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Sample records for elongation factor 1a

  1. Legionella pneumophila glucosyltransferase inhibits host elongation factor 1A

    PubMed Central

    Belyi, Yury; Niggeweg, Ricarda; Opitz, Bastian; Vogelsgesang, Martin; Hippenstiel, Stefan; Wilm, Matthias; Aktories, Klaus

    2006-01-01

    Legionella pneumophila, the causal agent of Legionnaires' disease, is an intracellular parasite and invades and proliferates within different eukaryotic cells, including human alveolar macrophages. After several 100-fold multiplication within host cells, the pathogens are released for new invasion by induction of apoptosis or necrosis. Here we report that L. pneumophila produces a glucosyltransferase, which selectively modifies an ≈50-kDa mammalian protein by using UDP-glucose as a cosubstrate. MS analysis identified the protein substrate as the mammalian elongation factor (EF)1A. Legionella glucosyltransferase modifies its eukaryotic protein substrate at serine-53, which is located in the GTPase domain of the EF. Glucosylation of EF1A results in inhibition of eukaryotic protein synthesis and death of target cells. Our findings show a mode of inhibition of protein synthesis by microbial pathogens and offer a perspective for understanding of the host-pathogen interaction of L. pneumophila. PMID:17068130

  2. Elongation factor-1A1 is a novel substrate of the protein phosphatase 1-TIMAP complex.

    PubMed

    Boratkó, Anita; Péter, Margit; Thalwieser, Zsófia; Kovács, Előd; Csortos, Csilla

    2015-12-01

    TIMAP (TGF-β inhibited membrane associated protein) is a protein phosphatase 1 (PP1) regulatory subunit highly abundant in endothelial cells and it is involved in the maintenance of pulmonary endothelial barrier function. It localizes mainly in the plasma membrane, but it is also present in the nuclei and cytoplasm. Direct interaction of TIMAP with the eukaryotic elongation factor 1 A1 (eEF1A1) is shown by pull-down, LC-MS/MS, Far-Western and immunoprecipitations. In connection with the so called moonlighting functions of the elongation factor, eEF1A is thought to establish protein-protein interactions through a transcription-dependent nuclear export motif, TD-NEM, and to aid nuclear export of TD-NEM containing proteins. We found that a TD-NEM-like motif of TIMAP has a critical role in its specific binding to eEF1A1. However, eEF1A1 is not or not exclusively responsible for the nuclear export of TIMAP. On the contrary, TIMAP seems to regulate membrane localization of eEF1A1 as the elongation factor co-localized with TIMAP in the plasma membrane fraction of control endothelial cells, but it has disappeared from the membrane in TIMAP depleted cells. It is demonstrated that membrane localization of eEF1A1 depends on the phosphorylation state of its Thr residue(s); and ROCK phosphorylated eEF1A1 is a novel substrate for TIMAP-PP1 underlining the complex regulatory role of TIMAP in the endothelium. The elongation factor seems to be involved in the regulation of endothelial cell attachment and spreading as silencing of eEF1A1 positively affected these processes which were monitored by transendothelial resistance measurements. PMID:26497934

  3. Molecular mechanism of elongation factor 1A inhibition by a Legionella pneumophila glycosyltransferase.

    PubMed

    Hurtado-Guerrero, Ramon; Zusman, Tal; Pathak, Shalini; Ibrahim, Adel F M; Shepherd, Sharon; Prescott, Alan; Segal, Gil; van Aalten, Daan M F

    2010-03-15

    Legionnaires' disease is caused by a lethal colonization of alveolar macrophages with the Gram-negative bacterium Legionella pneumophila. LpGT (L. pneumophila glucosyltransferase; also known as Lgt1) has recently been identified as a virulence factor, shutting down protein synthesis in the human cell by specific glucosylation of EF1A (elongation factor 1A), using an unknown mode of substrate recognition and a retaining mechanism for glycosyl transfer. We have determined the crystal structure of LpGT in complex with substrates, revealing a GT-A fold with two unusual protruding domains. Through structure-guided mutagenesis of LpGT, several residues essential for binding of the UDP-glucose-donor and EF1A-acceptor substrates were identified, which also affected L. pneumophila virulence as demonstrated by microinjection studies. Together, these results suggested that a positively charged EF1A loop binds to a negatively charged conserved groove on the LpGT structure, and that two asparagine residues are essential for catalysis. Furthermore, we showed that two further L. pneumophila glycosyltransferases possessed the conserved UDP-glucose-binding sites and EF1A-binding grooves, and are, like LpGT, translocated into the macrophage through the Icm/Dot (intracellular multiplication/defect in organelle trafficking) system. PMID:20030628

  4. Isolation and characterization of three cassava elongation factor 1 alpha (MeEF1A) promoters.

    PubMed

    Suhandono, Sony; Apriyanto, Ardha; Ihsani, Nisa

    2014-01-01

    In plant genetic engineering, the identification of gene promoters leading to particular expression patterns is crucial for the development of new genetically modified plant generations. This research was conducted in order to isolate and characterize several new promoters from cassava (Manihot esculenta Crantz) elongation factor 1 alpha (EF1A) gene family.Three promoters MeEF1A3, MeEF1A5 and MeEF1A6 were successfully isolated [corrected]. Sequence analyses showed that all of the promoters contain three conserved putative cis-acting elements which are located upstream of the transcription start site. These elements are included a TEF1, a TELO and TATA boxes. In addition, all of the promoters also have the 5'UTR intron but with a different lengths. These promoters were constructed translationally with gusA reporter gene (promoter::gusA fusion) in pBI-121 binary vector to build a new binary vector using Overlap Extension PCR Cloning (OEPC) technique. Transient expression assay that was done by using agroinfiltration method was used to show functionality of these promoters. Qualitative and quantitative analysis from GUS assay showed that these promoters were functional and conferred a specific activity in tobacco seedlings (Nicotiana tabacum), tomato fruits (Solanum lycopersicum) and banana fruits (Musa acuminata). We hypothesized that MeEF1A6 could be categorized as a constitutive promoter because it was able to drive the gene expression in all transformed tissue described in here and also comparable to CaMV35S. On the other hand, MeEF1A3 drove specific expression in the aerial parts of seedlings such as hypocotyl and cotyledon thus MeEF1A5 drove specific expression in fruit tissue. The results obtained from transient analysis showed that these promoters had a distinct activity although they came from same gene family. The DNA sequences identified here are new promoters potentially use for genetic engineering in cassava or other plants. PMID:24404183

  5. Treatment with didemnin B, an elongation factor 1A inhibitor, improves hepatic lipotoxicity in obese mice.

    PubMed

    Hetherington, Alexandra M; Sawyez, Cynthia G; Sutherland, Brian G; Robson, Debra L; Arya, Rigya; Kelly, Karen; Jacobs, René L; Borradaile, Nica M

    2016-09-01

    Eukaryotic elongation factor EEF1A1 is induced by oxidative and ER stress, and contributes to subsequent cell death in many cell types, including hepatocytes. We recently showed that blocking the protein synthesis activity of EEF1A1 with the peptide inhibitor, didemnin B, decreases saturated fatty acid overload-induced cell death in HepG2 cells. In light of this and other recent work suggesting that limiting protein synthesis may be beneficial in treating ER stress-related disease, we hypothesized that acute intervention with didemnin B would decrease hepatic ER stress and lipotoxicity in obese mice with nonalcoholic fatty liver disease (NAFLD). Hyperphagic male ob/ob mice were fed semipurified diet for 4 weeks, and during week 5 received i.p. injections of didemnin B or vehicle on days 1, 4, and 7. Interestingly, we observed that administration of this compound modestly decreased food intake without evidence of illness or distress, and thus included an additional control group matched for food consumption with didemnin B-treated animals. Treatment with didemnin B improved several characteristics of hepatic lipotoxicity to a greater extent than the effects of caloric restriction alone, including hepatic steatosis, and some hepatic markers of ER stress and inflammation (GRP78, Xbp1s, and Mcp1). Plasma lipid and lipoprotein profiles and histopathological measures of NAFLD, including lobular inflammation, and total NAFLD activity score were also improved by didemnin B. These data indicate that acute intervention with the EEF1A inhibitor, didemnin B, improves hepatic lipotoxicity in obese mice with NAFLD through mechanisms not entirely dependent on decreased food intake, suggesting a potential therapeutic strategy for this ER stress-related disease. PMID:27613825

  6. Heat tolerance and expression of protein synthesis elongation factors, EF-Tu and EF-1a, in spring wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein elongation factors, EF-Tu and EF-1a, have been implicated in cell response to heat stress. In spring wheat, EF-Tu displays chaperone activity and reduces thermal aggregation of Rubisco activase. Similarly, in mammalian cells, EF-1a displays chaperone-like activity and regulates the expressio...

  7. The in vivo dynamics of TCERG1, a factor that couples transcriptional elongation with splicing.

    PubMed

    Sánchez-Hernández, Noemí; Boireau, Stéphanie; Schmidt, Ute; Muñoz-Cobo, Juan Pablo; Hernández-Munain, Cristina; Bertrand, Edouard; Suñé, Carlos

    2016-04-01

    Coupling between transcription and RNA processing is key for gene regulation. Using live-cell photobleaching techniques, we investigated the factor TCERG1, which coordinates transcriptional elongation with splicing. We demonstrate that TCERG1 is highly mobile in the nucleoplasm and that this mobility is slightly decreased when it is associated with speckles. Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) but not α-amanitin treatment reduced the mobility of TCERG1, which suggests interaction with paused transcription elongation complexes. We found that TCERG1 mobility is rapid at the transcription site (TS) of a reporter that splices post-transcriptionally and that TCERG1 is recruited to the active TS independent of the CTD of RNAPII, thus excluding phosphorylated CTD as a requirement for recruiting this factor to the TS. Importantly, the mobility of TCERG1 is reduced when the reporter splices cotranscriptionally, which suggests that TCERG1 forms new macromolecular complexes when splicing occurs cotranscriptionally. In this condition, spliceostatin A has no effect, indicating that TCERG1 rapidly binds and dissociates from stalled spliceosomal complexes and that the mobility properties of TCERG1 do not depend on events occurring after the initial spliceosome formation. Taken together, these data suggest that TCERG1 binds independently to elongation and splicing complexes, thus performing their coupling by transient interactions rather than by stable association with one or the other complexes. This finding has conceptual implications for understanding the coupling between transcription and RNA processing. PMID:26873599

  8. Elongation Factor 1A-1 Is a Mediator of Hepatocyte Lipotoxicity Partly through Its Canonical Function in Protein Synthesis

    PubMed Central

    Stoianov, Alexandra M.; Robson, Debra L.; Hetherington, Alexandra M.; Sawyez, Cynthia G.; Borradaile, Nica M.

    2015-01-01

    Elongation factor 1A-1 (eEF1A-1) has non-canonical functions in regulation of the actin cytoskeleton and apoptosis. It was previously identified through a promoter-trap screen as a mediator of fatty acid-induced cell death (lipotoxicity), and was found to participate in this process downstream of ER stress. Since ER stress is implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD), we investigated the mechanism of action of eEF1A-1 in hepatocyte lipotoxicity. HepG2 cells were exposed to excess fatty acids, followed by assessments of ER stress, subcellular localization of eEF1A-1, and cell death. A specific inhibitor of eEF1A-1 elongation activity, didemnin B, was used to determine whether its function in protein synthesis is involved in lipotoxicity. Within 6 h, eEF1A-1 protein was modestly induced by high palmitate, and partially re-localized from its predominant location at the ER to polymerized actin at the cell periphery. This early induction and subcellular redistribution of eEF1A-1 coincided with the onset of ER stress, and was later followed by cell death. Didemnin B did not prevent the initiation of ER stress by high palmitate, as indicated by eIF2α phosphorylation. However, consistent with sustained inhibition of eEF1A-1-dependent elongation activity, didemnin B prevented the recovery of protein synthesis and increase in GRP78 protein that are normally associated with later phases of the response to ongoing ER stress. This resulted in decreased palmitate-induced cell death. Our data implicate eEF1A-1, and its function in protein synthesis, in hepatocyte lipotoxicity. PMID:26102086

  9. Ternatin and improved synthetic variants kill cancer cells by targeting the elongation factor-1A ternary complex

    PubMed Central

    Carelli, Jordan D; Sethofer, Steven G; Smith, Geoffrey A; Miller, Howard R; Simard, Jillian L; Merrick, William C; Jain, Rishi K; Ross, Nathan T; Taunton, Jack

    2015-01-01

    Cyclic peptide natural products have evolved to exploit diverse protein targets, many of which control essential cellular processes. Inspired by a series of cyclic peptides with partially elucidated structures, we designed synthetic variants of ternatin, a cytotoxic and anti-adipogenic natural product whose molecular mode of action was unknown. The new ternatin variants are cytotoxic toward cancer cells, with up to 500-fold greater potency than ternatin itself. Using a ternatin photo-affinity probe, we identify the translation elongation factor-1A ternary complex (eEF1A·GTP·aminoacyl-tRNA) as a specific target and demonstrate competitive binding by the unrelated natural products, didemnin and cytotrienin. Mutations in domain III of eEF1A prevent ternatin binding and confer resistance to its cytotoxic effects, implicating the adjacent hydrophobic surface as a functional hot spot for eEF1A modulation. We conclude that the eukaryotic elongation factor-1A and its ternary complex with GTP and aminoacyl-tRNA are common targets for the evolution of cytotoxic natural products. DOI: http://dx.doi.org/10.7554/eLife.10222.001 PMID:26651998

  10. Ternatin and improved synthetic variants kill cancer cells by targeting the elongation factor-1A ternary complex.

    PubMed

    Carelli, Jordan D; Sethofer, Steven G; Smith, Geoffrey A; Miller, Howard R; Simard, Jillian L; Merrick, William C; Jain, Rishi K; Ross, Nathan T; Taunton, Jack

    2015-01-01

    Cyclic peptide natural products have evolved to exploit diverse protein targets, many of which control essential cellular processes. Inspired by a series of cyclic peptides with partially elucidated structures, we designed synthetic variants of ternatin, a cytotoxic and anti-adipogenic natural product whose molecular mode of action was unknown. The new ternatin variants are cytotoxic toward cancer cells, with up to 500-fold greater potency than ternatin itself. Using a ternatin photo-affinity probe, we identify the translation elongation factor-1A ternary complex (eEF1A·GTP·aminoacyl-tRNA) as a specific target and demonstrate competitive binding by the unrelated natural products, didemnin and cytotrienin. Mutations in domain III of eEF1A prevent ternatin binding and confer resistance to its cytotoxic effects, implicating the adjacent hydrophobic surface as a functional hot spot for eEF1A modulation. We conclude that the eukaryotic elongation factor-1A and its ternary complex with GTP and aminoacyl-tRNA are common targets for the evolution of cytotoxic natural products. PMID:26651998

  11. Aptamer targeting of the elongation factor 1A impairs hepatocarcinoma cells viability and potentiates bortezomib and idarubicin effects.

    PubMed

    Scaggiante, Bruna; Farra, Rosella; Dapas, Barbara; Baj, Gabriele; Pozzato, Gabriele; Grassi, Mario; Zanconati, Fabrizio; Grassi, Gabriele

    2016-06-15

    The high morbidity and mortality of hepatocellular carcinoma (HCC) is mostly due to the limited efficacy of the available therapeutic approaches. Here we explore the anti-HCC potential of an aptamer targeting the elongation factor 1A (eEF1A), a protein implicated in the promotion of HCC. As delivery methods, we have compared the effectiveness of cationic liposome and cholesterol-mediated approaches. A75 nucleotide long aptamer containing GT repetition (GT75) was tested in three HCC cell lines, HepG2, HuH7 and JHH6. When delivered by liposomes, GT75 was able to effectively reducing HCC cells viability in a dose and time dependent fashion. Particular sensitive were JHH6 where increased apoptosis with no effects on cell cycle were observed. GT75 effect was likely due to the interference with eEF1A activity as neither the mRNA nor the protein levels were significantly affected. Notably, cholesterol-mediated delivery of GT75 abrogated its efficacy due to cellular mis-localization as proven by fluorescence and confocal microscopic analysis. Finally, liposome-mediated delivery of GT75 improved the therapeutic index of the anticancer drugs bortezomib and idarubicin. In conclusion, liposome but not cholesterol-mediated delivery of GT75 resulted in an effective delivery of GT75, causing the impairment of the vitality of a panel of HCC derived cells. PMID:27094354

  12. Saccharomyces cerevisiae Eukaryotic Elongation Factor 1A (eEF1A) Is Methylated at Lys-390 by a METTL21-Like Methyltransferase

    PubMed Central

    Jakobsson, Magnus E.; Davydova, Erna; Małecki, Jędrzej; Moen, Anders; Falnes, Pål Ø.

    2015-01-01

    The human methyltransferases (MTases) METTL21A and VCP-KMT (METTL21D) were recently shown to methylate single lysine residues in Hsp70 proteins and in VCP, respectively. The yet uncharacterized MTase encoded by the YNL024C gene in Saccharomyces cerevisiae shows high sequence similarity to METTL21A and VCP-KMT, as well as to their uncharacterized paralogues METTL21B and METTL21C. Despite being most similar to METTL21A, the Ynl024c protein does not methylate yeast Hsp70 proteins, which were found to be unmethylated on the relevant lysine residue. Eukaryotic translation elongation factor eEF1A in yeast has been reported to contain four methylated lysine residues (Lys30, Lys79, Lys318 and Lys390), and we here show that the YNL024C gene is required for methylation of eEF1A at Lys390, the only of these methylations for which the responsible MTase has not yet been identified. Lys390 was found in a partially monomethylated state in wild-type yeast cells but was exclusively unmethylated in a ynl024cΔ strain, and over-expression of Ynl024c caused a dramatic increase in Lys390 methylation, with trimethylation becoming the predominant state. Our results demonstrate that Ynl024c is the enzyme responsible for methylation of eEF1A at Lys390, and in accordance with prior naming of similar enzymes, we suggest that Ynl024c is renamed to Efm6 (Elongation factor MTase 6). PMID:26115316

  13. Translation elongation factor 1A mutants with altered actin bundling activity show reduced aminoacyl-tRNA binding and alter initiation via eIF2α phosphorylation.

    PubMed

    Perez, Winder B; Kinzy, Terri Goss

    2014-07-25

    Apart from its canonical function in translation elongation, eukaryotic translation elongation factor 1A (eEF1A) has been shown to interact with the actin cytoskeleton. Amino acid substitutions in eEF1A that reduce its ability to bind and bundle actin in vitro cause improper actin organization in vivo and reduce total translation. Initial in vivo analysis indicated the reduced translation was through initiation. The mutant strains exhibit increased levels of phosphorylated initiation factor 2α (eIF2α) dependent on the presence of the general control non-derepressible 2 (Gcn2p) protein kinase. Gcn2p causes downregulation of total protein synthesis at initiation in response to increases in deacylated tRNA levels in the cell. Increased levels of eIF2α phosphorylation are not due to a general reduction in translation elongation as eEF2 and eEF3 mutants do not exhibit this effect. Deletion of GCN2 from the eEF1A actin bundling mutant strains revealed a second defect in translation. The eEF1A actin-bundling proteins exhibit changes in their elongation activity at the level of aminoacyl-tRNA binding in vitro. These findings implicate eEF1A in a feedback mechanism for regulating translation at initiation. PMID:24936063

  14. Evidence That Eukaryotic Translation Elongation Factor 1A (eEF1A) Binds the Gcn2 Protein C Terminus and Inhibits Gcn2 Activity*♦

    PubMed Central

    Visweswaraiah, Jyothsna; Lageix, Sebastien; Castilho, Beatriz A.; Izotova, Lara; Kinzy, Terri Goss; Hinnebusch, Alan G.; Sattlegger, Evelyn

    2011-01-01

    The eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to the ribosomal A-site during protein synthesis. To ensure a continuous supply of amino acids, cells harbor the kinase Gcn2 and its effector protein Gcn1. The ultimate signal for amino acid shortage is uncharged tRNAs. We have proposed a model for sensing starvation, in which Gcn1 and Gcn2 are tethered to the ribosome, and Gcn1 is directly involved in delivering uncharged tRNAs from the A-site to Gcn2 for its subsequent activation. Gcn1 and Gcn2 are large proteins, and these proteins as well as eEF1A access the A-site, leading us to investigate whether there is a functional or physical link between these proteins. Using Saccharomyces cerevisiae cells expressing His6-eEF1A and affinity purification, we found that eEF1A co-eluted with Gcn2. Furthermore, Gcn2 co-immunoprecipitated with eEF1A, suggesting that they reside in the same complex. The purified GST-tagged Gcn2 C-terminal domain (CTD) was sufficient for precipitating eEF1A from whole cell extracts generated from gcn2Δ cells, independently of ribosomes. Purified GST-Gcn2-CTD and purified His6-eEF1A interacted with each other, and this was largely independent of the Lys residues in Gcn2-CTD known to be required for tRNA binding and ribosome association. Interestingly, Gcn2-eEF1A interaction was diminished in amino acid-starved cells and by uncharged tRNAs in vitro, suggesting that eEF1A functions as a Gcn2 inhibitor. Consistent with this possibility, purified eEF1A reduced the ability of Gcn2 to phosphorylate its substrate, eIF2α, but did not diminish Gcn2 autophosphorylation. These findings implicate eEF1A in the intricate regulation of Gcn2 and amino acid homeostasis. PMID:21849502

  15. Evidence that eukaryotic translation elongation factor 1A (eEF1A) binds the Gcn2 protein C terminus and inhibits Gcn2 activity.

    PubMed

    Visweswaraiah, Jyothsna; Lageix, Sebastien; Castilho, Beatriz A; Izotova, Lara; Kinzy, Terri Goss; Hinnebusch, Alan G; Sattlegger, Evelyn

    2011-10-21

    The eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to the ribosomal A-site during protein synthesis. To ensure a continuous supply of amino acids, cells harbor the kinase Gcn2 and its effector protein Gcn1. The ultimate signal for amino acid shortage is uncharged tRNAs. We have proposed a model for sensing starvation, in which Gcn1 and Gcn2 are tethered to the ribosome, and Gcn1 is directly involved in delivering uncharged tRNAs from the A-site to Gcn2 for its subsequent activation. Gcn1 and Gcn2 are large proteins, and these proteins as well as eEF1A access the A-site, leading us to investigate whether there is a functional or physical link between these proteins. Using Saccharomyces cerevisiae cells expressing His(6)-eEF1A and affinity purification, we found that eEF1A co-eluted with Gcn2. Furthermore, Gcn2 co-immunoprecipitated with eEF1A, suggesting that they reside in the same complex. The purified GST-tagged Gcn2 C-terminal domain (CTD) was sufficient for precipitating eEF1A from whole cell extracts generated from gcn2Δ cells, independently of ribosomes. Purified GST-Gcn2-CTD and purified His(6)-eEF1A interacted with each other, and this was largely independent of the Lys residues in Gcn2-CTD known to be required for tRNA binding and ribosome association. Interestingly, Gcn2-eEF1A interaction was diminished in amino acid-starved cells and by uncharged tRNAs in vitro, suggesting that eEF1A functions as a Gcn2 inhibitor. Consistent with this possibility, purified eEF1A reduced the ability of Gcn2 to phosphorylate its substrate, eIF2α, but did not diminish Gcn2 autophosphorylation. These findings implicate eEF1A in the intricate regulation of Gcn2 and amino acid homeostasis. PMID:21849502

  16. The eukaryotic translation elongation factor eEF1A2 induces neoplastic properties and mediates tumorigenic effects of ZNF217 in precursor cells of human ovarian carcinomas

    SciTech Connect

    Sun, Yu; Wong, Nicholas; Guan, Yinghui; Salamanca, Clara M.; Cheng, Jung Chien; Lee, Jonathan M.; Gray, Joe W.; Auersperg, Nelly

    2008-04-25

    Ovarian epithelial carcinomas (OEC) frequently exhibit amplifications at the 20q13 locus which is the site of several oncogenes, including the eukaryotic elongation factor EEF1A2 and the transcription factor ZNF217. We reported previously that overexpressed ZNF217 induces neoplastic characteristics in precursor cells of OEC. Unexpectedly, ZNF217, which is a transcriptional repressor, enhanced expression of eEF1A2. In this study, array comparative genomic hybridization, single nucleotide polymorphism and Affymetrix analysis of ZNF217-overexpressing cell lines confirmed consistently increased expression of eEF1A2 but not of other oncogenes, and revealed early changes in EEF1A2 gene copy numbers and increased expression at crisis during immortalization. We defined the influence of eEF1A2 overexpression on immortalized ovarian surface epithelial cells, and investigated interrelationships between effects of ZNF217 and eEF1A2 on cellular phenotypes. Lentivirally induced eEF1A2 overexpression caused delayed crisis, apoptosis resistance and increases in serum-independence, saturation densities, and anchorage independence. siRNA to eEF1A2 reversed apoptosis resistance and reduced anchorage independence in eEF1A2-overexpressing lines. Remarkably, siRNA to eEF1A2 was equally efficient in inhibiting both anchorage independence and resistance to apoptosis conferred by ZNF217 overexpression. Our data define neoplastic properties that are caused by eEF1A2 in nontumorigenic ovarian cancer precursor cells, and suggest that eEF1A2 plays a role in mediating ZNF217-induced neoplastic progression.

  17. MLIF Alleviates SH-SY5Y Neuroblastoma Injury Induced by Oxygen-Glucose Deprivation by Targeting Eukaryotic Translation Elongation Factor 1A2

    PubMed Central

    Liu, Yulan; Cheng, Hao; Wang, Jing; Zhang, Yue; Rui, Yaocheng; Li, Tiejun

    2016-01-01

    Monocyte locomotion inhibitory factor (MLIF), a heat-stable pentapeptide, has been shown to exert potent anti-inflammatory effects in ischemic brain injury. In this study, we investigated the neuroprotective action of MLIF against oxygen-glucose deprivation (OGD)-induced injury in human neuroblastoma SH-SY5Y cells. MTT assay was used to assess cell viability, and flow cytometry assay and Hoechst staining were used to evaluate apoptosis. LDH assay was used to exam necrosis. The release of inflammatory cytokines was detected by ELISA. Levels of the apoptosis associated proteins were measured by western blot analysis. To identify the protein target of MLIF, pull-down assay and mass spectrometry were performed. We observed that MLIF enhanced cell survival and inhibited apoptosis and necrosis by inhibiting p-JNK, p53, c-caspase9 and c-caspase3 expression. In the microglia, OGD-induced secretion of inflammatory cytokines was markedly reduced in the presence of MLIF. Furthermore, we found that eukaryotic translation elongation factor 1A2 (eEF1A2) is a downstream target of MLIF. Knockdown eEF1A2 using short interfering RNA (siRNA) almost completely abrogated the anti-apoptotic effect of MLIF in SH-SY5Y cells subjected to OGD, with an associated decrease in cell survival and an increase in expression of p-JNK and p53. These results indicate that MLIF ameliorates OGD-induced SH-SY5Y neuroblastoma injury by inhibiting the p-JNK/p53 apoptotic signaling pathway via eEF1A2. Our findings suggest that eEF1A2 may be a new therapeutic target for ischemic brain injury. PMID:26918757

  18. Loss of translation elongation factor (eEF1A2) expression in vivo differentiates between Wallerian degeneration and dying-back neuronal pathology

    PubMed Central

    Murray, Lyndsay M; Thomson, Derek; Conklin, Annalijn; Wishart, Thomas M; Gillingwater, Thomas H

    2008-01-01

    Wallerian degeneration and dying-back pathology are two well-known cellular pathways capable of regulating the breakdown and loss of axonal and synaptic compartments of neurons in vivo. However, the underlying mechanisms and molecular triggers of these pathways remain elusive. Here, we show that loss of translation elongation factor eEF1A2 expression in lower motor neurons and skeletal muscle fibres in homozygous Wasted mice triggered a dying-back neuropathy. Synaptic loss at the neuromuscular junction occurred in advance of axonal pathology and by a mechanism morphologically distinct from Wallerian degeneration. Dying-back pathology in Wasted mice was accompanied by reduced expression levels of the zinc finger protein ZPR1, as found in other dying-back neuropathies such as spinal muscular atrophy. Surprisingly, experimental nerve lesion revealed that Wallerian degeneration was significantly delayed in homozygous Wasted mice; morphological assessment revealed that ∼80% of neuromuscular junctions in deep lumbrical muscles at 24 h and ∼50% at 48 h had retained motor nerve terminals following tibial nerve lesion. This was in contrast to wild-type and heterozygous Wasted mice where < 5% of neuromuscular junctions had retained motor nerve terminals at 24 h post-lesion. These data show that eEF1A2 expression is required to prevent the initiation of dying-back pathology at the neuromuscular junction in vivo. In contrast, loss of eEF1A2 expression significantly inhibited the initiation and progression of Wallerian degeneration in vivo. We conclude that loss of eEF1A2 expression distinguishes mechanisms underlying dying-back pathology from those responsible for Wallerian degeneration in vivo and suggest that eEF1A2-dependent cascades may provide novel molecular targets to manipulate neurodegenerative pathways in lower motor neurons. PMID:19094180

  19. MicroRNA-33a-5p Modulates Japanese Encephalitis Virus Replication by Targeting Eukaryotic Translation Elongation Factor 1A1

    PubMed Central

    Chen, Zheng; Ye, Jing; Ashraf, Usama; Li, Yunchuan; Wei, Siqi; Wan, Shengfeng; Zohaib, Ali; Song, Yunfeng; Chen, Huanchun

    2016-01-01

    ABSTRACT Japanese encephalitis virus (JEV) is a typical mosquito-borne flavivirus responsible for acute encephalitis and meningitis in humans. However, the molecular mechanism for JEV pathogenesis is still unclear. MicroRNAs (miRNAs) are small noncoding RNAs that act as gene regulators. They are directly or indirectly involved in many cellular functions owing to their ability to target mRNAs for degradation or translational repression. However, how cellular miRNAs are regulated and their functions during JEV infection are largely unknown. In the present study, we found that JEV infection downregulated the expression of endogenous cellular miR-33a-5p. Notably, artificially transfecting with miR-33a-5p mimics led to a significant decrease in viral replication, suggesting that miR-33a-5p acts as a negative regulator of JEV replication. A dual-luciferase reporter assay identified eukaryotic translation elongation factor 1A1 (EEF1A1) as one of the miR-33a-5p target genes. Our study further demonstrated that EEF1A1 can interact with the JEV proteins NS3 and NS5 in replicase complex. Through this interaction, EEF1A1 can stabilize the components of viral replicase complex and thus facilitates viral replication during JEV infection. Taken together, these results suggest that miR-33a-5p is downregulated during JEV infection, which contributes to viral replication by increasing the intracellular level of EEF1A1, an interaction partner of JEV NS3 and NS5. This study provides a better understanding of the molecular mechanisms of JEV pathogenesis. IMPORTANCE MiRNAs are critical regulators of gene expression that utilize sequence complementarity to bind to and modulate the stability or translation efficiency of target mRNAs. Accumulating data suggest that miRNAs regulate a wide variety of molecular mechanisms in the host cells during viral infections. JEV, a neurotropic flavivirus, is one of the major causes of acute encephalitis in humans worldwide. The roles of cellular mi

  20. A Function for the hnRNP A1/A2 Proteins in Transcription Elongation

    PubMed Central

    Lemieux, Bruno; Blanchette, Marco; Monette, Anne; Mouland, Andrew J.; Wellinger, Raymund J.; Chabot, Benoit

    2015-01-01

    The hnRNP A1 and A2 proteins regulate processes such as alternative pre-mRNA splicing and mRNA stability. Here, we report that a reduction in the levels of hnRNP A1 and A2 by RNA interference or their cytoplasmic retention by osmotic stress drastically increases the transcription of a reporter gene. Based on previous work, we propose that this effect may be linked to a decrease in the activity of the transcription elongation factor P-TEFb. Consistent with this hypothesis, the transcription of the reporter gene was stimulated when the catalytic component of P-TEFb, CDK9, was inhibited with DRB. While low levels of A1/A2 stimulated the association of RNA polymerase II with the reporter gene, they also increased the association of CDK9 with the repressor 7SK RNA, and compromised the recovery of promoter-distal transcription on the Kitlg gene after the release of pausing. Transcriptome analysis revealed that more than 50% of the genes whose expression was affected by the siRNA-mediated depletion of A1/A2 were also affected by DRB. RNA polymerase II-chromatin immunoprecipitation assays on DRB-treated and A1/A2-depleted cells identified a common set of repressed genes displaying increased occupancy of polymerases at promoter-proximal locations, consistent with pausing. Overall, our results suggest that lowering the levels of hnRNP A1/A2 elicits defective transcription elongation on a fraction of P-TEFb-dependent genes, hence favoring the transcription of P-TEFb-independent genes. PMID:26011126

  1. A Complex Distribution of Elongation Family GTPases EF1A and EFL in Basal Alveolate Lineages

    PubMed Central

    Mikhailov, Kirill V.; Janouškovec, Jan; Tikhonenkov, Denis V.; Mirzaeva, Gulnara S.; Diakin, Andrei Yu.; Simdyanov, Timur G.; Mylnikov, Alexander P.; Keeling, Patrick J.; Aleoshin, Vladimir V.

    2014-01-01

    Translation elongation factor-1 alpha (EF1A) and the related GTPase EF-like (EFL) are two proteins with a complex mutually exclusive distribution across the tree of eukaryotes. Recent surveys revealed that the distribution of the two GTPases in even closely related taxa is frequently at odds with their phylogenetic relationships. Here, we investigate the distribution of EF1A and EFL in the alveolate supergroup. Alveolates comprise three major lineages: ciliates and apicomplexans encode EF1A, whereas dinoflagellates encode EFL. We searched transcriptome databases for seven early-diverging alveolate taxa that do not belong to any of these groups: colpodellids, chromerids, and colponemids. Current data suggest all seven are expected to encode EF1A, but we find three genera encode EFL: Colpodella, Voromonas, and the photosynthetic Chromera. Comparing this distribution with the phylogeny of alveolates suggests that EF1A and EFL evolution in alveolates cannot be explained by a simple horizontal gene transfer event or lineage sorting. PMID:25179686

  2. Nannocystin A: an Elongation Factor 1 Inhibitor from Myxobacteria with Differential Anti-Cancer Properties.

    PubMed

    Krastel, Philipp; Roggo, Silvio; Schirle, Markus; Ross, Nathan T; Perruccio, Francesca; Aspesi, Peter; Aust, Thomas; Buntin, Kathrin; Estoppey, David; Liechty, Brigitta; Mapa, Felipa; Memmert, Klaus; Miller, Howard; Pan, Xuewen; Riedl, Ralph; Thibaut, Christian; Thomas, Jason; Wagner, Trixie; Weber, Eric; Xie, Xiaobing; Schmitt, Esther K; Hoepfner, Dominic

    2015-08-24

    Cultivation of myxobacteria of the Nannocystis genus led to the isolation and structure elucidation of a class of novel cyclic lactone inhibitors of elongation factor 1. Whole genome sequence analysis and annotation enabled identification of the putative biosynthetic cluster and synthesis process. In biological assays the compounds displayed anti-fungal and cytotoxic activity. Combined genetic and proteomic approaches identified the eukaryotic translation elongation factor 1α (EF-1α) as the primary target for this compound class. Nannocystin A (1) displayed differential activity across various cancer cell lines and EEF1A1 expression levels appear to be the main differentiating factor. Biochemical and genetic evidence support an overlapping binding site of 1 with the anti-cancer compound didemnin B on EF-1α. This myxobacterial chemotype thus offers an interesting starting point for further investigations of the potential of therapeutics targeting elongation factor 1. PMID:26179970

  3. Gain and loss of elongation factor genes in green algae

    PubMed Central

    Cocquyt, Ellen; Verbruggen, Heroen; Leliaert, Frederik; Zechman, Frederick W; Sabbe, Koen; De Clerck, Olivier

    2009-01-01

    Background Two key genes of the translational apparatus, elongation factor-1 alpha (EF-1α) and elongation factor-like (EFL) have an almost mutually exclusive distribution in eukaryotes. In the green plant lineage, the Chlorophyta encode EFL except Acetabularia where EF-1α is found, and the Streptophyta possess EF-1α except Mesostigma, which has EFL. These results raise questions about evolutionary patterns of gain and loss of EF-1α and EFL. A previous study launched the hypothesis that EF-1α was the primitive state and that EFL was gained once in the ancestor of the green plants, followed by differential loss of EF-1α or EFL in the principal clades of the Viridiplantae. In order to gain more insight in the distribution of EF-1α and EFL in green plants and test this hypothesis we screened the presence of the genes in a large sample of green algae and analyzed their gain-loss dynamics in a maximum likelihood framework using continuous-time Markov models. Results Within the Chlorophyta, EF-1α is shown to be present in three ulvophycean orders (i.e., Dasycladales, Bryopsidales, Siphonocladales) and the genus Ignatius. Models describing gene gain-loss dynamics revealed that the presence of EF-1α, EFL or both genes along the backbone of the green plant phylogeny is highly uncertain due to sensitivity to branch lengths and lack of prior knowledge about ancestral states or rates of gene gain and loss. Model refinements based on insights gained from the EF-1α phylogeny reduce uncertainty but still imply several equally likely possibilities: a primitive EF-1α state with multiple independent EFL gains or coexistence of both genes in the ancestor of the Viridiplantae or Chlorophyta followed by differential loss of one or the other gene in the various lineages. Conclusion EF-1α is much more common among green algae than previously thought. The mutually exclusive distribution of EF-1α and EFL is confirmed in a large sample of green plants. Hypotheses about the gain

  4. Identification of autophosphorylation sites in eukaryotic elongation factor-2 kinase

    PubMed Central

    Pyr Dit Ruys, Sébastien; Wang, Xuemin; Smith, Ewan M.; Herinckx, Gaëtan; Hussain, Nusrat; Rider, Mark H.; Vertommen, Didier; Proud, Christopher G.

    2012-01-01

    eEF2K [eEF2 (eukaryotic elongation factor 2) kinase] phosphorylates and inactivates the translation elongation factor eEF2. eEF2K is not a member of the main eukaryotic protein kinase superfamily, but instead belongs to a small group of so-called α-kinases. The activity of eEF2K is normally dependent upon Ca2+ and calmodulin. eEF2K has previously been shown to undergo autophosphorylation, the stoichiometry of which suggested the existence of multiple sites. In the present study we have identified several autophosphorylation sites, including Thr348, Thr353, Ser366 and Ser445, all of which are highly conserved among vertebrate eEF2Ks. We also identified a number of other sites, including Ser78, a known site of phosphorylation, and others, some of which are less well conserved. None of the sites lies in the catalytic domain, but three affect eEF2K activity. Mutation of Ser78, Thr348 and Ser366 to a non-phosphorylatable alanine residue decreased eEF2K activity. Phosphorylation of Thr348 was detected by immunoblotting after transfecting wild-type eEF2K into HEK (human embryonic kidney)-293 cells, but not after transfection with a kinase-inactive construct, confirming that this is indeed a site of autophosphorylation. Thr348 appears to be constitutively autophosphorylated in vitro. Interestingly, other recent data suggest that the corresponding residue in other α-kinases is also autophosphorylated and contributes to the activation of these enzymes [Crawley, Gharaei, Ye, Yang, Raveh, London, Schueler-Furman, Jia and Cote (2011) J. Biol. Chem. 286, 2607–2616]. Ser366 phosphorylation was also detected in intact cells, but was still observed in the kinase-inactive construct, demonstrating that this site is phosphorylated not only autocatalytically but also in trans by other kinases. PMID:22216903

  5. Mammalian elongation factor 4 regulates mitochondrial translation essential for spermatogenesis.

    PubMed

    Gao, Yanyan; Bai, Xiufeng; Zhang, Dejiu; Han, Chunsheng; Yuan, Jing; Liu, Wenbin; Cao, Xintao; Chen, Zilei; Shangguan, Fugen; Zhu, Zhenyuan; Gao, Fei; Qin, Yan

    2016-05-01

    Elongation factor 4 (EF4) is a key quality-control factor in translation. Despite its high conservation throughout evolution, EF4 deletion in various organisms has not yielded a distinct phenotype. Here we report that genetic ablation of mitochondrial EF4 (mtEF4) in mice causes testis-specific dysfunction in oxidative phosphorylation, leading to male infertility. Deletion of mtEF4 accelerated mitochondrial translation at the cost of producing unstable proteins. Somatic tissues overcame this defect by activating mechanistic (mammalian) target of rapamycin (mTOR), thereby increasing rates of cytoplasmic translation to match rates of mitochondrial translation. However, in spermatogenic cells, the mTOR pathway was downregulated as part of the developmental program, and the resulting inability to compensate for accelerated mitochondrial translation caused cell-cycle arrest and apoptosis. We detected the same phenotype and molecular defects in germline-specific mtEF4-knockout mice. Thus, our study demonstrates cross-talk between mtEF4-dependent quality control in mitochondria and cytoplasmic mTOR signaling. PMID:27065197

  6. Functional interaction of yeast elongation factor 3 with yeast ribosomes.

    PubMed

    Chakraburtty, K

    1999-01-01

    Elongation factor 3 (EF-3) is a unique and essential requirement of the fungal translational apparatus. EF-3 is a monomeric protein with a molecular mass of 116,000. EF-3 is required by yeast ribosomes for in vitro translation and for in vivo growth. The protein stimulates the binding of EF-1 alpha :GTP:aa-tRNA ternary complex to the ribosomal A-site by facilitating release of deacylated-tRNA from the E-site. The reaction requires ATP hydrolysis. EF-3 contains two ATP-binding sequence motifs (NBS). NBSI is sufficient for the intrinsic ATPase function. NBSII is essential for ribosome-stimulated activity. By limited proteolysis, EF-3 was divided into two distinct functional domains. The N-terminal domain lacking the highly charged lysine blocks failed to bind ribosomes and was inactive in the ribosome-stimulated ATPase activity. The C-terminally derived lysine-rich fragment showed strong binding to yeast ribosomes. The purported S5 homology region of EF-3 at the N-terminal end has been reported to interact with 18S ribosomal RNA. We postulate that EF-3 contacts rRNA and/or protein(s) through the C-terminal end. Removal of these residues severely weakens its interaction mediated possibly through the N-terminal domain of the protein. PMID:10216951

  7. Negative elongation factor controls energy homeostasis in cardiomyocytes.

    PubMed

    Pan, Haihui; Qin, Kunhua; Guo, Zhanyong; Ma, Yonggang; April, Craig; Gao, Xiaoli; Andrews, Thomas G; Bokov, Alex; Zhang, Jianhua; Chen, Yidong; Weintraub, Susan T; Fan, Jian-Bing; Wang, Degeng; Hu, Yanfen; Aune, Gregory J; Lindsey, Merry L; Li, Rong

    2014-04-10

    Negative elongation factor (NELF) is known to enforce promoter-proximal pausing of RNA polymerase II (Pol II), a pervasive phenomenon observed across multicellular genomes. However, the physiological impact of NELF on tissue homeostasis remains unclear. Here, we show that whole-body conditional deletion of the B subunit of NELF (NELF-B) in adult mice results in cardiomyopathy and impaired response to cardiac stress. Tissue-specific knockout of NELF-B confirms its cell-autonomous function in cardiomyocytes. NELF directly supports transcription of those genes encoding rate-limiting enzymes in fatty acid oxidation (FAO) and the tricarboxylic acid (TCA) cycle. NELF also shares extensively transcriptional target genes with peroxisome proliferator-activated receptor α (PPARα), a master regulator of energy metabolism in the myocardium. Mechanistically, NELF helps stabilize the transcription initiation complex at the metabolism-related genes. Our findings strongly indicate that NELF is part of the PPARα-mediated transcription regulatory network that maintains metabolic homeostasis in cardiomyocytes. PMID:24656816

  8. Architecture and RNA binding of the human negative elongation factor

    PubMed Central

    Vos, Seychelle M; Pöllmann, David; Caizzi, Livia; Hofmann, Katharina B; Rombaut, Pascaline; Zimniak, Tomasz; Herzog, Franz; Cramer, Patrick

    2016-01-01

    Transcription regulation in metazoans often involves promoter-proximal pausing of RNA polymerase (Pol) II, which requires the 4-subunit negative elongation factor (NELF). Here we discern the functional architecture of human NELF through X-ray crystallography, protein crosslinking, biochemical assays, and RNA crosslinking in cells. We identify a NELF core subcomplex formed by conserved regions in subunits NELF-A and NELF-C, and resolve its crystal structure. The NELF-AC subcomplex binds single-stranded nucleic acids in vitro, and NELF-C associates with RNA in vivo. A positively charged face of NELF-AC is involved in RNA binding, whereas the opposite face of the NELF-AC subcomplex binds NELF-B. NELF-B is predicted to form a HEAT repeat fold, also binds RNA in vivo, and anchors the subunit NELF-E, which is confirmed to bind RNA in vivo. These results reveal the three-dimensional architecture and three RNA-binding faces of NELF. DOI: http://dx.doi.org/10.7554/eLife.14981.001 PMID:27282391

  9. Movement of Elongation Factor G between Compact and Extended Conformations

    PubMed Central

    Salsi, Enea; Farah, Elie; Netter, Zoe; Dann, Jillian; Ermolenko, Dmitri N.

