocyte aplasia, and approximately 70% of patients carry mutations in the genes encoding ribosomal proteins (RP). Here, we report the case of a male infant with DBA who presented with anemic crisis (hemoglobin [Hb] concentration 1.5 g/dL) at 58 days after birth. On admission, the infant was pale and had tachypnea, but recovered with intensive care, including red blood cell transfusions, and prednisolone. Based on the clinical diagnosis of DBA, the father of the infant had cyclosporine-A-dependent anemia. On analysis of RP genes when the infant was 6 months old, both the infant and the father, but not the mother, were found to harbor a mutation of RPS19 (c.167G > C, p. R56P). Therefore, genetic background search and early neonatal health check-ups are recommended for families with a history of inherited bone marrow failure syndromes.

  1. Combined heat shock protein 90 and ribosomal RNA sequence phylogeny supports multiple replacements of dinoflagellate plastids.

    PubMed

    Shalchian-Tabrizi, Kamran; Minge, Marianne A; Cavalier-Smith, Tom; Nedreklepp, Joachim M; Klaveness, Dag; Jakobsen, Kjetill S

    2006-01-01

    Dinoflagellates harbour diverse plastids obtained from several algal groups, including haptophytes, diatoms, cryptophytes, and prasinophytes. Their major plastid type with the accessory pigment peridinin is found in the vast majority of photosynthetic species. Some species of dinoflagellates have other aberrantly pigmented plastids. We sequenced the nuclear small subunit (SSU) ribosomal RNA (rRNA) gene of the "green" dinoflagellate Gymnodinium chlorophorum and show that it is sister to Lepidodinium viride, indicating that their common ancestor obtained the prasinophyte (or other green alga) plastid in one event. As the placement of dinoflagellate species that acquired green algal or haptophyte plastids is unclear from small and large subunit (LSU) rRNA trees, we tested the usefulness of the heat shock protein (Hsp) 90 gene for dinoflagellate phylogeny by sequencing it from four species with aberrant plastids (G. chlorophorum, Karlodinium micrum, Karenia brevis, and Karenia mikimotoi) plus Alexandrium tamarense, and constructing phylogenetic trees for Hsp90 and rRNAs, separately and together. Analyses of the Hsp90 and concatenated data suggest an ancestral origin of the peridinin-containing plastid, and two independent replacements of the peridinin plastid soon after the early radiation of the dinoflagellates. Thus, the Hsp90 gene seems to be a promising phylogenetic marker for dinoflagellate phylogeny.

  2. Structural basis for targeting the ribosomal protein S1 of Mycobacterium tuberculosis by pyrazinamide.

    PubMed

    Yang, Juanjuan; Liu, Yindi; Bi, Jing; Cai, Qixu; Liao, Xinli; Li, Wenqian; Guo, Chenyun; Zhang, Qian; Lin, Tianwei; Zhao, Yufen; Wang, Honghai; Liu, Jun; Zhang, Xuelian; Lin, Donghai

    2015-03-01

    Pyrazinamide (PZA) is a first-line drug for tuberculosis (TB) treatment and is responsible for shortening the duration of TB therapy. The mode of action of PZA remains elusive. RpsA, the ribosomal protein S1 of Mycobacterium tuberculosis (Mtb), was recently identified as a target of PZA based on its binding activity to pyrazinoic acid (POA), the active form of PZA. POA binding to RpsA led to the inhibition of trans-translation. However, the nature of the RpsA-POA interaction remains unknown. Key questions include why POA exhibits an exquisite specificity to RpsA of Mtb and how RpsA mutations confer PZA resistance. Here, we report the crystal structures of the C-terminal domain of RpsA of Mtb and its complex with POA, as well as the corresponding domains of two RpsA variants that are associated with PZA resistance. Structural analysis reveals that POA binds to RpsA through hydrogen bonds and hydrophobic interactions, mediated mainly by residues (Lys303, Phe307, Phe310 and Arg357) that are essential for tmRNA binding. Conformational changes induced by mutation or sequence variation at the C-terminus of RpsA abolish the POA binding activity. Our findings provide insights into the mode of action of PZA and molecular basis of PZA resistance associated with RpsA mutations.

  3. Ribosome Protein L4 is essential for Epstein–Barr Virus Nuclear Antigen 1 function

    PubMed Central

    Shen, Chih-Lung; Liu, Cheng-Der; You, Ren-In; Ching, Yung-Hao; Liang, Jun; Ke, Liangru; Chen, Ya-Lin; Chen, Hong-Chi; Hsu, Hao-Jen; Liou, Je-Wen; Kieff, Elliott; Peng, Chih-Wen

    2016-01-01

    Epstein–Barr Virus (EBV) Nuclear Antigen 1 (EBNA1)-mediated origin of plasmid replication (oriP) DNA episome maintenance is essential for EBV-mediated tumorigenesis. We have now found that EBNA1 binds to Ribosome Protein L4 (RPL4). RPL4 shRNA knockdown decreased EBNA1 activation of an oriP luciferase reporter, EBNA1 DNA binding in lymphoblastoid cell lines, and EBV genome number per lymphoblastoid cell line. EBV infection increased RPL4 expression and redistributed RPL4 to cell nuclei. RPL4 and Nucleolin (NCL) were a scaffold for an EBNA1-induced oriP complex. The RPL4 N terminus cooperated with NCL-K429 to support EBNA1 and oriP-mediated episome binding and maintenance, whereas the NCL C-terminal K380 and K393 induced oriP DNA H3K4me2 modification and promoted EBNA1 activation of oriP-dependent transcription. These observations provide new insights into the mechanisms by which EBV uses NCL and RPL4 to establish persistent B-lymphoblastoid cell infection. PMID:26858444

  4. Control of hematopoietic stem cell emergence by antagonistic functions of ribosomal protein paralogs.

    PubMed

    Zhang, Yong; Duc, Anne-Cécile E; Rao, Shuyun; Sun, Xiao-Li; Bilbee, Alison N; Rhodes, Michele; Li, Qin; Kappes, Dietmar J; Rhodes, Jennifer; Wiest, David L

    2013-02-25

    It remains controversial whether the highly homologous ribosomal protein (RP) paralogs found in lower eukaryotes have distinct functions and this has not been explored in vertebrates. Here we demonstrate that despite ubiquitous expression, the RP paralogs, Rpl22 and Rpl22-like1 (Rpl22l1) play essential, distinct, and antagonistic roles in hematopoietic development. Knockdown of Rpl22 in zebrafish embryos selectively blocks the development of T lineage progenitors after they have seeded the thymus. In contrast, knockdown of the Rpl22 paralog, Rpl22l1, impairs the emergence of hematopoietic stem cells (HSC) in the aorta-gonad-mesonephros by abrogating Smad1 expression and the consequent induction of essential transcriptional regulator, Runx1. Indeed, despite the ability of both paralogs to bind smad1 RNA, Rpl22 and Rpl22l1 have opposing effects on Smad1 expression. Accordingly, circumstances that tip the balance of these paralogs in favor of Rpl22 (e.g., Rpl22l1 knockdown or Rpl22 overexpression) result in repression of Smad1 and blockade of HSC emergence.

  5. Control of hematopoietic stem cell emergence by antagonistic functions of ribosomal protein paralogs

    PubMed Central

    Zhang, Yong; Duc, Anne-Cécile E.; Rao, Shuyun; Sun, Xiao-Li; Bilbee, Alison N.; Rhodes, Michele; Li, Qin; Kappes, Dietmar J.; Rhodes, Jennifer; Wiest, David L.

    2013-01-01

    Summary It remains controversial whether the highly-homologous ribosomal protein (RP) paralogs found in lower eukaryotes have distinct functions and this has not been explored in vertebrates. Here we demonstrate that despite ubiquitous expression, the RP paralogs, Rpl22 and Rpl22-like1 (Rpl22l1) play essential, distinct, and antagonistic roles in hematopoietic development. Knockdown of rpl22 in zebrafish embryos selectively blocks the development of T lineage progenitors after they have seeded the thymus. In contrast, knockdown of the rpl22 paralog, rpl22l1, impairs the emergence of hematopoietic stem cells (HSC) in the aorta-gonad-mesonephros by abrogating Smad1 expression and the consequent induction of essential transcriptional regulator, Runx1. Indeed, despite the ability of both paralogs to bind Smad1 RNA, Rpl22 and Rpl22l1 have opposing effects on Smad1 expression. Accordingly, circumstances that tip the balance of these paralogs in favor of Rpl22 (e.g., Rpl22l1 knockdown or Rpl22 overexpression) result in repression of Smad1 and blockade of HSC emergence. PMID:23449473

  6. Thiazolyl Peptide Antibiotic Biosynthesis: A Cascade of Post-translational Modifications on Ribosomal Nascent Proteins*

    PubMed Central

    Walsh, Christopher T.; Acker, Michael G.; Bowers, Albert A.

    2010-01-01

    Antibiotics of the thiocillin, GE2270A, and thiostrepton class, which block steps in bacterial protein synthesis, contain a trithiazolyl (tetrahydro)pyridine core that provides the architectural constraints for high affinity binding to either the 50 S ribosomal subunit or elongation factor Tu. These mature antibiotic scaffolds arise from a cascade of post-translational modifications on 50–60-residue prepeptide precursors that trim away the N-terminal leader sequences (∼40 residues) while the C-terminal 14–18 residues are converted into the mature scaffold. In the producing microbes, the genes encoding the prepeptide open reading frames are flanked in biosynthetic clusters by genes encoding post-translational modification enzymes that carry out lantibiotic-type dehydrations of Ser and Thr residues to dehydroamino acid side chains, cyclodehydration and oxidation of cysteines to thiazoles, and condensation of two dehydroalanine residues en route to the (tetrahydro)pyridine core. The trithiazolyl pyridine framework thus arises from post-translational modification of the peptide backbone of three Cys and two Ser residues of the prepeptide. PMID:20522549

  7. New Evidence for Differential Roles of L10 Ribosomal Proteins from Arabidopsis1[C][W][OPEN

    PubMed Central

    Falcone Ferreyra, María Lorena; Casadevall, Romina; Luciani, Marianela Dana; Pezza, Alejandro; Casati, Paula

    2013-01-01

    The RIBOSOMAL PROTEIN L10 (RPL10) is an integral component of the eukaryotic ribosome large subunit. Besides being a constituent of ribosomes and participating in protein translation, additional extraribosomal functions in the nucleus have been described for RPL10 in different organisms. Previously, we demonstrated that Arabidopsis (Arabidopsis thaliana) RPL10 genes are involved in development and translation under ultraviolet B (UV-B) stress. In this work, transgenic plants expressing ProRPL10:β-glucuronidase fusions show that, while AtRPL10A and AtRPL10B are expressed both in the female and male reproductive organs, AtRPL10C expression is restricted to pollen grains. Moreover, the characterization of double rpl10 mutants indicates that the three AtRPL10s differentially contribute to the total RPL10 activity in the male gametophyte. All three AtRPL10 proteins mainly accumulate in the cytosol but also in the nucleus, suggesting extraribosomal functions. After UV-B treatment, only AtRPL10B localization increases in the nuclei. We also here demonstrate that the three AtRPL10 genes can complement a yeast RPL10 mutant. Finally, the involvement of RPL10B and RPL10C in UV-B responses was analyzed by two-dimensional gels followed by mass spectrometry. Overall, our data provide new evidence about the nonredundant roles of RPL10 proteins in Arabidopsis. PMID:23886624

  8. Interaction of the cotranslational Hsp70 Ssb with ribosomal proteins and rRNA depends on its lid domain

    PubMed Central

    Gumiero, Andrea; Conz, Charlotte; Gesé, Genís Valentín; Zhang, Ying; Weyer, Felix Alexander; Lapouge, Karine; Kappes, Julia; von Plehwe, Ulrike; Schermann, Géza; Fitzke, Edith; Wölfle, Tina; Fischer, Tamás; Rospert, Sabine; Sinning, Irmgard

    2016-01-01

    Cotranslational chaperones assist in de novo folding of nascent polypeptides in all organisms. In yeast, the heterodimeric ribosome-associated complex (RAC) forms a unique chaperone triad with the Hsp70 homologue Ssb. We report the X-ray structure of full length Ssb in the ATP-bound open conformation at 2.6 Å resolution and identify a positively charged region in the α-helical lid domain (SBDα), which is present in all members of the Ssb-subfamily of Hsp70s. Mutational analysis demonstrates that this region is strictly required for ribosome binding. Crosslinking shows that Ssb binds close to the tunnel exit via contacts with both, ribosomal proteins and rRNA, and that specific contacts can be correlated with switching between the open (ATP-bound) and closed (ADP-bound) conformation. Taken together, our data reveal how Ssb dynamics on the ribosome allows for the efficient interaction with nascent chains upon RAC-mediated activation of ATP hydrolysis. PMID:27882919

  9. Interaction of the cotranslational Hsp70 Ssb with ribosomal proteins and rRNA depends on its lid domain.

    PubMed

    Gumiero, Andrea; Conz, Charlotte; Gesé, Genís Valentín; Zhang, Ying; Weyer, Felix Alexander; Lapouge, Karine; Kappes, Julia; von Plehwe, Ulrike; Schermann, Géza; Fitzke, Edith; Wölfle, Tina; Fischer, Tamás; Rospert, Sabine; Sinning, Irmgard

    2016-11-24

    Cotranslational chaperones assist in de novo folding of nascent polypeptides in all organisms. In yeast, the heterodimeric ribosome-associated complex (RAC) forms a unique chaperone triad with the Hsp70 homologue Ssb. We report the X-ray structure of full length Ssb in the ATP-bound open conformation at 2.6 Å resolution and identify a positively charged region in the α-helical lid domain (SBDα), which is present in all members of the Ssb-subfamily of Hsp70s. Mutational analysis demonstrates that this region is strictly required for ribosome binding. Crosslinking shows that Ssb binds close to the tunnel exit via contacts with both, ribosomal proteins and rRNA, and that specific contacts can be correlated with switching between the open (ATP-bound) and closed (ADP-bound) conformation. Taken together, our data reveal how Ssb dynamics on the ribosome allows for the efficient interaction with nascent chains upon RAC-mediated activation of ATP hydrolysis.

  10. Ribosomal protein S6 is highly expressed in non-Hodgkin lymphoma and associates with mRNA containing a 5' terminal oligopyrimidine tract.

    PubMed

    Hagner, P R; Mazan-Mamczarz, K; Dai, B; Balzer, E M; Corl, S; Martin, S S; Zhao, X F; Gartenhaus, R B

    2011-03-31

    The molecular mechanism(s) linking tumorigenesis and morphological alterations in the nucleolus are presently coming into focus. The nucleolus is the cellular organelle in which the formation of ribosomal subunits occurs. Ribosomal biogenesis occurs through the transcription of ribosomal RNA (rRNA), rRNA processing and production of ribosomal proteins. An error in any of these processes may lead to deregulated cellular translation, evident in multiple cancers and 'ribosomopathies'. Deregulated protein synthesis may be achieved through the overexpression of ribosomal proteins as seen in primary leukemic blasts with elevated levels of ribosomal proteins S11 and S14. In this study, we demonstrate that ribosomal protein S6 (RPS6) is highly expressed in primary diffuse large B-cell lymphoma (DLBCL) samples. Genetic modulation of RPS6 protein levels with specifically targeted short hairpin RNA (shRNA) lentiviruses led to a decrease in the actively proliferating population of cells compared with control shRNA. Low-dose rapamycin treatments have been shown to affect the translation of 5' terminal oligopyrimidine (5' TOP) tract mRNA, which encodes the translational machinery, implicating RPS6 in 5' TOP translation. Recently, it was shown that disruption of 40S ribosomal biogenesis through specific small inhibitory RNA knockdown of RPS6 defined RPS6 as a critical regulator of 5' TOP translation. For the first time, we show that RPS6 associates with multiple mRNAs containing a 5' TOP tract. These findings expand our understanding of the mechanism(s) involved in ribosomal biogenesis and deregulated protein synthesis in DLBCL.

  11. Cell-free compartmentalized protein synthesis inside double emulsion templated liposomes with in vitro synthesized and assembled ribosomes.

    PubMed

    Caschera, Filippo; Lee, Jin Woo; Ho, Kenneth K Y; Liu, Allen P; Jewett, Michael C

    2016-04-07

    A cell-free expression platform for making bacterial ribosomes encapsulated within giant liposomes was capable of synthesizing sfGFP. The liposomes were prepared using a double emulsion template, and compartmentalized in vitro protein synthesis was analysed using spinning disk confocal microscopy. Two different liposome phospholipid formulations were investigated to characterize their effects on the compartmentalized reaction kinetics. This study was performed as a necessary step towards the synthesis of minimal cells.

  12. Diversity and recombination of dispersed ribosomal DNA and protein coding genes in microsporidia.

    PubMed

    Ironside, Joseph Edward

    2013-01-01

    Microsporidian strains are usually classified on the basis of their ribosomal DNA (rDNA) sequences. Although rDNA occurs as multiple copies, in most non-microsporidian species copies within a genome occur as tandem arrays and are homogenised by concerted evolution. In contrast, microsporidian rDNA units are dispersed throughout the genome in some species, and on this basis are predicted to undergo reduced concerted evolution. Furthermore many microsporidian species appear to be asexual and should therefore exhibit reduced genetic diversity due to a lack of recombination. Here, DNA sequences are compared between microsporidia with different life cycles in order to determine the effects of concerted evolution and sexual reproduction upon the diversity of rDNA and protein coding genes. Comparisons of cloned rDNA sequences between microsporidia of the genus Nosema with different life cycles provide evidence of intragenomic variability coupled with strong purifying selection. This suggests a birth and death process of evolution. However, some concerted evolution is suggested by clustering of rDNA sequences within species. Variability of protein-coding sequences indicates that considerable intergenomic variation also occurs between microsporidian cells within a single host. Patterns of variation in microsporidian DNA sequences indicate that additional diversity is generated by intragenomic and/or intergenomic recombination between sequence variants. The discovery of intragenomic variability coupled with strong purifying selection in microsporidian rRNA sequences supports the hypothesis that concerted evolution is reduced when copies of a gene are dispersed rather than repeated tandemly. The presence of intragenomic variability also renders the use of rDNA sequences for barcoding microsporidia questionable. Evidence of recombination in the single-copy genes of putatively asexual microsporidia suggests that these species may undergo cryptic sexual reproduction, a

  13. A cytotoxic type-2 ribosome inactivating protein (from leafless mistletoe) lacking sugar binding activity.

    PubMed

    Das, Mrinal Kumar; Sharma, Radhey Shyam; Mishra, Vandana

    2011-12-01

    Articulatin-D, a 66 kDa ribosome inactivating protein (RIP) comprised of 29 kDa A-chain linked to 35 kDa B-chain, is purified from leafless mistletoe (Viscum articulatum) parasitic on Dalbergia sp. from Western Ghats (India). N-terminal sequence and LC-MS/MS analyses of A- and B-chain confirmed that articulatin-D is a type-2 RIP having high homology with other mistletoe lectins. Translation inhibition and diagnostic N-glycosidase activity of articulatin-D illustrate the presence of catalytically active A-chain. Its inability to: (i) bind to acid treated Sepharose CL-6B column, (ii) agglutinate trypsin-treated and untreated RBCs of human (A, B, O, AB), mice, rat, rabbit, buffalo, porcine, pigeon, cock, fish, sheep and goat even with 10mg/ml of purified articulatin-D, (iii) show change in circular dichroism spectra after addition of sugar to the native protein, (iv) bind to different sugars (galactose, lactose, gal-NAc, rhamnose, arabinose, fucose and mannose) immobilized on Sepharose 4B matrix, and (v) show change in enthalpy during titration with galactose confirm that the B-chain of articulatin-D lacks sugar binding activity. Despite this, articulatin-D is highly toxic as characterized with low IC(50) against different cancer cell lines (Jurkat: 0.31 ± 0.02 nM, MOLT-4: 0.51 ± 0.03 nM, U-937: 0.64 ± 0.07 nM, HL-60: 0.79 ± 0.11 nM, Raji: 1.45 ± 0.09 nM). Toxicity of RIPs has been ascribed to the absence/presence of B-chain with sugar binding activity. Identification of articulatin-D, the first cytotoxic RIP with B-chain lacking sugar binding activity opens new vistas in understanding cytotoxic action of RIPs.

