Science.gov

Sample records for elongation factor nelf

  1. Cellular dynamics of the negative transcription elongation factor NELF

    SciTech Connect

    Yung, Tetsu M.C.; Narita, Takashi; Komori, Toshiharu; Yamaguchi, Yuki; Handa, Hiroshi

    2009-06-10

    Negative Elongation Factor (NELF) is a transcription factor discovered based on its biochemical activity to suppress transcription elongation, and has since been implicated in various diseases ranging from neurological disorders to cancer. Besides its role in promoter-proximal pausing of RNA polymerase II during early stages of transcription, recently we found that it also plays important roles in the 3'-end processing of histone mRNA. Furthermore, NELF has been found to form a distinct subnuclear structure, which we named NELF bodies. These recent developments point to a wide range of potential functions for NELF, and, as most studies on NELF thus far had been carried out in vitro, here, we prepared a complete set of fusion protein constructs of NELF subunits and carried out a general cell biological study of the intracellular dynamics of NELF. Our data show that NELF subunits exhibit highly specific subcellular localizations, such as in NELF bodies or in midbodies, and some shuttle actively between the nucleus and cytoplasm. We further show that loss of NELF from cells can lead to enlarged and/or multiple nuclei. This work serves as a foundation and starting point for further cell biological investigations of NELF in the future.

  2. Transcription elongation factors DSIF and NELF: promoter-proximal pausing and beyond.

    PubMed

    Yamaguchi, Yuki; Shibata, Hirotaka; Handa, Hiroshi

    2013-01-01

    DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF) were originally identified as factors responsible for transcriptional inhibition by 5,6-dichloro-1-beta-d-ribofuranosyl-benzimidazole (DRB) and were later found to control transcription elongation, together with P-TEFb, at the promoter-proximal region. Although there is ample evidence that these factors play roles throughout the genome, other data also suggest gene- or tissue-specific roles for these factors. In this review, we discuss how these apparently conflicting data can be reconciled. In light of recent findings, we also discuss the detailed mechanism by which these factors control the elongation process at the molecular level. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.

  3. NELF Potentiates Gene Transcription in the Drosophila Embryo

    PubMed Central

    Wang, Xiaoling; Hang, Saiyu; Prazak, Lisa; Gergen, J. Peter

    2010-01-01

    A hallmark of genes that are subject to developmental regulation of transcriptional elongation is association of the negative elongation factor NELF with the paused RNA polymerase complex. Here we use a combination of biochemical and genetic experiments to investigate the in vivo function of NELF in the Drosophila embryo. NELF associates with different gene promoter regions in correlation with the association of RNA polymerase II (Pol II) and the initial activation of gene expression during the early stages of embryogenesis. Genetic experiments reveal that maternally provided NELF is required for the activation, rather than the repression of reporter genes that emulate the expression of key developmental control genes. Furthermore, the relative requirement for NELF is dictated by attributes of the flanking cis-regulatory information. We propose that NELF-associated paused Pol II complexes provide a platform for high fidelity integration of the combinatorial spatial and temporal information that is central to the regulation of gene expression during animal development. PMID:20634899

  4. 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.

  5. NF-κB-repressing factor phosphorylation regulates transcription elongation via its interactions with 5'→3' exoribonuclease 2 and negative elongation factor.

    PubMed

    Rother, Sascha; Bartels, Myriam; Schweda, Aike Torben; Resch, Klaus; Pallua, Norbert; Nourbakhsh, Mahtab

    2016-01-01

    NF-κB-repressing factor (NKRF) inhibits transcription elongation by binding to specific sequences in target promoters. Stimuli such as IL-1 have been shown to overcome this inhibitory action and enable the resumption of transcription elongation machinery by an unknown mechanism. Using mass spectrometry and in vitro phosphorylation analyses, we demonstrate that NKRF is phosphorylated within 3 different domains in unstimulated HeLa cells. Phosphoamino acid mapping and mutation analysis of NKRF further suggest that only Ser phosphorylation within aa 421-429 is regulated by IL-1 stimulation. In copurification studies, aa 421-429 is required for interactions between NKRF, 5'→3' exoribonuclease 2 (XRN2) and the negative elongation factor (NELF)-E in HeLa cells. Chromatin immunoprecipitation experiments further show that IL-1 stimulation leads to decrease in NKRF aa 421-429 phosphorylation and dissociation of NELF-E and XRN2 by concomitant resumption of transcription elongation of a synthetic reporter or the endogenous NKRF target gene, IL-8. Together, NKRF phosphorylation modulates promoter-proximal transcription elongation of NF-κB/NKRF-regulated genes via direct interactions with elongation complex in response to specific stimuli.

  6. NELF-mediated stalling of Pol II can enhance gene expression by blocking promoter-proximal nucleosome assembly

    PubMed Central

    Gilchrist, Daniel A.; Nechaev, Sergei; Lee, Chanhyo; Ghosh, Saikat Kumar B.; Collins, Jennifer B.; Li, Leping; Gilmour, David S.; Adelman, Karen

    2008-01-01

    The Negative Elongation Factor (NELF) is a transcription regulatory complex that induces stalling of RNA polymerase II (Pol II) during early transcription elongation and represses expression of several genes studied to date, including Drosophila Hsp70, mammalian proto-oncogene junB, and HIV RNA. To determine the full spectrum of NELF target genes in Drosophila, we performed a microarray analysis of S2 cells depleted of NELF and discovered that NELF RNAi affects many rapidly inducible genes involved in cellular responses to stimuli. Surprisingly, only one-third of NELF target genes were, like Hsp70, up-regulated by NELF-depletion, whereas the majority of target genes showed decreased expression levels upon NELF RNAi. Our data reveal that the presence of stalled Pol II at this latter group of genes enhances gene expression by maintaining a permissive chromatin architecture around the promoter-proximal region, and that loss of Pol II stalling at these promoters is accompanied by a significant increase in nucleosome occupancy and a decrease in histone H3 Lys 4 trimethylation. These findings identify a novel, positive role for stalled Pol II in regulating gene expression and suggest that there is a dynamic interplay between stalled Pol II and chromatin structure. PMID:18628398

  7. CTCF regulates NELF, DSIF and P-TEFb recruitment during transcription.

    PubMed

    Laitem, Clélia; Zaborowska, Justyna; Tellier, Michael; Yamaguchi, Yuki; Cao, Qingfu; Egloff, Sylvain; Handa, Hiroshi; Murphy, Shona

    2015-01-01

    CTCF is a versatile transcription factor with well-established roles in chromatin organization and insulator function. Recent findings also implicate CTCF in the control of elongation by RNA polymerase (RNAP) II. Here we show that CTCF knockdown abrogates RNAP II pausing at the early elongation checkpoint of c-myc by affecting recruitment of DRB-sensitivity-inducing factor (DSIF). CTCF knockdown also causes a termination defect on the U2 snRNA genes (U2), by affecting recruitment of negative elongation factor (NELF). In addition, CTCF is required for recruitment of positive elongation factor b (P-TEFb), which phosphorylates NELF, DSIF, and Ser2 of the RNAP II CTD to activate elongation of transcription of c-myc and recognition of the snRNA gene-specific 3' box RNA processing signal. These findings implicate CTCF in a complex network of protein:protein/protein:DNA interactions and assign a key role to CTCF in controlling RNAP II transcription through the elongation checkpoint of the protein-coding c-myc and the termination site of the non-coding U2, by regulating the recruitment and/or activity of key players in these processes.

  8. Elongation factors in protein synthesis.

    PubMed

    Kraal, B; Bosch, L; Mesters, J R; de Graaf, J M; Woudt, L P; Vijgenboom, E; Heinstra, P W; Zeef, L A; Boon, C

    1993-01-01

    Recent discoveries of elongation factor-related proteins have considerably complicated the simple textbook scheme of the peptide chain elongation cycle. During growth and differentiation the cycle may be regulated not only by factor modification but also factor replacement. In addition, rare tRNAs may have their own rare factor proteins. A special case is the acquisition of resistance by bacteria to elongation factor-directed antibiotics. Pertinent data from the literature and our own work with Escherichia coli and Streptomyces are discussed. The GTP-binding domain of EF-Tu has been studied extensively, but little molecular detail is available on the interactions with its other ligands or effectors, or on the way they are affected by the GTPase switch signal. A growing number of EF-Tu mutants obtained by ourselves and others are helping us in testing current ideas. We have found a synergistic effect between EF-Tu and EF-G in their uncoupled GTPase reactions on empty ribosomes. Only the EF-G reaction is perturbed by fluoroaluminates.

  9. Scatter factor corrections for elongated fields

    SciTech Connect

    Higgins, P.D.; Sohn, W.H.; Sibata, C.H.; McCarthy, W.A. )

    1989-09-01

    Measurements have been made to determine scatter factor corrections for elongated fields of Cobalt-60 and for nominal linear accelerator energies of 6 MV (Siemens Mevatron 67) and 18 MV (AECL Therac 20). It was found that for every energy the collimator scatter factor varies by 2% or more as the field length-to-width ratio increases beyond 3:1. The phantom scatter factor is independent of which collimator pair is elongated at these energies. For 18 MV photons it was found that the collimator scatter factor is complicated by field-size-dependent backscatter into the beam monitor.

  10. Scatter factor corrections for elongated fields.

    PubMed

    Higgins, P D; Sohn, W H; Sibata, C H; McCarthy, W A

    1989-01-01

    Measurements have been made to determine scatter factor corrections for elongated fields of Cobalt-60 and for nominal linear accelerator energies of 6 MV (Siemens Mevatron 67) and 18 MV (AECL Therac 20). It was found that for every energy the collimator scatter factor varies by 2% or more as the field length-to-width ratio increases beyond 3:1. The phantom scatter factor is independent of which collimator pair is elongated at these energies. For 18 MV photons it was found that the collimator scatter factor is complicated by field-size-dependent backscatter into the beam monitor.

  11. Functional specialization of transcription elongation factors

    PubMed Central

    Belogurov, Georgiy A; Mooney, Rachel A; Svetlov, Vladimir; Landick, Robert; Artsimovitch, Irina

    2009-01-01

    Elongation factors NusG and RfaH evolved from a common ancestor and utilize the same binding site on RNA polymerase (RNAP) to modulate transcription. However, although NusG associates with RNAP transcribing most Escherichia coli genes, RfaH regulates just a few operons containing ops, a DNA sequence that mediates RfaH recruitment. Here, we describe the mechanism by which this specificity is maintained. We observe that RfaH action is indeed restricted to those several operons that are devoid of NusG in vivo. We also show that RfaH and NusG compete for their effects on transcript elongation and termination in vitro. Our data argue that RfaH recognizes its DNA target even in the presence of NusG. Once recruited, RfaH remains stably associated with RNAP, thereby precluding NusG binding. We envision a pathway by which a specialized regulator has evolved in the background of its ubiquitous paralogue. We propose that RfaH and NusG may have opposite regulatory functions: although NusG appears to function in concert with Rho, RfaH inhibits Rho action and activates the expression of poorly translated, frequently foreign genes. PMID:19096362

  12. Maintenance of Transcription-Translation Coupling by Elongation Factor P

    PubMed Central

    Elgamal, Sara

    2016-01-01

    ABSTRACT Under conditions of tight coupling between translation and transcription, the ribosome enables synthesis of full-length mRNAs by preventing both formation of intrinsic terminator hairpins and loading of the transcription termination factor Rho. While previous studies have focused on transcription factors, we investigated the role of Escherichia coli elongation factor P (EF-P), an elongation factor required for efficient translation of mRNAs containing consecutive proline codons, in maintaining coupled translation and transcription. In the absence of EF-P, the presence of Rho utilization (rut) sites led to an ~30-fold decrease in translation of polyproline-encoding mRNAs. Coexpression of the Rho inhibitor Psu fully restored translation. EF-P was also shown to inhibit premature termination during synthesis and translation of mRNAs encoding intrinsic terminators. The effects of EF-P loss on expression of polyproline mRNAs were augmented by a substitution in RNA polymerase that accelerates transcription. Analyses of previously reported ribosome profiling and global proteomic data identified several candidate gene clusters where EF-P could act to prevent premature transcription termination. In vivo probing allowed detection of some predicted premature termination products in the absence of EF-P. Our findings support a model in which EF-P maintains coupling of translation and transcription by decreasing ribosome stalling at polyproline motifs. Other regulators that facilitate ribosome translocation through roadblocks to prevent premature transcription termination upon uncoupling remain to be identified. PMID:27624127

  13. Elongation factor-2: a useful gene for arthropod phylogenetics.

    PubMed

    Regier, J C; Shultz, J W

    2001-07-01

    Robust resolution of controversial higher-level groupings within Arthropoda requires additional sources of characters. Toward this end, elongation factor-2 sequences (1899 nucleotides) were generated from 17 arthropod taxa (5 chelicerates, 6 crustaceans, 3 hexapods, 3 myriapods) plus an onychophoran and a tardigrade as outgroups. Likelihood and parsimony analyses of nucleotide and amino acid data sets consistently recovered Myriapoda and major chelicerate groups with high bootstrap support. Crustacea + Hexapoda (= Pancrustacea) was recovered with moderate support, whereas the conflicting group Myriapoda + Hexapoda (= Atelocerata) was never recovered and bootstrap values were always <5%. With additional nonarthropod sequences included, one indel supports monophyly of Tardigrada, Onychophora, and Arthropoda relative to molluscan, annelidan, and mammalian outgroups. New and previously published sequences from RNA polymerase II (1038 nucleotides) and elongation factor-1alpha (1092 nucleotides) were analyzed for the same taxa. A comparison of bootstrap values from the three genes analyzed separately revealed widely varying values for some clades, although there was never strong support for conflicting groups. In combined analyses, there was strong bootstrap support for the generally accepted clades Arachnida, Arthropoda, Euchelicerata, Hexapoda, and Pycnogonida, and for Chelicerata, Myriapoda, and Pancrustacea, whose monophyly is more controversial. Recovery of some additional groups was fairly robust to method of analysis but bootstrap values were not high; these included Pancrustacea + Chelicerata, Hexapoda + Cephalocarida + Remipedia, Cephalocarida + Remipedia, and Malaocostraca + Cirripedia. Atelocerata (= Myriapoda + Hexapoda) was never recovered. Elongation factor-2 is now the second protein-encoding, nuclear gene (in addition to RNA polymerase II) to support Pancrustacea over Atelocerata. Atelocerata is widely cited in morphology-based analyses, and the

  14. Engineering the elongation factor Tu for efficient selenoprotein synthesis.

    PubMed

    Haruna, Ken-ichi; Alkazemi, Muhammad H; Liu, Yuchen; Söll, Dieter; Englert, Markus

    2014-09-01

    Selenocysteine (Sec) is naturally co-translationally incorporated into proteins by recoding the UGA opal codon with a specialized elongation factor (SelB in bacteria) and an RNA structural signal (SECIS element). We have recently developed a SECIS-free selenoprotein synthesis system that site-specifically--using the UAG amber codon--inserts Sec depending on the elongation factor Tu (EF-Tu). Here, we describe the engineering of EF-Tu for improved selenoprotein synthesis. A Sec-specific selection system was established by expression of human protein O(6)-alkylguanine-DNA alkyltransferase (hAGT), in which the active site cysteine codon has been replaced by the UAG amber codon. The formed hAGT selenoprotein repairs the DNA damage caused by the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine, and thereby enables Escherichia coli to grow in the presence of this mutagen. An EF-Tu library was created in which codons specifying the amino acid binding pocket were randomized. Selection was carried out for enhanced Sec incorporation into hAGT; the resulting EF-Tu variants contained highly conserved amino acid changes within members of the library. The improved UTu-system with EF-Sel1 raises the efficiency of UAG-specific Sec incorporation to >90%, and also doubles the yield of selenoprotein production.

  15. Doc Toxin Is a Kinase That Inactivates Elongation Factor Tu*

    PubMed Central

    Cruz, Jonathan W.; Rothenbacher, Francesca P.; Maehigashi, Tatsuya; Lane, William S.; Dunham, Christine M.; Woychik, Nancy A.

    2014-01-01

    The Doc toxin from bacteriophage P1 (of the phd-doc toxin-antitoxin system) has served as a model for the family of Doc toxins, many of which are harbored in the genomes of pathogens. We have shown previously that the mode of action of this toxin is distinct from the majority derived from toxin-antitoxin systems: it does not cleave RNA; in fact P1 Doc expression leads to mRNA stabilization. However, the molecular triggers that lead to translation arrest are not understood. The presence of a Fic domain, albeit slightly altered in length and at the catalytic site, provided a clue to the mechanism of P1 Doc action, as most proteins with this conserved domain inactivate GTPases through addition of an adenylyl group (also referred to as AMPylation). We demonstrated that P1 Doc added a single phosphate group to the essential translation elongation factor and GTPase, elongation factor (EF)-Tu. The phosphorylation site was at a highly conserved threonine, Thr-382, which was blocked when EF-Tu was treated with the antibiotic kirromycin. Therefore, we have established that Fic domain proteins can function as kinases. This distinct enzymatic activity exhibited by P1 Doc also solves the mystery of the degenerate Fic motif unique to the Doc family of toxins. Moreover, we have established that all characterized Fic domain proteins, even those that phosphorylate, target pivotal GTPases for inactivation through a post-translational modification at a single functionally critical acceptor site. PMID:24448800

  16. 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

  17. Structure and expression of elongation factor 1 alpha in tomato.

    PubMed Central

    Pokalsky, A R; Hiatt, W R; Ridge, N; Rasmussen, R; Houck, C M; Shewmaker, C K

    1989-01-01

    A full-length cDNA clone, LeEF-1, has been isolated from tomato for the alpha subunit of elongation factor 1 (EF-1 alpha), a polypeptide which plays a central role in protein synthesis. The 448 amino acid protein encoded by this cDNA appears highly homologous to other EF-1 alpha s having a high degree of similarity (75-78%) to EF1 alpha previously described from both lower eukaryotes and animals. Southern analysis indicated that EF-1 alpha belongs to a small multigene family of 4-8 members in tomato. The pattern of expression of EF-1 alpha mRNA in various tomato tissues was analyzed by Northern analysis, in vitro translation and in situ hybridization. EF-1 alpha mRNA is an abundant species and higher levels of mRNA were found in developing tissues such as young leaves and green fruit compared to the mRNA levels observed in older tissues. The increased levels of EF-1 alpha mRNA therefore appear to correlate with higher levels of protein synthesis in developing tissues. Images PMID:2748335

  18. Movement of elongation factor G between compact and extended conformations.

    PubMed

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

    2015-01-30

    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. 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 pretranslocation ribosomes or with 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.

  19. 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

  20. Structural outline of the detailed mechanism for elongation factor Ts-mediated guanine nucleotide exchange on elongation factor Tu.

    PubMed

    Thirup, Søren S; Van, Lan Bich; Nielsen, Tine K; Knudsen, Charlotte R

    2015-07-01

    Translation elongation factor EF-Tu belongs to the superfamily of guanine-nucleotide binding proteins, which play key cellular roles as regulatory switches. All G-proteins require activation via exchange of GDP for GTP to carry out their respective tasks. Often, guanine-nucleotide exchange factors are essential to this process. During translation, EF-Tu:GTP transports aminoacylated tRNA to the ribosome. GTP is hydrolyzed during this process, and subsequent reactivation of EF-Tu is catalyzed by EF-Ts. The reaction path of guanine-nucleotide exchange is structurally poorly defined for EF-Tu and EF-Ts. We have determined the crystal structures of the following reaction intermediates: two structures of EF-Tu:GDP:EF-Ts (2.2 and 1.8Å resolution), EF-Tu:PO4:EF-Ts (1.9Å resolution), EF-Tu:GDPNP:EF-Ts (2.2Å resolution) and EF-Tu:GDPNP:pulvomycin:Mg(2+):EF-Ts (3.5Å resolution). These structures provide snapshots throughout the entire exchange reaction and suggest a mechanism for the release of EF-Tu in its GTP conformation. An inferred sequence of events during the exchange reaction is presented.

  1. 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

    2016-07-05

    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.

  2. The interaction between bacterial transcription factors and RNA polymerase during the transition from initiation to elongation.

    PubMed

    Yang, Xiao; Lewis, Peter J

    2010-01-01

    There are three stages of transcription: initiation, elongation and termination, and traditionally there has been a clear distinction between the stages. The specificity factor sigma is completely released from bacterial RNA polymerase after initiation, and then recycled for another round of transcription. Elongation factors then associate with the polymerase followed by termination factors (where necessary). These factors dissociate prior to initiation of a new round of transcription. However, there is growing evidence suggesting that sigma factors can be retained in the elongation complex. The structure of bacterial RNAP in complex with an essential elongation factor NusA has recently been published, which suggested rather than competing for the major σ binding site, NusA binds to a discrete region on RNAP. A model was proposed to help explain the way in which both factors could be associated with RNAP during the transition from transcription initiation to elongation.

  3. Arabidopsis chromatin remodeling factor PICKLE interacts with transcription factor HY5 to regulate hypocotyl cell elongation.

    PubMed

    Jing, Yanjun; Zhang, Dong; Wang, Xin; Tang, Weijiang; Wang, Wanqing; Huai, Junling; Xu, Gang; Chen, Dongqin; Li, Yunliang; Lin, Rongcheng

    2013-01-01

    Photomorphogenesis is a critical plant developmental process that involves light-mediated transcriptome changes, histone modifications, and inhibition of hypocotyl growth. However, the chromatin-based regulatory mechanism underlying this process remains largely unknown. Here, we identify ENHANCED PHOTOMORPHOGENIC1 (EPP1), previously known as PICKLE (PKL), an ATP-dependent chromatin remodeling factor of the chromodomain/helicase/DNA binding family, as a repressor of photomorphogenesis in Arabidopsis thaliana. We show that PKL/EPP1 expression is repressed by light in the hypocotyls in a photoreceptor-dependent manner. Furthermore, we reveal that the transcription factor ELONGATED HYPOCOTYL5 (HY5) binds to the promoters of cell elongation-related genes and recruits PKL/EPP1 through their physical interaction. PKL/EPP1 in turn negatively regulates HY5 by repressing trimethylation of histone H3 Lys 27 at the target loci, thereby regulating the expression of these genes and, thus, hypocotyl elongation. We also show that HY5 possesses transcriptional repression activity. Our study reveals a crucial role for a chromatin remodeling factor in repressing photomorphogenesis and demonstrates that transcription factor-mediated recruitment of chromatin-remodeling machinery is important for plant development in response to changing light environments.

  4. Identification and Characterization of Elf1, a Conserved Transcription Elongation Factor in Saccharomyces cerevisiae

    PubMed Central

    Prather, Donald; Krogan, Nevan J.; Emili, Andrew; Greenblatt, Jack F.; Winston, Fred

    2005-01-01

    In order to identify previously unknown transcription elongation factors, a genetic screen was carried out to identify mutations that cause lethality when combined with mutations in the genes encoding the elongation factors TFIIS and Spt6. This screen identified a mutation in YKL160W, hereafter named ELF1 (elongation factor 1). Further analysis identified synthetic lethality between an elf1Δ mutation and mutations in genes encoding several known elongation factors, including Spt4, Spt5, Spt6, and members of the Paf1 complex. Genome-wide synthetic lethality studies confirmed that elf1Δ specifically interacts with mutations in genes affecting transcription elongation. Chromatin immunoprecipitation experiments show that Elf1 is cotranscriptionally recruited over actively transcribed regions and that this association is partially dependent on Spt4 and Spt6. Analysis of elf1Δ mutants suggests a role for this factor in maintaining proper chromatin structure in regions of active transcription. Finally, purification of Elf1 suggests an association with casein kinase II, previously implicated in roles in transcription. Together, these results suggest an important role for Elf1 in the regulation of transcription elongation. PMID:16260625

  5. Fusidic acid targets elongation factor G in several stages of translocation on the bacterial ribosome.

    PubMed

    Borg, Anneli; Holm, Mikael; Shiroyama, Ikue; Hauryliuk, Vasili; Pavlov, Michael; Sanyal, Suparna; Ehrenberg, Måns

    2015-02-06

    The antibiotic fusidic acid (FA) targets elongation factor G (EF-G) and inhibits ribosomal peptide elongation and ribosome recycling, but deeper mechanistic aspects of FA action have remained unknown. Using quench flow and stopped flow experiments in a biochemical system for protein synthesis and taking advantage of separate time scales for inhibited (10 s) and uninhibited (100 ms) elongation cycles, a detailed kinetic model of FA action was obtained. FA targets EF-G at an early stage in the translocation process (I), which proceeds unhindered by the presence of the drug to a later stage (II), where the ribosome stalls. Stalling may also occur at a third stage of translocation (III), just before release of EF-G from the post-translocation ribosome. We show that FA is a strong elongation inhibitor (K50% ≈ 1 μm), discuss the identity of the FA targeted states, and place existing cryo-EM and crystal structures in their functional context.

  6. Three-dimensional structure of the ribosomal translocase: elongation factor G from Thermus thermophilus.

    PubMed Central

    AEvarsson, A; Brazhnikov, E; Garber, M; Zheltonosova, J; Chirgadze, Y; al-Karadaghi, S; Svensson, L A; Liljas, A

    1994-01-01

    The crystal structure of Thermus thermophilus elongation factor G without guanine nucleotide was determined to 2.85 A. This GTPase has five domains with overall dimensions of 50 x 60 x 118 A. The GTP binding domain has a core common to other GTPases with a unique subdomain which probably functions as an intrinsic nucleotide exchange factor. Domains I and II are homologous to elongation factor Tu and their arrangement, both with and without GDP, is more similar to elongation factor Tu in complex with a GTP analogue than with GDP. Domains III and V show structural similarities to ribosomal proteins. Domain IV protrudes from the main body of the protein and has an extraordinary topology with a left-handed cross-over connection between two parallel beta-strands. Images PMID:8070397

  7. 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.

  8. Inhibition by Elongation Factor EF G of Aminoacyl-tRNA Binding to Ribosomes

    PubMed Central

    Cabrer, Bartolomé; Vázquez, David; Modolell, Juan

    1972-01-01

    Elongation factor G (EF G), bound to ribosomes either with GMPPCP or with fusidic acid and GDP, inhibits elongation factor Tu (EF Tu)-dependent binding of Phe-tRNA on the ribosome-poly(U) complex and binding of Ala-tRNA on the initiation complex formed with RNA from bacteriophage R17; GTP hydrolysis associated with Phe-tRNA binding is also inhibited. Moreover, nonenzymic binding of Phe-tRNA at high Mg++ concentration is completely blocked by EF G. Thus, EF G appears to bind at a site that overlaps or interacts with the ribosomal A-site. PMID:4551985

  9. 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.

  10. SHORT HYPOCOTYL1 Encodes a SMARCA3-Like Chromatin Remodeling Factor Regulating Elongation1[OPEN

    PubMed Central

    Bo, Kailiang; Behera, Tusar K.; Pandey, Sudhakar; Wen, Changlong; Wang, Yuhui; Simon, Philipp W.; Li, Yuhong

    2016-01-01

    In Arabidopsis (Arabidopsis thaliana), the UVR8-mediated signaling pathway is employed to attain UVB protection and acclimation to deal with low-dosage UVB (LDUVB)-induced stresses. Here, we identified SHORT HYPOCOTYL1 (SH1) in cucumber (Cucumis sativus), which regulates LDUVB-dependent hypocotyl elongation by modulating the UVR8 signaling pathway. We showed that hypocotyl elongation in cucumbers carrying the recessive sh1 allele was LDUVB insensitive and that Sh1 encoded a human SMARCA3-like chromatin remodeling factor. The allele frequency and distribution pattern at this locus among natural populations supported the wild cucumber origin of sh1 for local adaptation, which was under selection during domestication. The cultivated cucumber carries predominantly the Sh1 allele; the sh1 allele is nearly fixed in the semiwild Xishuangbanna cucumber, and the wild cucumber population is largely at Hardy-Weinberg equilibrium for the two alleles. The SH1 protein sequence was highly conserved among eukaryotic organisms, but its regulation of hypocotyl elongation in cucumber seems to be a novel function. While Sh1 expression was inhibited by LDUVB, its transcript abundance was highly correlated with hypocotyl elongation rate and the expression level of cell-elongation-related genes. Expression profiling of key regulators in the UVR8 signaling pathway revealed significant differential expression of CsHY5 between two near isogenic lines of Sh1. Sh1 and CsHY5 acted antagonistically at transcriptional level. A working model was proposed in which Sh1 regulates LDUVB-dependent hypocotyl elongation in cucumber through changing the chromatin states and thus the accessibility of CsHY5 in the UVR8 signaling pathway to promoters of LDUVB-responsive genes for hypocotyl elongation. PMID:27559036

  11. SHORT HYPOCOTYL1 Encodes a SMARCA3-Like Chromatin Remodeling Factor Regulating Elongation.

    PubMed

    Bo, Kailiang; Wang, Hui; Pan, Yupeng; Behera, Tusar K; Pandey, Sudhakar; Wen, Changlong; Wang, Yuhui; Simon, Philipp W; Li, Yuhong; Chen, Jinfeng; Weng, Yiqun

    2016-10-01

    In Arabidopsis (Arabidopsis thaliana), the UVR8-mediated signaling pathway is employed to attain UVB protection and acclimation to deal with low-dosage UVB (LDUVB)-induced stresses. Here, we identified SHORT HYPOCOTYL1 (SH1) in cucumber (Cucumis sativus), which regulates LDUVB-dependent hypocotyl elongation by modulating the UVR8 signaling pathway. We showed that hypocotyl elongation in cucumbers carrying the recessive sh1 allele was LDUVB insensitive and that Sh1 encoded a human SMARCA3-like chromatin remodeling factor. The allele frequency and distribution pattern at this locus among natural populations supported the wild cucumber origin of sh1 for local adaptation, which was under selection during domestication. The cultivated cucumber carries predominantly the Sh1 allele; the sh1 allele is nearly fixed in the semiwild Xishuangbanna cucumber, and the wild cucumber population is largely at Hardy-Weinberg equilibrium for the two alleles. The SH1 protein sequence was highly conserved among eukaryotic organisms, but its regulation of hypocotyl elongation in cucumber seems to be a novel function. While Sh1 expression was inhibited by LDUVB, its transcript abundance was highly correlated with hypocotyl elongation rate and the expression level of cell-elongation-related genes. Expression profiling of key regulators in the UVR8 signaling pathway revealed significant differential expression of CsHY5 between two near isogenic lines of Sh1 Sh1 and CsHY5 acted antagonistically at transcriptional level. A working model was proposed in which Sh1 regulates LDUVB-dependent hypocotyl elongation in cucumber through changing the chromatin states and thus the accessibility of CsHY5 in the UVR8 signaling pathway to promoters of LDUVB-responsive genes for hypocotyl elongation.

  12. Fruit flies with additional expression of the elongation factor EF-1 alpha live longer.

    PubMed Central

    Shepherd, J C; Walldorf, U; Hug, P; Gehring, W J

    1989-01-01

    In Drosophila melanogaster, the decrease in protein synthesis that accompanies aging is preceded by a decrease in elongation factor EF-1 alpha protein and mRNA. Here we show that Drosophila transformed with a P-element vector containing an EF-1 alpha gene under control of hsp70 regulatory sequences have a longer life-span than control flies. Images PMID:2508089

  13. Eukaryotic elongation factor 2 controls TNF-α translation in LPS-induced hepatitis

    PubMed Central

    González-Terán, Bárbara; Cortés, José R.; Manieri, Elisa; Matesanz, Nuria; Verdugo, ρngeles; Rodríguez, María E.; González-Rodríguez, ρgueda; Valverde, ρngela; Martín, Pilar; Davis, Roger J.; Sabio, Guadalupe

    2012-01-01

    Bacterial LPS (endotoxin) has been implicated in the pathogenesis of acute liver disease through its induction of the proinflammatory cytokine TNF-α. TNF-α is a key determinant of the outcome in a well-established mouse model of acute liver failure during septic shock. One possible mechanism for regulating TNF-α expression is through the control of protein elongation during translation, which would allow rapid cell adaptation to physiological changes. However, the regulation of translational elongation is poorly understood. We found that expression of p38γ/δ MAPK proteins is required for the elongation of nascent TNF-α protein in macrophages. The MKK3/6-p38γ/δ pathway mediated an inhibitory phosphorylation of eukaryotic elongation factor 2 (eEF2) kinase, which in turn promoted eEF2 activation (dephosphorylation) and subsequent TNF-α elongation. These results identify a new signaling pathway that regulates TNF-α production in LPS-induced liver damage and suggest potential cell-specific therapeutic targets for liver diseases in which TNF-α production is involved. PMID:23202732

  14. Transcriptional elongation factor ENL phosphorylated by ATM recruits polycomb and switches off transcription for DSB repair.

    PubMed

    Ui, Ayako; Nagaura, Yuko; Yasui, Akira

    2015-05-07

    Transcription is repressed if a DNA double-strand break (DSB) is introduced in close proximity to a transcriptional activation site at least in part by H2A-ubiquitination. While ATM signaling is involved, how it controls H2A-ubiquitination remains unclear. Here, we identify that, in response to DSBs, a transcriptional elongation factor, ENL (MLLT1), is phosphorylated by ATM at conserved SQ sites. This phosphorylation increases the interaction between ENL and the E3-ubiquitin-ligase complex of Polycomb Repressive Complex 1 (PRC1) via BMI1. This interaction promotes enrichment of PRC1 at transcription elongation sites near DSBs to ubiquitinate H2A leading to transcriptional repression. ENL SQ sites and BMI1 are necessary for KU70 accumulation at DSBs near active transcription sites and cellular resistance to DSBs. Our data suggest that ATM-dependent phosphorylation of ENL functions as switch from elongation to Polycomb-mediated repression to preserve genome integrity.

  15. Thiostrepton inhibits stable 70S ribosome binding and ribosome-dependent GTPase activation of elongation factor G and elongation factor 4

    PubMed Central

    Walter, Justin D.; Hunter, Margaret; Cobb, Melanie; Traeger, Geoff; Spiegel, P. Clint

    2012-01-01

    Thiostrepton, a macrocyclic thiopeptide antibiotic, inhibits prokaryotic translation by interfering with the function of elongation factor G (EF-G). Here, we have used 70S ribosome binding and GTP hydrolysis assays to study the effects of thiostrepton on EF-G and a newly described translation factor, elongation factor 4 (EF4). In the presence of thiostrepton, ribosome-dependent GTP hydrolysis is inhibited for both EF-G and EF4, with IC(50) values equivalent to the 70S ribosome concentration (0.15 µM). Further studies indicate the mode of thiostrepton inhibition is to abrogate the stable binding of EF-G and EF4 to the 70S ribosome. In support of this model, an EF-G truncation variant that does not possess domains IV and V was shown to possess ribosome-dependent GTP hydrolysis activity that was not affected by the presence of thiostrepton (>100 µM). Lastly, chemical footprinting was employed to examine the nature of ribosome interaction and tRNA movements associated with EF4. In the presence of non-hydrolyzable GTP, EF4 showed chemical protections similar to EF-G and stabilized a ratcheted state of the 70S ribosome. These data support the model that thiostrepton inhibits stable GTPase binding to 70S ribosomal complexes, and a model for the first step of EF4-catalyzed reverse-translocation is presented. PMID:21908407

  16. The elongation factor RfaH and the initiation factor σ bind to the same site on the transcription elongation complex

    PubMed Central

    Sevostyanova, Anastasiya; Svetlov, Vladimir; Vassylyev, Dmitry G.; Artsimovitch, Irina

    2008-01-01

    RNA polymerase is a target for numerous regulatory events in all living cells. Recent studies identified a few “hot spots” on the surface of bacterial RNA polymerase that mediate its interactions with diverse accessory proteins. Prominent among these hot spots, the β′ subunit clamp helices serve as a major binding site for the initiation factor σ and for the elongation factor RfaH. Furthermore, the two proteins interact with the nontemplate DNA strand in transcription complexes and thus may interfere with each other's activity. We show that RfaH does not inhibit transcription initiation but, once recruited to RNA polymerase, abolishes σ-dependent pausing. We argue that this apparent competition is due to a steric exclusion of σ by RfaH that is stably bound to the nontemplate DNA and clamp helices, both of which are necessary for the σ recruitment to the transcription complex. Our findings highlight the key regulatory role played by the clamp helices during both initiation and elongation stages of transcription. PMID:18195372

  17. 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

  18. Fusidic Acid Targets Elongation Factor G in Several Stages of Translocation on the Bacterial Ribosome*

    PubMed Central

    Borg, Anneli; Holm, Mikael; Shiroyama, Ikue; Hauryliuk, Vasili; Pavlov, Michael; Sanyal, Suparna; Ehrenberg, Måns

    2015-01-01

    The antibiotic fusidic acid (FA) targets elongation factor G (EF-G) and inhibits ribosomal peptide elongation and ribosome recycling, but deeper mechanistic aspects of FA action have remained unknown. Using quench flow and stopped flow experiments in a biochemical system for protein synthesis and taking advantage of separate time scales for inhibited (10 s) and uninhibited (100 ms) elongation cycles, a detailed kinetic model of FA action was obtained. FA targets EF-G at an early stage in the translocation process (I), which proceeds unhindered by the presence of the drug to a later stage (II), where the ribosome stalls. Stalling may also occur at a third stage of translocation (III), just before release of EF-G from the post-translocation ribosome. We show that FA is a strong elongation inhibitor (K50% ≈ 1 μm), discuss the identity of the FA targeted states, and place existing cryo-EM and crystal structures in their functional context. PMID:25451927

  19. 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

  20. 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.

  1. The elongation factor Spt4/5 regulates RNA polymerase II transcription through the nucleosome.

    PubMed

    Crickard, John B; Lee, Jaehyoun; Lee, Tae-Hee; Reese, Joseph C

    2017-04-03

    RNA polymerase II (RNAPII) passes through the nucleosome in a coordinated manner, generating several intermediate nucleosomal states as it breaks and then reforms histone-DNA contacts ahead of and behind it, respectively. Several studies have defined transcription-induced nucleosome intermediates using only RNA Polymerase. However, RNAPII is decorated with elongation factors as it transcribes the genome. One such factor, Spt4/5, becomes an integral component of the elongation complex, making direct contact with the 'jaws' of RNAPII and nucleic acids in the transcription scaffold. We have characterized the effect of incorporating Spt4/5 into the elongation complex on transcription through the 601R nucleosome. Spt4/5 suppressed RNAPII pausing at the major H3/H4-induced arrest point, resulting in downstream re-positioning of RNAPII further into the nucleosome. Using a novel single molecule FRET system, we found that Spt4/5 affected the kinetics of DNA re-wrapping and stabilized a nucleosomal intermediate with partially unwrapped DNA behind RNAPII. Comparison of nucleosomes of different sequence polarities suggest that the strength of the DNA-histone interactions behind RNAPII specifies the Spt4/5 requirement. We propose that Spt4/5 may be important to coordinate the mechanical movement of RNAPII through the nucleosome with co-transcriptional chromatin modifications during transcription, which is affected by the strength of histone-DNA interactions.

  2. RNA polymerase and transcription elongation factor Spt4/5 complex structure

    PubMed Central

    Klein, Brianna J.; Bose, Daniel; Baker, Kevin J.; Yusoff, Zahirah M.; Zhang, Xiaodong; Murakami, Katsuhiko S.

    2011-01-01

    Spt4/5 in archaea and eukaryote and its bacterial homolog NusG is the only elongation factor conserved in all three domains of life and plays many key roles in cotranscriptional regulation and in recruiting other factors to the elongating RNA polymerase. Here, we present the crystal structure of Spt4/5 as well as the structure of RNA polymerase-Spt4/5 complex using cryoelectron microscopy reconstruction and single particle analysis. The Spt4/5 binds in the middle of RNA polymerase claw and encloses the DNA, reminiscent of the DNA polymerase clamp and ring helicases. The transcription elongation complex model reveals that the Spt4/5 is an upstream DNA holder and contacts the nontemplate DNA in the transcription bubble. These structures reveal that the cellular RNA polymerases also use a strategy of encircling DNA to enhance its processivity as commonly observed for many nucleic acid processing enzymes including DNA polymerases and helicases. PMID:21187417

  3. Study of phosphorylation of translation elongation factor 2 (EF-2) from wheat germ.

    PubMed

    Smailov, S K; Lee, A V; Iskakov, B K

    1993-04-26

    Phosphorylation of elongation factor 2 (EF-2) by specific Ca2+/calmodulin-dependent kinase is considered as a possible mechanism of regulation of protein biosynthesis in animal cells at the level of polypeptide chain elongation. In this report we show that wheat germ EF-2 can be intensively phosphorylated by the rabbit reticulocyte EF-2 kinase. Phosphorylation results in inhibition of the activity of plant EF-2 in poly(U)-dependent cell-free translation system. Thus, the activity of EF-2 in plant cells can be potentially regulated by phosphorylation. However, we could not detect endogenous EF-2 kinase activity in wheat germ either in vitro or in vivo. Furthermore, EF-2 kinase activity is not displayed in different organs of wheat and other higher plants.