    2014-01-01

    Previous structural studies suggested that ribosomal translocation is accompanied by large interdomain rearrangements of elongation factor G (EF-G). Here, we follow the movement of domain IV of EF-G relative to domain II of EF-G using ensemble and single-molecule Förster resonance energy transfer (smFRET). Our results indicate that ribosome-free EF-G predominantly adopts a compact conformation that can also, albeit infrequently, transition into a more extended conformation in which domain IV moves away from domain II. By contrast, ribosome-bound EF-G predominantly adopts an extended conformation regardless of whether it is interacting with pre- or posttranslocation ribosomes. Our data suggest that ribosome-bound EF-G may also occasionally sample at least one more compact conformation. GTP hydrolysis catalyzed by EF-G does not affect the relative stability of the observed conformations in ribosome-free and ribosome-bound EF-G. Our data support a model suggesting that, upon binding to a pretranslocation ribosome, EF-G moves from a compact to a more extended conformation. This transition is not coupled to, but likely precedes both GTP hydrolysis and mRNA/tRNA translocation. PMID:25463439

  10. Elongation factor G initiates translocation through a power stroke.

    PubMed

    Chen, Chunlai; Cui, Xiaonan; Beausang, John F; Zhang, Haibo; Farrell, Ian; Cooperman, Barry S; Goldman, Yale E

    2016-07-01

    During the translocation step of prokaryotic protein synthesis, elongation factor G (EF-G), a guanosine triphosphatase (GTPase), binds to the ribosomal PRE-translocation (PRE) complex and facilitates movement of transfer RNAs (tRNAs) and messenger RNA (mRNA) by one codon. Energy liberated by EF-G's GTPase activity is necessary for EF-G to catalyze rapid and precise translocation. Whether this energy is used mainly to drive movements of the tRNAs and mRNA or to foster EF-G dissociation from the ribosome after translocation has been a long-lasting debate. Free EF-G, not bound to the ribosome, adopts quite different structures in its GTP and GDP forms. Structures of EF-G on the ribosome have been visualized at various intermediate steps along the translocation pathway, using antibiotics and nonhydolyzable GTP analogs to block translocation and to prolong the dwell time of EF-G on the ribosome. However, the structural dynamics of EF-G bound to the ribosome have not yet been described during normal, uninhibited translocation. Here, we report the rotational motions of EF-G domains during normal translocation detected by single-molecule polarized total internal reflection fluorescence (polTIRF) microscopy. Our study shows that EF-G has a small (∼10°) global rotational motion relative to the ribosome after GTP hydrolysis that exerts a force to unlock the ribosome. This is followed by a larger rotation within domain III of EF-G before its dissociation from the ribosome. PMID:27313204

  11. The ERF11 Transcription Factor Promotes Internode Elongation by Activating Gibberellin Biosynthesis and Signaling1[OPEN

    PubMed Central

    Zhou, Xin; Zhang, Zhong-Lin; Tyler, Ludmila; Yusuke, Jikumaru; Qiu, Kai; Lumba, Shelley; Desveaux, Darrell; McCourt, Peter; Sun, Tai-ping

    2016-01-01

    The phytohormone gibberellin (GA) plays a key role in promoting stem elongation in plants. Previous studies show that GA activates its signaling pathway by inducing rapid degradation of DELLA proteins, GA signaling repressors. Using an activation-tagging screen in a reduced-GA mutant ga1-6 background, we identified AtERF11 to be a novel positive regulator of both GA biosynthesis and GA signaling for internode elongation. Overexpression of AtERF11 partially rescued the dwarf phenotype of ga1-6. AtERF11 is a member of the ERF (ETHYLENE RESPONSE FACTOR) subfamily VIII-B-1a of ERF/AP2 transcription factors in Arabidopsis (Arabidopsis thaliana). Overexpression of AtERF11 resulted in elevated bioactive GA levels by up-regulating expression of GA3ox1 and GA20ox genes. Hypocotyl elongation assays further showed that overexpression of AtERF11 conferred elevated GA response, whereas loss-of-function erf11 and erf11 erf4 mutants displayed reduced GA response. In addition, yeast two-hybrid, coimmunoprecipitation, and transient expression assays showed that AtERF11 enhances GA signaling by antagonizing the function of DELLA proteins via direct protein-protein interaction. Interestingly, AtERF11 overexpression also caused a reduction in the levels of another phytohormone ethylene in the growing stem, consistent with recent finding showing that AtERF11 represses transcription of ethylene biosynthesis ACS genes. The effect of AtERF11 on promoting GA biosynthesis gene expression is likely via its repressive function on ethylene biosynthesis. These results suggest that AtERF11 plays a dual role in promoting internode elongation by inhibiting ethylene biosynthesis and activating GA biosynthesis and signaling pathways. PMID:27255484

  12. The ERF11 Transcription Factor Promotes Internode Elongation by Activating Gibberellin Biosynthesis and Signaling.

    PubMed

    Zhou, Xin; Zhang, Zhong-Lin; Park, Jeongmoo; Tyler, Ludmila; Yusuke, Jikumaru; Qiu, Kai; Nam, Edward A; Lumba, Shelley; Desveaux, Darrell; McCourt, Peter; Kamiya, Yuji; Sun, Tai-Ping

    2016-08-01

    The phytohormone gibberellin (GA) plays a key role in promoting stem elongation in plants. Previous studies show that GA activates its signaling pathway by inducing rapid degradation of DELLA proteins, GA signaling repressors. Using an activation-tagging screen in a reduced-GA mutant ga1-6 background, we identified AtERF11 to be a novel positive regulator of both GA biosynthesis and GA signaling for internode elongation. Overexpression of AtERF11 partially rescued the dwarf phenotype of ga1-6 AtERF11 is a member of the ERF (ETHYLENE RESPONSE FACTOR) subfamily VIII-B-1a of ERF/AP2 transcription factors in Arabidopsis (Arabidopsis thaliana). Overexpression of AtERF11 resulted in elevated bioactive GA levels by up-regulating expression of GA3ox1 and GA20ox genes. Hypocotyl elongation assays further showed that overexpression of AtERF11 conferred elevated GA response, whereas loss-of-function erf11 and erf11 erf4 mutants displayed reduced GA response. In addition, yeast two-hybrid, coimmunoprecipitation, and transient expression assays showed that AtERF11 enhances GA signaling by antagonizing the function of DELLA proteins via direct protein-protein interaction. Interestingly, AtERF11 overexpression also caused a reduction in the levels of another phytohormone ethylene in the growing stem, consistent with recent finding showing that AtERF11 represses transcription of ethylene biosynthesis ACS genes. The effect of AtERF11 on promoting GA biosynthesis gene expression is likely via its repressive function on ethylene biosynthesis. These results suggest that AtERF11 plays a dual role in promoting internode elongation by inhibiting ethylene biosynthesis and activating GA biosynthesis and signaling pathways. PMID:27255484

  13. Pleiohomeotic Interacts with the Core Transcription Elongation Factor Spt5 to Regulate Gene Expression in Drosophila

    PubMed Central

    Jennings, Barbara H.

    2013-01-01

    The early elongation checkpoint regulated by Positive Transcription Elongation Factor b (P-TEFb) is a critical control point for the expression of many genes. Spt5 interacts directly with RNA polymerase II and has an essential role in establishing this checkpoint, and also for further transcript elongation. Here we demonstrate that Drosophila Spt5 interacts both physically and genetically with the Polycomb Group (PcG) protein Pleiohomeotic (Pho), and the majority of Pho binding sites overlap with Spt5 binding sites across the genome in S2 cells. Our results indicate that Pho can interact with Spt5 to regulate transcription elongation in a gene specific manner. PMID:23894613

  14. Modes of Action of ADP-Ribosylated Elongation Factor 2 in Inhibiting the Polypeptide Elongation Cycle: A Modeling Study

    PubMed Central

    Chen, Kevin C.; Xie, Honglin; Cai, Yujie

    2013-01-01

    Despite the fact that ADP-ribosylation of eukaryotic elongation factor 2 (EF2) leads to inhibition of protein synthesis, the mechanism by which ADP-ribosylated EF2 (ADPR•EF2) causes this inhibition remains controversial. Here, we applied modeling approaches to investigate the consequences of various modes of ADPR•EF2 inhibitory actions on the two coupled processes, the polypeptide chain elongation and ADP-ribosylation of EF2. Modeling of experimental data indicates that ADPR•EF2 fully blocks the late-phase translocation of tRNAs; but the impairment in the translocation upstream process, mainly the GTP-dependent factor binding with the pretranslocation ribosome and/or the guanine nucleotide exchange in EF2, is responsible for the overall inhibition kinetics. The reduced ADPR•EF2-ribosome association spares the ribosome to bind and shield native EF2 against toxin attack, thereby deferring the inhibition of protein synthesis inhibition and inactivation of EF2. Minimum association with the ribosome also keeps ADPR•EF2 in an accessible state for toxins to catalyze the reverse reaction when nicotinamide becomes available. Our work underscores the importance of unveiling the interactions between ADPR•EF2 and the ribosome, and argues against that toxins inhibit protein synthesis through converting native EF2 to a competitive inhibitor to actively disable the ribosome. PMID:23861744

  15. The selenocysteine-specific elongation factor contains a novel and multi-functional domain.

    PubMed

    Gonzalez-Flores, Jonathan N; Gupta, Nirupama; DeMong, Louise W; Copeland, Paul R

    2012-11-01

    The selenocysteine (Sec)-specific eukaryotic elongation factor (eEFSec) delivers the aminoacylated selenocysteine-tRNA (Sec-tRNA(Sec)) to the ribosome and suppresses UGA codons that are upstream of Sec insertion sequence (SECIS) elements bound by SECIS-binding protein 2 (SBP2). Multiple studies have highlighted the importance of SBP2 forming a complex with the SECIS element, but it is not clear how this regulates eEFSec during Sec incorporation. Compared with the canonical elongation factor eEF1A, eEFSec has a unique C-terminal extension called Domain IV. To understand the role of Domain IV in Sec incorporation, we examined a series of mutant proteins for all of the known molecular functions for eEFSec: GTP hydrolysis, Sec-tRNA(Sec) binding, and SBP2/SECIS binding. In addition, wild-type and mutant versions of eEFSec were analyzed for Sec incorporation activity in a novel eEFSec-dependent translation extract. We have found that Domain IV is essential for both tRNA and SBP2 binding as well as regulating GTPase activity. We propose a model where the SBP2/SECIS complex activates eEFSec by directing functional interactions between Domain IV and the ribosome to promote Sec-tRNA(Sec) binding and accommodation into the ribosomal A-site. PMID:22992746

  16. Phosphoglycerylethanolamine Posttranslational Modification of Plant Eukaryotic Elongation Factor 1α1

    PubMed Central

    Ransom, Wendy D.; Lao, Pao-Chi; Gage, Douglas A.; Boss, Wendy F.

    1998-01-01

    Eukaryotic elongation factor 1α (eEF-1A) is a multifunctional protein. There are three known posttranslational modifications of eEF-1A that could potentially affect its function. Except for phosphorylation, the other posttranslational modifications have not been demonstrated in plants. Using matrix-assisted laser desorption/ionization-mass spectrometry and peptide mass mapping, we show that carrot (Daucus carota L.) eEF-1A contains a phosphoglycerylethanolamine (PGE) posttranslational modification. eEF-1A was the only protein labeled with [14C]ethanolamine in carrot cells and was the predominant ethanolamine-labeled protein in Arabidopsis seedlings and tobacco (Nicotiana tabacum L.) cell cultures. In vivo-labeling studies using [3H]glycerol, [32P]Pi, [14C]myristic acid, and [14C]linoleic acid indicated that the entire phospholipid phosphatidylethanolamine is covalently attached to the protein. The PGE lipid modification did not affect the partitioning of eEF-1A in Triton X-114 or its actin-binding activity in in vitro assays. Our in vitro data indicate that this newly characterized posttranslational modification alone does not affect the function of eEF-1A. Therefore, the PGE lipid modification may work in combination with other posttranslational modifications to affect the distribution and the function of eEF-1A within the cell. PMID:9662537

  17. Identification of the gene encoding the mitochondrial elongation factor G in mammals.

    PubMed Central

    Barker, C; Makris, A; Patriotis, C; Bear, S E; Tsichlis, P N

    1993-01-01

    Protein synthesis in cytosolic and rough endoplasmic reticulum associated ribosomes is directed by factors, many of which have been well characterized. Although these factors have been the subject of intense study, most of the corresponding factors regulating protein synthesis in the mitochondrial ribosomes remain unknown. In this report we present the cloning and initial characterization of the gene encoding the rat mitochondrial elongation factor-G (rEF-Gmt). The rat gene encoding EF-Gmt (rMef-g) maps to rat chromosome 2 and it is expressed in all tissues with highest levels in liver, thymus and brain. Its DNA sequence predicts a 752 amino acid protein exhibiting 72% homology to the yeast Saccharomyces cerevisiae mitochondrial elongation factor-G (YMEF-G), 62% and 61% homology to the Thermus thermophilus and E. coli elongation factor-G (EF-G) respectively and 52% homology to the rat elongation factor-2 (EF-2). The deduced amino acid sequence of EF-G contains characteristic motifs shared by all GTP binding proteins. Therefore, similarly to other elongation factors, the enzymatic function of EF-Gmt is predicted to depend on GTP binding and hydrolysis. EF-Gmt differs from its cytoplasmic homolog, EF-2, in that it contains an aspartic acid residue at amino acid position 621 which corresponds to the EF-2 histidine residue at position 715. Since this histidine residue, following posttranslational modification into diphthamide, appears to be the sole cellular target of diphtheria toxin and Pseudomonas aeruginosa endotoxin A, we conclude that EF-Gmt will not be inactivated by these toxins. The severe effects of these toxins on protein elongation in tissues expressing EF-Gmt suggest that EF-Gmt and EF-2 exhibit nonoverlapping functions. The cloning and characterization of the mammalian mitochondrial elongation factor G will permit us to address its role in the regulation of normal mitochondrial function and in disease states attributed to mitochondrial dysfunction. Images

  18. The initiation factor TFE and the elongation factor Spt4/5 compete for the RNAP clamp during transcription initiation and elongation.

    PubMed

    Grohmann, Dina; Nagy, Julia; Chakraborty, Anirban; Klose, Daniel; Fielden, Daniel; Ebright, Richard H; Michaelis, Jens; Werner, Finn

    2011-07-22

    TFIIE and the archaeal homolog TFE enhance DNA strand separation of eukaryotic RNAPII and the archaeal RNAP during transcription initiation by an unknown mechanism. We have developed a fluorescently labeled recombinant M. jannaschii RNAP system to probe the archaeal transcription initiation complex, consisting of promoter DNA, TBP, TFB, TFE, and RNAP. We have localized the position of the TFE winged helix (WH) and Zinc ribbon (ZR) domains on the RNAP using single-molecule FRET. The interaction sites of the TFE WH domain and the transcription elongation factor Spt4/5 overlap, and both factors compete for RNAP binding. Binding of Spt4/5 to RNAP represses promoter-directed transcription in the absence of TFE, which alleviates this effect by displacing Spt4/5 from RNAP. During elongation, Spt4/5 can displace TFE from the RNAP elongation complex and stimulate processivity. Our results identify the RNAP "clamp" region as a regulatory hot spot for both transcription initiation and transcription elongation. PMID:21777815

  19. The Dual Functions of WLIM1a in Cell Elongation and Secondary Wall Formation in Developing Cotton Fibers[C][W

    PubMed Central

    Han, Li-Bo; Li, Yuan-Bao; Wang, Hai-Yun; Wu, Xiao-Min; Li, Chun-Li; Luo, Ming; Wu, Shen-Jie; Kong, Zhao-Sheng; Pei, Yan; Jiao, Gai-Li; Xia, Gui-Xian

    2013-01-01

    LIN-11, Isl1 and MEC-3 (LIM)-domain proteins play pivotal roles in a variety of cellular processes in animals, but plant LIM functions remain largely unexplored. Here, we demonstrate dual roles of the WLIM1a gene in fiber development in upland cotton (Gossypium hirsutum). WLIM1a is preferentially expressed during the elongation and secondary wall synthesis stages in developing fibers. Overexpression of WLIM1a in cotton led to significant changes in fiber length and secondary wall structure. Compared with the wild type, fibers of WLIM1a-overexpressing plants grew longer and formed a thinner and more compact secondary cell wall, which contributed to improved fiber strength and fineness. Functional studies demonstrated that (1) WLIM1a acts as an actin bundler to facilitate elongation of fiber cells and (2) WLIM1a also functions as a transcription factor to activate expression of Phe ammonia lyase–box genes involved in phenylpropanoid biosynthesis to build up the secondary cell wall. WLIM1a localizes in the cytosol and nucleus and moves into the nucleus in response to hydrogen peroxide. Taken together, these results demonstrate that WLIM1a has dual roles in cotton fiber development, elongation, and secondary wall formation. Moreover, our study shows that lignin/lignin-like phenolics may substantially affect cotton fiber quality; this finding may guide cotton breeding for improved fiber traits. PMID:24220634

  20. Brevis plant1, a putative inositol polyphosphate 5-phosphatase, is required for internode elongation in maize

    PubMed Central

    Avila, Luis M.; Cerrudo, Diego; Swanton, Clarence

    2016-01-01

    In maize (Zea mays L.), as in other grass species, stem elongation occurs during growth and most noticeably upon the transition to flowering. Genes that reduce stem elongation have been important to reduce stem breakage, or lodging. Stem elongation has been mediated by dwarf and brachytic/brevis plant mutants that affect giberellic acid and auxin pathways, respectively. Maize brevis plant1 (bv1) mutants, first identified over 80 years ago, strongly resemble brachytic2 mutants that have shortened internodes, short internode cells, and are deficient in auxin transport. Here, we characterized two novel bv1 maize mutants. We found that an inositol polyphosphate 5-phosphatase orthologue of the rice gene dwarf50 was the molecular basis for the bv1 phenotype, implicating auxin-mediated inositol polyphosphate and/or phosphoinositide signalling in stem elongation. We suggest that auxin-mediated internode elongation involves processes that also contribute to stem gravitropism. Genes misregulated in bv1 mutants included genes important for cell wall synthesis, transmembrane transport, and cytoskeletal function. Mutant and wild-type plants were indistinguishable early in development, responded similarly to changes in light quality, had unaltered flowering times, and had normal flower development. These attributes suggest that breeding could utilize bv1 alleles to increase crop grain yields. PMID:26767748

  1. Brevis plant1, a putative inositol polyphosphate 5-phosphatase, is required for internode elongation in maize.

    PubMed

    Avila, Luis M; Cerrudo, Diego; Swanton, Clarence; Lukens, Lewis

    2016-03-01

    In maize (Zea mays L.), as in other grass species, stem elongation occurs during growth and most noticeably upon the transition to flowering. Genes that reduce stem elongation have been important to reduce stem breakage, or lodging. Stem elongation has been mediated by dwarf and brachytic/brevis plant mutants that affect giberellic acid and auxin pathways, respectively. Maize brevis plant1 (bv1) mutants, first identified over 80 years ago, strongly resemble brachytic2 mutants that have shortened internodes, short internode cells, and are deficient in auxin transport. Here, we characterized two novel bv1 maize mutants. We found that an inositol polyphosphate 5-phosphatase orthologue of the rice gene dwarf50 was the molecular basis for the bv1 phenotype, implicating auxin-mediated inositol polyphosphate and/or phosphoinositide signalling in stem elongation. We suggest that auxin-mediated internode elongation involves processes that also contribute to stem gravitropism. Genes misregulated in bv1 mutants included genes important for cell wall synthesis, transmembrane transport, and cytoskeletal function. Mutant and wild-type plants were indistinguishable early in development, responded similarly to changes in light quality, had unaltered flowering times, and had normal flower development. These attributes suggest that breeding could utilize bv1 alleles to increase crop grain yields. PMID:26767748

  2. Role of Sulf1A in Wnt1- and Wnt6-induced growth regulation and myoblast hyper-elongation.

    PubMed

    Hitchins, L; Fletcher, F; Allen, S; Dhoot, G K

    2013-01-01

    Sulf1A expression, which is a characteristic of embryonic muscle, is undetectable in mature muscle fibres and quiescent satellite cells, but is re-activated in vivo upon injury and in vitro following activation of satellite cells. Sulf1A is known to enhance canonical Wnt signalling, and its association with Wnt1-induced satellite cell proliferation in vitro in the present study further confirmed this. However, exogenous Wnt6 decreased satellite cell proliferation but promoted the adoption of a hyper-elongated cell morphology in myoblasts on isolated single fibres in culture. Such Wnt6-induced cellular hyper-elongation and inhibition of proliferation was found to be dependent upon Sulf1A, as treatment with Sulf1A neutralising antibodies abolished both these effects. This indicates that Sulf1A can regulate Wnt6 signalling and cellular differentiation in skeletal muscle. PMID:23772371

  3. A putative transcriptional elongation factor hIws1 is essential for mammalian cell proliferation

    SciTech Connect

    Liu Zhangguo; Zhou Zhongwei; Chen Guohong; Bao Shilai . E-mail: slbao@genetics.ac.cn

    2007-02-02

    Iws1 has been implicated in transcriptional elongation by interaction with RNA polymerase II (RNAP II) and elongation factor Spt6 in budding yeast Saccharomyces cerevisiae, and association with transcription factor TFIIS in mammalian cells, but its role in controlling cell growth and proliferation remains unknown. Here we report that the human homolog of Iws1, hIws1, physically interacts with protein arginine methyltransferases PRMT5 which methylates elongation factor Spt5 and regulates its interaction with RNA polymerase II. Gene-specific silencing of hIws1 by RNA interference reveals that hIws1 is essential for cell viability. GFP fusion protein expression approaches demonstrate that the hIws1 protein is located in the nucleus, subsequently, two regions harbored within the hIws1 protein are demonstrated to contain nuclear localization signals (NLSs). In addition, mouse homolog of hiws1 is found to express ubiquitously in various tissues.

  4. CHARACTERIZATION AND GENE EXPRESSION OF BABESIA BOVIS ELONGATION FACTOR-1ALPHA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Elongation factor 1 alpha (EF-1') is a constitutively expressed, abundant protein that is a key element in eukaryotic protein translation. Because of its high level of transcription, the EF-1''promoter has been utilized to drive exogenous gene expression in transfected cells. In this study, we ident...

  5. Heat-induced Accumulation of Chloroplast Protein Synthesis Elongation Factor, EF-TU, in Winter Wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in heat tolerance in maize (Zea mays L.). Chloroplast EF-Tu is highly conserved, and it is possible that this protein may be of importance to heat tolerance in other species including wheat (Triticum aestivum L.). In this ...

  6. Negative elongation factor NELF controls transcription of immediate early genes in a stimulus-specific manner

    SciTech Connect

    Fujita, Toshitsugu; Piuz, Isabelle; Schlegel, Werner

    2009-01-15

    The transcription rate of immediate early genes (IEGs) is controlled directly by transcription elongation factors at the transcription elongation step. Negative elongation factor (NELF) and 5,6-dichloro-1-{beta}-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF) stall RNA polymerase II (pol II) soon after transcription initiation. Upon induction of IEG transcription, DSIF is converted into an accelerator for pol II elongation. To address whether and how NELF as well as DSIF controls overall IEG transcription, its expression was reduced using stable RNA interference in GH4C1 cells. NELF knock-down reduced thyrotropin-releasing hormone (TRH)-induced transcription of the IEGs c-fos, MKP-1, and junB. In contrast, epidermal growth factor (EGF)-induced transcription of these IEGs was unaltered or even slightly increased by NELF knock-down. Thus, stable knock-down of NELF affects IEG transcription stimulation-specifically. Conversely, DSIF knock-down reduced both TRH- and EGF-induced transcription of the three IEGs. Interestingly, TRH-induced activation of the MAP kinase pathway, a pathway essential for transcription of the three IEGs, was down-regulated by NELF knock-down. Thus, stable knock-down of NELF, by modulating intracellular signaling pathways, caused stimulation-specific loss of IEG transcription. These observations indicate that NELF controls overall IEG transcription via multiple mechanisms both directly and indirectly.

  7. Divergence of a conserved elongation factor and transcription regulation in budding and fission yeast.

    PubMed

    Booth, Gregory T; Wang, Isabel X; Cheung, Vivian G; Lis, John T

    2016-06-01

    Complex regulation of gene expression in mammals has evolved from simpler eukaryotic systems, yet the mechanistic features of this evolution remain elusive. Here, we compared the transcriptional landscapes of the distantly related budding and fission yeast. We adapted the Precision Run-On sequencing (PRO-seq) approach to map the positions of RNA polymerase active sites genome-wide in Schizosaccharomyces pombe and Saccharomyces cerevisiae. Additionally, we mapped preferred sites of transcription initiation in each organism using PRO-cap. Unexpectedly, we identify a pause in early elongation, specific to S. pombe, that requires the conserved elongation factor subunit Spt4 and resembles promoter-proximal pausing in metazoans. PRO-seq profiles in strains lacking Spt4 reveal globally elevated levels of transcribing RNA Polymerase II (Pol II) within genes in both species. Messenger RNA abundance, however, does not reflect the increases in Pol II density, indicating a global reduction in elongation rate. Together, our results provide the first base-pair resolution map of transcription elongation in S. pombe and identify divergent roles for Spt4 in controlling elongation in budding and fission yeast. PMID:27197211

  8. Intrauterine growth restriction inhibits expression of eukaryotic elongation factor 2 kinase, a regulator of protein translation.

    PubMed

    McKnight, Robert A; Yost, Christian C; Zinkhan, Erin K; Fu, Qi; Callaway, Christopher W; Fung, Camille M

    2016-08-01

    Nutrient deprivation suppresses protein synthesis by blocking peptide elongation. Transcriptional upregulation and activation of eukaryotic elongation factor 2 kinase (eEF2K) blocks peptide elongation by phosphorylating eukaryotic elongation factor 2. Previous studies examining placentas from intrauterine growth restricted (IUGR) newborn infants show decreased eEF2K expression and activity despite chronic nutrient deprivation. However, the effect of IUGR on hepatic eEF2K expression in the fetus is unknown. We, therefore, examined the transcriptional regulation of hepatic eEF2K gene expression in a Sprague-Dawley rat model of IUGR. We found decreased hepatic eEF2K mRNA and protein levels in IUGR offspring at birth compared with control, consistent with previous placental observations. Furthermore, the CpG island within the eEF2K promoter demonstrated increased methylation at a critical USF 1/2 transcription factor binding site. In vitro methylation of this binding site caused near complete loss of eEF2K promoter activity, designating this promoter as methylation sensitive. The eEF2K promotor in IUGR offspring also lost the protective histone covalent modifications associated with unmethylated CGIs. In addition, the +1 nucleosome was displaced 3' and RNA polymerase loading was reduced at the IUGR eEF2K promoter. Our findings provide evidence to explain why IUGR-induced chronic nutrient deprivation does not result in the upregulation of eEF2K gene transcription. PMID:27317589

  9. Structure of the GTP Form of Elongation Factor 4 (EF4) Bound to the Ribosome.

    PubMed

    Kumar, Veerendra; Ero, Rya; Ahmed, Tofayel; Goh, Kwok Jian; Zhan, Yin; Bhushan, Shashi; Gao, Yong-Gui

    2016-06-17

    Elongation factor 4 (EF4) is a member of the family of ribosome-dependent translational GTPase factors, along with elongation factor G and BPI-inducible protein A. Although EF4 is highly conserved in bacterial, mitochondrial, and chloroplast genomes, its exact biological function remains controversial. Here we present the cryo-EM reconstitution of the GTP form of EF4 bound to the ribosome with P and E site tRNAs at 3.8-Å resolution. Interestingly, our structure reveals an unrotated ribosome rather than a clockwise-rotated ribosome, as observed in the presence of EF4-GDP and P site tRNA. In addition, we also observed a counterclockwise-rotated form of the above complex at 5.7-Å resolution. Taken together, our results shed light on the interactions formed between EF4, the ribosome, and the P site tRNA and illuminate the GTPase activation mechanism at previously unresolved detail. PMID:27137929

  10. Elongation in translation as a dynamic interaction among the ribosome, tRNA, and elongation factors EF-G and EF-Tu

    PubMed Central

    Agirrezabala, Xabier; Frank, Joachim

    2010-01-01

    The ribosome is a complex macromolecular machine that translates the message encoded in the messenger RNA and synthesizes polypeptides by linking the individual amino acids carried by the cognate transfer RNAs (tRNAs). The protein elongation cycle, during which the tRNAs traverse the ribosome in a coordinated manner along a path of more than 100 Å, is facilitated by large-scale rearrangements of the ribosome. These rearrangements go hand in hand with conformational changes of tRNA as well as elongation factors EF-Tu and EF-G – GTPases that catalyze tRNA delivery and translocation, respectively. This review focuses on the structural data related to the dynamics of the ribosomal machinery, which are the basis, in conjunction with existing biochemical, kinetic, and fluorescence resonance energy transfer data, of our knowledge of the decoding and translocation steps of protein elongation. PMID:20025795

  11. Elongation in translation as a dynamic interaction among the ribosome, tRNA, and elongation factors EF-G and EF-Tu.

    PubMed

    Agirrezabala, Xabier; Frank, Joachim

    2009-08-01

    The ribosome is a complex macromolecular machine that translates the message encoded in the messenger RNA and synthesizes polypeptides by linking the individual amino acids carried by the cognate transfer RNAs (tRNAs). The protein elongation cycle, during which the tRNAs traverse the ribosome in a coordinated manner along a path of more than 100 A, is facilitated by large-scale rearrangements of the ribosome. These rearrangements go hand in hand with conformational changes of tRNA as well as elongation factors EF-Tu and EF-G - GTPases that catalyze tRNA delivery and translocation, respectively. This review focuses on the structural data related to the dynamics of the ribosomal machinery, which are the basis, in conjunction with existing biochemical, kinetic, and fluorescence resonance energy transfer data, of our knowledge of the decoding and translocation steps of protein elongation. PMID:20025795

  12. Genetic interaction between transcription elongation factor TFIIS and RNA polymerase II.

    PubMed Central

    Archambault, J; Lacroute, F; Ruet, A; Friesen, J D

    1992-01-01

    Little is known about the regions of RNA polymerase II (RNAPII) that are involved in the process of transcript elongation and interaction with elongation factors. One elongation factor, TFIIS, stimulates transcript elongation by binding to RNAPII and facilitating its passage through intrinsic pausing sites in vitro. In Saccharomyces cerevisiae, TFIIS is encoded by the PPR2 gene. Deletion of PPR2 from the yeast genome is not lethal but renders cells sensitive to the uracil analog 6-azauracil (6AU). Here, we show that mutations conferring 6AU sensitivity can also be isolated in the gene encoding the largest subunit of S. cerevisiae RNAPII (RPO21). A screen for mutations in RPO21 that confer 6AU sensitivity identified seven mutations that had been generated by either linker-insertion or random chemical mutagenesis. All seven mutational alterations are clustered within one region of the largest subunit that is conserved among eukaryotic RNAPII. The finding that six of the seven rpo21 mutants failed to grow at elevated temperature underscores the importance of this region for the functional and/or structural integrity of RNAPII. We found that the 6AU sensitivity of the rpo21 mutants can be suppressed by increasing the dosage of the wild-type PPR2 gene, presumably as a result of overexpression of TFIIS. These results are consistent with the proposal that in the rpo21 mutants, the formation of the RNAPII-TFIIS complex is rate limiting for the passage of the mutant enzyme through pausing sites. In addition to implicating a region of the largest subunit of RNAPII in the process of transcript elongation, our observations provide in vivo evidence that TFIIS is involved in transcription by RNAPII. Images PMID:1508210

  13. Synthesis of reinitiated transcripts by mammalian RNA polymerase II is controlled by elongation factor SII.

    PubMed Central

    Szentirmay, M N; Sawadogo, M

    1993-01-01

    Previous studies have revealed that the in vitro synthesis of reinitiated transcripts by RNA polymerase II requires an additional activity, designated reinitiation transcription factor (RTF), which is distinct from all of the general class II initiation factors. While further characterizing this activity, it was found that RTF displays properties indistinguishable from those of the RNA polymerase II elongation factor SII. In addition, Western blot analysis using SII-specific antibodies revealed that human SII is a major component in purified RTF preparations. The functional equivalence of the two proteins was established using recombinant SII, which proved fully capable of substituting for RTF in the reinitiation assay. In these reconstituted reactions, transcription complexes resulting from reinitiation events required SII to proceed through a 400 bp G-free cassette, while complexes resulting from the first round of initiations were SII-independent. Reinitiations can take place in the absence of SII; however, addition of the elongation factor is essential for full extension of the reinitiated transcripts. These results suggest that events taking place at the promoter (e.g. first-round initiations versus reinitiations) can create marked differences in the properties of RNA polymerase II elongation complexes. Images PMID:8223477

  14. Interplay between GCN2 and GCN4 expression, translation elongation factor 1 mutations and translational fidelity in yeast.

    PubMed

    Magazinnik, Tanya; Anand, Monika; Sattlegger, Evelyn; Hinnebusch, Alan G; Kinzy, Terri Goss

    2005-01-01

    Genetic screens in Saccharomyces cerevisiae have identified the roles of ribosome components, tRNAs and translation factors in translational fidelity. These screens rely on the suppression of altered start codons, nonsense codons or frameshift mutations in genes involved in amino acid or nucleotide metabolism. Many of these genes are regulated by the General Amino Acid Control (GAAC) pathway. Upon amino acid starvation, the kinase GCN2 induces the GAAC cascade via increased translation of the transcriptional activator GCN4 controlled by upstream open reading frames (uORFs). Overexpression of the GCN2 or GCN4 genes enhances the sensitivity of translation fidelity assays that utilize genes regulated by GCN4, such as the suppression of a +1 insertion by S.cerevisiae translation elongation factor 1A (eEF1A) mutants. Paromomycin and the prion [PSI+], which reduce translational fidelity, do not increase GCN4 expression to induce the suppression phenotype and in fact reduce derepression. eEF1A mutations that reduce translation, however, reduce expression of GCN4 under non-starvation conditions. These eEF1A mutants also reduce HIS4 mRNA expression. Taken together, this system improves in vivo strategies for the analysis of translational fidelity and further provides new information on the interplay among translation fidelity, altered elongation and translational control via uORFs. PMID:16100380

  15. Interplay between GCN2 and GCN4 expression, translation elongation factor 1 mutations and translational fidelity in yeast

    PubMed Central

    Magazinnik, Tanya; Anand, Monika; Sattlegger, Evelyn; Hinnebusch, Alan G.; Kinzy, Terri Goss

    2005-01-01

    Genetic screens in Saccharomyces cerevisiae have identified the roles of ribosome components, tRNAs and translation factors in translational fidelity. These screens rely on the suppression of altered start codons, nonsense codons or frameshift mutations in genes involved in amino acid or nucleotide metabolism. Many of these genes are regulated by the General Amino Acid Control (GAAC) pathway. Upon amino acid starvation, the kinase GCN2 induces the GAAC cascade via increased translation of the transcriptional activator GCN4 controlled by upstream open reading frames (uORFs). Overexpression of the GCN2 or GCN4 genes enhances the sensitivity of translation fidelity assays that utilize genes regulated by GCN4, such as the suppression of a +1 insertion by S.cerevisiae translation elongation factor 1A (eEF1A) mutants. Paromomycin and the prion [PSI+], which reduce translational fidelity, do not increase GCN4 expression to induce the suppression phenotype and in fact reduce derepression. eEF1A mutations that reduce translation, however, reduce expression of GCN4 under non-starvation conditions. These eEF1A mutants also reduce HIS4 mRNA expression. Taken together, this system improves in vivo strategies for the analysis of translational fidelity and further provides new information on the interplay among translation fidelity, altered elongation and translational control via uORFs. PMID:16100380

  16. Elongation factor TFIIS contains three structural domains: solution structure of domain II.

    PubMed Central

    Morin, P E; Awrey, D E; Edwards, A M; Arrowsmith, C H

    1996-01-01

    Transcription elongation by RNA polymerase II is regulated by the general elongation factor TFIIS. This factor stimulates RNA polymerase II to transcribe through regions of DNA that promote the formation of stalled ternary complexes. Limited proteolytic digestion showed that yeast TFIIS is composed of three structural domains, termed I, II, and III. The two C-terminal domains (II and III) are required for transcription activity. The structure of domain III has been solved previously by using NMR spectroscopy. Here, we report the NMR-derived structure of domain II: a three-helix bundle built around a hydrophobic core composed largely of three tyrosines protruding from one face of the C-terminal helix. The arrangement of known inactivating mutations of TFIIS suggests that two surfaces of domain II are critical for transcription activity. Images Fig. 1 Fig. 2 Fig. 3 PMID:8855225

  17. Transcription initiation factor DksA has diverse effects on RNA chain elongation

    PubMed Central

    Furman, Ran; Sevostyanova, Anastasiya; Artsimovitch, Irina

    2012-01-01

    Bacterial transcription factors DksA and GreB belong to a family of coiled-coil proteins that bind within the secondarychannel of RNA polymerase (RNAP). These proteins display structural homology but play different regulatory roles. DksA disrupts RNAP interactions with promoter DNA and inhibits formation of initiation complexes, sensitizing rRNA synthesis to changes in concentrations of ppGpp and NTPs. Gre proteins remodel the RNAP active site and facilitate cleavage of the nascent RNA in elongation complexes. However, DksA and GreB were shown to have overlapping effects during initiation, and in vivo studies suggested that DksA may also function at post-initiation steps. Here we show that DksA has many features of an elongation factor: it inhibits both RNA chain extension and RNA shortening by exonucleolytic cleavage or pyrophosphorolysis and increases intrinsic termination in vitro and in vivo. However, DksA has no effect on Rho- or Mfd-mediated RNA release or nascent RNA cleavage in backtracked complexes, the regulatory target of Gre factors. Our results reveal that DksA effects on elongating RNAP are very different from those of GreB, suggesting that these regulators recognize distinct states of the transcription complex. PMID:22210857

  18. PEX11 family members are membrane elongation factors that coordinate peroxisome proliferation and maintenance.

    PubMed

    Koch, Johannes; Pranjic, Kornelija; Huber, Anja; Ellinger, Adolf; Hartig, Andreas; Kragler, Friedrich; Brocard, Cécile

    2010-10-01

    Dynamic changes of membrane structure are intrinsic to organelle morphogenesis and homeostasis. Ectopic expression of proteins of the PEX11 family from yeast, plant or human lead to the formation of juxtaposed elongated peroxisomes (JEPs),which is evocative of an evolutionary conserved function of these proteins in membrane tubulation. Microscopic examinations reveal that JEPs are composed of independent elongated peroxisomes with heterogeneous distribution of matrix proteins. We established the homo- and heterodimerization properties of the human PEX11 proteins and their interaction with the fission factor hFis1, which is known to recruit the GTPase DRP1 to the peroxisomal membrane. We show that excess of hFis1 but not of DRP1 is sufficient to fragment JEPs into normal round-shaped organelles, and illustrate the requirement of microtubules for JEP formation. Our results demonstrate that PEX11-induced JEPs represent intermediates in the process of peroxisome membrane proliferation and that hFis1 is the limiting factor for progression. Hence, we propose a model for a conserved role of PEX11 proteins in peroxisome maintenance through peroxisome polarization, membrane elongation and segregation. PMID:20826455

  19. An elongation factor G-induced ribosome rearrangement precedes tRNA-mRNA translocation.

    PubMed

    Savelsbergh, Andreas; Katunin, Vladimir I; Mohr, Dagmar; Peske, Frank; Rodnina, Marina V; Wintermeyer, Wolfgang

    2003-06-01

    The elongation cycle of protein synthesis is completed by translocation, a rearrangement during which two tRNAs bound to the mRNA move on the ribosome. The reaction is promoted by elongation factor G (EF-G) and accelerated by GTP hydrolysis. Here we report a pre-steady-state kinetic analysis of translocation. The kinetic model suggests that GTP hydrolysis drives a conformational rearrangement of the ribosome that precedes and limits the rates of tRNA-mRNA translocation and Pi release from EF-G.GDP.Pi. The latter two steps are intrinsically rapid and take place at random. These results indicate that the energy of GTP hydrolysis is utilized to promote the ribosome rearrangement and to bias spontaneous fluctuations within the ribosome-EF-G complex toward unidirectional movement of mRNA and tRNA. PMID:12820965

  20. Characterization and phylogeny of entomopathogenic Isaria spp. (Ascomycota: Hypocreales) using ITS1-5.8X-ITS2 and elongation factor 1-alpha sequences

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The elongation factor 1-alpha (EF1-a) and the internal transcribed spacer regions ITS1 and ITS2 (ITS1-5.8S-ITS2) sequences were used to characterize and identify Isaria isolates from Argentina and Brazil, as well as to study the phylogenetic relationships among these isolates and other related fungi...

  1. Control of cotton fibre elongation by a homeodomain transcription factor GhHOX3

    PubMed Central

    Shan, Chun-Min; Shangguan, Xiao-Xia; Zhao, Bo; Zhang, Xiu-Fang; Chao, Lu-men; Yang, Chang-Qing; Wang, Ling-Jian; Zhu, Hua-Yu; Zeng, Yan-Da; Guo, Wang-Zhen; Zhou, Bao-Liang; Hu, Guan-Jing; Guan, Xue-Ying; Chen, Z. Jeffrey; Wendel, Jonathan F.; Zhang, Tian-Zhen; Chen, Xiao-Ya

    2014-01-01

    Cotton fibres are unusually long, single-celled epidermal seed trichomes and a model for plant cell growth, but little is known about the regulation of fibre cell elongation. Here we report that a homeodomain-leucine zipper (HD-ZIP) transcription factor, GhHOX3, controls cotton fibre elongation. GhHOX3 genes are localized to the 12th homoeologous chromosome set of allotetraploid cotton cultivars, associated with quantitative trait loci (QTLs) for fibre length. Silencing of GhHOX3 greatly reduces (>80%) fibre length, whereas its overexpression leads to longer fibre. Combined transcriptomic and biochemical analyses identify target genes of GhHOX3 that also contain the L1-box cis-element, including two cell wall loosening protein genes GhRDL1 and GhEXPA1. GhHOX3 interacts with GhHD1, another homeodomain protein, resulting in enhanced transcriptional activity, and with cotton DELLA, GhSLR1, repressor of the growth hormone gibberellin (GA). GhSLR1 interferes with the GhHOX3–GhHD1 interaction and represses target gene transcription. Our results uncover a novel mechanism whereby a homeodomain protein transduces GA signal to promote fibre cell elongation. PMID:25413731

  2. GhCFE1A, a dynamic linker between the ER network and actin cytoskeleton, plays an important role in cotton fibre cell initiation and elongation

    PubMed Central

    Lv, Fenni; Wang, Haihai; Wang, Xinyu; Han, Libo; Ma, Yinping; Wang, Sen; Feng, Zhidi; Niu, Xiaowei; Cai, Caiping; Kong, Zhaosheng; Zhang, Tianzhen; Guo, Wangzhen

    2015-01-01

    Fibre cell initiation and elongation is critical for cotton fibre development. However, little is known about the regulation of initiation and elongation during fibre cell development. Here, the regulatory role of a novel protein GhCFE1A was uncovered. GhCFE1A is preferentially expressed at initiation and rapid elongation stages during fibre development; in addition, much higher expression of GhCFE1A was detected at the fibre initiation stage in fibreless cotton mutants than in the fibre-bearing TM-1 wild-type. Importantly, overexpression of GhCFE1A in cotton not only delayed fibre cell elongation but also significantly reduced the density of lint and fuzz fibre initials and stem trichomes. Yeast two-hybrid assay showed that GhCFE1A interacted with several actin proteins, and the interaction was further confirmed by co-sedimentation assay. Interestingly, a subcellular localization assay showed that GhCFE1A resided on the cortical endoplasmic reticulum (ER) network and co-localized with actin cables. Moreover, the density of F-actin filaments was shown to be reduced in GhCFE1A-overexpressing fibres at the rapid elongation stage compared with the wild-type control. Taken together, the results demonstrate that GhCFE1A probably functions as a dynamic linker between the actin cytoskeleton and the ER network, and plays an important role in fibre cell initiation and elongation during cotton fibre development. PMID:25609828

  3. GhCFE1A, a dynamic linker between the ER network and actin cytoskeleton, plays an important role in cotton fibre cell initiation and elongation.