  14. Diversity and Recombination of Dispersed Ribosomal DNA and Protein Coding Genes in Microsporidia

    PubMed Central

    Ironside, Joseph Edward

    2013-01-01

    Microsporidian strains are usually classified on the basis of their ribosomal DNA (rDNA) sequences. Although rDNA occurs as multiple copies, in most non-microsporidian species copies within a genome occur as tandem arrays and are homogenised by concerted evolution. In contrast, microsporidian rDNA units are dispersed throughout the genome in some species, and on this basis are predicted to undergo reduced concerted evolution. Furthermore many microsporidian species appear to be asexual and should therefore exhibit reduced genetic diversity due to a lack of recombination. Here, DNA sequences are compared between microsporidia with different life cycles in order to determine the effects of concerted evolution and sexual reproduction upon the diversity of rDNA and protein coding genes. Comparisons of cloned rDNA sequences between microsporidia of the genus Nosema with different life cycles provide evidence of intragenomic variability coupled with strong purifying selection. This suggests a birth and death process of evolution. However, some concerted evolution is suggested by clustering of rDNA sequences within species. Variability of protein-coding sequences indicates that considerable intergenomic variation also occurs between microsporidian cells within a single host. Patterns of variation in microsporidian DNA sequences indicate that additional diversity is generated by intragenomic and/or intergenomic recombination between sequence variants. The discovery of intragenomic variability coupled with strong purifying selection in microsporidian rRNA sequences supports the hypothesis that concerted evolution is reduced when copies of a gene are dispersed rather than repeated tandemly. The presence of intragenomic variability also renders the use of rDNA sequences for barcoding microsporidia questionable. Evidence of recombination in the single-copy genes of putatively asexual microsporidia suggests that these species may undergo cryptic sexual reproduction, a

  15. Transcriptional control of two ribosome-inactivating protein genes expressed in spinach (Spinacia oleracea) embryos.

    PubMed

    Kawade, Kensuke; Masuda, Kiyoshi

    2009-05-01

    SoRIP1 and SoRIP2 are ribosome-inactivating protein (RIP: EC 3.2.2.22) genes identified in spinach (Spinacia oleracea). They are differentially expressed in a development-dependent manner during spinach somatic embryogenesis. Here, we isolated genomic clones of SoRIP1 and SoRIP2. These two RIP genes have different genomic organization. Phylogenetic analysis of predicted amino acid sequences of RIPs in Caryophyllales plants revealed that they are divided into two major subfamilies, corresponding to SoRIP1 and SoRIP2. To gain further insight into the transcriptional control of SoRIP1 and SoRIP2, we obtained their 5'-flanking sequences by inverse PCR. Comparison of two 5'-flanking sequences revealed the characteristic cis elements in each region that confer differential transcriptional control. In the 5'-flanking region of SoRIP1, we found several motifs with functions related to embryonic development. The 5'-flanking region of SoRIP2 contains some defense-responsive motifs. Expression of SoRIP1 was detected in various tissues. In particular, SoRIP1 was highly expressed in the early immature fruits, and immunohistochemistry showed that SoRIP1 accumulated in the peripheral region of the immature embryo, with weaker expression in internal cells. During fruit development, the expression of SoRIP2 was low. However, the accumulation of SoRIP2 was conspicuous in the epidermis of the immature embryo. The expression of SoRIP2, but not SoRIP1, in leaves was induced by salicylic acid treatment. This differential transcriptional regulation of SoRIP1 and SoRIP2 suggests that the corresponding proteins may have different functions, one being related to embryonic development and the other to embryo defense.

  16. Differential expression of ribosome-inactivating protein genes during somatic embryogenesis in spinach (Spinacia oleracea).

    PubMed

    Kawade, Kensuke; Ishizaki, Takuma; Masuda, Kiyoshi

    2008-10-01

    Root segments from spinach (Spinacia oleracea L. cv. Jiromaru) seedlings form embryogenic callus (EC) that responded to exogenous GA(3) by accumulating a 31-kDa glycoprotein [BP31 or S. oleracea ribosome-inactivating protein (EC 3.2.2.22) (SoRIP1)] in association with the expression of embryogenic potential. Microsequencing of this protein revealed significant similarity with type 1 RIPs. We identified cDNAs for SoRIP1 and S. oleracea RIP2 (SoRIP2), a novel RIP having a consensus shiga/ricin toxic domain and performed a comparative analysis of the expression of SoRIPs during somatic embryogenesis. Western blotting and quantitative polymerase chain reaction analyses revealed that the expression of SoRIP1 in calli increased remarkably in association with the acquisition of embryogenic potential, although the expression in somatic embryos decreased moderately with their development. However, the expression of SoRIP2 in calli remained low and constant but increased markedly with the development of somatic embryos. Treatment of callus with GA(3) and/or ABA for 24 h, or with ABA for a longer period, failed to stimulate the expression of either gene. Immunohistochemistry showed that SoRIP1 preferentially accumulated in the proembryos and peripheral meristem of somatic embryos early in development. Appreciable expression of SoRIP2 was not detected in the callus, but intense expression was found in the epidermis of somatic embryos. These results suggest that the expression of spinach RIP genes is differentially regulated in a development-dependent fashion during somatic embryogenesis in spinach.

  17. Comparisons of Ribosomal Protein Gene Promoters Indicate Superiority of Heterologous Regulatory Sequences for Expressing Transgenes in Phytophthora infestans.

    PubMed

    Poidevin, Laetitia; Andreeva, Kalina; Khachatoorian, Careen; Judelson, Howard S

    2015-01-01

    Molecular genetics approaches in Phytophthora research can be hampered by the limited number of known constitutive promoters for expressing transgenes and the instability of transgene activity. We have therefore characterized genes encoding the cytoplasmic ribosomal proteins of Phytophthora and studied their suitability for expressing transgenes in P. infestans. Phytophthora spp. encode a standard complement of 79 cytoplasmic ribosomal proteins. Several genes are duplicated, and two appear to be pseudogenes. Half of the genes are expressed at similar levels during all stages of asexual development, and we discovered that the majority share a novel promoter motif named the PhRiboBox. This sequence is enriched in genes associated with transcription, translation, and DNA replication, including tRNA and rRNA biogenesis. Promoters from the three P. infestans genes encoding ribosomal proteins S9, L10, and L23 and their orthologs from P. capsici were tested for their ability to drive transgenes in stable transformants of P. infestans. Five of the six promoters yielded strong expression of a GUS reporter, but the stability of expression was higher using the P. capsici promoters. With the RPS9 and RPL10 promoters of P. infestans, about half of transformants stopped making GUS over two years of culture, while their P. capsici orthologs conferred stable expression. Since cross-talk between native and transgene loci may trigger gene silencing, we encourage the use of heterologous promoters in transformation studies.

  18. Ribosomal protein L18aB is required for both male gametophyte function and embryo development in Arabidopsis

    PubMed Central

    Yan, Hailong; Chen, Dan; Wang, Yifan; Sun, Yang; Zhao, Jing; Sun, Mengxiang; Peng, Xiongbo

    2016-01-01

    Ribosomal proteins are involved in numerous essential cell activities in plants. However, the regulatory role in specific plant developmental processes has not yet been fully elucidated. Here we identified the new ribosomal protein L18aB, which is specifically involved in sexual reproduction and plays a critical role in male gametophyte development and embryo pattern formation. In rpl18aB mutant plants, the mature pollen grains can germinate normally, but their competitiveness for growing in the style is significantly reduced. More interestingly, RPL18aB is required in early embryogenesis. rpl18aB embryos displayed irregular cell division orientations in the early pro-embryo and arrested at the globular stage with possible, secondary pattern formation defects. Further investigations revealed that the polar transportation of auxin is disturbed in the rpl18aB mutant embryos, which may explain the observed failure in embryo pattern formation. The cell type-specific complementation of RPL18aB in rpl18aB was not able to recover the phenotype, indicating that RPL18aB may play an essential role in early cell fate determination. This work unravels a novel role in embryo development for a ribosomal protein, and provides insight into regulatory mechanism of early embryogenesis. PMID:27502163

  19. Nuclear Protein Sam68 Interacts with the Enterovirus 71 Internal Ribosome Entry Site and Positively Regulates Viral Protein Translation

    PubMed Central

    Zhang, Hua; Song, Lei; Cong, Haolong

    2015-01-01

    ABSTRACT Enterovirus 71 (EV71) recruits various cellular factors to assist in the replication and translation of its genome. Identification of the host factors involved in the EV71 life cycle not only will enable a better understanding of the infection mechanism but also has the potential to be of use in the development of antiviral therapeutics. In this study, we demonstrated that the cellular factor 68-kDa Src-associated protein in mitosis (Sam68) acts as an internal ribosome entry site (IRES) trans-acting factor (ITAF) that binds specifically to the EV71 5′ untranslated region (5′UTR). Interaction sites in both the viral IRES (stem-loops IV and V) and the heterogeneous nuclear ribonucleoprotein K homology (KH) domain of Sam68 protein were further mapped using an electrophoretic mobility shift assay (EMSA) and biotin RNA pulldown assay. More importantly, dual-luciferase (firefly) reporter analysis suggested that overexpression of Sam68 positively regulated IRES-dependent translation of virus proteins. In contrast, both IRES activity and viral protein translation significantly decreased in Sam68 knockdown cells compared with the negative-control cells treated with short hairpin RNA (shRNA). However, downregulation of Sam68 did not have a significant inhibitory effect on the accumulation of the EV71 genome. Moreover, Sam68 was redistributed from the nucleus to the cytoplasm and interacts with cellular factors, such as poly(rC)-binding protein 2 (PCBP2) and poly(A)-binding protein (PABP), during EV71 infection. The cytoplasmic relocalization of Sam68 in EV71-infected cells may be involved in the enhancement of EV71 IRES-mediated translation. Since Sam68 is known to be a RNA-binding protein, these results provide direct evidence that Sam68 is a novel ITAF that interacts with EV71 IRES and positively regulates viral protein translation. IMPORTANCE The nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection

  20. GUN1 Controls Accumulation of the Plastid Ribosomal Protein S1 at the Protein Level and Interacts with Proteins Involved in Plastid Protein Homeostasis1

    PubMed Central

    Pesaresi, Paolo; Rossi, Fabio; Guljamow, Arthur; Sommer, Frederik; Mühlhaus, Timo; Schroda, Michael; Masiero, Simona; Rothbart, Maxi; Hedtke, Boris

    2016-01-01

    Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the plant cell. Such intracellular responses are associated with signals that originate in chloroplasts and convey information on their physiological status to the nucleus, which leads to large-scale changes in gene expression (retrograde signaling). A screen designed to identify components of retrograde signaling resulted in the discovery of the so-called genomes uncoupled (gun) mutants. Genetic evidence suggests that the chloroplast protein GUN1 integrates signals derived from perturbations in plastid redox state, plastid gene expression, and tetrapyrrole biosynthesis (TPB) in Arabidopsis (Arabidopsis thaliana) seedlings, exerting biogenic control of chloroplast functions. However, the molecular mechanism by which GUN1 integrates retrograde signaling in the chloroplast is unclear. Here we show that GUN1 also operates in adult plants, contributing to operational control of chloroplasts. The gun1 mutation genetically interacts with mutations of genes for the chloroplast ribosomal proteins S1 (PRPS1) and L11. Analysis of gun1 prps1 lines indicates that GUN1 controls PRPS1 accumulation at the protein level. The GUN1 protein physically interacts with proteins involved in chloroplast protein homeostasis based on coimmunoprecipitation experiments. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments suggest that GUN1 might transiently interact with several TPB enzymes, including Mg-chelatase subunit D (CHLD) and two other TPB enzymes known to activate retrograde signaling. Moreover, the association of PRPS1 and CHLD with protein complexes is modulated by GUN1. These findings allow us to speculate that retrograde signaling might involve GUN1-dependent formation of protein complexes. PMID:26823545

  1. GUN1 Controls Accumulation of the Plastid Ribosomal Protein S1 at the Protein Level and Interacts with Proteins Involved in Plastid Protein Homeostasis.

    PubMed

    Tadini, Luca; Pesaresi, Paolo; Kleine, Tatjana; Rossi, Fabio; Guljamow, Arthur; Sommer, Frederik; Mühlhaus, Timo; Schroda, Michael; Masiero, Simona; Pribil, Mathias; Rothbart, Maxi; Hedtke, Boris; Grimm, Bernhard; Leister, Dario

    2016-03-01

    Developmental or metabolic changes in chloroplasts can have profound effects on the rest of the plant cell. Such intracellular responses are associated with signals that originate in chloroplasts and convey information on their physiological status to the nucleus, which leads to large-scale changes in gene expression (retrograde signaling). A screen designed to identify components of retrograde signaling resulted in the discovery of the so-called genomes uncoupled (gun) mutants. Genetic evidence suggests that the chloroplast protein GUN1 integrates signals derived from perturbations in plastid redox state, plastid gene expression, and tetrapyrrole biosynthesis (TPB) in Arabidopsis (Arabidopsis thaliana) seedlings, exerting biogenic control of chloroplast functions. However, the molecular mechanism by which GUN1 integrates retrograde signaling in the chloroplast is unclear. Here we show that GUN1 also operates in adult plants, contributing to operational control of chloroplasts. The gun1 mutation genetically interacts with mutations of genes for the chloroplast ribosomal proteins S1 (PRPS1) and L11. Analysis of gun1 prps1 lines indicates that GUN1 controls PRPS1 accumulation at the protein level. The GUN1 protein physically interacts with proteins involved in chloroplast protein homeostasis based on coimmunoprecipitation experiments. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments suggest that GUN1 might transiently interact with several TPB enzymes, including Mg-chelatase subunit D (CHLD) and two other TPB enzymes known to activate retrograde signaling. Moreover, the association of PRPS1 and CHLD with protein complexes is modulated by GUN1. These findings allow us to speculate that retrograde signaling might involve GUN1-dependent formation of protein complexes.

  2. The Ribosomal Biogenesis Protein Utp21 Interacts with Hsp90 and Has Differing Requirements for Hsp90-Associated Proteins

    PubMed Central

    Tenge, Victoria R.; Knowles, Jared; Johnson, Jill L.

    2014-01-01

    The molecular chaperone Hsp90 buffers the effects of genetic variation by assisting the stabilization and folding of multiple clients critical for cell signaling and growth. We identified an interaction of Hsp90 and associated proteins with the essential nucleolar protein, Utp21, part of a large complex required for biogenesis of the small ribosomal subunit. The utp21-S602F mutation, which causes minor defects in otherwise wild-type yeast, exhibited severe or lethal growth defects when combined with mutations in Hsp90 or co-chaperones. WT Utp21 and Utp21-S602F exhibited similar interactions with Hsp90, and steady-state levels of WT Utp21 were reduced upon Hsp90 mutation or inhibition. Mutations in the human homolog of UTP21, WDR36, have been associated with adult-onset primary open-angle glaucoma, a leading cause of blindness worldwide. Three different mutant forms of Utp21 analogous to glaucoma-associated WDR36 mutations exhibit reduced levels in yeast cells expressing mutations in Hsp90 or associated chaperones, suggesting that Hsp90 and co-chaperones buffer the effects of those mutations. PMID:24647762

  3. Recombineering reveals a diverse collection of ribosomal proteins L4 and L22 that confer resistance to macrolide antibiotics

    PubMed Central

    Diner, Elie J.; Hayes, Christopher S.

    2009-01-01

    Summary Mutations in ribosomal proteins L4 and L22 confer resistance to erythromycin and other macrolide antibiotics in a variety of bacteria. L4 and L22 have elongated loops whose tips converge in the peptide exit tunnel near the macrolide binding site, and resistance mutations typically affect residues within these loops. Here, we use bacteriophage λ Red-mediated recombination, or “recombineering”, to uncover new L4 and L22 alleles that confer macrolide resistance in Escherichia coli. We randomized residues at the tips of the L4 and L22 loops using recombineered oligonucleotide libraries, and selected the mutagenized cells for erythromycin-resistant mutants. These experiments led to the identification of 341 different resistance mutations encoding 278 unique L4 and L22 proteins – the overwhelming majority of which are novel. Many resistance mutations were complex, involving multiple missense mutations, in-frame deletions, and insertions. Transfer of L4 and L22 mutations into wild-type cells by phage P1-mediated transduction demonstrated that each allele was sufficient to confer macrolide resistance. Although L4 and L22 mutants are typically resistant to most macrolides, selections carried out on different antibiotics revealed macrolide-specific resistance mutations. L22 Lys90Trp is one such allele, which confers resistance to erythromycin, but not tylosin or spiramycin. Purified L22 Lys90Trp ribosomes show reduced erythromycin binding, but have the same affinity for tylosin as wild-type ribosomes. Moreover, DMS methylation protection assays demonstrated that L22 Lys90Trp ribosomes bind tylosin more readily than erythromycin in vivo. This work underscores the exceptional functional plasticity of the L4 and L22 proteins, and highlights the utility of Red-mediated recombination in targeted genetic selections. PMID:19150357

  4. Inter-Protein Sequence Co-Evolution Predicts Known Physical Interactions in Bacterial Ribosomes and the Trp Operon.

    PubMed

    Feinauer, Christoph; Szurmant, Hendrik; Weigt, Martin; Pagnani, Andrea

    2016-01-01

    Interaction between proteins is a fundamental mechanism that underlies virtually all biological processes. Many important interactions are conserved across a large variety of species. The need to maintain interaction leads to a high degree of co-evolution between residues in the interface between partner proteins. The inference of protein-protein interaction networks from the rapidly growing sequence databases is one of the most formidable tasks in systems biology today. We propose here a novel approach based on the Direct-Coupling Analysis of the co-evolution between inter-protein residue pairs. We use ribosomal and trp operon proteins as test cases: For the small resp. large ribosomal subunit our approach predicts protein-interaction partners at a true-positive rate of 70% resp. 90% within the first 10 predictions, with areas of 0.69 resp. 0.81 under the ROC curves for all predictions. In the trp operon, it assigns the two largest interaction scores to the only two interactions experimentally known. On the level of residue interactions we show that for both the small and the large ribosomal subunit our approach predicts interacting residues in the system with a true positive rate of 60% and 85% in the first 20 predictions. We use artificial data to show that the performance of our approach depends crucially on the size of the joint multiple sequence alignments and analyze how many sequences would be necessary for a perfect prediction if the sequences were sampled from the same model that we use for prediction. Given the performance of our approach on the test data we speculate that it can be used to detect new interactions, especially in the light of the rapid growth of available sequence data.

  5. Inter-Protein Sequence Co-Evolution Predicts Known Physical Interactions in Bacterial Ribosomes and the Trp Operon

    PubMed Central

    Feinauer, Christoph; Szurmant, Hendrik; Weigt, Martin; Pagnani, Andrea

    2016-01-01

    Interaction between proteins is a fundamental mechanism that underlies virtually all biological processes. Many important interactions are conserved across a large variety of species. The need to maintain interaction leads to a high degree of co-evolution between residues in the interface between partner proteins. The inference of protein-protein interaction networks from the rapidly growing sequence databases is one of the most formidable tasks in systems biology today. We propose here a novel approach based on the Direct-Coupling Analysis of the co-evolution between inter-protein residue pairs. We use ribosomal and trp operon proteins as test cases: For the small resp. large ribosomal subunit our approach predicts protein-interaction partners at a true-positive rate of 70% resp. 90% within the first 10 predictions, with areas of 0.69 resp. 0.81 under the ROC curves for all predictions. In the trp operon, it assigns the two largest interaction scores to the only two interactions experimentally known. On the level of residue interactions we show that for both the small and the large ribosomal subunit our approach predicts interacting residues in the system with a true positive rate of 60% and 85% in the first 20 predictions. We use artificial data to show that the performance of our approach depends crucially on the size of the joint multiple sequence alignments and analyze how many sequences would be necessary for a perfect prediction if the sequences were sampled from the same model that we use for prediction. Given the performance of our approach on the test data we speculate that it can be used to detect new interactions, especially in the light of the rapid growth of available sequence data. PMID:26882169

  6. Topography and stoichiometry of acidic proteins in large ribosomal subunits from Artemia salina as determined by crosslinking

    SciTech Connect

    Uchiumi, T.; Wahba, A.J.; Traut, R.R.

    1987-08-01

    The 60S subunits isolated from Artemia salina ribosomes were treated with the crosslinking reagent 2-iminothiolane under mild conditions. Proteins were extracted and fractions containing crosslinked acidic proteins were obtained by stepwise elution from CM-cellulose. Each fraction was analyzed by diagonal (two-dimensional nonreducing-reducing) NaDodSO/sub 4//polyacrylamide gel electrophoresis. Crosslinked proteins below the diagonal were radioiodinated and identified by two-dimensional acidic urea-NaDodSO/sub 4/ gel electrophoresis. Each of the acidic proteins P1 and P2 was crosslinked individually to the same third protein, PO. The fractions containing acidic proteins were also analyzed by two-dimensional nonequilibrium isoelectric focusing-NaDodSO/sub 4//polyacrylamide gel electrophoresis. Two crosslinked complexes were observed that coincide in isoelectric positions with monomeric P1 and P2, respectively. Both P1 and P2 appear to form crosslinked homodimers. These results suggest the presence in the 60S subunit of (P1)/sub 2/ and (P2)/sub 2/ dimers, each of which is anchored to PO. Protein PO appears to play the same role as L10 in Escherichia coli ribosomes and may form a pentameric complex with the two dimers in the 60S subunits.