  4. Protein synthesis during cellular quiescence is inhibited by phosphorylation of a translational elongation factor.

    PubMed

    Pereira, Sandro F F; Gonzalez, Ruben L; Dworkin, Jonathan

    2015-06-23

    In nature, most organisms experience conditions that are suboptimal for growth. To survive, cells must fine-tune energy-demanding metabolic processes in response to nutrient availability. Here, we describe a novel mechanism by which protein synthesis in starved cells is down-regulated by phosphorylation of the universally conserved elongation factor Tu (EF-Tu). Phosphorylation impairs the essential GTPase activity of EF-Tu, thereby preventing its release from the ribosome. As a consequence, phosphorylated EF-Tu has a dominant-negative effect in elongation, resulting in the overall inhibition of protein synthesis. Importantly, this mechanism allows a quick and robust regulation of one of the most abundant cellular proteins. Given that the threonine that serves as the primary site of phosphorylation is conserved in all translational GTPases from bacteria to humans, this mechanism may have important implications for growth-rate control in phylogenetically diverse organisms.

  5. 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

  6. Vps factors are required for efficient transcription elongation in budding yeast.

    PubMed

    Gaur, Naseem A; Hasek, Jiri; Brickner, Donna Garvey; Qiu, Hongfang; Zhang, Fan; Wong, Chi-Ming; Malcova, Ivana; Vasicova, Pavla; Brickner, Jason H; Hinnebusch, Alan G

    2013-03-01

    There is increasing evidence that certain Vacuolar protein sorting (Vps) proteins, factors that mediate vesicular protein trafficking, have additional roles in regulating transcription factors at the endosome. We found that yeast mutants lacking the phosphatidylinositol 3-phosphate [PI(3)P] kinase Vps34 or its associated protein kinase Vps15 display multiple phenotypes indicating impaired transcription elongation. These phenotypes include reduced mRNA production from long or G+C-rich coding sequences (CDS) without affecting the associated GAL1 promoter activity, and a reduced rate of RNA polymerase II (Pol II) progression through lacZ CDS in vivo. Consistent with reported genetic interactions with mutations affecting the histone acetyltransferase complex NuA4, vps15Δ and vps34Δ mutations reduce NuA4 occupancy in certain transcribed CDS. vps15Δ and vps34Δ mutants also exhibit impaired localization of the induced GAL1 gene to the nuclear periphery. We found unexpectedly that, similar to known transcription elongation factors, these and several other Vps factors can be cross-linked to the CDS of genes induced by Gcn4 or Gal4 in a manner dependent on transcriptional induction and stimulated by Cdk7/Kin28-dependent phosphorylation of the Pol II C-terminal domain (CTD). We also observed colocalization of a fraction of Vps15-GFP and Vps34-GFP with nuclear pores at nucleus-vacuole (NV) junctions in live cells. These findings suggest that Vps factors enhance the efficiency of transcription elongation in a manner involving their physical proximity to nuclear pores and transcribed chromatin.

  7. The cauliflower Orange gene enhances petiole elongation by suppressing expression of eukaryotic release factor 1.

    PubMed

    Zhou, Xiangjun; Sun, Tian-Hu; Wang, Ning; Ling, Hong-Qing; Lu, Shan; Li, Li

    2011-04-01

    The cauliflower (Brassica oleracea var. botrytis) Orange (Or) gene affects plant growth and development in addition to conferring β-carotene accumulation. This study was undertaken to investigate the molecular basis for the effects of the Or gene mutation in on plant growth. The OR protein was found to interact with cauliflower and Arabidopsis eukaryotic release factor 1-2 (eRF1-2), a member of the eRF1 family, by yeast two-hybrid analysis and by bimolecular fluorescence complementation (BiFC) assay. Concomitantly, the Or mutant showed reduced expression of the BoeRF1 family genes. Transgenic cauliflower plants with suppressed expression of BoeRF1-2 and BoeRF1-3 were generated by RNA interference. Like the Or mutant, the BoeRF1 RNAi lines showed increased elongation of the leaf petiole. This long-petiole phenotype was largely caused by enhanced cell elongation, which resulted from increased cell length and elevated expression of genes involved in cell-wall loosening. These findings demonstrate that the cauliflower Or gene controls petiole elongation by suppressing the expression of eRF1 genes, and provide new insights into the molecular mechanism of leaf petiole regulation.

  8. Visualization of positive transcription elongation factor b (P-TEFb) activation in living cells.

    PubMed

    Fujinaga, Koh; Luo, Zeping; Schaufele, Fred; Peterlin, B Matija

    2015-01-16

    Regulation of transcription elongation by positive transcription elongation factor b (P-TEFb) plays a central role in determining the state of cell activation, proliferation, and differentiation. In cells, P-TEFb exists in active and inactive forms. Its release from the inactive 7SK small nuclear ribonucleoprotein complex is a critical step for P-TEFb to activate transcription elongation. However, no good method exists to analyze this P-TEFb equilibrium in living cells. Only inaccurate and labor-intensive cell-free biochemical assays are currently available. In this study, we present the first experimental system to monitor P-TEFb activation in living cells. We created a bimolecular fluorescence complementation assay to detect interactions between P-TEFb and its substrate, the C-terminal domain of RNA polymerase II. When cells were treated with suberoylanilide hydroxamic acid, which releases P-TEFb from the 7SK small nuclear ribonucleoprotein, they turned green. Other known P-TEFb-releasing agents, including histone deacetylase inhibitors, bromodomain and extraterminal bromodomain inhibitors, and protein kinase C agonists, also scored positive in this assay. Finally, we identified 5'-azacytidine as a new P-TEFb-releasing agent. This release of P-TEFb correlated directly with activation of human HIV and HEXIM1 transcription. Thus, our visualization of P-TEFb activation by fluorescent complementation assay could be used to find new P-TEFb-releasing agents, compare different classes of agents, and assess their efficacy singly and/or in combination.

  9. Elongation Factor 2 Kinase Is Regulated by Proline Hydroxylation and Protects Cells during Hypoxia.

    PubMed

    Moore, Claire E J; Mikolajek, Halina; Regufe da Mota, Sergio; Wang, Xuemin; Kenney, Justin W; Werner, Jörn M; Proud, Christopher G

    2015-05-01

    Protein synthesis, especially translation elongation, requires large amounts of energy, which is often generated by oxidative metabolism. Elongation is controlled by phosphorylation of eukaryotic elongation factor 2 (eEF2), which inhibits its activity and is catalyzed by eEF2 kinase (eEF2K), a calcium/calmodulin-dependent α-kinase. Hypoxia causes the activation of eEF2K and induces eEF2 phosphorylation independently of previously known inputs into eEF2K. Here, we show that eEF2K is subject to hydroxylation on proline-98. Proline hydroxylation is catalyzed by proline hydroxylases, oxygen-dependent enzymes which are inactivated during hypoxia. Pharmacological inhibition of proline hydroxylases also stimulates eEF2 phosphorylation. Pro98 lies in a universally conserved linker between the calmodulin-binding and catalytic domains of eEF2K. Its hydroxylation partially impairs the binding of calmodulin to eEF2K and markedly limits the calmodulin-stimulated activity of eEF2K. Neuronal cells depend on oxygen, and eEF2K helps to protect them from hypoxia. eEF2K is the first example of a protein directly involved in a major energy-consuming process to be regulated by proline hydroxylation. Since eEF2K is cytoprotective during hypoxia and other conditions of nutrient insufficiency, it may be a valuable target for therapy of poorly vascularized solid tumors.

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

    PubMed

    Kamikawa, Ryoma; Inagaki, Yuji; Sako, Yoshihiko

    2008-05-13

    Genes encoding elongation factor-like (EFL) proteins, which show high similarity to elongation factor-1alpha (EF-1alpha), have been found in phylogenetically distantly related eukaryotes. The sporadic distribution of "EFL-containing" lineages within "EF-1alpha-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-1alpha and EFL genes and secondary EFL gene loss in Phaeodactylum tricornutum, the complete genome of which encodes only the EF-1alpha 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-1alpha.

  11. 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

  12. 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

  13. Activation of contact-dependent antibacterial tRNase toxins by translation elongation factors.

    PubMed

    Jones, Allison M; Garza-Sánchez, Fernando; So, Jaime; Hayes, Christopher S; Low, David A

    2017-03-07

    Contact-dependent growth inhibition (CDI) is a mechanism by which bacteria exchange toxins via direct cell-to-cell contact. CDI systems are distributed widely among Gram-negative pathogens and are thought to mediate interstrain competition. Here, we describe tsf mutations that alter the coiled-coil domain of elongation factor Ts (EF-Ts) and confer resistance to the CdiA-CT(EC869) tRNase toxin from enterohemorrhagic Escherichia coli EC869. Although EF-Ts is required for toxicity in vivo, our results indicate that it is dispensable for tRNase activity in vitro. We find that CdiA-CT(EC869) binds to elongation factor Tu (EF-Tu) with high affinity and this interaction is critical for nuclease activity. Moreover, in vitro tRNase activity is GTP-dependent, suggesting that CdiA-CT(EC869) only cleaves tRNA in the context of translationally active GTP·EF-Tu·tRNA ternary complexes. We propose that EF-Ts promotes the formation of GTP·EF-Tu·tRNA ternary complexes, thereby accelerating substrate turnover for rapid depletion of target-cell tRNA.

  14. 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

  15. O-GlcNAcase Is an RNA Polymerase II Elongation Factor Coupled to Pausing Factors SPT5 and TIF1β.

    PubMed

    Resto, Melissa; Kim, Bong-Hyun; Fernandez, Alfonso G; Abraham, Brian J; Zhao, Keji; Lewis, Brian A

    2016-10-21

    We describe here the identification and functional characterization of the enzyme O-GlcNAcase (OGA) as an RNA polymerase II elongation factor. Using in vitro transcription elongation assays, we show that OGA activity is required for elongation in a crude nuclear extract system, whereas in a purified system devoid of OGA the addition of rOGA inhibited elongation. Furthermore, OGA is physically associated with the known RNA polymerase II (pol II) pausing/elongation factors SPT5 and TRIM28-KAP1-TIF1β, and a purified OGA-SPT5-TIF1β complex has elongation properties. Lastly, ChIP-seq experiments show that OGA maps to the transcriptional start site/5' ends of genes, showing considerable overlap with RNA pol II, SPT5, TRIM28-KAP1-TIF1β, and O-GlcNAc itself. These data all point to OGA as a component of the RNA pol II elongation machinery regulating elongation genome-wide. Our results add a novel and unexpected dimension to the regulation of elongation by the insertion of O-GlcNAc cycling into the pol II elongation regulatory dynamics.

  16. Allosteric control of the RNA polymerase by the elongation factor RfaH

    PubMed Central

    Svetlov, Vladimir; Belogurov, Georgiy A.; Shabrova, Elena; Vassylyev, Dmitry G.; Artsimovitch, Irina

    2007-01-01

    Efficient transcription of long polycistronic operons in bacteria frequently relies on accessory proteins but their molecular mechanisms remain obscure. RfaH is a cellular elongation factor that acts as a polarity suppressor by increasing RNA polymerase (RNAP) processivity. In this work, we provide evidence that RfaH acts by reducing transcriptional pausing at certain positions rather than by accelerating RNAP at all sites. We show that ‘fast’ RNAP variants are characterized by pause-free RNA chain elongation and are resistant to RfaH action. Similarly, the wild-type RNAP is insensitive to RfaH in the absence of pauses. In contrast, those enzymes that may be prone to falling into a paused state are hypersensitive to RfaH. RfaH inhibits pyrophosphorolysis of the nascent RNA and reduces the apparent Michaelis–Menten constant for nucleotides, suggesting that it stabilizes the post-translocated, active RNAP state. Given that the RfaH-binding site is located 75 Å away from the RNAP catalytic center, these results strongly indicate that RfaH acts allosterically. We argue that despite the apparent differences in the nucleic acid targets, the time of recruitment and the binding sites on RNAP, unrelated antiterminators (such as RfaH and λQ) utilize common strategies during both recruitment and anti-pausing modification of the transcription complex. PMID:17711918

  17. 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

  18. Adduct formation of 4-hydroxynonenal and malondialdehyde with elongation factor-2 in vitro and in vivo.

    PubMed

    Argüelles, Sandro; Machado, Alberto; Ayala, Antonio

    2009-08-01

    Protein synthesis is universally affected by aging in all organisms. There is no clear consensus about the mechanism underlying the decline of translation with aging. Previous reports from our laboratory have shown that the elongation step is especially affected with aging as a consequence of alterations in elongation factor-2 (eEF-2), the monomeric protein that catalyzes the movement of the ribosome along the mRNA during protein synthesis. eEF-2 seems to be specifically affected by lipid peroxidant compounds, which concomitantly produce several reactive, toxic aldehydes, such as MDA and HNE. These aldehydes are able to form adducts with proteins that lead to their inactivation. In this paper we studied the formation of adducts between MDA or HNE and eEF-2. The study was performed both in vitro, using liver homogenates treated with cumene hydroperoxide, and in vivo using young control rats, treated with the same oxidant, and 12-and 24-month-old rats. In all cases we found a decrease in the levels of eEF-2, an increase in the amount of lipid peroxidation, and a concomitant formation of adducts between eEF-2 and MDA or HNE. The results suggest that one possible mechanism responsible for the decline of protein synthesis during aging could be the alteration in eEF-2 levels, secondary to lipid peroxidation and adduct formation with these aldehydes.

  19. 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

  20. Characterisation of the rubber elongation factor from ammoniated latex by electrophoresis and mass spectrometry.

    PubMed

    Dürauer, A; Csaszar, E; Mechtler, K; Jungbauer, A; Schmid, E

    2000-08-18

    Rubber elongation factor (REF) is considered as one of the major allergens present in latex. An extraction and purification protocol for preparation of REF standards has been modified. A protein fraction was extracted from ammoniated latex sap and purified by gel filtration chromatography. The purified and concentrated proteins were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis into two major bands. These bands were further characterised by matrix-assisted laser desorption/ionisation time-of-flight and nano-electrospray ionization mass spectrometry. REF and a truncated form could be ascertained by the mass and fragmentation pattern of the tryptic peptides. In the faster migrating band an additional peptide could be identified. This peptide is also present in Hevb3 and a Mr 27000 latex allergen. Our findings indicate that conventional REF preparations as standards may contain additional allergenic proteins.

  1. Immunospecific responses to bacterial elongation factor Tu during Burkholderia infection and immunization.

    PubMed

    Nieves, Wildaliz; Heang, Julie; Asakrah, Saja; Höner zu Bentrup, Kerstin; Roy, Chad J; Morici, Lisa A

    2010-12-17

    Burkholderia pseudomallei is the etiological agent of melioidosis, a disease endemic in parts of Southeast Asia and Northern Australia. Currently there is no licensed vaccine against infection with this biological threat agent. In this study, we employed an immunoproteomic approach and identified bacterial Elongation factor-Tu (EF-Tu) as a potential vaccine antigen. EF-Tu is membrane-associated, secreted in outer membrane vesicles (OMVs), and immunogenic during Burkholderia infection in the murine model of melioidosis. Active immunization with EF-Tu induced antigen-specific antibody and cell-mediated immune responses in mice. Mucosal immunization with EF-Tu also reduced lung bacterial loads in mice challenged with aerosolized B. thailandensis. Our data support the utility of EF-Tu as a novel vaccine immunogen against bacterial infection.

  2. 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

  3. An interbacterial NAD(P)(+) glycohydrolase toxin requires elongation factor Tu for delivery to target cells.

    PubMed

    Whitney, John C; Quentin, Dennis; Sawai, Shin; LeRoux, Michele; Harding, Brittany N; Ledvina, Hannah E; Tran, Bao Q; Robinson, Howard; Goo, Young Ah; Goodlett, David R; Raunser, Stefan; Mougous, Joseph D

    2015-10-22

    Type VI secretion (T6S) influences the composition of microbial communities by catalyzing the delivery of toxins between adjacent bacterial cells. Here, we demonstrate that a T6S integral membrane toxin from Pseudomonas aeruginosa, Tse6, acts on target cells by degrading the universally essential dinucleotides NAD(+) and NADP(+). Structural analyses of Tse6 show that it resembles mono-ADP-ribosyltransferase proteins, such as diphtheria toxin, with the exception of a unique loop that both excludes proteinaceous ADP-ribose acceptors and contributes to hydrolysis. We find that entry of Tse6 into target cells requires its binding to an essential housekeeping protein, translation elongation factor Tu (EF-Tu). These proteins participate in a larger assembly that additionally directs toxin export and provides chaperone activity. Visualization of this complex by electron microscopy defines the architecture of a toxin-loaded T6S apparatus and provides mechanistic insight into intercellular membrane protein delivery between bacteria.

  4. "In vitro" effect of cumene hydroperoxide on hepatic elongation factor-2 and its protection by melatonin.

    PubMed

    Parrado, J; Absi, E H; Machado, A; Ayala, A

    2003-12-05

    We have examined by immunoblotting the effect of three oxidant compounds on the level of hepatic elongation factor-2 (eEF-2). Rat liver homogenates were exposed to cumene hydroperoxide (CH), 2-2'-azobis (2-aminopropane) dihydrochloride (AAPH) and H(2)O(2). Only CH treatment produced the disappearance of eEF-2, probably due to a phenomena of peptide bond cleavage. The direct implication of free radical species in this process is evident because of the fact that the inclusion of a free radical scavenger such as melatonin prevented the eEF-2 depletion. The results also suggest that the disappearance of eEF-2 induced by CH can be linked to a lipid peroxidant process, which could account for the decline of protein synthesis in aging and other circumstances where lipid peroxidation is high.

  5. "In vitro" effect of lipid peroxidation metabolites on elongation factor-2.

    PubMed

    Argüelles, Sandro; Machado, Alberto; Ayala, Antonio

    2006-03-01

    Elongation Factor-2 (eEF-2) is the protein that catalyzes the translocation of the ribosome through mRNA. Not all oxidants affect eEF-2, which is extremely sensitive to oxidative stress caused mainly by lipid peroxidant compounds such as cumene hydroperoxide and t-butyl hydroperoxide. Lipid peroxides constitute a potential hazard to living organisms because of their direct reactivity with a variety of biomolecules and the ability to decompose into free radicals and reactive aldehydes. In this "in vitro" study, we show the effect of three of these aldehydes on the levels of hepatic eEF-2. The results suggest that the toxicity associated with prooxidant-mediated hepatic lipid peroxidation on protein synthesis can originate from the interaction of the aldehydic end products of lipid peroxidation with eEF-2.

  6. Effect of aging and oxidative stress on elongation factor-2 in hypothalamus and hypophysis.

    PubMed

    Argüelles, Sandro; Cano, Mercedes; Machado, Alberto; Ayala, Antonio

    2011-01-01

    The hypothalamic-hypophysis system (HHS) secretes peptide hormones whose synthesis requires the integrity of the translation machinery. As the organisms age, a considerable diminution of the protein synthesis takes place in several tissues. Among the possible causes of the decline of translation in old animals are the modifications of elongation factor-2 (eEF-2). We studied whether the level of this protein was affected in the HHS in old animals. The effects of aging are compared to those of an oxidant compound (cumene hydroperoxide) administered to young rats. The results indicate that oxidative stress could be involved in the alterations of eEF-2, which forms adducts with malondialdehyde (MDA) and 4-hydroxynonenal (HNE). The alterations of eEF-2 levels, secondary to lipid peroxidation and adduct formation with these aldehydes could contribute to the suboptimal hormone production from these tissues during aging. Besides eEF-2, proteomic analysis shows that several other proteins are affected.

  7. The structure of the ribosome with elongation factor G trapped in the post-translocational state

    PubMed Central

    Gao, Yong-Gui; Selmer, Maria; Dunham, Christine M.; Weixlbaumer, Albert; Kelley, Ann C.; Ramakrishnan, V.

    2013-01-01

    Elongation factor G (EF-G) is a GTPase that plays a crucial role in the translocation of tRNAs and mRNA during translation by the ribosome. We report a crystal structure refined to 3.6 Å resolution of the ribosome trapped with EF-G in the post-translocational state using the antibiotic fusidic acid. Fusidic acid traps EF-G in a conformation intermediate between the GTP and GDP forms. The interaction of EF-G with ribosomal elements implicated in stimulating catalysis, such as the L10-L12 stalk and the L11 region, and of domain IV of EF-G with P-site tRNA and mRNA shed light on various aspects of EF-G function in catalysis and translocation. The stabilization of the mobile stalks of the ribosome also results in a more complete description of its structure. PMID:19833919

  8. 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

  9. Effect of oxidative stress, produced by cumene hydroperoxide, on the various steps of protein synthesis. Modifications of elongation factor-2.

    PubMed

    Ayala, A; Parrado, J; Bougria, M; Machado, A

    1996-09-20

    We have studied the effect of oxidative stress on protein synthesis in rat liver. Cumene hydroperoxide (CH) was used as an oxidant agent. The approach used was to determine the ribosomal state of aggregation and the time for assembly and release of polypeptide chains in the process of protein synthesis in rat liver in vivo. The results suggest that the elongation step is the most sensitive to CH treatment. The measurement of both carbonyl groups content and ADP-ribosylatable elongation factor 2 (EF-2), the main protein involved in the elongation step, indicates that under CH treatment EF-2 is oxidatively modified and a lower amount of active EF-2 is present. These results are corroborated by in vitro oxidation of EF-2 and could explain for the decline in the elongation step.

  10. Conserved discrimination against misacylated tRNAs by two mesophilic elongation factor Tu orthologs.

    PubMed

    Cathopoulis, Terry J T; Chuawong, Pitak; Hendrickson, Tamara L

    2008-07-22

    Elongation factor Tu (EF-Tu) binds and loads elongating aminoacyl-tRNAs (aa-tRNAs) onto the ribosome for protein biosynthesis. Many bacteria biosynthesize Gln-tRNA (Gln) and Asn-tRNA (Asn) by an indirect, two-step pathway that relies on the misacylated tRNAs Glu-tRNA (Gln) and Asp-tRNA (Asn) as intermediates. Previous thermodynamic and experimental analyses have demonstrated that Thermus thermophilus EF-Tu does not bind Asp-tRNA (Asn) and predicted a similar discriminatory response against Glu-tRNA (Gln) [Asahara, H., and Uhlenbeck, O. (2005) Biochemistry 46, 6194-6200; Roy, H., et al. (2007) Nucleic Acids Res. 35, 3420-3430]. By discriminating against these misacylated tRNAS, EF-Tu plays a direct role in preventing misincorporation of aspartate and glutamate into proteins at asparagine and glutamine codons. Here we report the characterization of two different mesophilic EF-Tu orthologs, one from Escherichia coli, a bacterium that does not utilize either Glu-tRNA (Gln) or Asp-tRNA (Asn), and the second from Helicobacter pylori, an organism in which both misacylated tRNAs are essential. Both EF-Tu orthologs discriminate against these misacylated tRNAs, confirming the prediction that Glu-tRNA (Gln), like Asp-tRNA (Asn), will not form a complex with EF-Tu. These results also demonstrate that the capacity of EF-Tu to discriminate against both of these aminoacyl-tRNAs is conserved even in bacteria like E. coli that do not generate either misacylated tRNA.

  11. Circadian clock regulation of mRNA translation through eukaryotic elongation factor eEF-2

    PubMed Central

    Caster, Stephen Z.; Castillo, Kathrina; Sachs, Matthew S.; Bell-Pedersen, Deborah

    2016-01-01

    The circadian clock has a profound effect on gene regulation, controlling rhythmic transcript accumulation for up to half of expressed genes in eukaryotes. Evidence also exists for clock control of mRNA translation, but the extent and mechanisms for this regulation are not known. In Neurospora crassa, the circadian clock generates daily rhythms in the activation of conserved mitogen-activated protein kinase (MAPK) pathways when cells are grown in constant conditions, including rhythmic activation of the well-characterized p38 osmosensing (OS) MAPK pathway. Rhythmic phosphorylation of the MAPK OS-2 (P-OS-2) leads to temporal control of downstream targets of OS-2. We show that osmotic stress in N. crassa induced the phosphorylation of a eukaryotic elongation factor-2 (eEF-2) kinase, radiation sensitivity complementing kinase-2 (RCK-2), and that RCK-2 is necessary for high-level phosphorylation of eEF-2, a key regulator of translation elongation. The levels of phosphorylated RCK-2 and phosphorylated eEF-2 cycle in abundance in wild-type cells but not in cells deleted for OS-2 or the core clock component FREQUENCY (FRQ). Translation extracts from cells grown in constant conditions show decreased translational activity in the late subjective morning, coincident with the peak in eEF-2 phosphorylation, and rhythmic translation of glutathione S-transferase (GST-3) from constitutive mRNA levels in vivo is dependent on circadian regulation of eEF-2 activity. In contrast, rhythms in phosphorylated eEF-2 levels are not necessary for rhythms in accumulation of the clock protein FRQ, indicating that clock control of eEF-2 activity promotes rhythmic translation of specific mRNAs. PMID:27506798

  12. Eukaryotic Elongation Factor 2 Kinase Activity Is Controlled by Multiple Inputs from Oncogenic Signaling

    PubMed Central

    Wang, Xuemin; Regufe da Mota, Sergio; Liu, Rui; Moore, Claire E.; Xie, Jianling; Lanucara, Francesco; Agarwala, Usha; Pyr dit Ruys, Sébastien; Vertommen, Didier; Rider, Mark H.; Eyers, Claire E.

    2014-01-01

    Eukaryotic elongation factor 2 kinase (eEF2K), an atypical calmodulin-dependent protein kinase, phosphorylates and inhibits eEF2, slowing down translation elongation. eEF2K contains an N-terminal catalytic domain, a C-terminal α-helical region and a linker containing several regulatory phosphorylation sites. eEF2K is expressed at high levels in certain cancers, where it may act to help cell survival, e.g., during nutrient starvation. However, it is a negative regulator of protein synthesis and thus cell growth, suggesting that cancer cells may possess mechanisms to inhibit eEF2K under good growth conditions, to allow protein synthesis to proceed. We show here that the mTORC1 pathway and the oncogenic Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway cooperate to restrict eEF2K activity. We identify multiple sites in eEF2K whose phosphorylation is regulated by mTORC1 and/or ERK, including new ones in the linker region. We demonstrate that certain sites are phosphorylated directly by mTOR or ERK. Our data reveal that glycogen synthase kinase 3 signaling also regulates eEF2 phosphorylation. In addition, we show that phosphorylation sites remote from the N-terminal calmodulin-binding motif regulate the phosphorylation of N-terminal sites that control CaM binding. Mutations in the former sites, which occur in cancer cells, cause the activation of eEF2K. eEF2K is thus regulated by a network of oncogenic signaling pathways. PMID:25182533

  13. Direct evidence of an elongation factor-Tu/Ts·GTP·Aminoacyl-tRNA quaternary complex.

    PubMed

    Burnett, Benjamin J; Altman, Roger B; Ferguson, Angelica; Wasserman, Michael R; Zhou, Zhou; Blanchard, Scott C

    2014-08-22

    During protein synthesis, elongation factor-Tu (EF-Tu) bound to GTP chaperones the entry of aminoacyl-tRNA (aa-tRNA) into actively translating ribosomes. In so doing, EF-Tu increases the rate and fidelity of the translation mechanism. Recent evidence suggests that EF-Ts, the guanosine nucleotide exchange factor for EF-Tu, directly accelerates both the formation and dissociation of the EF-Tu-GTP-Phe-tRNA(Phe) ternary complex (Burnett, B. J., Altman, R. B., Ferrao, R., Alejo, J. L., Kaur, N., Kanji, J., and Blanchard, S. C. (2013) J. Biol. Chem. 288, 13917-13928). A central feature of this model is the existence of a quaternary complex of EF-Tu/Ts·GTP·aa-tRNA(aa). Here, through comparative investigations of phenylalanyl, methionyl, and arginyl ternary complexes, and the development of a strategy to monitor their formation and decay using fluorescence resonance energy transfer, we reveal the generality of this newly described EF-Ts function and the first direct evidence of the transient quaternary complex species. These findings suggest that EF-Ts may regulate ternary complex abundance in the cell through mechanisms that are distinct from its guanosine nucleotide exchange factor functions.

  14. Eukaryotic elongation factor 2 kinase regulates the synthesis of microtubule-related proteins in neurons.

    PubMed

    Kenney, Justin W; Genheden, Maja; Moon, Kyung-Mee; Wang, Xuemin; Foster, Leonard J; Proud, Christopher G

    2016-01-01

    Modulation of the elongation phase of protein synthesis is important for numerous physiological processes in both neurons and other cell types. Elongation is primarily regulated via eukaryotic elongation factor 2 kinase (eEF2K). However, the consequence of altering eEF2K activity on the synthesis of specific proteins is largely unknown. Using both pharmacological and genetic manipulations of eEF2K combined with two protein-labeling techniques, stable isotope labeling of amino acids in cell culture and bio-orthogonal non-canonical amino acid tagging, we identified a subset of proteins whose synthesis is sensitive to inhibition of eEF2K in murine primary cortical neurons. Gene ontology (GO) analyses indicated that processes related to microtubules are particularly sensitive to eEF2K inhibition. Our findings suggest that eEF2K likely contributes to neuronal function by regulating the synthesis of microtubule-related proteins. Modulation of the elongation phase of protein synthesis is important for numerous physiological processes in neurons. Here, using labeling of new proteins coupled with proteomic techniques in primary cortical neurons, we find that the synthesis of microtubule-related proteins is up-regulated by inhibition of elongation. This suggests that translation elongation is a key regulator of cytoskeletal dynamics in neurons.

  15. SHORT HYPOCOTYL 1 encodes a SMARCA3-like chromatin remodeling factor regulating elongation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Understanding the mechanisms and control of hypocotyl elongation is important for greenhouse vegetable crop production. In this study, we identified SHORT HYPOCOTYL1 (SH1) in cucumber which regulates low-dosage ultraviolet B (LDUVB)-dependent hypocotyl elongation by recruiting the cucumber UVR8 sign...

  16. The Potyviral P3 Protein Targets Eukaryotic Elongation Factor 1A to Promote the Unfolded Protein Response and Viral Pathogenesis.

    PubMed

    Luan, Hexiang; Shine, M B; Cui, Xiaoyan; Chen, Xin; Ma, Na; Kachroo, Pradeep; Zhi, Haijan; Kachroo, Aardra

    2016-09-01

    The biochemical function of the potyviral P3 protein is not known, although it is known to regulate virus replication, movement, and pathogenesis. We show that P3, the putative virulence determinant of soybean mosaic virus (SMV), targets a component of the translation elongation complex in soybean. Eukaryotic elongation factor 1A (eEF1A), a well-known host factor in viral pathogenesis, is essential for SMV virulence and the associated unfolded protein response (UPR). Silencing GmEF1A inhibits accumulation of SMV and another ER-associated virus in soybean. Conversely, endoplasmic reticulum (ER) stress-inducing chemicals promote SMV accumulation in wild-type, but not GmEF1A-knockdown, plants. Knockdown of genes encoding the eEF1B isoform, which is important for eEF1A function in translation elongation, has similar effects on UPR and SMV resistance, suggesting a link to translation elongation. P3 and GmEF1A promote each other's nuclear localization, similar to the nuclear-cytoplasmic transport of eEF1A by the Human immunodeficiency virus 1 Nef protein. Our results suggest that P3 targets host elongation factors resulting in UPR, which in turn facilitates SMV replication and place eEF1A upstream of BiP in the ER stress response during pathogen infection.

  17. Evolutionarily Conserved Binding of Translationally Controlled Tumor Protein to Eukaryotic Elongation Factor 1B*

    PubMed Central

    Wu, Huiwen; Gong, Weibin; Yao, Xingzhe; Wang, Jinfeng; Perrett, Sarah; Feng, Yingang

    2015-01-01

    Translationally controlled tumor protein (TCTP) is an abundant protein that is highly conserved in eukaryotes. However, its primary function is still not clear. Human TCTP interacts with the metazoan-specific eukaryotic elongation factor 1Bδ (eEF1Bδ) and inhibits its guanine nucleotide exchange factor (GEF) activity, but the structural mechanism remains unknown. The interaction between TCTP and eEF1Bδ was investigated by NMR titration, structure determination, paramagnetic relaxation enhancement, site-directed mutagenesis, isothermal titration calorimetry, and HADDOCK docking. We first demonstrated that the catalytic GEF domain of eEF1Bδ is not responsible for binding to TCTP but rather a previously unnoticed central acidic region (CAR) domain in eEF1Bδ. The mutagenesis data and the structural model of the TCTP-eEF1Bδ CAR domain complex revealed the key binding residues. These residues are highly conserved in eukaryotic TCTPs and in eEF1B GEFs, including the eukaryotically conserved eEF1Bα, implying the interaction may be conserved in all eukaryotes. Interactions were confirmed between TCTP and the eEF1Bα CAR domain for human, fission yeast, and unicellular photosynthetic microalgal proteins, suggesting that involvement in protein translation through the conserved interaction with eEF1B represents a primary function of TCTP. PMID:25635048

  18. Cloning, overexpression and purification of Bacillus subtilis elongation factor Tu in Escherichia coli.

    PubMed

    Kim, S I; Kim, H Y; Kwak, J H; Kwon, S H; Lee, S Y

    2000-02-29

    To establish the overexpression and one-step purification system of Bacillus subtilis elongation factor-Tu (EF-Tu), the EF-Tu gene was amplified with or without own ribosome binding site (rbs) by PCR and the only PCR product without rbs was subcloned successfully. For the expression of the EF-Tu gene cloned after PCR amplification, a constitutive expression system and inducible expression system with His6 tag at N-terminus or C-terminus, or glutathione-S-transferase (GST) fusion system were examined in E. coli and B. subtilis. Except GST fusion system in E. coli, however, all other trials were unsuccessful at the step of plasmid construction for the EF-Tu expression. The GST/EF-Tu fusion proteins were highly expressed by IPTG induction and obtained as both soluble and insoluble form. From the soluble GST/EF-Tu fusion protein, EF-Tu was obtained to near homogeneity by one-step purification with glutathione-sepharose affinity column chromatography followed by factor Xa treatment. The purified EF-Tu showed high GDP binding activity. These results indicate that the GST/EF-Tu fusion system is favorable to overexpression and purification of B. subtilis EF-Tu.

  19. A Novel Protein Domain Induces High Affinity Selenocysteine Insertion Sequence Binding and Elongation Factor Recruitment*

    PubMed Central

    Donovan, Jesse; Caban, Kelvin; Ranaweera, Ruchira; Gonzalez-Flores, Jonathan N.; Copeland, Paul R.

    2008-01-01

    Selenocysteine (Sec) is incorporated at UGA codons in mRNAs possessing a Sec insertion sequence (SECIS) element in their 3′-untranslated region. At least three additional factors are necessary for Sec incorporation: SECIS-binding protein 2 (SBP2), Sec-tRNASec, and a Sec-specific translation elongation factor (eEFSec). The C-terminal half of SBP2 is sufficient to promote Sec incorporation in vitro, which is carried out by the concerted action of a novel Sec incorporation domain and an L7Ae RNA-binding domain. Using alanine scanning mutagenesis, we show that two distinct regions of the Sec incorporation domain are required for Sec incorporation. Physical separation of the Sec incorporation and RNA-binding domains revealed that they are able to function in trans and established a novel role of the Sec incorporation domain in promoting SECIS and eEFSec binding to the SBP2 RNA-binding domain. We propose a model in which SECIS binding induces a conformational change in SBP2 that recruits eEFSec, which in concert with the Sec incorporation domain gains access to the ribosomal A site. PMID:18948268

  20. Elongation factor-P at the crossroads of the host-endosymbiont interface

    PubMed Central

    Rajkovic, Andrei; Witzky, Anne; Navarre, William; Darwin, Andrew J.; Ibba, Michael

    2015-01-01

    Elongation factor P (EF-P) is an ancient bacterial translational factor that aids the ribosome in polymerizing oligo-prolines. EF-P structurally resembles tRNA and binds in-between the exit and peptidyl sites of the ribosome to accelerate the intrinsically slow reaction of peptidyl-prolyl bond formation. Recent studies have identified in separate organisms, two evolutionarily convergent EF-P post-translational modification systems (EPMS), split predominantly between gammaproteobacteria, and betaproteobacteria. In both cases EF-P receives a post-translational modification, critical for its function, on a highly conserved residue that protrudes into the peptidyl-transfer center of the ribosome. EPMSs are comprised of a gene(s) that synthesizes the precursor molecule used in modifying EF-P, and a gene(s) encoding an enzyme that reacts with the precursor molecule to catalyze covalent attachment to EF-P. However, not all organisms genetically encode a complete EPMS. For instance, some symbiotic bacteria harbor efp and the corresponding gene that enzymatically attaches the modification, but lack the ability to synthesize the substrate used in the modification reaction. Here we highlight the recent discoveries made regarding EPMSs, with a focus on how these incomplete modification pathways shape or have been shaped by the endosymbiont-host relationship. PMID:28357263

  1. Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis

    PubMed Central

    Villa, Elizabeth; Sengupta, Jayati; Trabuco, Leonardo G.; LeBarron, Jamie; Baxter, William T.; Shaikh, Tanvir R.; Grassucci, Robert A.; Nissen, Poul; Ehrenberg, Måns; Schulten, Klaus; Frank, Joachim

    2009-01-01

    In translation, elongation factor Tu (EF-Tu) molecules deliver aminoacyl-tRNAs to the mRNA-programmed ribosome. The GTPase activity of EF-Tu is triggered by ribosome-induced conformational changes of the factor that play a pivotal role in the selection of the cognate aminoacyl-tRNAs. We present a 6.7-Å cryo-electron microscopy map of the aminoacyl-tRNA·EF-Tu·GDP·kirromycin-bound Escherichia coli ribosome, together with an atomic model of the complex obtained through molecular dynamics flexible fitting. The model reveals the conformational changes in the conserved GTPase switch regions of EF-Tu that trigger hydrolysis of GTP, along with key interactions, including those between the sarcin-ricin loop and the P loop of EF-Tu, and between the effector loop of EF-Tu and a conserved region of the 16S rRNA. Our data suggest that GTP hydrolysis on EF-Tu is controlled through a hydrophobic gate mechanism. PMID:19122150

  2. 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

  3. Evolution of elongation factor-like (EFL) protein in Rhizaria is revised by radiolarian EFL gene sequences.

    PubMed

    Ishitani, Yoshiyuki; Kamikawa, Ryoma; Yabuki, Akinori; Tsuchiya, Masashi; Inagaki, Yuji; Takishita, Kiyotaka

    2012-01-01

    Elongation factor 1α (EF-1α) and elongation factor-like (EFL) proteins are considered to carry out equivalent functions in translation in eukaryotic cells. Elongation factor 1α and EFL genes are patchily distributed in the global eukaryotic tree, suggesting that the evolution of these elongation factors cannot be reconciled without multiple lateral gene transfer and/or ancestral co-occurrence followed by differential loss of either of the two factors. Our current understanding of the EF-1α/EFL evolution in the eukaryotic group Rhizaria, composed of Foraminifera, Radiolaria, Filosa, and Endomyxa, remains insufficient, as no information on EF-1α/EFL gene is available for any members of Radiolaria. In this study, EFL genes were experimentally isolated from four polycystine radiolarians (i.e. Dictyocoryne, Eucyrtidium, Collozoum, and Sphaerozoum), as well as retrieved from publicly accessible expressed sequence tag data of two acantharean radiolarians (i.e. Astrolonche and Phyllostaurus) and the endomyxan Gromia. The EFL homologs from radiolarians, foraminiferans, and Gromia formed a robust clade in both maximum-likelihood and Bayesian phylogenetic analyses, suggesting that EFL genes were vertically inherited from their common ancestor. We propose an updated model for EF-1α/EFL evolution in Rhizaria by incorporating new EFL data obtained in this study.

  4. 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

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

    PubMed

    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

    2014-09-16

    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.

  6. Inappropriate expression of the translation elongation factor 1A disrupts genome stability and metabolism

    PubMed Central

    Tarrant, Daniel J.; Stirpe, Mariarita; Rowe, Michelle; Howard, Mark J.