    PubMed

    Lv, Fenni; Wang, Haihai; Wang, Xinyu; Han, Libo; Ma, Yinping; Wang, Sen; Feng, Zhidi; Niu, Xiaowei; Cai, Caiping; Kong, Zhaosheng; Zhang, Tianzhen; Guo, Wangzhen

    2015-04-01

    Fibre cell initiation and elongation is critical for cotton fibre development. However, little is known about the regulation of initiation and elongation during fibre cell development. Here, the regulatory role of a novel protein GhCFE1A was uncovered. GhCFE1A is preferentially expressed at initiation and rapid elongation stages during fibre development; in addition, much higher expression of GhCFE1A was detected at the fibre initiation stage in fibreless cotton mutants than in the fibre-bearing TM-1 wild-type. Importantly, overexpression of GhCFE1A in cotton not only delayed fibre cell elongation but also significantly reduced the density of lint and fuzz fibre initials and stem trichomes. Yeast two-hybrid assay showed that GhCFE1A interacted with several actin proteins, and the interaction was further confirmed by co-sedimentation assay. Interestingly, a subcellular localization assay showed that GhCFE1A resided on the cortical endoplasmic reticulum (ER) network and co-localized with actin cables. Moreover, the density of F-actin filaments was shown to be reduced in GhCFE1A-overexpressing fibres at the rapid elongation stage compared with the wild-type control. Taken together, the results demonstrate that GhCFE1A probably functions as a dynamic linker between the actin cytoskeleton and the ER network, and plays an important role in fibre cell initiation and elongation during cotton fibre development. PMID:25609828

  4. Dual use of GTP hydrolysis by elongation factor G on the ribosome

    PubMed Central

    Cunha, Carlos E.; Belardinelli, Riccardo; Peske, Frank; Holtkamp, Wolf; Wintermeyer, Wolfgang; Rodnina, Marina V.

    2013-01-01

    Elongation factor G (EF-G) is a GTPase that catalyzes tRNA and mRNA translocation during the elongation cycle of protein synthesis. The GTP-bound state of the factor on the ribosome has been studied mainly with non-hydrolyzable analogs of GTP, which led to controversial conclusions about the role of GTP hydrolysis in translocation. Here we describe a mutant of EF-G in which the catalytic His91 is replaced with Ala. The mutant EF-G does not hydrolyze GTP, but binds GTP with unchanged affinity, allowing us to study the function of the authentic GTP-bound form of EF-G in translocation. Utilizing fluorescent reporter groups attached to the tRNAs, mRNA, and the ribosome we compile the velocity map of translocation seen from different perspectives. The data suggest that GTP hydrolysis accelerates translocation up to 30-fold and facilitates conformational rearrangements of both 30S subunit (presumably the backward rotation of the 30S head) and EF-G that lead to the dissociation of the factor. Thus, EF-G combines the energy regime characteristic for motor proteins, accelerating movement by a conformational change induced by GTP hydrolysis, with that of a switch GTPase, which upon Pi release switches the conformations of EF-G and the ribosome to low affinity, allowing the dissociation of the factor. PMID:26824016

  5. Dual use of GTP hydrolysis by elongation factor G on the ribosome.

    PubMed

    Cunha, Carlos E; Belardinelli, Riccardo; Peske, Frank; Holtkamp, Wolf; Wintermeyer, Wolfgang; Rodnina, Marina V

    2013-01-01

    Elongation factor G (EF-G) is a GTPase that catalyzes tRNA and mRNA translocation during the elongation cycle of protein synthesis. The GTP-bound state of the factor on the ribosome has been studied mainly with non-hydrolyzable analogs of GTP, which led to controversial conclusions about the role of GTP hydrolysis in translocation. Here we describe a mutant of EF-G in which the catalytic His91 is replaced with Ala. The mutant EF-G does not hydrolyze GTP, but binds GTP with unchanged affinity, allowing us to study the function of the authentic GTP-bound form of EF-G in translocation. Utilizing fluorescent reporter groups attached to the tRNAs, mRNA, and the ribosome we compile the velocity map of translocation seen from different perspectives. The data suggest that GTP hydrolysis accelerates translocation up to 30-fold and facilitates conformational rearrangements of both 30S subunit (presumably the backward rotation of the 30S head) and EF-G that lead to the dissociation of the factor. Thus, EF-G combines the energy regime characteristic for motor proteins, accelerating movement by a conformational change induced by GTP hydrolysis, with that of a switch GTPase, which upon Pi release switches the conformations of EF-G and the ribosome to low affinity, allowing the dissociation of the factor. PMID:26824016

  6. Direct phylogenetic evidence for lateral transfer of elongation factor-like gene

    PubMed Central

    Kamikawa, Ryoma; Inagaki, Yuji; Sako, Yoshihiko

    2008-01-01

    Genes encoding elongation factor-like (EFL) proteins, which show high similarity to elongation factor-1α (EF-1α), have been found in phylogenetically distantly related eukaryotes. The sporadic distribution of “EFL-containing” lineages within “EF-1α-containing” lineages indirectly, but strongly, suggests lateral gene transfer as the principal driving force in EFL evolution. However, one of the most critical aspects in the above hypothesis, the donor lineages in any putative cases of lateral EFL gene transfer, remained unclear. In this study, we provide direct evidence for lateral transfer of an EFL gene through the analyses of 10 diatom EFL genes. All diatom EFL homologues tightly clustered in phylogenetic analyses, suggesting acquisition of the exogenous EFL gene early in diatom evolution. Our survey additionally identified Thalassiosira pseudonana as a eukaryote bearing EF-1α and EFL genes and secondary EFL gene loss in Phaeodactylum tricornutum, the complete genome of which encodes only the EF-1α gene. Most importantly, the EFL phylogeny recovered a robust grouping of homologues from diatoms, the cercozoan Bigelowiella natans, and the foraminifer Planoglabratella opecularis, with the diatoms nested within the Bigelowiella plus Planoglabratella (Rhizaria) grouping. The particular relationships recovered are further consistent with two characteristic sequence motifs. The best explanation of our data analyses is an EFL gene transfer from a foraminifer to a diatom, the first case in which the donor–recipient relationship was clarified. Finally, based on a reverse transcriptase quantitative PCR assay and the genome information of Thalassiosira and Phaeodactylum, we propose the loss of elongation factor function in Thalassiosira EF-1α. PMID:18458344

  7. Arginine-rhamnosylation as new strategy to activate translation elongation factor P

    PubMed Central

    Lassak, Jürgen; Keilhauer, Eva C; Fürst, Maximilian; Wuichet, Kristin; Gödeke, Julia; Starosta, Agata L; Chen, Jhong-Min; Søgaard-Andersen, Lotte; Rohr, Jürgen; Wilson, Daniel N; Häussler, Susanne; Mann, Matthias; Jung, Kirsten

    2015-01-01

    Ribosome stalling at polyproline stretches is common and fundamental. In bacteria, translation elongation factor P (EF-P) rescues such stalled ribosomes, but only when it is post-translationally activated. In Escherichia coli, activation of EF-P is achieved by (R)-β-lysinylation and hydroxylation of a conserved lysine. Here we have unveiled a markedly different modification strategy in which a conserved arginine of EF-P is rhamnosylated by a glycosyltransferase (EarP) using dTDP-l-rhamnose as a substrate. This is to our knowledge the first report of N-linked protein glycosylation on arginine in bacteria and the first example in which a glycosylated side chain of a translation elongation factor is essential for function. Arginine-rhamnosylation of EF-P also occurs in clinically relevant bacteria such as Pseudomonas aeruginosa. We demonstrate that the modification is needed to develop pathogenicity, making EarP and dTDP-l-rhamnose-biosynthesizing enzymes ideal targets for antibiotic development. PMID:25686373

  8. Arginine-rhamnosylation as new strategy to activate translation elongation factor P.

    PubMed

    Lassak, Jürgen; Keilhauer, Eva C; Fürst, Maximilian; Wuichet, Kristin; Gödeke, Julia; Starosta, Agata L; Chen, Jhong-Min; Søgaard-Andersen, Lotte; Rohr, Jürgen; Wilson, Daniel N; Häussler, Susanne; Mann, Matthias; Jung, Kirsten

    2015-04-01

    Ribosome stalling at polyproline stretches is common and fundamental. In bacteria, translation elongation factor P (EF-P) rescues such stalled ribosomes, but only when it is post-translationally activated. In Escherichia coli, activation of EF-P is achieved by (R)-β-lysinylation and hydroxylation of a conserved lysine. Here we have unveiled a markedly different modification strategy in which a conserved arginine of EF-P is rhamnosylated by a glycosyltransferase (EarP) using dTDP-L-rhamnose as a substrate. This is to our knowledge the first report of N-linked protein glycosylation on arginine in bacteria and the first example in which a glycosylated side chain of a translation elongation factor is essential for function. Arginine-rhamnosylation of EF-P also occurs in clinically relevant bacteria such as Pseudomonas aeruginosa. We demonstrate that the modification is needed to develop pathogenicity, making EarP and dTDP-L-rhamnose-biosynthesizing enzymes ideal targets for antibiotic development. PMID:25686373

  9. Elongation factor Ts of Chlamydia trachomatis: structure of the gene and properties of the protein.

    PubMed

    Zhang, Y; Tao, J; Zhou, M; Meng, Q; Zhang, L; Shen, L; Klein, R; Miller, D L

    1997-08-01

    A putative structural gene cluster containing four open reading frames (ORFs) located downstream of the omp1 gene of Chlamydia trachomatis mouse pneumonitis (MoPn) was cloned and sequenced. A GenBank survey indicated that the identified cluster is similar to the rpsB-tsf-pyrH(smbA)-frr region of Escherichia coli. The second ORF was 846 bp encoding a 282-amino-acid polypeptide with a calculated M(r) 30,824. Alignment of this deduced protein sequence and E. coli elongation factor Ts (EF-Ts, product of tsf) demonstrated 34% identity and an additional 14% similarity. The putative chlamydial tsf gene was expressed in E. coli as a nonfusion protein and as a 6x His-tagged fusion protein. By SDS-PAGE analysis, the molecular weights of the nonfusion recombinant protein and a protein of chlamydial elementary bodies (EBs), which was recognized by monoclonal antibodies derived from the nonfusion recombinant protein, are 34 kDa. The purified recombinant 6x His-tagged fusion protein increased the rate of GDP exchange with both Chlamydia and E. coli elongation factor Tu (EF-Tu). These data show that the second gene of the identified cluster is tsf. Unlike EF-Ts from any other species, its activity was comparable to that of E. coli EF-Ts in exchange reaction with E. coli EF-Tu. PMID:9244380

  10. Activation of GTP hydrolysis in mRNA-tRNA translocation by elongation factor G

    PubMed Central

    Li, Wen; Liu, Zheng; Koripella, Ravi Kiran; Langlois, Robert; Sanyal, Suparna; Frank, Joachim

    2015-01-01

    During protein synthesis, elongation of the polypeptide chain by each amino acid is followed by a translocation step in which mRNA and transfer RNA (tRNA) are advanced by one codon. This crucial step is catalyzed by elongation factor G (EF-G), a guanosine triphosphatase (GTPase), and accompanied by a rotation between the two ribosomal subunits. A mutant of EF-G, H91A, renders the factor impaired in guanosine triphosphate (GTP) hydrolysis and thereby stabilizes it on the ribosome. We use cryogenic electron microscopy (cryo-EM) at near-atomic resolution to investigate two complexes formed by EF-G H91A in its GTP state with the ribosome, distinguished by the presence or absence of the intersubunit rotation. Comparison of these two structures argues in favor of a direct role of the conserved histidine in the switch II loop of EF-G in GTPase activation, and explains why GTP hydrolysis cannot proceed with EF-G bound to the unrotated form of the ribosome. PMID:26229983

  11. Conformationally restricted elongation factor G retains GTPase activity but is inactive in translocation on the ribosome.

    PubMed

    Peske, F; Matassova, N B; Savelsbergh, A; Rodnina, M V; Wintermeyer, W

    2000-08-01

    Elongation factor G (EF-G) from Escherichia coli is a large, five-domain GTPase that promotes tRNA translocation on the ribosome. Full activity requires GTP hydrolysis, suggesting that a conformational change of the factor is important for function. To restrict the intramolecular mobility, two cysteine residues were engineered into domains 1 and 5 of EF-G that spontaneously formed a disulfide cross-link. Cross-linked EF-G retained GTPase activity on the ribosome, whereas it was inactive in translocation as well as in turnover. Both activities were restored when the cross-link was reversed by reduction. These results strongly argue against a GTPase switch-type model of EF-G function and demonstrate that conformational mobility is an absolute requirement for EF-G function on the ribosome. PMID:10983996

  12. Elongated Structure of the Outer-Membrane Activator of Peptidoglycan Synthesis LpoA: Implications for PBP1A Stimulation

    PubMed Central

    Jean, Nicolas L.; Bougault, Catherine M.; Lodge, Adam; Derouaux, Adeline; Callens, Gilles; Egan, Alexander J.F.; Ayala, Isabel; Lewis, Richard J.; Vollmer, Waldemar; Simorre, Jean-Pierre

    2014-01-01

    Summary The bacterial cell envelope contains the stress-bearing peptidoglycan layer, which is enlarged during cell growth and division by membrane-anchored synthases guided by cytoskeletal elements. In Escherichia coli, the major peptidoglycan synthase PBP1A requires stimulation by the outer-membrane-anchored lipoprotein LpoA. Whereas the C-terminal domain of LpoA interacts with PBP1A to stimulate its peptide crosslinking activity, little is known about the role of the N-terminal domain. Herein we report its NMR structure, which adopts an all-α-helical fold comprising a series of helix-turn-helix tetratricopeptide-repeat (TPR)-like motifs. NMR spectroscopy of full-length LpoA revealed two extended flexible regions in the C-terminal domain and limited, if any, flexibility between the N- and C-terminal domains. Analytical ultracentrifugation and small-angle X-ray scattering results are consistent with LpoA adopting an elongated shape, with dimensions sufficient to span from the outer membrane through the periplasm to interact with the peptidoglycan synthase PBP1A. PMID:24954617

  13. The elongation factor Spt5 facilitates transcription initiation for rapid induction of inflammatory-response genes

    PubMed Central

    Diamant, Gil; Bahat, Anat; Dikstein, Rivka

    2016-01-01

    A subset of inflammatory-response NF-κB target genes is activated immediately following pro-inflammatory signal. Here we followed the kinetics of primary transcript accumulation after NF-κB activation when the elongation factor Spt5 is knocked down. While elongation rate is unchanged, the transcript synthesis at the 5′-end and at the earliest time points is delayed and reduced, suggesting an unexpected role in early transcription. Investigating the underlying mechanism reveals that the induced TFIID–promoter association is practically abolished by Spt5 depletion. This effect is associated with a decrease in promoter-proximal H3K4me3 and H4K5Ac histone modifications that are differentially required for rapid transcriptional induction. In contrast, the displacement of TFIIE and Mediator, which occurs during promoter escape, is attenuated in the absence of Spt5. Our findings are consistent with a central role of Spt5 in maintenance of TFIID–promoter association and promoter escape to support rapid transcriptional induction and re-initiation of inflammatory-response genes. PMID:27180651

  14. West syndrome caused by homozygous variant in the evolutionary conserved gene encoding the mitochondrial elongation factor GUF1.

    PubMed

    Alfaiz, Ali Abdullah; Müller, Verena; Boutry-Kryza, Nadia; Ville, Dorothée; Guex, Nicolas; de Bellescize, Julitta; Rivier, Clotilde; Labalme, Audrey; des Portes, Vincent; Edery, Patrick; Till, Marianne; Xenarios, Ioannis; Sanlaville, Damien; Herrmann, Johannes M; Lesca, Gaétan; Reymond, Alexandre

    2016-07-01

    West syndrome (WS), defined by the triad of infantile spasms, pathognomonic hypsarrhythmia and developmental regression, is a rare epileptic disease affecting about 1:3500 live births. To get better insights on the genetic of this pathology, we exome-sequenced the members of a consanguineous family affected with isolated WS. We identified a homozygous variant (c.1825G>T/p.(Ala609Ser)) in the GUF1 gene in the three affected siblings. GUF1 encodes a protein essential in conditions that counteract faithful protein synthesis: it is able to remobilize stuck ribosomes and transiently inhibit the elongation process to optimize protein synthesis. The variant identified in the WS family changes an alanine residue conserved in all eukaryotic organisms and positioned within the tRNA-binding moiety of this nuclear genome-encoded mitochondrial translational elongation factor. Yeast complementation assays show that the activity of GUF1(A609S) is modified in suboptimal environments. We suggest a new link between improper assembly of respiratory chain complexes and WS. PMID:26486472

  15. Elongation Factor-Tu (EF-Tu) proteins structural stability and bioinformatics in ancestral gene reconstruction

    NASA Astrophysics Data System (ADS)

    Dehipawala, Sunil; Nguyen, A.; Tremberger, G.; Cheung, E.; Schneider, P.; Lieberman, D.; Holden, T.; Cheung, T.

    2013-09-01

    A paleo-experimental evolution report on elongation factor EF-Tu structural stability results has provided an opportunity to rewind the tape of life using the ancestral protein sequence reconstruction modeling approach; consistent with the book of life dogma in current biology and being an important component in the astrobiology community. Fractal dimension via the Higuchi fractal method and Shannon entropy of the DNA sequence classification could be used in a diagram that serves as a simple summary. Results from biomedical gene research provide examples on the diagram methodology. Comparisons between biomedical genes such as EEF2 (elongation factor 2 human, mouse, etc), WDR85 in epigenetics, HAR1 in human specificity, DLG1 in cognitive skill, and HLA-C in mosquito bite immunology with EF Tu DNA sequences have accounted for the reported circular dichroism thermo-stability data systematically; the results also infer a relatively less volatility geologic time period from 2 to 3 Gyr from adaptation viewpoint. Comparison to Thermotoga maritima MSB8 and Psychrobacter shows that Thermus thermophilus HB8 EF-Tu calibration sequence could be an outlier, consistent with free energy calculation by NUPACK. Diagram methodology allows computer simulation studies and HAR1 shows about 0.5% probability from chimp to human in terms of diagram location, and SNP simulation results such as amoebic meningoencephalitis NAF1 suggest correlation. Extensions to the studies of the translation and transcription elongation factor sequences in Megavirus Chiliensis, Megavirus Lba and Pandoravirus show that the studied Pandoravirus sequence could be an outlier with the highest fractal dimension and lowest entropy, as compared to chicken as a deviant in the DNMT3A DNA methylation gene sequences from zebrafish to human and to the less than one percent probability in computer simulation using the HAR1 0.5% probability as reference. The diagram methodology would be useful in ancestral gene

  16. The Positive Transcription Elongation Factor b Is an Essential Cofactor for the Activation of Transcription by Myocyte Enhancer Factor 2

    PubMed Central

    Nojima, Masanori; Huang, Yehong; Tyagi, Mudit; Kao, Hung-Ying; Fujinaga, Koh

    2014-01-01

    The positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 and cyclin T1, stimulates the elongation of transcription by hyperphosphorylating the C-terminal region of RNA polymerase II. Aberrant activation of P-TEFb results in manifestations of cardiac hypertrophy in mice, suggesting that P-TEFb is an essential factor for cardiac myocyte function and development. Here, we present evidence that P-TEFb selectively activates transcription mediated by the myocyte enhancer factor 2 (MEF2) family of transcription factors, key regulatory factors for myocyte development. Knockdown of endogenous cyclin T1 in murine C2C12 cells abolishes MEF2-dependent reporter gene expression as well as transcription of endogenous MEF2 target genes, whereas overexpression of P-TEFb enhances MEF2-dependent transcription. P-TEFb interacts with MEF2 both in vitro and in vivo. Activation of MEF2-dependent transcription induced by serum starvation is mediated by a rapid dissociation of P-TEFb from its inhibitory subunit, HEXIM1, and a subsequent recruitment of P-TEFb to MEF2 binding sites in the promoter region of MEF2 target genes. These results indicate that recruitment of P-TEFb is a critical step for stimulation of MEF2-dependent transcription, therefore providing a fundamentally important regulatory mechanism underlying the transcriptional program in muscle cells. PMID:18662700

  17. TLR4-dependent activation of inflammatory cytokine response in macrophages by Francisella elongation factor Tu1

    PubMed Central

    Sharma, Jyotika; Mishra, Bibhuti B.; Li, Qun; Teale, Judy M.

    2011-01-01

    The bacterial determinants of pulmonary Francisella induced inflammatory responses and their interaction with host components are not clearly defined. In this study, proteomic and immunoblot analyses showed presence of a cytoplasmic protein elongation factor Tu (EF-Tu) in the membrane fractions of virulent F. novicida, LVS and SchuS4, but not in an attenuated F. novicida mutant. EF-Tu was immunodominant in mice vaccinated and protected from virulent F. novicida. Moreover, recombinant EF-Tu induced macrophages to produce inflammatory cytokines in a TLR4 dependent manner. This study shows immune stimulatory properties of a cytoplasmic protein EF-Tu expressed on the membrane of virulent Francisella strains. PMID:21497800

  18. Elongation factor 1 alpha concentration is highly correlated with the lysine content of maize endosperm.

    PubMed Central

    Habben, J E; Moro, G L; Hunter, B G; Hamaker, B R; Larkins, B A

    1995-01-01

    Lysine is the most limiting essential amino acid in cereals, and for many years plant breeders have attempted to increase its concentration to improve the nutritional quality of these grains. The opaque2 mutation in maize doubles the lysine content in the endosperm, but the mechanism by which this occurs is unknown. We show that elongation factor 1 alpha (EF-1 alpha) is overexpressed in opaque2 endosperm compared with its normal counterpart and that there is a highly significant correlation between EF-1 alpha concentration and the total lysine content of the endosperm. This relationship is also true for two other cereals, sorghum and barley. It appears that genetic selection for genotypes with a high concentration of EF-1 alpha can significantly improve the nutritional quality of maize and other cereals. Images Fig. 1 Fig. 2 PMID:7567989

  19. Elongation as a factor in artefacts of humans and other animals: an Acheulean example in comparative context

    PubMed Central

    Gowlett, J. A. J.

    2013-01-01

    Elongation is a commonly found feature in artefacts made and used by humans and other animals and can be analysed in comparative study. Whether made for use in hand or beak, the artefacts have some common properties of length, breadth, thickness and balance point, and elongation can be studied as a factor relating to construction or use of a long axis. In human artefacts, elongation can be traced through the archaeological record, for example in stone blades of the Upper Palaeolithic (traditionally regarded as more sophisticated than earlier artefacts), and in earlier blades of the Middle Palaeolithic. It is now recognized that elongation extends to earlier Palaeolithic artefacts, being found in the repertoire of both Neanderthals and more archaic humans. Artefacts used by non-human animals, including chimpanzees, capuchin monkeys and New Caledonian crows show selection for diameter and length, and consistent interventions of modification. Both chimpanzees and capuchins trim side branches from stems, and appropriate lengths of stave are selected or cut. In human artefacts, occasional organic finds show elongation back to about 0.5 million years. A record of elongation achieved in stone tools survives to at least 1.75 Ma (million years ago) in the Acheulean tradition. Throughout this tradition, some Acheulean handaxes are highly elongated, usually found with others that are less elongated. Finds from the million-year-old site of Kilombe and Kenya are given as an example. These findings argue that the elongation need not be integral to a design, but that artefacts may be the outcome of adjustments to individual variables. Such individual adjustments are seen in animal artefacts. In the case of a handaxe, the maker must balance the adjustments to achieve a satisfactory outcome in the artefact as a whole. It is argued that the need to make decisions about individual variables within multivariate objects provides an essential continuity across artefacts made by

  20. Elongation factor 2 kinase promotes cell survival by inhibiting protein synthesis without inducing autophagy

    PubMed Central

    Moore, Claire E.J.; Wang, Xuemin; Xie, Jianling; Pickford, Jo; Barron, John; Regufe da Mota, Sergio; Versele, Matthias; Proud, Christopher G.

    2016-01-01

    Eukaryotic elongation factor 2 kinase (eEF2K) inhibits the elongation stage of protein synthesis by phosphorylating its only known substrate, eEF2. eEF2K is tightly regulated by nutrient-sensitive signalling pathways. For example, it is inhibited by signalling through mammalian target of rapamycin complex 1 (mTORC1). It is therefore activated under conditions of nutrient deficiency. Here we show that inhibiting eEF2K or knocking down its expression renders cancer cells sensitive to death under nutrient-starved conditions, and that this is rescued by compounds that block protein synthesis. This implies that eEF2K protects nutrient-deprived cells by inhibiting protein synthesis. Cells in which signalling through mTORC1 is highly active are very sensitive to nutrient withdrawal. Inhibiting mTORC1 protects them. Our data reveal that eEF2K makes a substantial contribution to the cytoprotective effect of mTORC1 inhibition. eEF2K is also reported to promote another potentially cytoprotective process, autophagy. We have used several approaches to test whether inhibition or loss of eEF2K affects autophagy under a variety of conditions. We find no evidence that eEF2K is involved in the activation of autophagy in the cell types we have studied. We conclude that eEF2K protects cancer cells against nutrient starvation by inhibiting protein synthesis rather than by activating autophagy. PMID:26795954

  1. Distinct functions of elongation factor G in ribosome recycling and translocation

    PubMed Central

    Savelsbergh, Andreas; Rodnina, Marina V.; Wintermeyer, Wolfgang

    2009-01-01

    Elongation factor G (EF-G) promotes the translocation step in bacterial protein synthesis and, together with ribosome recycling factor (RRF), the disassembly of the post-termination ribosome. Unlike translocation, ribosome disassembly strictly requires GTP hydrolysis by EF-G. Here we report that ribosome disassembly is strongly inhibited by vanadate, an analog of inorganic phosphate (Pi), indicating that Pi release is required for ribosome disassembly. In contrast, the function of EF-G in single-round translocation is not affected by vanadate, while the turnover reaction is strongly inhibited. We also show that the antibiotic fusidic acid blocks ribosome disassembly by EF-G/RRF at a 1000-fold lower concentration than required for the inhibition of EF-G turnover in vitro and close to the effective inhibitory concentration in vivo, suggesting that the antimicrobial activity of fusidic acid is primarily due to the direct inhibition of ribosome recycling. Our results indicate that conformational coupling between EF-G and the ribosome is principally different in translocation and ribosome disassembly. Pi release is not required for the mechanochemical function of EF-G in translocation, whereas the interactions between RRF and EF-G introduce tight coupling between the conformational change of EF-G induced by Pi release and ribosome disassembly. PMID:19324963

  2. Elongation Factor P and Modifying Enzyme PoxA Are Necessary for Virulence of Shigella flexneri

    PubMed Central

    Marman, Hannah E.; Mey, Alexandra R.

    2014-01-01

    Elongation factor P (EF-P) is a universally conserved bacterial translation factor. In many bacteria, EF-P is posttranslationally modified by PoxA, which covalently attaches a β-lysine to a conserved lysine residue of EF-P. Here we show that both EF-P and PoxA are necessary for virulence of the human diarrheal pathogen Shigella flexneri. Loss of either EF-P or PoxA leads to an impaired ability of S. flexneri to invade epithelial cells and form plaques in an epithelial cell monolayer. Proteomic analysis of efp and poxA deletion mutants revealed decreased levels of several virulence effector proteins, including IpaA, -B, and -C and IcsA. Additionally, mRNA levels of virB and virF, which encode master virulence regulators, were decreased in the efp mutant. The reduction in virF transcription was at least partially due to decreased levels of CpxA, which activates virF through the response regulator CpxR. The role of CpxAR in reduced synthesis of VirF and its downstream effectors was indicated by restoration of invasion when a mutation resulting in constitutively activated CpxR was introduced into the efp mutant. Thus, modified EF-P is required for appropriate synthesis of proteins involved in the virulence of this bacterial pathogen. PMID:24935977

  3. Gene Expression in Mouse Thyrotrope Adenoma: Transcription Elongation Factor Stimulates Proliferation.

    PubMed

    Gergics, Peter; Christian, Helen C; Choo, Monica S; Ajmal, Adnan; Camper, Sally A

    2016-09-01

    Thyrotrope hyperplasia and hypertrophy are common responses to primary hypothyroidism. To understand the genetic regulation of these processes, we studied gene expression changes in the pituitaries of Cga(-/-) mice, which are deficient in the common α-subunit of TSH, LH, and FSH. These mice have thyrotrope hypertrophy and hyperplasia and develop thyrotrope adenoma. We report that cell proliferation is increased, but the expression of most stem cell markers is unchanged. The α-subunit is required for secretion of the glycoprotein hormone β-subunits, and mutants exhibit elevated expression of many genes involved in the unfolded protein response, consistent with dilation and stress of the endoplasmic reticulum. Mutants have elevated expression of transcription factors that are important in thyrotrope function, such as Gata2 and Islet 1, and those that stimulate proliferation, including Nupr1, E2f1, and Etv5. We characterized the expression and function of a novel, overexpressed gene, transcription elongation factor A (SII)-like 5 (Tceal5). Stable expression of Tceal5 in a pituitary progenitor cell line is sufficient to increase cell proliferation. Thus, Tceal5 may act as a proto-oncogene. This study provides a rich resource for comparing pituitary transcriptomes and an analysis of gene expression networks. PMID:27580811

  4. The primary σ factor in Escherichia coli can access the transcription elongation complex from solution in vivo

    PubMed Central

    Goldman, Seth R; Nair, Nikhil U; Wells, Christopher D; Nickels, Bryce E; Hochschild, Ann

    2015-01-01

    The σ subunit of bacterial RNA polymerase (RNAP) confers on the enzyme the ability to initiate promoter-specific transcription. Although σ factors are generally classified as initiation factors, σ can also remain associated with, and modulate the behavior of, RNAP during elongation. Here we establish that the primary σ factor in Escherichia coli, σ70, can function as an elongation factor in vivo by loading directly onto the transcription elongation complex (TEC) in trans. We demonstrate that σ70 can bind in trans to TECs that emanate from either a σ70-dependent promoter or a promoter that is controlled by an alternative σ factor. We further demonstrate that binding of σ70 to the TEC in trans can have a particularly large impact on the dynamics of transcription elongation during stationary phase. Our findings establish a mechanism whereby the primary σ factor can exert direct effects on the composition of the entire transcriptome, not just that portion that is produced under the control of σ70-dependent promoters. DOI: http://dx.doi.org/10.7554/eLife.10514.001 PMID:26371553

  5. Ribosomal Elongation Factor 4 Promotes Cell Death Associated with Lethal Stress

    PubMed Central

    Li, Liping; Hong, Yuzhi; Luan, Gan; Mosel, Michael; Malik, Muhammad; Drlica, Karl

    2014-01-01

    ABSTRACT Ribosomal elongation factor 4 (EF4) is highly conserved among bacteria, mitochondria, and chloroplasts. However, the EF4-encoding gene, lepA, is nonessential and its deficiency shows no growth or fitness defect. In purified systems, EF4 back-translocates stalled, posttranslational ribosomes for efficient protein synthesis; consequently, EF4 has a protective role during moderate stress. We were surprised to find that EF4 also has a detrimental role during severe stress: deletion of lepA increased Escherichia coli survival following treatment with several antimicrobials. EF4 contributed to stress-mediated lethality through reactive oxygen species (ROS) because (i) the protective effect of a ΔlepA mutation against lethal antimicrobials was eliminated by anaerobic growth or by agents that block hydroxyl radical accumulation and (ii) the ΔlepA mutation decreased ROS levels stimulated by antimicrobial stress. Epistasis experiments showed that EF4 functions in the same genetic pathway as the MazF toxin, a stress response factor implicated in ROS-mediated cell death. The detrimental action of EF4 required transfer-messenger RNA (tmRNA, which tags truncated proteins for degradation and is known to be inhibited by EF4) and the ClpP protease. Inhibition of a protective, tmRNA/ClpP-mediated degradative activity would allow truncated proteins to indirectly perturb the respiratory chain and thereby provide a potential link between EF4 and ROS. The connection among EF4, MazF, tmRNA, and ROS expands a pathway leading from harsh stress to bacterial self-destruction. The destructive aspect of EF4 plus the protective properties described previously make EF4 a bifunctional factor in a stress response that promotes survival or death, depending on the severity of stress. PMID:25491353

  6. Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation

    PubMed Central

    Scotti, John S.; Leung, Ivanhoe K. H.; Ge, Wei; Bentley, Michael A.; Paps, Jordi; Kramer, Holger B.; Lee, Joongoo; Aik, WeiShen; Choi, Hwanho; Paulsen, Steinar M.; Bowman, Lesley A. H.; Loik, Nikita D.; Horita, Shoichiro; Ho, Chia-hua; Kershaw, Nadia J.; Tang, Christoph M.; Claridge, Timothy D. W.; Preston, Gail M.; McDonough, Michael A.; Schofield, Christopher J.

    2014-01-01

    The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolyl-hydroxylation in human hypoxia sensing has ancient origins. PMID:25197067

  7. Elongation factor 1 β/δ of Echinococcus granulosus and allergic manifestations in human cystic echinococcosis

    PubMed Central

    Ortona, E; Margutti, P; Vaccari, S; Riganò, R; Profumo, E; Buttari, B; Chersi, A; Teggi, A; Siracusano, A

    2001-01-01

    Allergic reactions, such as urticaria, itching and anaphylactic shock, often complicate the course of cystic echinococcosis (CE). To investigate the role of the IgE-immunoreactive recombinant Echinococcus granulosus elongation factor-1 β/δ (EgEF-1 β/δ) in the allergic disorders during CE we determined humoral and cell-mediated responses to this antigen in patients with CE grouped according to the clinical presence or absence of allergic reactions. Immunoblotting analysis showed that serum IgE-binding reactivity to EgEF-1 β/δ differed significantly in patients with and without allergic reactions (38 of 42, 90% vs. 31 of 56, 56%; P < 10−4). EgEF-1 β/δ induced a proliferative response in 14 of 19 (74%) patients' peripheral blood mononuclear cells (PBMC) irrespective of the allergic manifestations and skewed Th1/Th2 cytokine activation towards a preferentially Th2 polarization. Epitope mapping identified an immunodominant epitope of 18 residues with 78% identity and 89% similarity with an IgE-immunoreactive Strongyloides stercoralis antigen. Overall these findings suggest that EgEF-1 β/δ is an allergenic molecule that may be a general marker of the intensity of CE immune response and that could lead to a deeper understanding of the specific antigen-induced mechanisms underlying allergic reactions in the human host. PMID:11472433

  8. Divergence among Genes Encoding the Elongation Factor Tu of Yersinia Species▿

    PubMed Central

    Isabel, Sandra; Leblanc, Éric; Boissinot, Maurice; Boudreau, Dominique K.; Grondin, Myrian; Picard, François J.; Martel, Eric A.; Parham, Nicholas J.; Chain, Patrick S. G.; Bader, Douglas E.; Mulvey, Michael R.; Bryden, Louis; Roy, Paul H.; Ouellette, Marc; Bergeron, Michel G.

    2008-01-01

    Elongation factor Tu (EF-Tu), encoded by tuf genes, carries aminoacyl-tRNA to the ribosome during protein synthesis. Duplicated tuf genes (tufA and tufB), which are commonly found in enterobacterial species, usually coevolve via gene conversion and are very similar to one another. However, sequence analysis of tuf genes in our laboratory has revealed highly divergent copies in 72 strains spanning the genus Yersinia (representing 12 Yersinia species). The levels of intragenomic divergence between tufA and tufB sequences ranged from 8.3 to 16.2% for the genus Yersinia, which is significantly greater than the 0.0 to 3.6% divergence observed for other enterobacterial genera. We further explored tuf gene evolution in Yersinia and other Enterobacteriaceae by performing directed sequencing and phylogenetic analyses. Phylogenetic trees constructed using concatenated tufA and tufB sequences revealed a monophyletic genus Yersinia in the family Enterobacteriaceae. Moreover, Yersinia strains form clades within the genus that mostly correlate with their phenotypic and genetic classifications. These genetic analyses revealed an unusual divergence between Yersinia tufA and tufB sequences, a feature unique among sequenced Enterobacteriaceae and indicative of a genus-wide loss of gene conversion. Furthermore, they provided valuable phylogenetic information for possible reclassification and identification of Yersinia species. PMID:18790860

  9. Translation Control of Swarming Proficiency in Bacillus subtilis by 5-Amino-pentanolylated Elongation Factor P.

    PubMed

    Rajkovic, Andrei; Hummels, Katherine R; Witzky, Anne; Erickson, Sarah; Gafken, Philip R; Whitelegge, Julian P; Faull, Kym F; Kearns, Daniel B; Ibba, Michael

    2016-05-20

    Elongation factor P (EF-P) accelerates diprolyl synthesis and requires a posttranslational modification to maintain proteostasis. Two phylogenetically distinct EF-P modification pathways have been described and are encoded in the majority of Gram-negative bacteria, but neither is present in Gram-positive bacteria. Prior work suggested that the EF-P-encoding gene (efp) primarily supports Bacillus subtilis swarming differentiation, whereas EF-P in Gram-negative bacteria has a more global housekeeping role, prompting our investigation to determine whether EF-P is modified and how it impacts gene expression in motile cells. We identified a 5-aminopentanol moiety attached to Lys(32) of B. subtilis EF-P that is required for swarming motility. A fluorescent in vivo B. subtilis reporter system identified peptide motifs whose efficient synthesis was most dependent on 5-aminopentanol EF-P. Examination of the B. subtilis genome sequence showed that these EF-P-dependent peptide motifs were represented in flagellar genes. Taken together, these data show that, in B. subtilis, a previously uncharacterized posttranslational modification of EF-P can modulate the synthesis of specific diprolyl motifs present in proteins required for swarming motility. PMID:27002156

  10. Elongation factor 1 gamma mRNA expression in oesophageal carcinoma.

    PubMed Central

    Mimori, K; Mori, M; Inoue, H; Ueo, H; Mafune, K; Akiyoshi, T; Sugimachi, K

    1996-01-01

    Elongation factor 1 gamma (EF1 gamma) is known to be a subunit of EF1, one of the G proteins that mediate the transport of aminoacyl tRNA to 80S ribosomes during translation. As little is known regarding the expression of EF1 gamma in human oesophageal carcinoma, this study looked at its expression using a northern blot analysis. Thirty six cases of oesophageal carcinoma and 15 oesophageal carcinoma cell lines were studied. The EF1 gamma mRNA overexpression at a level of twofold or more was seen in five (14%) of 36 carcinomatous tissues compared with the normal counterparts. All five overexpressed cases showed severe lymph node metastases compared with the non-overexpressed cases, and the difference was significant (p = 0.028). The stage of the disease of these five cases was far advanced compared with the nonoverexpressed cases (p = 0.012). All 15 oesophageal carcinoma cells expressed EF1 gamma mRNA relatively lower than the gastric or pancreatic carcinoma cell lines, in which EF1 gamma was originally isolated. As the expression of EF1 gamma mRNA could be detected even in the biopsy specimens, its overexpression in tumour tissue may provide preoperative useful information for predicting the aggressiveness of tumours. Images Figure 1 Figure 2 Figure 3 PMID:8566862

  11. Recombination between elongation factor 1α genes from distantly related archaeal lineages

    PubMed Central

    Inagaki, Yuji; Susko, Edward; Roger, Andrew J.

    2006-01-01

    Homologous recombination (HR) and lateral gene transfer are major processes in genome evolution. The combination of the two processes, HR between genes in different species, has been documented but is thought to be restricted to very similar sequences in relatively closely related organisms. Here we report two cases of interspecific HR in the gene encoding the core translational protein translation elongation factor 1α (EF-1α) between distantly related archaeal groups. Maximum-likelihood sliding window analyses indicate that a fragment of the EF-1α gene from the archaeal lineage represented by Methanopyrus kandleri was recombined into the orthologous gene in a common ancestor of the Thermococcales. A second recombination event appears to have occurred between the EF-1α gene of the genus Methanothermobacter and its ortholog in a common ancestor of the Methanosarcinales, a distantly related euryarchaeal lineage. These findings suggest that HR occurs across a much larger evolutionary distance than generally accepted and affects highly conserved essential “informational” genes. Although difficult to detect by standard whole-gene phylogenetic analyses, interspecific HR in highly conserved genes may occur at an appreciable frequency, potentially confounding deep phylogenetic inference and hypothesis testing. PMID:16537397

  12. Cloning and expression of Bombyx mori silk gland elongation factor 1gamma in Escherichia coli.

    PubMed

    Kamiie, Katsuyoshi; Nomura, Yoshitaka; Kobayashi, Satoru; Taira, Hideharu; Kobayashi, Kohmei; Yamashita, Tetsuro; Kidou, Shin-ichiro; Ejiri, Shin-ichiro

    2002-03-01

    Elongation factor 1 (EF-1) from the silk gland of Bombyx mori consists of alpha-, beta-, gamma-, and delta-subunits. EF-1alpha GTP catalyzes the binding of aminoacyl-tRNA to ribosomes concomitant with the hydrolysis of GTP. EF-1betagammadelta catalyzes the exchange of EF-1alpha-bound GDP for exogenous GTP and stimulates the EF-1alpha-dependent binding of aminoacyl-tRNA to ribosomes. EF-1gamma cDNA, which contains an open reading frame (ORF) encoding a polypeptide of 423 amino acid residues, was amplified and cloned by PCR from a silk gland cDNA library. The calculated molecular mass and predicted pI of the product were 48,388 Da and 5.84, respectively. The silk gland EF-1gamma shares 67.3% amino acid identity with Artemia salina EF-lgamma. The N-terminal domain (amino acid residues 1-211) of silk gland EF-lgamma is 29.3% identical to maize glutathione S-transferase. We demonstrated that silk gland EF-lgamma bound to glutathione Sepharose, suggesting that the N-terminal domain of EF-1gamma may have the capacity to bind to glutathione. PMID:12005049

  13. Molecular cloning and phylogenetic analysis of Clonorchis sinensis elongation factor-1alpha.

    PubMed

    Kim, Tae Yun; Cho, Pyo Yun; Na, Jong Won; Hong, Sung-Jong

    2007-11-01

    Elongation factor-1 (EF-1) plays a primary role in protein synthesis, e.g., in the regulation of cell growth, aging, motility, embryogenesis, and signal transduction. The authors identified a clone CsIH23 by immunoscreening a Clonorchis sinensis cDNA library. The cDNA of CsIH23 was found to have a putative open reading frame containing 461 amino acids with a predicted molecular mass of 50.5 kDa. Its polypeptide sequence was highly homologous with EF-1alpha of parasites and vertebrate animals. CsIH23 polypeptide contained three GTP/GDP-binding sites, one ribosome-binding domain, one actin-binding domain, one tRNA-binding domain, and two glyceryl-phosphoryl-ethanolamine attachment sites. Based on these primary and secondary structural similarities, it was concluded that CsIH23 cDNA encodes C. sinensis EF-1alpha (CsEF-1alpha). In a molecular phylogenic tree, CsEF-1alpha clustered with the EF-1alpha of helminthic parasites. Subsequently, CsEF-1alpha recombinant protein was bacterially overexpressed and purified by Ni-NTA affinity column chromatography. Immunoblotting using CsEF-1alpha recombinant protein produced positive signals for all serum samples tested from clonorchiasis, opisthorchiasis viverinii, and paragonimiasis westermani patients and normal healthy controls. These findings suggest that recombinant CsEF-1alpha is of limited usefulness as serodiagnostic antigen for clonorchiasis. PMID:17674047

  14. Translation Elongation Factor Tuf of Acinetobacter baumannii Is a Plasminogen-Binding Protein

    PubMed Central

    Koenigs, Arno; Zipfel, Peter F.; Kraiczy, Peter

    2015-01-01

    Acinetobacter baumannii is an important nosocomial pathogen, causing a variety of opportunistic infections of the skin, soft tissues and wounds, urinary tract infections, secondary meningitis, pneumonia and bacteremia. Over 63% of A. baumannii infections occurring in the United States are caused by multidrug resistant isolates, and pan-resistant isolates have begun to emerge that are resistant to all clinically relevant antibiotics. The complement system represents the first line of defense against invading pathogens. However, many A. baumannii isolates, especially those causing severe bacteremia are resistant to complement-mediated killing, though the underlying mechanisms remain poorly understood. Here we show for the first time that A. baumannii binds host-derived plasminogen and we identify the translation elongation factor Tuf as a moonlighting plasminogen-binding protein that is exposed on the outer surface of A. baumannii. Binding of plasminogen to Tuf is at least partly dependent on lysine residues and ionic interactions. Plasminogen, once bound to Tuf can be converted to active plasmin and proteolytically degrade fibrinogen as well as the key complement component C3b. Thus, Tuf acts as a multifunctional protein that may contribute to virulence of A. baumannii by aiding in dissemination and evasion of the complement system. PMID:26230848

  15. Eukaryotic elongation factor-1α 2 knockdown inhibits hepatocarcinogenesis by suppressing PI3K/Akt/NF-κB signaling

    PubMed Central

    Qiu, Fu-Nan; Huang, Yi; Chen, Dun-Yan; Li, Feng; Wu, Yan-An; Wu, Wen-Bing; Huang, Xiao-Li

    2016-01-01

    AIM: To assess the impact of eukaryotic elongation factor 1 alpha 2 (eEF1A2) on hepatocellular carcinoma (HCC) cell proliferation, apoptosis, migration and invasion, and determine the underlying mechanisms. METHODS: eEF1A2 levels were detected in 62 HCC tissue samples and paired pericarcinomatous specimens, and the human HCC cell lines SK-HEP-1, HepG2 and BEF-7402, by real-time PCR and immunohistochemistry. Experimental groups included eEF1A2 silencing in BEL-7402 cells with lentivirus eEF1A2-shRNA (KD group) and eEF1A2 overexpression in SK-HEP-1 cells with eEF1A2 plasmid (OE group). Non-transfected cells (control group) and lentivirus-based empty vector transfected cells (NC group) were considered control groups. Cell proliferation (MTT and colony formation assays), apoptosis (Annexin V-APC assay), cell cycle (DNA ploidy assay), and migration and invasion (Transwell assays) were assessed. Protein levels of PI3K/Akt/NF-κB signaling effectors were evaluated by Western blot. RESULTS: eEF1A2 mRNA and protein levels were significantly higher in HCC cancer tissue samples than in paired pericarcinomatous and normal specimens. SK-HEP-1 cells showed lower eEF1A2 mRNA levels; HepG2 and BEL-7402 cells showed higher eEF1A2 mRNA levels, with BEL-7402 cells displaying the highest amount. Efficient eEF1A2 silencing resulted in reduced cell proliferation, migration and invasion, increased apoptosis, and induced cell cycle arrest. The PI3K/Akt/NF-κB signaling pathway was notably inhibited. Inversely, eEF1A2 overexpression resulted in promoted cell proliferation, migration and invasion. CONCLUSION: eEF1A2, highly expressed in HCC, is a potential oncogene. Its silencing significantly decreases HCC tumorigenesis, likely by inhibiting PI3K/Akt/NF-κB signaling. PMID:27122673

  16. Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu and pyruvate:ferredoxin oxidoreductase of C. perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium-related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by react...