  7. NAM9 nuclear suppressor of mitochondrial ochre mutations in Saccharomyces cerevisiae codes for a protein homologous to S4 ribosomal proteins from chloroplasts, bacteria, and eucaryotes.

    PubMed Central

    Boguta, M; Dmochowska, A; Borsuk, P; Wrobel, K; Gargouri, A; Lazowska, J; Slonimski, P P; Szczesniak, B; Kruszewska, A

    1992-01-01

    We report the genetic characterization, molecular cloning, and sequencing of a novel nuclear suppressor, the NAM9 gene from Saccharomyces cerevisiae, which acts on mutations of mitochondrial DNA. The strain NAM9-1 was isolated as a respiration-competent revertant of a mitochondrial mit mutant which carries the V25 ochre mutation in the oxi1 gene. Genetic characterization of the NAM9-1 mutation has shown that it is a nuclear dominant omnipotent suppressor alleviating several mutations in all four mitochondrial genes tested and has suggested its informational, and probably ribosomal, character. The NAM9 gene was cloned by transformation of the recipient oxi1-V25 mutant to respiration competence by using a gene bank from the NAM9-1 rho o strain. Orthogonal-field alternation gel electrophoresis analysis and genetic mapping localized the NAM9 gene on the right arm of chromosome XIV. Sequence analysis of the NAM9 gene showed that it encodes a basic protein of 485 amino acids with a presequence that could target the protein to the mitochondrial matrix. The N-terminal sequence of 200 amino acids of the deduced NAM9 product strongly resembles the S4 ribosomal proteins from chloroplasts and bacteria. Significant although less extensive similarity was found with ribosomal cytoplasmic proteins from lower eucaryotes, including S. cerevisiae. Chromosomal inactivation of the NAM9+ gene is not lethal to the cell but leads to respiration deficiency and loss of mitochondrial DNA integrity. We conclude that the NAM9 gene product is a mitochondrial ribosomal counterpart of S4 ribosomal proteins found in other systems and that the suppressor acts through decreasing the fidelity of translation. Images PMID:1729612

  8. Crystallization and preliminary crystallographic studies of L30e, a ribosomal protein from Methanocaldococcus jannaschii (MJ1044)

    SciTech Connect

    Rangarajan, Sarani; Jeyakanthan, Jeyaraman; Mridula, Palappetty; Sakamoto, Keiko; Kitamura, Yoshiaki; Agari, Yoshihiro; Shinkai, Akeo; Ebihara, Akio; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Sekar, Kanagaraj E-mail: sekar@physics.iisc.ernet.in

    2008-02-01

    The ribosomal protein (L30e) from M. jannaschii was cloned from the gene MJ1044, expressed, purified and crystallized. The crystal belongs to the primitive tetragonal space group P4{sub 3} and diffracted to 1.9 Å resolution. In view of the biological significance of understanding the ribosomal machinery of both prokaryotes and eukaryotes, the L30e ribosomal protein from Methanocaldococcus jannaschii was cloned, overexpressed, purified and crystallized using the microbatch-under-oil method with the crystallization conditions 40% PEG 400, 0.1 M MES pH 6.0 and 5% PEG 3000 at 291 K. A diffraction-quality crystal (0.20 × 0.20 × 0.35 mm) was obtained that belonged to the primitive tetragonal space group P4{sub 3}, with unit-cell parameters a = 46.1, b = 46.1, c = 98.5 Å, and diffracted to a resolution of 1.9 Å. Preliminary calculations reveal that the asymmetric unit contains two monomers with a Matthews coefficient (V{sub M}) of 2.16 Å{sup 3} Da{sup −1}.

  9. Differential expression of a subset of ribosomal protein genes in cell lines derived from human nasopharyngeal epithelium.

    PubMed

    Sim, Edmund Ui Hang; Ang, Chow Hiang; Ng, Ching Ching; Lee, Choon Weng; Narayanan, Kumaran

    2010-02-01

    Extraribosomal functions of human ribosomal proteins (RPs) include the regulation of cellular growth and differentiation, and are inferred from studies that linked congenital disorders and cancer to the deregulated expression of RP genes. We have previously shown the upregulation and downregulation of RP genes in tumors of colorectal and nasopharyngeal carcinomas (NPCs), respectively. Herein, we show that a subset of RP genes for the large ribosomal subunit is differentially expressed among cell lines derived from the human nasopharyngeal epithelium. Three such genes (RPL27, RPL37a and RPL41) were found to be significantly downregulated in all cell lines derived from NPC tissues compared with a nonmalignant nasopharyngeal epithelial cell line. The expression of RPL37a and RPL41 genes in human nasopharyngeal tissues has not been reported previously. Our findings support earlier suspicions on the existence of NPC-associated RP genes, and indicate their importance in human nasopharyngeal organogenesis.

  10. Characterization of Zn(II)-responsive ribosomal proteins YkgM and L31 in E. coli.

    PubMed

    Hensley, M Patrick; Gunasekera, Thusitha S; Easton, J Allen; Sigdel, Tara K; Sugarbaker, Stacy A; Klingbeil, Lindsey; Breece, Robert M; Tierney, David L; Crowder, Michael W

    2012-06-01

    RT-PCR and DNA microarrays were used to probe for Zn(II)-responsive genes in E. coli cells that were made Zn(II) deficient. Microarray data revealed 114 genes were significantly up-regulated and 146 genes were significantly down-regulated in Zn(II) deficient conditions. The three most up-regulated genes were (1) znuA, which encodes for a periplasmic protein known to be involved with Zn(II) import, (2) yodA, which encodes for a periplasmic protein with unknown function, and (3) ykgM, which encodes for a ribosomal protein that is thought to be a paralog of ribosomal protein L31. YodA was over-expressed and purified as a maltose binding protein (MBP) fusion protein and shown to tightly bind 4 equivalents of Zn(II). Metal analyses showed that MBP-YkgM does not bind Zn(II). On the other hand, MBP-L31 tightly binds 1 equivalent of Zn(II). EXAFS studies on MBP-L31 suggest a ligand field of 1 histidine, 1 cysteine, and 2 additional N/O scatterers. Site-directed mutagenesis studies suggest that Cys16 coordinates Zn(II) in MBP-L31 and that the other three cysteines do not bind metal. These results are discussed in light of Zn(II) starvation model that has been postulated for B. subtilis.

  11. Mechanisms of the initiation of protein synthesis: in reading frame binding of ribosomes to mRNA.

    PubMed

    Nakamoto, Tokumasa

    2011-02-01

    The various mechanisms proposed to describe the initiation of protein synthesis are reviewed with a focus on their initiation signals. A characteristic feature of the various mechanisms is that each one of them postulates a distinct initiation signal. The signals of the Shine-Dalgarno (SD), the scanning and the internal ribosome entry site (IRES) mechanisms are all located exclusively in the 5' leader sequence, whereas, the signal of the cumulative specificity (CS) mechanism includes the entire initiation site (IS). Computer analysis of known E. coli IS sequences showed signal characteristics in the entire model IS consisting of 47 bases, in segments of the 5' leader and of the protein-coding regions. The proposal that eukaryotic translation actually occurs in two steps is scrutinized. In a first step, initiation factors (eIF4F) interact with the cap of the mRNA, thereby enhancing the accessibility of the IS. In the second step, initiation is by the conserved prokaryotic mechanism in which the ribosomes bind directly to the mRNA without ribosomal scanning. This binding occurs by the proposed process of in reading frame binding of ribosomes to mRNA, which is consistent with the CS mechanism. The basic CS mechanism is able to account for the initiation of translation of leaderless mRNAs, as well as for that of canonical mRNAs. The SD, the scanning and the IRES mechanisms, on the other hand, are inconsistent with the initiation of translation of leaderless mRNAs. Based on these and other observations, it is deemed that the CS mechanism is the universal initiation mechanism.

  12. Modular organization of residue-level contacts shape the selection pressure on individual amino acid sites of ribosomal proteins.

    PubMed

    Mallik, Saurav; Kundu, Sudip

    2017-02-22

    Understanding the molecular evolution of macromolecular complexes in the light of their structure, assembly and stability is of central importance. Here we address how the modular organization of native molecular contacts shapes the selection pressure on individual residue sites of ribosomal complexes. The bacterial ribosomal complex is represented as a residue contact network where nodes represent amino acid/nucleotide residues and edges represent their van der Waals interactions. We find statistically overrepresented native amino acid-nucleotide contacts (OaantC, one amino acid contacts one or multiple nucleotides, inter-nucleotide contacts are disregarded). Contact number is defined as the number of nucleotides contacted. Involvement of individual amino acids in OaantCs with smaller contact numbers is more random, while only a few amino acids significantly contribute to OaantCs with higher contact numbers. An investigation of structure, stability and assembly of bacterial ribosome depicts the involvement of these OaantCs in diverse biophysical interactions stabilizing the complex, including high-affinity protein-RNA contacts, inter-protein cooperativity, inter-subunit bridge, packing of multiple ribosomal RNA domains etc. Amino acid-nucleotide constituents of OaantCs with higher contact numbers are generally associated with significantly slower substitution rates compared to that of OaantCs with smaller contact numbers. This evolutionary rate heterogeneity emerges from the strong purifying selection pressure that conserves the respective amino acid physicochemical properties relevant to the stabilizing interaction with OaantC nucleotides. An analysis of relative molecular orientations of OaantC residues and their interaction energetics provides the biophysical ground of purifying selection conserving OaantC amino acid physicochemical properties.

  13. RPL39L is an example of a recently evolved ribosomal protein paralog that shows highly specific tissue expression patterns and is upregulated in ESCs and HCC tumors.

    PubMed

    Wong, Queenie Wing-Lei; Li, Jia; Ng, Sheng Rong; Lim, Seng Gee; Yang, Henry; Vardy, Leah A

    2014-01-01

    Ribosomal proteins (RPs) have been shown to be able to impart selectivity on the translating ribosome implicating them in gene expression control. Many ribosomal proteins are highly conserved and recently a number of ribosomal protein paralogs have been described in mammals. We examined the expression pattern of RPs in differentiating mouse Embryonic Stem Cells (ESCs), paying particular attention to the RP paralogs. We find the RP paralog Rpl39l is highly expressed in ESC and its expression strongly correlates with hepatocellular carcinoma tumor (HCC) samples with high tumor grading and alpha-fetoprotein level giving it diagnostic potential. We further screen the expression pattern of all RPs and their paralogs across 22 different tissues. We find that the more recently evolved RP paralogs show a much greater level of tissue-specific expression. We propose that these RP paralogs evolved more recently to provide a greater level of gene expression control to higher eukaryotes.

  14. Analysis of a sequence region of 5S RNA from E. coli cross-linked in situ to the ribosomal protein L25.

    PubMed Central

    Szymkowiak, C; Wagner, R

    1985-01-01

    70S ribosomes from E. coli were chemically cross-linked under conditions of in vitro protein biosynthesis. The ribosomal RNAs were extracted from reacted ribosomes and separated on sucrose gradients. The 5S RNA was shown to contain the ribosomal protein L25 covalently bound. After total RNase T1 hydrolysis of the covalent RNA-protein complex several high molecular weight RNA fragments were obtained and identified by sequencing. One fragment, sequence region U103 to U120, was shown to be directly linked to the protein first by protein specific staining of the particular fragment and second by phosphor cellulose chromatography of the covalent RNA-protein complex. The other two fragments, U89 to G106 and A34 to G51, could not be shown to be directly linked to L25 but were only formed under cross-linking conditions. While the fragment U89 to G106 may be protected from RNase T1 digestion because of a strong interaction with the covalent RNA-protein complex, the formation of the fragment A34 to G51 is very likely the result of a double monovalent modification of two neighbouring guanosines in the 5S RNA. The RNA sequences U103 to U120 established to be in direct contact to the protein L25 within the ribosome falls into the sequence region previously proposed as L25 binding site from studies with isolated 5S RNA-protein complexes. Images PMID:3892485

  15. An Evolutionary Strategy for All-Atom Folding of the 60-Amino-Acid Bacterial Ribosomal Protein L20

    PubMed Central

    Schug, A.; Wenzel, W.

    2006-01-01

    We have investigated an evolutionary algorithm for de novo all-atom folding of the bacterial ribosomal protein L20. We report results of two simulations that converge to near-native conformations of this 60-amino-acid, four-helix protein. We observe a steady increase of “native content” in both simulated ensembles and a large number of near-native conformations in their final populations. We argue that these structures represent a significant fraction of the low-energy metastable conformations, which characterize the folding funnel of this protein. These data validate our all-atom free-energy force field PFF01 for tertiary structure prediction of a previously inaccessible structural family of proteins. We also compare folding simulations of the evolutionary algorithm with the basin-hopping technique for the Trp-cage protein. We find that the evolutionary algorithm generates a dynamic memory in the simulated population, which leads to faster overall convergence. PMID:16565067

  16. Structures of proline-rich peptides bound to the ribosome reveal a common mechanism of protein synthesis inhibition

    PubMed Central

    Gagnon, Matthieu G.; Roy, Raktim N.; Lomakin, Ivan B.; Florin, Tanja; Mankin, Alexander S.; Steitz, Thomas A.

    2016-01-01

    With bacterial resistance becoming a serious threat to global public health, antimicrobial peptides (AMPs) have become a promising area of focus in antibiotic research. AMPs are derived from a diverse range of species, from prokaryotes to humans, with a mechanism of action that often involves disruption of the bacterial cell membrane. Proline-rich antimicrobial peptides (PrAMPs) are instead actively transported inside the bacterial cell where they bind and inactivate specific targets. Recently, it was reported that some PrAMPs, such as Bac71–35, oncocins and apidaecins, bind and inactivate the bacterial ribosome. Here we report the crystal structures of Bac71–35, Pyrrhocoricin, Metalnikowin and two oncocin derivatives, bound to the Thermus thermophilus 70S ribosome. Each of the PrAMPs blocks the peptide exit tunnel of the ribosome by simultaneously occupying three well characterized antibiotic-binding sites and interferes with the initiation step of translation, thereby revealing a common mechanism of action used by these PrAMPs to inactivate protein synthesis. Our study expands the repertoire of PrAMPs and provides a framework for designing new-generation therapeutics. PMID:26809677

  17. Structures of proline-rich peptides bound to the ribosome reveal a common mechanism of protein synthesis inhibition

    DOE PAGES

    Gagnon, Matthieu G.; Roy, Raktim N.; Lomakin, Ivan B.; ...

    2016-01-24

    Here, with bacterial resistance becoming a serious threat to global public health, antimicrobial peptides (AMPs) have become a promising area of focus in antibiotic research. AMPs are derived from a diverse range of species, from prokaryotes to humans, with a mechanism of action that often involves disruption of the bacterial cell membrane. Proline-rich antimicrobial peptides (PrAMPs) are instead actively transported inside the bacterial cell where they bind and inactivate specific targets. Recently, it was reported that some PrAMPs, such as Bac71–35, oncocins and apidaecins, bind and inactivate the bacterial ribosome. Here we report the crystal structures of Bac71–35, Pyrrhocoricin, Metalnikowinmore » and two oncocin derivatives, bound to the Thermus thermophilus 70S ribosome. Each of the PrAMPs blocks the peptide exit tunnel of the ribosome by simultaneously occupying three well characterized antibioticbinding sites and interferes with the initiation step of translation, thereby revealing a common mechanism of action used by these PrAMPs to inactivate protein synthesis. Our study expands the repertoire of PrAMPs and provides a framework for designing new-generation therapeutics.« less

  18. Structures of proline-rich peptides bound to the ribosome reveal a common mechanism of protein synthesis inhibition

    SciTech Connect

    Gagnon, Matthieu G.; Roy, Raktim N.; Lomakin, Ivan B.; Florin, Tanja; Mankin, Alexander S.; Steitz, Thomas A.

    2016-01-24

    Here, with bacterial resistance becoming a serious threat to global public health, antimicrobial peptides (AMPs) have become a promising area of focus in antibiotic research. AMPs are derived from a diverse range of species, from prokaryotes to humans, with a mechanism of action that often involves disruption of the bacterial cell membrane. Proline-rich antimicrobial peptides (PrAMPs) are instead actively transported inside the bacterial cell where they bind and inactivate specific targets. Recently, it was reported that some PrAMPs, such as Bac71–35, oncocins and apidaecins, bind and inactivate the bacterial ribosome. Here we report the crystal structures of Bac71–35, Pyrrhocoricin, Metalnikowin and two oncocin derivatives, bound to the Thermus thermophilus 70S ribosome. Each of the PrAMPs blocks the peptide exit tunnel of the ribosome by simultaneously occupying three well characterized antibioticbinding sites and interferes with the initiation step of translation, thereby revealing a common mechanism of action used by these PrAMPs to inactivate protein synthesis. Our study expands the repertoire of PrAMPs and provides a framework for designing new-generation therapeutics.

  19. Ribosomal protein genes are highly enriched among genes with allele-specific expression in the interspecific F1 hybrid catfish.

    PubMed

    Chen, Ailu; Wang, Ruijia; Liu, Shikai; Peatman, Eric; Sun, Luyang; Bao, Lisui; Jiang, Chen; Li, Chao; Li, Yun; Zeng, Qifan; Liu, Zhanjiang

    2016-06-01

    Interspecific hybrids provide a rich source for the analysis of allele-specific expression (ASE). In this work, we analyzed ASE in F1 hybrid catfish using RNA-Seq datasets. While the vast majority of genes were expressed with both alleles, 7-8 % SNPs exhibited significant differences in allele ratios of expression. Of the 66,251 and 177,841 SNPs identified from the datasets of the liver and gill, 5420 (8.2 %) and 13,390 (7.5 %) SNPs were identified as significant ASE-SNPs, respectively. With these SNPs, a total of 1519 and 3075 ASE-genes were identified. Gene Ontology analysis revealed that genes encoding cytoplasmic ribosomal proteins (RP) were highly enriched among ASE genes. Parent-of-origin was determined for 27 and 30 ASE RP genes in the liver and gill, respectively. The results indicated that genes from both channel catfish and blue catfish were involved in ASE. However, each RP gene appeared to be almost exclusively expressed from only one parent, indicating that ribosomes in the hybrid catfish were in the "hybrid" form. Overall representation of RP transcripts among the transcriptome appeared lower in the F1 hybrid catfish than in channel catfish or blue catfish, suggesting that the "hybrid" ribosomes may work more efficiently for translation in the F1 hybrid catfish.

  20. Identification, characterization and structure analysis of a type I ribosome-inactivating protein from Sapium sebiferum (Euphorbiaceae).

    PubMed

    Wu, Ying; Mao, Yingji; Jin, Shan; Hou, Jinyan; Du, Hua; Yang, Minglei; Wu, Lifang

    2015-08-07

    Ribosome-inactivating proteins (RIPs) are N-glycosidases (EC3.2.2.22) that universally inactivate the ribosome, thereby inhibiting protein biosynthesis. In this study, a novel type I RIPs named SEBIN was identified in Sapium sebiferum. Nuclear acid depurine experiment showed that SEBIN had rRNA N-Glycosidase activity. Further experiment indicated that SEBIN significantly inhibited Caenorhabditis elegans development as well as resulted in worm cell apoptosis. This is the first report to evaluate RIPs toxicity using C. elegans. We proposed that SEBIN may impaire C. elegans reproduction in a DNA-damage manner besides traditional protein synthesis inhibition approach. The predicted 3D structure was modeled using threading and ab initio modeling, and the r-RNA binding residue of SEBIN was identified through the protein-ligand docking approach. It showed the amino acid residues, Glu195, Asn81, Ala82, Tyr83, Glu164, Ser163, Ile159 and Arg167, played critical roles in catalytic process. Our results provided the theoretical foundation of structure-function relationships between enzymatic properties, toxicity and structural characterization of SEBIN.

  1. Dinoflagellate Phylogeny as Inferred from Heat Shock Protein 90 and Ribosomal Gene Sequences

    PubMed Central

    Hoppenrath, Mona; Leander, Brian S.

    2010-01-01

    Background Interrelationships among dinoflagellates in molecular phylogenies are largely unresolved, especially in the deepest branches. Ribosomal DNA (rDNA) sequences provide phylogenetic signals only at the tips of the dinoflagellate tree. Two reasons for the poor resolution of deep dinoflagellate relationships using rDNA sequences are (1) most sites are relatively conserved and (2) there are different evolutionary rates among sites in different lineages. Therefore, alternative molecular markers are required to address the deeper phylogenetic relationships among dinoflagellates. Preliminary evidence indicates that the heat shock protein 90 gene (Hsp90) will provide an informative marker, mainly because this gene is relatively long and appears to have relatively uniform rates of evolution in different lineages. Methodology/Principal Findings We more than doubled the previous dataset of Hsp90 sequences from dinoflagellates by generating additional sequences from 17 different species, representing seven different orders. In order to concatenate the Hsp90 data with rDNA sequences, we supplemented the Hsp90 sequences with three new SSU rDNA sequences and five new LSU rDNA sequences. The new Hsp90 sequences were generated, in part, from four additional heterotrophic dinoflagellates and the type species for six different genera. Molecular phylogenetic analyses resulted in a paraphyletic assemblage near the base of the dinoflagellate tree consisting of only athecate species. However, Noctiluca was never part of this assemblage and branched in a position that was nested within other lineages of dinokaryotes. The phylogenetic trees inferred from Hsp90 sequences were consistent with trees inferred from rDNA sequences in that the backbone of the dinoflagellate clade was largely unresolved. Conclusions/Significance The sequence conservation in both Hsp90 and rDNA sequences and the poor resolution of the deepest nodes suggests that dinoflagellates reflect an explosive

  2. Cold stress-induced protein Rbm3 binds 60S ribosomal subunits, alters microRNA levels, and enhances global protein synthesis.