    2016-01-01

    ABSTRACT The translation elongation factor eEF1A is one of the most abundant proteins found within cells, and its role within protein synthesis is well documented. Levels of eEF1A are tightly controlled, with inappropriate expression linked to oncogenesis. However, the mechanisms by which increased eEF1A expression alters cell behaviour are unknown. Our analyses in yeast suggest that elevation of eEF1A levels leads to stabilisation of the spindle pole body and changes in nuclear organisation. Elevation of the eEF1A2 isoform also leads to altered nuclear morphology in cultured human cells, suggesting a conserved role in maintaining genome stability. Gene expression and metabolomic analyses reveal that the level of eEF1A is crucial for the maintenance of metabolism and amino acid levels in yeast, most likely because of its role in the control of vacuole function. Increased eEF1A2 levels trigger lysosome biogenesis in cultured human cells, also suggesting a conserved role within metabolic control mechanisms. Taken together, our data suggest that the control of eEF1A levels is important for the maintenance of a number of cell functions beyond translation and that its de-regulation might contribute to its oncogenic properties. PMID:27807005

  7. Elongation factor 1-alpha is released into the culture medium during growth of Giardia intestinalis trophozoites.

    PubMed

    Skarin, Hanna; Ringqvist, Emma; Hellman, Ulf; Svärd, Staffan G

    2011-04-01

    The molecular pathogenesis of the intestinal parasite Giardia intestinalis is still not fully understood but excretory-secretory products have been suggested to be important during host-parasite interactions. Here we used SDS-PAGE gels and MALDI-TOF analysis to identify proteins released by Giardia trophozoites during in vitro growth. Serum proteins (mainly bovine serum albumin) in the growth medium, bind to the parasite surface and they are continuously released, which interfere with parasite secretome characterization. However, we identified two released Giardia proteins: elongation factor-1 alpha (EF-1α) and a 58 kDa protein, identified as arginine deiminase (ADI). This is the first description of EF-1α as a released/secreted Giardia protein, whereas ADI has been identified in an earlier secretome study. Two genes encoding EF-1α were detected in the Giardia WB genome 35 kbp apart with almost identical coding sequences but with different promoter and 3' regions. Promoter luciferase-fusions showed that both genes are transcribed in trophozoites. The EF-1α protein localizes to the nuclear region in trophozoites but it relocalizes to the cytoplasm during host-cell interaction. Recombinant EF-1α is recognized by serum from giardiasis patients. Our results suggest that released EF-1α protein can be important during Giardia infections.

  8. Identification and subcellular localization analysis of two rubber elongation factor isoforms on Hevea brasiliensis rubber particles.

    PubMed

    Dai, Longjun; Nie, Zhiyi; Kang, Guijuan; Li, Yu; Zeng, Rizhong

    2017-02-01

    Rubber elongation factor (REF) is the most abundant protein found on the rubber particles or latex from Hevea brasiliensis (the Para rubber tree) and is considered to play important roles in natural rubber (cis-polyisoprene) biosynthesis. 16 BAC (benzyldimethyl-n-hexadecylammonium chloride)/SDS-PAGE separations and mass spectrometric identification had revealed that two REF isoforms shared similar amino acid sequences and common C-terminal sequences. In this study, the gene sequences encoding these two REF isoforms (one is 23.6 kDa in size with 222 amino acid residues and the other is 27.3 kDa in size with 258 amino acid residues) were obtained. Their proteins were relatively enriched by sequential extraction of the rubber particle proteins and separated by 16 BAC/SDS-PAGE. The localization of these isoforms on the surfaces of rubber particles was further verified by western blotting and immunogold electron microscopy, which demonstrated that these two REF isoforms are mainly located on the surfaces of larger rubber particles and that they bind more tightly to rubber particles than the most abundant REF and SRPP (small rubber particle protein).

  9. Protein synthesis elongation factor EF-1 alpha expression and longevity in Drosophila melanogaster.

    PubMed Central

    Shikama, N; Ackermann, R; Brack, C

    1994-01-01

    It has been proposed that the decline in protein synthesis observed in aging organisms may result from a decrease in elongation factor EF-1 alpha. Transgenic Drosophila melanogaster flies carrying an additional copy of the EF-1 alpha gene under control of a heat-inducible promoter have an extended lifespan, further indicating that the EF-1 alpha gene may play an important role in determining longevity. To test this hypothesis, we have quantitated EF-1 alpha mRNA, EF-1 alpha protein, and the EF-1 alpha complex-formation activity in these transgenic flies. Furthermore, we have tested whether the transgene construct is functional--i.e., whether transgenic mRNA is induced when flies are grown at higher temperature. The results show that although there is a clear difference in mean lifespan between the EF-1 alpha transgenic (E) flies and the control transgenic (C) flies, E flies do not express more EF-1 alpha protein or mRNA than C flies kept at the same experimental conditions. Although the transgene can be induced when E flies are heat-shocked at 37 degrees C, transgenic mRNA is not detectable in E flies aged at 29 degrees C. In both lines, the loss in catalytic activity with age is the same. We conclude that the E flies examined here do not live longer because of overexpressing the EF-1 alpha gene. Images PMID:8183891

  10. Bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filament ends

    PubMed Central

    1996-01-01

    Elongation factor 1 alpha (EF1 alpha) is an abundant protein that binds aminoacyl-tRNA and ribosomes in a GTP-dependent manner. EF1 alpha also interacts with the cytoskeleton by binding and bundling actin filaments and microtubules. In this report, the effect of purified EF1 alpha on actin polymerization and depolymerization is examined. At molar ratios present in the cytosol, EF1 alpha significantly blocks both polymerization and depolymerization of actin filaments and increases the final extent of actin polymer, while at high molar ratios to actin, EF1 alpha nucleates actin polymerization. Although EF1 alpha binds actin monomer, this monomer-binding activity does not explain the effects of EF1 alpha on actin polymerization at physiological molar ratios. The mechanism for the inhibition of polymerization is related to the actin-bundling activity of EF1 alpha. Both ends of the actin filament are inhibited for polymerization and both bundling and the inhibition of actin polymerization are affected by pH within the same physiological range; at high pH both bundling and the inhibition of actin polymerization are reduced. Additionally, it is seen that the binding of aminoacyl-tRNA to EF1 alpha releases EF1 alpha's inhibiting effect on actin polymerization. These data demonstrate that EF1 alpha can alter the assembly of F-actin, a filamentous scaffold on which non- membrane-associated protein translation may be occurring in vivo. PMID:8947553

  11. Antisense-mediated FLC transcriptional repression requires the P-TEFb transcription elongation factor

    PubMed Central

    Wang, Zhi-Wei; Wu, Zhe; Raitskin, Oleg; Sun, Qianwen; Dean, Caroline

    2014-01-01

    The functional significance of noncoding transcripts is currently a major question in biology. We have been studying the function of a set of antisense transcripts called COOLAIR that encompass the whole transcription unit of the Arabidopsis floral repressor FLOWERING LOCUS C (FLC). Alternative polyadenylation of COOLAIR transcripts correlates with different FLC sense expression states. Suppressor mutagenesis aimed at understanding the importance of this sense–antisense transcriptional circuitry has identified a role for Arabidopsis cyclin-dependent kinase C (CDKC;2) in FLC repression. CDKC;2 functions in an Arabidopsis positive transcription elongation factor b (P-TEFb) complex and influences global RNA polymerase II (Pol II) Ser2 phosphorylation levels. CDKC;2 activity directly promotes COOLAIR transcription but does not affect an FLC transgene missing the COOLAIR promoter. In the endogenous gene context, however, the reduction of COOLAIR transcription by cdkc;2 disrupts a COOLAIR-mediated repression mechanism that increases FLC expression. This disruption then feeds back to indirectly increase COOLAIR expression. This tight interconnection between sense and antisense transcription, together with differential promoter sensitivity to P-TEFb, is central to quantitative regulation of this important floral repressor gene. PMID:24799695

  12. Mutational analysis of Glu272 in elongation factor 1A of E. coli.

    PubMed

    Mansilla, F; Knudsen, C R; Clark, B F

    1998-06-16

    In our previous work (Mansilla et al. (1997) Protein Eng. 10, 927-934) we showed that Arg7 of Escherichia coli elongation factor Tu (EF1A) plays an essential role in aminoacyl-tRNA (aa-tRNA) binding. Substitution of Arg7 by Ala or Glu lost this activity. We proposed that Arg7 forms a salt bridge with the charged conserved amino acid Glu272 (Asp284 in Thermus aquaticus) thereby binding the N-terminal region of the protein to domain 2 and thus completing the conformational rearrangement needed for binding aa-tRNA. In this work we have mutated Glu272 to arginine, either alone (Glu272Arg), or in combination with one of the above mentioned mutations (Arg7Glu/Glu272Arg) in order to test this hypothesis. Our results show that, in confirmation of our thesis based on structural knowledge, the substitution of Glu272 (Asp284) decreases the ability of EF1A:GTP to bind aa-tRNA.

  13. An interbacterial NAD(P)+ glycohydrolase toxin requires elongation factor Tu for delivery to target cells

    DOE PAGES

    Whitney, John C.; Quentin, Dennis; Sawai, Shin; ...

    2015-10-08

    Type VI secretion (T6S) influences the composition of microbial communities by catalyzing the delivery of toxins between adjacent bacterial cells. Here, we demonstrate that a T6S integral membrane toxin from Pseudomonas aeruginosa, Tse6, acts on target cells by degrading the universally essential dinucleotides NAD+ and NADP+. Structural analyses of Tse6 show that it resembles mono-ADP-ribosyltransferase proteins, such as diphtheria toxin, with the exception of a unique loop that both excludes proteinaceous ADP-ribose acceptors and contributes to hydrolysis. We find that entry of Tse6 into target cells requires its binding to an essential housekeeping protein, translation elongation factor Tu (EF-Tu). Thesemore » proteins participate in a larger assembly that additionally directs toxin export and provides chaperone activity. Lastly, visualization of this complex by electron microscopy defines the architecture of a toxin-loaded T6S apparatus and provides mechanistic insight into intercellular membrane protein delivery between bacteria.« less

  14. Mechanism of activation of elongation factor Tu by ribosome: catalytic histidine activates GTP by protonation.

    PubMed

    Aleksandrov, Alexey; Field, Martin

    2013-09-01

    Elongation factor Tu (EF-Tu) is central to prokaryotic protein synthesis as it has the role of delivering amino-acylated tRNAs to the ribosome. Release of EF-Tu, after correct binding of the EF-Tu:aa-tRNA complex to the ribosome, is initiated by GTP hydrolysis. This reaction, whose mechanism is uncertain, is catalyzed by EF-Tu, but requires activation by the ribosome. There have been a number of mechanistic proposals, including those spurred by a recent X-ray crystallographic analysis of a ribosome:EF-Tu:aa-tRNA:GTP-analog complex. In this work, we have investigated these and alternative hypotheses, using high-level quantum chemical/molecular mechanical simulations for the wild-type protein and its His85Gln mutant. For both proteins, we find previously unsuggested mechanisms as being preferred, in which residue 85, either His or Gln, directly assists in the reaction. Analysis shows that the RNA has a minor catalytic effect in the wild-type reaction, but plays a significant role in the mutant by greatly stabilizing the reaction's transition state. Given the similarity between EF-Tu and other members of the translational G-protein family, it is likely that these mechanisms of ribosome-activated GTP hydrolysis are pertinent to all of these proteins.

  15. Borrelia burgdorferi elongation factor EF-Tu is an immunogenic protein during Lyme borreliosis.

    PubMed

    Carrasco, Sebastian E; Yang, Youyun; Troxell, Bryan; Yang, Xiuli; Pal, Utpal; Yang, X Frank

    2015-09-02

    Borrelia burgdorferi, the etiological agent of Lyme disease, does not produce lipopolysaccharide but expresses a large number of lipoproteins on its cell surface. These outer membrane lipoproteins are highly immunogenic and have been used for serodiagnosis of Lyme disease. Recent studies have shown that highly conserved cytosolic proteins such as enolase and elongation factor Tu (EF-Tu) unexpectedly localized on the surface of bacteria including B. burgdorferi, and surface-localized enolase has shown to contribute to the enzootic cycle of B. burgdorferi. In this study, we studied the immunogenicity, surface localization, and function of B. burgdorferi EF-Tu. We found that EF-Tu is highly immunogenic in mice, and EF-Tu antibodies were readily detected in Lyme disease patients. On the other hand, active immunization studies showed that EF-Tu antibodies did not protect mice from infection when challenged with B. burgdorferi via either needle inoculation or tick bites. Borrelial mouse-tick cycle studies showed that EF-Tu antibodies also did not block B. burgdorferi migration and survival in ticks. Consistent with these findings, we found that EF-Tu primarily localizes in the protoplasmic cylinder of spirochetes and is not on the surface of B. burgdorferi. Taken together, our studies suggest that B. burgdorferi EF-Tu is not surfaced exposed, but it is highly immunogenic and is a potential serodiagnostic marker for Lyme borreliosis.

  16. 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.

  17. Exportin-5-mediated nuclear export of eukaryotic elongation factor 1A and tRNA.

    PubMed

    Calado, Angelo; Treichel, Nathalie; Müller, Eva-Christina; Otto, Albrecht; Kutay, Ulrike

    2002-11-15

    Transport of proteins and RNA into and out of the cell nucleus is mediated largely by a family of RanGTP-binding transport receptors. Export receptors (exportins) need to bind RanGTP for efficient loading of their export cargo. We have identified eukaryotic elongation factor 1A (eEF1A) and tRNA as RanGTP-dependent binding partners of exportin-5 (Exp5). Exp5 stimulates nuclear export of eEF1A when microinjected into the nucleus of Xenopus laevis oocytes. Surprisingly, the interaction between eEF1A and Exp5 is dependent on tRNA that can interact directly with Exp5 and, if aminoacylated, recruits eEF1A into the export complex. These data suggested to us that Exp5 might support tRNA export. Indeed, not only the canonical tRNA export receptor, exportin-t, but also Exp5 can drive nuclear export of tRNA. Taken together, we show that there exists an alternative tRNA export pathway which can be exploited to keep eEF1A out of the cell nucleus.

  18. Divergence among genes encoding the elongation factor Tu of Yersinia Species.

    PubMed

    Isabel, Sandra; Leblanc, Eric; 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

    2008-11-01

    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.

  19. 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

  20. Cloning, expression and functional study of translation elongation factor 2 (EF-2) in zebrafish.

    PubMed

    Zhang, Shu-Hong; Yao, Ji-Hua; Song, Huai-Dong; Wang, Lu; Xue, Jing-Lun

    2006-01-01

    We have identified translation elongation factor 2 (EF-2) in zebrafish (GenBank Accession No. AAQ91234). Analysis of the DNA sequence of zebrafish EF-2 shows that the 2826 bp cDNA spans an open reading frame between nucleotide 55 to 2631 and encodes a protein of 858 amino acids. Zebrafish EF-2 protein shares 92%, 93%, 93% and 92% identity with the corresponding amino acid sequence in human, mouse, Chinese hamster and Gallus EF-2, respectively. Whole-mount in situ hybridization showed that zebrafish EF-2 was a developmentally regulated gene and might play important roles during the early development of zebrafish embryos. Therefore, we further studied the function of EF-2 during early embryogenesis. Using morpholino antisense oligo knockdown assays, anti-MO injected embryos were found to display abnormal development. The yolk balls were larger than normal and the melanophores spreading on their bodies became fewer. Furthermore, their tails were incurvate and their lenses were much smaller than those of the normal embryos. However the EF-2 overexpression data showed that extra EF-2 protein had no obvious effect on zebrafish embryonic development.

  1. Cloning and expression of translation elongation factor 2 (EF-2) in zebrafish.

    PubMed

    Zhang, Shu-Hong; Yao, Ji-Hua; Song, Huai-Dong; Wang, Lu; Xue, Jing-Lun

    2008-02-01

    We have identified a developmentally regulated gene translation elongation factor 2 (EF-2) in zebrafish (GenBank Accession No. AAQ91234). Analysis of DNA sequence of zebrafish EF-2 shows that the 2826 bp cDNA spans an open reading frame from nucleotide 55 to 2631 and encodes a protein of 858 amino acids. It shares an identity of 92, 93, 93, 92, 79 and 80% in amino acid sequence to human, mouse, Chinese hamster, Gallus gullus, C. elegans and Drosophila EF-2, respectively. Zebrafish EF-2 protein has 16 conserved domains, GTP-binding domain is found in the NH2 terminus, and the ADP-ribosylation domain locates at the COOH terminus. Whole mount in situ hybridization on zebrafish embryos shows that the transcripts of EF-2 gene are detected during the early development of zebrafish embryo and constantly change from 5-somite stage to protruding-mouth stage. It expresses strongly throughout envelope at 5-somite stage. Then the stained cells concentrate strongly in the eyes, brain and muscle tissue. From prim-25 stage the stained cells only appear strongly in the lens and the anterior portion of the cerebellum.

  2. 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...

  3. Heat-induced accumulation of protein synthesis elongation factor 1A indicates an important role in heat tolerance in potato

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Heat stress substantially reduces crop productivity worldwide, and will become more severe due to global warming. Identification of proteins involved in heat stress response may help develop varieties for heat tolerance. Eukaryotic elongation factor 1A (eEF1A) is a cytosolic, multifunctional protei...

  4. 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...

  5. Initiation factor IF2, thiostrepton and micrococcin prevent the binding of elongation factor G to the Escherichia coli ribosome.

    PubMed

    Cameron, Dale M; Thompson, Jill; March, Paul E; Dahlberg, Albert E

    2002-05-24

    The bacterial translational GTPases (initiation factor IF2, elongation factors EF-G and EF-Tu and release factor RF3) are involved in all stages of translation, and evidence indicates that they bind to overlapping sites on the ribosome, whereupon GTP hydrolysis is triggered. We provide evidence for a common ribosomal binding site for EF-G and IF2. IF2 prevents the binding of EF-G to the ribosome, as shown by Western blot analysis and fusidic acid-stabilized EF-G.GDP.ribosome complex formation. Additionally, IF2 inhibits EF-G-dependent GTP hydrolysis on 70 S ribosomes. The antibiotics thiostrepton and micrococcin, which bind to part of the EF-G binding site and interfere with the function of the factor, also affect the function of IF2. While thiostrepton is a strong inhibitor of EF-G-dependent GTP hydrolysis, GTP hydrolysis by IF2 is stimulated by the drug. Micrococcin stimulates GTP hydrolysis by both factors. We show directly that these drugs act by destabilizing the interaction of EF-G with the ribosome, and provide evidence that they have similar effects on IF2.

  6. Interaction of Leptospira elongation factor Tu with plasminogen and complement factor H: a metabolic leptospiral protein with moonlighting activities.

    PubMed

    Wolff, Danielly G; Castiblanco-Valencia, Mónica M; Abe, Cecília M; Monaris, Denize; Morais, Zenaide M; Souza, Gisele O; Vasconcellos, Sílvio A; Isaac, Lourdes; Abreu, Patrícia A E; Barbosa, Angela S

    2013-01-01

    The elongation factor Tu (EF-Tu), an abundant bacterial protein involved in protein synthesis, has been shown to display moonlighting activities. Known to perform more than one function at different times or in different places, it is found in several subcellular locations in a single organism, and may serve as a virulence factor in a range of important human pathogens. Here we demonstrate that Leptospira EF-Tu is surface-exposed and performs additional roles as a cell-surface receptor for host plasma proteins. It binds plasminogen in a dose-dependent manner, and lysine residues are critical for this interaction. Bound plasminogen is converted to active plasmin, which, in turn, is able to cleave the natural substrates C3b and fibrinogen. Leptospira EF-Tu also acquires the complement regulator Factor H (FH). FH bound to immobilized EF-Tu displays cofactor activity, mediating C3b degradation by Factor I (FI). In this manner, EF-Tu may contribute to leptospiral tissue invasion and complement inactivation. To our knowledge, this is the first description of a leptospiral protein exhibiting moonlighting activities.

  7. Regulation of elongation factor-1 expression by vitamin E in diabetic rat kidneys.

    PubMed

    Al-Maghrebi, May; Cojocel, Constantin; Thompson, Mary S

    2005-05-01

    Translation elongation factor-1 (EF-1) forms a primary site of regulation of protein synthesis and has been implicated amongst others in tumorigenesis, diabetes and cell death. To investigate whether diabetes-induced oxidative stress affects EF-1 gene expression, we used a free radical scavenger, vitamin E. The following groups of rats (5/group) were studied: control, vitamin E control, diabetic and diabetic treated with vitamin E. Markers of hyperglycemia, kidney function, oxidative stress, and kidney hypertrophy were elevated in diabetic rats. Increased urinary protein excretion indicated early signs of glomerular and tubular dysfunction. The mRNA and protein levels of the three EF-1 subunits (A, Balpha, and Bgamma) were determined in renal cortex extracts using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), northern blot analysis and western blotting. EF-1A mRNA expression in renal cortex extracts was significantly increased by at least 2-fold (p < 0.002) in diabetic rats; however, there was no change in the mRNA levels of EF-1Balpha and EF-1Bgamma subunits. Similar results were observed at the protein level. Treatment of diabetic rats with vitamin E for 10 days suppressed both glycemic and oxidative stresses in renal cortex and kidney hypertrophy. EF-1A mRNA and protein levels were also reduced to control levels. In conclusion, EF-1A but not EF-1Balpha and EF-1Bgamma gene expression is significantly enhanced in the renal cortex of diabetic rats. Normalization of enhanced EF-1A expression by vitamin E treatment suggests a role for EF-1A during diabetes-induced oxidative stress.

  8. Rooting for the root of elongation factor-like protein phylogeny.

    PubMed

    Kamikawa, Ryoma; Sakaguchi, Miako; Matsumoto, Takuya; Hashimoto, Tetsuo; Inagaki, Yuji

    2010-09-01

    Lateral gene transfer (LGT) may play a pivotal role in the evolution of elongation factor-like (EFL) genes in eukaryotes. To date, numbers of putative cases for lateral transfer of EFL genes have been postulated based on unrooted EFL phylogenies. Nevertheless, the root position in EFL phylogeny is important to validate lateral EFL gene transfer: for instance, a clade of two EFL homologs from distantly related organisms in an unrooted EFL tree does not necessarily confirm the LGT, since the possibility that the root may locate in this clade cannot be excluded. Cocquyt et al. (2009, p. 39) recently demonstrated that a putative case of lateral EFL gene transfer, which was originally proposed based on an unrooted phylogeny, could not be endorsed by the corresponding rooted analysis. Although rooting EFL phylogeny is indispensable to elucidate various aspects in EFL gene evolution, we suspected that the outgroup clade comprised of EF-1alpha and eukaryote-specific EF-1alpha paralogs erroneously attached to long EFL branches in Cocquyt et al. (2009) - a typical long branch attraction (LBA) artifact. Here, we systematically assessed the putative LBA artifact between the branch leading to the outgroup clade and long ingroup branches by analyzing the original dataset used in Cocquyt et al. (2009) with and without modifying ingroup-sequence sampling. A series of the rooted EFL analyses indicated that the root inference was highly susceptible to presence and absence of long-branched ingroup-sequences, suggesting that the rooted EFL phylogenies cannot be free from severe LBA artifact. We also discussed a new aspect in EFL gene evolution in stramenopiles identified in the course of the EFL analyses described above. Finally, the relative timing of the first emergence of EFL gene in eukaryotes was contemplated based on the current EF-1alpha/EFL distribution.

  9. 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

  10. In vitro and in vivo activities of novel, semisynthetic thiopeptide inhibitors of bacterial elongation factor Tu.

    PubMed

    Leeds, J A; LaMarche, M J; Brewer, J T; Bushell, S M; Deng, G; Dewhurst, J M; Dzink-Fox, J; Gangl, E; Jain, A; Lee, L; Lilly, M; Manni, K; Mullin, S; Neckermann, G; Osborne, C; Palestrant, D; Patane, M A; Raimondi, A; Ranjitkar, S; Rann, E M; Sachdeva, M; Shao, J; Tiamfook, S; Whitehead, L; Yu, D

    2011-11-01

    Recently, we identified aminothiazole derivatives of GE2270 A. These novel semisynthetic congeners, like GE2270 A, target the essential bacterial protein elongation factor Tu (EF-Tu). Medicinal chemistry optimization of lead molecules led to the identification of preclinical development candidates 1 and 2. These cycloalklycarboxylic acid derivatives show activity against difficult to treat Gram-positive pathogens and demonstrate increased aqueous solubility compared to GE2270 A. We describe here the in vitro and in vivo activities of compounds 1 and 2 compared to marketed antibiotics. Compounds 1 and 2 were potent against clinical isolates of methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci (MIC(90) ≤ 0.25 μg/ml) but weaker against the streptococci (MIC(90) ≥ 4 μg/ml). Like GE2270 A, the derivatives inhibited bacterial protein synthesis and selected for spontaneous loss of susceptibility via mutations in the tuf gene, encoding EF-Tu. The mutants were not cross-resistant to other antibiotic classes. In a mouse systemic infection model, compounds 1 and 2 protected mice from lethal S. aureus infections with 50% effective doses (ED(50)) of 5.2 and 4.3 mg/kg, respectively. Similarly, compounds 1 and 2 protected mice from lethal systemic E. faecalis infections with ED(50) of 0.56 and 0.23 mg/kg, respectively. In summary, compounds 1 and 2 are active in vitro and in vivo activity against difficult-to-treat Gram-positive bacterial infections and represent a promising new class of antibacterials for use in human therapy.

  11. 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.

  12. A critical role for eukaryotic elongation factor 1A-1 in lipotoxic cell death.

    PubMed

    Borradaile, Nica M; Buhman, Kimberly K; Listenberger, Laura L; Magee, Carolyn J; Morimoto, Emiko T A; Ory, Daniel S; Schaffer, Jean E

    2006-02-01

    The deleterious consequences of fatty acid (FA) and neutral lipid accumulation in nonadipose tissues, such as the heart, contribute to the pathogenesis of type 2 diabetes. To elucidate mechanisms of FA-induced cell death, or lipotoxicity, we generated Chinese hamster ovary (CHO) cell mutants resistant to palmitate-induced death and isolated a clone with disruption of eukaryotic elongation factor (eEF) 1A-1. eEF1A-1 involvement in lipotoxicity was confirmed in H9c2 cardiomyoblasts, in which small interfering RNA-mediated knockdown also conferred palmitate resistance. In wild-type CHO and H9c2 cells, palmitate increased reactive oxygen species and induced endoplasmic reticulum (ER) stress, changes accompanied by increased eEF1A-1 expression. Disruption of eEF1A-1 expression rendered these cells resistant to hydrogen peroxide- and ER stress-induced death, indicating that eEF1A-1 plays a critical role in the cell death response to these stressors downstream of lipid overload. Disruption of eEF1A-1 also resulted in actin cytoskeleton defects under basal conditions and in response to palmitate, suggesting that eEF1A-1 mediates lipotoxic cell death, secondary to oxidative and ER stress, by regulating cytoskeletal changes critical for this process. Furthermore, our observations of oxidative stress, ER stress, and induction of eEF1A-1 expression in a mouse model of lipotoxic cardiomyopathy implicate this cellular response in the pathophysiology of metabolic disease.

  13. A Critical Role for Eukaryotic Elongation Factor 1A-1 in Lipotoxic Cell DeathD⃞

    PubMed Central

    Borradaile, Nica M.; Buhman, Kimberly K.; Listenberger, Laura L.; Magee, Carolyn J.; Morimoto, Emiko T.A.; Ory, Daniel S.; Schaffer, Jean E.

    2006-01-01

    The deleterious consequences of fatty acid (FA) and neutral lipid accumulation in nonadipose tissues, such as the heart, contribute to the pathogenesis of type 2 diabetes. To elucidate mechanisms of FA-induced cell death, or lipotoxicity, we generated Chinese hamster ovary (CHO) cell mutants resistant to palmitate-induced death and isolated a clone with disruption of eukaryotic elongation factor (eEF) 1A-1. eEF1A-1 involvement in lipotoxicity was confirmed in H9c2 cardiomyoblasts, in which small interfering RNA-mediated knockdown also conferred palmitate resistance. In wild-type CHO and H9c2 cells, palmitate increased reactive oxygen species and induced endoplasmic reticulum (ER) stress, changes accompanied by increased eEF1A-1 expression. Disruption of eEF1A-1 expression rendered these cells resistant to hydrogen peroxide- and ER stress-induced death, indicating that eEF1A-1 plays a critical role in the cell death response to these stressors downstream of lipid overload. Disruption of eEF1A-1 also resulted in actin cytoskeleton defects under basal conditions and in response to palmitate, suggesting that eEF1A-1 mediates lipotoxic cell death, secondary to oxidative and ER stress, by regulating cytoskeletal changes critical for this process. Furthermore, our observations of oxidative stress, ER stress, and induction of eEF1A-1 expression in a mouse model of lipotoxic cardiomyopathy implicate this cellular response in the pathophysiology of metabolic disease. PMID:16319173

  14. 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

  15. Mitochondrial translation factors of Trypanosoma brucei: elongation factor-Tu has a unique subdomain that is essential for its function.

    PubMed

    Cristodero, Marina; Mani, Jan; Oeljeklaus, Silke; Aeberhard, Lukas; Hashimi, Hassan; Ramrath, David J F; Lukeš, Julius; Warscheid, Bettina; Schneider, André

    2013-11-01

    Mitochondrial translation in the parasitic protozoan Trypanosoma brucei relies on imported eukaryotic-type tRNAs as well as on bacterial-type ribosomes that have the shortest known rRNAs. Here we have identified the mitochondrial translation elongation factors EF-Tu, EF-Ts, EF-G1 and release factor RF1 of trypanosomatids and show that their ablation impairs growth and oxidative phosphorylation. In vivo labelling experiments and a SILAC-based analysis of the global proteomic changes induced by EF-Tu RNAi directly link EF-Tu to mitochondrial translation. Moreover, EF-Tu RNAi reveals downregulation of many nuclear encoded subunits of cytochrome oxidase as well as of components of the bc1-complex, whereas most cytosolic ribosomal proteins were upregulated. Interestingly, T. brucei EF-Tu has a 30-amino-acid-long, highly charged subdomain, which is unique to trypanosomatids. A combination of RNAi and complementation experiments shows that this subdomain is essential for EF-Tu function, but that it can be replaced by a similar sequence found in eukaryotic EF-1a, the cytosolic counterpart of EF-Tu. A recent cryo-electron microscopy study revealed that trypanosomatid mitochondrial ribosomes have a unique intersubunit space that likely harbours the EF-Tu binding site. These findings suggest that the trypanosomatid-specific EF-Tu subdomain serves as an adaption for binding to these unusual mitochondrial ribosomes.

  16. Promoter Escape with Bacterial Two-component σ Factor Suggests Retention of σ Region Two in the Elongation Complex.

    PubMed

    Sengupta, Shreya; Prajapati, Ranjit Kumar; Mukhopadhyay, Jayanta

    2015-11-20

    The transition from the formation of the RNA polymerase (RNAP)-promoter open complex step to the productive elongation complex step involves "promoter escape" of RNAP. From the structure of RNAP, a promoter escape model has been proposed that suggests that the interactions between σR4 and RNAP and σR4 and DNA are destabilized upon transition to elongation. This accounts for the reduced affinity of σ to RNAP and stochastic release of σ. However, as the loss of interaction of σR4 with RNAP results in the release of intact σ, assessing this interaction remains challenging to be experimentally verified. Here we study the promoter escape model using a two-component σ factor YvrI and YvrHa from Bacillus subtilis that independently contributes to the functions of σR4 and σR2 in a RNAP-promoter complex. Our results show that YvrI, which mimics σR4, is released gradually as transcription elongation proceeds, whereas YvrHa, which mimics σR2 is retained throughout the elongation complexes. Thus our result validates the proposed model for promoter escape and also suggests that promoter escape involves little or no change in the interaction of σR2 with RNAP.

  17. Promoter Escape with Bacterial Two-component σ Factor Suggests Retention of σ Region Two in the Elongation Complex*

    PubMed Central

    Sengupta, Shreya; Prajapati, Ranjit Kumar; Mukhopadhyay, Jayanta

    2015-01-01

    The transition from the formation of the RNA polymerase (RNAP)-promoter open complex step to the productive elongation complex step involves “promoter escape” of RNAP. From the structure of RNAP, a promoter escape model has been proposed that suggests that the interactions between σR4 and RNAP and σR4 and DNA are destabilized upon transition to elongation. This accounts for the reduced affinity of σ to RNAP and stochastic release of σ. However, as the loss of interaction of σR4 with RNAP results in the release of intact σ, assessing this interaction remains challenging to be experimentally verified. Here we study the promoter escape model using a two-component σ factor YvrI and YvrHa from Bacillus subtilis that independently contributes to the functions of σR4 and σR2 in a RNAP-promoter complex. Our results show that YvrI, which mimics σR4, is released gradually as transcription elongation proceeds, whereas YvrHa, which mimics σR2 is retained throughout the elongation complexes. Thus our result validates the proposed model for promoter escape and also suggests that promoter escape involves little or no change in the interaction of σR2 with RNAP. PMID:26400263

  18. The interface between Escherichia coli elongation factor Tu and aminoacyl-tRNA.

    PubMed

    Yikilmaz, Emine; Chapman, Stephen J; Schrader, Jared M; Uhlenbeck, Olke C

    2014-09-09

    Nineteen of the highly conserved residues of Escherichia coli (E. coli) Elongation factor Tu (EF-Tu) that form the binding interface with aa-tRNA were mutated to alanine to better understand how modifying the thermodynamic properties of EF-Tu-tRNA interaction can affect the decoding properties of the ribosome. Comparison of ΔΔG(o) values for binding EF-Tu to aa-tRNA show that the majority of the interface residues stabilize the ternary complex and their thermodynamic contribution can depend on the tRNA species that is used. Experiments with a very tight binding mutation of tRNA(Tyr) indicate that interface amino acids distant from the tRNA mutation can contribute to the specificity. For nearly all of the mutations, the values of ΔΔG(o) were identical to those previously determined at the orthologous positions of Thermus thermophilus (T. thermophilus) EF-Tu indicating that the thermodynamic properties of the interface were conserved between distantly related bacteria. Measurement of the rate of GTP hydrolysis on programmed ribosomes revealed that nearly all of the interface mutations were able to function in ribosomal decoding. The only interface mutation with greatly impaired GTPase activity was R223A which is the only one that also forms a direct contact with the ribosome. Finally, the ability of the EF-Tu interface mutants to destabilize the EF-Tu-aa-tRNA interaction on the ribosome after GTP hydrolysis were evaluated by their ability to suppress the hyperstable T1 tRNA(Tyr) variant where EF-Tu release is sufficiently slow to limit the rate of peptide bond formation (kpep) . In general, interface mutations that destabilize EF-Tu binding are also able to stimulate kpep of T1 tRNA(Tyr), suggesting that the thermodynamic properties of the EF-Tu-aa-tRNA interaction on the ribosome are quite similar to those found in the free ternary complex.

  19. A non-catalytic N-terminal domain negatively influences the nucleotide exchange activity of translation elongation factor 1Bα.

    PubMed

    Trosiuk, Tetiana V; Shalak, Vyacheslav F; Szczepanowski, Roman H; Negrutskii, Boris S; El'skaya, Anna V

    2016-02-01

    Eukaryotic translation elongation factor 1Bα (eEF1Bα) is a functional homolog of the bacterial factor EF-Ts, and is a component of the macromolecular eEF1B complex. eEF1Bα functions as a catalyst of guanine nucleotide exchange on translation elongation factor 1A (eEF1A). The C-terminal domain of eEF1Bα is necessary and sufficient for its catalytic activity, whereas the N-terminal domain interacts with eukaryotic translation elongation factor 1Bγ (eEF1Bγ) to form a tight complex. However, eEF1Bγ has been shown to enhance the catalytic activity of eEF1Bα attributed to the C-terminal domain of eEF1Bα. This suggests that the N-terminal domain of eEF1Bα may in some way influence the guanine nucleotide exchange process. We have shown that full-length recombinant eEF1Bα and its truncated forms are non-globular proteins with elongated shapes. Truncation of the N-terminal domain of eEF1Bα, which is dispensable for catalytic activity, resulted in acceleration of the rate of guanine nucleotide exchange on eEF1A compared to full-length eEF1Bα. A similar effect on the catalytic activity of eEF1Bα was observed after its interaction with eEF1Bγ. We suggest that the non-catalytic N-terminal domain of eEF1Bα may interfere with eEF1A binding to the C-terminal catalytic domain, resulting in a decrease in the overall rate of the guanine nucleotide exchange reaction. Formation of a tight complex between the eEF1Bγ and eEF1Bα N-terminal domains abolishes this inhibitory effect.

  20. Thermoperiodic control of hypocotyl elongation depends on auxin-induced ethylene signaling that controls downstream PHYTOCHROME INTERACTING FACTOR3 activity.

    PubMed

    Bours, Ralph; Kohlen, Wouter; Bouwmeester, Harro J; van der Krol, Alexander

    2015-02-01

    We show that antiphase light-temperature cycles (negative day-night temperature difference [-DIF]) inhibit hypocotyl growth in Arabidopsis (Arabidopsis thaliana). This is caused by reduced cell elongation during the cold photoperiod. Cell elongation in the basal part of the hypocotyl under -DIF was restored by both 1-aminocyclopropane-1-carboxylic acid (ACC; ethylene precursor) and auxin, indicating limited auxin and ethylene signaling under -DIF. Both auxin biosynthesis and auxin signaling were reduced during -DIF. In addition, expression of several ACC Synthase was reduced under -DIF but could be restored by auxin application. In contrast, the reduced hypocotyl elongation of ethylene biosynthesis and signaling mutants could not be complemented by auxin, indicating that auxin functions upstream of ethylene. The PHYTOCHROME INTERACTING FACTORS (PIFs) PIF3, PIF4, and PIF5 were previously shown to be important regulators of hypocotyl elongation. We now show that, in contrast to pif4 and pif5 mutants, the reduced hypocotyl length in pif3 cannot be rescued by either ACC or auxin. In line with this, treatment with ethylene or auxin inhibitors reduced hypocotyl elongation in PIF4 overexpressor (PIF4ox) and PIF5ox but not PIF3ox plants. PIF3 promoter activity was strongly reduced under -DIF but could be restored by auxin application in an ACC Synthase-dependent manner. Combined, these results show that PIF3 regulates hypocotyl length downstream, whereas PIF4 and PIF5 regulate hypocotyl length upstream of an auxin and ethylene cascade. We show that, under -DIF, lower auxin biosynthesis activity limits the signaling in this pathway, resulting in low activity of PIF3 and short hypocotyls.

  1. 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

  2. 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

  3. Comparative study of the in vitro protective effects of several antioxidants on elongation factor 2 under oxidative stress conditions.

    PubMed

    Arguelles, Sandro; Cano, Mercedes; Machado, Alberto; Ayala, Antonio

    2010-01-01

    One of the biochemical pathways affected by aging in all organisms is protein synthesis. Previous reports from our laboratory have indicated that the elongation step is specially affected by aging as a consequence of alterations in elongation factor-2 (eEF-2). In the present work, we studied in vitro the effectiveness of several individual nutritional antioxidants in protecting the levels of hepatic eEF-2 subjected to oxidative stress induced by cumene hydroperoxide. The in vitro system employed consisted of rat liver homogenates treated with cumene hydroperoxide. The antioxidants used in this study were lipoic acid, coenzyme Q10, tethrahydrofolic acid, and N-tert-butyl-alpha-phenylnitrone. The results indicate that the antioxidants have different capacities to prevent eEF-2 loss, folic acid being the most effective. A comparison between the antioxidants used and their potential pro-oxidant activity is also discussed, on the basis of the oxidative stress parameters measured.

  4. Relation between elongation factor and angle of friction from various outcrops

    NASA Astrophysics Data System (ADS)

    Martins-Campina, B.; Lebourg, T.; Riss, J.; Benabderrazik, A.; Fabre, R.