  17. Do maise and wheat chloroplast protein synthesis elongation factor, EF-Tu, protect Rubisco activase from thermal aggregation and inactivation?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize (Zea mays L.) chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in the development of heat tolerance. The precursor of this protein (pre-EF-Tu) has been shown to display chaperone activity, as it protected heat labile citrate synthase and malate dehydrogenase from the...

  18. GhLTPG1, a cotton GPI-anchored lipid transfer protein, regulates the transport of phosphatidylinositol monophosphates and cotton fiber elongation.

    PubMed

    Deng, Ting; Yao, Hongyan; Wang, Jin; Wang, Jun; Xue, Hongwei; Zuo, Kaijing

    2016-01-01

    The cotton fibers are seed trichomes that elongate from the ovule epidermis. Polar lipids are required for the quick enlargement of cell membrane and fiber cell growth, however, how lipids are transported from the ovules into the developing fibers remains less known. Here, we reported the functional characterization of GhLTPG1, a GPI-anchored lipid transport protein, during cotton fiber elongation. GhLTPG1 was abundantly expressed in elongating cotton fibers and outer integument of the ovules, and GhLTPG1 protein was located on cell membrane. Biochemical analysis showed that GhLTPG1 specifically bound to phosphatidylinositol mono-phosphates (PtdIns3P, PtdIns4P and PtdIns5P) in vitro and transported PtdInsPs from the synthesis places to the plasma membranes in vivo. Expression of GhLTPG1 in Arabidopsis caused an increased number of trichomes, and fibers in GhLTPG1-knockdown cotton plants exhibited significantly reduced length, decreased polar lipid content, and repression of fiber elongation-related genes expression. These results suggested that GhLTPG1 protein regulates the cotton fiber elongation through mediating the transport of phosphatidylinositol monophosphates. PMID:27311358

  19. GhLTPG1, a cotton GPI-anchored lipid transfer protein, regulates the transport of phosphatidylinositol monophosphates and cotton fiber elongation

    PubMed Central

    Deng, Ting; Yao, Hongyan; Wang, Jin; Wang, Jun; Xue, Hongwei; Zuo, Kaijing

    2016-01-01

    The cotton fibers are seed trichomes that elongate from the ovule epidermis. Polar lipids are required for the quick enlargement of cell membrane and fiber cell growth, however, how lipids are transported from the ovules into the developing fibers remains less known. Here, we reported the functional characterization of GhLTPG1, a GPI-anchored lipid transport protein, during cotton fiber elongation. GhLTPG1 was abundantly expressed in elongating cotton fibers and outer integument of the ovules, and GhLTPG1 protein was located on cell membrane. Biochemical analysis showed that GhLTPG1 specifically bound to phosphatidylinositol mono-phosphates (PtdIns3P, PtdIns4P and PtdIns5P) in vitro and transported PtdInsPs from the synthesis places to the plasma membranes in vivo. Expression of GhLTPG1 in Arabidopsis caused an increased number of trichomes, and fibers in GhLTPG1-knockdown cotton plants exhibited significantly reduced length, decreased polar lipid content, and repression of fiber elongation-related genes expression. These results suggested that GhLTPG1 protein regulates the cotton fiber elongation through mediating the transport of phosphatidylinositol monophosphates. PMID:27311358

  20. The gene family encoding the Arabidopsis thaliana translation elongation factor EF-1 alpha: molecular cloning, characterization and expression.

    PubMed

    Axelos, M; Bardet, C; Liboz, T; Le Van Thai, A; Curie, C; Lescure, B

    1989-10-01

    The gene family encoding the Arabidopsis thaliana translation elongation factor (EF-1 alpha) was analysed. This family contains four genes (A1-A4) organized in a similar manner in different varieties of Arabidopsis. Based upon both their physical separation and a comparison of their sequences, it is suggested that the A4 gene and the A1, A2, and A3 genes constitute two distinct subfamilies within the genome. By introducing chimaeric gene constructs into Arabidopsis cells, we showed that the A1 gene promoter mediates a transient expression about twofold higher than that obtained using the CaMV 35 S promoter. This expression depends on a 348 bp DNA fragment extending from -982 to -634 bp upstream of the initiation codon. This element contains a characteristic telomeric sequence (AACCCTAA) which is also found in the promoters of the A2 and A4 genes as well as in the promoters of the Drosophila EF-1 alpha F1 gene and of several highly expressed plant genes. PMID:2615757

  1. Overexpression of Eukaryotic Translation Elongation Factor 3 Impairs Gcn2 Protein Activation*

    PubMed Central

    Visweswaraiah, Jyothsna; Lee, Su Jung; Hinnebusch, Alan G.; Sattlegger, Evelyn

    2012-01-01

    In eukaryotes, phosphorylation of translation initiation factor 2α (eIF2α) by the kinase Gcn2 (general control nonderepressible 2) is a key response to amino acid starvation. Sensing starvation requires that Gcn2 directly contacts its effector protein Gcn1, and both must contact the ribosome. We have proposed that Gcn2 is activated by uncharged tRNA bound to the ribosomal decoding (A) site, in a manner facilitated by ribosome-bound Gcn1. Protein synthesis requires cyclical association of eukaryotic elongation factors (eEFs) with the ribosome. Gcn1 and Gcn2 are large proteins, raising the question of whether translation and monitoring amino acid availability can occur on the same ribosome. Part of the ribosome-binding domain in Gcn1 has homology to one of the ribosome-binding domains in eEF3, suggesting that these proteins utilize overlapping binding sites on the ribosome and consequently cannot function simultaneously on the same ribosome. Supporting this idea, we found that eEF3 overexpression in Saccharomyces cerevisiae diminished growth on amino acid starvation medium (Gcn− phenotype) and decreased eIF2α phosphorylation, and that the growth defect associated with constitutively active Gcn2 was diminished by eEF3 overexpression. Overexpression of the eEF3 HEAT domain, or C terminus, was sufficient to confer a Gcn− phenotype, and both fragments have ribosome affinity. eEF3 overexpression did not significantly affect Gcn1-ribosome association, but it exacerbated the Gcn− phenotype of Gcn1-M7A that has reduced ribosome affinity. Together, this suggests that eEF3 blocks Gcn1 regulatory function on the ribosome. We propose that the Gcn1-Gcn2 complex only functions on ribosomes with A-site-bound uncharged tRNA, because eEF3 does not occupy these stalled complexes. PMID:22888004

  2. Overexpression of eukaryotic translation elongation factor 3 impairs Gcn2 protein activation.

    PubMed

    Visweswaraiah, Jyothsna; Lee, Su Jung; Hinnebusch, Alan G; Sattlegger, Evelyn

    2012-11-01

    In eukaryotes, phosphorylation of translation initiation factor 2α (eIF2α) by the kinase Gcn2 (general control nonderepressible 2) is a key response to amino acid starvation. Sensing starvation requires that Gcn2 directly contacts its effector protein Gcn1, and both must contact the ribosome. We have proposed that Gcn2 is activated by uncharged tRNA bound to the ribosomal decoding (A) site, in a manner facilitated by ribosome-bound Gcn1. Protein synthesis requires cyclical association of eukaryotic elongation factors (eEFs) with the ribosome. Gcn1 and Gcn2 are large proteins, raising the question of whether translation and monitoring amino acid availability can occur on the same ribosome. Part of the ribosome-binding domain in Gcn1 has homology to one of the ribosome-binding domains in eEF3, suggesting that these proteins utilize overlapping binding sites on the ribosome and consequently cannot function simultaneously on the same ribosome. Supporting this idea, we found that eEF3 overexpression in Saccharomyces cerevisiae diminished growth on amino acid starvation medium (Gcn(-) phenotype) and decreased eIF2α phosphorylation, and that the growth defect associated with constitutively active Gcn2 was diminished by eEF3 overexpression. Overexpression of the eEF3 HEAT domain, or C terminus, was sufficient to confer a Gcn(-) phenotype, and both fragments have ribosome affinity. eEF3 overexpression did not significantly affect Gcn1-ribosome association, but it exacerbated the Gcn(-) phenotype of Gcn1-M7A that has reduced ribosome affinity. Together, this suggests that eEF3 blocks Gcn1 regulatory function on the ribosome. We propose that the Gcn1-Gcn2 complex only functions on ribosomes with A-site-bound uncharged tRNA, because eEF3 does not occupy these stalled complexes. PMID:22888004

  3. Crystal structure of the full-length bacterial selenocysteine-specific elongation factor SelB

    PubMed Central

    Itoh, Yuzuru; Sekine, Shun-ichi; Yokoyama, Shigeyuki

    2015-01-01

    Selenocysteine (Sec), the 21st amino acid in translation, uses its specific tRNA (tRNASec) to recognize the UGA codon. The Sec-specific elongation factor SelB brings the selenocysteinyl-tRNASec (Sec-tRNASec) to the ribosome, dependent on both an in-frame UGA and a Sec-insertion sequence (SECIS) in the mRNA. The bacterial SelB binds mRNA through its C-terminal region, for which crystal structures have been reported. In this study, we determined the crystal structure of the full-length SelB from the bacterium Aquifex aeolicus, in complex with a GTP analog, at 3.2-Å resolution. SelB consists of three EF-Tu-like domains (D1–3), followed by four winged-helix domains (WHD1–4). The spacer region, connecting the N- and C-terminal halves, fixes the position of WHD1 relative to D3. The binding site for the Sec moiety of Sec-tRNASec is located on the interface between D1 and D2, where a cysteine molecule from the crystallization solution is coordinated by Arg residues, which may mimic Sec binding. The Sec-binding site is smaller and more exposed than the corresponding site of EF-Tu. Complex models of Sec-tRNASec, SECIS RNA, and the 70S ribosome suggest that the unique secondary structure of tRNASec allows SelB to specifically recognize tRNASec and characteristically place it at the ribosomal A-site. PMID:26304550

  4. Extensive proteomic remodeling is induced by eukaryotic translation elongation factor 1Bγ deletion in Aspergillus fumigatus.

    PubMed

    O'Keeffe, Grainne; Jöchl, Christoph; Kavanagh, Kevin; Doyle, Sean

    2013-11-01

    The opportunistic pathogen Aspergillus fumigatus is ubiquitous in the environment and predominantly infects immunocompromised patients. The functions of many genes remain unknown despite sequencing of the fungal genome. A putative translation elongation factor 1Bγ (eEF1Bγ, termed elfA; 750 bp) is expressed, and exhibits glutathione S-transferase activity, in A. fumigatus. Here, we demonstrate the role of ElfA in the oxidative stress response, as well as a possible involvement in translation and actin cytoskeleton organization, respectively. Comparative proteomics, in addition to phenotypic analysis, under basal and oxidative stress conditions, demonstrated a role for A. fumigatus elfA in the oxidative stress response. An elfA-deficient strain (A. fumigatus ΔelfA) was significantly more sensitive to the oxidants H2O2, diamide, and 4,4'-dipyridyl disulfide (DPS) than the wild-type. This was further supported with the identification of differentially expressed proteins of the oxidative stress response, including; mitochondrial peroxiredoxin Prx1, molecular chaperone Hsp70 and mitochondrial glycerol-3-phosphate dehydrogenase. Phenotypic analysis also revealed that A. fumigatus ΔelfA was significantly more tolerant to voriconazole than the wild-type. The differential expression of two aminoacyl-tRNA synthetases suggests a role for A. fumigatus elfA in translation, while the identification of actin-bundling protein Sac6 and vacuolar dynamin-like GTPase VpsA link A. fumigatus elfA to the actin cytoskeleton. Overall, this work highlights the diverse roles of A. fumigatus elfA, with respect to translation, oxidative stress and actin cytoskeleton organization. In addition to this, the strategy of combining targeted gene deletion with comparative proteomics for elucidating the role of proteins of unknown function is further revealed. PMID:24023013

  5. Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome

    PubMed Central

    Spiegel, P. Clint; Ermolenko, Dmitri N.; Noller, Harry F.

    2007-01-01

    Following peptide bond formation, transfer RNAs (tRNAs) and messenger RNA (mRNA) are translocated through the ribosome, a process catalyzed by elongation factor EF-G. Here, we have used a combination of chemical footprinting, peptidyl transferase activity assays, and mRNA toeprinting to monitor the effects of EF-G on the positions of tRNA and mRNA relative to the A, P, and E sites of the ribosome in the presence of GTP, GDP, GDPNP, and fusidic acid. Chemical footprinting experiments show that binding of EF-G in the presence of the non-hydrolyzable GTP analog GDPNP or GDP·fusidic acid induces movement of a deacylated tRNA from the classical P/P state to the hybrid P/E state. Furthermore, stabilization of the hybrid P/E state by EF-G compromises P-site codon–anticodon interaction, causing frame-shifting. A deacylated tRNA bound to the P site and a peptidyl-tRNA in the A site are completely translocated to the E and P sites, respectively, in the presence of EF-G with GTP or GDPNP but not with EF-G·GDP. Unexpectedly, translocation with EF-G·GTP leads to dissociation of deacylated tRNA from the E site, while tRNA remains bound in the presence of EF-G·GDPNP, suggesting that dissociation of tRNA from the E site is promoted by GTP hydrolysis and/or EF-G release. Our results show that binding of EF-G in the presence of GDPNP or GDP·fusidic acid stabilizes the ribosomal intermediate hybrid state, but that complete translocation is supported only by EF-G·GTP or EF-G·GDPNP. PMID:17630323

  6. Cyclic Rhamnosylated Elongation Factor P Establishes Antibiotic Resistance in Pseudomonas aeruginosa

    PubMed Central

    Rajkovic, Andrei; Erickson, Sarah; Witzky, Anne; Branson, Owen E.; Seo, Jin; Gafken, Philip R.; Frietas, Michael A.; Whitelegge, Julian P.; Faull, Kym F.; Navarre, William; Darwin, Andrew J.

    2015-01-01

    ABSTRACT Elongation factor P (EF-P) is a ubiquitous bacterial protein that is required for the synthesis of poly-proline motifs during translation. In Escherichia coli and Salmonella enterica, the posttranslational β-lysylation of Lys34 by the PoxA protein is critical for EF-P activity. PoxA is absent from many bacterial species such as Pseudomonas aeruginosa, prompting a search for alternative EF-P posttranslation modification pathways. Structural analyses of P. aeruginosa EF-P revealed the attachment of a single cyclic rhamnose moiety to an Arg residue at a position equivalent to that at which β-Lys is attached to E. coli EF-P. Analysis of the genomes of organisms that both lack poxA and encode an Arg32-containing EF-P revealed a highly conserved glycosyltransferase (EarP) encoded at a position adjacent to efp. EF-P proteins isolated from P. aeruginosa ΔearP, or from a ΔrmlC::acc1 strain deficient in dTDP-l-rhamnose biosynthesis, were unmodified. In vitro assays confirmed the ability of EarP to use dTDP-l-rhamnose as a substrate for the posttranslational glycosylation of EF-P. The role of rhamnosylated EF-P in translational control was investigated in P. aeruginosa using a Pro4-green fluorescent protein (Pro4GFP) in vivo reporter assay, and the fluorescence was significantly reduced in Δefp, ΔearP, and ΔrmlC::acc1 strains. ΔrmlC::acc1, ΔearP, and Δefp strains also displayed significant increases in their sensitivities to a range of antibiotics, including ertapenem, polymyxin B, cefotaxim, and piperacillin. Taken together, our findings indicate that posttranslational rhamnosylation of EF-P plays a key role in P. aeruginosa gene expression and survival. PMID:26060278

  7. The elongation factor Tu.kirromycin complex has two binding sites for tRNA molecules.

    PubMed Central

    van Noort, J M; Duisterwinkel, F J; Jonák, J; Sedlácek, J; Kraal, B; Bosch, L

    1982-01-01

    The interaction of the polypeptide chain elongation factor Tu (EF-Tu) with the antibiotic kirromycin and tRNA has been studied by measuring the extent of protein modification with N-tosyl-L-phenylalanine chloromethylketone (TPCK) and N-ethylmaleimide (NEM). Kirromycin protects both EF-Tu.GDP and EF-Tu.GTP against modification with TPCK. Binding of aminoacyl-tRNA added at increasing concentrations to a solution of 40 microM EF-Tu.GDP.kirromycin complex re-exposes the TPCK target site on the protein. However, when the aminoacyl-tRNA concentration is raised beyond 20 microM, TPCK labeling drops again and is blocked completely at approximately 300 microM aminoacyl-tRNA. By contrast, addition of uncharged tRNA or N- acetylaminoacyl -tRNA enhances TPCK labeling of the protein over the entire tRNA concentration range studied. These data strongly suggest that kirromycin induces in EF-Tu.GDP an additional tRNA binding site that can bind uncharged tRNA, aminoacyl-tRNA, and N- acetylaminoacyl -tRNA. Support for this assumption is provided by measuring the modification of EF-Tu.GDP with the sulfhydryl reagent NEM. Moreover, NEM modification also indicates an additional tRNA binding site on EF-Tu.GTP.kirromycin, which could not be detected with TPCK. Mapping of the tryptic peptides of EF-Tu.GDP labeled with [14C]TPCK revealed only one target site for this agent, i.e., cysteine-81. Modification occurred at the same site in the presence and in the absence of kirromycin and uncharged tRNA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:6765192

  8. Blood Translation Elongation Factor-1δ Is a Novel Marker for Cadmium Exposure

    PubMed Central

    Lu, Qian; Lei, Yi-Xiong; He, Chao-Cai; Lei, Zi-Ning

    2013-01-01

    Translation elongation factor-1δ (TEF-1δ) has been identified as a novel cadmium-responsive proto-oncogene. However, it is still unclear whether TEF-1δ could be a potential biomarker of cadmium exposure. Rats were treated with CdCl2 at different concentrations (high dose 1.225, mid-dose 0.612 and low dose 0.306 mg/kg body weight, respectively) for 14 weeks, and the cadmium levels, weight coefficients, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), serum creatinine (SCR), 24-h urine protein (24hPro), urinary creatinine (Cr) and pathological features were determined. The TEF-1δ expression in white blood cells and multiple organs were examined by reverse transcription polymerase chain reaction (PCR) and were also confirmed with fluorescence quantitative PCR. A cadmium dose-dependent increase (p < 0.05) of cadmium levels in blood, urine, liver, kidney, heart and lung, and the weight coefficients was observed. The liver and renal function indictors including AST, ALT, SCR, BUN and 24hPro, were elevated in a cadmium dose-dependent manner (p < 0.05). Significant pathological changes in liver, kidney, heart and lung were indicated. The TEF-1δ expression was up-regulated in both blood and organs (p < 0.05). Moreover, the expression level of blood TEF-1δ was positively correlated to TEF-1δ expression level, cadmium level and toxicity in the organs (p < 0.01). This study indicates that blood TEF-1δ is a novel valuable biomarker for cadmium exposure and its organ toxicity. PMID:23459232

  9. Crystal structure of the full-length bacterial selenocysteine-specific elongation factor SelB.

    PubMed

    Itoh, Yuzuru; Sekine, Shun-Ichi; Yokoyama, Shigeyuki

    2015-10-15

    Selenocysteine (Sec), the 21(st) amino acid in translation, uses its specific tRNA (tRNA(Sec)) to recognize the UGA codon. The Sec-specific elongation factor SelB brings the selenocysteinyl-tRNA(Sec) (Sec-tRNA(Sec)) to the ribosome, dependent on both an in-frame UGA and a Sec-insertion sequence (SECIS) in the mRNA. The bacterial SelB binds mRNA through its C-terminal region, for which crystal structures have been reported. In this study, we determined the crystal structure of the full-length SelB from the bacterium Aquifex aeolicus, in complex with a GTP analog, at 3.2-Å resolution. SelB consists of three EF-Tu-like domains (D1-3), followed by four winged-helix domains (WHD1-4). The spacer region, connecting the N- and C-terminal halves, fixes the position of WHD1 relative to D3. The binding site for the Sec moiety of Sec-tRNA(Sec) is located on the interface between D1 and D2, where a cysteine molecule from the crystallization solution is coordinated by Arg residues, which may mimic Sec binding. The Sec-binding site is smaller and more exposed than the corresponding site of EF-Tu. Complex models of Sec-tRNA(Sec), SECIS RNA, and the 70S ribosome suggest that the unique secondary structure of tRNA(Sec) allows SelB to specifically recognize tRNA(Sec) and characteristically place it at the ribosomal A-site. PMID:26304550

  10. Elongation Factor 1β′ Gene from Spodoptera exigua: Characterization and Function Identification through RNA Interference

    PubMed Central

    Zhao, Li-Na; Qin, Zi; Wei, Ping; Guo, Hong-Shuang; Dang, Xiang-Li; Wang, Shi-Gui; Tang, Bin

    2012-01-01

    Elongation factor (EF) is a key regulation factor for translation in many organisms, including plants, bacteria, fungi, animals and insects. To investigate the nature and function of elongation factor 1β′ from Spodoptera exigua (SeEF-1β′), its cDNA was cloned. This contained an open reading frame of 672 nucleotides encoding a protein of 223 amino acids with a predicted molecular weight of 24.04 kDa and pI of 4.53. Northern blotting revealed that SeEF-1β′ mRNA is expressed in brain, epidermis, fat body, midgut, Malpighian tubules, ovary and tracheae. RT-PCR revealed that SeEF-1β′ mRNA is expressed at different levels in fat body and whole body during different developmental stages. In RNAi experiments, the survival rate of insects injected with SeEF-1β′ dsRNA was 58.7% at 36 h after injection, which was significantly lower than three control groups. Other elongation factors and transcription factors were also influenced when EF-1β′ was suppressed. The results demonstrate that SeEF-1β′ is a key gene in transcription in S. exigua. PMID:22942694

  11. Effect of alpha-sarcin and ribosome-inactivating proteins on the interaction of elongation factors with ribosomes.

    PubMed

    Brigotti, M; Rambelli, F; Zamboni, M; Montanaro, L; Sperti, S

    1989-02-01

    alpha-Sarcin from Aspergillus giganteus and the ribosome-inactivating proteins (RIPs) from higher plants inactivate the 60 S ribosomal subunit. The former is an RNAase, whereas RIPs are N-glycosidases. The site of cleavage of RNA and that of N-glycosidic depurinization are at one nucleotide distance in 28 S rRNA [Endo & Tsurugi (1987) J. Biol. Chem. 262, 8128-8130]. The effect of alpha-sarcin and that of RIPs on the interaction of elongation factors with Artemia salina (brine shrimp) ribosomes have been investigated. alpha-Sarcin inhibits both the EF1 (elongation factor 1)-dependent binding of aminoacyl-tRNA and the GTP-dependent binding of EF2 (elongation factor 2) to ribosomes, whereas two of the RIPs tested, ricin from Ricinus communis (castor bean) and volkensin from Adenia volkensii (kilyambiti), inhibit only the latter reaction. EF2 protects ribosomes from inactivation by both alpha-sarcin and ricin. The EF1-binding site is affected only by alpha-sarcin. The sensitivity of this site to alpha-sarcin is increased by pretreatment of ribosomes with ricin. A. salina ribosomes were highly resistant to the third RIP tested, namely gelonin from Gelonium multiflorum. All four proteins tested have, however, a comparable activity on the rabbit reticulocyte-lysate system. PMID:2930482

  12. [Factors influencing the pulse character of RNA elongation in vitro by E. coli RNA polymerase].

    PubMed

    Aivazashvili, V A; Bibilashvili, R Sh; Vartikian, R M; Kutateladze, T V

    1981-01-01

    Pause location along primary structure of two RNA fragments each 200 nucleotide residues in the length synthesized from A1 promoters of T7 phage DNA and delta D111 T7 phage DNA was analyzed. No correlation between the location of pauses and GC-rich or self complementary regions of RNA were found. The location of pauses does not change upon the variation of the temperature or ionic strength. Concurrent variation of all four NTP concentrations also did not influence pausing pattern. However the distribution of pauses depends highly on the ratio of the individual substrate concentrations. Substitution of GTP by ITP changes the pausing pattern completely. Inorganic pyrophosphate (PPi) of inhibits RNA elongation preferentially in the regions: NAUN, CGUAG. The study of PPi action on RNA terminated with 3' OCH3-NMP suggest that the sequence-specific inhibition of RNA elongation may be a result of pyrophosphorolysis of terminal nucleotide residues of RNA. It was proposed that the pulse character of RNA elongation stems rather from differences in the kinetic constants of nucleotides attachment and pyrophosphorolysis from the 3'-termini of RNA than by termination signals encoded in the primary structure of DNA. The stable location of pauses in certain short oligonucleotides: AUG, AUU, AAU and some others is in favour of the hypothesis. PMID:6265762

  13. Structures and Functions of the Multiple KOW Domains of Transcription Elongation Factor Spt5

    PubMed Central

    Meyer, Peter A.; Li, Sheng; Zhang, Mincheng; Yamada, Kentaro; Takagi, Yuichiro; Hartzog, Grant A.

    2015-01-01

    The eukaryotic Spt4-Spt5 heterodimer forms a higher-order complex with RNA polymerase II (and I) to regulate transcription elongation. Extensive genetic and functional data have revealed diverse roles of Spt4-Spt5 in coupling elongation with chromatin modification and RNA-processing pathways. A mechanistic understanding of the diverse functions of Spt4-Spt5 is hampered by challenges in resolving the distribution of functions among its structural domains, including the five KOW domains in Spt5, and a lack of their high-resolution structures. We present high-resolution crystallographic results demonstrating that distinct structures are formed by the first through third KOW domains (KOW1-Linker1 [K1L1] and KOW2-KOW3) of Saccharomyces cerevisiae Spt5. The structure reveals that K1L1 displays a positively charged patch (PCP) on its surface, which binds nucleic acids in vitro, as shown in biochemical assays, and is important for in vivo function, as shown in growth assays. Furthermore, assays in yeast have shown that the PCP has a function that partially overlaps that of Spt4. Synthesis of our results with previous evidence suggests a model in which Spt4 and the K1L1 domain of Spt5 form functionally overlapping interactions with nucleic acids upstream of the transcription bubble, and this mechanism may confer robustness on processes associated with transcription elongation. PMID:26217010

  14. Nanoscale segregation of actin nucleation and elongation factors determines dendritic spine protrusion

    PubMed Central

    Chazeau, Anaël; Mehidi, Amine; Nair, Deepak; Gautier, Jérémie J; Leduc, Cécile; Chamma, Ingrid; Kage, Frieda; Kechkar, Adel; Thoumine, Olivier; Rottner, Klemens; Choquet, Daniel; Gautreau, Alexis; Sibarita, Jean-Baptiste; Giannone, Grégory

    2014-01-01

    Actin dynamics drive morphological remodeling of neuronal dendritic spines and changes in synaptic transmission. Yet, the spatiotemporal coordination of actin regulators in spines is unknown. Using single protein tracking and super-resolution imaging, we revealed the nanoscale organization and dynamics of branched F-actin regulators in spines. Branched F-actin nucleation occurs at the PSD vicinity, while elongation occurs at the tip of finger-like protrusions. This spatial segregation differs from lamellipodia where both branched F-actin nucleation and elongation occur at protrusion tips. The PSD is a persistent confinement zone for IRSp53 and the WAVE complex, an activator of the Arp2/3 complex. In contrast, filament elongators like VASP and formin-like protein-2 move outwards from the PSD with protrusion tips. Accordingly, Arp2/3 complexes associated with F-actin are immobile and surround the PSD. Arp2/3 and Rac1 GTPase converge to the PSD, respectively, by cytosolic and free-diffusion on the membrane. Enhanced Rac1 activation and Shank3 over-expression, both associated with spine enlargement, induce delocalization of the WAVE complex from the PSD. Thus, the specific localization of branched F-actin regulators in spines might be reorganized during spine morphological remodeling often associated with synaptic plasticity. PMID:25293574

  15. Plant Translation Elongation Factor 1Bβ Facilitates Potato Virus X (PVX) Infection and Interacts with PVX Triple Gene Block Protein 1.

    PubMed

    Hwang, JeeNa; Lee, Seonhee; Lee, Joung-Ho; Kang, Won-Hee; Kang, Jin-Ho; Kang, Min-Young; Oh, Chang-Sik; Kang, Byoung-Cheorl

    2015-01-01

    The eukaryotic translation elongation factor 1 (eEF1) has two components: the G-protein eEF1A and the nucleotide exchange factor eEF1B. In plants, eEF1B is itself composed of a structural protein (eEF1Bγ) and two nucleotide exchange subunits (eEF1Bα and eEF1Bβ). To test the effects of elongation factors on virus infection, we isolated eEF1A and eEF1B genes from pepper (Capsicum annuum) and suppressed their homologs in Nicotiana benthamiana using virus-induced gene silencing (VIGS). The accumulation of a green fluorescent protein (GFP)-tagged Potato virus X (PVX) was significantly reduced in the eEF1Bβ- or eEF1Bɣ-silenced plants as well as in eEF1A-silenced plants. Yeast two-hybrid and co-immunoprecipitation analyses revealed that eEF1Bα and eEF1Bβ interacted with eEF1A and that eEF1A and eEF1Bβ interacted with triple gene block protein 1 (TGBp1) of PVX. These results suggest that both eEF1A and eEF1Bβ play essential roles in the multiplication of PVX by physically interacting with TGBp1. Furthermore, using eEF1Bβ deletion constructs, we found that both N- (1-64 amino acids) and C-terminal (150-195 amino acids) domains of eEF1Bβ are important for the interaction with PVX TGBp1 and that the C-terminal domain of eEF1Bβ is involved in the interaction with eEF1A. These results suggest that eEF1Bβ could be a potential target for engineering virus-resistant plants. PMID:26020533

  16. Comparative protein profiling identifies elongation factor-1beta and tryparedoxin peroxidase as factors associated with metastasis in Leishmania guyanensis.

    PubMed

    Walker, John; Acestor, Nathalie; Gongora, Rafael; Quadroni, Manfredo; Segura, Iris; Fasel, Nicolas; Saravia, Nancy G

    2006-02-01

    Parasites of the Leishmania Viannia subgenus are major causative agents of mucocutaneous leishmaniasis (MCL), a disease characterised by parasite dissemination (metastasis) from the original cutaneous lesion to form debilitating secondary lesions in the nasopharyngeal mucosa. We employed a protein profiling approach to identify potential metastasis factors in laboratory clones of L. (V.) guyanensis with stable phenotypes ranging from highly metastatic (M+) through infrequently metastatic (M+/M-) to non-metastatic (M-). Comparison of the soluble proteomes of promastigotes by two-dimensional electrophoresis revealed two abundant protein spots specifically associated with M+ and M+/M- clones (Met2 and Met3) and two others exclusively expressed in M- parasites (Met1 and Met4). The association between clinical disease phenotype and differential expression of Met1-Met4 was less clear in L. Viannia strains from mucosal (M+) or cutaneous (M-) lesions of patients. Identification of Met1-Met4 by biological mass spectrometry (LC-ES-MS/MS) and bioinformatics revealed that M+ and M- clones express distinct acidic and neutral isoforms of both elongation factor-1 subunit beta (EF-1beta) and cytosolic tryparedoxin peroxidase (TXNPx). This interchange of isoforms may relate to the mechanisms by which the activities of EF-1beta and TXNPx are modulated, and/or differential post-translational modification of the gene product(s). The multiple metabolic functions of EF-1 and TXNPx support the plausibility of their participation in parasite survival and persistence and thereby, metastatic disease. Both polypeptides are active in resistance to chemical and oxidant stress, providing a basis for further elucidation of the importance of antioxidant defence in the pathogenesis underlying MCL. PMID:16325936

  17. Purification and Characterization of Tagless Recombinant Human Elongation Factor 2 Kinase (eEF-2K) Expressed in Escherichia coli

    PubMed Central

    Abramczyk, Olga; Tavares, Clint D. J.; Devkota, Ashwini K.; Ryazanov, Alexey G.; Turk, Benjamin E.; Riggs, Austen F.; Ozpolat, Bulent; Dalby, Kevin N.

    2012-01-01

    The eukaryotic elongation factor 2 kinase (eEF-2K) modulates the rate of protein synthesis by impeding the elongation phase of translation by inactivating the eukaryotic elongation factor 2 (eEF-2) via phosphorylation. eEF-2K is known to be activated by calcium and calmodulin, whereas the mTOR and MAPK pathways are suggested to negatively regulate kinase activity. Despite its pivotal role in translation regulation and potential role in tumor survival, the structure, function and regulation of eEF-2K have not been described in detail. This deficiency may result from the difficulty of obtaining the recombinant kinase in a form suitable for biochemical analysis. Here we report the purification and characterization of recombinant human eEF-2K expressed in the Escherichia coli strain Rosetta-gami 2(DE3). Successive chromatography steps utilizing Ni-NTA affinity, anion-exchange and gel filtration columns accomplished purification. Cleavage of the thioredoxin-His6-tag from the N-terminus of the expressed kinase with TEV protease yielded 9 mg of recombinant (G-D-I)-eEF-2K per liter of culture. Light scattering shows that eEF-2K is a monomer of ~ 85 kDa. In vitro kinetic analysis confirmed that recombinant human eEF-2K is able to phosphorylate wheat germ eEF-2 with kinetic parameters comparable to the mammalian enzyme. PMID:21605678

  18. A dynamic RNA loop in an IRES affects multiple steps of elongation factor-mediated translation initiation.

    PubMed

    Ruehle, Marisa D; Zhang, Haibo; Sheridan, Ryan M; Mitra, Somdeb; Chen, Yuanwei; Gonzalez, Ruben L; Cooperman, Barry S; Kieft, Jeffrey S

    2015-01-01

    Internal ribosome entry sites (IRESs) are powerful model systems to understand how the translation machinery can be manipulated by structured RNAs and for exploring inherent features of ribosome function. The intergenic region (IGR) IRESs from the Dicistroviridae family of viruses are structured RNAs that bind directly to the ribosome and initiate translation by co-opting the translation elongation cycle. These IRESs require an RNA pseudoknot that mimics a codon-anticodon interaction and contains a conformationally dynamic loop. We explored the role of this loop and found that both the length and sequence are essential for translation in different types of IGR IRESs and from diverse viruses. We found that loop 3 affects two discrete elongation factor-dependent steps in the IRES initiation mechanism. Our results show how the IRES directs multiple steps after 80S ribosome placement and highlights the often underappreciated significance of discrete conformationally dynamic elements within the context of structured RNAs. PMID:26523395

  19. The yeast transcription elongation factor Spt4/5 is a sequence-specific RNA binding protein.

    PubMed

    Blythe, Amanda J; Yazar-Klosinski, Berra; Webster, Michael W; Chen, Eefei; Vandevenne, Marylène; Bendak, Katerina; Mackay, Joel P; Hartzog, Grant A; Vrielink, Alice

    2016-09-01

    The heterodimeric transcription elongation factor Spt4/Spt5 (Spt4/5) tightly associates with RNAPII to regulate both transcriptional elongation and co-transcriptional pre-mRNA processing; however, the mechanisms by which Spt4/5 acts are poorly understood. Recent studies of the human and Drosophila Spt4/5 complexes indicate that they can bind nucleic acids in vitro. We demonstrate here that yeast Spt4/5 can bind in a sequence-specific manner to single stranded RNA containing AAN repeats. Furthermore, we show that the major protein determinants for RNA-binding are Spt4 together with the NGN domain of Spt5 and that the KOW domains are not required for RNA recognition. These findings attribute a new function to a domain of Spt4/5 that associates directly with RNAPII, making significant steps towards elucidating the mechanism behind transcriptional control by Spt4/5. PMID:27376968

  20. Analysis of a Splice Array Experiment Elucidates Roles of Chromatin Elongation Factor Spt4–5 in Splicing

    PubMed Central

    2005-01-01

    Splicing is an important process for regulation of gene expression in eukaryotes, and it has important functional links to other steps of gene expression. Two examples of these linkages include Ceg1, a component of the mRNA capping enzyme, and the chromatin elongation factors Spt4–5, both of which have recently been shown to play a role in the normal splicing of several genes in the yeast Saccharomyces cerevisiae. Using a genomic approach to characterize the roles of Spt4–5 in splicing, we used splicing-sensitive DNA microarrays to identify specific sets of genes that are mis-spliced in ceg1, spt4, and spt5 mutants. In the context of a complex, nested, experimental design featuring 22 dye-swap array hybridizations, comprising both biological and technical replicates, we applied five appropriate statistical models for assessing differential expression between wild-type and the mutants. To refine selection of differential expression genes, we then used a robust model-synthesizing approach, Differential Expression via Distance Synthesis, to integrate all five models. The resultant list of differentially expressed genes was then further analyzed with regard to select attributes: we found that highly transcribed genes with long introns were most sensitive to spt mutations. QPCR confirmation of differential expression was established for the limited number of genes evaluated. In this paper, we showcase splicing array technology, as well as powerful, yet general, statistical methodology for assessing differential expression, in the context of a real, complex experimental design. Our results suggest that the Spt4–Spt5 complex may help coordinate splicing with transcription under conditions that present kinetic challenges to spliceosome assembly or function. PMID:16172632

  1. The positive transcriptional elongation factor (P-TEFb) is required for neural crest specification.

    PubMed

    Hatch, Victoria L; Marin-Barba, Marta; Moxon, Simon; Ford, Christopher T; Ward, Nicole J; Tomlinson, Matthew L; Desanlis, Ines; Hendry, Adam E; Hontelez, Saartje; van Kruijsbergen, Ila; Veenstra, Gert Jan C; Münsterberg, Andrea E; Wheeler, Grant N

    2016-08-15

    Regulation of gene expression at the level of transcriptional elongation has been shown to be important in stem cells and tumour cells, but its role in the whole animal is only now being fully explored. Neural crest cells (NCCs) are a multipotent population of cells that migrate during early development from the dorsal neural tube throughout the embryo where they differentiate into a variety of cell types including pigment cells, cranio-facial skeleton and sensory neurons. Specification of NCCs is both spatially and temporally regulated during embryonic development. Here we show that components of the transcriptional elongation regulatory machinery, CDK9 and CYCLINT1 of the P-TEFb complex, are required to regulate neural crest specification. In particular, we show that expression of the proto-oncogene c-Myc and c-Myc responsive genes are affected. Our data suggest that P-TEFb is crucial to drive expression of c-Myc, which acts as a 'gate-keeper' for the correct temporal and spatial development of the neural crest. PMID:27343897

  2. Mast Cell-Derived Tumor Necrosis Factor Can Promote Nerve Fiber Elongation in the Skin during Contact Hypersensitivity in Mice

    PubMed Central

    Kakurai, Maki; Monteforte, Rossella; Suto, Hajime; Tsai, Mindy; Nakae, Susumu; Galli, Stephen J.

    2006-01-01

    In humans, lesions of contact eczema or atopic dermatitis can exhibit increases in epidermal nerves, but the mechanism resulting in such nerve elongation are not fully understood. We found that contact hypersensitivity reactions to oxazolone in mice were associated with significant increases in the length of nerves in the epidermis and dermis. Using genetically mast cell-deficient c-kit mutant mice selectively repaired of their dermal mast cell deficiency with either wild-type or tumor necrosis factor (TNF)-deficient mast cells, we found that mast cells, and mast cell-derived TNF, significantly contributed to the elongation of epidermal and dermal PGP 9.5+ nerves and dermal CGRP+ nerves, as well as to the inflammation observed at sites of contact hypersensitivity in response to oxazolone. Moreover, the percentage of mast cells in close proximity to dermal PGP 9.5+ nerve fibers was significantly higher in wild-type mice and in c-kit mutant mice repaired of their dermal mast cell deficiency by the adoptive transfer of wild-type mast cells than in TNF-deficient mice or in TNF−/− mast cell-engrafted c-kit mutant mice. These observations show that mast cells, and mast cell-derived TNF, can promote the elongation of cutaneous nerve fibers during contact hypersensitivity in the mouse. PMID:17071594

  3. The transcript elongation factor SPT4/SPT5 is involved in auxin-related gene expression in Arabidopsis

    PubMed Central

    Dürr, Julius; Lolas, Ihab B.; Sørensen, Brian B.; Schubert, Veit; Houben, Andreas; Melzer, Michael; Deutzmann, Rainer; Grasser, Marion; Grasser, Klaus D.

    2014-01-01

    The heterodimeric complex SPT4/SPT5 is a transcript elongation factor (TEF) that directly interacts with RNA polymerase II (RNAPII) to regulate messenger RNA synthesis in the chromatin context. We provide biochemical evidence that in Arabidopsis, SPT4 occurs in a complex with SPT5, demonstrating that the SPT4/SPT5 complex is conserved in plants. Each subunit is encoded by two genes SPT4-1/2 and SPT5-1/2. A mutant affected in the tissue-specifically expressed SPT5-1 is viable, whereas inactivation of the generally expressed SPT5-2 is homozygous lethal. RNAi-mediated downregulation of SPT4 decreases cell proliferation and causes growth reduction and developmental defects. These plants display especially auxin signalling phenotypes. Consistently, auxin-related genes, most strikingly AUX/IAA genes, are downregulated in SPT4–RNAi plants that exhibit an enhanced auxin response. In Arabidopsis nuclei, SPT5 clearly localizes to the transcriptionally active euchromatin, and essentially co-localizes with transcribing RNAPII. Typical for TEFs, SPT5 is found over the entire transcription unit of RNAPII-transcribed genes. In SPT4–RNAi plants, elevated levels of RNAPII and SPT5 are detected within transcribed regions (including those of downregulated genes), indicating transcript elongation defects in these plants. Therefore, SPT4/SPT5 acts as a TEF in Arabidopsis, regulating transcription during the elongation stage with particular impact on the expression of certain auxin-related genes. PMID:24497194

  4. Eukaryotic elongation factor 2 kinase as a drug target in cancer, and in cardiovascular and neurodegenerative diseases

    PubMed Central

    Liu, Rui; Proud, Christopher G

    2016-01-01

    Eukaryotic elongation factor 2 kinase (eEF2K) is an unusual protein kinase that regulates the elongation stage of protein synthesis by phosphorylating and inhibiting its only known substrate, eEF2. Elongation is a highly energy-consuming process, and eEF2K activity is tightly regulated by several signaling pathways. Regulating translation elongation can modulate the cellular energy demand and may also control the expression of specific proteins. Growing evidence links eEF2K to a range of human diseases, including cardiovascular conditions (atherosclerosis, via macrophage survival) and pulmonary arterial hypertension, as well as solid tumors, where eEF2K appears to play contrasting roles depending on tumor type and stage. eEF2K is also involved in neurological disorders and may be a valuable target in treating depression and certain neurodegenerative diseases. Because eEF2K is not required for mammalian development or cell viability, inhibiting its function may not elicit serious side effects, while the fact that it is an atypical kinase and quite distinct from the vast majority of other mammalian kinases suggests the possibility to develop it into compounds that inhibit eEF2K without affecting other important protein kinases. Further research is needed to explore these possibilities and there is an urgent need to identify and characterize potent and specific small-molecule inhibitors of eEF2K. In this article we review the recent evidence concerning the role of eEF2K in human diseases as well as the progress in developing small-molecule inhibitors of this enzyme. PMID:26806303

  5. Protein synthesis alongation factors EF-Tu and eEF1A: biosynthesis, functions and application in the improvement of heat tolerance in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein synthesis elongation factors EF-Tu and eEF1A (EFs) represent a group of highly conserved and abundant GTPases with an important role in transporting the aminoacyl-tRNA complex to the A site of the ribosome during elongation phase of translation. EF-Tu proteins are located in bacteria and, du...

  6. An Entamoeba histolytica ADP-ribosyl transferase from the diphtheria toxin family modifies the bacterial elongation factor Tu.