    PubMed

    Dresios, John; Aschrafi, Armaz; Owens, Geoffrey C; Vanderklish, Peter W; Edelman, Gerald M; Mauro, Vincent P

    2005-02-08

    The expression of Rbm3, a glycine-rich RNA-binding protein, is enhanced under conditions of mild hypothermia, and Rbm3 has been postulated to facilitate protein synthesis at colder temperatures. To investigate this possibility, Rbm3 was overexpressed as a c-Myc fusion protein in mouse neuroblastoma N2a cells. Cells expressing this fusion protein showed a 3-fold increase in protein synthesis at both 37 degrees C and 32 degrees C compared with control cells. Although polysome profiles of cells expressing the fusion protein and control cells were similar, several differences were noted, suggesting that Rbm3 might enhance the association of 40S and 60S ribosomal subunits at 32 degrees C. Studies to assess a direct interaction of Rbm3 with ribosomes showed that a fraction of Rbm3 was associated with 60S ribosomal subunits in an RNA-independent manner. It appeared unlikely that this association could explain the global enhancement of protein synthesis, however, because cells expressing the Rbm3 fusion protein showed no substantial increase in the size of their monosome and polysome peaks, suggesting that similar numbers of mRNAs were being translated at approximately the same rates. In contrast, a complex that sedimented between the top of the gradient and 40S subunits was less abundant in cells expressing recombinant Rbm3. Further analysis showed that the RNA component of this fraction was microRNA. We discuss the possibility that Rbm3 expression alters global protein synthesis by affecting microRNA levels and suggest that both Rbm3 and microRNAs are part of a homeostatic mechanism that regulates global levels of protein synthesis under normal and cold-stress conditions.

  3. Immunotoxins Constructed with Ribosome-Inactivating Proteins and their Enhancers: A Lethal Cocktail with Tumor Specific Efficacy

    PubMed Central

    Gilabert-Oriol, Roger; Weng, Alexander; von Mallinckrodt, Benedicta; Melzig, Matthias F; Fuchs, Hendrik; Thakur, Mayank

    2014-01-01

    The term ribosome-inactivating protein (RIP) is used to denominate proteins mostly of plant origin, which have N-glycosidase enzymatic activity leading to a complete destruction of the ribosomal function. The discovery of the RIPs was almost a century ago, but their usage has seen transition only in the last four decades. With the advent of antibody therapy, the RIPs have been a subject of extensive research especially in targeted tumor therapies, which is the primary focus of this review. In the present work we enumerate 250 RIPs, which have been identified so far. An attempt has been made to identify all the RIPs that have been used for the construction of immunotoxins, which are conjugates or fusion proteins of an antibody or ligand with a toxin. The data from 1960 onwards is reviewed in this paper and an extensive list of more than 450 immunotoxins is reported. The clinical reach of tumor-targeted toxins has been identified and detailed in the work as well. While there is a lot of potential that RIPs embrace for targeted tumor therapies, the success in preclinical and clinical evaluations has been limited mainly because of their inability to escape the endo/lysosomal degradation. Various strategies that can increase the efficacy and lower the required dose for targeted toxins have been compiled in this article. It is plausible that with the advancements in platform technologies or improved endosomal escape the usage of tumor targeted RIPs would see the daylight of clinical success. PMID:25341935

  4. β-Boswellic acid, a bioactive substance used in food supplements, inhibits protein synthesis by targeting the ribosomal machinery.

    PubMed

    Casapullo, A; Cassiano, C; Capolupo, A; Del Gaudio, F; Esposito, R; Tosco, A; Riccio, R; Monti, M C

    2016-09-01

    The Boswellia gum resin extracts have been used in traditional medicines because of their remarkable anti-inflammatory properties. Nowadays, these extracts are on the market as food supplements. β-Boswellic acid (βBA) is one of the main pentacyclic triterpene components, among the family of BAs, of the Boswellia gum resins. BAs have been broadly studied and are well known for their wide anti-inflammatory and potential anticancer properties. In this paper, a mass spectrometry-based chemoproteomic approach has been applied to characterize the whole βBA interacting profile. Among the large numbers of proteins fished out, proteasome, 14-3-3 and some ribosomal proteins were considered the most interesting targets strictly connected to the modulation of the cancer progression. In particular, because of their recent assessment as innovative chemotherapeutic targets, the ribosomal proteins were considered the most attractive βBA partners, and the biological role of their interaction with the natural compound has been evaluated. Copyright © 2016 John Wiley & Sons, Ltd.

  5. Effect of HIP/ribosomal protein L29 deficiency on mineral properties of murine bones and teeth.

    PubMed

    Sloofman, Laura G; Verdelis, Kostas; Spevak, Lyudmila; Zayzafoon, Majd; Yamauchi, Mistuo; Opdenaker, Lynn M; Farach-Carson, Mary C; Boskey, Adele L; Kirn-Safran, Catherine B

    2010-07-01

    Mice lacking HIP/RPL29, a component of the ribosomal machinery, display increased bone fragility. To understand the effect of sub-efficient protein synthetic rates on mineralized tissue quality, we performed dynamic and static histomorphometry and examined the mineral properties of both bones and teeth in HIP/RPL29 knock-out mice using Fourier transform infrared imaging (FTIRI). While loss of HIP/RPL29 consistently reduced total bone size, decreased mineral apposition rates were not significant, indicating that short stature is not primarily due to impaired osteoblast function. Interestingly, our microspectroscopic studies showed that a significant decrease in collagen crosslinking during maturation of HIP/RPL29-null bone precedes an overall enhancement in the relative extent of mineralization of both trabecular and cortical adult bones. This report provides strong genetic evidence that ribosomal insufficiency induces subtle organic matrix deficiencies which elevates calcification. Consistent with the HIP/RPL29-null bone phenotype, HIP/RPL29-deficient teeth also showed reduced geometric properties accompanied with relative increased mineral densities of both dentin and enamel. Increased mineralization associated with enhanced tissue fragility related to imperfection in organic phase microstructure evokes defects seen in matrix protein-related bone and tooth diseases. Thus, HIP/RPL29 mice constitute a new genetic model for studying the contribution of global protein synthesis in the establishment of organic and inorganic phases in mineral tissues.

  6. Selenocysteine insertion sequence (SECIS)-binding protein 2 alters conformational dynamics of residues involved in tRNA accommodation in 80 S ribosomes.

    PubMed

    Caban, Kelvin; Copeland, Paul R

    2012-03-23

    Sec-tRNA(Sec) is site-specifically delivered at defined UGA codons in selenoprotein mRNAs. This recoding event is specified by the selenocysteine insertion sequence (SECIS) element and requires the selenocysteine (Sec)-specific elongation factor, eEFSec, and the SECIS binding protein, SBP2. Sec-tRNA(Sec) is delivered to the ribosome by eEFSec-GTP, but this ternary complex is not sufficient for Sec incorporation, indicating that its access to the ribosomal A-site is regulated. SBP2 stably associates with ribosomes, and mutagenic analysis indicates that this interaction is essential for Sec incorporation. However, the ribosomal function of SBP2 has not been elucidated. To shed light on the functional relevance of the SBP2-ribosome interaction, we screened the functional centers of the 28 S rRNA in translationally competent 80 S ribosomes using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). We demonstrate that SBP2 specifically alters the reactivity of specific residues in Helix 89 (H89) and expansion segment 31 (ES31). These results are indicative of a conformational change in response to SBP2 binding. Based on the known functions of H89 during translation, we propose that SBP2 allows Sec incorporation by either promoting Sec-tRNA(Sec) accommodation into the peptidyltransferase center and/or by stimulating the ribosome-dependent GTPase activity of eEFSec.

  7. De novo translation initiation on membrane-bound ribosomes as a mechanism for localization of cytosolic protein mRNAs to the endoplasmic reticulum.

    PubMed

    Jagannathan, Sujatha; Reid, David W; Cox, Amanda H; Nicchitta, Christopher V

    2014-10-01

    The specialized protein synthesis functions of the cytosol and endoplasmic reticulum compartments are conferred by the signal recognition particle (SRP) pathway, which directs the cotranslational trafficking of signal sequence-encoding mRNAs from the cytosol to the endoplasmic reticulum (ER). Although subcellular mRNA distributions largely mirror the binary pattern predicted by the SRP pathway model, studies in mammalian cells, yeast, and Drosophila have also demonstrated that cytosolic protein-encoding mRNAs are broadly represented on ER-bound ribosomes. A mechanism for such noncanonical mRNA localization remains, however, to be identified. Here, we examine the hypothesis that de novo translation initiation on ER-bound ribosomes serves as a mechanism for localizing cytosolic protein-encoding mRNAs to the ER. As a test of this hypothesis, we performed single molecule RNA fluorescence in situ hybridization studies of subcellular mRNA distributions and report that a substantial fraction of mRNAs encoding the cytosolic protein GAPDH resides in close proximity to the ER. Consistent with these data, analyses of subcellular mRNA and ribosome distributions in multiple cell lines demonstrated that cytosolic protein mRNA-ribosome distributions were strongly correlated, whereas signal sequence-encoding mRNA-ribosome distributions were divergent. Ribosome footprinting studies of ER-bound polysomes revealed a substantial initiation codon read density enrichment for cytosolic protein-encoding mRNAs. We also demonstrate that eukaryotic initiation factor 2α is bound to the ER via a salt-sensitive, ribosome-independent mechanism. Combined, these data support ER-localized translation initiation as a mechanism for mRNA recruitment to the ER.

  8. Coexpression of Escherichia coli obgE, Encoding the Evolutionarily Conserved Obg GTPase, with Ribosomal Proteins L21 and L27

    PubMed Central

    Maouche, Rim; Burgos, Hector L.; My, Laetitia; Viala, Julie P.

    2016-01-01

    ABSTRACT Multiple essential small GTPases are involved in the assembly of the ribosome or in the control of its activity. Among them, ObgE (CgtA) has been shown recently to act as a ribosome antiassociation factor that binds to ppGpp, a regulator whose best-known target is RNA polymerase. The present study was aimed at elucidating the expression of obgE in Escherichia coli. We show that obgE is cotranscribed with ribosomal protein genes rplU and rpmA and with a gene of unknown function, yhbE. We show here that about 75% of the transcripts terminate before obgE, because there is a transcriptional terminator between rpmA and yhbE. As expected for ribosomal protein operons, expression was highest during exponential growth, decreased during entry into stationary phase, and became almost undetectable thereafter. Expression of the operon was derepressed in mutants lacking ppGpp or DksA. However, regulation by these factors appears to occur post-transcription initiation, since no effects of ppGpp and DksA on rplU promoter activity were observed in vitro. IMPORTANCE The conserved and essential ObgE GTPase binds to the ribosome and affects its assembly. ObgE has also been reported to impact chromosome segregation, cell division, resistance to DNA damage, and, perhaps most interestingly, persister formation and antibiotic tolerance. However, it is unclear whether these effects are related to its role in ribosome formation. Despite its importance, no studies on ObgE expression have been reported. We demonstrate here that obgE is expressed from an operon encoding two ribosomal proteins, that the operon's expression varies with the growth phase, and that it is dependent on the transcription regulators ppGpp and DksA. Our results thus demonstrate that obgE expression is coupled to ribosomal gene expression. PMID:27137500

  9. Isolation of ribosomes and polysomes.

    PubMed

    Rivera, Maria C; Maguire, Bruce; Lake, James A

    2015-03-02

    Here we describe a preparative differential centrifugation protocol for the isolation of ribosomes from a crude cell homogenate. The subcellular fraction obtained is enriched in ribosome monomers and polysomes. The protocol has been optimized for the homogenization and collection of the ribosomal fraction from prokaryotic cells, mammalian and plant tissues, reticulocytes, and chloroplasts. The quality of the ribosomal preparation is enhanced by the removal of the remaining cellular components and adsorbed proteins by pelleting through a sucrose cushion with a high concentration of monovalent salts, NH4Cl or KCl. The different components of the ribosomal fraction isolated using this protocol can be further purified by sucrose gradient centrifugation.

  10. Ordered phosphorylation of 40S ribosomal protein S6 after serum stimulation of quiescent 3T3 cells.

    PubMed Central

    Martin-Pérez, J; Thomas, G

    1983-01-01

    The amino acids and tryptic peptides that become phosphorylated in 40S ribosomal protein S6 after serum stimulation of quiescent 3T3 cells were examined by two-dimensional thin-layer electrophoresis. In the maximally phosphorylated form of the protein, most of the phosphate was incorporated into serine and a small amount, into threonine. Digestion of this form of the protein with trypsin revealed 10 major phosphopeptides. All 10 contained phosphoserine and 2 of the 10 also contained phosphothreonine. Next, the five forms of increasingly phosphorylated S6 were individually separated on two-dimensional polyacrylamide gels or total S6 was isolated from cells that were stimulated for only a short time and their phosphotryptic maps were analyzed. The results showed that, as larger amounts of phosphate were added to S6, the phosphopeptides appeared in a specific order. Images PMID:6573662

  11. An S18 ribosomal protein gene copy at the Arabidopsis PFL locus affects plant development by its specific expression in meristems.

    PubMed Central

    Van Lijsebettens, M; Vanderhaeghen, R; De Block, M; Bauw, G; Villarroel, R; Van Montagu, M

    1994-01-01

    In Arabidopsis, mutation at PFL causes pointed first leaves, reduced fresh weight and growth retardation. We have cloned the wild-type PFL gene by T-DNA tagging, and demonstrate that it complements the mutant phenotype. PFL codes for ribosomal protein S18, based on the high homology with rat S18 and on purification of S18-equivalent peptides from plant ribosomes. pfl represents the first mutation in eukaryotic S18 proteins or their S13 prokaryotic counterparts, involved in translation initiation. Arabidopsis contains three S18 gene copies dispersed in the genetic map; they are all transcribed and code for completely identical proteins. No transcript is detected from the mutated gene, S18A. The activity of the S18A promoter is restricted to meristems, with a markedly high expression at the embryonic heart stage, and to wounding sites. This means that plants activate an extra copy of this ribosomal protein gene in tissues with cell division activity. We postulate that in meristematic tissues plants use transcriptional control to synthesize extra ribosomes to increase translational efficiency. In analogy with this, an additional, developmentally regulated gene copy might be expected for all ribosomal proteins. Images PMID:7913892

  12. The Interaction Pattern between a Homology Model of 40S Ribosomal S9 Protein of Rhizoctonia solani and 1-Hydroxyphenaize by Docking Study

    PubMed Central

    Dharni, Seema; Sanchita; Sharma, Ashok; Patra, Dharani Dhar

    2014-01-01

    1-Hydroxyphenazine (1-OH-PHZ), a natural product from Pseudomonas aeruginosa strain SD12, was earlier reported to have potent antifungal activity against Rhizoctonia solani. In the present work, the antifungal activity of 1-OH-PHZ on 40S ribosomal S9 protein was validated by molecular docking approach. 1-OH-PHZ showed interaction with two polar contacts with residues, Arg69 and Phe19, which inhibits the synthesis of fungal protein. Our study reveals that 1-OH-PHZ can be a potent inhibitor of 40S ribosomal S9 protein of R. solani that may be a promising approach for the management of fungal diseases. PMID:24864254

  13. The interaction pattern between a homology model of 40S ribosomal S9 protein of Rhizoctonia solani and 1-hydroxyphenaize by docking study.

    PubMed

    Dharni, Seema; Sanchita; Samad, Abdul; Sharma, Ashok; Patra, Dharani Dhar

    2014-01-01

    1-Hydroxyphenazine (1-OH-PHZ), a natural product from Pseudomonas aeruginosa strain SD12, was earlier reported to have potent antifungal activity against Rhizoctonia solani. In the present work, the antifungal activity of 1-OH-PHZ on 40S ribosomal S9 protein was validated by molecular docking approach. 1-OH-PHZ showed interaction with two polar contacts with residues, Arg69 and Phe19, which inhibits the synthesis of fungal protein. Our study reveals that 1-OH-PHZ can be a potent inhibitor of 40S ribosomal S9 protein of R. solani that may be a promising approach for the management of fungal diseases.

  14. Ribosomal protein L11 is related to brain maturation during the adult phase in Apis cerana cerana (Hymenoptera, Apidae).

    PubMed

    Meng, Fei; Lu, Wenjing; Yu, Feifei; Kang, Mingjiang; Guo, Xingqi; Xu, Baohua

    2012-05-01

    Ribosomal proteins (RPs) play pivotal roles in developmental regulation. The loss or mutation of ribosomal protein L11 (RPL11) induces various developmental defects. However, few RPs have been functionally characterized in Apis cerana cerana. In this study, we isolated a single copy gene, AccRPL11, and characterized its connection to brain maturation. AccRPL11 expression was highly concentrated in the adult brain and was significantly induced by abiotic stresses such as pesticides and heavy metals. Immunofluorescence assays demonstrated that AccRPL11 was localized to the medulla, lobula and surrounding tissues of esophagus in the brain. The post-transcriptional knockdown of AccRPL11 gene expression resulted in a severe decrease in adult brain than in other tissues. The expression levels of other brain development-related genes, p38, ERK2, CacyBP and CREB, were also reduced. Immunofluorescence signal attenuation was also observed in AccRPL11-rich regions of the brain in dsAccRPL11-injected honeybees. Taken together, these results suggest that AccRPL11 may be functional in brain maturation in honeybee adults.

  15. Inactivation of ribosomal protein L22 promotes transformation by induction of the stemness factor, Lin28B

    PubMed Central

    Rao, Shuyun; Lee, Sang-Yun; Gutierrez, Alejandro; Perrigoue, Jacqueline; Thapa, Roshan J.; Tu, Zhigang; Jeffers, John R.; Rhodes, Michele; Anderson, Stephen; Oravecz, Tamas; Hunger, Stephen P.; Timakhov, Roman A.; Zhang, Rugang; Balachandran, Siddharth; Zambetti, Gerard P.; Testa, Joseph R.; Look, A. Thomas

    2012-01-01

    Ribosomal protein (RP) mutations in diseases such as 5q− syndrome both disrupt hematopoiesis and increase the risk of developing hematologic malignancy. However, the mechanism by which RP mutations increase cancer risk has remained an important unanswered question. We show here that monoallelic, germline inactivation of the ribosomal protein L22 (Rpl22) predisposes T-lineage progenitors to transformation. Indeed, RPL22 was found to be inactivated in ∼ 10% of human T-acute lymphoblastic leukemias. Moreover, monoallelic loss of Rpl22 accelerates development of thymic lymphoma in both a mouse model of T-cell malignancy and in acute transformation assays in vitro. We show that Rpl22 inactivation enhances transformation potential through induction of the stemness factor, Lin28B. Our finding that Rpl22 inactivation promotes transformation by inducing expression of Lin28B provides the first insight into the mechanistic basis by which mutations in Rpl22, and perhaps some other RP genes, increases cancer risk. PMID:22976955

  16. Erythromycin, carbomycin, and spiramycin inhibit protein synthesis by stimulating the dissociation of peptidyl-tRNA from ribosomes.

    PubMed

    Menninger, J R; Otto, D P

    1982-05-01

    In mutant Escherichia coli with temperature-sensitive peptidyl-tRNA hydrolase (aminoacyl-tRNA hydrolase; EC 3.1.1.29), peptidyl-tRNA accumulates at the nonpermissive temperature (40 degrees C), and the cells die. These consequences of high temperature were enhanced if the cells were first treated with erythromycin, carbomycin, or spiramycin at doses sufficient to inhibit protein synthesis in wild-type cells but not sufficient to kill either mutant or wild-type cells at the permissive temperature (30 degrees C). Since peptidyl-tRNA hydrolase in he mutant cells is inactivated rapidly and irreversibly at 40 degrees C, the enhanced accumulation of peptidyl-tRNA and killing were the result of enhanced dissociation, stimulated by the antibiotics, of peptidyl-tRNA from ribosomes. The implications of these findings for inhibition of cell growth and protein synthesis are discussed. Certain alternative interpretations are shown to be inconsistent with the relevant data. Previous conflicting observations on the effects of macrolide antibiotics are explained in terms of our observations. We conclude that erythromycin, carbomycin, and spiramycin (and probably all macrolides) have as a primary mechanism of action the stimulation of dissociation of peptidyl-tRNA from ribosomes, probably during translocation.