    2003-04-01

    The study of granular materials, and more particularly their mechanical behaviour, has made it possible to highlight, the influence of the grains shape on their mechanical behaviour. Recently when being concerned with till formations (glacial deposits) that cover mountain slopes and govern natural hazards, Lebourg (2000) has shown that there exists a simple linear regression of the internal angle of friction (phi) on a shape parameter (elongation factor IA ) based on the analysis of six tills formations coming from a paleoglacial valley (Vallée d Aspe, Pyrénées Occidentales : IGN map 1547 OT, 1/25 000 ; geological map URDOS 1/80 000). These results are in agreement with previous works where relation between shape and mechanical properties is assumed. Till-forming materials look like a heap of unsorted very heterogeneous material characterised by rock debris of all sizes from angular blocks of metric size to very fine rock. In addition to the block sizes, lithology, petrography and the spatial distribution of the blocks are also heterogeneous. Then it is hard, if not impossible, to collect a large sample of mechanical and physical data from the till in order to execute good simulations while running numerical programs. The validation of such a relation would be very interesting for other till formations and in any case of natural hazards such as landslides. So we propose new results based on the analysis of a set of samples collected on the site of La Clapière, a rocky landslide. The landslide at La Clapière, in south-eastern France, is located on the east side of the steep La Tinée river valley upstream of the village of Saint Etienne de Tinée. On the one hand, triaxial compression (test with simultaneous compression of a cylindrical sample and application of axisymetric confining pressure) has been performed on four sets of samples collected at La Clapière, then mechanical properties (E : Young modulus, phi: angle of internal friction, C : cohesion) were

  5. ADP-ribosylation of translation elongation factor 2 by diphtheria toxin in yeast inhibits translation and cell separation.

    PubMed

    Mateyak, Maria K; Kinzy, Terri Goss

    2013-08-23

    Eukaryotic translation elongation factor 2 (eEF2) facilitates the movement of the peptidyl tRNA-mRNA complex from the A site of the ribosome to the P site during protein synthesis. ADP-ribosylation (ADP(R)) of eEF2 by bacterial toxins on a unique diphthamide residue inhibits its translocation activity, but the mechanism is unclear. We have employed a hormone-inducible diphtheria toxin (DT) expression system in Saccharomyces cerevisiae which allows for the rapid induction of ADP(R)-eEF2 to examine the effects of DT in vivo. ADP(R) of eEF2 resulted in a decrease in total protein synthesis consistent with a defect in translation elongation. Association of eEF2 with polyribosomes, however, was unchanged upon expression of DT. Upon prolonged exposure to DT, cells with an abnormal morphology and increased DNA content accumulated. This observation was specific to DT expression and was not observed when translation elongation was inhibited by other methods. Examination of these cells by electron microscopy indicated a defect in cell separation following mitosis. These results suggest that expression of proteins late in the cell cycle is particularly sensitive to inhibition by ADP(R)-eEF2.

  6. 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.

  7. The Selenocysteine-Specific Elongation Factor Contains Unique Sequences That Are Required for Both Nuclear Export and Selenocysteine Incorporation

    PubMed Central

    Dubey, Aditi

    2016-01-01

    Selenocysteine (Sec) is a critical residue in at least 25 human proteins that are essential for antioxidant defense and redox signaling in cells. Sec is inserted into proteins cotranslationally by the recoding of an in-frame UGA termination codon to a Sec codon. In eukaryotes, this recoding event requires several specialized factors, including a dedicated, Sec-specific elongation factor called eEFSec, which binds Sec-tRNASec with high specificity and delivers it to the ribosome for selenoprotein production. Unlike most translation factors, including the canonical elongation factor eEF1A, eEFSec readily localizes to the nucleus of mammalian cells and shuttles between the cytoplasmic and nuclear compartments. The functional significance of eEFSec’s nuclear localization has remained unclear. In this study, we have examined the subcellular localization of eEFSec in the context of altered Sec incorporation to demonstrate that reduced selenoprotein production does not correlate with changes in the nuclear localization of eEFSec. In addition, we identify several novel sequences of the protein that are essential for localization as well as Sec insertion activity, and show that eEFSec utilizes CRM1-mediated nuclear export pathway. Our findings argue for two distinct pools of eEFSec in the cell, where the cytoplasmic pool participates in Sec incorporation and the nuclear pool may be involved in an as yet unknown function. PMID:27802322

  8. The distribution of Elongation Factor-1 Alpha (EF-1alpha), Elongation Factor-Like (EFL), and a non-canonical genetic code in the ulvophyceae: discrete genetic characters support a consistent phylogenetic framework.

    PubMed

    Gile, Gillian H; Novis, Philip M; Cragg, David S; Zuccarello, Giuseppe C; Keeling, Patrick J

    2009-01-01

    The systematics of the green algal class Ulvophyceae have been difficult to resolve with ultrastructural and molecular phylogenetic analyses. Therefore, we investigated relationships among ulvophycean orders by determining the distribution of two discrete genetic characters previously identified only in the order Dasycladales. First, Acetabularia acetabulum uses the core translation GTPase Elongation Factor 1alpha (EF-1alpha) while most Chlorophyta instead possess the related GTPase Elongation Factor-Like (EFL). Second, the nuclear genomes of dasycladaleans A. acetabulum and Batophora oerstedii use a rare non-canonical genetic code in which the canonical termination codons TAA and TAG instead encode glutamine. Representatives of Ulvales and Ulotrichales were found to encode EFL, while Caulerpales, Dasycladales, Siphonocladales, and Ignatius tetrasporus were found to encode EF-1alpha, in congruence with the two major lineages previously proposed for the Ulvophyceae. The EF-1alpha of I. tetrasporus supports its relationship with Caulerpales/Dasycladales/Siphonocladales, in agreement with ultrastructural evidence, but contrary to certain small subunit rRNA analyses that place it with Ulvales/Ulotrichales. The same non-canonical genetic code previously described in A. acetabulum was observed in EF-1alpha sequences from Parvocaulis pusillus (Dasycladales), Chaetomorpha coliformis, and Cladophora cf. crinalis (Siphonocladales), whereas Caulerpales use the universal code. This supports a sister relationship between Siphonocladales and Dasycladales and further refines our understanding of ulvophycean phylogeny.

  9. Production of an antiserum specific to the ADP-ribosylated form of elongation factor 2 from archaebacteria and eukaryotes.

    PubMed

    Siegmund, K D; Klink, F

    1992-11-09

    An antiserum to ADP-ribosylated elongation factor 2 (ADPR-EF-2) from S. acidocaldarius was raised in rabbits using stained, homogenized, ADPR-EF-2-containing slices from SDS-gels as a source of antigen. Elongation factor 2 (EF-2) from S. acidocaldarius was cloned in E. coli and the expressed gene product was used in order to adsorb all anti-EF-2 antibodies which do not contain the ADP-ribosyl group within their epitopes, as E. coli is unable to synthesize the ADP-ribosyl acceptor diphthamide. The remaining antibodies were specific to ADP-ribosylated EF-2 from Thermoplasma acidophilum, S. acidocaldarius and Desulfurococcus mucosus. ADP-ribosylated EF-2 from eukaryotic sources also reacted with the adsorbed antiserum as shown for EF-2 isolated from the killi-fish Cynolebias whitei, the mouse species BALB/c and Han/Wistar rats. The adsorbed antiserum did not cross-react with ADP-ribosylated actin or rho protein or with FAD-containing D-amino acid oxidase.

  10. 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

  11. 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

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

    PubMed

    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.

  13. Biphasic Stimulation of Translational Activity Correlates with Induction of Translation Elongation Factor 1 Subunit [alpha] upon Wounding in Potato Tubers.

    PubMed Central

    Morelli, J. K.; Shewmaker, C. K.; Vayda, M. E.

    1994-01-01

    Potato (Solanum tuberosum) tubers exhibit an increase in translational activity in response to mechanical wounding. The response is biphasic, with an initial stimulation apparent within the first 2 h after wounding and a second increase occurring 12 to 24 h after wounding. Increased activity is apparent by measurement of protein synthesis both in vivo and in vitro using a cell-free extract. Accumulation of the translational elongation factor 1 subunit [alpha] (EF-1[alpha]) parallels translational activity. Changes in the steady-state level of EF-1[alpha] mRNA, and expression of a chimeric EF-1[alpha] promoter/[beta]-glucuronidase construct in transgenic potato tubers, indicate that the gene encoding EF-1[alpha] is transcribed during both periods of translational stimulation. These results indicate that stimulation of translational activity is coordinated with increased expression and accumulation of translation factors. PMID:12232374

  14. The translation elongation factor eEF1A1 couples transcription to translation during heat shock response.

    PubMed

    Vera, Maria; Pani, Bibhusita; Griffiths, Lowri A; Muchardt, Christian; Abbott, Catherine M; Singer, Robert H; Nudler, Evgeny

    2014-09-16

    Translation elongation factor eEF1A has a well-defined role in protein synthesis. In this study, we demonstrate a new role for eEF1A: it participates in the entire process of the heat shock response (HSR) in mammalian cells from transcription through translation. Upon stress, isoform 1 of eEF1A rapidly activates transcription of HSP70 by recruiting the master regulator HSF1 to its promoter. eEF1A1 then associates with elongating RNA polymerase II and the 3'UTR of HSP70 mRNA, stabilizing it and facilitating its transport from the nucleus to active ribosomes. eEF1A1-depleted cells exhibit severely impaired HSR and compromised thermotolerance. In contrast, tissue-specific isoform 2 of eEF1A does not support HSR. By adjusting transcriptional yield to translational needs, eEF1A1 renders HSR rapid, robust, and highly selective; thus, representing an attractive therapeutic target for numerous conditions associated with disrupted protein homeostasis, ranging from neurodegeneration to cancer.

  15. Elevated eukaryotic elongation factor 2 expression is involved in proliferation and invasion of lung squamous cell carcinoma

    PubMed Central

    Hao, LiHong; Hu, Jun; Du, Sha; Zhou, Xin; Zhang, LiYuan; Liu, Lu; Gong, LinLin; Chi, XinMing; Liu, Qiang; Shao, ShuJuan

    2016-01-01

    Eukaryotic elongation factor 2 (EF2), is a critical enzyme solely responsible for catalyzing the translocation of the elongated peptidyl-tRNA from the A to P sites of the ribosome during the process of protein synthesis. EF2 is found to be highly expressed in a variety of malignant tumors and is correlated with cancer cell progression and recurrence. The present study was designed to uncover the function of EF2 on lung squamous cell carcinoma (LSCC) cancer cell growth and progression. Our results from clinical tissue studies showed that EF2 protein was significantly overexpressed in LSCC tissues, compared with the adjacent normal lung tissues, which was confirmed by western blotting and tissue microarray. Forced expression of EF2 resulted in the enhancement of lung squamous carcinoma NCI-H520 cells growth through promotion of G2/M progression in cell cycle, activating Akt and Cdc2/Cyclin B1. In nude mice cancer xenograft model, overexpression of EF2 significantly facilitated cell proliferation in vivo. Furthermore, forced expression of EF2 in the cells increased the capabilities of migration and invasion by changing the expressions of EMT-related proteins and genes. These results provided novel insights into the role of EF2 in tumorigenesis and progression in LSCC. EF2-targeted therapy could become a good strategy for the clinical treatment of LSCC. PMID:27542262

  16. Lack of discrimination against non-proteinogenic amino acid norvaline by elongation factor Tu from Escherichia coli.

    PubMed

    Cvetesic, Nevena; Akmacic, Irena; Gruic-Sovulj, Ita

    2013-01-01

    The GTP-bound form of elongation factor Tu (EF-Tu) brings aminoacylated tRNAs (aa-tRNA) to the A-site of the ribosome. EF-Tu binds all cognate elongator aa-tRNAs with highly similar affinities, and its weaker or tighter binding of misacylated tRNAs may discourage their participation in translation. Norvaline (Nva) is a non-proteinogenic amino acid that is activated and transferred to tRNA(Leu) by leucyl-tRNA synthetase (LeuRS). No notable accumulation of Nva-tRNA(Leu) has been observed in vitro, because of the efficient post-transfer hydrolytic editing activity of LeuRS. However, incorporation of norvaline into proteins in place of leucine does occur under certain conditions in vivo. Here we show that EF-Tu binds Nva-tRNA(Leu) and Leu-tRNA(Leu) with similar affinities, and that Nva-tRNA(Leu) and Leu-tRNA(Leu) dissociate from EF-Tu at comparable rates. The inability of EF-Tu to discriminate against norvaline may have driven evolution of highly efficient LeuRS editing as the main quality control mechanism against misincorporation of norvaline into proteins.

  17. 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

  18. Human cytomegalovirus pUL79 is an elongation factor of RNA polymerase II for viral gene transcription.

    PubMed

    Perng, Yi-Chieh; Campbell, Jessica A; Lenschow, Deborah J; Yu, Dong

    2014-08-01

    In this study, we have identified a unique mechanism in which human cytomegalovirus (HCMV) protein pUL79 acts as an elongation factor to direct cellular RNA polymerase II for viral transcription during late times of infection. We and others previously reported that pUL79 and its homologues are required for viral transcript accumulation after viral DNA synthesis. We hypothesized that pUL79 represented a unique mechanism to regulate viral transcription at late times during HCMV infection. To test this hypothesis, we analyzed the proteome associated with pUL79 during virus infection by mass spectrometry. We identified both cellular transcriptional factors, including multiple RNA polymerase II (RNAP II) subunits, and novel viral transactivators, including pUL87 and pUL95, as protein binding partners of pUL79. Co-immunoprecipitation (co-IP) followed by immunoblot analysis confirmed the pUL79-RNAP II interaction, and this interaction was independent of any other viral proteins. Using a recombinant HCMV virus where pUL79 protein is conditionally regulated by a protein destabilization domain ddFKBP, we showed that this interaction did not alter the total levels of RNAP II or its recruitment to viral late promoters. Furthermore, pUL79 did not alter the phosphorylation profiles of the RNAP II C-terminal domain, which was critical for transcriptional regulation. Rather, a nuclear run-on assay indicated that, in the absence of pUL79, RNAP II failed to elongate and stalled on the viral DNA. pUL79-dependent RNAP II elongation was required for transcription from all three kinetic classes of viral genes (i.e. immediate-early, early, and late) at late times during virus infection. In contrast, host gene transcription during HCMV infection was independent of pUL79. In summary, we have identified a novel viral mechanism by which pUL79, and potentially other viral factors, regulates the rate of RNAP II transcription machinery on viral transcription during late stages of HCMV infection.

  19. Crystal structures of the human elongation factor eEFSec suggest a non-canonical mechanism for selenocysteine incorporation

    PubMed Central

    Dobosz-Bartoszek, Malgorzata; Pinkerton, Mark H.; Otwinowski, Zbyszek; Chakravarthy, Srinivas; Söll, Dieter; Copeland, Paul R.; Simonović, Miljan

    2016-01-01

    Selenocysteine is the only proteinogenic amino acid encoded by a recoded in-frame UGA codon that does not operate as the canonical opal stop codon. A specialized translation elongation factor, eEFSec in eukaryotes and SelB in prokaryotes, promotes selenocysteine incorporation into selenoproteins by a still poorly understood mechanism. Our structural and biochemical results reveal that four domains of human eEFSec fold into a chalice-like structure that has similar binding affinities for GDP, GTP and other guanine nucleotides. Surprisingly, unlike in eEF1A and EF-Tu, the guanine nucleotide exchange does not cause a major conformational change in domain 1 of eEFSec, but instead induces a swing of domain 4. We propose that eEFSec employs a non-canonical mechanism involving the distinct C-terminal domain 4 for the release of the selenocysteinyl-tRNA during decoding on the ribosome. PMID:27708257

  20. Tau mRNA is present in axonal RNA granules and is associated with elongation factor 1A.

    PubMed

    Malmqvist, Tony; Anthony, Karen; Gallo, Jean-Marc

    2014-10-10

    The microtubule-associated protein tau is predominantly localized in the axonal compartment over the entire length of the axon in neurons. The mechanisms responsible for the localization of tau in axons at long distance from the cell body are not properly understood. Using fluorescence in situ hybridization, we show that tau mRNA is present in the central and distal parts of the axons of cultured rat cortical neurons. Axonal tau mRNA is associated with granules which are distributed throughout the entire length of the axon, including the growth cone. We also show that tau mRNA-containing axonal particles are associated with elongation factor 1A, a component of the protein translation machinery. The presence of tau mRNA in axons might be at least part of the process by which tau is localized to distal axons.

  1. Thiopental Inhibits Global Protein Synthesis by Repression of Eukaryotic Elongation Factor 2 and Protects from Hypoxic Neuronal Cell Death

    PubMed Central

    Schwer, Christian I.; Lehane, Cornelius; Guelzow, Timo; Zenker, Simone; Strosing, Karl M.; Spassov, Sashko; Erxleben, Anika; Heimrich, Bernd; Buerkle, Hartmut; Humar, Matjaz

    2013-01-01

    Ischemic and traumatic brain injury is associated with increased risk for death and disability. The inhibition of penumbral tissue damage has been recognized as a target for therapeutic intervention, because cellular injury evolves progressively upon ATP-depletion and loss of ion homeostasis. In patients, thiopental is used to treat refractory intracranial hypertension by reducing intracranial pressure and cerebral metabolic demands; however, therapeutic benefits of thiopental-treatment are controversially discussed. In the present study we identified fundamental neuroprotective molecular mechanisms mediated by thiopental. Here we show that thiopental inhibits global protein synthesis, which preserves the intracellular energy metabolite content in oxygen-deprived human neuronal SK-N-SH cells or primary mouse cortical neurons and thus ameliorates hypoxic cell damage. Sensitivity to hypoxic damage was restored by pharmacologic repression of eukaryotic elongation factor 2 kinase. Translational inhibition was mediated by calcium influx, activation of the AMP-activated protein kinase, and inhibitory phosphorylation of eukaryotic elongation factor 2. Our results explain the reduction of cerebral metabolic demands during thiopental treatment. Cycloheximide also protected neurons from hypoxic cell death, indicating that translational inhibitors may generally reduce secondary brain injury. In conclusion our study demonstrates that therapeutic inhibition of global protein synthesis protects neurons from hypoxic damage by preserving energy balance in oxygen-deprived cells. Molecular evidence for thiopental-mediated neuroprotection favours a positive clinical evaluation of barbiturate treatment. The chemical structure of thiopental could represent a pharmacologically relevant scaffold for the development of new organ-protective compounds to ameliorate tissue damage when oxygen availability is limited. PMID:24167567

  2. Omnipotent role of archaeal elongation factor 1 alpha (EF1α in translational elongation and termination, and quality control of protein synthesis.

    PubMed

    Saito, Kazuki; Kobayashi, Kan; Wada, Miki; Kikuno, Izumi; Takusagawa, Akira; Mochizuki, Masahiro; Uchiumi, Toshio; Ishitani, Ryuichiro; Nureki, Osamu; Ito, Koichi

    2010-11-09

    The molecular mechanisms of translation termination and mRNA surveillance in archaea remain unclear. In eukaryotes, eRF3 and HBS1, which are homologous to the tRNA carrier GTPase EF1α, respectively bind eRF1 and Pelota to decipher stop codons or to facilitate mRNA surveillance. However, genome-wide searches of archaea have failed to detect any orthologs to both GTPases. Here, we report the crystal structure of aRF1 from an archaeon, Aeropyrum pernix, and present strong evidence that the authentic archaeal EF1α acts as a carrier GTPase for aRF1 and for aPelota. The binding interface residues between aRF1 and aEF1α predicted from aRF1·aEF1α·GTP ternary structure model were confirmed by in vivo functional assays. The aRF1/eRF1 structural domain with GGQ motif, which corresponds to the CCA arm of tRNA, contacts with all three structural domains of aEF1α showing striking tRNA mimicry of aRF1/eRF1 and its GTPase-mediated catalysis for stop codon decoding. The multiple binding capacity of archaeal EF1α explains the absence of GTPase orthologs for eRF3 and HBS1 in archaea species and suggests that universal molecular mechanisms underlie translational elongation and termination, and mRNA surveillance pathways.

  3. A primary role for release factor 3 in quality control during translation elongation in Escherichia coli.

    PubMed

    Zaher, Hani S; Green, Rachel

    2011-10-14

    Release factor 3 (RF3) is a GTPase found in a broad range of bacteria where it is thought to play a critical "recycling" role in translation by facilitating the removal of class 1 release factors (RF1 and RF2) from the ribosome following peptide release. More recently, RF3 was shown in vitro to stimulate a retrospective editing reaction on the bacterial ribosome wherein peptides carrying mistakes are prematurely terminated during protein synthesis. Here, we examine the role of RF3 in the bacterial cell and show that the deletion of this gene sensitizes cells to other perturbations that reduce the overall fidelity of protein synthesis. We further document substantial effects on mRNA stability and protein expression using reporter systems, native mRNAs and proteins. We conclude that RF3 plays a primary role in vivo in specifying the fidelity of protein synthesis thus impacting overall protein quantity and quality.

  4. 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.

  5. The interaction surface of a bacterial transcription elongation factor required for complex formation with an antiterminator during transcription antitermination.

    PubMed

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

    2013-09-27

    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.

  6. 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

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

    PubMed

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

    2016-03-22

    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.

  8. 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...

  9. Regulation of the utilization of mRNA for eucaryotic elongation factor Tu in Friend erythroleukemia cells.

    PubMed Central

    Rao, T R; Slobin, L I

    1987-01-01

    When Friend erythroleukemia cells were allowed to grow to stationary phase (2 X 10(6) to 3 X 10(6) cells per ml), approximately 60% of the mRNA for eucaryotic elongation factor Tu (eEF-Tu) sedimented at less than or equal to 80S, and most of the remaining factor mRNA was associated with small polysomes. Under the same growth conditions, greater than 90% of the mRNA for eucaryotic initiation factor 4A remained associated with polysomes. The association of eEF-Tu mRNA with polysomes changed dramatically when stationary-phase cells were treated with fresh medium. After 1 h in fresh medium, approximately 90% of eEF-Tu mRNA in Friend cells was found in heavy polysomes. Associated with the shift of eEF-Tu mRNA into heavy polysomes, we found at least a 2.6-fold increase in the synthesis of eEF-Tu in vivo as well as a remarkable 40% decrease in the total amount of eEF-Tu mRNA per cell. Our data raise the possibility that eEF-Tu mRNA that has accumulated in ribonucleoprotein particles in stationary-phase cells is degraded rather than reutilized for eEF-Tu synthesis. Images PMID:2434834

  10. A transcription elongation factor that links signals from the reproductive system to lifespan extension in Caenorhabditis elegans.

    PubMed

    Ghazi, Arjumand; Henis-Korenblit, Sivan; Kenyon, Cynthia

    2009-09-01

    In Caenorhabditis elegans and Drosophila melanogaster, the aging of the soma is influenced by the germline. When germline-stem cells are removed, aging slows and lifespan is increased. The mechanism by which somatic tissues respond to loss of the germline is not well-understood. Surprisingly, we have found that a predicted transcription elongation factor, TCER-1, plays a key role in this process. TCER-1 is required for loss of the germ cells to increase C. elegans' lifespan, and it acts as a regulatory switch in the pathway. When the germ cells are removed, the levels of TCER-1 rise in somatic tissues. This increase is sufficient to trigger key downstream events, as overexpression of tcer-1 extends the lifespan of normal animals that have an intact reproductive system. Our findings suggest that TCER-1 extends lifespan by promoting the expression of a set of genes regulated by the conserved, life-extending transcription factor DAF-16/FOXO. Interestingly, TCER-1 is not required for DAF-16/FOXO to extend lifespan in animals with reduced insulin/IGF-1 signaling. Thus, TCER-1 specifically links the activity of a broadly deployed transcription factor, DAF-16/FOXO, to longevity signals from reproductive tissues.

  11. 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.

  12. Two genes encode related cytoplasmic elongation factors 1 alpha (EF-1 alpha) in Drosophila melanogaster with continuous and stage specific expression.

    PubMed Central

    Hovemann, B; Richter, S; Walldorf, U; Cziepluch, C

    1988-01-01

    We have characterized two previously cloned genes, F1 and F2 (1) that code for elongation factor EF - 1 alpha of Drosophila melanogaster. Genomic Southern blot hybridization revealed that they are the only gene copies present. We isolated cDNA clones of both transcripts from embryonal and pupal stage of development that cover the entire transcription unit. The 5' ends of both genes have been determined by primer extension and for F1 also by RNA sequencing. These start sites have been shown to be used consistently during development. Comparison of cDNA and genomic sequences revealed that EF - 1 alpha,F1 consists of two and EF - 1 alpha,F2 of five exons. The two described elongation factor genes exhibit several regions of strong sequence conservation when compared to five recently cloned eucaryotic elongation factors. Images PMID:3131735

  13. The Potyviral P3 Protein Targets Eukaryotic Elongation Factor 1A to Promote the Unfolded Protein Response and Viral Pathogenesis1[OPEN

    PubMed Central

    Shine, M.B.; Cui, Xiaoyan; Chen, Xin; Ma, Na; Kachroo, Pradeep; Zhi, Haijan; Kachroo, Aardra

    2016-01-01

    The biochemical function of the potyviral P3 protein is not known, although it is known to regulate virus replication, movement, and pathogenesis. We show that P3, the putative virulence determinant of soybean mosaic virus (SMV), targets a component of the translation elongation complex in soybean. Eukaryotic elongation factor 1A (eEF1A), a well-known host factor in viral pathogenesis, is essential for SMV virulence and the associated unfolded protein response (UPR). Silencing GmEF1A inhibits accumulation of SMV and another ER-associated virus in soybean. Conversely, endoplasmic reticulum (ER) stress-inducing chemicals promote SMV accumulation in wild-type, but not GmEF1A-knockdown, plants. Knockdown of genes encoding the eEF1B isoform, which is important for eEF1A function in translation elongation, has similar effects on UPR and SMV resistance, suggesting a link to translation elongation. P3 and GmEF1A promote each other’s nuclear localization, similar to the nuclear-cytoplasmic transport of eEF1A by the Human immunodeficiency virus 1 Nef protein. Our results suggest that P3 targets host elongation factors resulting in UPR, which in turn facilitates SMV replication and place eEF1A upstream of BiP in the ER stress response during pathogen infection. PMID:27356973

  14. Guanine-nucleotide exchange on ribosome-bound elongation factor G initiates the translocation of tRNAs

    PubMed Central

    Zavialov, Andrey V; Hauryliuk, Vasili V; Ehrenberg, Måns

    2005-01-01

    Background During the translation of mRNA into polypeptide, elongation factor G (EF-G) catalyzes the translocation of peptidyl-tRNA from the A site to the P site of the ribosome. According to the 'classical' model, EF-G in the GTP-bound form promotes translocation, while hydrolysis of the bound GTP promotes dissociation of the factor from the post-translocation ribosome. According to a more recent model, EF-G operates like a 'motor protein' and drives translocation of the peptidyl-tRNA after GTP hydrolysis. In both the classical and motor protein models, GDP-to-GTP exchange is assumed to occur spontaneously on 'free' EF-G even in the absence of a guanine-nucleotide exchange factor (GEF). Results We have made a number of findings that challenge both models. First, free EF-G in the cell is likely to be in the GDP-bound form. Second, the ribosome acts as the GEF for EF-G. Third, after guanine-nucleotide exchange, EF-G in the GTP-bound form moves the tRNA2-mRNA complex to an intermediate translocation state in which the mRNA is partially translocated. Fourth, subsequent accommodation of the tRNA2-mRNA complex in the post-translocation state requires GTP hydrolysis. Conclusion These results, in conjunction with previously published cryo-electron microscopy reconstructions of the ribosome in various functional states, suggest a novel mechanism for translocation of tRNAs on the ribosome by EF-G. Our observations suggest that the ribosome is a universal guanosine-nucleotide exchange factor for EF-G as previously shown for the class-II peptide-release factor 3. PMID:15985150

  15. Elongation factor P restricts Salmonella’s growth by controlling translation of a Mg2+ transporter gene during infection

    PubMed Central

    Choi, Eunna; Choi, Soomin; Nam, Daesil; Park, Shinae; Han, Yoontak; Lee, Jung-Shin; Lee, Eun-Jin

    2017-01-01

    When a ribosome translates mRNA sequences, the ribosome often stalls at certain codons because it is hard to translate. Consecutive proline codons are such examples that induce ribosome stalling and elongation factor P (EF-P) is required for the stalled ribosome to continue translation at those consecutive proline codons. We found that EF-P is required for translation of the mgtB gene encoding a Mg2+ transporter in the mgtCBR virulence operon from the intracellular pathogen Salmonella enterica serovar Typhimurium. Salmonella lacking EF-P decreases MgtB protein levels in a manner dependent on consecutive proline codons located in the mgtB coding region despite increasing transcription of the mgtCBR operon via the mgtP open reading frame in the leader RNA, resulting in an altered ratio between MgtC and MgtB proteins within the operon. Substitution of the consecutive proline codons to alanine codons eliminates EF-P-mediated control of the mgtB gene during infection and thus contributes to Salmonella’s survival inside macrophages where Salmonella experiences low levels of EF-P. This suggests that this pathogen utilizes a strategy to coordinate expression of virulence genes by an evolutionarily conserved translation factor. PMID:28181542

  16. 1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) Induces Phosphorylation of Eukaryotic Elongation Factor-2 (eEF2)

    PubMed Central

    Chen, Zehan; Gopalakrishnan, Sujatha M.; Bui, Mai-Ha; Soni, Niru B.; Warrior, Usha; Johnson, Eric F.; Donnelly, Jennifer B.; Glaser, Keith B.

    2011-01-01

    Eukaryotic elongation factor-2 kinase (eEF2K) relays growth and stress signals to protein synthesis through phosphorylation and inactivation of eukaryotic elongation factor 2 (eEF2). 1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) is a widely accepted inhibitor of mammalian eEF2K and an efficacious anti-proliferation agent against different cancer cells. It implied that eEF2K could be an efficacious anticancer target. However, eEF2K siRNA was ineffective against cancer cells including those sensitive to NH125. To test if pharmacological intervention differs from siRNA interference, we identified a highly selective small molecule eEF2K inhibitor A-484954. Like siRNA, A-484954 had little effect on cancer cell growth. We carefully examined the effect of NH125 and A-484954 on phosphorylation of eEF2, the known cellular substrate of eEF2K. Surprisingly, NH125 increased eEF2 phosphorylation, whereas A-484954 inhibited the phosphorylation as expected for an eEF2K inhibitor. Both A-484954 and eEF2K siRNA inhibited eEF2K and reduced eEF2 phosphorylation with little effect on cancer cell growth. These data demonstrated clearly that the anticancer activity of NH125 was more correlated with induction of eEF2 phosphorylation than inhibition of eEF2K. Actually, induction of eEF2 phosphorylation was reported to correlate with inhibition of cancer cell growth. We compared several known inducers of eEF2 phosphorylation including AMPK activators and an mTOR inhibitor. Interestingly, stronger induction of eEF2 phosphorylation correlated with more effective growth inhibition. We also explored signal transduction pathways leading to NH125-induced eEF2 phosphorylation. Preliminary data suggested that NH125-induced eEF2 phosphorylation was likely mediated through multiple pathways. These observations identified an opportunity for a new multipathway approach to anticancer therapies. PMID:22020937

  17. 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.

  18. Interaction of turnip yellow mosaic virus Val-RNA with eukaryotic elongation factor EF-1 [alpha]. Search for a function.

    PubMed

    Joshi, R L; Ravel, J M; Haenni, A L

    1986-06-01

    The 3'-terminal tRNA-like structure in turnip yellow mosaic virus (TYMV) RNA can be adenylated by tRNA nucleotidyltransferase and subsequently aminoacylated by valyl-tRNA synthetase. Here we present evidence that TYMV Val-RNA can form a stable complex with eukaryotic wheat germ elongation factor EF-1alpha and GTP: the Val-RNA is protected by EF-1alpha.. GTP against digestion by RNase A. By affinity chromatography of TYMV Val-RNA fragments on immobilized EF-1alpha . GTP, it has been established that the valylated aminoacyl RNA domain, which in TYMV RNA is formed by the 3' half of the tRNA-like region, is sufficient for complex formation with EF-1alpha . GTP. The aminoacyl RNA domain is equivalent in tRNAs to the continuous helix formed by the acceptor stem and the T stem and loop. In line with these results, the aminoacyl RNA domain in TYMV Val-RNA complexed to EF-1 alpha . GTP is resistant to digestion by RNase A. It is also shown that the TYMV RNA replicase (RNA-dependent RNA polymerase) isolated from TYMV-infected Chinese cabbage leaves does not contain tRNA nucleotidyltransferase, valyl-tRNA synthetase or EF-1alpha. This suggests that interaction of TYMV RNA with EF-1alpha is not mandatory for replicase activity.

  19. tRNA(Pro) -mediated downregulation of elongation factor P is required for mgtCBR expression during Salmonella infection.

    PubMed

    Nam, Daesil; Choi, Eunna; Shin, Dongwoo; Lee, Eun-Jin

    2016-10-01

    Bacterial ribosome requires elongation factor P to translate fragments harbouring consecutive proline codons. Given the abundance of ORFs with potential EF-P regulated sites, EF-P was assumed to be constitutively expressed. Here, we report that the intracellular pathogen Salmonella enterica serovar Typhimurium decreases efp mRNA levels during course of infection. We determined that the decrease in efp mRNA is triggered by low levels of charged tRNA(Pro) , a condition that Salmonella experiences when inside a macrophage phagosome. Surprisingly, downregulation of EF-P selectively promotes expression of the virulence mgtC gene and contributes to Salmonella's ability to survive inside macrophages. The decrease in EF-P levels induces ribosome stalling at the consecutive proline codons of the mgtP open reading frame in the mgtCBR leader RNA, and thus allows formation of a stem-loop structure promoting transcription of the mgtC gene. The substitution of proline codons in the mgtP gene eliminates EF-P-mediated mgtC expression and thus Salmonella's survival inside macrophages. Our findings indicate that Salmonella benefits virulence genes by decreasing EF-P levels and inducing the stringent response inside host.

  20. Host-cell positive transcription elongation factor b kinase activity is essential and limiting for HIV type 1 replication.

    PubMed

    Flores, O; Lee, G; Kessler, J; Miller, M; Schlief, W; Tomassini, J; Hazuda, D

    1999-06-22

    HIV-1 gene expression and viral replication require the viral transactivator protein Tat. The RNA polymerase II transcriptional elongation factor P-TEFb (cyclin-dependent kinase 9/cyclin T) is a cellular protein kinase that has recently been shown to be a key component of the Tat-transactivation process. For this report, we studied the requirement for P-TEFb in HIV-1 infection, and we now show that P-TEFb is both essential and limiting for HIV-1 replication. Attenuation of P-TEFb kinase activity either by expression of a dominant-negative cyclin-dependent kinase 9 transgene or through the use of small-molecule inhibitors suppresses HIV-1 gene expression and HIV-1 replication. Inhibition of HIV-1 replication is affected in a manner consistent with a direct and specific effect on P-TEFb and the known functional role of P-TEFb in Tat-activated transcription. Tat-activated expression of HIV-1 genes seems uniquely dependent on P-TEFb, as inhibition of P-TEFb activity and HIV-1 replication can be achieved without compromising cell viability or RNA polymerase II-dependent cellular gene transcription. Selective inhibition of the P-TEFb kinase may therefore provide a novel approach for developing chemotherapeutic agents against HIV-1.

  1. Protective effect of berberine on antioxidant enzymes and positive transcription elongation factor b expression in diabetic rat liver.

    PubMed

    Zhou, Ji-Yin; Zhou, Shi-Wen

    2011-03-01

    The protective effect of berberine against antioxidant, antilipid peroxidation in serum and liver tissue, and positive transcription elongation factor b (P-TEFb) expression in liver tissue of type 2 diabetic rats was investigated. Overnight fasted rats were intraperitoneally injected 35 mg/kg streptozotocin. Diabetic rats were admitted after 2 weeks and given a high-carbohydrate/high-fat diet to induce hyperlipidemias. From week 16, diabetic rats were treated with 75, 150, 300 mg/kg berberine, 100mg/kg fenofibrate or 4 mg/kg rosiglitazone for another 16 weeks. P-TEFb (composed of cyclin-dependent kinase 9 and cyclin T1) mRNA and protein expression in liver tissue were detected by real time PCR and immunohistochemistry, respectively. Berberine significantly up-regulated the declined cyclin-dependent kinase 9, cyclin T1 mRNA and protein expression in diabetic rat liver. Berberine obviously decreased malondialdehyde level and increased catalase, superoxide dismutase, glutathione peroxidase, and glutathione activities in liver tissue and serum of diabetic rats. These results suggest that the effects of berberine on up-regulation of P-TEFb expression, antioxidant and antilipid peroxidation may be related to its protective potential on diabetes.

  2. A non-canonical function of eukaryotic elongation factor 1A1: regulation of interleukin-6 expression.

    PubMed

    Schulz, Ingo; Engel, Claudia; Niestroj, André J; Kehlen, Astrid; Rahfeld, Jens-Ulrich; Kleinschmidt, Martin; Lehmann, Karola; Roßner, Steffen; Demuth, Hans-Ulrich

    2014-05-01

    Interleukin-6 is one of the most prominent triggers of inflammatory processes. We have shown recently that heteroarylketones (HAKs) interfere with stimulated interleukin-6 expression in astrocytes by suppression of STAT3 phosphorylation at serine 727. Surprisingly, this effect is not based on the inhibition of STAT3-relevant kinases. Therefore, we here used the structurally modified HAK compound biotin-HAK-3 in a reverse chemical approach to identify the relevant molecular target in UV-mediated cross-linking experiments. Employing streptavidin-specific 2D-immunoblotting followed by mass spectrometry we identified nine proteins putatively interacting with biotin-HAK-3. After co-immunoprecipitation, co-immunofluorescence, surface plasmon resonance analyses and RNAi-mediated knock-down, the eukaryotic elongation factor 1A1 (eEF1A1) was verified as the relevant target of HAK bioactivity. eEF1A1 forms complexes with STAT3 and PKCδ, which are crucial for STAT3(S727) phosphorylation and for NF-κB/STAT3-enhanced interleukin-6 expression. Furthermore, the intracellular HAK accumulation is strongly dependent on eEF1A1 expression. Taken together, the results reveal a novel molecular mechanism for a non-canonical role of eEF1A1 in signal transduction via direct modulation of kinase-dependent phosphorylation events.

  3. Regulation of the AEFG1 gene, a mitochondrial elongation factor G from the dimorphic yeast Arxula adeninivorans LS3.

    PubMed

    Wartmann, T; Gellissen, G; Kunze, G

    2001-10-01

    Oxygen influences the synthesis of mitochondrial proteins by alteration of the expression of mitochondrial genes and several nuclear genes. One of the genes localised in the nucleus is the EFG1 gene that encodes the mitochondrial elongation factor G (MEF-G). This unique gene (AEFG1) has been isolated from the non-conventional dimorphic yeast, Arxula adeninivorans LS3. The AEFG1 gene comprises a ORF of 2,274 bp, which corresponds to 757 amino acids. In the present study, the regulation of AEFG1 has been analysed for different morphological stages of A. adeninivorans and various culture conditions. It was demonstrated that the transfer of aerobically growing cultures to anaerobic conditions resulted in an accumulation of AEFG1 transcript, correlating with an increase in AMEF-G protein concentration. Since this regulation occurred in budding-cell culture growing at 30 degrees C and in both of the mycelial cultures grown at 45 degrees C and 30 degrees C, respectively, it was the oxygen level (but not the cultivation temperature or the morphological stage) which influenced the AEFG1 regulation.

  4. An interbacterial NAD(P)+ glycohydrolase toxin requires elongation factor Tu for delivery to target cells

    SciTech Connect

    Whitney, John C.; Quentin, Dennis; Sawai, Shin; LeRoux, Michele; Harding, Brittany N.; Ledvina, Hannah E.; Tran, Bao Q.; Robinson, Howard; Goo, Young Ah; Goodlett, David R.; Raunser, Stefan; Mougous, Joseph D.

    2015-10-08

    Type VI secretion (T6S) influences the composition of microbial communities by catalyzing the delivery of toxins between adjacent bacterial cells. Here, we demonstrate that a T6S integral membrane toxin from Pseudomonas aeruginosa, Tse6, acts on target cells by degrading the universally essential dinucleotides NAD+ and NADP+. Structural analyses of Tse6 show that it resembles mono-ADP-ribosyltransferase proteins, such as diphtheria toxin, with the exception of a unique loop that both excludes proteinaceous ADP-ribose acceptors and contributes to hydrolysis. We find that entry of Tse6 into target cells requires its binding to an essential housekeeping protein, translation elongation factor Tu (EF-Tu). These proteins participate in a larger assembly that additionally directs toxin export and provides chaperone activity. Lastly, visualization of this complex by electron microscopy defines the architecture of a toxin-loaded T6S apparatus and provides mechanistic insight into intercellular membrane protein delivery between bacteria.

  5. 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.

  6. Identification and characterisation of elongation factor Tu, a novel protein involved in Paracoccidioides brasiliensis-host interaction.