    PubMed

    Avila, Eva E; Rodriguez, Orlando I; Marquez, Jaqueline A; Berghuis, Albert M

    2016-06-01

    ADP-ribosyl transferases are enzymes involved in the post-translational modification of proteins; they participate in multiple physiological processes, pathogenesis and host-pathogen interactions. Several reports have characterized the functions of these enzymes in viruses, prokaryotes and higher eukaryotes, but few studies have reported ADP-ribosyl transferases in lower eukaryotes, such as parasites. The locus EHI_155600 from Entamoeba histolytica encodes a hypothetical protein that possesses a domain from the ADP-ribosylation superfamily; this protein belongs to the diphtheria toxin family according to a homology model using poly-ADP-ribosyl polymerase 12 (PARP12 or ARTD12) as a template. The recombinant protein expressed in Escherichia coli exhibited in vitro ADP-ribosylation activity that was dependent on the time and temperature. Unlabeled βNAD(+), but not ADP-ribose, competed in the enzymatic reaction using biotin-βNAD(+) as the ADP-ribose donor. The recombinant enzyme, denominated EhToxin-like, auto-ADP-ribosylated and modified an acceptor from E. coli that was identified by MS/MS as the elongation factor Tu (EF-Tu). To the best of our knowledge, this is the first report to identify an ADP-ribosyl transferase from the diphtheria toxin family in a protozoan parasite. The known toxins from this family (i.e., the diphtheria toxin, the Pseudomonas aeruginosa toxin Exo-A, and Cholix from Vibrio cholerae) modify eukaryotic elongation factor two (eEF-2), whereas the amoeba EhToxin-like modified EF-Tu, which is another elongation factor involved in protein synthesis in bacteria and mitochondria. PMID:27234208

  7. Structural elements defining elongation factor Tu mediated suppression of codon ambiguity

    PubMed Central

    Roy, Hervé; Becker, Hubert Dominique; Mazauric, Marie-Hélène; Kern, Daniel

    2007-01-01

    In most prokaryotes Asn-tRNAAsn and Gln-tRNAGln are formed by amidation of aspartate and glutamate mischarged onto tRNAAsn and tRNAGln, respectively. Coexistence in the organism of mischarged Asp-tRNAAsn and Glu-tRNAGln and the homologous Asn-tRNAAsn and Gln-tRNAGln does not, however, lead to erroneous incorporation of Asp and Glu into proteins, since EF-Tu discriminates the misacylated tRNAs from the correctly charged ones. This property contrasts with the canonical function of EF-Tu, which is to non-specifically bind the homologous aa-tRNAs, as well as heterologous species formed in vitro by aminoacylation of non-cognate tRNAs. In Thermus thermophilus that forms the Asp-tRNAAsn intermediate by the indirect pathway of tRNA asparaginylation, EF-Tu must discriminate the mischarged aminoacyl-tRNAs (aa-tRNA). We show that two base pairs in the tRNA T-arm and a single residue in the amino acid binding pocket of EF-Tu promote discrimination of Asp-tRNAAsn from Asn-tRNAAsn and Asp-tRNAAsp by the protein. Our analysis suggests that these structural elements might also contribute to rejection of other mischarged aa-tRNAs formed in vivo that are not involved in peptide elongation. Additionally, these structural features might be involved in maintaining a delicate balance of weak and strong binding affinities between EF-Tu and the amino acid and tRNA moieties of other elongator aa-tRNAs. PMID:17478519

  8. Yeast DEAD box protein Mss116p is a transcription elongation factor that modulates the activity of mitochondrial RNA polymerase.

    PubMed

    Markov, Dmitriy A; Wojtas, Ireneusz D; Tessitore, Kassandra; Henderson, Simmone; McAllister, William T

    2014-07-01

    DEAD box proteins have been widely implicated in regulation of gene expression. Here, we show that the yeast Saccharomyces cerevisiae DEAD box protein Mss116p, previously known as a mitochondrial splicing factor, also acts as a transcription factor that modulates the activity of the single-subunit mitochondrial RNA polymerase encoded by RPO41. Binding of Mss116p stabilizes paused mitochondrial RNA polymerase elongation complexes in vitro and favors the posttranslocated state of the enzyme, resulting in a lower concentration of nucleotide substrate required to escape the pause; this mechanism of action is similar to that of elongation factors that enhance the processivity of multisubunit RNA polymerases. In a yeast strain in which the RNA splicing-related functions of Mss116p are dispensable, overexpression of RPO41 or MSS116 increases cell survival from colonies that were exposed to low temperature, suggesting a role for Mss116p in enhancing the efficiency of mitochondrial transcription under stress conditions. PMID:24732805

  9. Elongation factor SII-dependent transcription by RNA polymerase II through a sequence-specific DNA-binding protein.

    PubMed Central

    Reines, D; Mote, J

    1993-01-01

    In eukaryotes the genetic material is contained within a coiled, protein-coated structure known as chromatin. RNA polymerases must recognize specific nucleoprotein assemblies and maintain contact with the underlying DNA duplex for many thousands of base pairs. Template-bound lac operon repressor from Escherichia coli arrests RNA polymerase II in vitro and in vivo [Kuhn, A., Bartsch, I. & Grummt, I. (1990) Nature (London) 344, 559-562; Deuschele, U., Hipskind, R. A. & Bujard, H. (1990) Science 248, 480-483]. We show that in a reconstituted transcription system, elongation factor SII enables RNA polymerase II to proceed through this blockage at high efficiency. lac repressor-arrested elongation complexes display an SII-activated transcript cleavage reaction, an activity associated with transcriptional read-through of a previously characterized region of bent DNA. This demonstrates factor-dependent transcription by RNA polymerase II through a sequence-specific DNA-binding protein. Nascent transcript cleavage may be a general mechanism by which RNA polymerase II can bypass many transcriptional impediments. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8446609

  10. Infantile Encephalopathy and Defective Mitochondrial DNA Translation in Patients with Mutations of Mitochondrial Elongation Factors EFG1 and EFTu

    PubMed Central

    Valente, Lucia; Tiranti, Valeria; Marsano, René Massimiliano; Malfatti, Edoardo; Fernandez-Vizarra, Erika; Donnini, Claudia; Mereghetti, Paolo; De Gioia, Luca; Burlina, Alberto; Castellan, Claudio; Comi, Giacomo P.; Savasta, Salvatore; Ferrero, Iliana; Zeviani, Massimo

    2007-01-01

    Mitochondrial protein translation is a complex process performed within mitochondria by an apparatus composed of mitochondrial DNA (mtDNA)–encoded RNAs and nuclear DNA–encoded proteins. Although the latter by far outnumber the former, the vast majority of mitochondrial translation defects in humans have been associated with mutations in RNA-encoding mtDNA genes, whereas mutations in protein-encoding nuclear genes have been identified in a handful of cases. Genetic investigation involving patients with defective mitochondrial translation led us to the discovery of novel mutations in the mitochondrial elongation factor G1 (EFG1) in one affected baby and, for the first time, in the mitochondrial elongation factor Tu (EFTu) in another one. Both patients were affected by severe lactic acidosis and rapidly progressive, fatal encephalopathy. The EFG1-mutant patient had early-onset Leigh syndrome, whereas the EFTu-mutant patient had severe infantile macrocystic leukodystrophy with micropolygyria. Structural modeling enabled us to make predictions about the effects of the mutations at the molecular level. Yeast and mammalian cell systems proved the pathogenic role of the mutant alleles by functional complementation in vivo. Nuclear-gene abnormalities causing mitochondrial translation defects represent a new, potentially broad field of mitochondrial medicine. Investigation of these defects is important to expand the molecular characterization of mitochondrial disorders and also may contribute to the elucidation of the complex control mechanisms, which regulate this fundamental pathway of mtDNA homeostasis. PMID:17160893

  11. Strand Transfer and Elongation of HIV-1 Reverse Transcription Is Facilitated by Cell Factors In Vitro

    PubMed Central

    Warrilow, David; Warren, Kylie; Harrich, David

    2010-01-01

    Recent work suggests a role for multiple host factors in facilitating HIV-1 reverse transcription. Previously, we identified a cellular activity which increases the efficiency of HIV-1 reverse transcription in vitro. Here, we describe aspects of the activity which shed light on its function. The cellular factor did not affect synthesis of strong-stop DNA but did improve downstream DNA synthesis. The stimulatory activity was isolated by gel filtration in a single fraction of the exclusion volume. Velocity-gradient purified HIV-1, which was free of detectable RNase activity, showed poor reverse transcription efficiency but was strongly stimulated by partially purified cell proteins. Hence, the cell factor(s) did not inactivate an RNase activity that might degrade the viral genomic RNA and block completion of reverse transcription. Instead, the cell factor(s) enhanced first strand transfer and synthesis of late reverse transcription suggesting it stabilized the reverse transcription complex. The factor did not affect lysis of HIV-1 by Triton X-100 in the endogenous reverse transcription (ERT) system, and ERT reactions with HIV-1 containing capsid mutations, which varied the biochemical stability of viral core structures and impeded reverse transcription in cells, showed no difference in the ability to be stimulated by the cell factor(s) suggesting a lack of involvement of the capsid in the in vitro assay. In addition, reverse transcription products were found to be resistant to exogenous DNase I activity when the active fraction was present in the ERT assay. These results indicate that the cell factor(s) may improve reverse transcription by facilitating DNA strand transfer and DNA synthesis. It also had a protective function for the reverse transcription products, but it is unclear if this is related to improved DNA synthesis. PMID:20949087

  12. A dynamic RNA loop in an IRES affects multiple steps of elongation factor-mediated translation initiation

    PubMed Central

    Ruehle, Marisa D; Zhang, Haibo; Sheridan, Ryan M; Mitra, Somdeb; Chen, Yuanwei; Gonzalez, Ruben L; Cooperman, Barry S; Kieft, Jeffrey S

    2015-01-01

    Internal ribosome entry sites (IRESs) are powerful model systems to understand how the translation machinery can be manipulated by structured RNAs and for exploring inherent features of ribosome function. The intergenic region (IGR) IRESs from the Dicistroviridae family of viruses are structured RNAs that bind directly to the ribosome and initiate translation by co-opting the translation elongation cycle. These IRESs require an RNA pseudoknot that mimics a codon-anticodon interaction and contains a conformationally dynamic loop. We explored the role of this loop and found that both the length and sequence are essential for translation in different types of IGR IRESs and from diverse viruses. We found that loop 3 affects two discrete elongation factor-dependent steps in the IRES initiation mechanism. Our results show how the IRES directs multiple steps after 80S ribosome placement and highlights the often underappreciated significance of discrete conformationally dynamic elements within the context of structured RNAs. DOI: http://dx.doi.org/10.7554/eLife.08146.001 PMID:26523395

  13. Molecular Mechanism for the Control of Eukaryotic Elongation Factor 2 Kinase by pH: Role in Cancer Cell Survival

    PubMed Central

    Xie, Jianling; Mikolajek, Halina; Pigott, Craig R.; Hooper, Kelly J.; Mellows, Toby; Moore, Claire E.; Mohammed, Hafeez; Werner, Jörn M.; Thomas, Gareth J.

    2015-01-01

    Acidification of the extracellular and/or intracellular environment is involved in many aspects of cell physiology and pathology. Eukaryotic elongation factor 2 kinase (eEF2K) is a Ca2+/calmodulin-dependent kinase that regulates translation elongation by phosphorylating and inhibiting eEF2. Here we show that extracellular acidosis elicits activation of eEF2K in vivo, leading to enhanced phosphorylation of eEF2. We identify five histidine residues in eEF2K that are crucial for the activation of eEF2K during acidosis. Three of them (H80, H87, and H94) are in its calmodulin-binding site, and their protonation appears to enhance the ability of calmodulin to activate eEF2K. The other two histidines (H227 and H230) lie in the catalytic domain of eEF2K. We also identify His108 in calmodulin as essential for activation of eEF2K. Acidification of cancer cell microenvironments is a hallmark of malignant solid tumors. Knocking down eEF2K in cancer cells attenuated the decrease in global protein synthesis when cells were cultured at acidic pH. Importantly, activation of eEF2K is linked to cancer cell survival under acidic conditions. Inhibition of eEF2K promotes cancer cell death under acidosis. PMID:25776553

  14. Drosophila translational elongation factor-1gamma is modified in response to DOA kinase activity and is essential for cellular viability.

    PubMed

    Fan, Yujie; Schlierf, Michael; Gaspar, Ana Cuervo; Dreux, Catherine; Kpebe, Arlette; Chaney, Linda; Mathieu, Aurelie; Hitte, Christophe; Grémy, Olivier; Sarot, Emeline; Horn, Mark; Zhao, Yunlong; Kinzy, Terri Goss; Rabinow, Leonard

    2010-01-01

    Drosophila translational elongation factor-1gamma (EF1gamma) interacts in the yeast two-hybrid system with DOA, the LAMMER protein kinase of Drosophila. Analysis of mutant EF1gamma alleles reveals that the locus encodes a structurally conserved protein essential for both organismal and cellular survival. Although no genetic interactions were detected in combinations with mutations in EF1alpha, an EF1gamma allele enhanced mutant phenotypes of Doa alleles. A predicted LAMMER kinase phosphorylation site conserved near the C terminus of all EF1gamma orthologs is a phosphorylation site in vitro for both Drosophila DOA and tobacco PK12 LAMMER kinases. EF1gamma protein derived from Doa mutant flies migrates with altered mobility on SDS gels, consistent with it being an in vivo substrate of DOA kinase. However, the aberrant mobility appears to be due to a secondary protein modification, since the mobility of EF1gamma protein obtained from wild-type Drosophila is unaltered following treatment with several nonspecific phosphatases. Expression of a construct expressing a serine-to-alanine substitution in the LAMMER kinase phosphorylation site into the fly germline rescued null EF1gamma alleles but at reduced efficiency compared to a wild-type construct. Our data suggest that EF1gamma functions in vital cellular processes in addition to translational elongation and is a LAMMER kinase substrate in vivo. PMID:19841092

  15. Binding of Tat to TAR and Recruitment of Positive Transcription Elongation Factor b Occur Independently in Bovine Immunodeficiency Virus

    PubMed Central

    Barboric, Matjaz; Taube, Ran; Nekrep, Nada; Fujinaga, Koh; Peterlin, B. Matija

    2000-01-01

    Transcriptional transactivators (Tat) from many lentiviruses interact with their cognate transactivation response RNA structures (TAR) to increase rates of elongation rather than initiation of transcription. For several of them, the complex of Tat and a species-specific cyclin T1 must be formed before the binding to TAR can occur with high affinity and specificity. In sharp contrast, Tat from the bovine immunodeficiency virus (BIV) binds to its TAR without the help of the cyclin T1. This binding depends on the upper stem and 5′ bulge, but not the central loop in TAR. Moreover, cyclins T1 from different species can mediate effects of this Tat in cells. Unlike the situation with other lentiviruses, Tat transactivation can be rescued simply by linking a heterologous promoter to TAR in permissive cells. Thus, lentiviruses have evolved different strategies to recruit Tat and the positive transcription elongation factor b to their promoters, and interactions between Tat and TAR are independent from those between Tat and the cyclin T1 in BIV. PMID:10846086

  16. Relationships Between RNA Polymerase II Activity and Spt Elongation Factors to Spt- Phenotype and Growth in Saccharomyces cerevisiae

    PubMed Central

    Cui, Ping; Jin, Huiyan; Vutukuru, Manjula Ramya; Kaplan, Craig D.

    2016-01-01

    The interplay between adjacent transcription units can result in transcription-dependent alterations in chromatin structure or recruitment of factors that determine transcription outcomes, including the generation of intragenic or other cryptic transcripts derived from cryptic promoters. Mutations in a number of genes in Saccharomyces cerevisiae confer both cryptic intragenic transcription and the Suppressor of Ty (Spt-) phenotype for the lys2-128∂ allele of the LYS2 gene. Mutants that suppress lys2-128∂ allow transcription from a normally inactive Ty1 ∂ promoter, conferring a LYS+ phenotype. The arrangement of transcription units at lys2-128∂ is reminiscent of genes containing cryptic promoters within their open reading frames. We set out to examine the relationship between RNA Polymerase II (Pol II) activity, functions of Spt elongation factors, and cryptic transcription because of the previous observation that increased-activity Pol II alleles confer an Spt- phenotype. We identify both cooperating and antagonistic genetic interactions between Pol II alleles and alleles of elongation factors SPT4, SPT5, and SPT6. We find that cryptic transcription at FLO8 and STE11 is distinct from that at lys2-128∂, though all show sensitivity to reduction in Pol II activity, especially the expression of lys2-128∂ found in Spt- mutants. We determine that the lys2-128∂ Spt- phenotypes for spt6-1004 and increased activity rpo21/rpb1 alleles each require transcription from the LYS2 promoter. Furthermore, we identify the Ty1 transcription start site (TSS) within the ∂ element as the position of Spt- transcription in tested Spt- mutants. PMID:27261007

  17. Relationships Between RNA Polymerase II Activity and Spt Elongation Factors to Spt- Phenotype and Growth in Saccharomyces cerevisiae.

    PubMed

    Cui, Ping; Jin, Huiyan; Vutukuru, Manjula Ramya; Kaplan, Craig D

    2016-01-01

    The interplay between adjacent transcription units can result in transcription-dependent alterations in chromatin structure or recruitment of factors that determine transcription outcomes, including the generation of intragenic or other cryptic transcripts derived from cryptic promoters. Mutations in a number of genes in Saccharomyces cerevisiae confer both cryptic intragenic transcription and the Suppressor of Ty (Spt(-)) phenotype for the lys2-128∂ allele of the LYS2 gene. Mutants that suppress lys2-128∂ allow transcription from a normally inactive Ty1 ∂ promoter, conferring a LYS(+) phenotype. The arrangement of transcription units at lys2-128∂ is reminiscent of genes containing cryptic promoters within their open reading frames. We set out to examine the relationship between RNA Polymerase II (Pol II) activity, functions of Spt elongation factors, and cryptic transcription because of the previous observation that increased-activity Pol II alleles confer an Spt(-) phenotype. We identify both cooperating and antagonistic genetic interactions between Pol II alleles and alleles of elongation factors SPT4, SPT5, and SPT6 We find that cryptic transcription at FLO8 and STE11 is distinct from that at lys2-128∂, though all show sensitivity to reduction in Pol II activity, especially the expression of lys2-128∂ found in Spt(-) mutants. We determine that the lys2-128∂ Spt(-) phenotypes for spt6-1004 and increased activity rpo21/rpb1 alleles each require transcription from the LYS2 promoter. Furthermore, we identify the Ty1 transcription start site (TSS) within the ∂ element as the position of Spt(-) transcription in tested Spt(-) mutants. PMID:27261007

  18. The transcription elongation factor CA150 interacts with RNA polymerase II and the pre-mRNA splicing factor SF1.

    PubMed

    Goldstrohm, A C; Albrecht, T R; Suñé, C; Bedford, M T; Garcia-Blanco, M A

    2001-11-01

    CA150 represses RNA polymerase II (RNAPII) transcription by inhibiting the elongation of transcripts. The FF repeat domains of CA150 bind directly to the phosphorylated carboxyl-terminal domain of the largest subunit of RNAPII. We determined that this interaction is required for efficient CA150-mediated repression of transcription from the alpha(4)-integrin promoter. Additional functional determinants, namely, the WW1 and WW2 domains of CA150, were also required for efficient repression. A protein that interacted directly with CA150 WW1 and WW2 was identified as the splicing-transcription factor SF1. Previous studies have demonstrated a role for SF1 in transcription repression, and we found that binding of the CA150 WW1 and WW2 domains to SF1 correlated exactly with the functional contribution of these domains for repression. The binding specificity of the CA150 WW domains was found to be unique in comparison to known classes of WW domains. Furthermore, the CA150 binding site, within the carboxyl-terminal half of SF1, contains a novel type of proline-rich motif that may be recognized by the CA150 WW1 and WW2 domains. These results support a model for the recruitment of CA150 to repress transcription elongation. In this model, CA150 binds to the phosphorylated CTD of elongating RNAPII and SF1 targets the nascent transcript. PMID:11604498

  19. Exploratory factor analysis for differentiating sensory and mechanical variables related to muscle-tendon unit elongation

    PubMed Central

    Chagas, Mauro H.; Magalhães, Fabrício A.; Peixoto, Gustavo H. C.; Pereira, Beatriz M.; Andrade, André G. P.; Menzel, Hans-Joachim K.

    2016-01-01

    ABSTRACT Background Stretching exercises are able to promote adaptations in the muscle-tendon unit (MTU), which can be tested through physiological and biomechanical variables. Identifying the key variables in MTU adaptations is crucial to improvements in training. Objective To perform an exploratory factor analysis (EFA) involving the variables often used to evaluate the response of the MTU to stretching exercises. Method Maximum joint range of motion (ROMMAX), ROM at first sensation of stretching (FSTROM), peak torque (torqueMAX), passive stiffness, normalized stiffness, passive energy, and normalized energy were investigated in 36 participants during passive knee extension on an isokinetic dynamometer. Stiffness and energy values were normalized by the muscle cross-sectional area and their passive mode assured by monitoring the EMG activity. Results EFA revealed two major factors that explained 89.68% of the total variance: 53.13% was explained by the variables torqueMAX, passive stiffness, normalized stiffness, passive energy, and normalized energy, whereas the remaining 36.55% was explained by the variables ROMMAX and FSTROM. Conclusion This result supports the literature wherein two main hypotheses (mechanical and sensory theories) have been suggested to describe the adaptations of the MTU to stretching exercises. Contrary to some studies, in the present investigation torqueMAX was significantly correlated with the variables of the mechanical theory rather than those of the sensory theory. Therefore, a new approach was proposed to explain the behavior of the torqueMAX during stretching exercises. PMID:27437715

  20. The Interaction Surface of a Bacterial Transcription Elongation Factor Required for Complex Formation with an Antiterminator during Transcription Antitermination*

    PubMed Central

    Mishra, Saurabh; Mohan, Shalini; Godavarthi, Sapna; Sen, Ranjan

    2013-01-01

    The bacterial transcription elongation factor, NusA, functions as an antiterminator when it is bound to the lambdoid phage derived antiterminator protein, N. The mode of N-NusA interaction is unknown, knowledge of which is essential to understand the antitermination process. It was reported earlier that in the absence of the transcription elongation complex (EC), N interacts with the C-terminal AR1 domain of NusA. However, the functional significance of this interaction is obscure. Here we identified mutations in NusA N terminus (NTD) specifically defective for N-mediated antitermination. These are located at a convex surface of the NusA-NTD, situated opposite its concave RNA polymerase (RNAP) binding surface. These NusA mutants disrupt the N-nut site interactions on the nascent RNA emerging out of a stalled EC. In the N/NusA-modified EC, a Cys-53 (S53C) from the convex surface of the NusA-NTD forms a specific disulfide (S-S) bridge with a Cys-39 (S39C) of the NusA binding region of the N protein. We conclude that when bound to the EC, the N interaction surface of NusA shifts from the AR1 domain to its NTD domain. This occurred due to a massive away-movement of the adjacent AR2 domain of NusA upon binding to the EC. We propose that the close proximity of this altered N-interaction site of NusA to its RNAP binding surface, enables N to influence the NusA-RNAP interaction during transcription antitermination that in turn facilitates the conversion of NusA into an antiterminator. PMID:23913688

  1. Berberine regulates peroxisome proliferator-activated receptors and positive transcription elongation factor b expression in diabetic adipocytes.

    PubMed

    Zhou, Jiyin; Zhou, Shiwen

    2010-12-15

    Berberine has hypoglycemic and hypolipidemic effects on diabetic rats. This study investigated the relationship between hypoglycemic and hypolipidemic effects of berberine and peroxisome proliferator-activated receptors (PPARs) and positive transcription elongation factor b (P-TEFb) (including cyclin-dependent kinase 9 (CDK9) and cyclin T1) in white adipose tissue of diabetic rats and RNA interference-treated 3T3-L1 cells. Berberine promoted differentiation and inhibited lipid accumulation of 3T3-L1 cells, further decreased PPARα/δ/γ, CDK9 and cyclin T1 mRNA and protein expression and decreased tumor necrosis factor α content in supernatants of both control and RNA interference-treated 3T3-L1 cells. After a 16-week induction with 35 mg/kg streptozotocin (i.p.) and high-carbohydrate/high-fat diet, diabetic rats were treated with 75, 150 and 300 mg/kg berberine and 100 mg/kg fenofibrate or 4 mg/kg rosiglitazone for another 16 weeks. Berberine decreased white adipose tissue to body weight ratio and adipocyte size and increased adipocyte number. Berberine upregulated PPARα/δ/γ, CDK9 and cyclin T1 mRNA and protein expression in adipose tissue, decreased tumor necrosis factor α and free fatty acid content and increased lipoprotein lipase activity in serum and adipose tissue. Berberine modulated metabolic related PPARs expression and differentiation related P-TEFb expression in adipocytes, which are associated with its hypoglycemic and hypolipidemic effects. PMID:20868663

  2. Depletion of elongation initiation factor 4E binding proteins by CRISPR/Cas9 genome editing enhances antiviral response in porcine cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Type I interferons (IFN) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor 4E binding proteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF7), the master regulator of IFN transcription. The role of 4EBPs in the negat...

  3. Visualization of two transfer RNAs trapped in transit during elongation factor G-mediated translocation

    PubMed Central

    Ramrath, David J. F.; Lancaster, Laura; Sprink, Thiemo; Mielke, Thorsten; Loerke, Justus; Noller, Harry F.; Spahn, Christian M. T.

    2013-01-01

    During protein synthesis, coupled translocation of messenger RNAs (mRNA) and transfer RNAs (tRNA) through the ribosome takes place following formation of each peptide bond. The reaction is facilitated by large-scale conformational changes within the ribosomal complex and catalyzed by elongtion factor G (EF-G). Previous structural analysis of the interaction of EF-G with the ribosome used either model complexes containing no tRNA or only a single tRNA, or complexes where EF-G was directly bound to ribosomes in the posttranslocational state. Here, we present a multiparticle cryo-EM reconstruction of a translocation intermediate containing two tRNAs trapped in transit, bound in chimeric intrasubunit ap/P and pe/E hybrid states. The downstream ap/P-tRNA is contacted by domain IV of EF-G and P-site elements within the 30S subunit body, whereas the upstream pe/E-tRNA maintains tight interactions with P-site elements of the swiveled 30S head. Remarkably, a tight compaction of the tRNA pair can be seen in this state. The translocational intermediate presented here represents a previously missing link in understanding the mechanism of translocation, revealing that the ribosome uses two distinct molecular ratchets, involving both intra- and intersubunit rotational movements, to drive the synchronous movement of tRNAs and mRNA. PMID:24324168

  4. A SHORT INTERNODES (SHI) family transcription factor gene regulates awn elongation and pistil morphology in barley.

    PubMed

    Yuo, Takahisa; Yamashita, Yuko; Kanamori, Hiroyuki; Matsumoto, Takashi; Lundqvist, Udda; Sato, Kazuhiro; Ichii, Masahiko; Jobling, Stephen A; Taketa, Shin

    2012-09-01

    The awn, an apical extension from the lemma of the spikelet, plays important roles in seed dispersal, burial, and photosynthesis. Barley typically has long awns, but short-awn variants exist. The short awn 2 (lks2) gene, which produces awns about 50% shorter than normal, is a natural variant that is restricted to Eastern Asia. Positional cloning revealed that Lks2 encodes a SHI-family transcription factor. Allelism tests showed that lks2 is allelic to unbranched style 4 (ubs4) and breviaristatum-d (ari-d), for which the phenotypes are very short awn and sparse stigma hairs. The gene identity was validated by 25 mutant alleles with lesions in the Lks2 gene. Of these, 17 affected either or both conserved regions: the zinc-binding RING-finger motif and the IGGH domain. Lks2 is highly expressed in awns and pistils. Histological observations of longitudinal awn sections showed that the lks2 short-awn phenotype resulted from reduced cell number. Natural variants of lks2 were classified into three types, but all shared a single-nucleotide polymorphism (SNP) that causes a proline-to-leucine change at position 245 in the IGGH domain. All three lks2 natural variants were regarded as weak alleles because their awn and pistil phenotypes are mild compared with those of the 25 mutant alleles. Natural variants of lks2 found in the east of China and the Himalayas had considerably different sequences in the regions flanking the critical SNP, suggesting independent origins. The available results suggest that the lks2 allele might have a selective advantage in the adaptation of barley to high-precipitation areas of Eastern Asia. PMID:22791834

  5. Elongation factor eEF1B modulates functions of the release factors eRF1 and eRF3 and the efficiency of translation termination in yeast

    PubMed Central

    Valouev, Igor A; Fominov, Gleb V; Sokolova, Elizaveta E; Smirnov, Vladimir N; Ter-Avanesyan, Michael D

    2009-01-01

    Background Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRF1 and eRF3. While eRF1 recognizes nonsense codons, eRF3 facilitates polypeptide chain release from the ribosome in a GTP-dependent manner. Besides termination, both release factors have essential, but poorly characterized functions outside of translation. Results To characterize further the functions of yeast eRF1 and eRF3, a genetic screen for their novel partner proteins was performed. As a result, the genes for γ (TEF4 and TEF3/CAM1) and α (TEF5/EFB1) subunits of the translation elongation factor eEF1B, known to catalyze the exchange of bound GDP for GTP on eEF1A, were revealed. These genes act as dosage suppressors of a synthetic growth defect caused by some mutations in the SUP45 and SUP35 genes encoding eRF1 and eRF3, respectively. Extra copies of TEF5 and TEF3 can also suppress the temperature sensitivity of some sup45 and sup35 mutants and reduce nonsense codon readthrough caused by these omnipotent suppressors. Besides, overproduction of eEF1Bα reduces nonsense codon readthrough in the strain carrying suppressor tRNA. Such effects were not shown for extra copies of TEF2, which encodes eEF1A, thus indicating that they were not due to eEF1A activation. Conclusion The data obtained demonstrate involvement of the translation elongation factor eEF1B in modulating the functions of translation termination factors and suggest its possible role in GDP for GTP exchange on eRF3. PMID:19545407

  6. Distinct XPPX sequence motifs induce ribosome stalling, which is rescued by the translation elongation factor EF-P

    PubMed Central

    Peil, Lauri; Starosta, Agata L.; Lassak, Jürgen; Atkinson, Gemma C.; Virumäe, Kai; Spitzer, Michaela; Tenson, Tanel; Jung, Kirsten; Remme, Jaanus; Wilson, Daniel N.

    2013-01-01

    Ribosomes are the protein synthesizing factories of the cell, polymerizing polypeptide chains from their constituent amino acids. However, distinct combinations of amino acids, such as polyproline stretches, cannot be efficiently polymerized by ribosomes, leading to translational stalling. The stalled ribosomes are rescued by the translational elongation factor P (EF-P), which by stimulating peptide-bond formation allows translation to resume. Using metabolic stable isotope labeling and mass spectrometry, we demonstrate in vivo that EF-P is important for expression of not only polyproline-containing proteins, but also for specific subsets of proteins containing diprolyl motifs (XPP/PPX). Together with a systematic in vitro and in vivo analysis, we provide a distinct hierarchy of stalling triplets, ranging from strong stallers, such as PPP, DPP, and PPN to weak stallers, such as CPP, PPR, and PPH, all of which are substrates for EF-P. These findings provide mechanistic insight into how the characteristics of the specific amino acid substrates influence the fundamentals of peptide bond formation. PMID:24003132

  7. Monoclonal antibodies specific for elongation factor Tu and complete nucleotide sequence of the tuf gene in Mycobacterium tuberculosis.

    PubMed Central

    Carlin, N I; Löfdahl, S; Magnusson, M

    1992-01-01

    Monoclonal antibodies against mycobacterial antigens were produced by immunizing LOU/C rats with live Mycobacterium bovis BCG. The antibodies were characterized by an enzyme-linked immunosorbent assay and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting (immunoblotting). One antibody, MAMB 2, reactive with a 47-kDa protein was used to screen a lambda gt11 M. tuberculosis gene library (R. A. Young, B. R. Bloom, C. M. Grosskinsky, J. Ivanji, D. Thomas, and R. W. Davis, Proc. Natl. Acad. Sci. USA 82:2583-2587, 1985). Three recombinant phages reactive with MAMB 2 in plaque lysates were isolated, and part of the insert was sequenced. The mycobacterial inserts were all expressed as proteins fused with beta-galactosidase when the phages were induced as lysogens in Escherichia coli. The entire M. tuberculosis tuf gene was obtained by screening the lambda gt11 library with a DNA probe specific for the primary clones. A phage isolated from this screening was able to express the native protein in E. coli when introduced as a lysogen. A comparison of the entire gene sequence and the deduced protein sequence with the EMBL DNA and Swiss-Prot protein data libraries revealed strong homologies with elongation factors of bacteria, yeast mitochondria, and a plant chloroplast. Images PMID:1639483

  8. Enhancement of innate immune system in monocot rice by transferring the dicotyledonous elongation factor Tu receptor EFR.

    PubMed

    Lu, Fen; Wang, Huiqin; Wang, Shanzhi; Jiang, Wendi; Shan, Changlin; Li, Bin; Yang, Jun; Zhang, Shiyong; Sun, Wenxian

    2015-07-01

    The elongation factor Tu (EF-Tu) receptor (EFR) in cruciferous plants specifically recognizes the N-terminal acetylated elf18 region of bacterial EF-Tu and thereby activates plant immunity. It has been demonstrated that Arabidopsis EFR confers broad-spectrum bacterial resistance in the EFR transgenic solanaceous plants. Here, the transgenic rice plants (Oryza sativa L. ssp. japonica cv. Zhonghua 17) and cell cultures with constitutive expression of AtEFR were developed to investigate whether AtEFR senses EF-Tu and thus enhances bacterial resistance in the monocot plants. We demonstrated that the Xanthomonas oryzae-derived elf18 peptide induced oxidative burst and mitogen-activated protein kinase activation in the AtEFR transgenic rice cells and plants, respectively. Pathogenesis-related genes, such as OsPBZ1, were upregulated dramatically in transgenic rice plant and cell lines in response to elf18 stimulation. Importantly, pretreatment with elf18 triggered strong resistance to X. oryzae pv. oryzae in the transgenic plants, which was largely dependent on the AtEFR expression level. These plants also exhibited enhanced resistance to rice bacterial brown stripe, but not to rice fungal blast. Collectively, the results indicate that the rice plants with heterologous expression of AtEFR recognize bacterial EF-Tu and exhibit enhanced broad-spectrum bacterial disease resistance and that pattern recognition receptor-mediated immunity may be manipulated across the two plant classes, dicots and monocots. PMID:25358295

  9. The real factor for polypeptide elongation in Dictyostelium cells is EF-2B, not EF-2A

    SciTech Connect

    Yoshino, Tomoko; Maeda, Yasuo; Amagai, Aiko . E-mail: aiamagai@mail.tains.tohoku.ac.jp

    2007-08-03

    Polypeptide elongation factor 2 (EF-2) plays an essential role in protein synthesis and is believed to be indispensable for cell proliferation. Recently, it has been demonstrated that there are two kinds of EF-2 (EF-2A and EF-2B with 76.6% of sequence identity at the amino acid level) in Dictyostelium discoideum. Although the knockout of EF-2A slightly impaired cytokinesis, EF-2A null cells exhibited almost normal protein synthesis and cell growth, suggesting that there is another molecule capable of compensating for EF-2 function. Since EF-2B is the most likely candidate, we examined its function using ef-2b knockdown cells prepared by the RNAi method. Our results strongly suggest that EF-2B is required for protein synthesis and cell proliferation, functioning as the real EF-2. Interestingly, the expressions of ef-2a and ef-2b mRNAs during development are reversely regulated, and the ef-2b expression is greatly augmented in ef-2a null cells.

  10. Genes encoding isoforms of transcription elongation factor TFIIS in Xenopus and the use of multiple unusual RNA processing signals.

    PubMed Central

    Plant, K E; Hair, A; Morgan, G T

    1996-01-01

    We have identified cDNAs encoding three related forms of transcription elongation factor TFIIS (S-II) in Xenopus laevis ovary. Comparison of Xenopus and mammalian sequences identifies likely diagnostic amino acids that distinguish classes of vertebrate TFIIS. The diversity of TFIIS polypeptides in Xenopus is due partly to the presence of two diverged genes in this tetraploid genome. We isolated genomic clones containing one of the genes, xTFIIS.oA, and, unlike a previously described vertebrate TFIIS gene, found that it contains introns. Alternative splicing at a CAG/CAG motif containing the 3' splice site of intron 4 produces the third form of xTFIIS, which differs from one of the others simply in lacking Ser109. Intron 6 of xTFIIS.oA contains splice and branch site consensus sequences conforming to those of the minor class of AT-AC introns and this was confirmed for the homeologous xTFIIS.oB gene by genomic PCR. Other unusual but functional variants of RNA processing signals were found in xTFIIS genes at the 5' splice site of intron 8 and the polyadenylation hexanucleotides. Utilization of multiple unusual processing signals may make the generation of mature xTFIIS.o mRNAs inefficient and the possible regulatory consequences of this are discussed. PMID:8836176

  11. Identification of flavopiridol analogues that selectively inhibit positive transcription elongation factor (P-TEFb) and block HIV-1 replication.

    PubMed

    Ali, Akbar; Ghosh, Animesh; Nathans, Robin S; Sharova, Natalia; O'Brien, Siobhan; Cao, Hong; Stevenson, Mario; Rana, Tariq M

    2009-08-17

    The positive transcription elongation factor (P-TEFb; CDK9/cyclin T1) regulates RNA polymerase II-dependent transcription of cellular and integrated viral genes. It is an essential cofactor for HIV-1 Tat transactivation, and selective inhibition of P-TEFb blocks HIV-1 replication without affecting cellular transcription; this indicates that P-TEFb could be a potential target for developing anti-HIV-1 therapeutics. Flavopiridol, a small molecule CDK inhibitor, blocks HIV-1 Tat transactivation and viral replication by inhibiting P-TEFb kinase activity, but it is highly cytotoxic. In the search for selective and less cytotoxic P-TEFb inhibitors, we prepared a series of flavopiridol analogues and evaluated their kinase inhibitory activity against P-TEFb and CDK2/cyclin A, and tested their cellular antiviral potency and cytotoxicity. We identified several analogues that selectively inhibit P-TEFb kinase activity in vitro and show antiviral potency comparable to that of flavopiridol, but with significantly reduced cytotoxicity. These compounds are valuable molecular probes for understanding P-TEFb-regulated cellular and HIV-1 gene transcription and provide potential anti-HIV-1 therapeutics. PMID:19603446

  12. The nuclear elongation factor-1α gene: a promising marker for phylogenetic studies of Triatominae (Hemiptera: Reduviidae).

    PubMed

    Díaz, Sebastián; Triana-Chávez, Omar; Gómez-Palacio, Andrés

    2016-09-01

    Molecular systematics is a remarkable approach for understanding the taxonomic traits and allows the exploration of the inter-population dynamics of several species in the Triatominae subfamily that are involved in Trypanosoma cruzi transmission. Compared to other relevant species that transmit vector-borne diseases, such as some species of the Diptera, there are relatively few nuclear genetic markers available for systematic studies in the Triatominae subfamily. Molecular systematic studies performed on Triatominae are based on mitochondrial gene fragments and, less frequently, on nuclear ribosomal genes or spacers. Due to the fact that these markers can occasionally present problems such as nuclear mitochondrial genes (NUMTs) or intra-genomic variation for high gene copy numbers, it is necessary to use additional nuclear markers to more reliably address the molecular evolution of Triatominae. In this study, we performed phylogenetic analysis using the nuclear elongation factor-1 alpha (EF-1α) gene in individuals from 12 species belonging to the Triatomini and Rhodniini tribes. Genetic diversities and phylogenetic topologies were compared with those obtained for the mitochondrial 16S rRNA and Cytochrome b (cyt b) genes, as well as for the D2 variable region of the ribosomal 28S rRNA gene. These results indicate that the EF-1α marker exhibits an intermediate level of diversity compared to mitochondrial and nuclear ribosomal genes, and that phylogenetic analysis based on EF-1α is highly informative for resolving deep phylogenetic relationships in Triatominae, such as tribe or genera. PMID:27268149

  13. Synchronous tRNA movements during translocation on the ribosome are orchestrated by elongation factor G and GTP hydrolysis.

    PubMed

    Holtkamp, Wolf; Wintermeyer, Wolfgang; Rodnina, Marina V

    2014-10-01

    The translocation of tRNAs through the ribosome proceeds through numerous small steps in which tRNAs gradually shift their positions on the small and large ribosomal subunits. The most urgent questions are: (i) whether these intermediates are important; (ii) how the ribosomal translocase, the GTPase elongation factor G (EF-G), promotes directed movement; and (iii) how the energy of GTP hydrolysis is coupled to movement. In the light of recent advances in biophysical and structural studies, we argue that intermediate states of translocation are snapshots of dynamic fluctuations that guide the movement. In contrast to current models of stepwise translocation, kinetic evidence shows that the tRNAs move synchronously on the two ribosomal subunits in a rapid reaction orchestrated by EF-G and GTP hydrolysis. EF-G combines the energy regimes of a GTPase and a motor protein and facilitates tRNA movement by a combination of directed Brownian ratchet and power stroke mechanisms. PMID:25118068

  14. Allosteric collaboration between elongation factor G and the ribosomal L1 stalk directs tRNA movements during translation

    PubMed Central

    Fei, Jingyi; Bronson, Jonathan E.; Hofman, Jake M.; Srinivas, Rathi L.; Wiggins, Chris H.; Gonzalez, Ruben L.

    2009-01-01

    Determining the mechanism by which tRNAs rapidly and precisely transit through the ribosomal A, P, and E sites during translation remains a major goal in the study of protein synthesis. Here, we report the real-time dynamics of the L1 stalk, a structural element of the large ribosomal subunit that is implicated in directing tRNA movements during translation. Within pretranslocation ribosomal complexes, the L1 stalk exists in a dynamic equilibrium between open and closed conformations. Binding of elongation factor G (EF-G) shifts this equilibrium toward the closed conformation through one of at least two distinct kinetic mechanisms, where the identity of the P-site tRNA dictates the kinetic route that is taken. Within posttranslocation complexes, L1 stalk dynamics are dependent on the presence and identity of the E-site tRNA. Collectively, our data demonstrate that EF-G and the L1 stalk allosterically collaborate to direct tRNA translocation from the P to the E sites, and suggest a model for the release of E-site tRNA. PMID:19717422

  15. The role of guanine nucleotides in the interaction between aminoacyl-tRNA and elongation factor 1 of Artemia salina.

    PubMed

    Roobol, K; Möller, W

    1978-10-16

    The low-molecular-weight form of elongation factor 1 (EF-1L) of the cysts of the brine shrimp Artemia salina and [3H]phenylalanyl-tRNA are able to form a stable complex which can be isolated on a Sephacryl S200 column. The formation of this complex is inhibited by increasing concentrations of magnesium acetate and KCl. Furthermore, the formation of this complex is independent of the presence of guanine nucleotides. Complex formation between EF-1L and phenylalanyl-tRNA appears to be specific, since acylation of the tRNA is a necessity for this interaction. Although EF-1L alone binds GDP somewhat more strongly than GTP, the complex between EF-1L and phenylalanyl-tRNA binds GTP exclusively. Our results support the idea that complex formation between EF-1L and aminoacyl-tRNA precedes the enzymatic binding of aminoacyl-tRNA to the 80-S ribosome. Subsequently to this binding, release of EF-1L from the ribosome occurs. PMID:251131

  16. Identification of a Taraxacum brevicorniculatum rubber elongation factor protein that is localized on rubber particles and promotes rubber biosynthesis.

    PubMed

    Laibach, Natalie; Hillebrand, Andrea; Twyman, Richard M; Prüfer, Dirk; Schulze Gronover, Christian

    2015-05-01

    Two protein families required for rubber biosynthesis in Taraxacum brevicorniculatum have recently been characterized, namely the cis-prenyltransferases (TbCPTs) and the small rubber particle proteins (TbSRPPs). The latter were shown to be the most abundant proteins on rubber particles, where rubber biosynthesis takes place. Here we identified a protein designated T. brevicorniculatum rubber elongation factor (TbREF) by using mass spectrometry to analyze rubber particle proteins. TbREF is homologous to the TbSRPPs but has a molecular mass that is atypical for the family. The promoter was shown to be active in laticifers, and the protein itself was localized on the rubber particle surface. In TbREF-silenced plants generated by RNA interference, the rubber content was significantly reduced, correlating with lower TbCPT protein levels and less TbCPT activity in the latex. However, the molecular mass of the rubber was not affected by TbREF silencing. The colloidal stability of rubber particles isolated from TbREF-silenced plants was also unchanged. This was not surprising because TbREF depletion did not affect the abundance of TbSRPPs, which are required for rubber particle stability. Our findings suggest that TbREF is an important component of the rubber biosynthesis machinery in T. brevicorniculatum, and may play a role in rubber particle biogenesis and influence rubber production. PMID:25809497

  17. Endogenous ADP-ribosylation of elongation factor 2 in polyoma virus-transformed baby hamster kidney cells

    SciTech Connect

    Fendrick, J.L.; Iglewski, W.J. )

    1989-01-01

    Polyoma virus-transformed baby hamster kidney (pyBHK) cells were cultured in medium containing ({sup 32}P)orthophosphate and 105 (vol/vol) fetal bovine serum. A {sup 32}P-labeled protein with an apparent molecular mass of 97 kDa was immunoprecipitated from cell lysates with antiserum to ADP-ribosylated elongation factor 2 (EF-2). The {sup 32}P labeling of the protein was enhanced by culturing cells in medium containing 2% serum instead of 10% serum. The {sup 32}P label was completely removed from the protein by treatment with snake venom phosphodiesterase and the digestion product was identified as ({sup 32}P)AMP, indicating the protein was mono-ADP-ribosylated. HPLC analysis of tryptic peptides of the {sup 32}P-labeled 97-kDa protein and purified EF-2, which was ADP-ribosylated in vitro with diphtheria toxin fragment A and ({sup 32}P)NAD, demonstrated an identical labeled peptide in the two proteins. The data strongly suggest that EF-2 was endogenously ADP-ribosylated in pyBHK cells. Maximum incorporation of radioactivity in EF-2 occurred by 12 hr and remained constant over the subsequent 12 hr. It was estimated that 30-35% of the EF-2 was ADP-ribosylated in cells cultured in medium containing 2% serum. When {sup 32}P-labeled cultures were incubated in medium containing unlabeled phosphate, the {sup 32}P label was lost from the EF-2 within 30 min.