  17. The folded and disordered domains of human ribosomal protein SA have both idiosyncratic and shared functions as membrane receptors.

    PubMed

    Zidane, Nora; Ould-Abeih, Mohamed B; Petit-Topin, Isabelle; Bedouelle, Hugues

    2012-12-20

    The human RPSA [ribosomal protein SA; also known as LamR1(laminin receptor 1)] belongs to the ribosome but is also a membrane receptor for laminin, growth factors, prion, pathogens and the anticarcinogen EGCG (epigallocatechin-gallate). It contributes to the crossing of the blood-brain barrier by neurotropic viruses and bacteria, and is a biomarker of metastasis. RPSA includes an N-terminal domain, which is folded and homologous to the prokaryotic RPS2, and a C-terminal extension, which is intrinsically disordered and conserved in vertebrates. We used recombinant derivatives of RPSA and its N- and C-domains to quantify its interactions with ligands by in-vitro immunochemical and spectrofluorimetric methods. Both N- and C-domains bound laminin with K(D) (dissociation constants) of 300 nM. Heparin bound only to the N-domain and competed for binding to laminin with the negatively charged C-domain, which therefore mimicked heparin. EGCG bound only to the N-domain with a K(D) of 100 nM. Domain 3 of the envelope protein from yellow fever virus and serotypes-1 and -2 of dengue virus bound preferentially to the C-domain whereas that from West Nile virus bound only to the N-domain. Our quantitative in-vitro approach should help clarify the mechanisms of action of RPSA, and ultimately fight against cancer and infectious agents.

  18. Ribosomal protein L11 is related to brain maturation during the adult phase in Apis cerana cerana (Hymenoptera, Apidae)

    NASA Astrophysics Data System (ADS)

    Meng, Fei; Lu, Wenjing; Yu, Feifei; Kang, Mingjiang; Guo, Xingqi; Xu, Baohua

    2012-05-01

    Ribosomal proteins (RPs) play pivotal roles in developmental regulation. The loss or mutation of ribosomal protein L11 ( RPL11) induces various developmental defects. However, few RPs have been functionally characterized in Apis cerana cerana. In this study, we isolated a single copy gene, AccRPL11, and characterized its connection to brain maturation. AccRPL11 expression was highly concentrated in the adult brain and was significantly induced by abiotic stresses such as pesticides and heavy metals. Immunofluorescence assays demonstrated that AccRPL11 was localized to the medulla, lobula and surrounding tissues of esophagus in the brain. The post-transcriptional knockdown of AccRPL11 gene expression resulted in a severe decrease in adult brain than in other tissues. The expression levels of other brain development-related genes, p38, ERK2, CacyBP and CREB, were also reduced. Immunofluorescence signal attenuation was also observed in AccRPL11-rich regions of the brain in ds AccRPL11-injected honeybees. Taken together, these results suggest that AccRPL11 may be functional in brain maturation in honeybee adults.

  19. Arabidopsis ribosomal proteins control vacuole trafficking and developmental programs through the regulation of lipid metabolism

    SciTech Connect

    Li, Ruixi; Sun, Ruobai; Hicks, Glenn R.; Raikhel, Natasha V.

    2014-12-22

    The vacuole is the most prominent compartment in plant cells and is important for ion and protein storage. In our effort to search for key regulators in the plant vacuole sorting pathway, ribosomal large subunit 4 (rpl4d) was identified as a translational mutant defective in both vacuole trafficking and normal development. Polysome profiling of the rpl4d mutant showed reduction in polysome-bound mRNA compared with wild-type, but no significant change in the general mRNA distribution pattern. Ribsomal profiling data indicated that genes in the lipid metabolism pathways were translationally down-regulated in the rpl4d mutant. Live imaging studies by Nile red staining suggested that both polar and nonpolar lipid accumulation was reduced in meristem tissues of rpl4d mutants. Pharmacological evidence showed that sterol and sphingolipid biosynthetic inhibitors can phenocopy the defects of the rpl4d mutant, including an altered vacuole trafficking pattern. Genetic evidence from lipid biosynthetic mutants indicates that alteration in the metabolism of either sterol or sphingolipid biosynthesis resulted in vacuole trafficking defects, similar to the rpl4d mutant. Tissue-specific complementation with key enzymes from lipid biosynthesis pathways can partially rescue both vacuole trafficking and auxin-related developmental defects in the rpl4d mutant. These results indicate that lipid metabolism modulates auxin-mediated tissue differentiation and endomembrane trafficking pathways downstream of ribosomal protein function.

  20. Common Pharmacophore of Structurally Distinct Small-Molecule Inhibitors of Intracellular Retrograde Trafficking of Ribosome Inactivating Proteins

    NASA Astrophysics Data System (ADS)

    Yu, Shichao; Park, Jewn Giew; Kahn, Jennifer Nielsen; Tumer, Nilgun E.; Pang, Yuan-Ping

    2013-12-01

    We reported previously (+/-)-2-(5-methylthiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one [(+/-)-Retro-2cycl] as the chemical structure of Retro-2 that showed mouse protection against ricin, a notorious ribosome inactivating protein (RIP). Herein we report our chemical resolution of (+/-)-Retro-2cycl, analog synthesis, and cell-based evaluation showing that the two optically pure enantiomers and their achiral analog have nearly the same degree of cell protection against ricin as (+/-)-Retro-2cycl. We also report our computational studies explaining the lack of stereo preference and revealing a common pharmacophore of structurally distinct inhibitors of intracellular retrograde trafficking of RIPs. This pharmacophore comprises a central aromatic ring o-substituted by an aromatic ring and a moiety bearing an O or S atom attached to sp2 C atom(s). These results offer new insights into lead identification and optimization for RIP antidote development to minimize the global health threat caused by ribosome-inactivating proteins.

  1. Posttranscriptional regulation of ribosomal protein S20 and stability of the S20 mRNA species.

    PubMed Central

    Mackie, G A

    1987-01-01

    I have tested whether selective degradation of mRNA for ribosomal protein S20 of Escherichia coli occurs under conditions for which the expression of S20 is regulated posttranscriptionally. Blot hybridization of total RNA extracted from cultures at different times after addition of rifampin has permitted the estimation of relative levels of the two S20 mRNA species and their half-lives. In a strain harboring a plasmid containing the complete gene for S20, including the transcriptional terminator, moderate posttranscriptional repression of S20 synthesis is accompanied by a substantial increase in the half-lives of both S20 mRNAs relative to those in the haploid parental strain. In an otherwise identical strain in which the transcriptional terminator is deleted from the plasmid-borne S20 genes, the half-life of total S20 mRNA declines more than twofold relative to that in the haploid parent. Thus accelerated decay of the mRNAs for ribosomal protein S20 appears to be an artifact of deletion of the transcriptional terminator, rather than a physiologically significant consequence of translational repression. Images PMID:2438268

  2. The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation.

    PubMed

    Khoshnevis, Sohail; Askenasy, Isabel; Johnson, Matthew C; Dattolo, Maria D; Young-Erdos, Crystal L; Stroupe, M Elizabeth; Karbstein, Katrin

    2016-06-01

    DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed "helicases," their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked.

  3. The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation

    PubMed Central

    Khoshnevis, Sohail; Askenasy, Isabel; Dattolo, Maria D.; Young-Erdos, Crystal L.; Stroupe, M. Elizabeth; Karbstein, Katrin

    2016-01-01

    DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed “helicases,” their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked. PMID:27280440

  4. Preparation and proteomic analysis of chloroplast ribosomes.

    PubMed

    Yamaguchi, Kenichi

    2011-01-01

    Proteomics of chloroplast ribosomes in spinach and Chlamydomonas revealed unique protein composition and structures of plastid ribosomes. These studies have suggested the presence of some ribosomal proteins unique to plastid ribosomes which may be involved in plastid-unique translation regulation. Considering the strong background of genetic analysis and molecular biology in Arabidopsis, the in-depth proteomic characterization of Arabidopsis plastid ribosomes would facilitate further understanding of plastid translation in higher plants. Here, I describe simple and rapid methods for the preparation of plastid ribosomes from Chlamydomonas and Arabidopsis using sucrose gradients. I also describe purity criteria and methods for yield estimation of the purified plastid ribosomes and subunits, methods for the preparation of plastid ribosomal proteins, as well as the identification of some Arabidopsis plastid ribosomal proteins by matrix-assisted laser desorption/ionization mass spectrometry.

  5. cDNA Cloning, expression and characterization of an allergenic 60s ribosomal protein of almond (prunus dulcis).

    PubMed

    Abolhassani, Mohsen; Roux, Kenneth H

    2009-06-01

    Tree nuts, including almond (prunus dulcis) are a source of food allergens often associated with life-threatening allergic reactions in susceptible individuals. Although the proteins in almonds have been biochemically characterized, relatively little has been reported regarding the identity of the allergens involved in almond sensitivity. The present study was undertaken to identify the allergens of the almond by cDNA library approach. cDNA library of almond seeds was constructed in Uni-Zap XR lamda vector and expressed in E. coli XL-1 blue. Plaques were immunoscreened with pooled sera of allergic patients. The cDNA clone reacting significantly with specific IgE antibodies was selected and subcloned and subsequently expressed in E. coli. The amino acids deducted from PCR product of clone showed homology to 60s acidic ribosomal protein of almond. The expressed protein was 11,450 Dalton without leader sequence. Immunoreactivity of the recombinant 60s ribosomal protein (r60sRP) was evaluated with dot blot analysis using pooled and individual sera of allergic patients. The data showed that r60sRP and almond extract (as positive control) possess the ability to bind the IgE antibodies. The results showed that expressed protein is an almond allergen.Whether this r60sRP represents a major allergen of almond needs to be further studied which requires a large number of sera from the almond atopic patients and also need to determine the IgE-reactive frequencies of each individual allergen.

  6. Major Trypanosoma cruzi antigenic determinant in Chagas' heart disease shares homology with the systemic lupus erythematosus ribosomal P protein epitope.

    PubMed Central

    Mesri, E A; Levitus, G; Hontebeyrie-Joskowicz, M; Dighiero, G; Van Regenmortel, M H; Levin, M J

    1990-01-01

    A Trypanosoma cruzi lambda gt11 cDNA clone, JL5, expressed a recombinant protein which was found to react predominantly with chronic Chagas' heart disease sera. The cloned 35-residue-long peptide was identified as the carboxyl-terminal portion of a T. cruzi ribosomal P protein. The JL5 13 carboxyl-terminal residues shared a high degree of homology with the systemic lupus erythematosus (SLE) ribosomal P protein epitope. Synthetic peptides comprising the 13 (R-13), 10 (R-10), and 7 (R-7) carboxyl-terminal residues of the JL5 protein were used to study, by enzyme-linked immunosorbent assay, the specificity of the Chagas' disease anti-JL5 and SLE anti-P antibodies. The R-13 peptide defined a linear antigenic determinant of the JL5 recombinant protein. As was proved for JL5, R-13 defined antibody specificities which were significantly increased in chronic Chagas' heart disease patients. Only SLE anti-P positive sera were found to react with JL5 and R-13. Fine epitope mapping showed that Chagas' disease anti-JL5 and SLE anti-P antibodies define similar epitopes within the R-13 peptide. The binding of the SLE sera to JL5 was completely blocked by the R-13 peptide, indicating that the shared specificity between anti-JL5 and anti-P autoantibodies was exclusively limited to the conserved linear epitope(s) within the R-13 peptide. The prevalence of high anti-R-13 antibody titers in Chagas' heart disease patients supports the hypothesis that postulates the existence of autoimmune disorders in Chagas' heart disease. PMID:1696282

  7. Hypophosphorylation of ribosomal protein S6 is a molecular mechanism underlying ischemic tolerance induced by either hibernation or preconditioning.

    PubMed

    Miyake, Shin-ichi; Wakita, Hideaki; Bernstock, Joshua D; Castri, Paola; Ruetzler, Christl; Miyake, Junko; Lee, Yang-Ja; Hallenbeck, John M

    2015-12-01

    Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) have an extraordinary capacity to withstand prolonged and profound reductions in blood flow and oxygen delivery to the brain without incurring any cellular damage. As such, the hibernation torpor of I. tridecemlineatus provides a valuable model of tolerance to ischemic stress. Herein, we report that during hibernation torpor, a marked reduction in the phosphorylation of the ribosomal protein S6 (rpS6) occurs within the brains of I. tridecemlineatus. Of note, rpS6 phosphorylation was shown to increase in the brains of rats that underwent an occlusion of the middle cerebral artery. However, such an increase was attenuated after the implementation of an ischemic preconditioning paradigm. In addition, cultured cortical neurons treated with the rpS6 kinase (S6K) inhibitors, D-glucosamine or PF4708671, displayed a decrease in rpS6 phosphorylation and a subsequent increase in tolerance to oxygen/glucose deprivation, an in vitro model of ischemic stroke. Collectively, such evidence suggests that the down-regulation of rpS6 signal transduction may account for a substantial part of the observed increase in cellular tolerance to brain ischemia that occurs during hibernation torpor and after ischemic preconditioning. Further identification and characterization of the mechanisms used by hibernating species to increase ischemic tolerance may eventually clarify how the loss of homeostatic control that occurs during and after cerebral ischemia in the clinic can ultimately be minimized and/or prevented. Mammalian hibernation provides a valuable model of tolerance to ischemic stress. Herein, we demonstrate that marked reductions in the phosphorylation of ribosomal protein S6 (rpS6), extracellular signal-regulated kinase family of mitogen-activated protein (MAP) kinase p44/42 (p44/42MAPK) and ribosomal protein S6 kinase (S6K) occur within the brains of both hibernating squirrels and rats, which have undergone an ischemic

  8. The Proximity of Ribosomal Protein Genes to oriC Enhances Vibrio cholerae Fitness in the Absence of Multifork Replication

    PubMed Central

    Soler-Bistué, Alfonso; Timmermans, Michaël

    2017-01-01

    ABSTRACT Recent works suggest that bacterial gene order links chromosome structure to cell homeostasis. Comparative genomics showed that, in fast-growing bacteria, ribosomal protein genes (RP) locate near the replication origin (oriC). We recently showed that Vibrio cholerae employs this positional bias as a growth optimization strategy: under fast-growth conditions, multifork replication increases RP dosage and expression. However, RP location may provide advantages in a dosage-independent manner: for example, the physical proximity of the many ribosomal components, in the context of a crowded cytoplasm, may favor ribosome biogenesis. To uncover putative dosage-independent effects, we studied isogenic V. cholerae derivatives in which the major RP locus, S10-spc-α (S10), was relocated to alternative genomic positions. When bacteria grew fast, bacterial fitness was reduced according to the S10 relative distance to oriC. The growth of wild-type V. cholerae could not be improved by additional copies of the locus, suggesting a physiologically optimized genomic location. Slow growth is expected to uncouple RP position from dosage, since multifork replication does not occur. Under these conditions, we detected a fitness impairment when S10 was far from oriC. Deep sequencing followed by marker frequency analysis in the absence of multifork replication revealed an up to 30% S10 dosage reduction associated with its relocation that closely correlated with fitness alterations. Hence, the impact of S10 location goes beyond a growth optimization strategy during feast periods. RP location may be important during the whole life cycle of this pathogen. PMID:28246358

  9. Structural analysis of the Drosophila rpA1 gene, a member of the eucaryotic 'A' type ribosomal protein family.

    PubMed Central

    Qian, S; Zhang, J Y; Kay, M A; Jacobs-Lorena, M

    1987-01-01

    The expression of ribosomal protein (r-protein) genes is uniquely regulated at the translational level during early development of Drosophila. Here we report results of a detailed analysis of the r-protein rpA1 gene. A cloned DNA sequence coding for rpA1 has been identified by hybrid-selected translation and amino acid composition analysis. The rpA1 gene was localized to polytene chromosome band 53CD. The nucleotide sequence of the rpA1 gene and its cDNA have been determined. rpA1 is a single copy gene and sequence comparison between the gene and its cDNA indicates that this r-protein gene is intronless. Allelic restriction site polymorphisms outside of the gene were observed, while the coding sequence is well conserved between two Drosophila strains. The protein has unusual domains rich in Ala and charged residues. The rpA1 is homologous to the "A" family of eucaryotic acidic r-proteins which are known to play a key role in the initiation and elongation steps of protein synthesis. Images PMID:3103101

  10. Rmt1 catalyzes zinc-finger independent arginine methylation of the small ribosomal protein Rps2 in Saccharomyces cerevisiae

    PubMed Central

    Lipson, Rebecca S.; Webb, Kristofor J.; Clarke, Steven G.

    2010-01-01

    Rps2/rpS2 is a well conserved protein of the eukaryotic ribosomal small subunit. Rps2 has previously been shown to contain asymmetric dimethylarginine residues, the addition of which is catalyzed by zinc-finger-containing arginine methyltransferase 3 (Rmt3) in the fission yeast Schizosaccharomyces pombe and protein arginine methyltransferase 3 (PRMT3) in mammalian cells. Here we demonstrate that despite the lack of a zinc-finger-containing homolog of Rmt3/PRMT3 in the budding yeast Saccharomyces cerevisiae, Rps2 is partially modified to generate asymmetric dimethylarginine and monomethylarginine residues. We find that this modification of Rps2 is dependent upon the major arginine methyltransferase 1 (Rmt1) in S. cerevisiae. These results are suggestive of a role for Rmt1 in modifying the function of Rps2 in a manner distinct from that occurring in S. pombe and mammalian cells. PMID:20035717

  11. Ribosome-inactivating Proteins from Root Tubers and Seeds of Trichosan-thes kirilowii and Other Trichosanthes Species.

    PubMed

    Ye, Xiujuan; Ng, Charlene C W; Wong, Jack H; Ng, Tzi B; Chan, Gabriel H H; Guan, Suzhen; Sha, Ou

    2016-01-01

    Ribosome-inactivating proteins have been isolated from Trichosanthes kirilowii root tubers and seeds, including trichosanthin, karasurin and T 33 from root tubers and trichosanthrip, trichokirin, alpha-kirilowin, beta-kirilowin and trichoanguin from seeds. The aforementioned proteins show structural and functional similarities. Among them trichosanthin is the best known and most intensely studied. Trichosanthin manifests anticancer activity in vitro and in tumor bearing mice against a variety of cancers/cancer cell lines. It also exhibits anti-HIV-1 and anti-HSV-1 activities. Trichosanthin has been found to be useful for treatment of cesarean scar pregnancies and ectopic pregnancy, and for preventing acute rejection of major histocompatibility complex-mismatched mouse skin allograft. Trichosanthin selectively lesions some neurons and thus can be used in neuroscience research.

  12. Rmt1 catalyzes zinc-finger independent arginine methylation of ribosomal protein Rps2 in Saccharomyces cerevisiae

    SciTech Connect

    Lipson, Rebecca S.; Webb, Kristofor J.; Clarke, Steven G.

    2010-01-22

    Rps2/rpS2 is a well conserved protein of the eukaryotic ribosomal small subunit. Rps2 has previously been shown to contain asymmetric dimethylarginine residues, the addition of which is catalyzed by zinc-finger-containing arginine methyltransferase 3 (Rmt3) in the fission yeast Schizosaccharomyces pombe and protein arginine methyltransferase 3 (PRMT3) in mammalian cells. Here, we demonstrate that despite the lack of a zinc-finger-containing homolog of Rmt3/PRMT3 in the budding yeast Saccharomyces cerevisiae, Rps2 is partially modified to generate asymmetric dimethylarginine and monomethylarginine residues. We find that this modification of Rps2 is dependent upon the major arginine methyltransferase 1 (Rmt1) in S. cerevisiae. These results are suggestive of a role for Rmt1 in modifying the function of Rps2 in a manner distinct from that occurring in S. pombe and mammalian cells.

  13. A proteogenomics approach integrating proteomics and ribosome profiling increases the efficiency of protein identification and enables the discovery of alternative translation start sites.

    PubMed

    Koch, Alexander; Gawron, Daria; Steyaert, Sandra; Ndah, Elvis; Crappé, Jeroen; De Keulenaer, Sarah; De Meester, Ellen; Ma, Ming; Shen, Ben; Gevaert, Kris; Van Criekinge, Wim; Van Damme, Petra; Menschaert, Gerben

    2014-12-01

    Next-generation transcriptome sequencing is increasingly integrated with MS to enhance MS-based protein and peptide identification. Recently, a breakthrough in transcriptome analysis was achieved with the development of ribosome profiling (ribo-seq). This technology is based on the deep sequencing of ribosome-protected mRNA fragments, thereby enabling the direct observation of in vivo protein synthesis at the transcript level. In order to explore the impact of a ribo-seq-derived protein sequence search space on MS/MS spectrum identification, we performed a comprehensive proteome study on a human cancer cell line, using both shotgun and N-terminal proteomics, next to ribosome profiling, which was used to delineate (alternative) translational reading frames. By including protein-level evidence of sample-specific genetic variation and alternative translation, this strategy improved the identification score of 69 proteins and identified 22 new proteins in the shotgun experiment. Furthermore, we discovered 18 new alternative translation start sites in the N-terminal proteomics data and observed a correlation between the quantitative measures of ribo-seq and shotgun proteomics with a Pearson correlation coefficient ranging from 0.483 to 0.664. Overall, this study demonstrated the benefits of ribosome profiling for MS-based protein and peptide identification and we believe this approach could develop into a common practice for next-generation proteomics.