    PubMed

    Marcos, Caroline Maria; de Oliveira, Haroldo Cesar; da Silva, Julhiany de Fátima; Assato, Patricia Akemi; Yamazaki, Daniella Sayuri; da Silva, Rosângela Aparecida Moraes; Santos, Cláudia Tavares; Santos-Filho, Norival Alves; Portuondo, Deivys Leandro; Mendes-Giannini, Maria José Soares; Fusco-Almeida, Ana Marisa

    2016-11-01

    Paracoccidioides spp., which are temperature-dependent dimorphic fungi, are responsible for the most prevalent human systemic mycosis in Latin America, the paracoccidioidomycosis. The aim of this study was to characterise the involvement of elongation factor Tu (EF-Tu) in Paracoccidioides brasiliensis-host interaction. Adhesive properties were examined using recombinant PbEF-Tu proteins and the respective polyclonal anti-rPbEF-Tu antibody. Immunogold analysis demonstrated the surface location of EF-Tu in P. brasiliensis. Moreover, PbEF-Tu was found to bind to fibronectin and plasminogen by enzyme-linked immunosorbent assay, and it was determined that the binding to plasminogen is at least partly dependent on lysine residues and ionic interactions. To verify the participation of EF-Tu in the interaction of P. brasiliensis with pneumocytes, we blocked the respective protein with an anti-rPbEF-Tu antibody and evaluated the consequences on the interaction index by flow cytometry. During the interaction, we observed a decrease of 2- and 3-fold at 8 and 24 h, respectively, suggesting the contribution of EF-Tu in fungal adhesion/invasion.

  7. 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.

  8. 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.

  9. Phylogeny of the Enterobacteriaceae based on genes encoding elongation factor Tu and F-ATPase beta-subunit.

    PubMed

    Paradis, Sonia; Boissinot, Maurice; Paquette, Nancy; Bélanger, Simon D; Martel, Eric A; Boudreau, Dominique K; Picard, François J; Ouellette, Marc; Roy, Paul H; Bergeron, Michel G

    2005-09-01

    The phylogeny of enterobacterial species commonly found in clinical samples was analysed by comparing partial sequences of their elongation factor Tu gene (tuf) and of their F-ATPase beta-subunit gene (atpD). An 884 bp fragment for tuf and an 884 or 871 bp fragment for atpD were sequenced for 96 strains representing 78 species from 31 enterobacterial genera. The atpD sequence analysis exhibited an indel specific to Pantoea and Tatumella species, showing, for the first time, a tight phylogenetic affiliation between these two genera. Comprehensive tuf and atpD phylogenetic trees were constructed and are in agreement with each other. Monophyletic genera are Cedecea, Edwardsiella, Proteus, Providencia, Salmonella, Serratia, Raoultella and Yersinia. Analogous trees based on 16S rRNA gene sequences available from databases were also reconstructed. The tuf and atpD phylogenies are in agreement with the 16S rRNA gene sequence analysis, and distance comparisons revealed that the tuf and atpD genes provide better discrimination for pairs of species belonging to the family Enterobacteriaceae. In conclusion, phylogeny based on tuf and atpD conserved genes allows discrimination between species of the Enterobacteriaceae.

  10. Cloning, expression and evolution of the gene encoding the elongation factor 1alpha from a low thermophilic Sulfolobus solfataricus strain.

    PubMed

    Masullo, Mariorosario; Cantiello, Piergiuseppe; Lamberti, Annalisa; Longo, Olimpia; Fiengo, Antonio; Arcari, Paolo

    2003-01-28

    The gene encoding the elongation factor 1alpha (EF-1alpha) from the archaeon Sulfolobus solfataricus strain MT3 (optimum growth temperature 75 degrees C) was cloned, sequenced and expressed in Escherichia coli. The structural and biochemical properties of the purified enzyme were compared to those of EF-1alpha isolated from S. solfataricus strain MT4 (optimum growth temperature 87 degrees C). Only one amino acid change (Val15-->Ile) was found. Interestingly, the difference was in the first guanine nucleotide binding consensus sequence G(13)HIDHGK and was responsible for a reduced efficiency in protein synthesis, which was accompanied by an increased affinity for both guanosine diphosphate (GDP) and guanosine triphosphate (GTP), and an increased efficiency in the intrinsic GTPase activity. Despite the different thermophilicities of the two microorganisms, only very marginal effects on the thermal properties of the enzyme were observed. Molecular evolution among EF-1alpha genes from Sulfolobus species showed that the average rate of nucleotide substitution per site per year (0.0312x10(-9)) is lower than that reported for other functional genes.

  11. [Expression of elongation factor-1 alpha-A and beta-actin promoters in embryos of transgenic Medaka (Oryzias latipes)].

    PubMed

    Long, Hua

    2003-06-01

    Two expression vectors with the promoter of either Medaka (Oryzias latipes) elongation factor gene or beta-actin gene were constructed based on pBluescript SK+. Both of them are linked with green-fluorescent protein (GFP) gene. And they are named as pB-EF and pB-BA, respectively. The microinjection experiments were conducted with fertilized Medaka eggs at one-cell stage. The expression of two vectors, pB-EF and pB-BA, was observed under stereo-fluorescence microscope. The detection results showed that both EF-1 alpha-A promoter and beta-actin promoter are strong. In the process of embryo development, the activity of beta-actin promoter became stronger while that of EF-1 alpha-A promoter weaker gradually. beta-actin promoter was but EF-1 alpha-A promoter distributed throughout fish body uniformly. The expression rate of two vectors, pB-EF and pB-BA, are 8.23% and 6.10%, respectively.

  12. 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...

  13. Characterization of two malaria parasite organelle translation elongation factor G proteins: the likely targets of the anti-malarial fusidic acid.

    PubMed

    Johnson, Russell A; McFadden, Geoffrey I; Goodman, Christopher D

    2011-01-01

    Malaria parasites harbour two organelles with bacteria-like metabolic processes that are the targets of many anti-bacterial drugs. One such drug is fusidic acid, which inhibits the translation component elongation factor G. The response of P. falciparum to fusidic acid was characterised using extended SYBR-Green based drug trials. This revealed that fusidic acid kills in vitro cultured P. falciparum parasites by immediately blocking parasite development. Two bacterial-type protein translation elongation factor G genes are identified as likely targets of fusidic acid. Sequence analysis suggests that these proteins function in the mitochondria and apicoplast and both should be sensitive to fusidic acid. Microscopic examination of protein-reporter fusions confirm the prediction that one elongation factor G is a component of parasite mitochondria whereas the second is a component of the relict plastid or apicoplast. The presence of two putative targets for a single inhibitory compound emphasizes the potential of elongation factor G as a drug target in malaria.

  14. 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...

  15. Targeting elongation factor-2 kinase (eEF-2K) induces apoptosis in human pancreatic cancer cells.

    PubMed

    Ashour, Ahmed A; Abdel-Aziz, Abdel-Aziz H; Mansour, Ahmed M; Alpay, S Neslihan; Huo, Longfei; Ozpolat, Bulent

    2014-01-01

    Pancreatic cancer (PaCa) is one of the most aggressive, apoptosis-resistant and currently incurable cancers with a poor survival rate. Eukaryotic elongation factor-2 kinase (eEF-2K) is an atypical kinase, whose role in PaCa survival is not yet known. Here, we show that eEF-2K is overexpressed in PaCa cells and its down-regulation induces apoptotic cell death. Rottlerin (ROT), a polyphenolic compound initially identified as a PKC-δ inhibitor, induces apoptosis and autophagy in a variety of cancer cells including PaCa cells. We demonstrated that ROT induces intrinsic apoptosis, with dissipation of mitochondrial membrane potential (ΔΨm), and stimulates extrinsic apoptosis with concomitant induction of TNF-related apoptosis inducing ligand (TRAIL) receptors, DR4 and DR5, with caspase-8 activation, in PANC-1 and MIAPaCa-2 cells. Notably, while none of these effects were dependent on PKC-δ inhibition, ROT down-regulates eEF-2K at mRNA level, and induce eEF-2K protein degradation through ubiquitin-proteasome pathway. Down-regulation of eEF-2K recapitulates the events observed after ROT treatment, while its over-expression suppressed the ROT-induced apoptosis. Furthermore, eEF-2K regulates the expression of tissue transglutaminase (TG2), an enzyme previously implicated in proliferation, drug resistance and survival of cancer cells. Inhibition of eEF-2K/TG2 axis leads to caspase-independent apoptosis which is associated with induction of apoptosis-inducing factor (AIF). Collectively, these results indicate, for the first time, that the down-regulation of eEF-2K leads to induction of intrinsic, extrinsic as well as AIF-dependent apoptosis in PaCa cells, suggesting that eEF-2K may represent an attractive therapeutic target for the future anticancer agents in PaCa.

  16. Elucidation of eukaryotic elongation factor-2 contact sites within the catalytic domain of Pseudomonas aeruginosa exotoxin A.

    PubMed Central

    Yates, Susan P; Merrill, Allan R

    2004-01-01

    Pseudomonas aeruginosa produces the virulence factor, ETA (exotoxin A), which catalyses an ADP-ribosyltransferase reaction of its target protein, eEF2 (eukaryotic elongation factor-2). Currently, this protein-protein interaction is poorly characterized and this study was aimed at identifying the contact sites between eEF2 and the catalytic domain of ETA (PE24H, an ETA from P. aeruginosa, a 24 kDa C-terminal fragment containing a His6 tag). Single-cysteine residues were introduced into the toxin at 21 defined surface-exposed sites and labelled with the fluorophore, IAEDANS [5-(2-iodoacetylaminoethylamino)-1-napthalenesulphonic acid]. Fluorescence quenching studies using acrylamide, and fluorescence lifetime and wavelength emission maxima analyses were conducted in the presence and absence of eEF2. Large changes in the microenvironment of the AEDANS [5-(2-aminoethylamino)-1-naphthalenesulphonic acid] probe after eEF2 binding were not observed as dictated by both fluorescence lifetime and wavelength emission maxima values. This supported the proposed minimal contact model, which suggests that only small, discrete contacts occur between these proteins. As dictated by the bimolecular quenching constant (k(q)) for acrylamide, binding of eEF2 with toxin caused the greatest change in acrylamide accessibility (>50%) when the fluorescence label was near the active site or was located within a known catalytic loop. All mutant proteins showed a decrease in accessibility to acrylamide once eEF2 bound, although the relative change varied for each labelled protein. From these data, a low-resolution model of the toxin-eEF2 complex was constructed based on the minimal contact model with the intention of enhancing our knowledge on the mode of inactivation of the ribosome translocase by the Pseudomonas toxin. PMID:14733615

  17. 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.

  18. 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.

  19. 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

  20. Translation Elongation Factor eEF1A2 is a Novel Anticancer Target for the Marine Natural Product Plitidepsin

    PubMed Central

    Losada, Alejandro; Muñoz-Alonso, María José; García, Carolina; Sánchez-Murcia, Pedro A.; Martínez-Leal, Juan Fernando; Domínguez, Juan Manuel; Lillo, M. Pilar; Gago, Federico; Galmarini, Carlos M.

    2016-01-01

    eEF1A2 is one of the isoforms of the alpha subunit of the eukaryotic Elongation Factor 1. It is overexpressed in human tumors and is endowed with oncogenic properties, favoring tumor cell proliferation while inhibiting apoptosis. We demonstrate that plitidepsin, an antitumor agent of marine origin that has successfully completed a phase-III clinical trial for multiple myeloma, exerts its antitumor activity by targeting eEF1A2. The drug interacts with eEF1A2 with a KD of 80 nM and a target residence time of circa 9 min. This protein was also identified as capable of binding [14C]-plitidepsin in a cell lysate from K-562 tumor cells. A molecular modelling approach was used to identify a favorable binding site for plitidepsin at the interface between domains 1 and 2 of eEF1A2 in the GTP conformation. Three tumor cell lines selected for at least 100-fold more resistance to plitidepsin than their respective parental cells showed reduced levels of eEF1A2 protein. Ectopic expression of eEF1A2 in resistant cells restored the sensitivity to plitidepsin. FLIM-phasor FRET experiments demonstrated that plitidepsin localizes in tumor cells sufficiently close to eEF1A2 as to suggest the formation of drug-protein complexes in living cells. Altogether, our results strongly suggest that eEF1A2 is the primary target of plitidepsin. PMID:27713531

  1. Toxoplasma gondii Elongation Factor 1-Alpha (TgEF-1α) Is a Novel Vaccine Candidate Antigen against Toxoplasmosis.

    PubMed

    Wang, Shuai; Zhang, Zhenchao; Wang, Yujian; Gadahi, Javaid A; Xu, Lixin; Yan, Ruofeng; Song, Xiaokai; Li, Xiangrui

    2017-01-01

    Toxoplasma gondii (T. gondii) is an obligate intracellular parasite which can infect almost all warm-blood animals, leading to toxoplasmosis. Screening and discovery of an effective vaccine candidate or new drug target is crucial for the control of this disease. In this study, the recombinant T. gondii elongation factor 1-alpha (rTgEF-1α) was successfully expressed in in Escherichia coli. Passive immunization of mice with anti-rTgEF-1α polyclonal antibody following challenge with a lethal dose of tachyzoites significantly increased the survival time compared with PBS control group. The survival time of mice challenged with tachyzoites pretreated with anti-rTgEF-1α PcAb also was significantly increased. Invasion of tachyzoites into mouse macrophages was significantly inhibited in the anti-rTgEF-1α PcAb pretreated group. Mice vaccinated with rTgEF-1α induced a high level of specific anti-T. gondii antibodies and production of IFN-gamma, interleukin-4. The expression levels of MHC-I and MHC-II molecules as well as the percentages of CD4(+) and CD8(+) T cells in mice vaccinated with rTgEF-1α was significantly increased, respectively (P < 0.05), compared with all the controls. Immunization with rTgEF-1α significantly (P < 0.05) prolonged survival time (14.53 ± 1.72 days) after challenge infection with the virulent T. gondii RH strain. These results indicate that T. gondii EF-1α plays an essential role in mediating host cell invasion by the parasite and, as such, could be a candidate vaccine antigen against toxoplasmosis.

  2. Toxoplasma gondii Elongation Factor 1-Alpha (TgEF-1α) Is a Novel Vaccine Candidate Antigen against Toxoplasmosis

    PubMed Central

    Wang, Shuai; Zhang, Zhenchao; Wang, Yujian; Gadahi, Javaid A.; Xu, Lixin; Yan, Ruofeng; Song, Xiaokai; Li, Xiangrui

    2017-01-01

    Toxoplasma gondii (T. gondii) is an obligate intracellular parasite which can infect almost all warm-blood animals, leading to toxoplasmosis. Screening and discovery of an effective vaccine candidate or new drug target is crucial for the control of this disease. In this study, the recombinant T. gondii elongation factor 1-alpha (rTgEF-1α) was successfully expressed in in Escherichia coli. Passive immunization of mice with anti-rTgEF-1α polyclonal antibody following challenge with a lethal dose of tachyzoites significantly increased the survival time compared with PBS control group. The survival time of mice challenged with tachyzoites pretreated with anti-rTgEF-1α PcAb also was significantly increased. Invasion of tachyzoites into mouse macrophages was significantly inhibited in the anti-rTgEF-1α PcAb pretreated group. Mice vaccinated with rTgEF-1α induced a high level of specific anti-T. gondii antibodies and production of IFN-gamma, interleukin-4. The expression levels of MHC-I and MHC-II molecules as well as the percentages of CD4+ and CD8+ T cells in mice vaccinated with rTgEF-1α was significantly increased, respectively (P < 0.05), compared with all the controls. Immunization with rTgEF-1α significantly (P < 0.05) prolonged survival time (14.53 ± 1.72 days) after challenge infection with the virulent T. gondii RH strain. These results indicate that T. gondii EF-1α plays an essential role in mediating host cell invasion by the parasite and, as such, could be a candidate vaccine antigen against toxoplasmosis. PMID:28243226

  3. Molecular control of the amount, subcellular location, and activity state of translation elongation factor 2 in neurons experiencing stress.

    PubMed

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

    2013-08-01

    Eukaryotic elongation factor 2 (eEF-2) is an important regulator of the protein translation machinery whereby 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 by 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 nonphosphorylated (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 after 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.

  4. Mapping the human translation elongation factor eEF1H complex using the yeast two-hybrid system.

    PubMed Central

    Mansilla, Francisco; Friis, Irene; Jadidi, Mandana; Nielsen, Karen M; Clark, Brian F C; Knudsen, Charlotte R

    2002-01-01

    In eukaryotes, the eukaryotic translation elongation factor eEF1A responsible for transporting amino-acylated tRNA to the ribosome forms a higher-order complex, eEF1H, with its guanine-nucleotide-exchange factor eEF1B. In metazoans, eEF1B consists of three subunits: eEF1B alpha, eEF1B eta and eEF1B gamma. The first two subunits possess the nucleotide-exchange activity, whereas the role of the last remains poorly defined. In mammals, two active tissue-specific isoforms of eEF1A have been identified. The reason for this pattern of differential expression is unknown. Several models on the basis of in vitro experiments have been proposed for the macromolecular organization of the eEF1H complex. However, these models differ in various aspects. This might be due to the difficulties of handling, particularly the eEF1B beta and eEF1B gamma subunits in vitro. Here, the human eEF1H complex is for the first time mapped using the yeast two-hybrid system, which is a powerful in vivo technique for analysing protein-protein interactions. The following complexes were observed: eEF1A1:eEF1B alpha, eEF1A1:eEF1B beta, eEF1B beta:eEF1B beta, eEF1B alpha:eEF1B gamma, eEF1B beta:eEF1B gamma and eEF1B alpha:eEF1B gamma:eEF1B beta, where the last was observed using a three-hybrid approach. Surprisingly, eEF1A2 showed no or only little affinity for the guanine-nucleotide-exchange factors. Truncated versions of the subunits of eEF1B were used to orientate these subunits within the resulting model. The model unit is a pentamer composed of two molecules of eEF1A, each interacting with either eEF1B alpha or eEF1B beta held together by eEF1B gamma. These units can dimerize via eEF1B beta. Our model is compared with other models, and structural as well as functional aspects of the model are discussed. PMID:11985494

  5. Activity and regulation by growth factors of calmodulin-dependent protein kinase III (elongation factor 2-kinase) in human breast cancer

    PubMed Central

    Parmer, T G; Ward, M D; Yurkow, E J; Vyas, V H; Kearney, T J; Hait, W N

    1999-01-01

    Calmodulin-dependent protein kinase III (CaM kinase III, elongation factor-2 kinase) is a unique member of the Ca2+/CaM-dependent protein kinase family. Activation of CaM kinase III leads to the selective phosphorylation of elongation factor 2 (eEF-2) and transient inhibition of protein synthesis. Recent cloning and sequencing of CaM kinase III revealed that this enzyme represents a new superfamily of protein kinases. The activity of CaM kinase III is selectively activated in proliferating cells; inhibition of the kinase blocked cells in G0/G1-S and decreased viability. To determine the significance of CaM kinase III in breast cancer, we measured the activity of the kinase in human breast cancer cell lines as well as in fresh surgical specimens. The specific activity of CaM kinase III in human breast cancer cell lines was equal to or greater than that seen in a variety of cell lines with similar rates of proliferation. The specific activity of CaM kinase III was markedly increased in human breast tumour specimens compared with that of normal adjacent breast tissue. The activity of this enzyme was regulated by breast cancer mitogens. In serum-deprived MDA-MB-231 cells, the combination of insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) stimulated cell proliferation and activated CaM kinase III to activities observed in the presence of 10% serum. Inhibition of enzyme activity blocked cell proliferation induced by growth factors. In MCF-7 cells separated by fluorescence-activated cell sorting, CaM kinase III was increased in S-phase over that of other phases of the cell cycle. In summary, the activity of Ca2+/CaM-dependent protein kinase III is controlled by breast cancer mitogens and appears to be constitutively activated in human breast cancer. These results suggest that CaM kinase III may contribute an important link between growth factor/receptor interactions, protein synthesis and the induction of cellular proliferation in human breast

  6. Secretomic Analysis of Host-Pathogen Interactions Reveals That Elongation Factor-Tu Is a Potential Adherence Factor of Helicobacter pylori during Pathogenesis.

    PubMed

    Chiu, Kuo-Hsun; Wang, Ling-Hui; Tsai, Tsung-Ting; Lei, Huan-Yao; Liao, Pao-Chi

    2017-01-06

    The secreted proteins of bacteria are usually accompanied by virulence factors, which can cause inflammation and damage host cells. Identifying the secretomes arising from the interactions of bacteria and host cells could therefore increase understanding of the mechanisms during initial pathogenesis. The present study used a host-pathogen coculture system of Helicobacter pylori and monocytes (THP-1 cells) to investigate the secreted proteins associated with initial H. pylori pathogenesis. The secreted proteins from the conditioned media from H. pylori, THP-1 cells, and the coculture were collected and analyzed using SDS-PAGE and LC-MS/MS. Results indicated the presence of 15 overexpressed bands in the coculture. Thirty-one proteins were identified-11 were derived from THP-1 cells and 20 were derived from H. pylori. A potential adherence factor from H. pylori, elongation factor-Tu (EF-Tu), was selected for investigation of its biological function. Results from confocal microscopic and flow cytometric analyses indicated the contribution of EF-Tu to the binding ability of H. pylori in THP-1. The data demonstrated that fluorescence of EF-Tu on THP-1 cells increased after the addition of the H. pylori-conditioned medium. This study reports a novel secretory adherence factor in H. pylori, EF-Tu, and further elucidates mechanisms of H. pylori adaptation for host-pathogen interaction during pathogenesis.

  7. A major substrate for MPF: cDNA cloning and expression of polypeptide chain elongation factor 1 gamma from goldfish (Carassius auratus).

    PubMed

    Tokumoto, Mika; Nagahama, Yoshitaka; Tokumoto, Toshinobu

    2002-02-01

    One of the eukaryotic polypeptide chain elongation factors, EF-1 beta gamma delta complex, is involved in polypeptide chain elongation via the GDP/GTP exchange activity of EF-1 alpha. In the complex, EF-1 gamma has been reported to be a major substrate for maturation promoting factor (MPF). Here, we present the cloning, sequencing and expression analysis of goldfish, Carassius auratus, EF-1 gamma from the goldfish ovary. The cloned cDNA was 1490 bp in length and encoded 442 amino acids. The deduced amino acid sequence was highly homologous to EF-1 gamma from other species. Although, the phosphorylation site identified in Xenopus EF-1 gamma was not conserved in the goldfish homologue, phosphorylation analysis showed that the goldfish EF-1 gamma was phosphorylated by MPF. We concluded that EF-1 gamma is a substrate for MPF during oocyte maturation in goldfish.

  8. A single amino acid substitution in elongation factor Tu disrupts interaction between the ternary complex and the ribosome.

    PubMed Central

    Tubulekas, I; Hughes, D

    1993-01-01

    Elongation factor Tu (EF-Tu).GTP has the primary function of promoting the efficient and correct interaction of aminoacyl-tRNA with the ribosome. Very little is known about the elements in EF-Tu involved in this interaction. We describe a mutant form of EF-Tu, isolated in Salmonella typhimurium, that causes a severe defect in the interaction of the ternary complex with the ribosome. The mutation causes the substitution of Val for Gly-280 in domain II of EF-Tu. The in vivo growth and translation phenotypes of strains harboring this mutation are indistinguishable from those of strains in which the same tuf gene is insertionally inactivated. Viable cells are not obtained when the other tuf gene is inactivated, showing that the mutant EF-Tu alone cannot support cell growth. We have confirmed, by partial protein sequencing, that the mutant EF-Tu is present in the cells. In vitro analysis of the natural mixture of wild-type and mutant EF-Tu allows us to identify the major defect of this mutant. Our data shows that the EF-Tu is homogeneous and competent with respect to guanine nucleotide binding and exchange, stimulation of nucleotide exchange by EF-Ts, and ternary complex formation with aminoacyl-tRNA. However various measures of translational efficiency show a significant reduction, which is associated with a defective interaction between the ribosome and the mutant EF-Tu.GTP.aminoacyl-tRNA complex. In addition, the antibiotic kirromycin, which blocks translation by binding EF-Tu on the ribosome, fails to do so with this mutant EF-Tu, although it does form a complex with EF-Tu. Our results suggest that this region of domain II in EF-Tu has an important function and influences the binding of the ternary complex to the codon-programmed ribosome during protein synthesis. Models involving either a direct or an indirect effect of the mutation are discussed. Images PMID:8416899

  9. 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

  10. 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.

  11. Release of positive transcription elongation factor b (P-TEFb) from 7SK small nuclear ribonucleoprotein (snRNP) activates hexamethylene bisacetamide-inducible protein (HEXIM1) transcription.

    PubMed

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

    2014-04-04

    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.

  12. [Analysis of protein-on-DNA binding profiles, detected with chIP-seq method, reveals possible interaction of specific transcription factors with RNA polymerase II in the process of transcription elongation].

    PubMed

    Belostotskiĭ, A A

    2012-01-01

    It is thought that in the course of mRNA transcription almost all transcription factors stay on a promoter while RNA polymerase II "clears" the promoter and "proceeds" to elongation. However, analysis of some specific transcription factors and RNA polymerase II binding profiles on DNA, detected with ChIP-seq method, revealed the possibility of interaction between transcription factors and RNA polymerase II in the process of transcription elongation.

  13. Elongation factor Tu D138N, a mutant with modified substrate specificity, as a tool to study energy consumption in protein biosynthesis.

    PubMed

    Weijland, A; Parlato, G; Parmeggiani, A

    1994-09-06

    Substitution Asp138-->Asn changes the substrate specificity of elongation factor (EF) Tu from GTP to XTP [Hwang & Miller (1987) J. Biol. Chem. 262, 13081-13085]. This mutated EF-Tu (EF-Tu D138N) was used to show that 2 XTP molecules are hydrolyzed for each elongation cycle [Weijland & Parmeggiani (1993) Science 259, 1311-1313]. Here we extend the study of the properties of this EF-Tu mutant and its function in the elongation process. In poly(U)-directed poly(phenylalanine) synthesis, the number of peptide chains synthesized using EF-Tu D138N.XTP was 30% higher than with EF-Tu wild type (wt).GTP. However, since in the former case the average peptide chain length was correspondingly reduced, the number of the residues incorporated turned out to be nearly the same in both systems. The K'd values of the XTP and XDP complexes of EF-Tu D138N were similar to those of the GTP and GDP complexes of EF-Tu wt. The extent of leucine misincorporation and the kirromycin effect were also comparable to those in the EF-Tu wt/GTP system. The hydrolysis of two XTP molecules, very likely as part of two EF-Tu D138N.XTP complexes, for each elongation cycle was found to be independent of (i) MgCl2 concentration, (ii) ribosome concentration, and (iii) temperature (5-40 degrees C). With rate-limiting amounts of XTP the K'm of its XTPase activity corresponded to the K'm for XTP of poly(phenylalanine) synthesis (0.3-0.6 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

  14. 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

    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.

  15. 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

  16. Mutation in subdomain G' of mitochondrial elongation factor G1 is associated with combined OXPHOS deficiency in fibroblasts but not in muscle

    PubMed Central

    Smits, Paulien; Antonicka, Hana; van Hasselt, Peter M; Weraarpachai, Woranontee; Haller, Wolfram; Schreurs, Marieke; Venselaar, Hanka; Rodenburg, Richard J; Smeitink, Jan A; van den Heuvel, Lambert P

    2011-01-01

    The mitochondrial translation system is responsible for the synthesis of 13 proteins required for oxidative phosphorylation (OXPHOS), the major energy-generating process of our cells. Mitochondrial translation is controlled by various nuclear encoded proteins. In 27 patients with combined OXPHOS deficiencies, in whom complex II (the only complex that is entirely encoded by the nuclear DNA) showed normal activities, and mutations in the mitochondrial genome as well as polymerase gamma were excluded, we screened all mitochondrial translation factors for mutations. Here, we report a mutation in mitochondrial elongation factor G1 (GFM1) in a patient affected by severe, rapidly progressive mitochondrial encephalopathy. This mutation is predicted to result in an Arg250Trp substitution in subdomain G' of the elongation factor G1 protein and is presumed to hamper ribosome-dependent GTP hydrolysis. Strikingly, the decrease in enzyme activities of complex I, III and IV detected in patient fibroblasts was not found in muscle tissue. The OXPHOS system defects and the impairment in mitochondrial translation in fibroblasts were rescued by overexpressing wild-type GFM1, establishing the GFM1 defect as the cause of the fatal mitochondrial disease. Furthermore, this study evinces the importance of a thorough diagnostic biochemical analysis of both muscle tissue and fibroblasts in patients suspected to suffer from a mitochondrial disorder, as enzyme deficiencies can be selectively expressed. PMID:21119709

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

    PubMed

    Xie, Chuan-Ming; Liu, Xiao-Yu; Sham, Kathy W Y; Lai, Josie M Y; Cheng, Christopher H K

    2014-09-01

    EEF2K (eukaryotic elongation factor-2 kinase), also known as Ca (2+)/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.

  18. 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.

  19. 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.

  20. Characterization of the Human Transcription Elongation Factor Rtf1: Evidence for Nonoverlapping Functions of Rtf1 and the Paf1 Complex.

    PubMed

    Cao, Qing-Fu; Yamamoto, Junichi; Isobe, Tomoyasu; Tateno, Shumpei; Murase, Yuki; Chen, Yexi; Handa, Hiroshi; Yamaguchi, Yuki

    2015-10-01

    Restores TBP function 1 (Rtf1) is generally considered to be a subunit of the Paf1 complex (PAF1C), a multifunctional protein complex involved in histone modification and transcriptional or posttranscriptional regulation. Rtf1, however, is not stably associated with the PAF1C in most species except Saccharomyces cerevisiae, and its biochemical functions are not well understood. Here, we show that human Rtf1 is a transcription elongation factor that may function independently of the PAF1C. Rtf1 requires "Rtf1 coactivator" activity, which is most likely unrelated to the PAF1C or DSIF, for transcriptional activation in vitro. A mutational study revealed that the Plus3 domain of human Rtf1 is critical for its coactivator-dependent function. Transcriptome sequencing (RNA-seq) and chromatin immunoprecipitation studies in HeLa cells showed that Rtf1 and the PAF1C play distinct roles in regulating the expression of a subset of genes. Moreover, contrary to the finding in S. cerevisiae, the PAF1C was apparently recruited to the genes examined in an Rtf1-independent manner. The present study establishes a role for human Rtf1 as a transcription elongation factor and highlights the similarities and differences between the S. cerevisiae and human Rtf1 proteins.

  1. Transcription elongation regulator 1 is a co-integrator of the cell fate determination factor Dachshund homolog 1.

    PubMed

    Zhou, Jie; Liu, Yang; Zhang, Wei; Popov, Vladimir M; Wang, Min; Pattabiraman, Nagarajan; Suñé, Carlos; Cvekl, Ales; Wu, Kongming; Jiang, Jie; Wang, Chenguang; Pestell, Richard G

    2010-12-17

    DACH1 (Dachshund homolog 1) is a key component of the retinal determination gene network and regulates gene expression either indirectly as a co-integrator or through direct DNA binding. The current studies were conducted to understand, at a higher level of resolution, the mechanisms governing DACH1-mediated transcriptional repression via DNA sequence-specific binding. DACH1 repressed gene transcription driven by the DACH1-responsive element (DRE). Recent genome-wide ChIP-Seq analysis demonstrated DACH1 binding sites co-localized with Forkhead protein (FOX) binding sites. Herein, DACH1 repressed, whereas FOX proteins enhanced, both DRE and FOXA-responsive element-driven gene expression. Reduced DACH1 expression using a shRNA approach enhanced FOX protein activity. As DACH1 antagonized FOX target gene expression and attenuated FOX signaling, we sought to identify limiting co-integrator proteins governing DACH1 signaling. Proteomic analysis identified transcription elongation regulator 1 (TCERG1) as the transcriptional co-regulator of DACH1 activity. The FF2 domain of TCERG1 was required for DACH1 binding, and the deletion of FF2 abolished DACH1 trans-repression function. The carboxyl terminus of DACH1 was necessary and sufficient for TCERG1 binding. Thus, DACH1 represses gene transcription through direct DNA binding to the promoter region of target genes by recruiting the transcriptional co-regulator, TCERG1.

  2. Structure of the Kti11/Kti13 heterodimer and its double role in modifications of tRNA and eukaryotic elongation factor 2.

    PubMed

    Glatt, Sebastian; Zabel, Rene; Vonkova, Ivana; Kumar, Amit; Netz, Daili J; Pierik, Antonio J; Rybin, Vladimir; Lill, Roland; Gavin, Anne-Claude; Balbach, Jochen; Breunig, Karin D; Müller, Christoph W

    2015-01-06

    The small, highly conserved Kti11 alias Dph3 protein encoded by the Kluyveromyces lactis killer toxin insensitive gene KTI11/DPH3 is involved in the diphthamide modification of eukaryotic elongation factor 2 and, together with Kti13, in Elongator-dependent tRNA wobble base modifications, thereby affecting the speed and accuracy of protein biosynthesis through two distinct mechanisms. We have solved the crystal structures of Saccharomyces cerevisiae Kti13 and the Kti11/Kti13 heterodimer at 2.4 and 2.9 Å resolution, respectively, and validated interacting residues through mutational analysis in vitro and in vivo. We show that metal coordination by Kti11 and its heterodimerization with Kti13 are essential for both translational control mechanisms. Our structural and functional analyses identify Kti13 as an additional component of the diphthamide modification pathway and provide insight into the molecular mechanisms that allow the Kti11/Kti13 heterodimer to coregulate two consecutive steps in ribosomal protein synthesis.

  3. 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

  4. 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%.

  5. F-actin sequesters elongation factor 1alpha from interaction with aminoacyl-tRNA in a pH-dependent reaction

    PubMed Central

    1996-01-01

    The machinery of eukaryotic protein synthesis is found in association with the actin cytoskeleton. A major component of this translational apparatus, which is involved in the shuttling of aa-tRNA, is the actin- binding protein elongation factor 1alpha (EF-1alpha). To investigate the consequences for translation of the interaction of EF-1alpha with F- actin, we have studied the effect of F-actin on the ability of EF- 1alpha to bind to aa-tRNA. We demonstrate that binding of EF-1alpha:GTP to aa-tRNA is not pH sensitive with a constant binding affinity of approximately 0.2 microM over the physiological range of pH. However, the sharp pH dependence of binding of EF-1alpha to F-actin is sufficient to shift the binding of EF-1alpha from F-actin to aa-tRNA as pH increases. The ability of EF-1alpha to bind either F-actin or aa- tRNA in competition binding experiments is also consistent with the observation that EF-1alpha's binding to F-actin and aa-tRNA is mutually exclusive. Two pH-sensitive actin-binding sequences in EF-1alpha are identified and are predicted to overlap with the aa-tRNA-binding sites. Our results suggest that pH-regulated recruitment and release of EF- 1alpha from actin filaments in vivo will supply a high local concentration of EF-1alpha to facilitate polypeptide elongation by the F-actin-associated translational apparatus. PMID:8922379

  6. Effect of elongation factor 1alpha promoter and SUMF1 over in vitro expression of N-acetylgalactosamine-6-sulfate sulfatase.

    PubMed

    Alméciga-Díaz, Carlos J; Rueda-Paramo, Maria A; Espejo, Angela J; Echeverri, Olga Y; Montaño, Adriana; Tomatsu, Shunji; Barrera, Luis A

    2009-09-01

    Morquio A is an autosomal recessive disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to the lysosomal accumulation of keratan-sulfate and chondroitin-6-sulfate. We evaluated in HEK293 cells the effect of the cytomegalovirus immediate early enhancer/promoter (CMV) or the elongation factor 1alpha (EF1alpha) promoters, and the coexpression with the sulfatase modifying factor 1 (SUMF1) on GALNS activity. Four days postransfection GALNS activity in transfected cells with CMV-pIRES-GALNS reached a plateau, whereas in cells transfected with EF1alpha-pIRES-GALNS continued to increase until day 8. Co-transfection with pCXN-SUMF1 showed an increment up to 2.6-fold in GALNS activity. Finally, computational analysis of transcription factor binding-sites and CpG islands showed that EF1alpha promoter has long CpG islands and high-density binding-sites for Sp1 compared to CMV. These results show the advantage of the SUMF1 coexpression on GALNS activity and indicate a considerable effect on the expression stability using EF1alpha promoter compared to CMV.

  7. Immobilized nerve growth factor and microtopography have distinct effects on polarization versus axon elongation in hippocampal cells in culture.

    PubMed

    Gomez, Natalia; Lu, Yi; Chen, Shaochen; Schmidt, Christine E

    2007-01-01

    Cell interfacing with biomaterial surfaces dictates important aspects of cell behavior. In particular, axon extension in neurons is effectively influenced by surface properties, both for the initial formation of an axon as well as for the maintenance of axon growth. Here, we investigated how neurons behaved on poly(dimethyl siloxane) (PDMS) surfaces decorated with biochemical and physical cues presented individually or in combination. In particular, nerve growth factor (NGF) was covalently tethered to PDMS to create a bioactive surface, and microtopography was introduced to the material in the form of microchannels. Embryonic hippocampal neurons were used to investigate the impact of these surface cues on polarization (i.e., axon initiation or axogenesis) and overall axon length. We found that topography had a more pronounced effect on polarization (68% increase over controls) compared to immobilized NGF (0.1 ng/mm(2)) (27% increase). However, the effect of NGF was negligible when both types of stimuli were simultaneously presented on the biomaterial surface. In addition to axon formation, chemical and physical cues are also involved in axon growth following the initiation process. Interestingly, for the same studies described above, the effects of microchannels and NGF were opposite from the effects on polarization; the most evident effect was for the immobilized growth factor (10% increase in axon length with respect to controls) whereas there was no effect in general for the microtopography. More importantly, when the two surface stimuli were presented in combination, a synergistic increase in axon length was detected (25% increase with respect to controls), which could be a result of faster polarization triggered by topography plus enhanced growth from NGF. Additionally, axon orientation was also analyzed and we found the well-known tendency of perpendicular or parallel axonal alignment to be dependent on the width and depth of the channels. This investigation

  8. 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.

  9. Elongation factor 2 diphthamide is critical for translation of two IRES-dependent protein targets, XIAP and FGF2, under oxidative stress conditions.

    PubMed

    Argüelles, Sandro; Camandola, Simonetta; Cutler, Roy G; Ayala, Antonio; Mattson, Mark P

    2014-02-01

    Elongation factor-2 (eEF2) catalyzes the movement of the ribosome along the mRNA. A single histidine residue in eEF2 (H715) is modified to form diphthamide. A role for eEF2 in the cellular stress response is highlighted by the fact that eEF2 is sensitive to oxidative stress and that it must be active to drive the synthesis of proteins that help cells to mitigate the adverse effects of oxidative stress. Many of these proteins are encoded by mRNAs containing a sequence called an "internal ribosomal entry site" (IRES). Under high oxidative stress conditions diphthamide-deficient cells were significantly more sensitive to cell death. These results suggest that diphthamide may play a role in protection against the degradation of eEF2. This protection is especially important in those situations in which eEF2 is necessary for the reprogramming of translation from global to IRES synthesis. Indeed, we found that the expression of X-linked inhibitor of apoptosis (XIAP) and fibroblast growth factor 2 (FGF2), two proteins synthesized from mRNAs with IRESs that promote cell survival, is deregulated in diphthamide-deficient cells. Our findings therefore suggest that eEF2 diphthamide controls the selective translation of IRES-dependent protein targets XIAP and FGF2, critical for cell survival under conditions of oxidative stress.

  10. Haploinsufficiency for translation elongation factor eEF1A2 in aged mouse muscle and neurons is compatible with normal function.

    PubMed

    Griffiths, Lowri A; Doig, Jennifer; Churchhouse, Antonia M D; Davies, Faith C J; Squires, Charlotte E; Newbery, Helen J; Abbott, Catherine M

    2012-01-01

    Translation elongation factor isoform eEF1A2 is expressed in muscle and neurons. Deletion of eEF1A2 in mice gives rise to the neurodegenerative phenotype "wasted" (wst). Mice homozygous for the wasted mutation die of muscle wasting and neurodegeneration at four weeks post-natal. Although the mutation is said to be recessive, aged heterozygous mice have never been examined in detail; a number of other mouse models of motor neuron degeneration have recently been shown to have similar, albeit less severe, phenotypic abnormalities in the heterozygous state. We therefore examined the effects of ageing on a cohort of heterozygous +/wst mice and control mice, in order to establish whether a presumed 50% reduction in eEF1A2 expression was compatible with normal function. We evaluated the grip strength assay as a way of distinguishing between wasted and wild-type mice at 3-4 weeks, and then performed the same assay in older +/wst and wild-type mice. We also used rotarod performance and immunohistochemistry of spinal cord sections to evaluate the phenotype of aged heterozygous mice. Heterozygous mutant mice showed no deficit in neuromuscular function or signs of spinal cord pathology, in spite of the low levels of eEF1A2.