  18. A conserved proline triplet in Val-tRNA synthetase and the origin of elongation factor P

    PubMed Central

    Starosta, Agata L.; Lassak, Jürgen; Peil, Lauri; Atkinson, Gemma C.; Woolstenhulme, Christopher J.; Virumäe, Kai; Buskirk, Allen; Tenson, Tanel; Remme, Jaanus; Jung, Kirsten; Wilson, Daniel N.

    2016-01-01

    Bacterial ribosomes stall on polyproline stretches and require the elongation factor P (EF-P) to relieve the arrest. Yet it remains unclear why evolution has favored the development of EF-P, rather than selecting against the occurrence of polyproline stretches in proteins. We have discovered that only a single polyproline stretch is invariant across all domains of life, namely, a proline triplet in ValS, the tRNA synthetase that charges tRNAVal with valine. Here we show that expression of ValS in vivo and in vitro requires EF-P and demonstrate that the proline triplet located in the active site of ValS is important for efficient charging of tRNAVal with valine, preventing formation of mischarged Thr-tRNAVal, as well as for efficient growth of E. coli in vivo. We suggest that the critical role of the proline triplet for ValS activity may explain why bacterial cells co-evolved the EF-P rescue system. PMID:25310979

  19. Identification and cloning of two immunogenic C. perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of Clostridium perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic C. perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by reaction with...

  20. Synthesis of Elongated Microcapsules

    NASA Technical Reports Server (NTRS)

    Li, Wenyan; Buhrow, Jerry; Calle, Luz M.

    2011-01-01

    One of the factors that influence the effectiveness of self-healing in functional materials is the amount of liquid healing agents that can be delivered to the damaged area. The use of hollow tubes or fibers and the more sophisticated micro-vascular networks has been proposed as a way to increase the amount of healing agents that can be released when damage is inflicted. Although these systems might be effective in some specific applications, they are not practical for coatings applications. One possible practical way to increase the healing efficiency is to use microcapsules with high-aspect-ratios, or elongated microcapsules. It is understood that elongated microcapsules will be more efficient because they can release more healing agent than a spherical microcapsule when a crack is initiated in the coating. Although the potential advantage of using elongated microcapsules for self healing applications is clear, it is very difficult to make elongated microcapsules from an emulsion system because spherical microcapsules are normally formed due to the interfacial tension between the dispersed phase and the continuous phase. This paper describes the two methods that have been developed by the authors to synthesize elongated microcapsules. The first method involves the use of an emulsion with intermediate stability and the second involves the application of mechanical shear conditions to the emulsion.

  1. TEF-7A, a transcript elongation factor gene, influences yield-related traits in bread wheat (Triticum aestivum L.)

    PubMed Central

    Zheng, Jun; Liu, Hong; Wang, Yuquan; Wang, Lanfen; Chang, Xiaoping; Jing, Ruilian; Hao, Chenyang; Zhang, Xueyong

    2014-01-01

    In this study, TaTEF-7A, a member of the transcript elongation factor gene family, and its flanking sequences were isolated. TaTEF-7A was located on chromosome 7A and was flanked by markers Xwmc83 and XP3156.3. Subcellular localization revealed that TaTEF-7A protein was localized in the nucleus. This gene was expressed in all organs, but the highest expression occurred in young spikes and developing seeds. Overexpression of TaTEF-7A in Arabidopsis thaliana produced pleiotropic effects on vegetative and reproductive development that enhanced grain length, silique number, and silique length. No diversity was found in the coding region of TaTEF-7A, but 16 single nucleotide polymorphisms and Indels were detected in the promoter regions of different cultivars. Markers based on sequence variations in the promoter regions (InDel-629 and InDel-604) were developed, and three haplotypes were identified based on those markers. Haplotype–trait association analysis of the Chinese wheat mini core collection revealed that TaTEF-7A was significantly associated with grain number per spike. Phenotyping of near-isogenic lines (NILs) confirmed that TaTEF-7A increases potential grain yield and yield-related traits. Frequency changes in favoured haplotypes gradually increased in cultivars released in China from the 1940s. Geographic distributions of favoured haplotypes were characterized in six major wheat production regions worldwide. The presence of Hap-7A-3, the favoured haplotype, showed a positive correlation with yield in a global set of breeding lines. These results suggest that TaTEF-7A is a functional regulatory factor for grain number per spike and provide a basis for marker-assisted selection. PMID:25056774

  2. The transcription factor TFIIS zinc ribbon dipeptide Asp-Glu is critical for stimulation of elongation and RNA cleavage by RNA polymerase II.

    PubMed Central

    Jeon, C; Yoon, H; Agarwal, K

    1994-01-01

    The eukaryotic transcription factor TFIIS enhances elongation and nascent transcript cleavage activities of RNA polymerase II in a stalled elongation complex. By site-directed mutagenesis, we have demonstrated that invariant residues Asp-261 and Glu-262 of the nucleic acid-binding TFIIS Zn ribbon are critical for stimulation of both elongation and RNA cleavage activities of RNA polymerase II. Substitution of either of these residues inactivates both TFIIS functions, suggesting a related role in both activities. These acidic residues may participate in phosphoryl transfer reactions by a two-metal-ion mechanism in a manner analogous to Klenow fragment. The RNA polymerase II itself may contain a Zn ribbon, in as much as the polymerase's 15-kDa subunit contains a sequence that aligns well with the TFIIS Zn ribbon sequence, including a similarly placed pair of acidic residues. Images PMID:8090778

  3. Cis and trans-acting elements involved in the activation of Arabidopsis thaliana A1 gene encoding the translation elongation factor EF-1 alpha.

    PubMed

    Curie, C; Liboz, T; Bardet, C; Gander, E; Médale, C; Axelos, M; Lescure, B

    1991-03-25

    In A. thaliana the translation elongation factor EF-1 alpha is encoded by a small multigenic family of four members (A1-A4). The A1 gene promoter has been dissected and examined in a transient expression system using the GUS reporter gene. Deletion analysis has shown that several elements are involved in the activation process. One cis-acting domain, the TEF 1 box, has been accurately mapped 100 bp upstream of the transcription initiation site. This domain is the target for trans-acting factors identified in nuclear extracts prepared from A. thaliana. Homologies are found between the TEF 1 box and sequences present at the same location within the A2, A3 and A4 promoters. This observation, together with those obtained from gel retardation assays performed using DNA fragments from the A4 promoter, suggest that the activation process mediated by the TEF 1 element is conserved among the A. thaliana EF-1 alpha genes. Analysis of nearly full length cDNA clones has shown that in addition to a single intron located within the coding region, the A1 gene contains a second intron located within the 5' non coding region. Such an intron is also present within the A2, A3 and A4 genes. This 5' intervening sequence appears to be essential to obtain a maximum GUS activity driven by the A1 gene promoter. PMID:1840652

  4. Cis and trans-acting elements involved in the activation of Arabidopsis thaliana A1 gene encoding the translation elongation factor EF-1 alpha.

    PubMed Central

    Curie, C; Liboz, T; Bardet, C; Gander, E; Médale, C; Axelos, M; Lescure, B

    1991-01-01

    In A. thaliana the translation elongation factor EF-1 alpha is encoded by a small multigenic family of four members (A1-A4). The A1 gene promoter has been dissected and examined in a transient expression system using the GUS reporter gene. Deletion analysis has shown that several elements are involved in the activation process. One cis-acting domain, the TEF 1 box, has been accurately mapped 100 bp upstream of the transcription initiation site. This domain is the target for trans-acting factors identified in nuclear extracts prepared from A. thaliana. Homologies are found between the TEF 1 box and sequences present at the same location within the A2, A3 and A4 promoters. This observation, together with those obtained from gel retardation assays performed using DNA fragments from the A4 promoter, suggest that the activation process mediated by the TEF 1 element is conserved among the A. thaliana EF-1 alpha genes. Analysis of nearly full length cDNA clones has shown that in addition to a single intron located within the coding region, the A1 gene contains a second intron located within the 5' non coding region. Such an intron is also present within the A2, A3 and A4 genes. This 5' intervening sequence appears to be essential to obtain a maximum GUS activity driven by the A1 gene promoter. Images PMID:1840652

  5. Stoichiometry and Change of the mRNA Closed-Loop Factors as Translating Ribosomes Transit from Initiation to Elongation.

    PubMed

    Wang, Xin; Xi, Wen; Toomey, Shaun; Chiang, Yueh-Chin; Hasek, Jiri; Laue, Thomas M; Denis, Clyde L

    2016-01-01

    Protein synthesis is a highly efficient process and is under exacting control. Yet, the actual abundance of translation factors present in translating complexes and how these abundances change during the transit of a ribosome across an mRNA remains unknown. Using analytical ultracentrifugation with fluorescent detection we have determined the stoichiometry of the closed-loop translation factors for translating ribosomes. A variety of pools of translating polysomes and monosomes were identified, each containing different abundances of the closed-loop factors eIF4E, eIF4G, and PAB1 and that of the translational repressor, SBP1. We establish that closed-loop factors eIF4E/eIF4G dissociated both as ribosomes transited polyadenylated mRNA from initiation to elongation and as translation changed from the polysomal to monosomal state prior to cessation of translation. eIF4G was found to particularly dissociate from polyadenylated mRNA as polysomes moved to the monosomal state, suggesting an active role for translational repressors in this process. Consistent with this suggestion, translating complexes generally did not simultaneously contain eIF4E/eIF4G and SBP1, implying mutual exclusivity in such complexes. For substantially deadenylated mRNA, however, a second type of closed-loop structure was identified that contained just eIF4E and eIF4G. More than one eIF4G molecule per polysome appeared to be present in these complexes, supporting the importance of eIF4G interactions with the mRNA independent of PAB1. These latter closed-loop structures, which were particularly stable in polysomes, may be playing specific roles in both normal and disease states for specific mRNA that are deadenylated and/or lacking PAB1. These analyses establish a dynamic snapshot of molecular abundance changes during ribosomal transit across an mRNA in what are likely to be critical targets of regulation. PMID:26953568

  6. Stoichiometry and Change of the mRNA Closed-Loop Factors as Translating Ribosomes Transit from Initiation to Elongation

    PubMed Central

    Wang, Xin; Xi, Wen; Toomey, Shaun; Chiang, Yueh-Chin; Hasek, Jiri; Laue, Thomas M.; Denis, Clyde L.

    2016-01-01

    Protein synthesis is a highly efficient process and is under exacting control. Yet, the actual abundance of translation factors present in translating complexes and how these abundances change during the transit of a ribosome across an mRNA remains unknown. Using analytical ultracentrifugation with fluorescent detection we have determined the stoichiometry of the closed-loop translation factors for translating ribosomes. A variety of pools of translating polysomes and monosomes were identified, each containing different abundances of the closed-loop factors eIF4E, eIF4G, and PAB1 and that of the translational repressor, SBP1. We establish that closed-loop factors eIF4E/eIF4G dissociated both as ribosomes transited polyadenylated mRNA from initiation to elongation and as translation changed from the polysomal to monosomal state prior to cessation of translation. eIF4G was found to particularly dissociate from polyadenylated mRNA as polysomes moved to the monosomal state, suggesting an active role for translational repressors in this process. Consistent with this suggestion, translating complexes generally did not simultaneously contain eIF4E/eIF4G and SBP1, implying mutual exclusivity in such complexes. For substantially deadenylated mRNA, however, a second type of closed-loop structure was identified that contained just eIF4E and eIF4G. More than one eIF4G molecule per polysome appeared to be present in these complexes, supporting the importance of eIF4G interactions with the mRNA independent of PAB1. These latter closed-loop structures, which were particularly stable in polysomes, may be playing specific roles in both normal and disease states for specific mRNA that are deadenylated and/or lacking PAB1. These analyses establish a dynamic snapshot of molecular abundance changes during ribosomal transit across an mRNA in what are likely to be critical targets of regulation. PMID:26953568

  7. Functional Characterization of a Gene in Sedum alfredii Hance Resembling Rubber Elongation Factor Endowed with Functions Associated with Cadmium Tolerance.

    PubMed

    Liu, Mingying; Qiu, Wenming; He, Xuelian; Zheng, Liu; Song, Xixi; Han, Xiaojiao; Jiang, Jing; Qiao, Guirong; Sang, Jian; Liu, Mingqing; Zhuo, Renying

    2016-01-01

    Cadmium is a major toxic heavy-metal pollutant considering their bioaccumulation potential and persistence in the environment. The hyperaccumulating ecotype of Sedum alfredii Hance is a Zn/Cd co-hyperaccumulator inhabiting in a region of China with soils rich in Pb/Zn. Investigations into the underlying molecular regulatory mechanisms of Cd tolerance are of substantial interest. Here, library screening for genes related to cadmium tolerance identified a gene resembling the rubber elongation factor gene designated as SaREFl. The heterologous expression of SaREFl rescued the growth of a transformed Cd-sensitive strain (ycf1). Furthermore, SaREFl-expressing Arabidopsis plants were more tolerant to cadmium stress compared with wild type by measuring parameters of root length, fresh weight and physiological indexes. When under four different heavy metal treatments, we found that SaREFl responded most strongly to Cd and the root was the plant organ most sensitive to this heavy metal. Yeast two-hybrid screening of SaREFl as a bait led to the identification of five possible interacting targets in Sedum alfredii Hance. Among them, a gene annotated as prenylated Rab acceptor 1 (PRA1) domain protein was detected with a high frequency. Moreover, subcellular localization of SaREF1-GFP fusion protein revealed some patchy spots in cytosol suggesting potential association with organelles for its cellular functions. Our findings would further enrich the connotation of REF-like genes and provide theoretical assistance for the application in breeding heavy metal-tolerant plants. PMID:27446189

  8. Conformational changes of the small ribosomal subunit during elongation factor G-dependent tRNA-mRNA translocation.

    PubMed

    Peske, Frank; Savelsbergh, Andreas; Katunin, Vladimir I; Rodnina, Marina V; Wintermeyer, Wolfgang

    2004-11-01

    Translocation, a coordinated movement of two tRNAs together with mRNA on the ribosome, is catalyzed by elongation factor G (EF-G). The reaction is accompanied by conformational rearrangements of the ribosome that are, as yet, not well characterized. Here, we analyze those rearrangements by restricting the conformational flexibility of the ribosome by antibiotics binding to specific sites of the ribosome. Paromomycin (Par), viomycin (Vio), spectinomycin (Spc), and hygromycin B (HygB) inhibited the tRNA-mRNA movement, while the other partial reactions of translocation, including the unlocking rearrangement of the ribosome that precedes tRNA-mRNA movement, were not affected. The functional cycle of EF-G, i.e. binding of EF-G.GTP to the ribosome, GTP hydrolysis, Pi release, and dissociation of EF-G.GDP from the ribosome, was not affected either, indicating that EF-G turnover is not coupled directly to tRNA-mRNA movement. The inhibition of translocation by Par and Vio is attributed to the stabilization of tRNA binding in the A site, whereas Spc and HygB had a direct inhibitory effect on tRNA-mRNA movement. Streptomycin (Str) had essentially no effect on translocation, although it caused a large increase in tRNA affinity to the A site. These results suggest that conformational changes in the vicinity of the decoding region at the binding sites of Spc and HygB are important for tRNA-mRNA movement, whereas Str seems to stabilize a conformation of the ribosome that is prone to rapid translocation, thereby compensating the effect on tRNA affinity. PMID:15491605

  9. Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha

    NASA Technical Reports Server (NTRS)

    Wang, W.; Poovaiah, B. W.

    1999-01-01

    A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.

  10. Multiple Orientia tsutsugamushi Ankyrin Repeat Proteins Interact with SCF1 Ubiquitin Ligase Complex and Eukaryotic Elongation Factor 1 α

    PubMed Central

    Min, Chan-Ki; Kwon, Ye-Jin; Ha, Na-Young; Cho, Bon-A; Kim, Jo-Min; Kwon, Eun-Kyung; Kim, Yeon-Sook; Choi, Myung-Sik; Kim, Ik-Sang; Cho, Nam-Hyuk

    2014-01-01

    Background Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular bacterium. Previously, a large number of genes that encode proteins containing eukaryotic protein-protein interaction motifs such as ankyrin-repeat (Ank) domains were identified in the O. tsutsugamushi genome. However, little is known about the Ank protein function in O. tsutsugamushi. Methodology/Principal Findings To characterize the function of Ank proteins, we investigated a group of Ank proteins containing an F-box–like domain in the C-terminus in addition to the Ank domains. All nine selected ank genes were expressed at the transcriptional level in host cells infected with O. tsutsugamushi, and specific antibody responses against three Ank proteins were detected in the serum from human patients, indicating an active expression of the bacterial Ank proteins post infection. When ectopically expressed in HeLa cells, the Ank proteins of O. tsutsugamushi were consistently found in the nucleus and/or cytoplasm. In GST pull-down assays, multiple Ank proteins specifically interacted with Cullin1 and Skp1, core components of the SCF1 ubiquitin ligase complex, as well as the eukaryotic elongation factor 1 α (EF1α). Moreover, one Ank protein co-localized with the identified host targets and induced downregulation of EF1α potentially via enhanced ubiquitination. The downregulation of EF1α was observed consistently in diverse host cell types infected with O. tsutsugamushi. Conclusion/Significance These results suggest that conserved targeting and subsequent degradation of EF1α by multiple O. tsutsugamushi Ank proteins could be a novel bacterial strategy for replication and/or pathogenesis during mammalian host infection. PMID:25166298

  11. Functional Characterization of a Gene in Sedum alfredii Hance Resembling Rubber Elongation Factor Endowed with Functions Associated with Cadmium Tolerance

    PubMed Central

    Liu, Mingying; Qiu, Wenming; He, Xuelian; Zheng, Liu; Song, Xixi; Han, Xiaojiao; Jiang, Jing; Qiao, Guirong; Sang, Jian; Liu, Mingqing; Zhuo, Renying

    2016-01-01

    Cadmium is a major toxic heavy-metal pollutant considering their bioaccumulation potential and persistence in the environment. The hyperaccumulating ecotype of Sedum alfredii Hance is a Zn/Cd co-hyperaccumulator inhabiting in a region of China with soils rich in Pb/Zn. Investigations into the underlying molecular regulatory mechanisms of Cd tolerance are of substantial interest. Here, library screening for genes related to cadmium tolerance identified a gene resembling the rubber elongation factor gene designated as SaREFl. The heterologous expression of SaREFl rescued the growth of a transformed Cd-sensitive strain (ycf1). Furthermore, SaREFl-expressing Arabidopsis plants were more tolerant to cadmium stress compared with wild type by measuring parameters of root length, fresh weight and physiological indexes. When under four different heavy metal treatments, we found that SaREFl responded most strongly to Cd and the root was the plant organ most sensitive to this heavy metal. Yeast two-hybrid screening of SaREFl as a bait led to the identification of five possible interacting targets in Sedum alfredii Hance. Among them, a gene annotated as prenylated Rab acceptor 1 (PRA1) domain protein was detected with a high frequency. Moreover, subcellular localization of SaREF1-GFP fusion protein revealed some patchy spots in cytosol suggesting potential association with organelles for its cellular functions. Our findings would further enrich the connotation of REF-like genes and provide theoretical assistance for the application in breeding heavy metal-tolerant plants. PMID:27446189

  12. The surface-associated elongation factor Tu is concealed for antibody binding on viable pneumococci and meningococci.

    PubMed

    Kolberg, Jan; Hammerschmidt, Sven; Frank, Ronald; Jonák, Jirí; Sanderová, Hana; Aase, Audun

    2008-07-01

    Proteome analyses revealed that elongation factor-Tu (EF-Tu) is associated with cytoplasmic membranes of Gram-positive bacteria and outer membranes of Gram-negative bacteria. It is still debatable whether EF-Tu is located on the external side or the internal side of the membranes. Here, we have generated two new monoclonal antibodies (mAbs) and polyclonal rabbit antibodies against pneumococcal EF-Tu. These antibodies were used to investigate the amount of surface-exposed EF-Tu on viable bacteria using a flow cytometric analysis. The control antibodies recognizing the pneumococcal surface protein A and phosphorylcholine showed a significant binding to viable pneumococci. In contrast, anti-EF-Tu antibodies did not recognize pneumococcal EF-Tu. However, heat killing of pneumococci lacking capsular polysaccharides resulted in specific antibody binding to EF-Tu and, moreover, increased the exposure of recognized phosphorylcholine epitopes. Similarly, our EF-Tu-specific antibodies did not recognize EF-Tu of viable Neisseria meningitidis. However, pretreatment of meningococci with ethanol resulted in specific antibody binding to EF-Tu on outer membranes. Importantly, these treatments did not destroy the membrane integrity as analysed with control mAbs directed against cytoplasmic proteins. In conclusion, our flow cytrometric assays emphasize the importance of using viable bacteria and not heat-killed or ethanol-treated bacteria for surface-localization experiments of proteins, because these treatments modulate the cytoplasmic and outer membranes of bacteria and the binding results may not reflect the situation under physiological conditions. PMID:18462389

  13. Systematic Analysis Reveals Elongation Factor 2 and α-Enolase as Novel Interaction Partners of AKT2.

    PubMed

    Bottermann, Katharina; Reinartz, Michael; Barsoum, Marian; Kötter, Sebastian; Gödecke, Axel

    2013-01-01

    AKT2 is one of the three isoforms of the protein kinase AKT being involved in the modulation of cellular metabolism. Since protein-protein interactions are one possibility to convey specificity in signal transduction, we performed AKT2-protein interaction analysis to elucidate their relevance for AKT2-dependent cellular functions. We identified heat shock protein 90 kDa (HSP90), Cdc37, heat shock protein 70 kDa (HSP70), 78 kDa glucose regulated protein (GRP78), tubulin, GAPDH, α-enolase and elongation factor 2 (EF2) as AKT2-interacting proteins by a combination of tandem affinity purification and mass spectrometry in HEK293T cells. Quantitative MS-analysis using stable isotope labeling by amino acids in cell culture (SILAC) revealed that only HSP90 and Cdc37 interact stably with AKT2, whereas the other proteins interact with low affinity with AKT2. The interactions of AKT2 with α-enolase and EF2 were further analyzed in order to uncover the functional relevance of these newly discovered binding partners. Despite the interaction of AKT2 and α-enolase, which was additionally validated by proximity ligation assay (PLA), no significant impact of AKT on α-enolase activity was detected in activity measurements. AKT stimulation via insulin and/or inhibition with the ATP-competitive inhibitor CCT128930 did not alter enzymatic activity of α-enolase. Interestingly, the direct interaction of AKT2 and EF2 was found to be dynamically regulated in embryonic rat cardiomyocytes. Treatment with the PI3-kinase inhibitor LY294002 before stimulation with several hormones stabilized the complex, whereas stimulation alone led to complex dissociation which was analyzed in situ with PLA. Taken together, these findings point to new aspects of AKT2-mediated signal transduction in protein synthesis and glucose metabolism. PMID:23823123

  14. The role of GTP in transient splitting of 70S ribosomes by RRF (ribosome recycling factor) and EF-G (elongation factor G)

    PubMed Central

    Hirokawa, Go; Iwakura, Nobuhiro; Kaji, Akira; Kaji, Hideko

    2008-01-01

    Ribosome recycling factor (RRF), elongation factor G (EF-G) and GTP split 70S ribosomes into subunits. Here, we demonstrated that the splitting was transient and the exhaustion of GTP resulted in re-association of the split subunits into 70S ribosomes unless IF3 (initiation factor 3) was present. However, the splitting was observed with sucrose density gradient centrifugation (SDGC) without IF3 if RRF, EF-G and GTP were present in the SDGC buffer. The splitting of 70S ribosomes causes the decrease of light scattering by ribosomes. Kinetic constants obtained from the light scattering studies are sufficient to account for the splitting of 70S ribosomes by RRF and EF-G/GTP during the lag phase for activation of ribosomes for the log phase. As the amount of 70S ribosomes increased, more RRF, EF-G and GTP were necessary to split 70S ribosomes. In the presence of a physiological amount of polyamines, GTP and factors, even 0.6 μM 70S ribosomes (12 times higher than the 70S ribosomes for routine assay) were split. Spermidine (2 mM) completely inhibited anti-association activity of IF3, and the RRF/EF-G/GTP-dependent splitting of 70S ribosomes. PMID:18948280

  15. Neisseria meningitidis Translation Elongation Factor P and Its Active-Site Arginine Residue Are Essential for Cell Viability

    PubMed Central

    Yanagisawa, Tatsuo; Takahashi, Hideyuki; Suzuki, Takehiro; Masuda, Akiko; Dohmae, Naoshi; Yokoyama, Shigeyuki

    2016-01-01

    Translation elongation factor P (EF-P), a ubiquitous protein over the entire range of bacterial species, rescues ribosomal stalling at consecutive prolines in proteins. In Escherichia coli and Salmonella enterica, the post-translational β-lysyl modification of Lys34 of EF-P is important for the EF-P activity. The β-lysyl EF-P modification pathway is conserved among only 26–28% of bacteria. Recently, it was found that the Shewanella oneidensis and Pseudomonas aeruginosa EF-P proteins, containing an Arg residue at position 32, are modified with rhamnose, which is a novel post-translational modification. In these bacteria, EF-P and its Arg modification are both dispensable for cell viability, similar to the E. coli and S. enterica EF-P proteins and their Lys34 modification. However, in the present study, we found that EF-P and Arg32 are essential for the viability of the human pathogen, Neisseria meningitidis. We therefore analyzed the modification of Arg32 in the N. meningitidis EF-P protein, and identified the same rhamnosyl modification as in the S. oneidensis and P. aeruginosa EF-P proteins. N. meningitidis also has the orthologue of the rhamnosyl modification enzyme (EarP) from S. oneidensis and P. aeruginosa. Therefore, EarP should be a promising target for antibacterial drug development specifically against N. meningitidis. The pair of genes encoding N. meningitidis EF-P and EarP suppressed the slow-growth phenotype of the EF-P-deficient mutant of E. coli, indicating that the activity of N. meningitidis rhamnosyl–EF-P for rescuing the stalled ribosomes at proline stretches is similar to that of E. coli β-lysyl–EF-P. The possible reasons for the unique requirement of rhamnosyl–EF-P for N. meningitidis cells are that more proline stretch-containing proteins are essential and/or the basal ribosomal activity to synthesize proline stretch-containing proteins in the absence of EF-P is lower in this bacterium than in others. PMID:26840407

  16. Crystallization and preliminary X-ray analysis of the mRNA-binding domain of elongation factor SelB in complex with RNA

    SciTech Connect

    Rasubala, Linda; Fourmy, Dominique; Ose, Toyoyuki; Kohda, Daisuke; Maenaka, Katsumi Yoshizawa, Satoko

    2005-03-01

    The mRNA-binding domain of M. thermoacetica selenocysteine-specific elongation factor SelB (residues 512–634, SelB-M) was overproduced in E. coli and its cognate mRNA ligand, 23 nucleotides of the SECIS RNA hairpin, was chemically prepared. The purified SelB-M–SECIS RNA complex has been crystallized in space group P2{sub 1}2{sub 1}2 and diffracted to 2.3 Å.

  17. Structural studies on the RNA-recognition motif of NELF E, a cellular negative transcription elongation factor involved in the regulation of HIV transcription

    PubMed Central

    Rao, Jampani N.; Neumann, Liane; Wenzel, Sabine; Schweimer, Kristian; Rösch, Paul; Wöhrl, Birgitta M.

    2006-01-01

    The elongation of transcription of HIV RNA at the TAR (transactivation-response element) is highly regulated by positive and negative factors. The cellular negative transcription elongation factor NELF (negative elongation factor) was suggested to be involved in transcriptional regulation of HIV-1 (HIV type 1) by binding to the stem of the viral TAR RNA which is synthesized by cellular RNA polymerase II at the viral long terminal repeat. NELF is a heterotetrameric protein consisting of NELF A, B, C or the splice variant D, and E. In the present study, we determined the solution structure of the RRM (RNA-recognition motif) of the RNA-binding subunit NELF E and studied its interaction with the viral TAR RNA. Our results show that the separately expressed recombinant NELF E RRM has α-helical and β-strand elements adopting a βαββαβ fold and is able to bind to TAR RNA. Fluorescence equilibrium titrations with fluorescently labelled double- and single-stranded oligoribonucleotides representing the TAR RNA stem imply that NELF E RRM binds to the single-stranded TAR RNAs with Kd values in the low-micromolar range. PMID:16898873

  18. The Rho guanine exchange factor RHGF-2 acts through the Rho-binding kinase LET-502 to mediate embryonic elongation in C. elegans.

    PubMed

    Chan, Benjamin G; Rocheleau, Simon K; Smit, Ryan B; Mains, Paul E

    2015-09-15

    Morphogenesis allows an organism to develop its final body shape. In Caenorhabditis elegans, a smooth muscle-like contraction of an actin/myosin network in the epidermis mediates the elongation of the worm embryo from a ball of cells into a long, thin worm. This process is controlled by two redundant pathways, one involving the small GTPase RHO-1 and its downstream effectors LET-502/Rho-binding kinase and MEL-11/myosin phosphatase, and another involving PAK-1/p21 activated kinase and FEM-2/PP2c phosphatase. Contraction occurs primarily in the lateral epidermal cells during elongation while the dorsal and ventral epidermal cells have a more passive role, and localized activity of a Rho GEF (guanine exchange factor) could contribute to this asymmetry. We found that loss of the C. elegans Rho GEF encoded by rhgf-2 results in arrest during early elongation. Genetically, rhgf-2 acts as an activator of let-502/Rho-binding kinase, in parallel to fem-2/PP2c phosphatase. Although expressed throughout the embryo, lateral cell-specific RHGF-2 expression can mediate elongation. The Rho GTPase activating protein (GAP) RGA-2 is known to inhibit contraction in the dorsal and ventral epidermis. Although rhgf-2 and rga-2 are individually lethal, the double mutant is viable with elongation still occurring in a let-502 dependent fashion. This indicates that LET-502/Rho-binding kinase has activity independent of the GEF and GAP. Finally, maternal LET-502 and MEL-11 are known to regulate the rate of cleavage furrow ingression in the early embryo and we show that maternal RHGF-2 also influences cleavage but RGA-2 does not. Thus while the LET-502/MEL-11 pathway is employed multiple times during embryogenesis, regulation by GEFs and GAPs differs at different points of the life cycle and fine tunes contractile function. PMID:26188247

  19. A new purification scheme for elongation factor 1 from rabbit reticulocytes and investigation of the homology of the subunits with those of initiation factor 2.

    PubMed

    Moretti, S; Staehelin, T; Trachsel, H; Gordon, J

    1979-07-01

    The aim of this work was to compare the subunits of the elongation factor EF-1 and the initiation factor eIF-2 from rabbit reticulocytes. We devised a simple procedure for the purification of EF-1: stepwise chromatography on heparin-Sepharose, separation of the heavy form by sucrose gradient centrifugation, and a final step of stepwise chromatography on RNA-Sepharose. The heparin-Sepharose column also clearly separated EF-1 and EF-2 within one chromatographic step. The EF-1 was 350-fold puried and the yield was 10%. This preparation showed after electrophoresis on polyacylamide gels in the presence of sodium dodecyl sulfate three bands corresponding to those described by others as the subunits, with Mr of 54000, 49000 and 29200. An additional band of Mr 34000 was present but no others. The 49000-Mr and 34000-Mr bands corresponded exactly in molecular weight to two of three subunits of eIF-2. A more detailed comparison was therefore made of all subunits of EF-1 and eIF-2. This was done by examination of chymotryptic fingerprints on polyacrylamide gel electrophoresis. No evidence for homology between EF-1 and eIF-2 was found. However, the two larger subunits of eIF-2 had a majority of chymotryptic fragments in common, thus indicating some homology between these polypeptides. PMID:467435

  20. Oxidation of a Cysteine Residue in Elongation Factor EF-Tu Reversibly Inhibits Translation in the Cyanobacterium Synechocystis sp. PCC 6803.

    PubMed

    Yutthanasirikul, Rayakorn; Nagano, Takanori; Jimbo, Haruhiko; Hihara, Yukako; Kanamori, Takashi; Ueda, Takuya; Haruyama, Takamitsu; Konno, Hiroki; Yoshida, Keisuke; Hisabori, Toru; Nishiyama, Yoshitaka

    2016-03-11

    Translational elongation is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803, and elongation factor G has been identified as a target of oxidation by ROS. In the present study we examined the sensitivity to oxidation by ROS of another elongation factor, EF-Tu. The structure of EF-Tu changes dramatically depending on the bound nucleotide. Therefore, we investigated the sensitivity to oxidation in vitro of GTP- and GDP-bound EF-Tu as well as that of nucleotide-free EF-Tu. Assays of translational activity with a reconstituted translation system from Escherichia coli revealed that GTP-bound and nucleotide-free EF-Tu were sensitive to oxidation by H2O2, whereas GDP-bound EF-Tu was resistant to H2O2. The inactivation of EF-Tu was the result of oxidation of Cys-82, a single cysteine residue, and subsequent formation of both an intermolecular disulfide bond and sulfenic acid. Replacement of Cys-82 with serine rendered EF-Tu resistant to inactivation by H2O2, confirming that Cys-82 was a target of oxidation. Furthermore, oxidized EF-Tu was reduced and reactivated by thioredoxin. Gel-filtration chromatography revealed that some of the oxidized nucleotide-free EF-Tu formed large complexes of >30 molecules. Atomic force microscopy revealed that such large complexes dissociated into several smaller aggregates upon the addition of dithiothreitol. Immunological analysis of the redox state of EF-Tu in vivo showed that levels of oxidized EF-Tu increased under strong light. Thus, resembling elongation factor G, EF-Tu appears to be sensitive to ROS via oxidation of a cysteine residue, and its inactivation might be reversed in a redox-dependent manner. PMID:26786107

  1. Release of Positive Transcription Elongation Factor b (P-TEFb) from 7SK Small Nuclear Ribonucleoprotein (snRNP) Activates Hexamethylene Bisacetamide-inducible Protein (HEXIM1) Transcription*

    PubMed Central

    Liu, Pingyang; Xiang, Yanhui; Fujinaga, Koh; Bartholomeeusen, Koen; Nilson, Kyle A.; Price, David H.; Peterlin, B. Matija

    2014-01-01

    By phosphorylating negative elongation factors and the C-terminal domain of RNA polymerase II (RNAPII), positive transcription elongation factor b (P-TEFb), which is composed of CycT1 or CycT2 and CDK9, activates eukaryotic transcription elongation. In growing cells, it is found in active and inactive forms. In the former, free P-TEFb is a potent transcriptional coactivator. In the latter, it is inhibited by HEXIM1 or HEXIM2 in the 7SK small nuclear ribonucleoprotein (snRNP), which contains, additionally, 7SK snRNA, methyl phosphate-capping enzyme (MePCE), and La-related protein 7 (LARP7). This P-TEFb equilibrium determines the state of growth and proliferation of the cell. In this study, the release of P-TEFb from the 7SK snRNP led to increased synthesis of HEXIM1 but not HEXIM2 in HeLa cells, and this occurred only from an unannotated, proximal promoter. ChIP with sequencing revealed P-TEFb-sensitive poised RNA polymerase II at this proximal but not the previously annotated distal HEXIM1 promoter. Its immediate upstream sequences were fused to luciferase reporters and were found to be responsive to many P-TEFb-releasing compounds. The superelongation complex subunits AF4/FMR2 family member 4 (AFF4) and elongation factor RNA polymerase II 2 (ELL2) were recruited to this proximal promoter after P-TEFb release and were required for its transcriptional effects. Thus, P-TEFb regulates its own equilibrium in cells, most likely to maintain optimal cellular homeostasis. PMID:24515107

  2. OsSNDP1, a Sec14-nodulin domain-containing protein, plays a critical role in root hair elongation in rice.

    PubMed

    Huang, Jin; Kim, Chul Min; Xuan, Yuan-hu; Park, Soon Ju; Piao, Hai Long; Je, Byoung Il; Liu, Jingmiao; Kim, Tae Ho; Kim, Bo-Kyeong; Han, Chang-Deok

    2013-05-01

    Rice is cultivated in water-logged paddy lands. Thus, rice root hairs on the epidermal layers are exposed to a different redox status of nitrogen species, organic acids, and metal ions than root hairs growing in drained soil. To identify genes that play an important role in root hair growth, a forward genetics approach was used to screen for short-root-hair mutants. A short-root-hair mutant was identified and isolated by using map-based cloning and sequencing. The mutation arose from a single amino acid substitution of OsSNDP1 (Oryza sativa Sec14-nodulin domain protein), which shows high sequence homology with Arabidopsis COW1/AtSFH1 and encodes a phosphatidylinositol transfer protein (PITP). By performing complementation assays with Atsfh1 mutants, we demonstrated that OsSNDP1 is involved in growth of root hairs. Cryo-scanning electron microscopy was utilized to further characterize the effect of the Ossndp1 mutation on root hair morphology. Aberrant morphogenesis was detected in root hair elongation and maturation zones. Many root hairs were branched and showed irregular shapes due to bulged nodes. Many epidermal cells also produced dome-shaped root hairs, which indicated that root hair elongation ceased at an early stage. These studies showed that PITP-mediated phospholipid signaling and metabolism is critical for root hair elongation in rice. PMID:23456248

  3. Molecular characterization and expression analysis of five different elongation factor 1 alpha genes in the flatfish Senegalese sole (Solea senegalensis Kaup): Differential gene expression and thyroid hormones dependence during metamorphosis

    PubMed Central

    Infante, Carlos; Asensio, Esther; Cañavate, José Pedro; Manchado, Manuel

    2008-01-01

    Background Eukaryotic elongation factor 1 alpha (eEF1A) is one of the four subunits composing eukaryotic translation elongation factor 1. It catalyzes the binding of aminoacyl-tRNA to the A-site of the ribosome in a GTP-dependent manner during protein synthesis, although it also seems to play a role in other non-translational processes. Currently, little information is still available about its expression profile and regulation during flatfish metamorphosis. With regard to this, Senegalese sole (Solea senegalensis) is a commercially important flatfish in which eEF1A gene remains to be characterized. Results The development of large-scale genomics of Senegalese sole has facilitated the identification of five different eEF1A genes, referred to as SseEF1A1, SseEF1A2, SseEF1A3, SseEF1A4, and Sse42Sp50. Main characteristics and sequence identities with other fish and mammalian eEF1As are described. Phylogenetic and tissue expression analyses allowed for the identification of SseEF1A1 and SseEF1A2 as the Senegalese sole counterparts of mammalian eEF1A1 and eEF1A2, respectively, and of Sse42Sp50 as the ortholog of Xenopus laevis and teleost 42Sp50 gene. The other two elongation factors, SseEF1A3 and SseEF1A4, represent novel genes that are mainly expressed in gills and skin. The expression profile of the five genes was also studied during larval development, revealing different behaviours. To study the possible regulation of SseEF1A gene expressions by thyroid hormones (THs), larvae were exposed to the goitrogen thiourea (TU). TU-treated larvae exhibited lower SseEF1A4 mRNA levels than untreated controls at both 11 and 15 days after treatment, whereas transcripts of the other four genes remained relatively unchanged. Moreover, addition of exogenous T4 hormone to TU-treated larvae increased significantly the steady-state levels of SseEF1A4 with respect to untreated controls, demonstrating that its expression is up-regulated by THs. Conclusion We have identified five

  4. CsAGP1, a Gibberellin-Responsive Gene from Cucumber Hypocotyls, Encodes a Classical Arabinogalactan Protein and Is Involved in Stem Elongation

    PubMed Central

    Park, Me Hea; Suzuki, Yoshihito; Chono, Makiko; Knox, J. Paul; Yamaguchi, Isomaro

    2003-01-01

    Fluorescence differential display was used to isolate the gibberellin (GA)-responsive gene, CsAGP1, from cucumber (Cucumis sativus) hypocotyls. A sequence analysis of CsAGP1 indicated that the gene putatively encodes a “classical” arabinogalactan protein (AGP) in cucumber. Transgenic tobacco (Nicotiana tabacum) plants overexpressing CsAGP1 under the control of the cauliflower mosaic virus 35S promoter produced a Y(βGlc)3-reactive proteoglycan in addition to AGPs present in wild-type tobacco plants. Immuno-dot blotting of the product, using anti-AGP antibodies, showed that the CsAGP1 protein had the AGP epitopes common to AGP families. The transcription level of CsAGP1 in cucumber hypocotyls increased in response not only to GA but also to indole-3-acetic acid. Although CsAGP1 is expressed in most vegetative tissues of cucumber, including the shoot apices and roots, the GA treatment resulted in an increase in the mRNA level of CsAGP1 only in the upper part of the hypocotyls. Y(βGlc)3, which selectively binds AGPs, inhibited the hormone-promoted elongation of cucumber seedling hypocotyls. Transgenic plants ectopically expressing CsAGP1 showed a taller stature and earlier flowering than the wild-type plants. These observations suggest that CsAGP1 is involved in stem elongation. PMID:12644694

  5. Identification and characterization of two novel human mitochondrial elongation factor genes, hEFG2 and hEFG1, phylogenetically conserved through evolution.

    PubMed

    Hammarsund, M; Wilson, W; Corcoran, M; Merup, M; Einhorn, S; Grandér, D; Sangfelt, O

    2001-11-01

    Rapid progress in the sequencing of the genome of man and other species allows for the comparative analysis of their genetic structure and content. We have used a combined biochemical and computer-based approach to characterize a 146 kb human genomic bacterial artificial chromosome clone from chromosome 5q13 and discovered a novel human elongation-factor gene, hEFG2. The complete human EFG2 cDNA sequence is 3033 bp and contains 21 exons with conserved exon-intron splice junctions encompassing 45 kb of the genomic sequence with its 5'-end residing within a CpG island, characteristic of a housekeeping gene. The complete size of the hEFG2 cDNA was confirmed by Northern blot and reverse transcription/polymerase chain reaction analysis, which showed a single transcript of 3.2 kb ubiquitously expressed in various human tissues. The hEFG2 protein shows significant homology to several bacterial EF-G proteins, including that of Thermus thermophilus, and to the yeast Saccharomyces cerevisiae mitochondrial elongation factor-G ( MEF2). Multiple alignments reveal a novel gene family of mitochondrial EF-G proteins that can by divided into two subgroups, EF-G1 and EF-G2, in several eukaryotic species including S. pombe, Caenorhabditis elegans and Drosophila melanogaster. Using the information contained in the public databases, we also identified and cloned the complete coding sequence of the human EFG1 gene on chromosome 3q25. The cloning and characterization of these human mitochondrial elongation factor genes should permit us to address their role in the regulation of normal mitochondrial function and in various disease states. PMID:11735030

  6. Dictyostelium ribosomal protein genes and the elongation factor 1B gene show coordinate developmental regulation which is under post-transcriptional control.