  14. Mitochondrial Ribosomal Protein L12 Is Required for POLRMT Stability and Exists as Two Forms Generated by Alternative Proteolysis during Import*

    PubMed Central

    Nouws, Jessica; Goswami, Arvind V.; Bestwick, Megan; McCann, Beverly Jo; Surovtseva, Yulia V.; Shadel, Gerald S.

    2016-01-01

    To translate the 13 mtDNA-encoded mRNAs involved in oxidative phosphorylation (OXPHOS), mammalian mitochondria contain a dedicated set of ribosomes comprising rRNAs encoded by the mitochondrial genome and mitochondrial ribosomal proteins (MRPs) that are encoded by nuclear genes and imported into the matrix. In addition to their role in the ribosome, several MRPs have auxiliary functions or have been implicated in other cellular processes like cell cycle regulation and apoptosis. For example, we have shown that human MRPL12 binds and activates mitochondrial RNA polymerase (POLRMT), and hence has distinct functions in the ribosome and mtDNA transcription. Here we provide concrete evidence that there are two mature forms of mammalian MRPL12 that are generated by a two-step cleavage during import, involving efficient cleavage by mitochondrial processing protease and a second inefficient or regulated cleavage by mitochondrial intermediate protease. We also show that knock-down of MRPL12 by RNAi results in instability of POLRMT, but not other primary mitochondrial transcription components, and a corresponding decrease in mitochondrial transcription rates. Knock-down of MRPL10, the binding partner of MRPL12 in the ribosome, results in selective degradation of the mature long form of MRPL12, but has no effect on POLRMT. We propose that the two forms of MRPL12 are involved in homeostatic regulation of mitochondrial transcription and ribosome biogenesis that likely contribute to cell cycle, growth regulation, and longevity pathways to which MRPL12 has been linked. PMID:26586915

  15. Mitochondrial Ribosomal Protein L12 Is Required for POLRMT Stability and Exists as Two Forms Generated by Alternative Proteolysis during Import.

    PubMed

    Nouws, Jessica; Goswami, Arvind V; Bestwick, Megan; McCann, Beverly Jo; Surovtseva, Yulia V; Shadel, Gerald S

    2016-01-08

    To translate the 13 mtDNA-encoded mRNAs involved in oxidative phosphorylation (OXPHOS), mammalian mitochondria contain a dedicated set of ribosomes comprising rRNAs encoded by the mitochondrial genome and mitochondrial ribosomal proteins (MRPs) that are encoded by nuclear genes and imported into the matrix. In addition to their role in the ribosome, several MRPs have auxiliary functions or have been implicated in other cellular processes like cell cycle regulation and apoptosis. For example, we have shown that human MRPL12 binds and activates mitochondrial RNA polymerase (POLRMT), and hence has distinct functions in the ribosome and mtDNA transcription. Here we provide concrete evidence that there are two mature forms of mammalian MRPL12 that are generated by a two-step cleavage during import, involving efficient cleavage by mitochondrial processing protease and a second inefficient or regulated cleavage by mitochondrial intermediate protease. We also show that knock-down of MRPL12 by RNAi results in instability of POLRMT, but not other primary mitochondrial transcription components, and a corresponding decrease in mitochondrial transcription rates. Knock-down of MRPL10, the binding partner of MRPL12 in the ribosome, results in selective degradation of the mature long form of MRPL12, but has no effect on POLRMT. We propose that the two forms of MRPL12 are involved in homeostatic regulation of mitochondrial transcription and ribosome biogenesis that likely contribute to cell cycle, growth regulation, and longevity pathways to which MRPL12 has been linked.

  16. Cryo-EM structure of the tetracycline resistance protein TetM in complex with a translating ribosome at 3.9-Å resolution

    PubMed Central

    Arenz, Stefan; Nguyen, Fabian; Beckmann, Roland; Wilson, Daniel N.

    2015-01-01

    Ribosome protection proteins (RPPs) confer resistance to tetracycline by binding to the ribosome and chasing the drug from its binding site. Current models for RPP action are derived from 7.2- to 16-Å resolution structures of RPPs bound to vacant or nontranslating ribosomes. Here we present a cryo-electron microscopy reconstruction of the RPP TetM in complex with a translating ribosome at 3.9-Å resolution. The structure reveals the contacts of TetM with the ribosome, including interaction between the conserved and functionally critical C-terminal extension of TetM with a unique splayed conformation of nucleotides A1492 and A1493 at the decoding center of the small subunit. The resolution enables us to unambiguously model the side chains of the amino acid residues comprising loop III in domain IV of TetM, revealing that the tyrosine residues Y506 and Y507 are not responsible for drug-release as suggested previously but rather for intrafactor contacts that appear to stabilize the conformation of loop III. Instead, Pro509 at the tip of loop III is located directly within the tetracycline binding site where it interacts with nucleotide C1054 of the 16S rRNA, such that RPP action uses Pro509, rather than Y506/Y507, to directly dislodge and release tetracycline from the ribosome. PMID:25870267

  17. Organization and nucleotide sequences of the Spiroplasma citri genes for ribosomal protein S2, elongation factor Ts, spiralin, phosphofructokinase, pyruvate kinase, and an unidentified protein.

    PubMed Central

    Chevalier, C; Saillard, C; Bové, J M

    1990-01-01

    The gene for spiralin, the major membrane protein of the helical mollicute Spiroplasma citri, was cloned in Escherichia coli as a 5-kilobase-pair (kbp) DNA fragment. The complete nucleotide sequence of the 5.0-kbp spiroplasmal DNA fragment was determined (GenBank accession no. M31161). The spiralin gene was identified by the size and amino acid composition of its translational product. Besides the spiralin gene, the spiroplasmal DNA fragment was found to contain five additional open reading frames (ORFs). The translational products of four of these ORFs were identified by their amino acid sequence homologies with known proteins: ribosomal protein S2, elongation factor Ts, phosphofructokinase, and pyruvate kinase, respectively encoded by the genes rpsB, tsf, pfk, and pyk. The product of the fifth ORF remains to be identified and was named protein X (X gene). The order of the above genes was tsf--X--spiralin gene--pfk--pyk. These genes were transcribed in one direction, while the gene for ribosomal protein S2 (rpsB) was transcribed in the opposite direction. Images PMID:2139649

  18. Dengue virus NS1 protein interacts with the ribosomal protein RPL18: this interaction is required for viral translation and replication in Huh-7 cells.

    PubMed

    Cervantes-Salazar, Margot; Angel-Ambrocio, Antonio H; Soto-Acosta, Ruben; Bautista-Carbajal, Patricia; Hurtado-Monzon, Arianna M; Alcaraz-Estrada, Sofia L; Ludert, Juan E; Del Angel, Rosa M

    2015-10-01

    Given dengue virus (DENV) genome austerity, it uses cellular molecules and structures for virion entry, translation and replication of the genome. NS1 is a multifunctional protein key to viral replication and pathogenesis. Identification of cellular proteins that interact with NS1 may help in further understanding the functions of NS1. In this paper we isolated a total of 64 proteins from DENV infected human hepatic cells (Huh-7) that interact with NS1 by affinity chromatography and immunoprecipitation assays. The subcellular location and expression levels during infection of the ribosomal proteins RPS3a, RPL7, RPL18, RPL18a plus GAPDH were determined. None of these proteins changed their expression levels during infection; however, RPL-18 was redistributed to the perinuclear region after 48hpi. Silencing of the RPL-18 does not affect cell translation efficiency or viability, but it reduces significantly viral translation, replication and viral yield, suggesting that the RPL-18 is required during DENV replicative cycle.

  19. On the Spatial Organization of mRNA, Plasmids, and Ribosomes in a Bacterial Host Overexpressing Membrane Proteins

    PubMed Central

    Robinson, Andrew; Poolman, Bert; Kok, Jan

    2016-01-01

    By using fluorescence imaging, we provide a time-resolved single-cell view on coupled defects in transcription, translation, and growth during expression of heterologous membrane proteins in Lactococcus lactis. Transcripts encoding poorly produced membrane proteins accumulate in mRNA-dense bodies at the cell poles, whereas transcripts of a well-expressed homologous membrane protein show membrane-proximal localization in a translation-dependent fashion. The presence of the aberrant polar mRNA foci correlates with cessation of cell division, which is restored once these bodies are cleared. In addition, activation of the heat-shock response and a loss of nucleoid-occluded ribosomes are observed. We show that the presence of a native-like N-terminal domain is key to SRP-dependent membrane localization and successful production of membrane proteins. The work presented gives new insights and detailed understanding of aberrant membrane protein biogenesis, which can be used for strategies to optimize membrane protein production. PMID:27977669

  20. Substitution of histidine-137 by glutamine abolishes the catalytic activity of the ribosome-inactivating protein alpha-sarcin.

    PubMed Central

    Lacadena, J; Mancheño, J M; Martinez-Ruiz, A; Martínez del Pozo, A; Gasset, M; Oñaderra, M; Gavilanes, J G

    1995-01-01

    The alpha-sarcin cytotoxin is an extracellular fungal protein that inhibits protein biosynthesis by specifically cleaving one phosphodiester bond of the 28 S rRNA. The His137 residue of alpha-sarcin is suggested to be involved in the catalytic activity of this protein, based on the observed sequence similarity with some fungal ribonucleases. Replacement of this residue by Gln (H137Q mutant variant of alpha-sarcin) abolishes the ribonuclease activity of the protein. This has been demonstrated for an homogeneous preparation of the H137Q alpha-sarcin by measuring its effect against both intact rabbit ribosomes and the homopolymer poly(A). The conformation of H137Q alpha-sarcin is highly similar to that of the wild-type protein, which has been analysed by CD and fluorescence spectroscopy. Both H137Q and wild-type alpha-sarcin exhibit identical CD spectra in the peptide-bond region, indicating that no changes at the level of the secondary structure are produced upon mutation. Only minor differences are observed in both near-UV CD and fluorescence emission spectra in comparison to those of the wild-type protein. Moreover, H137Q alpha-sarcin interacts with phospholipid vesicles, promoting the same effects as the native cytotoxin. Therefore, we propose that His137 is part of the ribonucleolytic active site of the cytotoxin alpha-sarcin. Images Figure 4 PMID:7626023

  1. Role of the ribosome-associated protein PY in the cold-shock response of Escherichia coli

    PubMed Central

    Di Pietro, Fabio; Brandi, Anna; Dzeladini, Nadire; Fabbretti, Attilio; Carzaniga, Thomas; Piersimoni, Lolita; Pon, Cynthia L; Giuliodori, Anna Maria

    2013-01-01

    Protein Y (PY) is an Escherichia coli cold-shock protein which has been proposed to be responsible for the repression of bulk protein synthesis during cold adaptation. Here, we present in vivo and in vitro data which clarify the role of PY and its mechanism of action. Deletion of yfiA, the gene encoding protein PY, demonstrates that this protein is dispensable for cold adaptation and is not responsible for the shutdown of bulk protein synthesis at the onset of the stress, although it is able to partially inhibit translation. In vitro assays reveal that the extent of PY inhibition changes with different mRNAs and that this inhibition is related to the capacity of PY of binding 30S subunits with a fairly strong association constant, thus stimulating the formation of 70S monomers. Furthermore, our data provide evidence that PY competes with the other ribosomal ligands for the binding to the 30S subunits. Overall these results suggest an alternative model to explain PY function during cold shock and to reconcile the inhibition caused by PY with the active translation observed for some mRNAs during cold shock. PMID:23420694

  2. Selective translational control and nonspecific posttranscriptional regulation of ribosomal protein gene expression during development and regeneration of rat liver.

    PubMed Central

    Aloni, R; Peleg, D; Meyuhas, O

    1992-01-01

    Mammalian liver development is accompanied by a transition from rapid growth in the fetus to a quiescent state in the adult. However, extensive proliferation can be induced in the adult liver by partial hepatectomy. In this study, we examined the regulation of ribosomal protein (rp) gene expression in the developing and regenerating rat liver. Our results indicate that the translation of rp mRNAs is selectively repressed by about 70% upon development from fetal to adult life, as illustrated by the decrease in ribosomal loading. In addition, the relative abundance of these mRNAs, like that of several other, but not all, housekeeping mRNAs, declines during development through a posttranscriptional mechanism. When liver cells commence growth following partial hepatectomy, translation of rp mRNAs is resumed to near-maximal capacity, as judged by their very efficient recruitment into polysomes. The concomitant increase in the abundance rp mRNAs under these circumstances is achieved by a posttranscriptional mechanism. The apparent fluctuations in the translation efficiency of rp mRNAs are accompanied by parallel changes in the expression of the genes encoding the initiation factors eIF-4E and eIF-4A. Our results indicate that selective translational control of rp mRNAs in mammals is not confined to manipulated cells in culture but constitutes an important regulatory mechanism operating in vivo in the course of liver development and regeneration. Images PMID:1373810

  3. TORC1 and TORC2 work together to regulate ribosomal protein S6 phosphorylation in Saccharomyces cerevisiae

    PubMed Central

    Yerlikaya, Seda; Meusburger, Madeleine; Kumari, Romika; Huber, Alexandre; Anrather, Dorothea; Costanzo, Michael; Boone, Charles; Ammerer, Gustav; Baranov, Pavel V.; Loewith, Robbie

    2016-01-01

    Nutrient-sensitive phosphorylation of the S6 protein of the 40S subunit of the eukaryote ribosome is highly conserved. However, despite four decades of research, the functional consequences of this modification remain unknown. Revisiting this enigma in Saccharomyces cerevisiae, we found that the regulation of Rps6 phosphorylation on Ser-232 and Ser-233 is mediated by both TOR complex 1 (TORC1) and TORC2. TORC1 regulates phosphorylation of both sites via the poorly characterized AGC-family kinase Ypk3 and the PP1 phosphatase Glc7, whereas TORC2 regulates phosphorylation of only the N-terminal phosphosite via Ypk1. Cells expressing a nonphosphorylatable variant of Rps6 display a reduced growth rate and a 40S biogenesis defect, but these phenotypes are not observed in cells in which Rps6 kinase activity is compromised. Furthermore, using polysome profiling and ribosome profiling, we failed to uncover a role of Rps6 phosphorylation in either global translation or translation of individual mRNAs. Taking the results together, this work depicts the signaling cascades orchestrating Rps6 phosphorylation in budding yeast, challenges the notion that Rps6 phosphorylation plays a role in translation, and demonstrates that observations made with Rps6 knock-ins must be interpreted cautiously. PMID:26582391

  4. Molecular dissection of the silkworm ribosomal stalk complex: the role of multiple copies of the stalk proteins

    PubMed Central

    Baba, Kentaro; Tumuraya, Kazuhiro; Tanaka, Isao; Yao, Min; Uchiumi, Toshio

    2013-01-01

    In animal ribosomes, two stalk proteins P1 and P2 form a heterodimer, and the two dimers, with the anchor protein P0, constitute a pentameric complex crucial for recruitment of translational GTPase factors to the ribosome. To investigate the functional contribution of each copy of the stalk proteins, we constructed P0 mutants, in which one of the two C-terminal helices, namely helix I (N-terminal side) or helix II (C-terminal side) were unable to bind the P1–P2 dimer. We also constructed ‘one-C-terminal domain (CTD) stalk dimers’, P1–P2ΔC and P1ΔC–P2, composed of intact P1/P2 monomer and a CTD-truncated partner. Through combinations of P0 and P1–P2 variants, various complexes were reconstituted and their function tested in eEF-2-dependent GTPase and eEF-1α/eEF-2-dependent polyphenylalanine synthesis assays in vitro. Double/single-CTD dimers bound to helix I showed higher activity than that bound to helix II. Despite low polypeptide synthetic activity by a single one-CTD dimer, its binding to both helices considerably increased activity, suggesting that two stalk dimers cooperate, particularly in polypeptide synthesis. This promotion of activity by two stalk dimers was lost upon mutation of the conserved YPT sequence connecting the two helices of P0, suggesting a role for this sequence in cooperativity of two stalk dimers. PMID:23376928

  5. GTPases involved in bacterial ribosome maturation.

    PubMed

    Goto, Simon; Muto, Akira; Himeno, Hyouta

    2013-05-01

    The ribosome is an RNA- and protein-based macromolecule having multiple functional domains to facilitate protein synthesis, and it is synthesized through multiple steps including transcription, stepwise cleavages of the primary transcript, modifications of ribosomal proteins and RNAs and assemblies of ribosomal proteins with rRNAs. This process requires dozens of trans-acting factors including GTP- and ATP-binding proteins to overcome several energy-consuming steps. Despite accumulation of genetic, biochemical and structural data, the entire process of bacterial ribosome synthesis remains elusive. Here, we review GTPases involved in bacterial ribosome maturation.

  6. Involvement of the N Terminus of Ribosomal Protein L11 in Regulation of the RelA Protein of Escherichia coli†

    PubMed Central

    Yang, Xiaoming; Ishiguro, Edward E.

    2001-01-01

    Amino acid-deprived rplK (previously known as relC) mutants of Escherichia coli cannot activate (p)ppGpp synthetase I (RelA) and consequently exhibit relaxed phenotypes. The rplK gene encodes ribosomal protein L11, suggesting that L11 is involved in regulating the activity of RelA. To investigate the role of L11 in the stringent response, a derivative of rplK encoding L11 lacking the N-terminal 36 amino acids (designated ′L11) was constructed. Bacteria overexpressing ′L11 exhibited a relaxed phenotype, and this was associated with an inhibition of RelA-dependent (p)ppGpp synthesis during amino acid deprivation. In contrast, bacteria overexpressing normal L11 exhibited a typical stringent response. The overexpressed ′L11 was incorporated into ribosomes and had no effect on the ribosome-binding activity of RelA. By several methods (yeast two-hybrid, affinity blotting, and copurification), no direct interaction was observed between the C-terminal ribosome-binding domain of RelA and L11. To determine whether the proline-rich helix of L11 was involved in RelA regulation, the Pro-22 residue was replaced with Leu by site-directed mutagenesis. The overexpression of the Leu-22 mutant derivative of L11 resulted in a relaxed phenotype. These results indicate that the proline-rich helix in the N terminus of L11 is involved in regulating the activity of RelA. PMID:11673421

  7. Ribosomal Protein S12 and Aminoglycoside Antibiotics Modulate A-site mRNA Cleavage and Transfer-Messenger RNA Activity in Escherichia coli*

    PubMed Central

    Holberger, Laura E.; Hayes, Christopher S.

    2009-01-01

    Translational pausing in Escherichia coli can lead to mRNA cleavage within the ribosomal A-site. A-site mRNA cleavage is thought to facilitate transfer-messenger RNA (tmRNA)·SmpB- mediated recycling of stalled ribosome complexes. Here, we demonstrate that the aminoglycosides paromomycin and streptomycin inhibit A-site cleavage of stop codons during inefficient translation termination. Aminoglycosides also induced stop codon read-through, suggesting that these antibiotics alleviate ribosome pausing during termination. Streptomycin did not inhibit A-site cleavage in rpsL mutants, which express streptomycin-resistant variants of ribosomal protein S12. However, rpsL strains exhibited reduced A-site mRNA cleavage compared with rpsL+ cells. Additionally, tmRNA·SmpB-mediated SsrA peptide tagging was significantly reduced in several rpsL strains but could be fully restored in a subset of mutants when treated with streptomycin. The streptomycin-dependent rpsL(P90K) mutant also showed significantly lower levels of A-site cleavage and tmRNA·SmpB activity. Mutations in rpsD (encoding ribosomal protein S4), which suppressed streptomycin dependence, were able to partially restore A-site cleavage to rpsL(P90K) cells but failed to increase tmRNA·SmpB activity. Taken together, these results show that perturbations to A-site structure and function modulate A-site mRNA cleavage and tmRNA·SmpB activity. We propose that tmRNA·SmpB binds to streptomycin-resistant rpsL ribosomes less efficiently, leading to a partial loss of ribosome rescue function in these mutants. PMID:19776006

  8. Enhanced resistance to blast fungus in rice (Oryza sativa L.) by expressing the ribosome-inactivating protein α-momorcharin.