  11. veA-dependent RNA-pol II transcription elongation factor like protein, RtfA, is associated with secondary metabolism and morphological development in Aspergillus nidulans

    PubMed Central

    Ramamoorthy, Vellaisamy; Shantappa, Sourabha; Dhingra, Sourabh; Calvo, Ana M.

    2012-01-01

    In Aspergillus nidulans the global regulatory gene veA is necessary for the biosynthesis of several secondary metabolites, including the mycotoxin sterigmatocystin (ST). In order to identify additional veA-dependent genetic elements involved in regulating ST production, we performed a mutagenesis on a deletion veA (ΔveA) strain to obtain revertant mutants (RM) that regained the capability to produce toxin. Genetic analysis and molecular characterization of one of the revertant mutants, RM3, revealed that a point mutation occurred at the coding region of the rtfA gene, encoding a RNA-pol II transcription elongation factor like protein, similar to Saccharomyces cerevisiae Rtf1. The A. nidulans rtfA gene product accumulates in nuclei. Deletion of rtfA gene in a ΔveA background restored mycotoxin production in a medium-dependent manner. rtfA also affects the production of other metabolites including penicillin. Biosynthesis of this antibiotic decreased in the absence of rtfA. Furthermore, rtfA is necessary for normal morphological development. Deletion of the rtfA gene in wild-type strains (veA+) resulted in a slight decrease in growth rate, drastic reduction in conidiation, and complete loss of sexual development. This is the first study of an Rtf1 like gene in filamentous fungi. We found rtfA putative orthologs extensively conserved in numerous fungal species. PMID:22783880

  12. The RACK1 signal anchor protein from Trypanosoma brucei associates with eukaryotic elongation factor 1A: a role for translational control in cytokinesis

    PubMed Central

    Regmi, Sandesh; Rothberg, Karen G; Hubbard, James G; Ruben, Larry

    2008-01-01

    RACK1 is a WD-repeat protein that forms signal complexes at appropriate locations in the cell. RACK1 homologues are core components of ribosomes from yeast, plants and mammals. In contrast, a cryo-EM analysis of trypanosome ribosomes failed to detect RACK1, thus eliminating an important translational regulatory mechanism. Here we report that TbRACK1 from Trypanosoma brucei associates with eukaryotic translation elongation factor-1a (eEF1A) as determined by tandem MS of TAP-TbRACK1 affinity eluates, co-sedimentation in a sucrose gradient, and co-precipitation assays. Consistent with these observations, sucrose gradient purified 80S monosomes and translating polysomes each contained TbRACK1. When RNAi was used to deplete cells of TbRACK1, a shift in the polysome profile was observed, while the phosphorylation of a ribosomal protein increased. Under these conditions, cell growth became hypersensitive to the translational inhibitor anisomycin. The kinetoplasts and nuclei were misaligned in the postmitotic cells, resulting in partial cleavage furrow ingression during cytokinesis. Overall, these findings identify eEF1A as a novel TbRACK1 binding partner and establish TbRACK1 as a component of the trypanosome translational apparatus. The synergy between anisomycin and TbRACK1 RNAi suggests that continued translation is required for complete ingression of the cleavage furrow. PMID:18786142

  13. A Leader Intron of a Soybean Elongation Factor 1A (eEF1A) Gene Interacts with Proximal Promoter Elements to Regulate Gene Expression in Synthetic Promoters.

    PubMed

    Zhang, Ning; McHale, Leah K; Finer, John J

    2016-01-01

    Introns, especially the first intron in the 5' untranslated region (5'UTR), can significantly impact gene expression via intron-mediated enhancement (IME). In this study, we demonstrate the leader intron of a soybean elongation factor 1A (eEF1A) gene (GmScreamM8) was essential for the high activity of the native promoter. Furthermore, the interaction of the GmScreamM8 leader intron with regulatory element sequences from several soybean eEF1A promoters was studied using synthetic promoters, which consisted of element tetramers upstream of a core promoter used to regulate a green fluorescent protein (gfp) reporter gene. Element tetramers, placed upstream of a GmScreamM8 core promoter, showed very high activity using both transient expression in lima bean cotyledons and stable expression in soybean hairy roots, only if the native leader intron was included, suggesting an interaction between intronic sequences and promoter elements. Partial deletions of the leader intron showed that a 222 bp intronic sequence significantly contributed to very high levels of GFP expression. Generation of synthetic intron variants with a monomeric or trimeric repeat of the 222 bp intronic sequence, yielded almost two-fold higher expression compared to the original intron, while partial deletion of the 222 bp intronic repeated sequence significantly decreased gene expression, indicating that this intronic sequence was essential for the intron-element interaction enhancement.

  14. Developmental regulation of elongation factor-1 delta in sea urchin suggests appearance of a mechanism for alternative poly(A) site selection in gastrulae.

    PubMed

    Delalande, C; Monnier, A; Minella, O; Genevière, A M; Mulner-Lorillon, O; Bellé, R; Cormier, P

    1998-07-10

    Elongation factor-1 delta gene expression was analyzed during sea urchin development. EF-1 delta mRNA is present as a single 2.7-kb transcript in unfertilized eggs and in rapidly dividing cleavage stage embryos. It decreases rapidly 6 h after fertilization and then reappears at the gastrula stage as two transcripts of 2.7 and 2.0 kb. cDNA clones encoding the 2.7- and 2.0-kb transcripts were isolated from a sea urchin embryos library. The two cDNAs originate from alternative poly(A) site selection from a unique precursor. Both cDNAs are terminated by a poly(A) tail and were shown to encode for the same protein identified as EF-1 delta. Thus, EF-1 delta gene expression undergoes developmental regulation in early embryos leading to the presence of two poly(A) forms of the transcript. Since the 2.0-kb polyadenylated form of the EF-1 delta transcript appears at gastrula stage, our results suggest that a mechanism for alternative poly(A) site selection of the EF-1 delta transcript appears during embryonic development.

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

    PubMed

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

    2014-09-01

    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.

  16. Development of a polymerase chain reaction-restriction fragment length polymorphism method for identification of the Fusarium genus using the transcription elongation factor-1α gene.

    PubMed

    Zarrin, Majid; Ganj, Farzaneh; Faramarzi, Sama

    2016-12-01

    Fusarium species are well-known plant pathogens and food contaminants that have also appeared as one of the most important groups of medically significant fungi. The sequences of the translation elongation factor (TEF)-1α gene have been broadly employed for species detection. A total of 50 strains of Fusarium spp., including environmental, clinical and reference isolates were used for the current study. The primer sets, Fu3f and Fu3r, were used to amplify an ~420-bp DNA fragment of the TEF-1α gene. Double digestion with two restriction enzymes, XhoI and SduI was used for discrimination of the Fusarium species in the TEF-1α gene fragment. Double digestion of the TEF-1α gene fragment from five clinically important Fusarium species were clearly differentiated from each other: The F. solani species complex, F. oxysporum species complex, F. verticillioides, F. proliferatum and F. fujikuroi. This method facilitates detection and enables verification of the Fusarium genus; therefore, it may be applied for disease control.

  17. A Leader Intron of a Soybean Elongation Factor 1A (eEF1A) Gene Interacts with Proximal Promoter Elements to Regulate Gene Expression in Synthetic Promoters

    PubMed Central

    Zhang, Ning; McHale, Leah K.; Finer, John J.

    2016-01-01

    Introns, especially the first intron in the 5’ untranslated region (5’UTR), can significantly impact gene expression via intron-mediated enhancement (IME). In this study, we demonstrate the leader intron of a soybean elongation factor 1A (eEF1A) gene (GmScreamM8) was essential for the high activity of the native promoter. Furthermore, the interaction of the GmScreamM8 leader intron with regulatory element sequences from several soybean eEF1A promoters was studied using synthetic promoters, which consisted of element tetramers upstream of a core promoter used to regulate a green fluorescent protein (gfp) reporter gene. Element tetramers, placed upstream of a GmScreamM8 core promoter, showed very high activity using both transient expression in lima bean cotyledons and stable expression in soybean hairy roots, only if the native leader intron was included, suggesting an interaction between intronic sequences and promoter elements. Partial deletions of the leader intron showed that a 222 bp intronic sequence significantly contributed to very high levels of GFP expression. Generation of synthetic intron variants with a monomeric or trimeric repeat of the 222 bp intronic sequence, yielded almost two-fold higher expression compared to the original intron, while partial deletion of the 222 bp intronic repeated sequence significantly decreased gene expression, indicating that this intronic sequence was essential for the intron-element interaction enhancement. PMID:27806110

  18. 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

  19. Elongation factor-1 alpha occurs as two copies in bees: implications for phylogenetic analysis of EF-1 alpha sequences in insects.

    PubMed

    Danforth, B N; Ji, S

    1998-03-01

    We report the complete sequence of a paralogous copy of elongation factor-1 alpha (EF-1 alpha) in the honeybee, Apis mellifera (Hymenoptera: Apidae). This copy differs from a previously described copy in the positions of five introns and in 25% of the nucleotide sites in the coding regions. The existence of two paralogous copies of EF-1 alpha in Drosophila and Apis suggests that two copies of EF-1 alpha may be widespread in the holometabolous insect orders. To distinguish between a single, ancient gene duplication and parallel, independent fly and bee gene duplications, we performed a phylogenetic analysis of hexapod EF-1 alpha sequences. Unweighted parsimony analysis of nucleotide sequences suggests an ancient gene duplication event, whereas weighted parsimony analysis of nucleotides and unweighted parsimony analysis of amino acids suggests the contrary: that EF-1 alpha underwent parallel gene duplications in the Diptera and the Hymenoptera. The hypothesis of parallel gene duplication is supported both by congruence among nucleotide and amino acid data sets and by topology-dependent permutation tail probability (T-PTP) tests. The resulting tree topologies are also congruent with current views on the relationships among the holometabolous orders included in this study (Diptera, Hymenoptera, and Lepidoptera). More sequences, from diverse orders of holometabolous insects, will be needed to more accurately assess the historical patterns of gene duplication in EF-1 alpha.

  20. 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

    2004-03-05

    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.

  1. Structure of the Acinetobacter baumannii dithiol oxidase DsbA bound to elongation factor EF-Tu reveals a novel protein interaction site.

    PubMed

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

    2014-07-18

    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.

  2. Development of a polymerase chain reaction-restriction fragment length polymorphism method for identification of the Fusarium genus using the transcription elongation factor-1α gene

    PubMed Central

    Zarrin, Majid; Ganj, Farzaneh; Faramarzi, Sama

    2016-01-01

    Fusarium species are well-known plant pathogens and food contaminants that have also appeared as one of the most important groups of medically significant fungi. The sequences of the translation elongation factor (TEF)-1α gene have been broadly employed for species detection. A total of 50 strains of Fusarium spp., including environmental, clinical and reference isolates were used for the current study. The primer sets, Fu3f and Fu3r, were used to amplify an ~420-bp DNA fragment of the TEF-1α gene. Double digestion with two restriction enzymes, XhoI and SduI was used for discrimination of the Fusarium species in the TEF-1α gene fragment. Double digestion of the TEF-1α gene fragment from five clinically important Fusarium species were clearly differentiated from each other: The F. solani species complex, F. oxysporum species complex, F. verticillioides, F. proliferatum and F. fujikuroi. This method facilitates detection and enables verification of the Fusarium genus; therefore, it may be applied for disease control. PMID:28105337

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

    PubMed

    Lee, Kyungwoo; Lillehoj, Hyun S; Li, Guangxing; Park, Myeong-Seon; Jang, Seung I; Jeong, Wooseog; Jeoung, Hye-Young; An, Dong-Jun; Lillehoj, Erik P

    2011-12-01

    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 reaction with immune sera from commercial meat-type chickens with clinical outbreak of Clostridium infections. In addition to the genes encoding EF-Tu and PFO, C. perfringens alpha-toxin and necrotic enteritis B-like (NetB) toxin were also expressed in Escherichia coli and their corresponding recombinant proteins were purified. Using the four recombinant proteins as target antigens in ELISA immunoassays, high serum antibody titers were observed not only in chickens with clinical signs of Clostridium infections, but also in apparently healthy animals from the same disease-endemic farm. By contrast, no antibodies against any of the proteins were present in the serum of a specific pathogen-free bird. In ELISA using recombinant proteins of C. perfringens, the levels of anti-bacterial protein antibodies were also higher in chickens which were experimentally induced to show NE clinical signs after co-infection with C. perfringens and Eimeria maxima compared with uninfected controls. These results show that two antigenic C. perfringens proteins, EF-Tu and PFO can be useful detection antigens for C. perfringens-afflicted infections in commercial poultry.

  4. Characterization and phylogeny of Isaria spp. strains (Ascomycota: Hypocreales) using ITS1-5.8S-ITS2 and elongation factor 1-alpha sequences.

    PubMed

    D'Alessandro, Celeste P; Jones, Leandro R; Humber, Richard A; López Lastra, Claudia C; Sosa-Gomez, Daniel R

    2014-07-01

    The elongation factor 1-alpha (EF1-α) and the internal transcribed spacer (ITS) regions ITS1 and ITS2 (ITS1-5.8S-ITS2) sequences were used to characterize and to identify Isaria isolates from Argentina, Mexico, and Brazil, as well as to study the phylogenetic relationships among these isolates and other related fungi from the order Hypocreales. The molecular characterization, which was performed by PCR-RFLP of EF1-α and ITS1-5.8-ITS2 genes, was useful for resolving representative isolates of Isaria fumosorosea, Isaria farinosa, and Isaria tenuipes and to confirm the taxonomic identity of fungi from Argentina, Mexico, and Brazil. The phylogenetic analyses showed three clades corresponding to three families of Hypocreales. The genus Isaria was confirmed as polyphyletic and in family Cordycipitaceae, Isaria species were related to anamorphic species of Beauveria, Lecanicillium, and Simplicillium and to teleomorphic Cordyceps and Torrubiella. Therefore, EF1-α and ITS1-5.8S-ITS2 genes were found to be powerful tools for improving the characterization, identification, and phylogenetic relationship of the Isaria species and other entomopathogenic fungi.

  5. Aging and Oxidative Stress Decrease Pineal Elongation Factor 2: In Vivo Protective Effect of Melatonin in Young Rats Treated With Cumene Hydroperoxide.

    PubMed

    Muñoz, Mario F; Argüelles, Sandro; Cano, Mercedes; Marotta, Francesco; Ayala, Antonio

    2017-01-01

    We studied the alterations of Elongation Factor 2 (eEF2) in the pineal gland of aged rats as well as the possible protective role of exogenous melatonin on these changes in young rats treated with cumene hydroperoxide (CH), a compound that promotes lipid peroxidation and inhibits protein synthesis. The study was performed using male Wistar rats of 3 (control), 12, and 24 months and 3-month-old rats treated with CH, melatonin, and CH plus melatonin. We found that pineal eEF-2 is affected by aging and CH, these changes being prevented by exogenous melatonin in the case of CH-treated rats. The proteomic studies show that many other proteins are affected by aging and oxidative stress in the pineal gland. The results suggest that one of the possible mechanisms underlying pineal gland dysfunction during aging is the effect of lipid peroxidation on eEF-2, which is a key component of protein synthesis machinery. J. Cell. Biochem. 118: 182-190, 2017. © 2016 Wiley Periodicals, Inc.

  6. tRNA is entrapped in similar, but distinct, nuclear and cytoplasmic ribonucleoprotein complexes, both of which contain vigilin and elongation factor 1 alpha.

    PubMed Central

    Kruse, C; Grünweller, A; Willkomm, D K; Pfeiffer, T; Hartmann, R K; Müller, P K

    1998-01-01

    Vigilin, which is found predominantly in cells and tissues with high levels of protein biosynthesis, was isolated in its native form from human HEp-2 cells (A.T.C.C. CCL23) by immunoaffinity chromatography. Here we demonstrate that vigilin is part of a novel large tRNA-binding ribonucleoprotein complex (tRNP), found not only in the cytoplasm, but also in the nuclei of human cells. Compositional differences in the protein pattern were detected between the nuclear and cytoplasmic tRNPs, although some properties of the purified nuclear tRNP, such as tRNA protection against nuclease attack, were identical with those of the cytoplasmic tRNP. By using either a pool of total human nuclear RNA or radioactively labelled yeast tRNAAsp in rebinding experiments, we could show that tRNA is specifically recaptured by the RNA-depleted, vigilin-containing nuclear complex. We could also show that vigilin is capable of binding tRNA in vitro. Another tRNA-binding protein is elongation factor 1 alpha, which appears to be enriched in the cytoplasmic and nuclear tRNP complexes. This suggests that the cytoplasmic tRNP may be involved in the channelled tRNA cycle in the cytoplasm of eukaryotic cells. Our results also suggest that the nuclear vigilin-containing tRNP may be related to the nuclear export of tRNA. PMID:9445390

  7. 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

  8. 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

  9. Dissecting the role of the elongation factor 1A isoforms in hepatocellular carcinoma cells by liposome-mediated delivery of siRNAs.

    PubMed

    Farra, Rossella; Scaggiante, Bruna; Guerra, Chiara; Pozzato, Gabriele; Grassi, Mario; Zanconati, Fabrizio; Perrone, Francesca; Ferrari, Cinzia; Trotta, Francesco; Grassi, Gabriele; Dapas, Barbara

    2017-02-13

    Eukaryotic elongation factor 1A (eEF1A), a protein involved in protein synthesis, has two major isoforms, eEF1A1 and eEF1A2. Despite the evidences of their involvement in hepatocellular carcinoma (HCC), the quantitative contribution of each of the two isoforms to the disease is unknown. We depleted the two isoforms by means of siRNAs and studied the effects in three different HCC cell lines. Particular care was dedicated to select siRNAs able to target each of the two isoform without affecting the other one. This is not a trivial aspect due to the high sequence homology between eEF1A1 and eEF1A2. The selected siRNAs can specifically deplete either eEF1A1 or eEF1A2. This, in turn, results in an impairment of cell vitality, growth and arrest in the G1/G0 phase of the cell cycle. Notably, these effects are quantitatively superior following eEF1A1 than eEF1A2 depletion. Moreover, functional tests revealed that the G1/G0 block induced by eEF1A1 depletion depends on the down-regulation of the transcription factor E2F1, a known player in HCC. In conclusion, our data indicate that the independent targeting of the two eEF1A isoforms is effective in reducing HCC cell growth and that eEF1A1 depletion may result in a more evident effect.

  10. In vivo characterization of the role of tissue-specific translation elongation factor 1A2 in protein synthesis reveals insights into muscle atrophy.

    PubMed

    Doig, Jennifer; Griffiths, Lowri A; Peberdy, David; Dharmasaroja, Permphan; Vera, Maria; Davies, Faith J C; Newbery, Helen J; Brownstein, David; Abbott, Catherine M

    2013-12-01

    Translation elongation factor 1A2 (eEF1A2), uniquely among translation factors, is expressed specifically in neurons and muscle. eEF1A2-null mutant wasted mice develop an aggressive, early-onset form of neurodegeneration, but it is unknown whether the wasting results from denervation of the muscles, or whether the mice have a primary myopathy resulting from loss of translation activity in muscle. We set out to establish the relative contributions of loss of eEF1A2 in the different tissues to this postnatal lethal phenotype. We used tissue-specific transgenesis to show that correction of eEF1A2 levels in muscle fails to ameliorate the overt phenotypic abnormalities or time of death of wasted mice. Molecular markers of muscle atrophy such as Fbxo32 were dramatically upregulated at the RNA level in wasted mice, both in the presence and in the absence of muscle-specific expression of eEF1A2, but the degree of upregulation at the protein level was significantly lower in those wasted mice without transgene-derived expression of eEF1A2 in muscle. This provides the first in vivo confirmation that eEF1A2 plays an important role in translation. In spite of the inability of the nontransgenic wasted mice to upregulate key atrogenes at the protein level in response to denervation to the same degree as their transgenic counterparts, there were no measurable differences between transgenic and nontransgenic wasted mice in terms of weight loss, grip strength, or muscle pathology. This suggests that a compromised ability fully to execute the atrogene pathway in denervated muscle does not affect the process of muscle atrophy in the short term.

  11. Heterogeneous nuclear ribonucleoprotein L-like (hnRNPLL) and elongation factor, RNA polymerase II, 2 (ELL2) are regulators of mRNA processing in plasma cells

    PubMed Central

    Benson, Micah J.; Äijö, Tarmo; Chang, Xing; Gagnon, John; Pape, Utz J.; Anantharaman, Vivek; Aravind, L.; Pursiheimo, Juha-Pekka; Oberdoerffer, Shalini; Liu, X. Shirley; Lahesmaa, Riitta; Lähdesmäki, Harri; Rao, Anjana

    2012-01-01

    B cells and plasma cells possess distinct RNA processing environments that respectively promote the expression of membrane-associated Ig by B cells versus the secretion of Ig by plasma cells. Through a combination of transcriptional profiling and screening using a lentiviral short-hairpin RNA interference library, we show that both the splicing factor hnRNPLL and the transcription elongation factor ELL2 modulate the ratio of secreted versus membrane-encoding Ighg2b transcripts in MPC11 plasmacytoma cell lines. hnRNPLL and ELL2 are both highly expressed in primary plasma cells relative to B cells, but hnRNPLL binds Ighg2b mRNA transcripts and promotes an increase in levels of the membrane-encoding Ighg2b isoform at the expense of the secreted Ighg2b isoform, whereas ELL2 counteracts this effect and drives Ig secretion by increasing the frequency of the secreted Ighg2b isoform. As in T cells, hnRNPLL also alters the splicing pattern of mRNA encoding the adhesion receptor CD44, promoting exon inclusion, and decreasing the overall level of CD44 expression. Further characterization of ELL2-dependent transcription by RNA-Seq revealed that ∼12% of transcripts expressed by plasma cells were differentially processed because of the activities of ELL2, including B-cell maturation antigen BCMA, a receptor with a defined role in plasma cell survival. Taken together, our data identify hnRNPLL and ELL2 as regulators of pre-mRNA processing in plasma cells. PMID:22991471

  12. 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.

  13. MicroRNA 603 acts as a tumor suppressor and inhibits triple-negative breast cancer tumorigenesis by targeting elongation factor 2 kinase.

    PubMed

    Bayraktar, Recep; Pichler, Martin; Kanlikilicer, Pinar; Ivan, Cristina; Bayraktar, Emine; Kahraman, Nermin; Aslan, Burcu; Oguztuzun, Serpil; Ulasli, Mustafa; Arslan, Ahmet; Calin, George; Lopez-Berestein, Gabriel; Ozpolat, Bulent

    2016-12-27

    Triple negative breast cancer (TNBC) is an aggressive type of breast cancer characterized by the absence of defined molecular targets, including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and is associated with high rates of relapse and distant metastasis despite surgery and adjuvant chemotherapy. The lack of effective targeted therapies for TNBC represents an unmet therapeutic challenge. Eukaryotic elongation factor 2 kinase (eEF2K) is an atypical calcium/calmodulin-dependent serine/threonine kinase that promotes TNBC tumorigenesis, progression, and drug resistance, representing a potential novel molecular target. However, the mechanisms regulating eEF2K expression are unknown. Here, we report that eEF2K protein expression is highly up-regulated in TNBC cells and patient tumors and it is associated with poor patient survival and clinical outcome. We found that loss/reduced expression of miR-603 leads to eEF2K overexpression in TNBC cell lines. Its expression results in inhibition of eEF2K by directly targeting the 3-UTR and the inhibition of tumor cell growth, migration and invasion in TNBC. In vivo therapeutic gene delivery of miR-603 into TNBC xenograft mouse models by systemic administration of miR-603-nanoparticles led to a significant inhibition of eEF2K expression and tumor growth, which was associated with decreased activity of the downstream targets of eEF2K, including Src, Akt, cyclin D1 and c-myc. Our findings suggest that miR-603 functions as a tumor suppressor and loss of miR-603 expression leads to increase in eEF2K expression and contributes to the growth, invasion, and progression of TNBC. Taken together, our data suggest that miR-603-based gene therapy is a potential strategy against TNBC.

  14. Characterization of the snowy cotyledon 1 mutant of Arabidopsis thaliana: the impact of chloroplast elongation factor G on chloroplast development and plant vitality.

    PubMed

    Albrecht, Verónica; Ingenfeld, Anke; Apel, Klaus

    2006-03-01

    During seedling development chloroplast formation marks the transition from heterotrophic to autotrophic growth. The development and activity of chloroplasts may differ in cotyledons that initially serve as a storage organ and true leaves whose primary function is photosynthesis. A genetic screen was used for the identification of genes that affect selectively chloroplast function in cotyledons of Arabidopsis thaliana. Several mutants exhibiting pale cotyledons and green true leaves were isolated and dubbed snowy cotyledon (sco). One of the mutants, sco1, was characterized in more detail. The mutated gene was identified using map-based cloning. The mutant contains a point mutation in a gene encoding the chloroplast elongation factor G, leading to an amino acid exchange within the predicted 70S ribosome-binding domain. The mutation results in a delay in the onset of germination. At this early developmental stage embryos still contain undifferentiated proplastids, whose proper function seems necessary for seed germination. In light-grown sco1 seedlings the greening of cotyledons is severely impaired, whereas the following true leaves develop normally as in wild-type plants. Despite this apparent similarity of chloroplast development in true leaves of mutant and wild-type plants various aspects of mature plant development are also affected by the sco1 mutation such as the onset of flowering, the growth rate, and seed production. The onset of senescence in the mutant and the wild-type plants occurs, however, at the same time, suggesting that in the mutant this particular developmental step does not seem to suffer from reduced protein translation efficiency in chloroplasts.

  15. Spectrophotometric and kinetic studies on the interaction of antibiotic X5108, the N-methylated derivative of kirromycin, with elongation factor Tu from Escherichia coli.

    PubMed

    Eccleston, J F

    1981-04-10

    The absorption spectrum of antibiotic X5108, the N-methylated derivative of kirromycin, has been found to be decreased in intensity on binding to elongation factor (EF)-Tu . GDP, EF-Tu . GTP, and nucleotide-free EF-Tu. This has allowed the binding of X5108 to be studied directly. In agreement with previous studies, a 1:1 stoichiometry is observed, with a dissociation constant of less than 1 microM. Identical results were obtained with all three EF-Tu species. The absorption spectrum of X5108 in increasing concentrations of isopropyl alcohol first intensifies and then decreases, 80% isopropyl alcohol giving the same spectrum as that of X5108 bound to EF-Tu. This result is interpreted as showing that the chromophoric moiety of X5108 is bound in a highly hydrophobic environment on EF-Tu. The rate of binding of X5108 to EF-Tu . GDP was measured using a stopped flow spectrophotometer. This rate was proportional to the concentration of X5108, giving a second order binding rate constant of 4.8 X 10(3) M-1 s-1. Since this is several orders of magnitude too slow for a diffusion-controlled reaction, the results are interpreted based on a two-step binding process. A half-time of about 10 min is calculated for the dissociation of X5108 from EF-Tu . GDP. The fact that X5108 bound to EF-Tu is not in rapid equilibrium with X5108 free in solution needs to be considered in studies on the effect of X5108 and kirromycin on partial reactions of protein biosynthesis.

  16. 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

  17. 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

  18. In vitro and in vivo protection by melatonin against the decline of elongation factor-2 caused by lipid peroxidation: preservation of protein synthesis.

    PubMed

    Argüelles, Sandro; Muñoz, Mario F; Cano, Mercedes; Machado, Alberto; Ayala, Antonio

    2012-08-01

    As organisms age, a considerable decrease in protein synthesis takes place in all tissues. Among the possible causes of the decline of translation in old animals are the modifications of elongation factor-2 (eEF-2). eEF-2 occupies an essential role in protein synthesis where it catalyzes the ribosomal translocation reaction. eEF-2 is particularly sensitive to increased oxidative stress. However, all oxidants do not affect eEF-2, only compounds that increase lipid peroxidation. As peroxides are unstable compounds, they decompose and generate a series of highly reactive compounds, including aldehydes malondialdehyde (MDA) and 4-hydroxynoenal (HNE). We have previously reported that hepatic eEF-2 forms adducts with low-molecular weight aldehydes, MDA and HNE. Therefore, the protection of eEF-2 must be specifically carried out by a compound with lipoperoxyl radical-scavenging features such as melatonin. In this article, we show the ability of melatonin to protect against the changes that occur in the eEF-2 under conditions of lipid peroxidation induced by cumene hydroperoxide (CH), a compound used experimentally to induce lipid breakdown. As experimental models, we used cultured cells and rats treated with this oxidant compound. eEF-2 levels, adduct formation of this protein with MDA and HNE, and lipid peroxides were determined. In the cultured cells, protein synthesis rate was also measured. Our results show that melatonin prevented the molecular changes in eEF-2 and the decline in protein synthesis rate secondary to lipid peroxidation. The results also show that serum levels of several hormones were affected by CH-induced oxidative stress, which was partially or totally prevented by melatonin.

  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.

  20. Abrogation of the Brd4-Positive Transcription Elongation Factor b Complex by Papillomavirus E2 Protein Contributes to Viral Oncogene Repression▿

    PubMed Central

    Yan, Junpeng; Li, Qing; Lievens, Sam; Tavernier, Jan; You, Jianxin

    2010-01-01

    The cellular bromodomain protein Brd4 is a major interacting partner of the papillomavirus (PV) E2 protein. Interaction of E2 with Brd4 contributes to viral episome maintenance. The E2-Brd4 interaction also plays an important role in repressing viral oncogene expression from the integrated viral genome in human PV (HPV)-positive cancer cells. However, the underlying mechanism is not clearly understood. In host cells, Brd4 recruits positive transcription elongation factor b (P-TEFb) to stimulate RNA polymerase II phosphorylation during cellular and viral gene expression. P-TEFb associates with the C terminus of Brd4, which largely overlaps with the E2 binding site on Brd4. In this study, we demonstrate that E2 binding to Brd4 inhibits the interaction of endogenous Brd4 and P-TEFb. P-TEFb is essential for viral oncogene E6/E7 transcription in both HeLa and CaSki cells that contain integrated HPV genomes. E2 binding to Brd4 abrogates the recruitment of P-TEFb to the integrated viral chromatin template, leading to inactivation of P-TEFb and repression of the viral oncogene E6/E7. Furthermore, dissociation of the Brd4-P-TEFb complex from the integrated viral chromatin template using a Brd4 bromodomain dominant-negative inhibitor also hampers HPV E6/E7 oncogene expression. Our data support that Brd4 recruitment of P-TEFb to the viral chromatin template is essential for viral oncogene expression. Abrogation of the interaction between P-TEFb and Brd4 thus provides a mechanism for E2-mediated repression of the viral oncogenes from the integrated viral genomes in cancer cells. PMID:19846528

  1. The translation elongation factor eEF2 is a novel tumor-associated antigen overexpressed in various types of cancers

    PubMed Central

    OJI, YUSUKE; TATSUMI, NAOYA; FUKUDA, MARI; NAKATSUKA, SHIN-ICHI; AOYAGI, SAYAKA; HIRATA, ERIKA; NANCHI, ISAMU; FUJIKI, FUMIHIRO; NAKAJIMA, HIROKO; YAMAMOTO, YUMIKO; SHIBATA, SYOHEI; NAKAMURA, MICHIYO; HASEGAWA, KANA; TAKAGI, SAYAKA; FUKUDA, IKUYO; HOSHIKAWA, TOMOKO; MURAKAMI, YUI; MORI, MASAHIDE; INOUE, MASAYOSHI; NAKA, TETSUJI; TOMONAGA, TAKESHI; SHIMIZU, YOSHIFUMI; NAKAGAWA, MASASHI; HASEGAWA, JUNICHI; NEZU, RIICHIRO; INOHARA, HIDENORI; IZUMOTO, SHUICHI; NONOMURA, NORIO; YOSHIMINE, TOSHIKI; OKUMURA, MEINOSHIN; MORII, EIICHI; MAEDA, HAJIME; NISHIDA, SUMIYUKI; HOSEN, NAOKI; TSUBOI, AKIHIRO; OKA, YOSHIHIRO; SUGIYAMA, HARUO

    2014-01-01

    Recent studies have shown that cancer immunotherapy could be a promising therapeutic approach for the treatment of cancer. In the present study, to identify novel tumor-associated antigens (TAAs), the proteins expressed in a panel of cancer cells were serologically screened by immunoblot analysis and the eukaryotic elongation factor 2 (eEF2) was identified as an antigen that was recognized by IgG autoantibody in sera from a group of patients with head and neck squamous cell carcinoma (HNSCC) or colon cancer. Enzyme-linked immunosorbent assay showed that serum eEF2 IgG Ab levels were significantly higher in colorectal and gastric cancer patients compared to healthy individuals. Immunohistochemistry experiments showed that the eEF2 protein was overexpressed in the majority of lung, esophageal, pancreatic, breast and prostate cancers, HNSCC, glioblastoma multiforme and non-Hodgkin’s lymphoma (NHL). Knockdown of eEF2 by short hairpin RNA (shRNA) significantly inhibited the growth in four eEF2-expressing cell lines, PC14 lung cancer, PCI6 pancreatic cancer, HT1080 fibrosarcoma and A172 glioblastoma cells, but not in eEF2-undetectable MCF7 cells. Furthermore, eEF2-derived 9-mer peptides, EF786 (eEF2 786–794 aa) and EF292 (eEF2 292–300 aa), elicited cytotoxic T lymphocyte (CTL) responses in peripheral blood mononuclear cells (PBMCs) from an HLA-A*24:02- and an HLA-A*02:01-positive healthy donor, respectively, in an HLA-A-restricted manner. These results indicated that the eEF2 gene is overexpressed in the majority of several types of cancers and plays an oncogenic role in cancer cell growth. Moreover, the eEF2 gene product is immunogenic and a promising target molecule of cancer immunotherapy for several types of cancers. PMID:24589652

  2. Succinate dehydrogenase assembly factor 2 is needed for assembly and activity of mitochondrial complex II and for normal root elongation in Arabidopsis.

    PubMed

    Huang, Shaobai; Taylor, Nicolas L; Ströher, Elke; Fenske, Ricarda; Millar, A Harvey

    2013-02-01

    Mitochondria complex II (succinate dehydrogenase, SDH) plays a central role in respiratory metabolism as a component of both the electron transport chain and the tricarboxylic acid cycle. We report the identification of an SDH assembly factor by analysis of T-DNA insertions in At5g51040, a protein with unknown function that was identified by mass spectrometry analysis as a low abundance mitochondrial protein. This gene is co-expressed with a number of genes encoding mitochondrial proteins, including SDH1-1, and has low partial sequence similarity to human SDHAF2, a protein required for flavin-adenine dinucleotide (FAD) insertion into SDH. In contrast to observations of other SDH deficient lines in Arabidopsis, the sdhaf2 line did not affect photosynthetic rate or stomatal conductance, but instead showed inhibition of primary root elongation with early lateral root emergence, presumably due to the low SDH activity caused by the reduced abundance of SDHAF2. Both roots and leaves showed succinate accumulation but different responses in the abundance of other organic acids and amino acids assayed. Isolated mitochondria showed lowered SDH1 protein abundance, lowered maximal SDH activity and less protein-bound flavin-adenine dinucleotide (FAD) at the molecular mass of SDH1 in the gel separation. The short root phenotype and SDH function of sdhaf2 was fully complemented by transformation with SDHAF2. Application of the SDH inhibitor, malonate, phenocopied the sdhaf2 root architecture in WT. Whole root respiratory assays showed no difference between WT and sdhaf2, but micro-respirometry of the tips of roots clearly showed low oxygen consumption in sdhaf2 which could explain a metabolic deficit responsible for root tip growth.

  3. Expression of mRNA encoding the macrophage colony-stimulating factor receptor (c-fms) is controlled by a constitutive promoter and tissue-specific transcription elongation.

    PubMed Central

    Yue, X; Favot, P; Dunn, T L; Cassady, A I; Hume, D A

    1993-01-01

    The gene encoding the receptor for macrophage colony-stimulating factor 1 (CSF-1), the c-fms protooncogene, is selectively expressed in immature and mature mononuclear phagocytes and trophoblasts. Exon 1 is expressed only in trophoblasts. Isolation and sequencing of genomic DNA flanking exon 2 of the murine c-fms gene revealed a TATA-less promoter with significant homology to human c-fms. Reverse transcriptase primer extension analysis using exon 2 primers identified multiple clustered transcription initiation sites. Their position was confirmed by RNase protection. The same primer extension products were detected in equal abundance from macrophage or nonmacrophage sources of RNA. c-fms mRNA is acutely down-regulated in primary macrophages by CSF-1, bacterial lipopolysaccharide (LPS), and phorbol myristate acetate (PMA). Each of these agents reduced the abundance of c-fms RNA detectable by primer extension using an exon 3 primer without altering the abundance of presumptive short c-fms transcripts detected with exon 2 primers. Primer extension analysis with an intron 2 primer detected products at greater abundance in nonmacrophages. Templates detected with the intronic primer were induced in macrophages by LPS, PMA, and CSF-1, suggesting that each of the agents caused a shift from full-length c-fms mRNA production to production of unspliced, truncated transcripts. The c-fms promoter functioned constitutively in the RAW264 macrophage cell line, the B-cell line MOPC.31C, and several nonhematopoietic cell lines. Macrophage-specific expression and responsiveness to selective repression by LPS and PMA was achieved by the incorporation of intron 2 into the c-fms promoter-reporter construct. The results suggest that expression of the c-fms gene in macrophages is controlled by sequences in intron 2 that act by regulating transcription elongation. Images PMID:8497248

  4. LIM kinase 1 (LIMK1) interacts with tropomyosin-related kinase B (TrkB) and Mediates brain-derived neurotrophic factor (BDNF)-induced axonal elongation.

    PubMed

    Dong, Qing; Ji, Yun-Song; Cai, Chang; Chen, Zhe-Yu

    2012-12-07

    BDNF/TrkB signaling plays critical roles in axonal outgrowth of neurons, the process of which requires the remodeling of the cytoskeleton structure, including microtubules and filamentous actin. However, the mechanism by which BDNF/TrkB signaling regulates cytoskeleton reorganization is still unclear. Here, we identified a novel interaction between LIMK1 and TrkB, which is required for the BDNF-induced axonal elongation. We demonstrated that BDNF-induced TrkB dimerization led to LIMK1 dimerization and transphosphorylation independent of TrkB kinase activity, which could further enhance the activation and stabilization of LIMK1. Moreover, activated LIMK1 translocated to the membrane fraction and phosphorylated its substrate cofilin, thus promoting actin polymerization and axonal elongation. Our findings provided evidence of a novel mechanism for the BDNF-mediated signal transduction leading to axonal elongation.

  5. 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...

  6. Molecular characterization, expression analysis and RNAi knock-down of elongation factor 1α and 1γ from Nilaparvata lugens and its yeast-like symbiont.