    PubMed

    Agarwal, A K; Blumberg, D D

    1999-06-01

    Starvation for amino acids initiates the developmental program in the cellular slime mold, Dictyostelium discoideum [19, 20]. One of the earliest developmental events is the decline in ribosomal protein synthesis [2, 17, 29, 30]. The ribosomal protein mRNAs are excluded from polysomes with 20 min to 1 h following the removal of nutrients, and their mRNA levels decline sharply at about 9 h into the 24-h developmental cycle [28, 31, 35, 36]. It has been generally assumed that the decline in r-protein mRNA levels during late development reflected a decline in the transcription rate [12, 32, 43]. Here we demonstrate that this is not the case. The transcription rates of three ribosomal protein genes, rpL11, rpL23 and rpS9 as well as an elongation factor 1B gene have been determined during growth and development in Dictyostelium. Throughout growth and development the transcription rate of the ribosomal protein genes remains relatively constant at 0.2%-0.5% of the rate of rRNA transcription while the elongation factor 1B gene is transcribed at 0.4%-0.6% of the rRNA rate. This low but constant transcription rate is in contrast to a spore coat protein gene Psp D, which is transcribed at 6% of the rRNA rate in late developing cells. The elongation factor 1B gene appears to be co-regulated with the ribosomal protein genes both in terms of its transcription rate and mRNA accumulation. Dictyostelium has been a popular model for understanding signal transduction and the growth to differentiation transition, thus it is of significance that the regulation of ribosome biosynthesis in Dictyostelium resembles that of higher eukaryotes in being regulated largely at the post-transcriptional level in response to starvation as opposed to yeasts where the regulation is largely transcriptional [27]. PMID:10374261

  7. Improved elongation factor-1 alpha-based vectors for stable high-level expression of heterologous proteins in Chinese hamster ovary cells

    PubMed Central

    2014-01-01

    Background Establishing highly productive clonal cell lines with constant productivity over 2–3 months of continuous culture remains a tedious task requiring the screening of tens of thousands of clonal colonies. In addition, long-term cultivation of many candidate lines derived in the absence of drug selection pressure is necessary. Expression vectors based on the elongation factor-1 alpha (EEF1A) gene and the dihydrofolate reductase (DHFR) selection marker (with separate promoters) can be used to obtain highly productive populations of stably transfected cells in the selection medium, but they have not been tested for their ability to support target gene amplification under gradually increasing methotrexate pressure. Results We have modified EEF1A-based vectors by linking the DHFR selection marker to the target gene in the bicistronic RNA, shortening the overall plasmid size, and adding an Epstein-Barr virus terminal repeat fragment (EBVTR) element. Presence of the EBVTR element increased the rate of stable transfection by the plasmid by 24 times that of the EBVTR-minus control and improved the rate of methotrexate-driven gene amplification. The mean expression level of the enhanced green fluorescent protein (eGFP) used herein as a model protein, increased up to eight-fold using a single round of amplification in the case of adherent colonies formation and up to 4.5-fold in the case of suspension polyclonal cultures. Several eGFP-expressing cell populations produced using vectors with antibiotic resistance markers instead of the DHFR marker were compared with each other. Stable transfection of Chinese hamster ovary (CHO) DG44 cells by the p1.2-Hygro-eGFP plasmid (containing a hygromycin resistance marker) generated highest eGFP expression levels of up to 8.9% of the total cytoplasmic protein, with less than 5% of the cell population being eGFP-negative. Conclusions The p1.1 vector was very effective for stable transfection of CHO cells and capable of rapid MTX

  8. Knockdown of Selenocysteine-Specific Elongation Factor in Amblyomma maculatum Alters the Pathogen Burden of Rickettsia parkeri with Epigenetic Control by the Sin3 Histone Deacetylase Corepressor Complex

    PubMed Central

    Adamson, Steven W.; Browning, Rebecca E.; Budachetri, Khemraj; Ribeiro, José M. C.; Karim, Shahid

    2013-01-01

    Selenocysteine is the 21st naturally-occurring amino acid. Selenoproteins have diverse functions and many remain uncharacterized, but they are typically associated with antioxidant activity. The incorporation of selenocysteine into the nascent polypeptide chain recodes the TGA stop codon and this process depends upon a number of essential factors including the selenocysteine elongation factor (SEF). The transcriptional expression of SEF did not change significantly in tick midguts throughout the blood meal, but decreased in salivary glands to 20% at the end of the fast feeding phase. Since selenoprotein translation requires this specialized elongation factor, we targeted this gene for knockdown by RNAi to gain a global view of the role selenoproteins play in tick physiology. We found no significant differences in tick engorgement and embryogenesis but detected no antioxidant capacity in tick saliva. The transcriptional profile of selenoproteins in R. parkeri-infected Amblyomma maculatum revealed declined activity of selenoprotein M and catalase and increased activity of selenoprotein O, selenoprotein S, and selenoprotein T. Furthermore, the pathogen burden was significantly altered in SEF-knockdowns. We then determined the global impact of SEF-knockdown by RNA-seq, and mapped huge shifts in secretory gene expression that could be the result of downregulation of the Sin3 histone deacetylase corepressor complex. PMID:24282621

  9. Ubiquitin fusion constructs allow the expression and purification of multi-KOW domain complexes of the Saccharomyces cerevisiae transcription elongation factor Spt4/5.

    PubMed

    Blythe, Amanda; Gunasekara, Sanjika; Walshe, James; Mackay, Joel P; Hartzog, Grant A; Vrielink, Alice

    2014-08-01

    Spt4/5 is a hetero-dimeric transcription elongation factor that can both inhibit and promote transcription elongation by RNA polymerase II (RNAPII). However, Spt4/5's mechanism of action remains elusive. Spt5 is an essential protein and the only universally-conserved RNAP-associated transcription elongation factor. The protein contains multiple Kyrpides, Ouzounis and Woese (KOW) domains. These domains, in other proteins, are thought to bind RNA although there is little direct evidence in the literature to support such a function in Spt5. This could be due, at least in part, to difficulties in expressing and purifying recombinant Spt5. When expressed in Escherichia coli (E. coli), Spt5 is innately insoluble. Here we report a new approach for the successful expression and purification of milligram quantities of three different multi-KOW domain complexes of Saccharomyces cerevisiae Spt4/5 for use in future functional studies. Using the E. coli strain Rosetta2 (DE3) we have developed strategies for co-expression of Spt4 and multi-KOW domain Spt5 complexes from the bi-cistronic pET-Duet vector. In a second strategy, Spt4/5 was expressed via co-transformation of Spt4 in the vector pET-M11 with Spt5 ubiquitin fusion constructs in the vector pHUE. We characterized the multi-KOW domain Spt4/5 complexes by Western blot, limited proteolysis, circular dichroism, SDS-PAGE and size exclusion chromatography-multiangle light scattering and found that the proteins are folded with a Spt4:Spt5 hetero-dimeric stoichiometry of 1:1. These expression constructs encompass a larger region of Spt5 than has previously been reported, and will provide the opportunity to elucidate the biological function of the multi-KOW containing Spt5. PMID:24859675

  10. Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells

    PubMed Central

    Xie, Chuan-Ming; Liu, Xiao-Yu; Sham, Kathy WY; Lai, Josie MY; Cheng, Christopher HK

    2014-01-01

    EEF2K (eukaryotic elongation factor-2 kinase), also known as Ca2+/calmodulin-dependent protein kinase III, functions in downregulating peptide chain elongation through inactivation of EEF2 (eukaryotic translation elongation factor 2). Currently, there is a limited amount of information on the promotion of autophagic survival by EEF2K in breast and glioblastoma cell lines. However, the precise role of EEF2K in carcinogenesis as well as the underlying mechanism involved is still poorly understood. In this study, contrary to the reported autophagy-promoting activity of EEF2K in certain cancer cells, EEF2K is shown to negatively regulate autophagy in human colon cancer cells as indicated by the increase of LC3-II levels, the accumulation of LC3 dots per cell, and the promotion of autophagic flux in EEF2K knockdown cells. EEF2K negatively regulates cell viability, clonogenicity, cell proliferation, and cell size in colon cancer cells. Autophagy induced by EEF2K silencing promotes cell survival and does not potentiate the anticancer efficacy of the AKT inhibitor MK-2206. In addition, autophagy induced by silencing of EEF2K is attributed to induction of protein synthesis and activation of the AMPK-ULK1 pathway, independent of the suppression of MTOR activity and ROS generation. Knockdown of AMPK or ULK1 significantly abrogates EEF2K silencing-induced increase of LC3-II levels, accumulation of LC3 dots per cell as well as cell proliferation in colon cancer cells. In conclusion, silencing of EEF2K promotes autophagic survival via activation of the AMPK-ULK1 pathway in colon cancer cells. This finding suggests that upregulation of EEF2K activity may constitute a novel approach for the treatment of human colon cancer. PMID:24955726

  11. The transcription elongation factor NusA is required for stress-induced mutagenesis in Escherichia coli.

    PubMed

    Cohen, Susan E; Walker, Graham C

    2010-01-12

    Stress-induced mutagenesis describes the accumulation of mutations that occur in nongrowing cells, in contrast to mutagenesis that occurs in actively dividing populations, and has been referred to as stationary-phase or adaptive mutagenesis. The most widely studied system for stress-induced mutagenesis involves monitoring the appearance of Lac(+) revertants of the strain FC40 under starvation conditions in Escherichia coli. The SOS-inducible translesion DNA polymerase DinB plays an important role in this phenomenon. Loss of DinB (DNA pol IV) function results in a severe reduction of Lac(+) revertants. We previously reported that NusA, an essential component of elongating RNA polymerases, interacts with DinB. Here we report our unexpected observation that wild-type NusA function is required for stress-induced mutagenesis. We present evidence that this effect is unlikely to be due to defects in transcription of lac genes but rather is due to an inability to adapt and mutate in response to environmental stress. Furthermore, we extended our analysis to the formation of stress-induced mutants in response to antibiotic treatment, observing the same striking abolition of mutagenesis under entirely different conditions. Our results are the first to implicate NusA as a crucial participant in the phenomenon of stress-induced mutagenesis. PMID:20036541

  12. The transcription elongation factor ELL2 is specifically upregulated in HTLV-1-infected T-cells and is dependent on the viral oncoprotein Tax

    SciTech Connect

    Mann, Melanie C. Strobel, Sarah Fleckenstein, Bernhard Kress, Andrea K.

    2014-09-15

    The oncoprotein Tax of human T-cell leukemia virus type 1 (HTLV-1) is a potent transactivator of viral and cellular transcription. Here, we identified ELL2 as the sole transcription elongation factor to be specifically upregulated in HTLV-1-/Tax-transformed T-cells. Tax contributes to regulation of ELL2, since transient transfection of Tax increases ELL2 mRNA, Tax transactivates the ELL2 promoter, and repression of Tax results in decrease of ELL2 in transformed T-lymphocytes. However, we also measured upregulation of ELL2 in HTLV-1-transformed cells exhibiting undetectable amounts of Tax, suggesting that ELL2 can still be maintained independent of continuous Tax expression. We further show that Tax and ELL2 synergistically activate the HTLV-1 promoter, indicating that ELL2 cooperates with Tax in viral transactivation. This is supported by our findings that Tax and ELL2 accumulate in nuclear fractions and that they co-precipitate upon co-expression in transiently-transfected cells. Thus, upregulation of ELL2 could contribute to HTLV-1 gene regulation. - Highlights: • ELL2, a transcription elongation factor, is upregulated in HTLV-1-positive T-cells. • Tax transactivates the ELL2 promoter. • Tax and ELL2 synergistically activate the HTLV-1 promoter. • Tax and ELL2 interact in vivo.

  13. The Ability of Positive Transcription Elongation Factor b To Transactivate Human Immunodeficiency Virus Transcription Depends on a Functional Kinase Domain, Cyclin T1, and Tat

    PubMed Central

    Fujinaga, Koh; Cujec, Thomas P.; Peng, Junmin; Garriga, Judit; Price, David H.; Graña, Xavier; Peterlin, B. Matija

    1998-01-01

    By binding to the transactivation response element (TAR) RNA, the transcriptional transactivator (Tat) from the human immunodeficiency virus increases rates of elongation rather than initiation of viral transcription. Two cyclin-dependent serine/threonine kinases, CDK7 and CDK9, which phosphorylate the C-terminal domain of RNA polymerase II, have been implicated in Tat transactivation in vivo and in vitro. In this report, we demonstrate that CDK9, which is the kinase component of the positive transcription elongation factor b (P-TEFb) complex, can activate viral transcription when tethered to the heterologous Rev response element RNA via the regulator of expression of virion proteins (Rev). The kinase activity of CDK9 and cyclin T1 is essential for these effects. Moreover, P-TEFb binds to TAR only in the presence of Tat. We conclude that Tat–P-TEFb complexes bind to TAR, where CDK9 modifies RNA polymerase II for the efficient copying of the viral genome. PMID:9696809

  14. The Caenorhabditis elegans Protein FIC-1 Is an AMPylase That Covalently Modifies Heat-Shock 70 Family Proteins, Translation Elongation Factors and Histones

    PubMed Central

    Truttmann, Matthias C.; Guo, Xuanzong; Engert, Christoph; Schwartz, Thomas U.; Ploegh, Hidde L.

    2016-01-01

    Protein AMPylation by Fic domain-containing proteins (Fic proteins) is an ancient and conserved post-translational modification of mostly unexplored significance. Here we characterize the Caenorhabditis elegans Fic protein FIC-1 in vitro and in vivo. FIC-1 is an AMPylase that localizes to the nuclear surface and modifies core histones H2 and H3 as well as heat shock protein 70 family members and translation elongation factors. The three-dimensional structure of FIC-1 is similar to that of its human ortholog, HYPE, with 38% sequence identity. We identify a link between FIC-1-mediated AMPylation and susceptibility to the pathogen Pseudomonas aeruginosa, establishing a connection between AMPylation and innate immunity in C. elegans. PMID:27138431

  15. Higher-level phylogeny of the Therevidae (Diptera: insecta) based on 28S ribosomal and elongation factor-1 alpha gene sequences.

    PubMed

    Yang, L; Wiegmann, B M; Yeates, D K; Irwin, M E

    2000-06-01

    Therevidae (stilleto flies) are a little-known family of asiloid brachyceran Diptera (Insecta). Separate and combined phylogenetic analyses of 1200 bases of the 28S ribosomal DNA and 1100 bases of elongation factor-1alpha were used to infer phylogenetic relationships within the family. The position of the enigmatic taxon Apsilocephala Kröber is evaluated in light of the molecular evidence. In all analyses, molecular data strongly support the monophyly of Therevidae, excluding Apsilocephala, and the division of Therevidae into two main clades corresponding to a previous classification of the family into the subfamilies Phycinae and Therevinae. Despite strong support for some relationships within these groups, relationships at the base of the two main clades are weakly supported. Short branch lengths for Australasian clades at the base of the Therevinae may represent a rapid radiation of therevids in Australia. PMID:10860652

  16. The Caenorhabditis elegans Protein FIC-1 Is an AMPylase That Covalently Modifies Heat-Shock 70 Family Proteins, Translation Elongation Factors and Histones.

    PubMed

    Truttmann, Matthias C; Cruz, Victor E; Guo, Xuanzong; Engert, Christoph; Schwartz, Thomas U; Ploegh, Hidde L

    2016-05-01

    Protein AMPylation by Fic domain-containing proteins (Fic proteins) is an ancient and conserved post-translational modification of mostly unexplored significance. Here we characterize the Caenorhabditis elegans Fic protein FIC-1 in vitro and in vivo. FIC-1 is an AMPylase that localizes to the nuclear surface and modifies core histones H2 and H3 as well as heat shock protein 70 family members and translation elongation factors. The three-dimensional structure of FIC-1 is similar to that of its human ortholog, HYPE, with 38% sequence identity. We identify a link between FIC-1-mediated AMPylation and susceptibility to the pathogen Pseudomonas aeruginosa, establishing a connection between AMPylation and innate immunity in C. elegans. PMID:27138431

  17. Crystallization and preliminary X-ray analysis of the mRNA-binding domain of elongation factor SelB from Escherichia coli in complex with RNA

    SciTech Connect

    Soler, Nicolas; Fourmy, Dominique; Yoshizawa, Satoko

    2007-05-01

    The mRNA-binding domain of E. coli selenocysteine-specific elongation factor SelB (residues 478–614; SelB-WH3/4) was overproduced in E. coli and its cognate mRNA ligand, 23 nucleotides of the SECIS RNA hairpin, was prepared by in vitro transcription. The purified SelB-WH3/4–SECIS RNA complex crystallized in space group C2 and diffracted to 2.3 Å. In bacteria, selenocysteine (the 21st amino acid) is incorporated into proteins via machinery that includes SelB, a specific translational elongation factor. SelB binds to an mRNA hairpin called the selenocysteine-insertion sequence (SECIS) and delivers selenocysteyl-tRNA{sup Sec} to the ribosomal A site. The minimum C-terminal fragment (residues 478–614) of Escherichia coli SelB (SelB-WH3/4) required for SECIS binding has been overexpressed and purified. This protein was crystallized in complex with 23 nucleotides of the SECIS hairpin at 294 K using the hanging-drop vapour-diffusion method. A data set was collected to 2.3 Å resolution from a single crystal at 100 K using ESRF beamline BM-30. The crystal belongs to space group C2, with unit-cell parameters a = 103.50, b = 56.51, c = 48.41 Å. The asymmetric unit contains one WH3/4-domain–RNA complex. The Matthews coefficient was calculated to be 3.37 Å{sup 3} Da{sup −1} and the solvent content was estimated to be 67.4%.

  18. Elongation factor 1Bgamma (eEF1Bgamma) expression during the molting cycle and cold acclimation in the crayfish Procambarus clarkii.

    PubMed

    Gillen, Christopher M; Gao, Yongping; Niehaus-Sauter, Margaret M; Wylde, Meredith R; Wheatly, Michele G

    2008-06-01

    Eukaryotic elongation factor 1Bgamma (eEF1Bgamma) is a subunit of elongation factor 1 (EF1), which regulates the recruitment of amino acyl-tRNAs to the ribosome during protein synthesis in eukaryotes. In addition to structural roles within eEF1, eEF1Bgamma has properties which suggest sensory or regulatory activities. We have cloned eEF1Bgamma from axial abdominal muscle of freshwater crayfish, Procambarus clarkii. The predicted amino acid sequence has 66% identity to Locusta migratoria eEF1Bgamma and 65% identity to Artemia salina eEF1Bgamma. We measured eEF1Bgamma expression by real-time PCR, using the relative quantification method with 18s ribosomal RNA as an internal calibrator. eEF1Bgamma expression was lowest in gill, axial abdominal muscle, and hepatopancreas, and was highest in the antennal gland (5.7-fold above hepatopancreas) and cardiac muscle (7.8-fold above hepatopancreas). In axial abdominal muscle, eEF1Bgamma expression was 4.4-fold higher in premolt and 11.9 higher in postmolt compared to intermolt. In contrast, eEF1Bgamma was decreased or unchanged in epithelial tissues during pre- and postmolt. eEF1Bgamma expression in the hepatopancreas was 3.5-fold higher during intermolt compared to premolt and was unchanged in gill and antennal gland. No significant differences in eEF1Bgamma were found after 1 week of acclimation to 4 degrees C. These results show that eEF1Bgamma is regulated at the mRNA level with tissue-specific differences in expression patterns. PMID:18407536

  19. Eukaryotic release factor 1-2 is involved in GA signaling pathway and regulates cell elongation in petioles

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Eukaryotic release factor 1 (eRF1) is responsible for recognition of the stop codons in mRNAs during protein synthesis. Accumulating evidence indicates that eRF1 functions in other processes in addition to translation termination. The physiological role of eRF1-2, a member of eRF1 family, was examin...

  20. Site of ADP-ribosylation and the RNA-binding site are situated in different domains of the elongation factor EF-2

    SciTech Connect

    Davydova, E.K.

    1987-01-01

    One of the proteins participating in the process of elongation of polypeptide chains - elongation factor 2 (EF-2) - can be ADP-ribosylated at a unique amino acid residue - diphthamide. Since the ADP-ribosylation of EF-2 at dipthamide leads to a loss of affinity of the factor for RNA while the presence of RNA inhibits the ADP-ribosylation reaction, it seemed probable to the authors that diphthamide participated directly in the binding of EF-2 to DNA. The experiments presented in this article showed that this was not the case: diphthamide and the RNA-binding site are situated on different domains of EF-2. Thus, ADP-ribosylation of factor EF-2 in one domain leads to a loss of the ability to bind to RNA in the other. The authors investigated the mutual arrangement of diphthamide and the RNA-binding site on the EF-2 molecule by preparing a factor from rabbit reticulocytes and subjecting it to proteolytic digestion with elastase. The factor was incubated with elastase for 15 min at 37/sup 0/C at an enzyme:substrate ratio of 1:100 in buffer solution containing 20 mM Tris-HCl, pH 7.6, 10 mM KCl, 1 mM MgCl/sub 2/, and 2 mM dithiothreitol. The reaction was stopped by adding para-methylsulfonyl fluoride to 50 micro-M. The authors obtained a preparation as a result of proteolysis and applied it on a column with RNA-Sepharose and separated into two fractions: RNA-binding and without affinity for RNA. The initial preparation and its fractions were subjected to exhaustive ADP-ribosylation in the presence of diphtheria toxin and (U-/sup 14/C) nicotinaide adenine dinucleotide ((/sup 14/C)NAD) (296 mCi/mmole). The samples were analyzed electrophoretically in a polyacrylamide gel gradient in the presence of sodium dodecyl sulfate. For the detection of (/sup 14/C) ADP-ribosylated components, the gels were dried and exposed with RM-V x-ray film.

  1. Elongation factor G-induced structural change in helix 34 of 16S rRNA related to translocation on the ribosome.

    PubMed Central

    Matassova, A B; Rodnina, M V; Wintermeyer, W

    2001-01-01

    During the translocation step of the elongation cycle, two tRNAs together with the mRNA move synchronously and rapidly on the ribosome. The movement is catalyzed by the binding of elongation factor G (EF-G) and driven by GTP hydrolysis. Here we study structural changes of the ribosome related to EF-G binding and translocation by monitoring the accessibility of ribosomal RNA (rRNA) for chemical modification by dimethyl sulfate or cleavage by hydroxyl radicals generated by Fe(II)-EDTA. In the state of the ribosome that is formed upon binding of EF-G but before the movement of the tRNAs takes place, residues 1054,1196, and 1201 in helix 34 in 16S rRNA are strongly protected. The protections depend on EF-G binding, but do not require GTP hydrolysis, and are lost upon translocation. Mutants of EF-G, which are active in ribosome binding and GTP hydrolysis but impaired in translocation, do not bring about the protections. According to cryo-electron microscopy (Stark et al., Cell, 2000, 100:301-309), there is no contact of EF-G with the protected residues of helix 34 in the pretranslocation state, suggesting that the observed protections are due to an induced conformational change. Thus, the present results indicate that EF-G binding to the pretranslocation ribosome induces a structural change of the head of the 30S subunit that is essential for subsequent tRNA-mRNA movement in translocation. PMID:11780642

  2. Direct observation of von Willebrand factor elongation and fiber formation on collagen during acute whole blood exposure to pathological flow

    PubMed Central

    Colace, T. V.; Diamond, S. L.

    2013-01-01

    Objective In severe stenosis, von Willebrand Factor (vWF) experiences millisecond exposures to pathological wall shear rates (γw). We sought to evaluate the deposition of vWF onto collagen surfaces under flow in these environments. Methods and Results Distinct from shear experiments that last many seconds, we deployed microfluidic devices for single-pass perfusion of whole blood or platelet free plasma (PFP) over fibrillar type 1 collagen (< 50 msec transit time) at pathological γw or spatial wall shear rate gradient (grad γw). Using fluorescent anti-vWF, long thick vWF fibers (>20 μm) bound to collagen were visualized at constant γw > 30,000 s−1 during perfusion of PFP, a process enhanced by EDTA. Rapid acceleration or deceleration of EDTA-PFP at grad γw = ± 5.5 × 105 to 4.3 × 107 s−1/cm did not promote vWF deposition. At 19,400 s−1, EDTA-blood perfusion resulted in rolling vWF-platelet nets, while blood perfusion (normal Ca2+) generated large vWF/platelet deposits that repeatedly embolized and were blocked by anti-GPIb or the α IIbβ3 inhibitor GR144053 and did not require grad γw. Blood perfusion at venous shear rate (200 s−1) produced a stable platelet deposit that was a substrate for massive but unstable vWF-platelet aggregates when flow was increased to 7800 s−1. Conclusion Triggered by collagen and enhanced by platelet GPIb and α IIbβ3, vWF fiber formation occurred during acute exposures to pathological γw and did not require gradients in wall shear rate. PMID:23104847

  3. Depletion of elongation initiation factor 4E binding proteins by CRISPR/Cas9 enhances the antiviral response in porcine cells.

    PubMed

    Ramírez-Carvajal, Lisbeth; Singh, Neetu; de los Santos, Teresa; Rodríguez, Luis L; Long, Charles R

    2016-01-01

    Type I interferons (IFNs) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor 4E binding proteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF-7), the "master regulator" of IFN transcription. Previous studies have suggested that mouse cells depleted of 4E-BPs are more sensitive to IFNβ treatment and had lower viral loads as compared to wild type (WT) cells. However, such approach has not been tested as an antiviral strategy in livestock species. In this study, we tested the antiviral activity of porcine cells depleted of 4E-BP1 by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome engineering system. We found that 4E-BP1 knockout (KO) porcine cells had increased expression of IFNα and β, IFN stimulated genes, and significant reduction in vesicular stomatitis virus titer as compare to WT cells. No phenotypical changes associated with CRISPR/Cas9 manipulation were observed in 4E-BP1 KO cells. This work highlights the use of the CRISPR/Cas9 system to enhance the antiviral response in porcine cells. PMID:26592975

  4. Identification and Characterization of a Novel Evolutionarily Conserved Lysine-specific Methyltransferase Targeting Eukaryotic Translation Elongation Factor 2 (eEF2)*

    PubMed Central

    Davydova, Erna; Ho, Angela Y. Y.; Malecki, Jedrzej; Moen, Anders; Enserink, Jorrit M.; Jakobsson, Magnus E.; Loenarz, Christoph; Falnes, Pål Ø.

    2014-01-01

    The components of the cellular protein translation machinery, such as ribosomal proteins and translation factors, are subject to numerous post-translational modifications. In particular, this group of proteins is frequently methylated. However, for the majority of these methylations, the responsible methyltransferases (MTases) remain unknown. The human FAM86A (family with sequence similarity 86) protein belongs to a recently identified family of protein MTases, and we here show that FAM86A catalyzes the trimethylation of eukaryotic elongation factor 2 (eEF2) on Lys-525. Moreover, we demonstrate that the Saccharomyces cerevisiae MTase Yjr129c, which displays sequence homology to FAM86A, is a functional FAM86A orthologue, modifying the corresponding residue (Lys-509) in yeast eEF2, both in vitro and in vivo. Finally, Yjr129c-deficient yeast cells displayed phenotypes related to eEF2 function (i.e. increased frameshifting during protein translation and hypersensitivity toward the eEF2-specific drug sordarin). In summary, the present study establishes the function of the previously uncharacterized MTases FAM86A and Yjr129c, demonstrating that these enzymes introduce a functionally important lysine methylation in eEF2. Based on the previous naming of similar enzymes, we have redubbed FAM86A and Yjr129c as eEF2-KMT and Efm3, respectively. PMID:25231979

  5. What doesn’t kill them makes them stronger: an association between elongation factor 1-α overdominance in the sea star Pisaster ochraceus and “sea star wasting disease”

    PubMed Central

    Schiebelhut, Lauren M.

    2016-01-01

    In recent years, a massive mortality event has killed millions of sea stars, of many different species, along the Pacific coast of North America. This disease event, known as ‘sea star wasting disease’ (SSWD), is linked to viral infection. In one affected sea star (Pisaster ochraceus), previous work had identified that the elongation factor 1-α locus (EF1A) harbored an intronic insertion allele that is lethal when homozygous yet appears to be maintained at moderate frequency in populations through increased fitness for heterozygotes. The environmental conditions supporting this increased fitness are unknown, but overdominance is often associated with disease. Here, we evaluate populations of P. ochraceus to identify the relationship between SSWD and EF1A genotype. Our data suggest that there may be significantly decreased occurrence of SSWD in individuals that are heterozygous at this locus. These results suggest further studies are warranted to understand the functional relationship between diversity at EF1A and survival in P. ochraceus. PMID:27069810

  6. Auxin and Cellular Elongation.

    PubMed

    Velasquez, Silvia Melina; Barbez, Elke; Kleine-Vehn, Jürgen; Estevez, José M

    2016-03-01

    Auxin is a crucial growth regulator in plants. However, a comprehensive understanding of how auxin induces cell expansion is perplexing, because auxin acts in a concentration- and cell type-dependent manner. Consequently, it is desirable to focus on certain cell types to exemplify the underlying growth mechanisms. On the other hand, plant tissues display supracellular growth (beyond the level of single cells); hence, other cell types might compromise the growth of a certain tissue. Tip-growing cells do not display neighbor-induced growth constraints and, therefore, are a valuable source of information for growth-controlling mechanisms. Here, we focus on auxin-induced cellular elongation in root hairs, exposing a mechanistic view of plant growth regulation. We highlight a complex interplay between auxin metabolism and transport, steering root hair development in response to internal and external triggers. Auxin signaling modules and downstream cascades of transcription factors define a developmental program that appears rate limiting for cellular growth. With this knowledge in mind, the root hair cell is a very suitable model system in which to dissect cellular effectors required for cellular expansion. PMID:26787325

  7. 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

  8. Chloroplast Elongation Factor Ts Pro-Protein Is an Evolutionarily Conserved Fusion with the S1 Domain-Containing Plastid-Specific Ribosomal Protein-7

    PubMed Central

    Beligni, María Verónica; Yamaguchi, Kenichi; Mayfield, Stephen P.

    2004-01-01

    The components of chloroplast translation are similar to those of prokaryotic translation but contain some additional unique features. Proteomic analysis of the Chlamydomonas reinhardtii chloroplast ribosome identified an S1-like protein, plastid-specific ribosomal protein-7 (PSRP-7), as a stoichiometric component of the 30S subunit. Here, we report that PSRP-7 is part of a polyprotein that contains PSRP-7 on its amino end and two translation elongation factor Ts (EF-Ts) domains at the carboxy end. We named this polyprotein PETs (for polyprotein of EF-Ts). Pets is a single-copy gene containing the only chloroplast PSRP-7 and EF-Ts sequences found in the C. reinhardtii genome. The pets precursor transcript undergoes alternative splicing to generate three mRNAs with open reading frames (ORFs) of 1.68, 1.8, and 3 kb. A 110-kD pro-protein is translated from the 3-kb ORF, and the majority of this protein is likely posttranslationally processed into the 65-kD protein PSRP-7 and a 55-kD EF-Ts. PETs homologs are found in Arabidopsis thaliana and rice (Oryza sativa). The conservation of the 110-kD PETs polyprotein in the plant kingdom suggests that PSRP-7 and EF-Ts function together in some aspects of chloroplast translation and that the PETs pro-protein may have a novel function as a whole. PMID:15548736

  9. Molecular cloning of the black tiger shrimp (Penaeus monodon) elongation factor 2 (EF-2): sequence analysis and its expression on the ovarian maturation stage.

    PubMed

    Qiu, Lihua; Jiang, Shigui; Zhou, Falin; Zhang, Dianchang; Huang, Jianhua; Guo, Yihui

    2008-09-01

    The techniques of homology cloning and anchored PCR were used to clone the elongation factor 2 (EF-2) gene from black tiger shrimp (Penaeus monodon). The full length cDNA of black tiger shrimp EF-2 (btsEF-2) contained a 5' untranslated region (UTR) of 73 bp, an ORF of 2541 bp encoding a polypeptide of 846 amino acids with an estimated molecular mass of 95 kDa, and a 3( UTR of 112 bp. The searches for protein sequence similarities with BLAST analysis indicated that the deduced amino acid sequence of btsEF-2 was homological to the EF-2 of other species and even the mammalians. The conserved signature sequence of EF-2 gene family, GTPase effector domain and ADP-ribosylation domain were found in the btsEF-2 deduced amino acid sequence. The temporal expressions of gene in the different ovarian stages were measured by real time PCR. The mRNA expressions of the gene were constitutively expressed in ovary and different during the maturation stages. The result indicated that EF-2 gene was constitutively expressed and could play a critical role in the ovarian maturation stage. PMID:17629788

  10. BnEPFL6, an EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) secreted peptide gene, is required for filament elongation in Brassica napus.

    PubMed

    Huang, Yi; Tao, Zhangsheng; Liu, Qiong; Wang, Xinfa; Yu, Jingyin; Liu, Guihua; Wang, Hanzhong

    2014-07-01

    Inflorescence architecture, pedicel length and stomata patterning in Arabidopsis thaliana are specified by inter-tissue communication mediated by ERECTA and its signaling ligands in the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family of secreted cysteine-rich peptides. Here, we identified and characterized BnEPFL6 from Brassica napus. Heterologous expression of this gene under the double enhanced CaMV promoter (D35S) in Arabidopsis resulted in shortened stamen filaments, filaments degradation, and reduced filament cell size that displayed down-regulated expression of AHK2, in which phenotypic variation of ahk2-1 mutant presented highly consistent with that of BnEPFL6 transgenic lines. Especially, the expression level of BnEPFL6 in the shortened filaments of four B. napus male sterile lines (98A, 86A, SA, and Z11A) was similar to that of BnEPFL6 in the transgenic Arabidopsis lines. The activity of pBnEPFL6.2::GUS was intensive in the filaments of transgenic lines. These observations reveal that BnEPFL6 plays an important role in filament elongation and may also affect organ morphology and floral organ specification via a BnEPFL6-mediated cascade. PMID:24838654

  11. Interaction of helix D of elongation factor Tu with helices 4 and 5 of protein L7/12 on the ribosome.

    PubMed

    Kothe, Ute; Wieden, Hans-Joachim; Mohr, Dagmar; Rodnina, Marina V

    2004-03-01

    Elongation factor Tu (EF-Tu) promotes binding of aminoacyl-tRNA to the A site of the ribosome. Here, we report the effects of mutations in helix D of EF-Tu and in the C-terminal domain of L7/12 on the kinetics of A-site binding. Reaction rates were measured by stopped-flow and quench-flow techniques. The rates of A-site binding were decreased by mutations at positions 144, 145, 148, and 152 in helix D of EF-Tu as well as at positions 65, 66, 69, 70, 73, and 84 in helices 4 and 5 of L7/12. The effect was due primarily to the lower association rate constant of ternary complex binding to the ribosome. These results suggest that helix D of EF-Tu is involved in an initial transient contact with helices 4 and 5 of L7/12 that promotes ternary complex binding to the ribosome. By analogy to the interaction of helix D of EF-Tu with the N-terminal domain of EF-Ts, the contact area is likely to consist of a hydrophobic patch flanked by two salt-bridges. PMID:15037065

  12. Glucocorticoids Induce Cardiac Fibrosis via Mineralocorticoid Receptor in Oxidative Stress: Contribution of Elongation Factor Eleven-Nineteen Lysine-Rich Leukemia (ELL)

    PubMed Central

    Omori, Yosuke; Mano, Toshiaki; Ohtani, Tomohito; Sakata, Yasushi; Takeda, Yasuharu; Tamaki, Shunsuke; Tsukamoto, Yasumasa; Miwa, Takeshi; Yamamoto, Kazuhiro; Komuro, Issei

    2014-01-01

    Background Cardiac fibrosis is considered to be a crucial factor in the development of heart failure. Blockade of the mineralocorticoid receptor (MR) attenuated cardiac fibrosis and improved the prognosis of patients with chronic heart failure but the ligand for MR and the regulatory mechanism of MR pathway in the diseased heart are unclear. Here, we investigated whether glucocorticoids can promote cardiac fibrosis through MR in oxidative stress and the involvement of elongation factor eleven-nineteen lysine-rich leukemia (ELL), a co-activator of MR, in this pathway. Methods and Results The MR antagonist eplerenone attenuated corticosterone-induced collagen synthesis assessed by [3H]proline incorporation in rat neonatal cultured cardiac fibroblasts in the presence of H2O2, as an oxidative stress but not in the absence of H2O2. H2O2 increased the ELL expression levels and MR-bound ELL. ELL expression levels and MR-bound ELL were also increased in the left ventricle of heart failure model rats with significant fibrosis and enhanced oxidative stress. Eplerenone did not attenuate corticosterone-induced increase of [3H]proline incorporation in the presence of H2O2 after knockdown of ELL expression using small interfering RNA in cardiac fibroblasts. Conclusion Glucocorticoids can promote cardiac fibrosis via MR in oxidative stress, and oxidative stress modulates MR response to glucocorticoids through the interaction with ELL. Preventing cardiac fibrosis by modulating glucocorticoid-MR-ELL pathway may become a new therapeutic strategy for heart failure. PMID:25349466

  13. Function of the Bacillus subtilis transcription elongation factor NusG in hairpin-dependent RNA polymerase pausing in the trp leader.

    PubMed

    Yakhnin, Alexander V; Yakhnin, Helen; Babitzke, Paul

    2008-10-21

    NusA and NusG are transcription elongation factors that bind to RNA polymerase (RNAP) after sigma subunit release. Escherichia coli NusA (NusA(Ec)) stimulates intrinsic termination and RNAP(Ec) pausing, whereas NusG(Ec) promotes Rho-dependent termination and pause escape. Both Nus factors also participate in the formation of RNAP(Ec) antitermination complexes. We showed that Bacillus subtilis NusA (NusA(Bs)) stimulates intrinsic termination and RNAP(Bs) pausing at U107 and U144 in the trpEDCFBA operon leader. Pausing at U107 and U144 participates in the transcription attenuation and translational control mechanisms, respectively, presumably by providing additional time for trp RNA-binding attenuation protein (TRAP) to bind to the nascent trp leader transcript. Here, we show that NusG(Bs) causes modest pause stimulation at U107 and dramatic pause stimulation at U144. NusA(Bs) and NusG(Bs) act synergistically to increase the U107 and U144 pause half-lives. NusG(Bs)-stimulated pausing at U144 requires RNAP(Bs), whereas NusA(Bs) stimulates pausing of RNAP(Bs) and RNAP(Ec) at the U144 and E. coli his pause sites. Although NusG(Ec) does not stimulate pausing at U144, it competes with NusG(Bs)-stimulated pausing, suggesting that both proteins bind to the same surface of RNAP(Bs). Inactivation of nusG results in the loss of RNAP pausing at U144 in vivo and elevated trp operon expression, whereas plasmid-encoded NusG complements the mutant defects. Overexpression of nusG reduces trp operon expression to a larger extent than overexpression of nusA. PMID:18852477

  14. FOXM1 regulates expression of eukaryotic elongation factor 2 kinase and promotes proliferation, invasion and tumorgenesis of human triple negative breast cancer cells

    PubMed Central

    Hamurcu, Zuhal; Ashour, Ahmed; Kahraman, Nermin; Ozpolat, Bulent

    2016-01-01

    Eukaryotic elongation factor 2 kinase (eEF2K), an emerging molecular target for cancer therapy, contributes to cancer proliferation, cell survival, tumorigenesis, and invasion, disease progression and drug resistance. Although eEF2K is highly up-regulated in various cancers, the mechanism of gene regulation has not been elucidated. In this study, we examined the role of Forkhead Box M1 (FOXM1) proto-oncogenic transcription factor in triple negative breast cancer (TNBC) cells and the regulation of eEF2K. We found that FOXM1 is highly upregulated in TNBC and its knockdown by RNA interference (siRNA) significantly inhibited eEF2K expression and suppressed cell proliferation, colony formation, migration, invasion and induced apoptotic cell death, recapitulating the effects of eEF2K inhibition. Knockdown of FOXM1 inhibited regulators of cell cycle, migration/invasion and survival, including cyclin D1, Src and MAPK-ERK signaling pathways, respectively. We also demonstrated that FOXM1 (1B and 1C isoforms) directly binds to and transcriptionally regulates eEF2K gene expression by chromatin immunoprecipitation (ChIP) and luciferase gene reporter assays. Furthermore, in vivo inhibition of FOXM1 by liposomal siRNA-nanoparticles suppressed growth of MDA-MB-231 TNBC tumor xenografts in orthotopic models. In conclusion, our study provides the first evidence about the transcriptional regulation of eEF2K in TNBC and the role of FOXM1 in mediating breast cancer cell proliferation, survival, migration/invasion, progression and tumorgenesis and highlighting the potential of FOXM1/eEF2K axis as a molecular target in breast and other cancers. PMID:26918606

  15. The submergence tolerance regulator Sub1A mediates stress-responsive expression of AP2/ERF transcription factors.

    PubMed

    Jung, Ki-Hong; Seo, Young-Su; Walia, Harkamal; Cao, Peijian; Fukao, Takeshi; Canlas, Patrick E; Amonpant, Fawn; Bailey-Serres, Julia; Ronald, Pamela C

    2010-03-01

    We previously characterized the rice (Oryza sativa) Submergence1 (Sub1) locus encoding three ethylene-responsive factor (ERF) transcriptional regulators. Genotypes carrying the Sub1A-1 allele are tolerant of prolonged submergence. To elucidate the mechanism of Sub1A-1-mediated tolerance, we performed transcriptome analyses comparing the temporal submergence response of Sub1A-1-containing tolerant M202(Sub1) with the intolerant isoline M202 lacking this gene. We identified 898 genes displaying Sub1A-1-dependent regulation. Integration of the expression data with publicly available metabolic pathway data identified submergence tolerance-associated pathways governing anaerobic respiration, hormone responses, and antioxidant systems. Of particular interest were a set of APETALA2 (AP2)/ERF family transcriptional regulators that are associated with the Sub1A-1-mediated response upon submergence. Visualization of expression patterns of the AP2/ERF superfamily members in a phylogenetic context resolved 12 submergence-regulated AP2/ERFs into three putative functional groups: (1) anaerobic respiration and cytokinin-mediated delay in senescence via ethylene accumulation during submergence (three ERFs); (2) negative regulation of ethylene-dependent gene expression (five ERFs); and (3) negative regulation of gibberellin-mediated shoot elongation (four ERFs). These results confirm that the presence of Sub1A-1 impacts multiple pathways of response to submergence. PMID:20107022

  16. Genome-Wide Analysis of Factors Affecting Transcription Elongation and DNA Repair: A New Role for PAF and Ccr4-Not in Transcription-Coupled Repair

    PubMed Central

    Gaillard, Hélène; Tous, Cristina; Botet, Javier; González-Aguilera, Cristina; Quintero, Maria José; Viladevall, Laia; García-Rubio, María L.; Rodríguez-Gil, Alfonso; Marín, Antonio; Ariño, Joaquín; Revuelta, José Luis; Chávez, Sebastián; Aguilera, Andrés

    2009-01-01

    RNA polymerases frequently deal with a number of obstacles during transcription elongation that need to be removed for transcription resumption. One important type of hindrance consists of DNA lesions, which are removed by transcription-coupled repair (TC-NER), a specific sub-pathway of nucleotide excision repair. To improve our knowledge of transcription elongation and its coupling to TC-NER, we used the yeast library of non-essential knock-out mutations to screen for genes conferring resistance to the transcription-elongation inhibitor mycophenolic acid and the DNA-damaging agent 4-nitroquinoline-N-oxide. Our data provide evidence that subunits of the SAGA and Ccr4-Not complexes, Mediator, Bre1, Bur2, and Fun12 affect transcription elongation to different extents. Given the dependency of TC-NER on RNA Polymerase II transcription and the fact that the few proteins known to be involved in TC-NER are related to transcription, we performed an in-depth TC-NER analysis of a selection of mutants. We found that mutants of the PAF and Ccr4-Not complexes are impaired in TC-NER. This study provides evidence that PAF and Ccr4-Not are required for efficient TC-NER in yeast, unraveling a novel function for these transcription complexes and opening new perspectives for the understanding of TC-NER and its functional interconnection with transcription elongation. PMID:19197357

  17. Elongation Factor-1α Is a Novel Protein Associated with Host Cell Invasion and a Potential Protective Antigen of Cryptosporidium parvum *

    PubMed Central

    Matsubayashi, Makoto; Teramoto-Kimata, Isao; Uni, Shigehiko; Lillehoj, Hyun S.; Matsuda, Haruo; Furuya, Masaru; Tani, Hiroyuki; Sasai, Kazumi

    2013-01-01

    The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess an apical complex, a unique assembly of organelles localized to the anterior end of the cell and involved in host cell invasion. Previously, we generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina sporozoites. This antigen was highly conserved among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium. In the present study, we identified the apical cytoskeletal antigen of Cryptosporidium parvum (C. parvum) and further characterized this antigen in C. parvum to assess its potential as a target molecule against cryptosporidiosis. Indirect immunofluorescence demonstrated that the reactivity of 6D-12-G10 with C. parvum sporozoites was similar to those of anti-β- and anti-γ-tubulins antibodies. Immunoelectron microscopy with the 6D-12-G10 mAb detected the antigen both on the sporozoite surface and underneath the inner membrane at the apical region of zoites. The 6D-12-G10 mAb significantly inhibited in vitro host cell invasion by C. parvum. MALDI-TOF/MS and LC-MS/MS analysis of tryptic peptides revealed that the mAb 6D-12-G10 target antigen was elongation factor-1α (EF-1α). These results indicate that C. parvum EF-1α plays an essential role in mediating host cell entry by the parasite and, as such, could be a candidate vaccine antigen against cryptosporidiosis. PMID:24085304

  18. Molecular Control of the Amount, Subcellular Location and Activity State of Translation Elongation Factor 2 (eEF-2) in Neurons Experiencing Stress

    PubMed Central

    Argüelles-Castilla, Sandro; Camandola, Simonetta; Hutchison, Emmette R.; Cutler, Roy G.; Ayala, Antonio; Mattson, Mark P.