    PubMed

    Qian, Qian; Huang, Lin; Yi, Rong; Wang, Shuzhen; Ding, Yi

    2014-03-01

    Rice blast caused by Magnaporthe grisea is one of the three major diseases that seriously affect the rice production. Alpha-momorcharin (α-MC), a ribosome-inactivating protein (RIP) isolated from Momordica charantia seeds, has antifungal effects in vitro. In this study, the α-MC gene was constitutively expressed under the control of the 2×35S promoter in transgenic rice (Oryza sativa L.) using an Agrobacterium tumefaciens-mediated method. The nine transgenic plants were obtained and confirmed by PCR and RT-PCR, and the four (B2, B4, B7 and B9) of them whose copy numbers were 1, 2, 3 and 3, respectively, were shown to express the α-MC protein by Western blot. The molecular weight of α-MC in transgenic plants was approximately 38 kDa larger than the purified α-MC protein (28 kDa) in vitro. When the confirmed T1 generations were inoculated with a suspension of M. grisea spores for ten days, the lesions on leaves of transgenic plants were much lesser than those found on wild type (WT). According to the criteria of International Rice Research Institute standard, the mean values for morbidity and disease index numbers were 29.8% and 14.9%, respectively, which were lower than for WT. It is unclear whether RIPs could impact plant fitness and however our results suggest that the α-MC protein is an effective antifungal protein preventing rice blast in transgenic rice.

  9. Ribosome recycling induces optimal translation rate at low ribosomal availability.

    PubMed

    Marshall, E; Stansfield, I; Romano, M C

    2014-09-06

    During eukaryotic cellular protein synthesis, ribosomal translation is made more efficient through interaction between the two ends of the messenger RNA (mRNA). Ribosomes reaching the 3' end of the mRNA can thus recycle and begin translation again on the same mRNA, the so-called 'closed-loop' model. Using a driven diffusion lattice model of translation, we study the effects of ribosome recycling on the dynamics of ribosome flow and density on the mRNA. We show that ribosome recycling induces a substantial increase in ribosome current. Furthermore, for sufficiently large values of the recycling rate, the lattice does not transition directly from low to high ribosome density, as seen in lattice models without recycling. Instead, a maximal current phase becomes accessible for much lower values of the initiation rate, and multiple phase transitions occur over a wide region of the phase plane. Crucially, we show that in the presence of ribosome recycling, mRNAs can exhibit a peak in protein production at low values of the initiation rate, beyond which translation rate decreases. This has important implications for translation of certain mRNAs, suggesting that there is an optimal concentration of ribosomes at which protein synthesis is maximal, and beyond which translational efficiency is impaired.

  10. Crystallization of the two-domain N-terminal fragment of the archaeal ribosomal protein L10(P0) in complex with a specific fragment of 23S rRNA

    SciTech Connect

    Kravchenko, O. V.; Mitroshin, I. V.; Gabdulkhakov, A. G.; Nikonov, S. V.; Garber, M. B.

    2011-07-15

    Lateral L12-stalk (P1-stalk in Archaea, P1/P2-stalk in eukaryotes) is an obligatory morphological element of large ribosomal subunits in all organisms studied. This stalk is composed of the complex of ribosomal proteins L10(P0) and L12(P1) and interacts with 23S rRNA through the protein L10(P0). L12(P1)-stalk is involved in the formation of GTPase center of the ribosome and plays an important role in the ribosome interaction with translation factors. High mobility of this stalk puts obstacles in determination of its structure within the intact ribosome. Crystals of a two-domain N-terminal fragment of ribosomal protein L10(P0) from the archaeon Methanococcus jannaschii in complex with a specific fragment of rRNA from the same organism have been obtained. The crystals diffract X-rays at 3.2 Angstrom-Sign resolution.

  11. Nuclear-encoded chloroplast ribosomal protein L12 of Nicotiana tabacum: characterization of mature protein and isolation and sequence analysis of cDNA clones encoding its cytoplasmic precursor.

    PubMed Central

    Elhag, G A; Thomas, F J; McCreery, T P; Bourque, D P

    1992-01-01

    Poly(A)+ mRNA isolated from Nicotiana tabacum (cv. Petite Havana) leaves was used to prepare a cDNA library in the expression vector lambda gt11. Recombinant phage containing cDNAs coding for chloroplast ribosomal protein L12 were identified and sequenced. Mature tobacco L12 protein has 44% amino acid identity with ribosomal protein L7/L12 of Escherichia coli. The longest L12 cDNA (733 nucleotides) codes for a 13,823 molecular weight polypeptide with a transit peptide of 53 amino acids and a mature protein of 133 amino acids. The transit peptide and mature protein share 43% and 79% amino acid identity, respectively, with corresponding regions of spinach chloroplast ribosomal protein L12. The predicted amino terminus of the mature protein was confirmed by partial sequence analysis of HPLC-purified tobacco chloroplast ribosomal protein L12. A single L12 mRNA of about 0.8 kb was detected by hybridization of L12 cDNA to poly(A)+ and total leaf RNA. Hybridization patterns of restriction fragments of tobacco genomic DNA probed with the L12 cDNA suggested the existence of more than one gene for ribosomal protein L12. Characterization of a second cDNA with an identical L12 coding sequence but a different 3'-noncoding sequence provided evidence that at least two L12 genes are expressed in tobacco. Images PMID:1542565

  12. Glucose triggers different global responses in yeast, depending on the strength of the signal, and transiently stabilizes ribosomal protein mRNAs.

    PubMed

    Yin, Zhikang; Wilson, Séan; Hauser, Nicole C; Tournu, Helene; Hoheisel, Jörg D; Brown, Alistair J P

    2003-05-01

    Glucose exerts profound effects upon yeast physiology. In general, the effects of high glucose concentrations (>1%) upon Saccharomyces cerevisiae have been studied. In this paper, we have characterized the global responses of yeast cells to very low (0.01%), low (0.1%) and high glucose signals (1.0%) by transcript profiling. We show that yeast is more sensitive to very low glucose signals than was previously thought, and that yeast displays different responses to these different glucose signals. Genes involved in central metabolic pathways respond rapidly to very low glucose signals, whereas genes involved in the biogenesis of cytoplasmic ribosomes generally respond only to glucose concentrations of> 0.1%. We also show that cytoplasmic ribosomal protein mRNAs are transiently stabilized by glucose, indicating that both transcriptional and post-transcriptional mechanisms combine to accelerate the accumulation of ribosomal protein mRNAs. Presumably, this facilitates rapid ribosome biogenesis after exposure to glucose. However, our data indicate that yeast activates ribosome biogenesis only when sufficient glucose is available to make this metabolic investment worthwhile. In contrast, the regulation of metabolic functions in response to very low glucose signals presumably ensures that yeast can exploit even minute amounts of this preferred nutrient.

  13. Lyar, a cell growth-regulating zinc finger protein, was identified to be associated with cytoplasmic ribosomes in male germ and cancer cells.

    PubMed

    Yonezawa, Kahori; Sugihara, Yoshihiko; Oshima, Kenzi; Matsuda, Tsukasa; Nadano, Daita

    2014-10-01

    Translational control is a basic mechanism for gene regulation in cells and important for tissue growth and development in mammals. Deregulation of the mechanism thus causes diseases such as cancer. Considering the importance of the ribosome as a factory of polypeptide synthesis, some new factors have been expected to be associated with the ribosome and involved in translational control. Our proteomic survey for these factors identified a zinc finger protein, Lyar, in cytoplasmic ribosomes of the rodent testis. Subcellular fractionation of the testis provided data supporting association of Lyar with ribosomes. Lyar was then suggested to be included in the 60S large subunit, but not in polysomes, by ultracentrifugation of testicular ribosomes. While analysis of tissue distribution of Lyar has indicated its testis-predominant expression, Lyar mRNA was expressed in the cancer cells originated from tissues other than testis, and Lyar promoted proliferation of NIH-3T3 cells. Furthermore, translation was increased by Lyar in vitro, pointing out the first experimental link between this protein and translation. Taken together, Lyar seems to be a new player in translational control and a potential target for cancer therapy.

  14. Structural insights into ribosome translocation

    PubMed Central

    Ling, Clarence

    2016-01-01

    During protein synthesis, tRNA and mRNA are translocated from the A to P to E sites of the ribosome thus enabling the ribosome to translate one codon of mRNA after the other. Ribosome translocation along mRNA is induced by the universally conserved ribosome GTPase, elongation factor G (EF‐G) in bacteria and elongation factor 2 (EF‐2) in eukaryotes. Recent structural and single‐molecule studies revealed that tRNA and mRNA translocation within the ribosome is accompanied by cyclic forward and reverse rotations between the large and small ribosomal subunits parallel to the plane of the intersubunit interface. In addition, during ribosome translocation, the ‘head’ domain of small ribosomal subunit undergoes forward‐ and back‐swiveling motions relative to the rest of the small ribosomal subunit around the axis that is orthogonal to the axis of intersubunit rotation. tRNA/mRNA translocation is also coupled to the docking of domain IV of EF‐G into the A site of the small ribosomal subunit that converts the thermally driven motions of the ribosome and tRNA into the forward translocation of tRNA/mRNA inside the ribosome. Despite recent and enormous progress made in the understanding of the molecular mechanism of ribosome translocation, the sequence of structural rearrangements of the ribosome, EF‐G and tRNA during translocation is still not fully established and awaits further investigation. WIREs RNA 2016, 7:620–636. doi: 10.1002/wrna.1354 For further resources related to this article, please visit the WIREs website. PMID:27117863

  15. Ribosomal acidic phosphoproteins P1 and P2 are not required for cell viability but regulate the pattern of protein expression in Saccharomyces cerevisiae.

    PubMed Central

    Remacha, M; Jimenez-Diaz, A; Bermejo, B; Rodriguez-Gabriel, M A; Guarinos, E; Ballesta, J P

    1995-01-01

    Saccharomyces cerevisiae strains with either three inactivated genes (triple disruptants) or four inactivated genes (quadruple disruptants) encoding the four acidic ribosomal phosphoproteins, YP1 alpha, YP1 beta, YP2 alpha, and YP2 beta, present in this species have been obtained. Ribosomes from the triple disruptants and, obviously, those from the quadruple strain do not have bound P proteins. All disrupted strains are viable; however, they show a cold-sensitive phenotype, growing very poorly at 23 degrees C. Cell extracts from the quadruple-disruptant strain are about 30% as active as the control in protein synthesis assays and are stimulated by the addition of free acidic P proteins. Strains lacking acidic proteins do not have a higher suppressor activity than the parental strains, and cell extracts derived from the quadruple disruptant do not show a higher degree of misreading, indicating that the absence of acidic proteins does not affect the accuracy of the ribosomes. However, the patterns of protein expressed in the cells as well as in the cell-free protein system are affected by the absence of P proteins from the particles; a wild-type pattern is restored upon addition of exogenous P proteins to the cell extract. In addition, strains carrying P-protein-deficient ribosomes are unable to sporulate but recover this capacity upon transformation with one of the missing genes. These results indicate that acidic proteins are not an absolute requirement for protein synthesis but regulate the activity of the 60S subunit, affecting the translation of certain mRNAs differently. PMID:7651393

  16. Ribosomal Protein S3 Negatively Regulates Unwinding Activity of RecQ-like Helicase 4 through Their Physical Interaction.

    PubMed

    Patil, Ajay Vitthal; Hsieh, Tao-Shih

    2017-03-10

    Human RecQ-like helicase 4 (RECQL4) plays crucial roles in replication initiation and DNA repair; however, the contextual regulation of its unwinding activity is not fully described. Mutations in RECQL4 have been linked to three diseases including Rothmund-Thomson syndrome, which is characterized by osteoskeletal deformities, photosensitivity, and increased osteosarcoma susceptibility. Understanding regulation of RECQL4 helicase activity by interaction partners will allow deciphering its role as an enzyme and a signaling cofactor in different cellular contexts. We became interested in studying the interaction of RECQL4 with ribosomal protein S3 (RPS3) because previous studies have shown that RPS3 activity is sometimes associated with phenotypes mimicking those of mutated RECQL4. RPS3 is a small ribosomal protein that also has extraribosomal functions, including apurnic-apyrimidinic endonuclease-like activity suggested to be important during DNA repair. Here, we report a functional and physical interaction between RPS3 and RECQL4 and show that this interaction may be enhanced during cellular stress. We show that RPS3 inhibits ATPase, DNA binding, and helicase activities of RECQL4 through their direct interaction. Further domain analysis shows that N-terminal 1-320 amino acids of RECQL4 directly interact with the C-terminal 94-244 amino acids of RPS3 (C-RPS3). Biochemical analysis of C-RPS3 revealed that it comprises a standalone apurnic-apyrimidinic endonuclease-like domain. We used U2OS cells to show that oxidative stress and UV exposure could enhance the interaction between nuclear RPS3 and RECQL4. Regulation of RECQL4 biochemical activities by RPS3 along with nuclear interaction during UV and oxidative stress may serve to modulate active DNA repair.

  17. Retention of functional genes for S19 ribosomal protein in both the mitochondrion and nucleus for over 60 million years.

    PubMed

    Atluri, Sruthi; Rampersad, Sarah N; Bonen, Linda

    2015-12-01

    Ribosomal protein genes occasionally undergo successful migration from the mitochondrion to the nucleus in flowering plants and we previously presented evidence that the S19 ribosomal protein gene (rps19) had been transferred to the nucleus in the common ancestor of Poaceae grasses. In many lineages, the mitochondrial copy was subsequently lost or pseudogenized, although in rice it was retained and the nuclear copy lost. We have now determined that functional rps19 genes are present in both the mitochondrion and nucleus in brome grass (Bromus inermis). The mitochondrion-located rps19 gene, which is immediately downstream of an rpl2 pseudogene, is transcribed and edited. The nuclear-located rps19 gene is also actively expressed and it possesses the same intron-containing hsp70-type presequence as its counterparts in other grasses, as well as shared derived amino acids within the S19 core. We conclude that this brome rps19 gene is derived from the same transfer event that occurred in the common ancestor of grasses at least 60 million years ago. In the oat lineage, a subsequent exon shuffling-type event has resulted in novel amino-terminal sequences replacing part of the hsp70 presequence, and in the barley lineage, there has been an additional DNA-mediated transfer of the mitochondrial rps19 gene and its flanking sequences, followed by relatively recent loss of the mitochondrion-located copy. The prolonged persistence of functional copies in both compartments, as evidenced by present-day brome, raises interesting questions about their respective roles.

  18. Sequence comparison and phylogenetic analysis by the Maximum Likelihood method of ribosome-inactivating proteins from angiosperms.

    PubMed

    Di Maro, Antimo; Citores, Lucía; Russo, Rosita; Iglesias, Rosario; Ferreras, José Miguel

    2014-08-01

    Ribosome-inactivating proteins (RIPs) from angiosperms are rRNA N-glycosidases that have been proposed as defence proteins against virus and fungi. They have been classified as type 1 RIPs, consisting of single-chain proteins, and type 2 RIPs, consisting of an A chain with RIP properties covalently linked to a B chain with lectin properties. In this work we have carried out a broad search of RIP sequence data banks from angiosperms in order to study their main structural characteristics and phylogenetic evolution. The comparison of the sequences revealed the presence, outside of the active site, of a novel structure that might be involved in the internal protein dynamics linked to enzyme catalysis. Also the B-chains presented another conserved structure that might function either supporting the beta-trefoil structure or in the communication between both sugar-binding sites. A systematic phylogenetic analysis of RIP sequences revealed that the most primitive type 1 RIPs were similar to that of the actual monocots (Poaceae and Asparagaceae). The primitive RIPs evolved to the dicot type 1 related RIPs (like those from Caryophyllales, Lamiales and Euphorbiales). The gene of a type 1 RIP related with the actual Euphorbiaceae type 1 RIPs fused with a double beta trefoil lectin gene similar to the actual Cucurbitaceae lectins to generate the type 2 RIPs and finally this gene underwent deletions rendering either type 1 RIPs (like those from Cucurbitaceae, Rosaceae and Iridaceae) or lectins without A chain (like those from Adoxaceae).

  19. Hypoxia Strongly Affects Mitochondrial Ribosomal Proteins and Translocases, as Shown by Quantitative Proteomics of HeLa Cells.

    PubMed

    Bousquet, Paula A; Sandvik, Joe Alexander; Arntzen, Magnus Ø; Jeppesen Edin, Nina F; Christoffersen, Stine; Krengel, Ute; Pettersen, Erik O; Thiede, Bernd

    2015-01-01

    Hypoxia is an important and common characteristic of many human tumors. It is a challenge clinically due to the correlation with poor prognosis and resistance to radiation and chemotherapy. Understanding the biochemical response to hypoxia would facilitate the development of novel therapeutics for cancer treatment. Here, we investigate alterations in gene expression in response to hypoxia by quantitative proteome analysis using stable isotope labeling with amino acids in cell culture (SILAC) in conjunction with LCMS/MS. Human HeLa cells were kept either in a hypoxic environment or under normoxic conditions. 125 proteins were found to be regulated, with maximum alteration of 18-fold. In particular, three clusters of differentially regulated proteins were identified, showing significant upregulation of glycolysis and downregulation of mitochondrial ribosomal proteins and translocases. This interaction is likely orchestrated by HIF-1. We also investigated the effect of hypoxia on the cell cycle, which shows accumulation in G1 and a prolonged S phase under these conditions. Implications. This work not only improves our understanding of the response to hypoxia, but also reveals proteins important for malignant progression, which may be targeted in future therapies.

  20. A functional RNAi screen links O-GlcNAc modification of ribosomal proteins to stress granule and processing body assembly

    PubMed Central

    Ohn, Takbum; Kedersha, Nancy; Hickman, Tyler; Tisdale, Sarah; Anderson, Paul

    2015-01-01

    Stress granules (SGs) and processing bodies (PBs) are microscopically visible ribonucleoprotein granules that cooperatively regulate the translation and decay of messenger RNA1–3. Using an RNA-mediated interference-based screen, we identify 101 human genes required for SG assembly, 39 genes required for PB assembly, and 31 genes required for coordinate SG and PB assembly. Although 51 genes encode proteins involved in mRNA translation, splicing and transcription, most are not obviously associated with RNA metabolism. We find that several components of the hexosamine biosynthetic pathway, which reversibly modifies proteins with O-linked N-acetylglucosamine (O-GlcNAc) in response to stress, are required for SG and PB assembly. O-GlcNAc-modified proteins are prominent components of SGs but not PBs, and include RACK1 (receptor for activated C kinase 1), prohibitin-2, glyceraldehyde-3-phosphate dehydrogenase and numerous ribosomal proteins. Our results suggest that O-GlcNAc modification of the translational machinery is required for aggregation of untranslated messenger ribonucleoproteins into SGs. The lack of enzymes of the hexosamine biosynthetic pathway in budding yeast may contribute to differences between mammalian SGs and related yeast EGP (eIF4E, 4G and Pab1 containing) bodies. PMID:18794846

  1. Brucella ribosomal protein L7/L12 is a major component in the antigenicity of brucellin INRA for delayed-type hypersensitivity in brucella-sensitized guinea pigs.

    PubMed Central

    Bachrach, G; Banai, M; Bardenstein, S; Hoida, G; Genizi, A; Bercovier, H

    1994-01-01

    A delayed-type hypersensitivity (DTH) reaction in the course of brucellosis in humans and animals can be revealed by the brucellin INRA (Brucellergen) skin test. Brucellergen is composed of more than 20 proteins of different molecular weights. A 12-kDa protein eliciting DTH in Brucella melitensis Rev1-sensitized guinea pigs was found to be a significant component for the allergenic properties of Brucellergen. Sequencing of the gene encoding this protein identified it as the L7/L12 ribosomal protein. The L7/L12 gene of B. melitensis was amplified by PCR and subcloned in the Escherichia coli pQE30 plasmid. The resulting recombinant protein did not produce a DTH reaction in sensitized animals. It was used to raise specific antibodies in a rabbit. Affinity chromatography with these antibodies was used to isolate a single protein from Brucellergen and from B. melitensis cytosol preparations which produced a DTH reaction in guinea pigs sensitized with B. melitensis Rev1. N-terminal amino acid sequencing of the protein confirmed that it was the L7/L12 ribosomal protein. This is the first complete report on the involvement of a defined bacterial ribosomal protein in the DTH response of animals infected with intracellularly multiplying bacteria. Images PMID:7960115

  2. gar2 is a nucleolar protein from Schizosaccharomyces pombe required for 18S rRNA and 40S ribosomal subunit accumulation.