    PubMed

    Wang, W X; Zhu, T H; Li, K L; Chen, L F; Lai, F X; Fu, Q

    2016-11-04

    In the present paper, four cDNAs encoding the alpha and gamma subunits of elongation factor 1 (EF-1) were cloned and sequenced from Nilaparvata lugens, named NlEF-1α, NlEF-1γ, and its yeast-like symbiont (YLS), named YsEF-1α and YsEF-1γ, respectively. Comparisons with sequences from other species indicated a greater conservation for EF-1α than for EF-1γ. NlEF-1α has two identical copies. The deduced amino acid sequence homology of NlEF-1α and NlEF-1γ is 96 and 64%, respectively, compared with Homalodisca vitripennis and Locusta migratoria. The deduced amino acid sequence homology of YsEF-1α and YsEF-1γ is 96 and 74%, respectively, compared with Metarhizium anisopliae and Ophiocordyceps sinensis. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis revealed that the expression level of NlEF-1α and NlEF-1γ mRNA in hemolymph, ovary, fat body and salivary glands were higher than the midgut and leg tissue. YsEF-1α and YsEF-1γ was highly expressed in fat body. The expression level of NlEF-1α was higher than that of NlEF-1γ. Through RNA interference (RNAi) of the two genes, the mortality of nymph reached 92.2% at the 11th day after treatment and the ovarian development was severely hindered. The RT-qPCR analysis verified the correlation between mortality, sterility and the down-regulation of the target genes. The expression and synthesis of vitellogenin (Vg) protein in insects injected with NlEF-1α and NlEF-1γ double-stranded RNA (dsRNA) was significantly lower than control groups. Attempts to knockdown the YsEF-1 genes in the YLS was unsuccessful. However, the phenotype of N. lugens injected with YsEF-1α dsRNA was the same as that injected with NlEF-1α dsRNA, possibly due to the high similarity (up to 71.9%) in the nucleotide sequences between NlEF-1α and YsEF-1α. We demonstrated that partial silencing of NlEF-1α and NlEF-1γ genes caused lethal and sterility effect on N. lugens. NlEF-1γ shares low identity with that of

  7. Optic Nerve Elongation

    PubMed Central

    Alvi, Aijaz; Janecka, Ivo P.; Kapadia, Silloo; Johnson, Bruce L.; McVay, William

    1996-01-01

    The length of the optic nerves is a reflection of normal postnatal cranio-orbital development. Unilateral elongation of an optic nerve has been observed in two patients with orbital and skull base neoplasms. In the first case as compared to the patient's opposite, normal optic nerve, an elongated length of the involved optic nerve of 45 mm was present. The involved optic nerve in the second patient was 10 mm longer than the normal opposite optic nerve. The visual and extraocular function was preserved in the second patient. The first patient had only light perception in the affected eye. In this paper, the embryology, anatomy, and physiology of the optic nerve and its mechanisms of stretch and repair are discussed. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6Figure 7Figure 8Figure 9Figure 10Figure 11Figure 13 PMID:17170975

  8. Regulation of Transcript Elongation

    PubMed Central

    Belogurov, Georgiy A.; Artsimovitch, Irina

    2015-01-01

    Bacteria lack subcellular compartments and harbor a single RNA polymerase that synthesizes both structural and protein-coding RNAs, which are cotranscriptionally processed by distinct pathways. Nascent rRNAs fold into elaborate secondary structures and associate with ribosomal proteins, whereas nascent mRNAs are translated by ribosomes. During elongation, nucleic acid signals and regulatory proteins modulate concurrent RNA-processing events, instruct RNA polymerase where to pause and terminate transcription, or act as roadblocks to the moving enzyme. Communications among complexes that carry out transcription, translation, repair, and other cellular processes ensure timely execution of the gene expression program and survival under conditions of stress. This network is maintained by auxiliary proteins that act as bridges between RNA polymerase, ribosome, and repair enzymes, blurring boundaries between separate information-processing steps and making assignments of unique regulatory functions meaningless. Understanding the regulation of transcript elongation thus requires genome-wide approaches, which confirm known and reveal new regulatory connections. PMID:26132790

  9. Elongation of Flare Ribbons

    NASA Astrophysics Data System (ADS)

    Qiu, Jiong; Longcope, Dana W.; Cassak, Paul A.; Priest, Eric R.

    2017-03-01

    We present an analysis of the apparent elongation motion of flare ribbons along the polarity inversion line (PIL), as well as the shear of flare loops in several two-ribbon flares. Flare ribbons and loops spread along the PIL at a speed ranging from a few to a hundred km s‑1. The shear measured from conjugate footpoints is consistent with the measurement from flare loops, and both show the decrease of shear toward a potential field as a flare evolves and ribbons and loops spread along the PIL. Flares exhibiting fast bidirectional elongation appear to have a strong shear, which may indicate a large magnetic guide field relative to the reconnection field in the coronal current sheet. We discuss how the analysis of ribbon motion could help infer properties in the corona where reconnection takes place.

  10. 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.

  11. Seeds' physicochemical traits and mucilage protection against aluminum effect during germination and root elongation as important factors in a biofuel seed crop (Ricinus communis).

    PubMed

    Silva, Giovanni Eustáquio Alves; Ramos, Flávia Toledo; de Faria, Ana Paula; França, Marcel Giovanni Costa

    2014-10-01

    We determined the length, volume, dry biomass, and density in seeds of five castor bean cultivars and verified notable physicochemical trait differences. Seeds were then subjected to different toxic aluminum (Al) concentrations to evaluate germination, relative root elongation, and the role of root apices' rhizosphere mucilage layer. Seeds' physicochemical traits were associated with Al toxicity responses, and the absence of Al in cotyledons near to the embryo was revealed by Al-hematoxylin staining, indicating that Al did not induce significant germination reduction rates between cultivars. However, in the more sensitive cultivar, Al was found around the embryo, contributing to subsequent growth inhibition. After this, to investigate the role of mucilage in Al tolerance, an assay was conducted using NH4Cl to remove root mucilage before or after exposure to different Al concentrations. Sequentially, the roots were stained with hematoxylin and a quantitative analysis of staining intensity was obtained. These results revealed the significant contribution of the mucilage layer to Al toxicity responses in castor bean seedlings. Root growth elongation under Al toxicity confirmed the role of the mucilage layer, which jointly indicated the differential Al tolerance between cultivars and an efficient Al-exclusion mechanism in the tolerant cultivar.

  12. Sea urchin elongation factor 1delta (EF1delta) and evidence for cell cycle-directed localization changes of a sub-fraction of the protein at M phase.

    PubMed

    Boulben, S; Monnier, A; Le Breton, M; Morales, J; Cormier, P; Bellé, R; Mulner-Lorillon, O

    2003-10-01

    Eukaryotic elongation factor 1 (eEF1) is a translational multimolecular complex reported in higher eukaryotes to be a target of CDK1/cyclin B, the universal regulator of M phase, but whose role in the cell cycle remains to be determined. A specific polyclonal antibody was produced and used to characterize the delta subunit of sea urchin elongation factor 1 (SgEF1delta) in early embryos, a powerful model for investigating cell cycle regulation. The SgEF1delta protein was present in unfertilized eggs as two isoforms of 35 and 37 kDa, issued from two different mRNAs. The two canonical eEF1delta partners, eEF1gamma and eEF1beta, were shown to co-immunoprecipitate with the SgEF1delta isoforms. Both isoforms were associated in a macromolecular complex, which resolved upon gel filtration chromatography at a molecular weight > 400 kDa, suggesting association with other yet unidentified partners. After fertilization, the amount as well as the ratio of both SgEF1delta isoforms remained constant during the first cell division as judged by Western blotting. Immunofluorescence analysis showed that a pool of the protein concentrated as a ring at the embryo nuclear location around the period of nuclear envelope breakdown and was visualized later as two large spheres around the mitotic spindle poles. Thus, the eEF1delta protein shows cell cycle-specific localization changes in sea urchin embryos.

  13. Elongation factor 2 and fragile X mental retardation protein control the dynamic translation of Arc/Arg3.1 essential for mGluR-LTD.

    PubMed

    Park, Sungjin; Park, Joo Min; Kim, Sangmok; Kim, Jin-Ah; Shepherd, Jason D; Smith-Hicks, Constance L; Chowdhury, Shoaib; Kaufmann, Walter; Kuhl, Dietmar; Ryazanov, Alexey G; Huganir, Richard L; Linden, David J; Worley, Paul F

    2008-07-10

    Group I metabotropic glutamate receptors (mGluR) induce long-term depression (LTD) that requires protein synthesis. Here, we demonstrate that Arc/Arg3.1 is translationally induced within 5 min of mGluR activation, and this response is essential for mGluR-dependent LTD. The increase in Arc/Arg3.1 translation requires eEF2K, a Ca(2+)/calmodulin-dependent kinase that binds mGluR and dissociates upon mGluR activation, whereupon it phosphorylates eEF2. Phospho-eEF2 acts to slow the elongation step of translation and inhibits general protein synthesis but simultaneously increases Arc/Arg3.1 translation. Genetic deletion of eEF2K results in a selective deficit of rapid mGluR-dependent Arc/Arg3.1 translation and mGluR-LTD. This rapid translational mechanism is disrupted in the fragile X disease mouse (Fmr1 KO) in which mGluR-LTD does not require de novo protein synthesis but does require Arc/Arg3.1. We propose a model in which eEF2K-eEF2 and FMRP coordinately control the dynamic translation of Arc/Arg3.1 mRNA in dendrites that is critical for synapse-specific LTD.

  14. 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.

  15. Elongated mandibular coronoid process as a cause of mandibular hypomobility.

    PubMed

    Kursoglu, Pinar; Capa, Nuray

    2006-07-01

    There are multiple factors in cases of mandibular hypomobility. One of these factors is elongated coronoid process. Two cases are presented to illustrate elongated coronoid process leading to mandibular hypomobility to help prevent misdiagnosis by clinicians. Coronoid process elongation is a rare condition. Both cases reported here had pulpitis on the teeth, however endodontic treatment could not be performed due to the restricted mouth opening in both cases. There were clinical findings of restricted range of motion, especially during protrusive movements. The restrictive movements did not cause pain for either patient, and the patients were not aware of their restricted mouth opening. Panoromic radiographs were taken and evaluated. The radiographs showed elongated coronoid process bilaterally. Three-dimensional computerized tomography was taken in one case only, due to the patient's financial restrictions. In cases of restricted mandibular opening, elongated coronoid process must be considered when diagnosing the cause.

  16. 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...

  17. 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

  18. 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.

  19. Post-meal responses of elongation factor 2 (eEF2) and adenosine monophosphate-activated protein kinase (AMPK) to leucine and carbohydrate supplements for regulating protein synthesis duration and energy homeostasis in rat skeletal muscle.

    PubMed

    Wilson, Gabriel J; Moulton, Christopher J; Garlick, Peter J; Anthony, Tracy G; Layman, Donald K

    2012-11-13

    Previous research demonstrates that the anabolic response of muscle protein synthesis (MPS) to a meal is regulated at the level of translation initiation with signals derived from leucine (Leu) and insulin to activate mTORC1 signaling. Recent evidence suggests that the duration of the meal response is limited by energy status of the cell and inhibition of translation elongation factor 2 (eEF2). This study evaluates the potential to extend the anabolic meal response with post-meal supplements of Leu or carbohydrates. Adult (~256 g) male Sprague-Dawley rats were food deprived for 12 h, then either euthanized before a standard meal (time 0) or at 90 or 180 min post-meal. At 135 min post-meal, rats received one of five oral supplements: 270 mg leucine (Leu270), 80:40:40 mg leucine, isoleucine, and valine (Leu80), 2.63 g carbohydrates (CHO2.6), 1 g carbohydrates (CHO1.0), or water (Sham control). Following the standard meal, MPS increased at 90 min then declined to pre-meal baseline at 180 min. Rats administered Leu270, Leu80, CHO2.6, or CHO1.0 maintained elevated rates of MPS at 180 min, while Sham controls declined from peak values. Leu80 and CHO1.0 treatments maintained MPS, but with values intermediate between Sham controls and Leu270 and CHO2.6 supplements. Consistent with MPS findings, the supplements maintained elongation activity and cellular energy status by preventing increases in AMP/ATP and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), acetyl-CoA carboxylase ACC and eEF2. The impact of the supplements on MPS and cellular energy status was in proportion to the energy content within the individual treatments (i.e., Leu270 > Leu80; CHO2.6 > CHO1.0), but the Leu supplements produced a disproportionate anabolic stimulation of MPS, eEF2 and energy status with significantly lower energy content. In summary, the incongruity between MPS and translation initiation at 180 min reflects a block in translation elongation due to reduced

  20. Presence of translation elongation factor-1A (eEF1A) in the excitatory postsynaptic density of rat cerebral cortex.

    PubMed

    Cho, Sun-Jung; Jung, Jae-Seob; Ko, Bok Hyun; Jin, IngNyol; Moon, Il Soo

    2004-08-05

    The postsynaptic density (PSD) is a proteinaceous cellular structure that is specialized for postsynaptic signal transduction. Here, we show that eukaryotic translation factor-1A (eEF1A; formerly known as eEF-1alpha) is associated with the excitatory PSD in rat forebrain. Immunoblot analysis showed that eEF1A in the PSD fraction is enriched over homogenate. Salt (1.0M NaCl), but not non-ionic detergents such as Triton X-100 (1.0%) and n-octyl glucoside (1.0%), could dissociate eEF1A from the PSD core. In cultured cortical neurons, eEF1A was colocalized with postsynaptic markers (PSD95 and SynGAPalpha), but not with a presynaptic marker (synaptophysin). These results indicate that eEF1A is present in the PSD of the excitatory synapses.

  1. Control of Transcriptional Elongation by RNA Polymerase II: A Retrospective.

    PubMed

    Brannan, Kris; Bentley, David L

    2012-01-01

    The origins of our current understanding of control of transcription elongation lie in pioneering experiments that mapped RNA polymerase II on viral and cellular genes. These studies first uncovered the surprising excess of polymerase molecules that we now know to be situated at the at the 5' ends of most genes in multicellular organisms. The pileup of pol II near transcription start sites reflects a ubiquitous bottle-neck that limits elongation right at the start of the transcription elongation. Subsequent seminal work identified conserved protein factors that positively and negatively control the flux of polymerase through this bottle-neck, and make a major contribution to control of gene expression.

  2. 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

  3. Grain Size Dependence of Uniform Elongation in Single-Phase FCC/BCC Metals

    NASA Astrophysics Data System (ADS)

    Liu, Haiting; Shen, Yao; Ma, Jiawei; Zheng, Pengfei; Zhang, Lei

    2016-09-01

    We studied the dependence of uniform elongation on grain size in the range of submicron to millimeter for single-phase FCC/BCC metals by reviewing recent experimental results and applying crystal plasticity finite element method simulation. In the order of increasing grain size, uniform elongation can be divided into three stages, namely low elongation stage, nearly constant elongation stage, and decreased elongation with large scatters stage. Low elongation stage features a dramatic increase near the critical grain size at the end of the stage, which is primarily attributed to the emergence of dislocation cell size transition from ultrafine to mid-size grain. Other factors can be neglected due to their negligible influence on overall variation trend. In nearly constant elongation stage, uniform elongation remains unchanged at a high level in general. As grain size keeps growing, uniform elongation starts decreasing and becomes scattered upon a certain grain size, indicating the initiation of decreased elongation with large scatters stage. It is shown that the increase is not linear or smooth but rather sharp at the end of low elongation stage, leading to a wider range in nearly constant elongation stage. The grain size dependence of uniform elongation can serve as a guiding principle for designing small uniaxial tensile specimens for mechanical testing, where size effect matters in most cases.

  4. 1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) induces phosphorylation of eukaryotic elongation factor-2 (eEF2): a cautionary note on the anticancer mechanism of an eEF2 kinase inhibitor.

    PubMed

    Chen, Zehan; Gopalakrishnan, Sujatha M; Bui, Mai-Ha; Soni, Niru B; Warrior, Usha; Johnson, Eric F; Donnelly, Jennifer B; Glaser, Keith B

    2011-12-23

    Eukaryotic elongation factor-2 kinase (eEF2K) relays growth and stress signals to protein synthesis through phosphorylation and inactivation of eukaryotic elongation factor 2 (eEF2). 1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) is a widely accepted inhibitor of mammalian eEF2K and an efficacious anti-proliferation agent against different cancer cells. It implied that eEF2K could be an efficacious anticancer target. However, eEF2K siRNA was ineffective against cancer cells including those sensitive to NH125. To test if pharmacological intervention differs from siRNA interference, we identified a highly selective small molecule eEF2K inhibitor A-484954. Like siRNA, A-484954 had little effect on cancer cell growth. We carefully examined the effect of NH125 and A-484954 on phosphorylation of eEF2, the known cellular substrate of eEF2K. Surprisingly, NH125 increased eEF2 phosphorylation, whereas A-484954 inhibited the phosphorylation as expected for an eEF2K inhibitor. Both A-484954 and eEF2K siRNA inhibited eEF2K and reduced eEF2 phosphorylation with little effect on cancer cell growth. These data demonstrated clearly that the anticancer activity of NH125 was more correlated with induction of eEF2 phosphorylation than inhibition of eEF2K. Actually, induction of eEF2 phosphorylation was reported to correlate with inhibition of cancer cell growth. We compared several known inducers of eEF2 phosphorylation including AMPK activators and an mTOR inhibitor. Interestingly, stronger induction of eEF2 phosphorylation correlated with more effective growth inhibition. We also explored signal transduction pathways leading to NH125-induced eEF2 phosphorylation. Preliminary data suggested that NH125-induced eEF2 phosphorylation was likely mediated through multiple pathways. These observations identified an opportunity for a new multipathway approach to anticancer therapies.

  5. Development of a rubber elongation factor, surface-imprinted polymer-quartz crystal microbalance sensor, for quantitative determination of Hev b1 rubber latex allergens present in natural rubber latex products.

    PubMed

    Sontimuang, Chonlatid; Suedee, Roongnapa; Canyuk, Bhutorn; Phadoongsombut, Narubodee; Dickert, Franz L

    2011-02-21

    Molecularly imprinted polymers (MIPs) for screening to detect rubber latex allergens (Hev b1) in natural rubber based products were designed as artificial recognition polymeric materials coated onto a quartz crystal microbalance (QCM). The polymers were prepared using a stamp imprinting procedure after mixing optimum amounts of methacrylic acid-vinylpyrrolidone-dihydroxyethylene bisacrylamide and Hev b1 latex allergen proteins, obtained from rubber gloves. QCM measurements showed that the resulting polymer layers after removal of the proteins used in their preparation could incorporate structures and features down to nanometer scale of protein templates into the imprinted polymer much better than a non-specific control polymer under controlled sensor conditions and an optimized polymerization process. This selective polymer but not the non-selective polymer clearly distinguished between the latex allergen Hev b1 and proteins such as lysozyme, ovalbumin and bovine serum albumin, with a selectivity factor of from 2 to 4, and the response of the rubber elongation factors by an astonishing factor of 12. The imprinted cavities recognized specific binding sites and could distinguish among related hevein latex allergenic proteins isolated from fresh natural rubber latex; Hev b1, Hev b2, and Hev b3 with a selectivity factor of from 4 to 6. The different QCM measurements obtained presumably reflected slightly different conformations and affinities to the MIP binding sites. The sensor layers selectively adsorbed Hev b1 within minutes in amounts ranging from 10 to 1500 μg L⁻¹ and with a detection limit of 1 μg L⁻¹. This work has demonstrated that this new sensor provides a fast and reliable response to natural rubber latex protein, even after being extracted from the matrix of rubber gloves.

  6. Plasma column changes at small solar elongations

    NASA Technical Reports Server (NTRS)

    Callahan, P. S.

    1973-01-01

    The differenced range versus integrated Doppler (DRVID) technique was used to study charged particle changes in the ray path between earth and Mariner 9. For plasma activity near the sun, DRVID data were obtained from August 10 to October 24, 1972, surrounding the Mariner 9 superior conjunction on September 7. If the records are viewed in terms of range change or range change rate, the day-to-day fluctuations in these quantities mask the changes expected due to the varying solar elongation. Thus, while the steady-state total electron content varies by a factor of 4, the columnar content changes show no systematic variations.

  7. Direct Characterization of Transcription Elongation by RNA Polymerase I

    PubMed Central

    Ucuncuoglu, Suleyman; Engel, Krysta L.; Purohit, Prashant K.; Dunlap, David D.; Schneider, David A.

    2016-01-01

    RNA polymerase I (Pol I) transcribes ribosomal DNA and is responsible for more than 60% of transcription in a growing cell. Despite this fundamental role that directly impacts cell growth and proliferation, the kinetics of transcription by Pol I are poorly understood. This study provides direct characterization of S. Cerevisiae Pol I transcription elongation using tethered particle microscopy (TPM). Pol I was shown to elongate at an average rate of approximately 20 nt/s. However, the maximum speed observed was, in average, about 60 nt/s, comparable to the rate calculated based on the in vivo number of active genes, the cell division rate and the number of engaged polymerases observed in EM images. Addition of RNA endonucleases to the TPM elongation assays enhanced processivity. Together, these data suggest that additional transcription factors contribute to efficient and processive transcription elongation by RNA polymerase I in vivo. PMID:27455049

  8. Amyloid-like fibril elongation follows michaelis-menten kinetics.

    PubMed

    Milto, Katazyna; Botyriute, Akvile; Smirnovas, Vytautas

    2013-01-01

    A number of proteins can aggregate into amyloid-like fibrils. It was noted that fibril elongation has similarities to an enzymatic reaction, where monomers or oligomers would play a role of substrate and nuclei/fibrils would play a role of enzyme. The question is how similar these processes really are. We obtained experimental data on insulin amyloid-like fibril elongation at the conditions where other processes which may impact kinetics of fibril formation are minor and fitted it using Michaelis-Menten equation. The correlation of the fit is very good and repeatable. It speaks in favour of enzyme-like model of fibril elongation. In addition, obtained [Formula: see text] and [Formula: see text] values at different conditions may help in better understanding influence of environmental factors on the process of fibril elongation.

  9. The elongation, termination, and recycling phases of translation in eukaryotes.

    PubMed

    Dever, Thomas E; Green, Rachel

    2012-07-01

    This work summarizes our current understanding of the elongation and termination/recycling phases of eukaryotic protein synthesis. We focus here on recent advances in the field. In addition to an overview of translation elongation, we discuss unique aspects of eukaryotic translation elongation including eEF1 recycling, eEF2 modification, and eEF3 and eIF5A function. Likewise, we highlight the function of the eukaryotic release factors eRF1 and eRF3 in translation termination, and the functions of ABCE1/Rli1, the Dom34:Hbs1 complex, and Ligatin (eIF2D) in ribosome recycling. Finally, we present some of the key questions in translation elongation, termination, and recycling that remain to be answered.

  10. Utility of the nuclear protein-coding gene, elongation factor-1 gamma (EF-1gamma), for spider systematics, emphasizing family level relationships of tarantulas and their kin (Araneae: Mygalomorphae).

    PubMed

    Ayoub, Nadia A; Garb, Jessica E; Hedin, Marshal; Hayashi, Cheryl Y

    2007-02-01

    Spider systematics has overwhelmingly relied on morphological characters to resolve higher-level phylogenetic questions. Molecular phylogenetic studies of spiders above the genus level have been rare, partly because of a paucity of characterized genes available for amplification and sequencing. Here we show the phylogenetic utility of a new molecular marker, elongation factor-1 gamma (EF-1gamma) for discerning family level relationships in the spider infraorder, Mygalomorphae. We included genomic sequences from 26 mygalomorph genera in 14 families as well as cDNA sequences from 10 families in the infraorder Araneomorphae. We found strong support for the traditional split of mygalomorphs into atypoids (Antrodiaetidae, Atypidae, and Mecicobothriidae) and non-atypoids (all other families). Some families with multiple generic representatives were found to be polyphyletic or paraphyletic, such as the Nemesiidae, Ctenizidae, and Hexathelidae. A small portion of genomic EF-1gamma that could be amplified from araneomorphs contained a short intron, suggesting that longer genomic sequences could not be amplified due to the presence of introns. This intron may be useful for intra-familial araneomorph relationships. A tentative timeline for spider evolution is proposed using the evolutionary rate of EF-1gamma, estimated to be approximately 0.22% pairwise divergence per million years based on a non-parametric smoothing method (NPRS) and fossil constraints.

  11. 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

  12. Comparison of genomes of Brucella melitensis M28 and the B. melitensis M5-90 derivative vaccine strain highlights the translation elongation factor Tu gene tuf2 as an attenuation-related gene.

    PubMed

    Wang, Fangkun; Qiao, Zujian; Hu, Sen; Liu, Wenxing; Zheng, Huajun; Liu, Sidang; Zhao, Xiaomin; Bu, Zhigao

    2013-08-01

    Brucella melitensis causes brucellosis, a disease affecting sheep, cattle, and sometimes humans. Attenuated B. melitensis strain M5-90, derived from virulent strain M28, is widely used as a live vaccine in ruminants in China. Genetic differences between the strains may cast light on the mechanism of attenuation. We recently reported the complete genomic sequences of M28 and M5-90. Genome organization is highly conserved between these isolates, and also with virulent strains 16 M and ATCC 23457. Analysis revealed 23 open reading frames (ORFs) with consistent differences between M5-90 and the virulent strains. Notably, the tuf2 gene encoding translation elongation factor EF-Tu from M5-90 contained 50 single nucleotide polymorphisms (SNPs) and 9 gaps (indels) compared to tuf2 of M28 or of the other virulent strains. There were no changes in tuf1. To evaluate the potential role of EF-Tu in pathogenesis, tuf1 and tuf2 mutants of M28 and an M5-90 strain harboring wild-type tuf2 were constructed, and their virulence/attenuation was evaluated in vivo. We report that the tuf2 gene plays an important role in the attenuation of M5-90 virulence.

  13. LMM5.1 and LMM5.4, two eukaryotic translation elongation factor 1A-like gene family members, negatively affect cell death and disease resistance in rice.

    PubMed

    Zhao, Jiying; Liu, Pengcheng; Li, Chunrong; Wang, Yanyan; Guo, Lequn; Jiang, Guanghuai; Zhai, Wenxue

    2017-02-20

    Lesion mimic mutant (LMM) genes, stimulating lesion formation in the absence of pathogens, play significant roles in immune response. In this study, we characterized a rice lesion mimic mutant, lmm5, which displayed light-dependent spontaneous lesions. Additionally, lmm5 plants exhibited enhanced resistance to all of the tested races of Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae (Xoo) by increasing the expression of defense-related genes and the accumulation of hydrogen peroxide. Genetic analysis showed that the lesion mimic phenotype of lmm5 was controlled by two genes, lmm5.1 and lmm5.4, which were isolated with a map-based cloning strategy. Remarkably, LMM5.1 and LMM5.4 share a 97.4% amino acid sequence identity, and they each encode a eukaryotic translation elongation factor 1A (eEF1A)-like protein. Besides, LMM5.1 and LMM5.4 were expressed in a tissue-specific and an indica-specific manner, respectively. In addition, high-throughput mRNA sequencing analysis confirmed that the basal immunity was constitutively activated in the lmm5 mutant. Taken together, these results suggest that the homologous eEF1A-like genes, LMM5.1 and LMM5.4, negatively affect cell death and disease resistance in rice.

  14. Hack's Law: Sinuosity, convexity, elongation

    NASA Astrophysics Data System (ADS)

    Willemin, James H.

    2000-11-01

    Hack's law, an empirical, power law relationship between drainage basin area and the length of the main stream channel, has long been taken to imply that drainage basins become more elongate (relatively longer and narrower) with increasing basin size. A study of the geometry of 38 basins from three distinct geomorphic settings shows that this geometric interpretation of Hack's law is only occasionally true: Even though Hack's power law relationship holds between basin area and main channel length, these basins do not necessarily become more elongate with increasing size. Rather, Hack's law is an expression of a balance between changes in basin shape and changes in channel planform geometry. For the basins in this study, changes in channel sinuosity play the most important role in this balance; changes in basin shape are far less regular. Local conditions appear to determine the partitioning of importance between changes in basin shape and channel sinuosity.

  15. Phage display biopanning identifies the translation initiation and elongation factors (IF1α-3 and eIF-3) as components of Hsp70-peptide complexes in breast tumour cells.

    PubMed

    Siebke, Christina; James, Tharappel C; Cummins, Robert; O'Grady, Tony; Kay, Elaine; Bond, Ursula

    2012-03-01

    The heat shock protein, HSP70, is over-expressed in many tumours and acts at the crossroads of key intracellular processes in its role as a molecular chaperone. HSP70 associates with a vast array of peptides, some of which are antigenic and can mount adaptive immune responses against the tumour from which they are derived. The pool of peptides associated with HSP70 represents a unique barcode of protein metabolism in tumour cells. With a view to identifying unique protein targets that may be developed as tumour biomarkers, we used purified HSP70 and its associated peptide pool (HSP70-peptide complexes, HSP70-PCs) from different human breast tumour cell lines as targets for phage display biopanning. Our results show that HSP70-PCs from each cell line interact with unique sets of peptides within the phage display library. One of the peptides, termed IST, enriched in the biopanning process, was used in a 'pull-down' assay to identify the original protein from which the HSP70-associated peptides may have been derived. The eukaryotic translation initiation factor 3 (eIF-3), a member of the elongation factor EF1α family, and the HSP GRP78, were pulled down by the IST peptide. All of these proteins are known to be up-regulated in cancer cells. Immunohistochemical staining of tumour tissue microarrays showed that the peptide co-localised with HSP70 in breast tumour tissue. The data indicate that the reservoir of peptides associated with HSP70 can act as a unique indicator of cellular protein activity and a novel source of potential tumour biomarkers.

  16. How slow RNA polymerase II elongation favors alternative exon skipping.

    PubMed

    Dujardin, Gwendal; Lafaille, Celina; de la Mata, Manuel; Marasco, Luciano E; Muñoz, Manuel J; Le Jossic-Corcos, Catherine; Corcos, Laurent; Kornblihtt, Alberto R

    2014-05-22

    Splicing is functionally coupled to transcription, linking the rate of RNA polymerase II (Pol II) elongation and the ability of splicing factors to recognize splice sites (ss) of various strengths. In most cases, slow Pol II elongation allows weak splice sites to be recognized, leading to higher inclusion of alternative exons. Using CFTR alternative exon 9 (E9) as a model, we show here that slowing down elongation can also cause exon skipping by promoting the recruitment of the negative factor ETR-3 onto the UG-repeat at E9 3' splice site, which displaces the constitutive splicing factor U2AF65 from the overlapping polypyrimidine tract. Weakening of E9 5' ss increases ETR-3 binding at the 3' ss and subsequent E9 skipping, whereas strengthening of the 5' ss usage has the opposite effect. This indicates that a delay in the cotranscriptional emergence of the 5' ss promotes ETR-3 recruitment and subsequent inhibition of E9 inclusion.

  17. METHOD OF FORMING ELONGATED COMPACTS

    DOEpatents

    Larson, H.F.

    1959-05-01

    A powder compacting procedure and apparatus which produces elongated compacts of Be is described. The powdered metal is placed in a thin metal tube which is chemically compatible to lubricant, powder, atmosphere, and die material and will undergo a high degree of plastic deformation and have intermediate hardness. The tube is capped and placed in the die, and punches are applied to the ends. During the compacting stroke the powder seizes the tube and a thickening and shortening of the tube occurs. The tube is easily removed from the die, split, and peeled from the compact. (T.R.H.)

  18. Inhibition of the outgrowth and elongation of neurites from pheochromocytoma cells by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and preventive effects of dimethylsulfoniopropionate in the presence of nerve growth factor.

    PubMed

    Nakajima, Kenji; Minematsu, Masaharu; Miyamoto, Yuuichi

    2008-04-01

    The combined effects of dimethylsulfoniopropionate (DMSP) (10(-3), 10(-4) and 10(-5) M) with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (5 ng/mL) and the nerve growth factor (NGF) (5 ng/mL) on the outgrowth and elongation of neurites from pheochromocytoma (PC12) cells were examined on RPMI medium containing fetal bovine serum and horse serum with penicillin and streptomycin in collagen-coated dishes for 5 d. The growth was higher in increasing order of the DMSP (10(-3) M), MPTP and NGF, the DMSP (10(-5) M), MPTP and NGF, the MPTP and NGF group and the control group up to 3 d, but not in the NGF and the DMSP (10(-4) M), MPTP and NGF groups. The growth in all the experimental groups showed plateaus from days 4 to 5. The appearance of neurites from the cells in all the groups showed maxima on the 3rd day. The administration of NGF significantly stimulated the outgrowth of neurites from the cells, while the supplementation of MPTP noticeably inhibited the appearance of neurites even in the presence of NGF up to 5 d. However, the addition of DMSP (10(-3 )and 10(-4) M) to the latter group completely prevented the inhibition of the MPTP. These facts were significantly supported by the photographs of neurite-bearing cells on the 3rd day and also by the photometric analyses examining the reaction of MPTP to DMSP, NGF or Collagen IV.

  19. The photomorphogenic factors UV-B RECEPTOR 1, ELONGATED HYPOCOTYL 5, and HY5 HOMOLOGUE are part of the UV-B signalling pathway in grapevine and mediate flavonol accumulation in response to the environment.

    PubMed

    Loyola, Rodrigo; Herrera, Daniela; Mas, Abraham; Wong, Darren Chern Jan; Höll, Janine; Cavallini, Erika; Amato, Alessandra; Azuma, Akifumi; Ziegler, Tobias; Aquea, Felipe; Castellarin, Simone Diego; Bogs, Jochen; Tornielli, Giovanni Battista; Peña-Neira, Alvaro; Czemmel, Stefan; Alcalde, José Antonio; Matus, José Tomás; Arce-Johnson, Patricio

    2016-10-01

    Grapevine (Vitis vinifera L.) is a species well known for its adaptation to radiation. However, photomorphogenic factors related to UV-B responses have not been molecularly characterized. We cloned and studied the role of UV-B RECEPTOR (UVR1), ELONGATED HYPOCOTYL 5 (HY5), and HY5 HOMOLOGUE (HYH) from V. vinifera We performed gene functional characterizations, generated co-expression networks, and tested them in different environmental conditions. These genes complemented the Arabidopsis uvr8 and hy5 mutants in morphological and secondary metabolic responses to radiation. We combined microarray and RNA sequencing (RNA-seq) data with promoter inspections to identify HY5 and HYH putative target genes and their DNA binding preferences. Despite sharing a large set of common co-expressed genes, we found different hierarchies for HY5 and HYH depending on the organ and stress condition, reflecting both co-operative and partially redundant roles. New candidate UV-B gene markers were supported by the presence of HY5-binding sites. These included a set of flavonol-related genes that were up-regulated in a HY5 transient expression assay. We irradiated in vitro plantlets and fruits from old potted vines with high and low UV-B exposures and followed the accumulation of flavonols and changes in gene expression in comparison with non-irradiated conditions. UVR1, HY5, and HYH expression varied with organ, developmental stage, and type of radiation. Surprisingly, UVR1 expression was modulated by shading and temperature in berries, but not by UV-B radiation. We propose that the UV-B response machinery favours berry flavonol accumulation through the activation of HY5 and HYH at different developmental stages at both high and low UV-B exposures.

  20. The photomorphogenic factors UV-B RECEPTOR 1, ELONGATED HYPOCOTYL 5, and HY5 HOMOLOGUE are part of the UV-B signalling pathway in grapevine and mediate flavonol accumulation in response to the environment

    PubMed Central

    Loyola, Rodrigo; Herrera, Daniela; Mas, Abraham; Wong, Darren Chern Jan; Höll, Janine; Cavallini, Erika; Amato, Alessandra; Azuma, Akifumi; Ziegler, Tobias; Aquea, Felipe; Castellarin, Simone Diego; Bogs, Jochen; Tornielli, Giovanni Battista; Peña-Neira, Alvaro; Czemmel, Stefan; Alcalde, José Antonio; Matus, José Tomás; Arce-Johnson, Patricio

    2016-01-01

    Grapevine (Vitis vinifera L.) is a species well known for its adaptation to radiation. However, photomorphogenic factors related to UV-B responses have not been molecularly characterized. We cloned and studied the role of UV-B RECEPTOR (UVR1), ELONGATED HYPOCOTYL 5 (HY5), and HY5 HOMOLOGUE (HYH) from V. vinifera. We performed gene functional characterizations, generated co-expression networks, and tested them in different environmental conditions. These genes complemented the Arabidopsis uvr8 and hy5 mutants in morphological and secondary metabolic responses to radiation. We combined microarray and RNA sequencing (RNA-seq) data with promoter inspections to identify HY5 and HYH putative target genes and their DNA binding preferences. Despite sharing a large set of common co-expressed genes, we found different hierarchies for HY5 and HYH depending on the organ and stress condition, reflecting both co-operative and partially redundant roles. New candidate UV-B gene markers were supported by the presence of HY5-binding sites. These included a set of flavonol-related genes that were up-regulated in a HY5 transient expression assay. We irradiated in vitro plantlets and fruits from old potted vines with high and low UV-B exposures and followed the accumulation of flavonols and changes in gene expression in comparison with non-irradiated conditions. UVR1, HY5, and HYH expression varied with organ, developmental stage, and type of radiation. Surprisingly, UVR1 expression was modulated by shading and temperature in berries, but not by UV-B radiation. We propose that the UV-B response machinery favours berry flavonol accumulation through the activation of HY5 and HYH at different developmental stages at both high and low UV-B exposures. PMID:27543604

  1. Quantitative Analysis of Transcription Elongation by RNA Polymerase I In Vitro

    PubMed Central

    Schneider, David Alan

    2016-01-01

    The elongation step in transcription has gained attention for its roles in regulation of eukaryotic gene expression and for its influence on RNA processing. Sophisticated genetic analyses have identified factors and/or conditions that may affect transcription elongation rate or processivity; however, differentiation of direct and indirect effects on transcription is difficult using in vivo strategies. Therefore, effective, reproducible in vitro assays have been developed to test whether a given factor or condition can have a direct effect on the kinetics of transcription elongation. We have adapted a fully reconstituted transcription system for RNA polymerase I (Pol I) for kinetic analysis of transcription elongation rate in vitro. The assay described here has proven to be effective in the characterization of defects or enhancement of wild-type transcription elongation by RNA Pol I. Since transcription elongation by RNA Pol I has only recently gained significant attention, this assay will be a valuable resource for years to come. PMID:22113301

  2. Locomotion in elongate fishes: A contact sport.

    PubMed

    Ward, Andrea B; Costa, Alyssa; Monroe, Stephanie L; Aluck, Robert J; Mehta, Rita S

    2015-10-01

    Despite the physical differences between water and air, a number of fish lineages are known to make terrestrial excursions on land. Many of these fishes exhibit an elongate body plan. Elongation of the body can occur in several ways, the most common of which is increasing the number of vertebrae in one or both regions of the axial skeleton--precaudal and/or caudal. Elongate species are often found in three-dimensionally complex habitats. It has been hypothesized that elongate fishes use this structure to their locomotor advantage. In this study, we consider how elongation and differences in vertebral regionalization correspond with the use of wooden pegs, which are provided as analogs to vertically oriented substrate, structures that protrude above the ground. We compare aquatic and terrestrial locomotor behaviors of Polypterus senegalus, Erpetoichthys calabaricus, and Gymnallabes typus as they move through a peg array. When considering axial elongation we find that the highly elongate species, E. calabaricus and G. typus, contact more pegs but on average move slower in both environments than P. senegalus. When considering axial regionalization, we find that the precaudally elongate species, P. senegalus and E. calabaricus, differ in the patterns of peg contact between the two environments whereas the caudally elongate species, G. typus, exhibits similar peg contact between the two environments. Our study highlights the importance of incorporating body shape and vertebral regionalization to understand how elongate fishes move in water and on land.

  3. Th1 stimulatory proteins of Leishmania donovani: comparative cellular and protective responses of rTriose phosphate isomerase, rProtein disulfide isomerase and rElongation factor-2 in combination with rHSP70 against visceral leishmaniasis.

    PubMed

    Jaiswal, Anil Kumar; Khare, Prashant; Joshi, Sumit; Kushawaha, Pramod Kumar; Sundar, Shyam; Dube, Anuradha

    2014-01-01

    In visceral leishmaniasis, the recovery from the disease is always associated with the generation of Th1-type of cellular responses. Based on this, we have previously identified several Th1-stimulatory proteins of Leishmania donovani -triose phosphate isomerase (TPI), protein disulfide isomerase (PDI) and elongation factor-2 (EL-2) etc. including heat shock protein 70 (HSP70) which induced Th1-type of cellular responses in both cured Leishmania patients/hamsters. Since, HSPs, being the logical targets for vaccines aimed at augmenting cellular immunity and can be early targets in the immune response against intracellular pathogens; they could be exploited as vaccine/adjuvant to induce long-term immunity more effectively. Therefore, in this study, we checked whether HSP70 can further enhance the immunogenicity and protective responses of the above said Th1-stimulatory proteins. Since, in most of the studies, immunogenicity of HSP70 of L. donovani was assessed in native condition, herein we generated recombinant HSP70 and tested its potential to stimulate immune responses in lymphocytes of cured Leishmania infected hamsters as well as in the peripheral blood mononuclear cells (PBMCs) of cured patients of VL either individually or in combination with above mentioned recombinant proteins. rLdHSP70 alone elicited strong cellular responses along with remarkable up-regulation of IFN-γ and IL-12 cytokines and extremely lower level of IL-4 and IL-10. Among the various combinations, rLdHSP70 + rLdPDI emerged as superior one augmenting improved cellular responses followed by rLdHSP70 + rLdEL-2. These combinations were further evaluated for its protective potential wherein rLdHSP70 + rLdPDI again conferred utmost protection (∼80%) followed by rLdHSP70 + rLdEL-2 (∼75%) and generated a strong cellular immune response with significant increase in the levels of iNOS transcript as well as IFN-γ and IL-12 cytokines which was further supported by the high level of IgG2 antibody

  4. Hox genes control vertebrate body elongation by collinear Wnt repression.

    PubMed

    Denans, Nicolas; Iimura, Tadahiro; Pourquié, Olivier

    2015-02-26

    In vertebrates, the total number of vertebrae is precisely defined. Vertebrae derive from embryonic somites that are continuously produced posteriorly from the presomitic mesoderm (PSM) during body formation. We show that in the chicken embryo, activation of posterior Hox genes (paralogs 9-13) in the tail-bud correlates with the slowing down of axis elongation. Our data indicate that a subset of progressively more posterior Hox genes, which are collinearly activated in vertebral precursors, repress Wnt activity with increasing strength. This leads to a graded repression of the Brachyury/T transcription factor, reducing mesoderm ingression and slowing down the elongation process. Due to the continuation of somite formation, this mechanism leads to the progressive reduction of PSM size. This ultimately brings the retinoic acid (RA)-producing segmented region in close vicinity to the tail bud, potentially accounting for the termination of segmentation and axis elongation.