    2013-01-01

    Eukaryotic elongation factor 2 (eEF-2) is an important regulator of the protein translation machinery wherein it controls the movement of the ribosome along the mRNA. The activity of eEF-2 is regulated by changes in cellular energy status and nutrient availability, and posttranslational modifications such as phosphorylation and mono-ADP-ribosylation. However, the mechanisms regulating protein translation under conditions of cellular stress in neurons are unknown. Here we show that when rat hippocampal neurons experience oxidative stress (lipid peroxidation induced by exposure to cumene hydroperoxide; CH), eEF-2 is hyperphosphorylated and ribosylated resulting in reduced translational activity. The degradation of eEF-2 requires calpain proteolytic activity and is accompanied by accumulation of eEF-2 in the nuclear compartment. The subcellular localization of both native and phosphorylated forms of eEF-2 is influenced by CRM1 and 14.3.3, respectively. In hippocampal neurons p53 interacts with non-phosphorylated (active) eEF-2, but not with its phosphorylated form. The p53 – eEF-2 complexes are present in cytoplasm and nucleus, and their abundance increases when neurons experience oxidative stress. The nuclear localization of active eEF-2 depends upon its interaction with p53, as cells lacking p53 contain less active eEF-2 in the nuclear compartment. Overexpression of eEF-2 in hippocampal neurons results in increased nuclear levels of eEF-2, and decreased cell death following exposure to CH. Our results reveal novel molecular mechanisms controlling the differential subcellular localization and activity state of eEF-2 that may influence the survival status of neurons during periods of elevated oxidative stress. PMID:23542375

  19. Elongation Factor Tu and Heat Shock Protein 70 Are Membrane-Associated Proteins from Mycoplasma ovipneumoniae Capable of Inducing Strong Immune Response in Mice.

    PubMed

    Jiang, Fei; He, Jinyan; Navarro-Alvarez, Nalu; Xu, Jian; Li, Xia; Li, Peng; Wu, Wenxue

    2016-01-01

    Chronic non-progressive pneumonia, a disease that has become a worldwide epidemic has caused considerable loss to sheep industry. Mycoplasma ovipneumoniae (M. ovipneumoniae) is the causative agent of interstitial pneumonia in sheep, goat and bighorn. We here have identified by immunogold and immunoblotting that elongation factor Tu (EF-Tu) and heat shock protein 70 (HSP 70) are membrane-associated proteins on M. ovipneumonaiea. We have evaluated the humoral and cellular immune responses in vivo by immunizing BALB/c mice with both purified recombinant proteins rEF-Tu and rHSP70. The sera of both rEF-Tu and rHSP70 treated BALB/c mice demonstrated increased levels of IgG, IFN-γ, TNF-α, IL-12(p70), IL-4, IL-5 and IL-6. In addition, ELISPOT assay showed significant increase in IFN-γ+ secreting lymphocytes in the rHSP70 group when compared to other groups. Collectively our study reveals that rHSP70 induces a significantly better cellular immune response in mice, and may act as a Th1 cytokine-like adjuvant in immune response induction. Finally, growth inhibition test (GIT) of M. ovipneumoniae strain Y98 showed that sera from rHSP70 or rEF-Tu-immunized mice inhibited in vitro growth of M. ovipneumoniae. Our data strongly suggest that EF-Tu and HSP70 of M. ovipneumoniae are membrane-associated proteins capable of inducing antibody production, and cytokine secretion. Therefore, these two proteins may be potential candidates for vaccine development against M. ovipneumoniae infection in sheep. PMID:27537186

  20. A Cyclin T1 point mutation that abolishes positive transcription elongation factor (P-TEFb) binding to Hexim1 and HIV tat

    PubMed Central

    2014-01-01

    Background The positive transcription elongation factor b (P-TEFb) plays an essential role in activating HIV genome transcription. It is recruited to the HIV LTR promoter through an interaction between the Tat viral protein and its Cyclin T1 subunit. P-TEFb activity is inhibited by direct binding of its subunit Cyclin T (1 or 2) with Hexim (1 or 2), a cellular protein, bound to the 7SK small nuclear RNA. Hexim1 competes with Tat for P-TEFb binding. Results Mutations that impair human Cyclin T1/Hexim1 interaction were searched using systematic mutagenesis of these proteins coupled with a yeast two-hybrid screen for loss of protein interaction. Evolutionary conserved Hexim1 residues belonging to an unstructured peptide located N-terminal of the dimerization domain, were found to be critical for P-TEFb binding. Random mutagenesis of the N-terminal region of Cyclin T1 provided identification of single amino-acid mutations that impair Hexim1 binding in human cells. Furthermore, conservation of critical residues supported the existence of a functional Hexim1 homologue in nematodes. Conclusions Single Cyclin T1 amino-acid mutations that impair Hexim1 binding are located on a groove between the two cyclin folds and define a surface overlapping the HIV-1 Tat protein binding surface. One residue, Y175, in the centre of this groove was identified as essential for both Hexim1 and Tat binding to P-TEFb as well as for HIV transcription. PMID:24985203

  1. Elongation Factor Tu Prevents Misediting of Gly-tRNA(Gly) Caused by the Design Behind the Chiral Proofreading Site of D-Aminoacyl-tRNA Deacylase

    PubMed Central

    Routh, Satya Brata; Ahmad, Sadeem; Suma, Katta; Kumar, Mantu; Kuncha, Santosh Kumar; Yadav, Kranthikumar; Kruparani, Shobha P; Sankaranarayanan, Rajan

    2016-01-01

    D-aminoacyl-tRNA deacylase (DTD) removes D-amino acids mischarged on tRNAs and is thus implicated in enforcing homochirality in proteins. Previously, we proposed that selective capture of D-aminoacyl-tRNA by DTD’s invariant, cross-subunit Gly-cisPro motif forms the mechanistic basis for its enantioselectivity. We now show, using nuclear magnetic resonance (NMR) spectroscopy-based binding studies followed by biochemical assays with both bacterial and eukaryotic systems, that DTD effectively misedits Gly-tRNAGly. High-resolution crystal structure reveals that the architecture of DTD’s chiral proofreading site is completely porous to achiral glycine. Hence, L-chiral rejection is the only design principle on which DTD functions, unlike other chiral-specific enzymes such as D-amino acid oxidases, which are specific for D-enantiomers. Competition assays with elongation factor thermo unstable (EF-Tu) and DTD demonstrate that EF-Tu precludes Gly-tRNAGly misediting at normal cellular concentrations. However, even slightly higher DTD levels overcome this protection conferred by EF-Tu, thus resulting in significant depletion of Gly-tRNAGly. Our in vitro observations are substantiated by cell-based studies in Escherichia coli that show that overexpression of DTD causes cellular toxicity, which is largely rescued upon glycine supplementation. Furthermore, we provide direct evidence that DTD is an RNA-based catalyst, since it uses only the terminal 2′-OH of tRNA for catalysis without the involvement of protein side chains. The study therefore provides a unique paradigm of enzyme action for substrate selection/specificity by DTD, and thus explains the underlying cause of DTD’s activity on Gly-tRNAGly. It also gives a molecular and functional basis for the necessity and the observed tight regulation of DTD levels, thereby preventing cellular toxicity due to misediting. PMID:27224426

  2. Elongation Factor Tu Prevents Misediting of Gly-tRNA(Gly) Caused by the Design Behind the Chiral Proofreading Site of D-Aminoacyl-tRNA Deacylase.

    PubMed

    Routh, Satya Brata; Pawar, Komal Ishwar; Ahmad, Sadeem; Singh, Swati; Suma, Katta; Kumar, Mantu; Kuncha, Santosh Kumar; Yadav, Kranthikumar; Kruparani, Shobha P; Sankaranarayanan, Rajan

    2016-05-01

    D-aminoacyl-tRNA deacylase (DTD) removes D-amino acids mischarged on tRNAs and is thus implicated in enforcing homochirality in proteins. Previously, we proposed that selective capture of D-aminoacyl-tRNA by DTD's invariant, cross-subunit Gly-cisPro motif forms the mechanistic basis for its enantioselectivity. We now show, using nuclear magnetic resonance (NMR) spectroscopy-based binding studies followed by biochemical assays with both bacterial and eukaryotic systems, that DTD effectively misedits Gly-tRNAGly. High-resolution crystal structure reveals that the architecture of DTD's chiral proofreading site is completely porous to achiral glycine. Hence, L-chiral rejection is the only design principle on which DTD functions, unlike other chiral-specific enzymes such as D-amino acid oxidases, which are specific for D-enantiomers. Competition assays with elongation factor thermo unstable (EF-Tu) and DTD demonstrate that EF-Tu precludes Gly-tRNAGly misediting at normal cellular concentrations. However, even slightly higher DTD levels overcome this protection conferred by EF-Tu, thus resulting in significant depletion of Gly-tRNAGly. Our in vitro observations are substantiated by cell-based studies in Escherichia coli that show that overexpression of DTD causes cellular toxicity, which is largely rescued upon glycine supplementation. Furthermore, we provide direct evidence that DTD is an RNA-based catalyst, since it uses only the terminal 2'-OH of tRNA for catalysis without the involvement of protein side chains. The study therefore provides a unique paradigm of enzyme action for substrate selection/specificity by DTD, and thus explains the underlying cause of DTD's activity on Gly-tRNAGly. It also gives a molecular and functional basis for the necessity and the observed tight regulation of DTD levels, thereby preventing cellular toxicity due to misediting. PMID:27224426

  3. Structure of the Acinetobacter baumannii Dithiol Oxidase DsbA Bound to Elongation Factor EF-Tu Reveals a Novel Protein Interaction Site

    PubMed Central

    Premkumar, Lakshmanane; Kurth, Fabian; Duprez, Wilko; Grøftehauge, Morten K.; King, Gordon J.; Halili, Maria A.; Heras, Begoña; Martin, Jennifer L.

    2014-01-01

    The multidrug resistant bacterium Acinetobacter baumannii is a significant cause of nosocomial infection. Biofilm formation, that requires both disulfide bond forming and chaperone-usher pathways, is a major virulence trait in this bacterium. Our biochemical characterizations show that the periplasmic A. baumannii DsbA (AbDsbA) enzyme has an oxidizing redox potential and dithiol oxidase activity. We found an unexpected non-covalent interaction between AbDsbA and the highly conserved prokaryotic elongation factor, EF-Tu. EF-Tu is a cytoplasmic protein but has been localized extracellularly in many bacterial pathogens. The crystal structure of this complex revealed that the EF-Tu switch I region binds to the non-catalytic surface of AbDsbA. Although the physiological and pathological significance of a DsbA/EF-Tu association is unknown, peptides derived from the EF-Tu switch I region bound to AbDsbA with submicromolar affinity. We also identified a seven-residue DsbB-derived peptide that bound to AbDsbA with low micromolar affinity. Further characterization confirmed that the EF-Tu- and DsbB-derived peptides bind at two distinct sites. These data point to the possibility that the non-catalytic surface of DsbA is a potential substrate or regulatory protein interaction site. The two peptides identified in this work together with the newly characterized interaction site provide a novel starting point for inhibitor design targeting AbDsbA. PMID:24860094

  4. Elongation Factor Tu and Heat Shock Protein 70 Are Membrane-Associated Proteins from Mycoplasma ovipneumoniae Capable of Inducing Strong Immune Response in Mice

    PubMed Central

    Jiang, Fei; He, Jinyan; Navarro-Alvarez, Nalu; Xu, Jian; Li, Xia; Li, Peng; Wu, Wenxue

    2016-01-01

    Chronic non-progressive pneumonia, a disease that has become a worldwide epidemic has caused considerable loss to sheep industry. Mycoplasma ovipneumoniae (M. ovipneumoniae) is the causative agent of interstitial pneumonia in sheep, goat and bighorn. We here have identified by immunogold and immunoblotting that elongation factor Tu (EF-Tu) and heat shock protein 70 (HSP 70) are membrane-associated proteins on M. ovipneumonaiea. We have evaluated the humoral and cellular immune responses in vivo by immunizing BALB/c mice with both purified recombinant proteins rEF-Tu and rHSP70. The sera of both rEF-Tu and rHSP70 treated BALB/c mice demonstrated increased levels of IgG, IFN-γ, TNF-α, IL-12(p70), IL-4, IL-5 and IL-6. In addition, ELISPOT assay showed significant increase in IFN-γ+ secreting lymphocytes in the rHSP70 group when compared to other groups. Collectively our study reveals that rHSP70 induces a significantly better cellular immune response in mice, and may act as a Th1 cytokine-like adjuvant in immune response induction. Finally, growth inhibition test (GIT) of M. ovipneumoniae strain Y98 showed that sera from rHSP70 or rEF-Tu-immunized mice inhibited in vitro growth of M. ovipneumoniae. Our data strongly suggest that EF-Tu and HSP70 of M. ovipneumoniae are membrane-associated proteins capable of inducing antibody production, and cytokine secretion. Therefore, these two proteins may be potential candidates for vaccine development against M. ovipneumoniae infection in sheep. PMID:27537186

  5. Sulpiride, but not SCH23390, modifies cocaine-induced conditioned place preference and expression of tyrosine hydroxylase and elongation factor 1α in zebrafish.

    PubMed

    Darland, Tristan; Mauch, Justin T; Meier, Ellen M; Hagan, Shannon J; Dowling, John E; Darland, Diane C

    2012-12-01

    Finding genetic polymorphisms and mutations linked to addictive behavior can provide important targets for pharmaceutical and therapeutic interventions. Forward genetic approaches in model organisms such as zebrafish provide a potentially powerful avenue for finding new target genes. In order to validate this use of zebrafish, the molecular nature of its reward system must be characterized. We have previously reported the use of cocaine-induced conditioned place preference (CPP) as a reliable method for screening mutagenized fish for defects in the reward pathway. Here we test if CPP in zebrafish involves the dopaminergic system by co-treating fish with cocaine and dopaminergic antagonists. Sulpiride, a potent D2 receptor (DR2) antagonist, blocked cocaine-induced CPP, while the D1 receptor (DR1) antagonist SCH23390 had no effect. Acute cocaine exposure also induced a rise in the expression of tyrosine hydroxylase (TH), an important enzyme in dopamine synthesis, and a significant decrease in the expression of elongation factor 1α (EF1α), a housekeeping gene that regulates protein synthesis. Cocaine selectively increased the ratio of TH/EF1α in the telencephalon, but not in other brain regions. The cocaine-induced change in TH/EF1α was blocked by co-treatment with sulpiride, but not SCH23390, correlating closely with the action of these drugs on the CPP behavioral response. Immunohistochemical analysis revealed that the drop in EF1α was selective for the dorsal nucleus of the ventral telencephalic area (Vd), a region believed to be the teleost equivalent of the striatum. Examination of TH mRNA and EF1α transcripts suggests that regulation of expression is post-transcriptional, but this requires further examination. These results highlight important similarities and differences between zebrafish and more traditional mammalian model organisms. PMID:22910534

  6. Abiotic and biotic stress tolerance in Arabidopsis overexpressing the multiprotein bridging factor 1a (MBF1a) transcriptional coactivator gene.

    PubMed

    Kim, Min-Jung; Lim, Gah-Hyun; Kim, Eun-Seon; Ko, Chang-Beom; Yang, Kwang-Yeol; Jeong, Jin-An; Lee, Myung-Chul; Kim, Cheol Soo

    2007-03-01

    We conducted a genetic yeast screen to identify salt tolerance (SAT) genes in a maize kernel cDNA library. During the screening, we identified a maize clone (SAT41) that seemed to confer elevated salt tolerance in comparison to control cells. SAT41 cDNA encodes a 16-kDa protein which is 82.4% identical to the Arabidopsis Multiprotein bridging factor 1a (MBF1a) transcriptional coactivator gene. To further examine salinity tolerance in Arabidopsis, we functionally characterized the MBF1a gene and found that dehydration as well as heightened glucose (Glc) induced MBF1a expression. Constitutive expression of MBF1a in Arabidopsis led to elevated salt tolerance in transgenic lines. Interestingly, plants overexpressing MBF1a exhibited insensitivity to Glc and resistance to fungal disease. Our results suggest that MBF1a is involved in stress tolerance as well as in ethylene and Glc signaling in Arabidopsis. PMID:17234157

  7. Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of C. perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium-related diseases such as gangrenous dermatitis (GD) and necrotic enteritis (NE) are increasingly emerging as major diseases in recent years with high economic loss around the world. In this report, we characterized two immunogenic Clostridium perfringens (CP) proteins (e.g., elongation f...

  8. The phylogenetic analysis of variable-length sequence data: elongation factor-1alpha introns in European populations of the parasitoid wasp genus Pauesia (Hymenoptera: Braconidae: Aphidiinae).

    PubMed

    Sanchis, A; Michelena, J M; Latorre, A; Quicke, D L; Gärdenfors, U; Belshaw, R

    2001-06-01

    Elongation factor-1alpha (EF-1alpha) is a highly conserved nuclear coding gene that can be used to investigate recent divergences due to the presence of rapidly evolving introns. However, a universal feature of intron sequences is that even closely related species exhibit insertion and deletion events, which cause variation in the lengths of the sequences. Indels are frequently rich in evolutionary information, but most investigators ignore sites that fall within these variable regions, largely because the analytical tools and theory are not well developed. We examined this problem in the taxonomically problematic parasitoid wasp genus Pauesia (Hymenoptera: Braconidae: Aphidiinae) using congruence as a criterion for assessing a range of methods for aligning such variable-length EF-1alpha intron sequences. These methods included distance- and parsimony-based multiple-alignment programs (CLUSTAL W and MALIGN), direct optimization (POY), and two "by eye" alignment strategies. Furthermore, with one method (CLUSTAL W) we explored in detail the robustness of results to changes in the gap cost parameters. Phenetic-based alignments ("by eye" and CLUSTAL W) appeared, under our criterion, to perform as well as more readily defensible, but computationally more demanding, methods. In general, all of our alignment and tree-building strategies recovered the same basic topological structure, which means that an underlying phylogenetic signal remained regardless of the strategy chosen. However, several relationships between clades were sensitive both to alignment and to tree-building protocol. Further alignments, considering only sequences belonging to the same group, allowed us to infer a range of phylogenetic relationships that were highly robust to tree-building protocol. By comparing these topologies with those obtained by varying the CLUSTAL parameters, we generated the distribution area of congruence and taxonomic compatibility. Finally, we present the first robust estimate

  9. Generation of plasmid vectors expressing FLAG-tagged proteins under the regulation of human elongation factor-1α promoter using Gibson assembly.

    PubMed

    Grozdanov, Petar N; MacDonald, Clinton C

    2015-01-01

    Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1α) promoter to drive gene expression. Plasmid vectors containing hEF1α are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1α promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1α promoter part 1, hEF1α promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification. PMID:25742071

  10. RICE SALT SENSITIVE3 Forms a Ternary Complex with JAZ and Class-C bHLH Factors and Regulates Jasmonate-Induced Gene Expression and Root Cell Elongation[C][W

    PubMed Central

    Toda, Yosuke; Tanaka, Maiko; Ogawa, Daisuke; Kurata, Kyo; Kurotani, Ken-ichi; Habu, Yoshiki; Ando, Tsuyu; Sugimoto, Kazuhiko; Mitsuda, Nobutaka; Katoh, Etsuko; Abe, Kiyomi; Miyao, Akio; Hirochika, Hirohiko; Hattori, Tsukaho; Takeda, Shin

    2013-01-01

    Plasticity of root growth in response to environmental cues and stresses is a fundamental characteristic of land plants. However, the molecular basis underlying the regulation of root growth under stressful conditions is poorly understood. Here, we report that a rice nuclear factor, RICE SALT SENSITIVE3 (RSS3), regulates root cell elongation during adaptation to salinity. Loss of function of RSS3 only moderately inhibits cell elongation under normal conditions, but it provokes spontaneous root cell swelling, accompanied by severe root growth inhibition, under saline conditions. RSS3 is preferentially expressed in the root tip and forms a ternary complex with class-C basic helix-loop-helix (bHLH) transcription factors and JASMONATE ZIM-DOMAIN proteins, the latter of which are the key regulators of jasmonate (JA) signaling. The mutated protein arising from the rss3 allele fails to interact with bHLH factors, and the expression of a significant portion of JA-responsive genes is upregulated in rss3. These results, together with the known roles of JAs in root growth regulation, suggest that RSS3 modulates the expression of JA-responsive genes and plays a crucial role in a mechanism that sustains root cell elongation at appropriate rates under stressful conditions. PMID:23715469

  11. Super elongation complex contains a TFIIF-related subcomplex.

    PubMed

    Knutson, Bruce A; Smith, Marissa L; Walker-Kopp, Nancy; Xu, Xia

    2016-08-01

    Super elongation complex (SEC) belongs to a family of RNA polymerase II (Pol II) elongation factors that has similar properties as TFIIF, a general transcription factor that increases the transcription elongation rate by reducing pausing. Although SEC has TFIIF-like functional properties, it apparently lacks sequence and structural homology. Using HHpred, we find that SEC contains an evolutionarily related TFIIF-like subcomplex. We show that the SEC subunit ELL interacts with the Pol II Rbp2 subunit, as expected for a TFIIF-like factor. These findings suggest a new model for how SEC functions as a Pol II elongation factor and how it suppresses Pol II pausing. PMID:27223670

  12. Investigating the kinetic mechanism of inhibition of elongation factor 2 kinase by NH125: evidence of a common in vitro artifact.

    PubMed

    Devkota, Ashwini K; Tavares, Clint D J; Warthaka, Mangalika; Abramczyk, Olga; Marshall, Kyle D; Kaoud, Tamer S; Gorgulu, Kivanc; Ozpolat, Bulent; Dalby, Kevin N

    2012-03-13

    Evidence that elongation factor 2 kinase (eEF-2K) has potential as a target for anticancer therapy and possibly for the treatment of depression is emerging. Here the steady-state kinetic mechanism of eEF-2K is presented using a peptide substrate and is shown to conform to an ordered sequential mechanism with ATP binding first. Substrate inhibition by the peptide was observed and revealed to be competitive with ATP, explaining the observed ordered mechanism. Several small molecules are reported to inhibit eEF-2K activity with the most notable being the histidine kinase inhibitor NH125, which has been used in a number of studies to characterize eEF-2K activity in cells. While NH125 was previously reported to inhibit eEF-2K in vitro with an IC(50) of 60 nM, its mechanism of action was not established. Using the same kinetic assay, the ability of an authentic sample of NH125 to inhibit eEF-2K was assessed over a range of substrate and inhibitor concentrations. A typical dose-response curve for the inhibition of eEF-2K by NH125 is best fit to an IC(50) of 18 ± 0.25 μM and a Hill coefficient of 3.7 ± 0.14, suggesting that NH125 is a weak inhibitor of eEF-2K under the experimental conditions of a standard in vitro kinase assay. To test the possibility that NH125 is a potent inhibitor of eEF2 phosphorylation, we assessed its ability to inhibit the phosphorylation of eEF2. Under standard kinase assay conditions, NH125 exhibits a similar weak ability to inhibit the phosphorylation of eEF2 by eEF-2K. Notably, the activity of NH125 is severely abrogated by the addition of 0.1% Triton to the kinase assay through a process that can be reversed upon dilution. These studies suggest that NH125 is a nonspecific colloidal aggregator in vitro, a notion further supported by the observation that NH125 inhibits other protein kinases, such as ERK2 and TRPM7 in a manner similar to that of eEF-2K. As NH125 is reported to inhibit eEF-2K in a cellular environment, its ability to inhibit e

  13. Elongated Microcapsules and Their Formation

    NASA Technical Reports Server (NTRS)

    Calle, Luz M. (Inventor); Li, Wenyan N. (Inventor); Buhrow, Jerry W. (Inventor); Perusich, Stephen A. (Inventor); Jolley, Scott T. (Inventor); Gibson, Tracy L. (Inventor); Williams, Martha K. (Inventor)

    2015-01-01

    Elongated microcapsules, such as elongated hydrophobic-core and hydrophilic-core microcapsules, may be formed by pulse stirring an emulsion or shearing an emulsion between two surfaces moving at different velocities. The elongated microcapsules may be dispersed in a coating formulation, such as paint.

  14. Single Molecule Transcription Elongation

    PubMed Central

    Galburt, Eric A.; Grill, Stephan W.; Bustamante, Carlos

    2009-01-01

    Single molecule optical trapping assays have now been applied to a great number of macromolecular systems including DNA, RNA, cargo motors, restriction enzymes, DNA helicases, chromosome remodelers, DNA polymerases and both viral and bacterial RNA polymerases. The advantages of the technique are the ability to observe dynamic, unsynchronized molecular processes, to determine the distributions of experimental quantities and to apply force to the system while monitoring the response over time. Here, we describe the application of these powerful techniques to study the dynamics of transcription elongation by RNA polymerase II from Saccharomyces cerevisiae. PMID:19426807

  15. Elongation Factor-1a is a novel protein associated with host cell invasion and a potential protective antigen of Cryptosporidium parvum*

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess a unique complex of organelles at the anterior end, referred to as the apical complex, which is involved in host cell invasion. Previously, we generated the chicken m...

  16. Cell division versus cell elongation: the control of radicle elongation during thermoinhibition of Tagetes minuta achenes.

    PubMed

    Taylor, Nicky J; Hills, Paul N; van Staden, Johannes

    2007-12-01

    Endogenous embryo factors, which act mainly in the radicle, prevent germination in Tagetes minuta at high temperatures. These factors act to prevent cell elongation, which is critical for radicle protrusion under optimal conditions. Once the radicle has emerged both cell elongation and cell division are required for post-germination growth. Germination can be induced at high temperatures by fusicoccin, which rapidly stimulates cell elongation. In addition, priming seeds at 25 degrees C on polyethylene glycol (PEG) 6000 and mannitol could also induce germination on water at 36 degrees C, indicating that priming prevents radicle protrusion at a point subsequent to the point of control in thermoinhibited achenes. Flow cytometry studies revealed that DNA synthesis occurs during thermoinhibition and the inhibition of DNA synthesis during this process inhibits subsequent germination on water under optimal conditions, suggesting a protective role for DNA synthesis in thermoinhibited achenes of T. minuta. PMID:17360069

  17. A Role for the Chromatin-Remodeling Factor BAZ1A in Neurodevelopment.

    PubMed

    Zaghlool, Ammar; Halvardson, Jonatan; Zhao, Jin J; Etemadikhah, Mitra; Kalushkova, Antonia; Konska, Katarzyna; Jernberg-Wiklund, Helena; Thuresson, Ann-Charlotte; Feuk, Lars

    2016-09-01

    Chromatin-remodeling factors are required for a wide range of cellular and biological processes including development and cognition, mainly by regulating gene expression. As these functions would predict, deregulation of chromatin-remodeling factors causes various disease syndromes, including neurodevelopmental disorders. Recent reports have linked mutations in several genes coding for chromatin-remodeling factors to intellectual disability (ID). Here, we used exome sequencing and identified a nonsynonymous de novo mutation in BAZ1A (NM_182648.2:c.4043T > G, p.Phe1348Cys), encoding the ATP-utilizing chromatin assembly and remodeling factor 1 (ACF1), in a patient with unexplained ID. ACF1 has been previously reported to bind to the promoter of the vitamin D receptor (VDR)-regulated genes and suppress their expression. Our results show that the patient displays decreased binding of ACF1 to the promoter of the VDR-regulated gene CYP24A1. Using RNA sequencing, we find that the mutation affects the expression of genes involved in several pathways including vitamin D metabolism, Wnt signaling and synaptic formation. RNA sequencing of BAZ1A knockdown cells and Baz1a knockout mice revealed that BAZ1A carry out distinctive functions in different tissues. We also demonstrate that BAZ1A depletion influence the expression of genes important for nervous system development and function. Our data point to an important role for BAZ1A in neurodevelopment, and highlight a possible link for BAZ1A to ID. PMID:27328812

  18. Stunt or elongate? Two opposite strategies by which rice adapts to floods.

    PubMed

    Nagai, Keisuke; Hattori, Yoko; Ashikari, Motoyuki

    2010-05-01

    Expansion of habitat is important for the perpetuation of species. In particular, plants which are sedentary must evolve specialized functions to adapt itself to new environment. Deepwater rice is cultivated mainly in the lowland areas of South and Southeast Asia that are flooded during the rainy season. The internodes of deepwater rice elongates in response to increasing water level to keep its leaves above the water surface and avoid anoxia. This elongation is stimulated by ethylene-regulated genes, Snorkel1 and Snorkel2. In contrast, when a flash flood occurs at the seedling stage, submergence-tolerant rice, which carries Submergence-1A, remains stunted and survives in water for a few weeks to avoid the energy consumption associated with plant elongation, and restarts its growth using its conserved energy after the water recedes. Interestingly, both Snorkel genes and Submergence-1A encode ethylene-responsive factor-type transcription factor and are connected to gibberellin biosynthesis or signal transduction. However, deepwater and submergence-tolerant rice seem to have opposite flooding response; namely, escape by elongation or remain stunted under water until flood recedes. PMID:20354754

  19. Morphological and Chemical Mechanisms of Elongated Mineral Particle Toxicities

    EPA Science Inventory

    Much of our understanding regarding the mechanisms for induction of disease following inhalation of respirable elongated mineral particles (REMPs) is based on studies involving the biological effects of asbestos fibers. The factors governing the disease potential of an exposure i...

  20. Elongational rheology of polyethylene melts

    NASA Astrophysics Data System (ADS)

    Seyfzadeh, Bijan

    Elongational melt flow behavior is an important and fundamental concept underlying many industrial plastics operations which involve a rapid change of shape as for example fiber spinning and stretching, bottle blow molding, and film blowing and stretching. Under high process loads polymeric materials experience enormous stresses causing the molecular structure to gain considerable orientation. This event has significant effects on the melt flow behavior and can be measured in terms of elongational viscosity and changes in enthalpy and entropy. Different polymeric materials with unique molecular characteristics are expected to respond uniquely to the elongational deformation; hence, molecular parameters such as molecular weight and degree of branching were related to the measurable elongational flow variables. Elongational viscosities were measured for high and low density polyethylenes using an advanced capillary extrusion rheometer fitted with semi-hyperbolic dies. Said dies establish a purely elongational. flow field at constant elongational strain rate. The elongational viscosities were evaluated under influence of process strain rate, Hencky strain (natural logarithm of area reduction of the extrusion die), and temperature. Enthalpy and entropy changes associated with the orientation development of semi-hyperbolic processed melts were also determined. Results showed that elongational viscosities were primarily affected by differences in weight average molecular weight rather than degree of branching. This effect was process strain rate as well as temperature dependent. An investigation of melt relaxation and the associated first decay time constants revealed that with increasing strain rate the molecular field of the melt asymptotically gained orientation in approaching a limit. As a result of this behavior molecular uniqueness vanished at high process strain rates, yielding to orientation development and the associated restructuring of the melt's molecular

  1. Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein*

    PubMed Central

    García-Márquez, Adrián; Gijsbers, Abril; de la Mora, Eugenio; Sánchez-Puig, Nuria

    2015-01-01

    Ribosome biogenesis is orchestrated by the action of several accessory factors that provide time and directionality to the process. One such accessory factor is the GTPase EFL1 involved in the cytoplasmic maturation of the ribosomal 60S subunit. EFL1 and SBDS, the protein mutated in the Shwachman-Diamond syndrome (SBDS), release the anti-association factor eIF6 from the surface of the ribosomal subunit 60S. Here we report a kinetic analysis of fluorescent guanine nucleotides binding to EFL1 alone and in the presence of SBDS using fluorescence stopped-flow spectroscopy. Binding kinetics of EFL1 to both GDP and GTP suggests a two-step mechanism with an initial binding event followed by a conformational change of the complex. Furthermore, the same behavior was observed in the presence of the SBDS protein irrespective of the guanine nucleotide evaluated. The affinity of EFL1 for GTP is 10-fold lower than that calculated for GDP. Association of EFL1 to SBDS did not modify the affinity for GTP but dramatically decreased that for GDP by increasing the dissociation rate of the nucleotide. Thus, SBDS acts as a guanine nucleotide exchange factor (GEF) for EFL1 promoting its activation by the release of GDP. Finally, fluorescence anisotropy measurements showed that the S143L mutation present in the Shwachman-Diamond syndrome altered a surface epitope for EFL1 and largely decreased the affinity for it. These results suggest that loss of interaction between these proteins due to mutations in the disease consequently prevents the nucleotide exchange regulation the SBDS exerts on EFL1. PMID:25991726

  2. The activation process of Arabidopsis thaliana A1 gene encoding the translation elongation factor EF-1 alpha is conserved among angiosperms.

    PubMed

    Curie, C; Liboz, T; Montané, M H; Rouan, D; Axelos, M; Lescure, B

    1992-04-01

    In Arabidopsis thaliana, the activation process of the A1 EF-1 alpha gene depends on several elements. Using the GUS reporter gene, transient expression experiments have shown that mutations of upstream cis-acting elements of the A1 promoter, or the deletion of an intron located within the 5' non-coding region, similarly affect expression in dicot or monocot protoplasts. The results reported here strongly suggest that this 5' intron is properly spliced in Zea mays. We show that two trans-acting factors, specifically interacting with an upstream activating sequence (the TEF 1 box), are present in nuclear extracts prepared from A. thaliana, Brassica rapa, Nicotiana tabacum and Z. mays. In addition, a DNA sequence homologous to the TEF 1 box, found at approximately the same location within a Lycopersicon esculentum EF-1 alpha promoter, interacts with the same trans-acting factors. Homologies found between the A. thaliana and L. esculentum TEF 1 box sequences have allowed us to define mutations of this upstream element which affect the interaction with the corresponding trans-acting factors. These results support the notion that the activation processes of A. thaliana EF-1 alpha genes have been conserved among angiosperms and provide interesting data on the functional structure of the TEF 1 box. PMID:1600144

  3. High elongation elastomers

    NASA Technical Reports Server (NTRS)

    Brady, V. L.; Reed, R.; Merwin, L.; Nissan, R.

    1994-01-01

    A new class of liquid curable elastomers with unusual strength and elasticity has been developed at the Naval Air Warfare Center Weapons Division, China Lake. Over the years, studies have been conducted on polymer structure and its influence on the mechanical properties of the ensuing composites. Different tools, including nuclear magnetic resonance, have been used. This paper presents a summary of the factors controlling the mechanical behavior of composites produced with the new liquid curable elastomers, including the effects of plasticizers. It also provides an overview of the nuclear magnetic resonance study on polymer structure, the composition and properties of some live and inert formulations produced at China Lake, and some possible peace-time applications for these new elastomeric materials.

  4. Plasma DYRK1A as a novel risk factor for Alzheimer's disease.

    PubMed

    Janel, N; Sarazin, M; Corlier, F; Corne, H; de Souza, L C; Hamelin, L; Aka, A; Lagarde, J; Blehaut, H; Hindié, V; Rain, J-C; Arbones, M L; Dubois, B; Potier, M C; Bottlaender, M; Delabar, J M

    2014-01-01

    To determine whether apparent involvement of DYRK1A in Alzheimer's disease (AD) pathology makes it a candidate plasma biomarker for diagnosis, we developed a method to quantify plasma DYRK1A by immunoblot in transgenic mouse models having different gene dosages of Dyrk1a, and, consequently, different relative protein expression. Then, we measured plasma DYRK1A levels in 26 patients with biologically confirmed AD and 25 controls (negative amyloid imaging available on 13). DYRK1A was detected in transgenic mouse brain and plasma samples, and relative levels of DYRK1A correlated with the gene copy number. In plasma from AD patients, DYRK1A levels were significantly lower compared with controls (P<0.0001). Results were similar when we compared AD patients with the subgroup of controls confirmed by negative amyloid imaging. In a subgroup of patients with early AD (CDR=0.5), lower DYRK1A expression was confirmed. In contrast, no difference was found in levels of DYRK1B, the closest relative of DYRK1A, between AD patients and controls. Further, AD patients exhibited a positive correlation between plasma DYRK1A levels and cerebrospinal fluid tau and phosphorylated-tau proteins, but no correlation with amyloid-β42 levels and Pittsburgh compound B cortical binding. DYRK1A levels detected in lymphoblastoid cell lines from AD patients were also lower when compared with cells from age-matched controls. These findings suggest that reduced DYRK1A expression might be a novel plasma risk factor for AD. PMID:25116835

  5. E1A inhibits transforming growth factor-beta signaling through binding to Smad proteins.

    PubMed

    Nishihara, A; Hanai, J; Imamura, T; Miyazono, K; Kawabata, M

    1999-10-01

    Smads form a recently identified family of proteins that mediate intracellular signaling of the transforming growth factor (TGF)-beta superfamily. Smads bind to DNA and act as transcriptional regulators. Smads interact with a variety of transcription factors, and the interaction is likely to determine the target specificity of gene induction. Smads also associate with transcriptional coactivators such as p300 and CBP. E1A, an adenoviral oncoprotein, inhibits TGF-beta-induced transactivation, and the ability of E1A to bind p300/CBP is required for the inhibition. Here we determined the Smad interaction domain (SID) in p300 and found that two adjacent regions are required for the interaction. One of the regions is the C/H3 domain conserved between p300 and CBP, and the other is a nonconserved region. p300 mutants containing SID inhibit transactivation by TGF-beta in a dose-dependent manner. E1A inhibits the interaction of Smad3 with a p300 mutant that contains SID but lacks the E1A binding domain. We found that E1A interacts specifically with receptor-regulated Smads, suggesting a novel mechanism whereby E1A antagonizes TGF-beta signaling. PMID:10497242

  6. Arabidopsis MICROTUBULE DESTABILIZING PROTEIN40 Is Involved in Brassinosteroid Regulation of Hypocotyl Elongation[C][W][OA

    PubMed Central

    Wang, Xianling; Zhang, Jin; Yuan, Ming; Ehrhardt, David W.; Wang, Zhiyong; Mao, Tonglin

    2012-01-01

    The brassinosteroid (BR) phytohormones play crucial roles in regulating plant cell growth and morphogenesis, particularly in hypocotyl cell elongation. The microtubule cytoskeleton is also known to participate in the regulation of hypocotyl elongation. However, it is unclear if BR regulation of hypocotyl elongation involves the microtubule cytoskeleton. In this study, we demonstrate that BRs mediate hypocotyl cell elongation by influencing the orientation and stability of cortical microtubules. Further analysis identified the previously undiscovered Arabidopsis thaliana MICROTUBULE DESTABILIZING PROTEIN40 (MDP40) as a positive regulator of hypocotyl cell elongation. BRASSINAZOLE-RESISTANT1, a key transcription factor in the BR signaling pathway, directly targets and upregulates MDP40. Overexpression of MDP40 partially rescued the shorter hypocotyl phenotype in BR-deficient mutant de-etiolated-2 seedlings. Reorientation of the cortical microtubules in the cells of MDP40 RNA interference transgenic lines was less sensitive to BR. These findings demonstrate that MDP40 is a key regulator in BR regulation of cortical microtubule reorientation and mediates hypocotyl growth. This study reveals a mechanism involving BR regulation of microtubules through MDP40 to mediate hypocotyl cell elongation. PMID:23115248

  7. Wolbachia Transcription Elongation Factor “Wol GreA” Interacts with α2ββ′σ Subunits of RNA Polymerase through Its Dimeric C-Terminal Domain

    PubMed Central

    Nag, Jeetendra Kumar; Shrivastava, Nidhi; Chahar, Dhanvantri; Gupta, Chhedi Lal; Bajpai, Preeti; Misra-Bhattacharya, Shailja

    2014-01-01

    Objectives Wolbachia, an endosymbiont of filarial nematode, is considered a promising target for therapy against lymphatic filariasis. Transcription elongation factor GreA is an essential factor that mediates transcriptional transition from abortive initiation to productive elongation by stimulating the escape of RNA polymerase (RNAP) from native prokaryotic promoters. Upon screening of 6257 essential bacterial genes, 57 were suggested as potential future drug targets, and GreA is among these. The current study emphasized the characterization of Wol GreA with its domains. Methodology/Principal Findings Biophysical characterization of Wol GreA with its N-terminal domain (NTD) and C-terminal domain (CTD) was performed with fluorimetry, size exclusion chromatography, and chemical cross-linking. Filter trap and far western blotting were used to determine the domain responsible for the interaction with α2ββ′σ subunits of RNAP. Protein-protein docking studies were done to explore residual interaction of RNAP with Wol GreA. The factor and its domains were found to be biochemically active. Size exclusion and chemical cross-linking studies revealed that Wol GreA and CTD exist in a dimeric conformation while NTD subsists in monomeric conformation. Asp120, Val121, Ser122, Lys123, and Ser134 are the residues of CTD through which monomers of Wol GreA interact and shape into a dimeric conformation. Filter trap, far western blotting, and protein-protein docking studies revealed that dimeric CTD of Wol GreA through Lys82, Ser98, Asp104, Ser105, Glu106, Tyr109, Glu116, Asp120, Val121, Ser122, Ser127, Ser129, Lys140, Glu143, Val147, Ser151, Glu153, and Phe163 residues exclusively participates in binding with α2ββ′σ subunits of polymerase. Conclusions/Significance To the best of our knowledge, this research is the first documentation of the residual mode of action in wolbachial mutualist. Therefore, findings may be crucial to understanding the transcription mechanism of

  8. Negative Elongation Factor Is Required for the Maintenance of Proviral Latency but Does Not Induce Promoter-Proximal Pausing of RNA Polymerase II on the HIV Long Terminal Repeat

    PubMed Central

    Jadlowsky, Julie K.; Wong, Julian Y.; Graham, Amy C.; Dobrowolski, Curtis; Devor, Renee L.; Adams, Mark D.; Fujinaga, Koh

    2014-01-01

    The role of the negative elongation factor (NELF) in maintaining HIV latency was investigated following small hairpin RNA (shRNA) knockdown of the NELF-E subunit, a condition that induced high levels of proviral transcription in latently infected Jurkat T cells. Chromatin immunoprecipitation (ChIP) assays showed that latent proviruses accumulate RNA polymerase II (RNAP II) on the 5′ long terminal repeat (LTR) but not on the 3′ LTR. NELF colocalizes with RNAP II, and its level increases following proviral induction. RNAP II pause sites on the HIV provirus were mapped to high resolution by ChIP with high-throughput sequencing (ChIP-Seq). Like cellular promoters, RNAP II accumulates at around position +30, but HIV also shows additional pausing at +90, which is immediately downstream of a transactivation response (TAR) element and other distal sites on the HIV LTR. Following NELF-E knockdown or tumor necrosis factor alpha (TNF-α) stimulation, promoter-proximal RNAP II levels increase up to 3-fold, and there is a dramatic increase in RNAP II levels within the HIV genome. These data support a kinetic model for proviral transcription based on continuous replacement of paused RNAP II during both latency and productive transcription. In contrast to most cellular genes, HIV is highly activated by the combined effects of NELF-E depletion and activation of initiation by TNF-α, suggesting that opportunities exist to selectively activate latent HIV proviruses. PMID:24636995

  9. Negative elongation factor is required for the maintenance of proviral latency but does not induce promoter-proximal pausing of RNA polymerase II on the HIV long terminal repeat.

    PubMed

    Jadlowsky, Julie K; Wong, Julian Y; Graham, Amy C; Dobrowolski, Curtis; Devor, Renee L; Adams, Mark D; Fujinaga, Koh; Karn, Jonathan

    2014-06-01

    The role of the negative elongation factor (NELF) in maintaining HIV latency was investigated following small hairpin RNA (shRNA) knockdown of the NELF-E subunit, a condition that induced high levels of proviral transcription in latently infected Jurkat T cells. Chromatin immunoprecipitation (ChIP) assays showed that latent proviruses accumulate RNA polymerase II (RNAP II) on the 5' long terminal repeat (LTR) but not on the 3' LTR. NELF colocalizes with RNAP II, and its level increases following proviral induction. RNAP II pause sites on the HIV provirus were mapped to high resolution by ChIP with high-throughput sequencing (ChIP-Seq). Like cellular promoters, RNAP II accumulates at around position +30, but HIV also shows additional pausing at +90, which is immediately downstream of a transactivation response (TAR) element and other distal sites on the HIV LTR. Following NELF-E knockdown or tumor necrosis factor alpha (TNF-α) stimulation, promoter-proximal RNAP II levels increase up to 3-fold, and there is a dramatic increase in RNAP II levels within the HIV genome. These data support a kinetic model for proviral transcription based on continuous replacement of paused RNAP II during both latency and productive transcription. In contrast to most cellular genes, HIV is highly activated by the combined effects of NELF-E depletion and activation of initiation by TNF-α, suggesting that opportunities exist to selectively activate latent HIV proviruses. PMID:24636995

  10. Transcriptional regulation of the human TR2 orphan receptor gene by nuclear factor 1-A

    SciTech Connect

    Lin, Y.-L.; Wang, Y.-H.; Lee, H.-J. . E-mail: hjlee@mail.ndhu.edu.tw

    2006-11-17

    The human testicular receptor 2 (TR2), a member of the nuclear hormone receptor superfamily, has no identified ligand yet. Previous evidence demonstrated that a 63 bp DNA fragment, named the promoter activating cis-element (PACE), has been identified as a positive regulatory region in the 5' promoter region of the human TR2 gene. In the present report, the human nuclear factor 1-A (NF1-A) was identified as a transcriptional activator to recognize the center of the PACE, called the PACE-C. NF1-A could bind to the 18 bp PACE-C region, and enhance about 13- to 17-fold of the luciferase reporter gene activity via the PACE-C in dose-dependent and orientation-independent manners. This transcriptional activation was further confirmed by real-time RT-PCR assay. In conclusion, our results indicated that NF1-A transcription factor plays an important role in the transcriptional activation of the TR2 gene expression via the PACE-C in the minimal promoter region.