    PubMed Central

    Gulli, M P; Girard, J P; Zabetakis, D; Lapeyre, B; Melese, T; Caizergues-Ferrer, M

    1995-01-01

    Several nucleolar proteins, such as nucleolin, NOP1/fibrillarin, SSB1, NSR1 and GAR1 share a common glycine and arginine rich structural motif called the GAR domain. To identify novel nucleolar proteins from fission yeast we screened Schizosaccharomyces pombe genomic DNA libraries with a probe encompassing the GAR structural motif. Here we report the identification and characterization of a S.pombe gene coding for a novel nucleolar protein, designated gar2. The structure of the fission yeast gar2 is reminiscent of that of nucleolin from vertebrates and NSR1 from Saccharomyces cerevisiae. In addition, like these proteins, gar2 has a nucleolar localisation. The disruption of the gar2+ gene affects normal cell growth, leads to an accumulation of 35S pre-rRNA and a decrease of mature 18S rRNA steady state levels. Moreover, ribosomal profiles of the mutant show an increase of free 60S ribosomal subunits and an absence of free 40S ribosomal subunits. gar2 is able to rescue a S.cerevisiae mutant lacking NSR1, thus establishing gar2 as a functional homolog of NSR1. We propose that gar2 helps the assembly of pre-ribosomal particles containing 18S rRNA. Images PMID:7596817

  3. Revealing pancrustacean relationships: Phylogenetic analysis of ribosomal protein genes places Collembola (springtails) in a monophyletic Hexapoda and reinforces the discrepancy between mitochondrial and nuclear DNA markers

    PubMed Central

    2008-01-01

    Background In recent years, several new hypotheses on phylogenetic relations among arthropods have been proposed on the basis of DNA sequences. One of the challenged hypotheses is the monophyly of hexapods. This discussion originated from analyses based on mitochondrial DNA datasets that, due to an unusual positioning of Collembola, suggested that the hexapod body plan evolved at least twice. Here, we re-evaluate the position of Collembola using ribosomal protein gene sequences. Results In total 48 ribosomal proteins were obtained for the collembolan Folsomia candida. These 48 sequences were aligned with sequence data on 35 other ecdysozoans. Each ribosomal protein gene was available for 25% to 86% of the taxa. However, the total sequence information was unequally distributed over the taxa and ranged between 4% and 100%. A concatenated dataset was constructed (5034 inferred amino acids in length), of which ~66% of the positions were filled. Phylogenetic tree reconstructions, using Maximum Likelihood, Maximum Parsimony, and Bayesian methods, resulted in a topology that supports monophyly of Hexapoda. Conclusion Although ribosomal proteins in general may not evolve independently, they once more appear highly valuable for phylogenetic reconstruction. Our analyses clearly suggest that Hexapoda is monophyletic. This underpins the inconsistency between nuclear and mitochondrial datasets when analyzing pancrustacean relationships. Caution is needed when applying mitochondrial markers in deep phylogeny. PMID:18366624

  4. Human AATF/Che-1 forms a nucleolar protein complex with NGDN and NOL10 required for 40S ribosomal subunit synthesis

    PubMed Central

    Bammert, Lukas; Jonas, Stefanie; Ungricht, Rosemarie; Kutay, Ulrike

    2016-01-01

    Mammalian AATF/Che-1 is essential for embryonic development, however, the underlying molecular mechanism is unclear. By immunoprecipitation of human AATF we discovered that AATF forms a salt-stable protein complex together with neuroguidin (NGDN) and NOL10, and demonstrate that the AATF-NGDN-NOL10 (ANN) complex functions in ribosome biogenesis. All three ANN complex members localize to nucleoli and display a mutual dependence with respect to protein stability. Mapping of protein-protein interaction domains revealed the importance of both the evolutionary conserved WD40 repeats in NOL10 and the UTP3/SAS10 domain in NGDN for complex formation. Functional analysis showed that the ANN complex supports nucleolar steps of 40S ribosomal subunit biosynthesis. All complex members were required for 18S rRNA maturation and their individual depletion affected the same nucleolar cleavage steps in the 5′ETS and ITS1 regions of the ribosomal RNA precursor. Collectively, we identified the ANN complex as a novel functional module supporting the nucleolar maturation of 40S ribosomal subunits. Our data help to explain the described role of AATF in cell proliferation during mouse development as well as its requirement for malignant tumor growth. PMID:27599843

  5. The integrity of the G2421-C2395 base pair in the ribosomal E-site is crucial for protein synthesis

    PubMed Central

    Koch, Miriam; Clementi, Nina; Rusca, Nicola; Vögele, Paul; Erlacher, Matthias; Polacek, Norbert

    2015-01-01

    During the elongation cycle of protein biosynthesis, tRNAs traverse through the ribosome by consecutive binding to the 3 ribosomal binding sites (A-, P-, and E- sites). While the ribosomal A- and P-sites have been functionally well characterized in the past, the contribution of the E-site to protein biosynthesis is still poorly understood in molecular terms. Previous studies suggested an important functional interaction of the terminal residue A76 of E-tRNA with the nucleobase of the universally conserved 23S rRNA residue C2394. Using an atomic mutagenesis approach to introduce non-natural nucleoside analogs into the 23S rRNA, we could show that removal of the nucleobase or the ribose 2'-OH at C2394 had no effect on protein synthesis. On the other hand, our data disclose the importance of the highly conserved E-site base pair G2421-C2395 for effective translation. Ribosomes with a disrupted G2421-C2395 base pair are defective in tRNA binding to the E-site. This results in an impaired translation of genuine mRNAs, while homo-polymeric templates are not affected. Cumulatively our data emphasize the importance of E-site tRNA occupancy and in particular the intactness of the 23S rRNA base pair G2421-C2395 for productive protein biosynthesis. PMID:25826414

  6. Arginine residues on the opposite side of the active site stimulate the catalysis of ribosome depurination by ricin A chain by interacting with the P-protein stalk.

    PubMed

    Li, Xiao-Ping; Kahn, Peter C; Kahn, Jennifer Nielsen; Grela, Przemyslaw; Tumer, Nilgun E

    2013-10-18

    Ricin inhibits protein synthesis by depurinating the α-sarcin/ricin loop (SRL). Ricin holotoxin does not inhibit translation unless the disulfide bond between the A (RTA) and B (RTB) subunits is reduced. Ricin holotoxin did not bind ribosomes or depurinate them but could depurinate free RNA. When RTA is separated from RTB, arginine residues located at the interface are exposed to the solvent. Because this positively charged region, but not the active site, is blocked by RTB, we mutated arginine residues at or near the interface of RTB to determine if they are critical for ribosome binding. These variants were structurally similar to wild type RTA but could not bind ribosomes. Their K(m) values and catalytic rates (k(cat)) for an SRL mimic RNA were similar to those of wild type, indicating that their activity was not altered. However, they showed an up to 5-fold increase in K(m) and up to 38-fold decrease in kcat toward ribosomes. These results suggest that the stalk binding stimulates the catalysis of ribosome depurination by RTA. The mutated arginines have side chains behind the active site cleft, indicating that the ribosome binding surface of RTA is on the opposite side of the surface that interacts with the SRL. We propose that stalk binding stimulates the catalysis of ribosome depurination by orienting the active site of RTA toward the SRL and thereby allows docking of the target adenine into the active site. This model may apply to the translation factors that interact with the stalk.

  7. Overexpression, purification, and pharmacologic evaluation of anticancer activity of ribosomal protein L24 from the giant panda (Ailuropoda melanoleuca).

    PubMed

    Hou, Y L; Ding, X; Hou, W; Song, B; Wang, T; Wang, F; Li, J; Zhong, J; Xu, T; Ma, B X; Zhu, H Q; Li, J H; Zhong, J C

    2013-10-18

    The ribosomal protein L24 (RPL24) belongs to the L24E family of ribosomal proteins and is located in the cytoplasm. The purpose of this study was to investigate the structure and anti-cancer function of RPL24 of the giant panda (Ailuropoda melanoleuca). The complementary DNA of RPL24 was cloned successfully using reverse transcription-polymerase chain reaction technology. We constructed a recombinant expression vector containing RPL24 complementary DNA and overexpressed it in Escherichia coli using pET28a plasmids. The expression product obtained was purified using Ni-chelating affinity chromatography. The results indicated that the length of the fragment cloned is 509 bp, and it contains an open-reading frame of 474 bp encoding 157 amino acids. Primary structure analysis revealed that the molecular weight of the putative RPL24 protein is 17.78 kDa with a theoretical isoelectric point of 11.86. The RPL24 gene is readily expressed in E. coli, and the RPL24 fused with the N-terminal histidine-tagged protein to give rise to the accumulation of an expected 23.51-kDa polypeptide. The inhibitory rate in mice treated with 0.1 mg/mL RPL24, the highest of 3 doses administered, can reach 67.662%, which may be comparable to the response to mannatide. The histology of organs with tumors showed that the tissues in the RPL24 group displayed a looser arrangement compared with that in the control group. Furthermore, no obvious damage was apparent in other organs, such as heart, lung, and kidney. The data showed that the recombinant RPL24 had time and dose dependency on the cell growth inhibition rate. Human laryngeal carcinoma Hep-2 cells treated with 0.3125-10 µg/mL RPL24 for 24 h displayed significant cell growth inhibition (P < 0.05; N = 6) in assays using 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide compared with that in control (untreated) cells. By contrast, human hepatoma Hep G-2 cells displayed no significant change (P > 0.05; N = 6) from control

  8. Genome-Wide Analysis of Drosophila RBf2 Protein Highlights the Diversity of RB Family Targets and Possible Role in Regulation of Ribosome Biosynthesis.

    PubMed

    Wei, Yiliang; Mondal, Shamba S; Mouawad, Rima; Wilczyński, Bartek; Henry, R William; Arnosti, David N

    2015-05-20

    RBf2 is a recently evolved retinoblastoma family member in Drosophila that differs from RBf1, especially in the C-terminus. To investigate whether the unique features of RBf2 contribute to diverse roles in gene regulation, we performed chromatin immunoprecipitation sequencing for both RBf2 and RBf1 in embryos. A previous model for RB-E2F interactions suggested that RBf1 binds dE2F1 or dE2F2, whereas RBf2 is restricted to binding to dE2F2; however, we found that RBf2 targets approximately twice as many genes as RBf1. Highly enriched among the RBf2 targets were ribosomal protein genes. We tested the functional significance of this finding by assessing RBf activity on ribosomal protein promoters and the endogenous genes. RBf1 and RBf2 significantly repressed expression of some ribosomal protein genes, although not all bound genes showed transcriptional effects. Interestingly, many ribosomal protein genes are similarly targeted in human cells, indicating that these interactions may be relevant for control of ribosome biosynthesis and growth. We carried out bioinformatic analysis to investigate the basis for differential targeting by these two proteins and found that RBf2-specific promoters have distinct sequence motifs, suggesting unique targeting mechanisms. Association of RBf2 with these promoters appears to be independent of dE2F2/dDP, although promoters bound by both RBf1 and RBf2 require dE2F2/dDP. The presence of unique RBf2 targets suggest that evolutionary appearance of this corepressor represents the acquisition of potentially novel roles in gene regulation for the RB family.

  9. Ribosomes exist in large excess over the apparent demand for protein synthesis during carbon starvation in marine Vibrio sp. strain CCUG 15956.

    PubMed Central

    Flärdh, K; Cohen, P S; Kjelleberg, S

    1992-01-01

    Carbon starvation induces the development of a starvation- and stress-resistant cell state in marine Vibrio sp. strain S14 (CCUG 15956). The starved cells remain highly responsive to nutrients during prolonged starvation and exhibit instantaneous severalfold increases in the rates of protein synthesis and RNA synthesis when substrate is added. In order to elucidate the physiological basis for the survival of cells that are starved for a long time, as well as the capacity of these cells for rapid and efficient recovery, we analyzed the ribosome content of carbon-starved Vibrio sp. strain S14 cells. By using direct chemical measurements of the amounts of ribosomal particles in carbon-starved cultures, we demonstrated that ribosomes were lost relatively slowly (half life, 79 h) and that they existed in large excess over the apparent demand for protein synthesis. After 24 h of starvation the total rate of protein synthesis was 2.3% of the rate during growth, and after 3 days this rate was 0.7% of the rate during growth; the relative amounts of ribosomal particles at these times were 81 and 52%, respectively. The ribosome population consisted of 90% 70S monoribosomes, and no polyribosomes were detected in the starved cells. The 70S monoribosomes were responsible for the bulk of the protein synthesis during carbon starvation; some activity was also detected in the polyribosome size region on sucrose density gradients. We suggest that nongrowing carbon-starved Vibrio sp. strain S14 cells possess an excess protein synthesis capacity, which may be essential for their ability to immediately initiate an upshift program when substrate is added. PMID:1383195

  10. Genome-Wide Analysis of Drosophila RBf2 Protein Highlights the Diversity of RB Family Targets and Possible Role in Regulation of Ribosome Biosynthesis

    PubMed Central

    Wei, Yiliang; Mondal, Shamba S.; Mouawad, Rima; Wilczyński, Bartek; Henry, R. William; Arnosti, David N.

    2015-01-01

    RBf2 is a recently evolved retinoblastoma family member in Drosophila that differs from RBf1, especially in the C-terminus. To investigate whether the unique features of RBf2 contribute to diverse roles in gene regulation, we performed chromatin immunoprecipitation sequencing for both RBf2 and RBf1 in embryos. A previous model for RB−E2F interactions suggested that RBf1 binds dE2F1 or dE2F2, whereas RBf2 is restricted to binding to dE2F2; however, we found that RBf2 targets approximately twice as many genes as RBf1. Highly enriched among the RBf2 targets were ribosomal protein genes. We tested the functional significance of this finding by assessing RBf activity on ribosomal protein promoters and the endogenous genes. RBf1 and RBf2 significantly repressed expression of some ribosomal protein genes, although not all bound genes showed transcriptional effects. Interestingly, many ribosomal protein genes are similarly targeted in human cells, indicating that these interactions may be relevant for control of ribosome biosynthesis and growth. We carried out bioinformatic analysis to investigate the basis for differential targeting by these two proteins and found that RBf2-specific promoters have distinct sequence motifs, suggesting unique targeting mechanisms. Association of RBf2 with these promoters appears to be independent of dE2F2/dDP, although promoters bound by both RBf1 and RBf2 require dE2F2/dDP. The presence of unique RBf2 targets suggest that evolutionary appearance of this corepressor represents the acquisition of potentially novel roles in gene regulation for the RB family. PMID:25999584

  11. Atomic resolution structure of cucurmosin, a novel type 1 ribosome-inactivating protein from the sarcocarp of Cucurbita moschata

    SciTech Connect

    Hou, Xiaomin; Meehan, Edward J.; Xie, Jieming; Huang, Mingdong; Chen, Minghuang; Chen, Liqing

    2008-10-27

    A novel type 1 ribosome-inactivating protein (RIP) designated cucurmosin was isolated from the sarcocarp of Cucurbita moschata (pumpkin). Besides rRNA N-glycosidase activity, cucurmosin exhibits strong cytotoxicities to three cancer cell lines of both human and murine origins, but low toxicity to normal cells. Plant genomic DNA extracted from the tender leaves was amplified by PCR between primers based on the N-terminal sequence and X-ray sequence of the C-terminal. The complete mature protein sequence was obtained from N-terminal protein sequencing and partial DNA sequencing, confirmed by high resolution crystal structure analysis. The crystal structure of cucurmosin has been determined at 1.04 {angstrom}, a resolution that has never been achieved before for any RIP. The structure contains two domains: a large N-terminal domain composed of seven {alpha}-helices and eight {beta}-strands, and a smaller C-terminal domain consisting of three {alpha}-helices and two {beta}-strands. The high resolution structure established a glycosylation pattern of GlcNAc{sub 2}Man3Xyl. Asn225 was identified as a glycosylation site. Residues Tyr70, Tyr109, Glu158 and Arg161 define the active site of cucurmosin as an RNA N-glycosidase. The structural basis of cytotoxicity difference between cucurmosin and trichosanthin is discussed.

  12. Role of horizontal gene transfer as a control on the coevolution of ribosomal proteins and the genetic code

    SciTech Connect

    Woese, Carl R.; Goldenfeld, Nigel; Luthey-Schulten, Zaida

    2011-03-31

    Our main goal is to develop the conceptual and computational tools necessary to understand the evolution of the universal processes of translation and replication and to identify events of horizontal gene transfer that occurred within the components. We will attempt to uncover the major evolutionary transitions that accompanied the development of protein synthesis by the ribosome and associated components of the translation apparatus. Our project goes beyond standard genomic approaches to explore homologs that are represented at both the structure and sequence level. Accordingly, use of structural phylogenetic analysis allows us to probe further back into deep evolutionary time than competing approaches, permitting greater resolution of primitive folds and structures. Specifically, our work focuses on the elements of translation, ranging from the emergence of the canonical genetic code to the evolution of specific protein folds, mediated by the predominance of horizontal gene transfer in early life. A unique element of this study is the explicit accounting for the impact of phenotype selection on translation, through a coevolutionary control mechanism. Our work contributes to DOE mission objectives through: (1) sophisticated computer simulation of protein dynamics and evolution, and the further refinement of techniques for structural phylogeny, which complement sequence information, leading to improved annotation of genomic databases; (2) development of evolutionary approaches to exploring cellular function and machinery in an integrated way; and (3) documentation of the phenotype interaction with translation over evolutionary time, reflecting the system response to changing selection pressures through horizontal gene transfer.

  13. Structural domains within the HIV-1 mRNA and the ribosomal protein S25 influence cap-independent translation initiation.

    PubMed

    Carvajal, Felipe; Vallejos, Maricarmen; Walters, Beth; Contreras, Nataly; Hertz, Marla I; Olivares, Eduardo; Cáceres, Carlos J; Pino, Karla; Letelier, Alejandro; Thompson, Sunnie R; López-Lastra, Marcelo

    2016-07-01

    The 5' leader of the HIV-1 genomic RNA is a multifunctional region that folds into secondary/tertiary structures that regulate multiple processes during viral replication including translation initiation. In this work, we examine the internal ribosome entry site (IRES) located in the 5' leader that drives translation initiation of the viral Gag protein under conditions that hinder cap-dependent translation initiation. We show that activity of the HIV-1 IRES relies on ribosomal protein S25 (eS25). Additionally, a mechanistic and mutational analysis revealed that the HIV-1 IRES is modular in nature and that once the 40S ribosomal subunit is recruited to the IRES, translation initiates without the need of ribosome scanning. These findings elucidate a mechanism of initiation by the HIV-1 IRES whereby a number of highly structured sites present within the HIV-1 5' leader leads to the recruitment of the 40S subunit directly at the site of initiation of protein synthesis.

  14. Study of several factors in RNA-protein cross-link formation induced by ionizing radiations within 70S ribosomes of E. coli MRE 600.

    PubMed

    Ekert, B; Giocanti, N; Sabattier, R

    1986-09-01

    The induction of RNA-protein crosslinks in E. coli 70S ribosomes by gamma-irradiation was studied by measuring the dependence of cross-link formation on ribosome concentration. The inverse dependence of cross-link percentage upon concentration up to at least 20 A260 nm units ml-1 indicate that indirect effects seem to play a more major part than direct effects for these ribosome concentrations. The effect of various gases and free radical scavengers was used to determine the roles of the radicals H., CO2-., OH. and e-aq and to estimate their relative efficiencies for cross-links. They were found to be: 7.2(H.), 6(CO2-.), 2(OH.) and 1(e-aq). The extent of RNA-protein cross-link production in 70S ribosomes induced by gamma-rays and neutrons in the presence and absence of oxygen was also investigated. Cross-link formation was estimated by separation of linked and unlinked material on nitrocellulose filters or after separation by SDS-sucrose gradient centrifugation under dissociating conditions. Oxygen inhibited cross-link formation by both neutrons and gamma-rays. However, very few cross-links were formed in de-aerated solutions by exposure to neutrons, compared to those produced by gamma-rays under the same conditions. This suggests that molecular oxygen generated along the secondary particle track can reduce the formation of RNA-protein cross-links.

  15. Preliminary structural studies of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi, a part of the P0/P1/P2 complex.

    PubMed

    Ayub, Maximiliano Juri; Barroso, Juan A; Levin, Mariano J; Aguilar, Carlos F

    2005-08-01

    The Trypanosoma cruzi ribosomal P0 protein (TcP0) is part of the ribosomal stalk, which is an elongated lateral protuberance of the large ribosomal subunit involved in the translocation step of protein synthesis. The TcP0 C-terminal peptide is highly antigenic and a major target of the antibody response in patients with systemic lupus erythematosus and patients suffering chronic heart disease produced by Trypanosoma cruzi infection. The structural properties of TcP0 have been explored by circular dichroism, tryptophan fluorescence and limited proteolysis experiments. These studies were complemented by secondary structure consensus prediction analysis. The results suggest that the tertiary structure of TcP0 could be described as a compact, stable, trypsin-resistant, 200 residues long N-terminal domain belonging to the alpha/beta class and a more flexible, degradable, helical, 123 residues long C-terminal domain which could be involved in the formation of an unusual hydrophobic zipper with the ribosomal P1/P2 proteins to form the P0/P1/P2 complex.

  16. Immunogenicity of recombinant L7/L12 ribosomal protein of Neisseria meningitidis--high prevalence of specific antibodies in humans but limited immunogenicity for T c