  5. Bilateral elongated mandibular coronoid process in an Anatolian skull

    PubMed Central

    Çorumlu, Ufuk; Demir, Mehmet Tevfik; Pirzirenli, Mennan Ece

    2016-01-01

    Elongation or hyperplasia of coronoid process of mandible is rare condition characterized by abnormal bone development which cause malocclusion and the limited mouth opening. In this study, in an Anatolian skull, a case of bilateral elongation of mandibular coronoid process was presented. Levandoski panographic analysis was performed on the panoramic radiographie to determine the hyperplasia of the coronoid process. The right condylar process was exactly hyperplastic. The measurements of Kr-Go/Cd-Go were 95.10 mm/79.03 mm on right side and 97.53 mm/87.80 mm on left side. The ratio of Kr-Go/Cd-Go on the right side was 1.20. Elongated coronoid process is one of the factors cause mandibular hypomobility, it as reported here might lead to limited mouth opening. The knowledge of this variation or abnormality can be useful for the radiologist and surgeons and prevent misdiagnosis. PMID:27722017

  6. Experimental and Theoretical Investigations of Phonation Threshold Pressure as a Function of Vocal Fold Elongation

    PubMed Central

    Tao, Chao; Regner, Michael F.; Zhang, Yu; Jiang, Jack J.

    2014-01-01

    Summary The relationship between the vocal fold elongation and the phonation threshold pressure (PTP) was experimentally and theoretically investigated. The PTP values of seventeen excised canine larynges with 0% to 15% bilateral vocal fold elongations in 5% elongation steps were measured using an excised larynx phonation system. It was found that twelve larynges exhibited a monotonic relationship between PTP and elongation; in these larynges, the 0% elongation condition had the lowest PTP. Five larynges exhibited a PTP minimum at 5% elongation. To provide a theoretical explanation of these phenomena, a two-mass model was modified to simulate vibration of the elongated vocal folds. Two pairs of longitudinal springs were used to represent the longitudinal elastin in the vocal folds. This model showed that when the vocal folds were elongated, the increased longitudinal tension would increase the PTP value and the increased vocal fold length would decrease the PTP value. The antagonistic effects contributed by these two factors were found to be able to cause either a monotonic or a non-monotonic relationship between PTP and elongation, which were consistent with experimental observations. Because PTP describes the ease of phonation, this study suggests that there may exist a nonzero optimal vocal fold elongation for the greatest ease for phonation in some larynges. PMID:25530744

  7. Translational Control of Cell Division by Elongator

    PubMed Central

    Bauer, Fanelie; Matsuyama, Akihisa; Candiracci, Julie; Dieu, Marc; Scheliga, Judith; Wolf, Dieter A.; Yoshida, Minoru; Hermand, Damien

    2012-01-01

    SUMMARY Elongator is required for the synthesis of the mcm5s2 modification found on tRNAs recognizing AA-ending codons. In order to obtain a global picture of the role of Elongator in translation, we used reverse protein arrays to screen the fission yeast proteome for translation defects. Unexpectedly, this revealed that Elongator inactivation mainly affected three specific functional groups including proteins implicated in cell division. The absence of Elongator results in a delay in mitosis onset and cytokinesis defects. We demonstrate that the kinase Cdr2, which is a central regulator of mitosis and cytokinesis, is under translational control by Elongator due to the Lysine codon usage bias of the cdr2 coding sequence. These findings uncover a mechanism by which the codon usage, coupled to tRNA modifications, fundamentally contributes to gene expression and cellular functions. PMID:22768388

  8. Activation of the transcription factor nuclear factor-kappa B in uterine luminal epithelial cells by interleukin 1 Beta 2: a novel interleukin 1 expressed by the elongating pig conceptus.

    PubMed

    Mathew, Daniel J; Newsom, Emily M; Guyton, Jennifer M; Tuggle, Christopher K; Geisert, Rodney D; Lucy, Matthew C

    2015-04-01

    Conceptus mortality is greatest in mammals during the peri-implantation period, a time when conceptuses appose and attach to the uterine surface epithelium while releasing proinflammatory molecules. Interleukin 1 beta (IL1B), a master proinflammatory cytokine, is released by the primate, rodent, and pig blastocyst during the peri-implantation period and is believed to be essential for establishment of pregnancy. The gene encoding IL1B has duplicated in the pig, resulting in a novel gene. Preliminary observations indicate that the novel IL1B is specifically expressed by pig conceptuses during the peri-implantation period. To verify this, IL1B was cloned from mRNA isolated from Day 12 pig conceptuses and compared with IL1B cloned from mRNA isolated from pig peripheral blood leukocytes (PBLs). The pig conceptuses, but not the PBLs, expressed a novel IL1B, referred to here as interleukin 1 beta 2 (IL1B2). Porcine endometrium was treated with recombinant porcine interleukin 1 beta 1 (IL1B1), the prototypical cytokine, and IL1B2 proteins. Immunohistochemistry and real-time RT-PCR were used to measure activation of nuclear factor-kappa B (NFKB) and NFKB-regulated transcripts, respectively, within the endometrium. Both IL1B1 and IL1B2 activated NFKB in the uterine luminal epithelium within 4 h. The NFKB activation and related gene expression, however, were lower in endometrium treated with IL1B2, suggesting that the conceptus-derived cytokine may have reduced activity within the uterus. In conclusion, the peri-implantation pig conceptus expresses a novel IL1B that can activate NFKB within the uterine surface epithelium, likely creating a proinflammatory microenvironment during establishment of pregnancy in the pig.

  9. Structural insights into NusG regulating transcription elongation

    PubMed Central

    Liu, Bin; Steitz, Thomas A.

    2017-01-01

    NusG is an essential transcription factor that plays multiple key regulatory roles in transcription elongation, termination and coupling translation and transcription. The core role of NusG is to enhance transcription elongation and RNA polymerase processivity. Here, we present the structure of Escherichia coli RNA polymerase complexed with NusG. The structure shows that the NusG N-terminal domain (NGN) binds at the central cleft of RNA polymerase surrounded by the β' clamp helices, the β protrusion, and the β lobe domains to close the promoter DNA binding channel and constrain the β' clamp domain, but with an orientation that is different from the one observed in the archaeal β' clamp–Spt4/5 complex. The structure also allows us to construct a reliable model of the complete NusG-associated transcription elongation complex, suggesting that the NGN domain binds at the upstream fork junction of the transcription elongation complex, similar to σ2 in the transcription initiation complex, to stabilize the junction, and therefore enhances transcription processivity. PMID:27899640

  10. Effects of elongation delay in transcription dynamics.

    PubMed

    Zhang, Xuan; Jin, Huiqin; Yang, Zhuoqin; Lei, Jinzhi

    2014-12-01

    In the transcription process, elongation delay is induced by the movement of RNA polymerases (RNAP) along the DNA sequence, and can result in changes in the transcription dynamics. This paper studies the transcription dynamics that involved the elongation delay and effects of cell division and DNA replication. The stochastic process of gene expression is modeled with delay chemical master equation with periodic coefficients, and is studied numerically through the stochastic simulation algorithm with delay. We show that the average transcription level approaches to a periodic dynamics over cell cycles at homeostasis, and the elongation delay can reduce the transcription level and increase the transcription noise. Moreover, the transcription elongation can induce bimodal distribution of mRNA levels that can be measured by the techniques of flow cytometry.

  11. Elongated Deposits in Southern Elysium Planitia, Mars

    NASA Astrophysics Data System (ADS)

    Nussbaumer, J. W.

    2012-03-01

    In the Elysium Planitia region, deposits have elongated elevations that resemble terrestrial drumlins or yardangs. Drumlins and drumlin clusters are glacial landforms that have been extensively studied. In contrast, Yardangs are formed by wind.

  12. A review of penile elongation surgery

    PubMed Central

    Gillis, Joshua

    2017-01-01

    Penile elongation surgery is less commonly performed in the public sector, but involves a collaborative approach between urology and plastic surgery. Congenital and acquired micropenis are the classic surgical indications for penile elongation surgery. The goal of intervention in these patients is to restore a functional penis size in order to allow normal standing micturition, enable satisfying sexual intercourse and improve patient quality of life. Many men seeking elongation actually have normal length penises, but perceive themselves to be small, a psychologic condition termed ‘penile dysmorphophobia’. This paper will review the anatomy and embryology of congenital micropenis and discuss both conservative and surgical management options for men seeking penile elongation therapy. PMID:28217452

  13. Numerical Experiments with Flows of Elongated Granules

    DTIC Science & Technology

    1992-01-01

    NASA AVSCOM Technical Memorandum 105567 Technical Report 91- C- 006 𔃼e- 0ok, Numerical Experiments With Flows of Elongated Granules AD-A251 853 DTIC...EXPERIMENTS WITH FLOWS OF ELONGATED GRANULES H.G. Elrod 14 Cromwell Court Old Saybrook, Connecticut 06475 and D.E. Brewe Propulsion Directorate U.S. Army...granular flows (1) between two infinite, counter-moving, parallel, roughened walls, and (2) for an infinitely-wide slider. Each granule is simulated by a

  14. Mutual interdependence of splicing and transcription elongation.

    PubMed

    Brzyżek, Grzegorz; Świeżewski, Szymon

    2015-01-01

    Transcription and splicing are intrinsically linked, as splicing needs a pre-mRNA substrate to commence. The more nuanced view is that the rate of transcription contributes to splicing regulation. On the other hand there is accumulating evidence that splicing has an active role in controlling transcription elongation by DNA-dependent RNA polymerase II (RNAP II). We briefly review those mechanisms and propose a unifying model where splicing controls transcription elongation to provide an optimal timing for successive rounds of splicing.

  15. Visualization of large elongated DNA molecules.

    PubMed

    Lee, Jinyong; Kim, Yongkyun; Lee, Seonghyun; Jo, Kyubong

    2015-09-01

    Long and linear DNA molecules are the mainstream single-molecule analytes for a variety of biochemical analysis within microfluidic devices, including functionalized surfaces and nanostructures. However, for biochemical analysis, large DNA molecules have to be unraveled, elongated, and visualized to obtain biochemical and genomic information. To date, elongated DNA molecules have been exploited in the development of a number of genome analysis systems as well as for the study of polymer physics due to the advantage of direct visualization of single DNA molecule. Moreover, each single DNA molecule provides individual information, which makes it useful for stochastic event analysis. Therefore, numerous studies of enzymatic random motions have been performed on a large elongated DNA molecule. In this review, we introduce mechanisms to elongate DNA molecules using microfluidics and nanostructures in the beginning. Secondly, we discuss how elongated DNA molecules have been utilized to obtain biochemical and genomic information by direct visualization of DNA molecules. Finally, we reviewed the approaches used to study the interaction of proteins and large DNA molecules. Although DNA-protein interactions have been investigated for many decades, it is noticeable that there have been significant achievements for the last five years. Therefore, we focus mainly on recent developments for monitoring enzymatic activity on large elongated DNA molecules.

  16. The marine polyketide myriaporone 3/4 stalls translation by targeting the elongation phase.

    PubMed

    Muthukumar, Yazh; Roy, Myriam; Raja, Aruna; Taylor, Richard E; Sasse, Florenz

    2013-01-21

    Myriaporone 3/4, a cytotoxic polyketide, has been reported as an inhibitor of eukaryotic protein synthesis. However, the mechanism by which it inhibits translation was unknown. Here we show that myriaporone 3/4 stalls protein synthesis in the elongation phase by inducing phosphorylation of eukaryotic elongation factor 2. The phosphorylation results from direct binding of myriaporone 3/4 to eukaryotic elongation factor 2 kinase. Our study also shows that myriaporone 3/4 in the nanomolar range inhibits in vitro tube formation by endothelial cells without being cytotoxic. In general, myriaporone 3/4 was at least 300 times less toxic to primary cells than to tumor cells.

  17. Transcriptional activation domains stimulate initiation and elongation at different times and via different residues.

    PubMed Central

    Brown, S A; Weirich, C S; Newton, E M; Kingston, R E

    1998-01-01

    Transcriptional activators can stimulate multiple steps in the transcription process. We have used GAL4 fusion proteins to characterize the ability of different transcriptional activation domains to stimulate transcriptional elongation on the hsp70 gene in vitro. Stimulation of elongation apparently occurs via a mechanistic pathway different from that of stimulation of initiation: the herpes simplex virus VP16, heat shock factor 1 (HSF1) and amphipathic helix (AH) activation domains all stimulate initiation, but only VP16 and HSF1 stimulate elongation; and mutations in hydrophobic residues of the HSF1 activation domains impair stimulation of elongation but not of initiation, while mutations in adjacent acidic residues impair stimulation of initiation more than of elongation. Experiments in which activators were exchanged between initiation and elongation demonstrate that the elongation function of HSF1 will stimulate RNA polymerase that has initiated and is transcriptionally engaged. Transcriptional activators thus appear to have at least two distinct functions that reside in the same domain, and that act at different times to stimulate initiation and elongation. PMID:9606196

  18. A Conserved Nuclear Cyclophilin Is Required for Both RNA Polymerase II Elongation and Co-transcriptional Splicing in Caenorhabditis elegans

    PubMed Central

    Ahn, Jeong H.; Rechsteiner, Andreas; Strome, Susan; Kelly, William G.

    2016-01-01

    The elongation phase of transcription by RNA Polymerase II (Pol II) involves numerous events that are tightly coordinated, including RNA processing, histone modification, and chromatin remodeling. RNA splicing factors are associated with elongating Pol II, and the interdependent coupling of splicing and elongation has been documented in several systems. Here we identify a conserved, multi-domain cyclophilin family member, SIG-7, as an essential factor for both normal transcription elongation and co-transcriptional splicing. In embryos depleted for SIG-7, RNA levels for over a thousand zygotically expressed genes are substantially reduced, Pol II becomes significantly reduced at the 3’ end of genes, marks of transcription elongation are reduced, and unspliced mRNAs accumulate. Our findings suggest that SIG-7 plays a central role in both Pol II elongation and co-transcriptional splicing and may provide an important link for their coordination and regulation. PMID:27541139

  19. Elongation growth and gravitropic curvature in the Flammulina velutipes (Agaricales) fruiting body.

    PubMed

    Haindl, E; Monzer, J

    1994-06-01

    Differential elongation of stipe hyphae drives the gravitropic reorientation of Flammulina velutipes (Agaricales) fruiting bodies. The gravitropic curvature is strictly dependent on the presence of the transition zone between pileus and stipe. Elongation growth, providing the driving force for curvature, is also promoted by the pileus. Gravitropic curvature is successfully suppressed by clinostatic rotation, but the elongation rate is not affected. Explantation of fruiting body stipes lowers curvature and elongation rates corresponding to explant size reduction. In Flammulina, 25 mm length of transition zone explants is an efficient size for reproducible curvature and elongation during 48- to 72-h curvature tests. Submersion of specimens in aqueous medium causes cessation of the gravitropic curvature, but does not affect elongation. Thus the involvement of a diffusible factor in transmission of the curvature signal is probable. Splitting the fruiting body stipe in segments of 1/8 diameter does not suppress the gravitropic response, and the segments are individually reoriented to the vertical. It is concluded that the graviresponse of the Flammulina fruiting body is based on cellular perception of the gravistimulus and that a differential growth signal is transmitted in the stipe by a soluble factor that regulates hyphal elongation.

  20. Architecture of the yeast Elongator complex.

    PubMed

    Dauden, Maria I; Kosinski, Jan; Kolaj-Robin, Olga; Desfosses, Ambroise; Ori, Alessandro; Faux, Celine; Hoffmann, Niklas A; Onuma, Osita F; Breunig, Karin D; Beck, Martin; Sachse, Carsten; Séraphin, Bertrand; Glatt, Sebastian; Müller, Christoph W

    2017-02-01

    The highly conserved eukaryotic Elongator complex performs specific chemical modifications on wobble base uridines of tRNAs, which are essential for proteome stability and homeostasis. The complex is formed by six individual subunits (Elp1-6) that are all equally important for its tRNA modification activity. However, its overall architecture and the detailed reaction mechanism remain elusive. Here, we report the structures of the fully assembled yeast Elongator and the Elp123 sub-complex solved by an integrative structure determination approach showing that two copies of the Elp1, Elp2, and Elp3 subunits form a two-lobed scaffold, which binds Elp456 asymmetrically. Our topological models are consistent with previous studies on individual subunits and further validated by complementary biochemical analyses. Our study provides a structural framework on how the tRNA modification activity is carried out by Elongator.

  1. Interplay between DNA supercoiling and transcription elongation.

    PubMed

    Ma, Jie; Wang, Michelle

    2014-01-01

    Transcription-coupled DNA supercoiling has been shown to be an important regulator of transcription that is broadly present in the cell. Here we review experimental work which shows that RNA polymerase is a powerful torsional motor that can alter DNA topology and structure, and DNA supercoiling in turn directly affects transcription elongation.

  2. Saccharomyces cerevisiae Elongator mutations confer resistance to the Kluyveromyces lactis zymocin

    PubMed Central

    Frohloff, Frank; Fichtner, Lars; Jablonowski, Daniel; Breunig, Karin D.; Schaffrath, Raffael

    2001-01-01

    Kluyveromyces lactis killer strains secrete a zymocin complex that inhibits proliferation of sensitive yeast genera including Saccharomyces cerevisiae. In search of the putative toxin target (TOT), we used mTn3:: tagging to isolate zymocin-resistant tot mutants from budding yeast. Of these we identified the TOT1, TOT2 and TOT3 genes (isoallelic with ELP1, ELP2 and ELP3, respectively) coding for the histone acetyltransferase (HAT)-associated Elongator complex of RNA polymerase II holoenzyme. Other than the typical elp ts-phenotype, tot phenocopies hypersensitivity towards caffeine and Calcofluor White as well as slow growth and a G1 cell cycle delay. In addition, TOT4 and TOT5 (isoallelic with KTI12 and IKI1, respectively) code for components that associate with Elongator. Intriguingly, strains lacking non-Elongator HATs (gcn5Δ, hat1Δ, hpa3Δ and sas3Δ) or non-Elongator transcription elongation factors TFIIS (dst1Δ) and Spt4p (spt4Δ) cannot confer resistance towards the K.lactis zymocin, thus providing evidence that Elongator equals TOT and that Elongator plays an important role in signalling toxicity of the K.lactis zymocin. PMID:11296232

  3. The Effects of High Steady State Auxin Levels on Root Cell Elongation in Brachypodium[OPEN

    PubMed Central

    Pacheco-Villalobos, David; Tamaki, Takayuki; Gujas, Bojan; Jaspert, Nina; Oecking, Claudia; Bulone, Vincent; Hardtke, Christian S.

    2016-01-01

    The long-standing Acid Growth Theory of plant cell elongation posits that auxin promotes cell elongation by stimulating cell wall acidification and thus expansin action. To date, the paucity of pertinent genetic materials has precluded thorough analysis of the importance of this concept in roots. The recent isolation of mutants of the model grass species Brachypodium distachyon with dramatically enhanced root cell elongation due to increased cellular auxin levels has allowed us to address this question. We found that the primary transcriptomic effect associated with elevated steady state auxin concentration in elongating root cells is upregulation of cell wall remodeling factors, notably expansins, while plant hormone signaling pathways maintain remarkable homeostasis. These changes are specifically accompanied by reduced cell wall arabinogalactan complexity but not by increased proton excretion. On the contrary, we observed a tendency for decreased rather than increased proton extrusion from root elongation zones with higher cellular auxin levels. Moreover, similar to Brachypodium, root cell elongation is, in general, robustly buffered against external pH fluctuation in Arabidopsis thaliana. However, forced acidification through artificial proton pump activation inhibits root cell elongation. Thus, the interplay between auxin, proton pump activation, and expansin action may be more flexible in roots than in shoots. PMID:27169463

  4. Real time determination of bacterial in vivo ribosome translation elongation speed based on LacZα complementation system

    PubMed Central

    Zhu, Manlu; Dai, Xiongfeng; Wang, Yi-Ping

    2016-01-01

    Bacterial growth significantly depends on protein synthesis catalyzed by ribosome. Ribosome translation elongation speed is a key factor determining the bacterial protein synthesis rate. However, existing methods for determining translation elongation speed have limited applications. Here we developed a simple and convenient method for measuring bacterial translation elongation speed based on LacZα complementation system. It enables the measurement of in vivo translation elongation speed of different individual genes. Tests related to ribosome translation elongation speed under various growth perturbations including different nutrient conditions, low temperature, a low-speed ribosome mutant, and fusidic acid treatment, were performed to quantitatively validate this method. Using this approach, we further found that nutrient starvation caused a remarkable slow-down of ribosome translation of Escherichia coli (E. coli). We also studied the dynamic change of translation elongation speed during the process of nutrient up-shift. This method will boost the quantitative understanding of bacterial ribosome translation capacity and growth. PMID:27903884

  5. Synthetic lethal interactions suggest a role for the Saccharomyces cerevisiae Rtf1 protein in transcription elongation.

    PubMed Central

    Costa, P J; Arndt, K M

    2000-01-01

    Strong evidence indicates that transcription elongation by RNA polymerase II (pol II) is a highly regulated process. Here we present genetic results that indicate a role for the Saccharomyces cerevisiae Rtf1 protein in transcription elongation. A screen for synthetic lethal mutations was carried out with an rtf1 deletion mutation to identify factors that interact with Rtf1 or regulate the same process as Rtf1. The screen uncovered mutations in SRB5, CTK1, FCP1, and POB3. These genes encode an Srb/mediator component, a CTD kinase, a CTD phosphatase, and a protein involved in the regulation of transcription by chromatin structure, respectively. All of these gene products have been directly or indirectly implicated in transcription elongation, indicating that Rtf1 may also regulate this process. In support of this view, we show that RTF1 functionally interacts with genes that encode known elongation factors, including SPT4, SPT5, SPT16, and PPR2. We also show that a deletion of RTF1 causes sensitivity to 6-azauracil and mycophenolic acid, phenotypes correlated with a transcription elongation defect. Collectively, our results suggest that Rtf1 may function as a novel transcription elongation factor in yeast. PMID:11014804

  6. Tbx1 is Necessary for Palatal Elongation and Elevation

    PubMed Central

    Goudy, Steven; Law, Amy; Sanchez, Gabriela; Baldwin, H. Scott; Brown, Christopher

    2010-01-01

    The transcription factor TBX1 is a key mediator of developmental abnormalities associated with DiGeorge/Velocardiofacial Syndrome. Studies in mice have demonstrated that decreased dosage of Tbx1 results in defects in pharyngeal arch, cardiovascular, and craniofacial development. The role of Tbx1 in cardiac development has been intensely studied; however, its role in palatal development is poorly understood. By studying the Tbx1-/- mice we found defects during the critical points of palate elongation and elevation. The intrinsic palate defects in the Tbx1-/- mice were determined by measuring changes in palate shelf length, proliferation, apoptosis, expression of relevant growth factors, and in palate fusion assays. Tbx1-/- embryos exhibit cleft palate with failed palate elevation in 100% and abnormal palatal-oral fusions in 50%. In the Tbx1-/- mice the palate shelf length was reduced and tongue height was greater, demonstrating a physical impediment to palate elevation and apposition. In vitro palate fusion assays demonstrate that Tbx1-/- palate shelves are capable of fusion but a roller culture assay showed that the null palatal shelves were unable to elongate. Diminished hyaluronic acid production in the Tbx1-/- palate shelves may explain failed palate shelf elevation. In addition, cell proliferation and apoptosis were perturbed in Tbx1-/- palates. A sharp decrease of Fgf8 expression was detected in the Tbx1-/- palate shelves, suggesting that Fgf8 is dependent on Tbx1 in the palate. Fgf10 is also up-regulated in the Tbx1-/- palate shelves and tongue. These data demonstrate that Tbx1 is a critical transcription factor that guides palatal elongation and elevation and that Fgf8 expression in the palate is Tbx1-dependent. PMID:20214979

  7. Fluorescent Methods to Study Transcription Initiation and Transition into Elongation

    PubMed Central

    Deshpande, Aishwarya P.; Sultana, Shemaila

    2015-01-01

    The DNA-dependent RNA polymerases induce specific conformational changes in the promoter DNA during transcription initiation. Fluorescence spectroscopy sensitively monitors these DNA conformational changes in real time and at equilibrium providing powerful ways to estimate interactions in transcriptional complexes and to assess how transcription is regulated by the promoter DNA sequence, transcription factors, and small ligands. Ensemble fluorescence methods described here probe the individual steps of promoter binding, bending, opening, and transition into the elongation using T7 phage and mitochondrial transcriptional systems as examples. PMID:25095993

  8. Plant Elongator regulates auxin-related genes during RNA polymerase II transcription elongation.

    PubMed

    Nelissen, Hilde; De Groeve, Steven; Fleury, Delphine; Neyt, Pia; Bruno, Leonardo; Bitonti, Maria Beatrice; Vandenbussche, Filip; Van der Straeten, Dominique; Yamaguchi, Takahiro; Tsukaya, Hirokazu; Witters, Erwin; De Jaeger, Geert; Houben, Andreas; Van Lijsebettens, Mieke

    2010-01-26

    In eukaryotes, transcription of protein-encoding genes is strongly regulated by posttranslational modifications of histones that affect the accessibility of the DNA by RNA polymerase II (RNAPII). The Elongator complex was originally identified in yeast as a histone acetyltransferase (HAT) complex that activates RNAPII-mediated transcription. In Arabidopsis thaliana, the Elongator mutants elo1, elo2, and elo3 with decreased leaf and primary root growth due to reduced cell proliferation identified homologs of components of the yeast Elongator complex, Elp4, Elp1, and Elp3, respectively. Here we show that the Elongator complex was purified from plant cell cultures as a six-component complex. The role of plant Elongator in transcription elongation was supported by colocalization of the HAT enzyme, ELO3, with euchromatin and the phosphorylated form of RNAPII, and reduced histone H3 lysine 14 acetylation at the coding region of the SHORT HYPOCOTYL 2 auxin repressor and the LAX2 auxin influx carrier gene with reduced expression levels in the elo3 mutant. Additional auxin-related genes were down-regulated in the transcriptome of elo mutants but not targeted by the Elongator HAT activity showing specificity in target gene selection. Biological relevance was apparent by auxin-related phenotypes and marker gene analysis. Ethylene and jasmonic acid signaling and abiotic stress responses were up-regulated in the elo transcriptome and might contribute to the pleiotropic elo phenotype. Thus, although the structure of Elongator and its substrate are conserved, target gene selection has diverged, showing that auxin signaling and influx are under chromatin control.

  9. Vibrational modes of elongated sessile liquid droplets.

    PubMed

    Temperton, Robert H; Sharp, James S

    2013-04-16

    Vibrations of small (microliter) sessile liquid droplets were studied using a simple optical deflection technique. The droplets were made to elongate in one direction by taking advantage of the anisotropic wetting of the liquids on structured diffraction grating surfaces. They were vibrated by applying a puff of nitrogen gas. Motion of the droplets was monitored by scattering laser light from their surfaces. The scattered light was collected using a photodiode, and the resulting time-dependent intensity signals were Fourier-transformed to obtain the vibrational response of the drops. The vibrational spectra of elongated sessile drops were observed to contain two closely spaced peaks. A simple model that considers the frequency of capillary wave fluctuations on the surfaces of the drops was used to show that the vibrational frequencies of these peaks correspond to standing wave states that exist along the major and minor profile lengths of the droplets.

  10. Vertically stabilized elongated cross-section tokamak

    DOEpatents

    Sheffield, George V.

    1977-01-01

    This invention provides a vertically stabilized, non-circular (minor) cross-section, toroidal plasma column characterized by an external separatrix. To this end, a specific poloidal coil means is added outside a toroidal plasma column containing an endless plasma current in a tokamak to produce a rectangular cross-section plasma column along the equilibrium axis of the plasma column. By elongating the spacing between the poloidal coil means the plasma cross-section is vertically elongated, while maintaining vertical stability, efficiently to increase the poloidal flux in linear proportion to the plasma cross-section height to achieve a much greater plasma volume than could be achieved with the heretofore known round cross-section plasma columns. Also, vertical stability is enhanced over an elliptical cross-section plasma column, and poloidal magnetic divertors are achieved.

  11. Measuring Hole Elongation in Bolted Joints

    NASA Technical Reports Server (NTRS)

    Wichorek, G. R.

    1986-01-01

    Measurement does not affect joint parameters. Verification of analytical and strength-prediction methods for bolted composite joints based generally on data obtained experimentally from double-lap-joint specimens. In mechanically fastened joints, stresses maximal at fastener holes. Ability to measure accurately hole elongations without affecting joint parameters provides better understanding of elastic and plastic behavior of joint material leading to failure mechanisms in mechanically fastened joints required for design of more-efficient, lightweight composite joints.

  12. Elongational viscosity of photo-oxidated LDPE

    NASA Astrophysics Data System (ADS)

    Rolón-Garrido, Víctor H.; Wagner, Manfred H.

    2014-05-01

    Sheets of low-density polyethylene (LDPE) were photo-oxidatively treated at room temperature, and subsequently characterized rheologically in the melt state by shear and uniaxial extensional experiments. For photo-oxidation, a xenon lamp was used to irradiate the samples for times between 1 day and 6 weeks. Linear-viscoelastic characterization was performed in a temperature range of 130 to 220°C to obtain the master curve at 170°C, the reference temperature at which the elongational viscosities were measured. Linear viscoelasticity is increasingly affected by increasing photo-oxidation due to crosslinking of LDPE, as corroborated by an increasing gel fraction as determined by a solvent extraction method. The elongational measurements reveal a strong enhancement of strain hardening until a saturation level is achieved. The elongational data are analyzed in the frame work of two constitutive equations, the rubber-like liquid and the molecular stress function models. Within the experimental window, timedeformation separability is confirmed for all samples, independent of the degree of photo-oxidation.

  13. Elongational viscosity of photo-oxidated LDPE

    SciTech Connect

    Rolón-Garrido, Víctor H. E-mail: manfred.wagner@tu-berlin.de; Wagner, Manfred H. E-mail: manfred.wagner@tu-berlin.de

    2014-05-15

    Sheets of low-density polyethylene (LDPE) were photo-oxidatively treated at room temperature, and subsequently characterized rheologically in the melt state by shear and uniaxial extensional experiments. For photo-oxidation, a xenon lamp was used to irradiate the samples for times between 1 day and 6 weeks. Linear-viscoelastic characterization was performed in a temperature range of 130 to 220°C to obtain the master curve at 170°C, the reference temperature at which the elongational viscosities were measured. Linear viscoelasticity is increasingly affected by increasing photo-oxidation due to crosslinking of LDPE, as corroborated by an increasing gel fraction as determined by a solvent extraction method. The elongational measurements reveal a strong enhancement of strain hardening until a saturation level is achieved. The elongational data are analyzed in the frame work of two constitutive equations, the rubber-like liquid and the molecular stress function models. Within the experimental window, timedeformation separability is confirmed for all samples, independent of the degree of photo-oxidation.

  14. Quasigeostrohpic flow over isolated elongated topography

    NASA Astrophysics Data System (ADS)

    Johnson, E. R.

    1982-09-01

    The finite amplitude perturbations to a uniform stream caused by the presence of elongated topography is considered using two simple models. The first considers elliptic seamounts with scales L and l ( L ⪖ l) and gives a smooth interpolation between axisymmetric models L ; l at one extreme and infinite ridges of fixed cross-section L å l at the other. Basing the Rossby number of the flow on the shorter scale gives blocking heights of order unity for all elongations, whereas it is the longer scale that determines the horizontal extent of the region affected by the topography. The second model considers greatly elongated topography (L å l) of variable cross section showing that the topographic velocity parallel to the ridges is given by ƒ A∗/2d , where A∗ is the local cross-section area, d the depth, and f the Coriolis parameter. The component perpendicular to the ridge is obtained directly from the parallel component via a linear transform. Topographically generated velocities may thus be obtained rapidly from contours of bottom topography and an example is given using the seamount 'Brontosaurus Bump' from GOULD, HENDRY AND HUPPERT (Deep-Sea Research, 28, 409-440, 1981).

  15. Arabidopsis microtubule destabilizing protein40 is involved in brassinosteroid regulation of hypocotyl elongation.

    PubMed

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

    2012-10-01

    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.

  16. Structure–function studies of the RNA polymerase II elongation complex

    SciTech Connect

    Brueckner, Florian; Armache, Karim-Jean; Cheung, Alan; Damsma, Gerke E.; Kettenberger, Hubert; Lehmann, Elisabeth; Sydow, Jasmin; Cramer, Patrick

    2009-02-01

    X-ray crystallographic and complementary functional studies have contributed significantly to the current understanding of gene transcription. Here, recent structure–function studies on various aspects of the elongation phase of transcription are summarized. RNA polymerase II (Pol II) is the eukaryotic enzyme that is responsible for transcribing all protein-coding genes into messenger RNA (mRNA). The mRNA-transcription cycle can be divided into three stages: initiation, elongation and termination. During elongation, Pol II moves along a DNA template and synthesizes a complementary RNA chain in a processive manner. X-ray structural analysis has proved to be a potent tool for elucidating the mechanism of Pol II elongation. Crystallographic snapshots of different functional states of the Pol II elongation complex (EC) have elucidated mechanistic details of nucleotide addition and Pol II translocation. Further structural studies in combination with in vitro transcription experiments led to a mechanistic understanding of various additional features of the EC, including its inhibition by the fungal toxin α-amanitin, the tunability of the active site by the elongation factor TFIIS, the recognition of DNA lesions and the use of RNA as a template.

  17. Limited Correlation between Expansin Gene Expression and Elongation Growth Rate1

    PubMed Central

    Caderas, Doina; Muster, Matthias; Vogler, Hannes; Mandel, Therese; Rose, Jocelyn K.C.; McQueen-Mason, Simon; Kuhlemeier, Cris

    2000-01-01

    The aim of this work was to study the role of the cell wall protein expansin in elongation growth. Expansins increase cell wall extensibility in vitro and are thought to be involved in cell elongation. Here, we studied the regulation of two tomato (Lycopersicon esculentum cv Moneymaker) expansin genes, LeExp2 and LeExp18, in rapidly expanding tissues. LeExp2 was strongly expressed in the elongation zone of hypocotyls and in the faster growing stem part during gravitropic stimulation. LeExp18 expression did not correlate with elongation growth. Exogenous application of hormones showed a substantial auxin-stimulation of LeExp2 mRNA in etiolated hypocotyls and a weaker auxin-stimulation of LeExp18 mRNA in stem tissue. Analysis of transcript accumulation revealed higher levels of LeExp2 and LeExp18 in light-treated, slow-growing tissue than in dark-treated, rapidly elongating tissue. Expansin protein levels and cell wall extension activities were similar in light- and dark-grown hypocotyl extracts. The results show a strong correlation between expansin gene expression and growth rate, but this correlation is not absolute. We conclude that elongation growth is likely to be controlled by expansin acting in concert with other factors that may limit growth under some physiological conditions. PMID:10938357

  18. Fruiting Branch K+ Level Affects Cotton Fiber Elongation Through Osmoregulation

    PubMed Central

    Yang, Jiashuo; Hu, Wei; Zhao, Wenqing; Chen, Binglin; Wang, Youhua; Zhou, Zhiguo; Meng, Yali

    2016-01-01

    Potassium (K) deficiency in cotton plants results in reduced fiber length. As one of the primary osmotica, K+ contributes to an increase in cell turgor pressure during fiber elongation. Therefore, it is hypothesized that fiber length is affected by K deficiency through an osmotic pathway, so in 2012 and 2013, an experiment was conducted to test this hypothesis by imposing three potassium supply regimes (0, 125, 250 kg K ha-1) on a low-K-sensitive cultivar, Siza 3, and a low-K-tolerant cultivar, Simian 3. We found that fibers were longer in the later season bolls than in the earlier ones in cotton plants grown under normal growth conditions, but later season bolls showed a greater sensitivity to low-K stress, especially the low-K sensitive genotype. We also found that the maximum velocity of fibre elongation (Vmax) is the parameter that best reflects the change in fiber elongation under K deficiency. This parameter mostly depends on cell turgor, so the content of the osmotically active solutes was analyzed accordingly. Statistical analysis showed that K+ was the major osmotic factor affecting fiber length, and malate was likely facilitating K+ accumulation into fibers, which enabled the low-K-tolerant genotype to cope with low-K stress. Moreover, the low-K-tolerant genotype tended to have greater K+ absorptive capacities in the upper fruiting branches. Based on our findings, we suggest a fertilization scheme for Gossypium hirsutum that adds extra potash fertilizer or distributes it during the development of late season bolls to mitigate K deficiency in the second half of the growth season and to enhance fiber length in late season bolls. PMID:26834777

  19. Morphological and Chemical Mechanisms of Elongated Mineral Particle Toxicities

    PubMed Central

    Aust, Ann E.; Cook, Philip M.; Dodson, Ronald F.

    2011-01-01

    Much of our understanding regarding the mechanisms for induction of disease following inhalation of respirable elongated mineral particles (REMP) is based on studies involving the biological effects of asbestos fibers. The factors governing the disease potential of an exposure include duration and frequency of exposures; tissue-specific dose over time; impacts on dose persistence from in vivo REMP dissolution, comminution, and clearance; individual susceptibility; and the mineral type and surface characteristics. The mechanisms associated with asbestos particle toxicity involve two facets for each particle's contribution: (1) the physical features of the inhaled REMP, which include width, length, aspect ratio, and effective surface area available for cell contact; and (2) the surface chemical composition and reactivity of the individual fiber/elongated particle. Studies in cell-free systems and with cultured cells suggest an important way in which REMP from asbestos damage cellular molecules or influence cellular processes. This may involve an unfortunate combination of the ability of REMP to chemically generate potentially damaging reactive oxygen species, through surface iron, and the interaction of the unique surfaces with cell membranes to trigger membrane receptor activation. Together these events appear to lead to a cascade of cellular events, including the production of damaging reactive nitrogen species, which may contribute to the disease process. Thus, there is a need to be more cognizant of the potential impact that the total surface area of REMP contributes to the generation of events resulting in pathological changes in biological systems. The information presented has applicability to inhaled dusts, in general, and specifically to respirable elongated mineral particles. PMID:21534085

  20. A random cell motility gradient downstream of FGF controls elongation of an amniote embryo

    PubMed Central

    Bénazéraf, Bertrand; Francois, Paul; Baker, Ruth E.; Denans, Nicolas; Little, Charles D.; Pourquie, Olivier

    2011-01-01

    Vertebrate embryos are characterized by an elongated antero-posterior (AP) body axis, which forms by progressive cell deposition from a posterior growth zone in the embryo. Here, we used tissue ablation in the chicken embryo to demonstrate that the caudal presomitic mesoderm (PSM) plays a key role in axis elongation. Using time-lapse microscopy, we analysed the movements of fluorescently labelled cells in the PSM during embryo elongation which revealed a clear posterior-to-anterior gradient of cell motility and directionality in the PSM. We tracked the movement of the PSM extracellular matrix in parallel with the labelled cells and subtracted the extracellular matrix movement from the global motion of cells. After subtraction, cell motility remained graded but lacked directionality, indicating that the posterior cell movements associated with axis elongation in the PSM are not intrinsic but reflect tissue deformation. The gradient of cell motion along the PSM parallels the fibroblast growth factor (FGF)/mitogen-activated protein kinase (MAPK) gradient 1, which has been implicated in the control of cell motility in this tissue2. Both FGF signalling gain- and loss-of-function experiments lead to disruption of the motility gradient and a slowing down of axis elongation. Furthermore, embryos treated with cell movement inhibitors (Blebbistatin or RhoK inhibitor), but not cell cycle inhibitors, show a slower axis elongation rate. We propose that the gradient of random cell motility downstream of FGF signalling in the PSM controls posterior elongation in the amniote embryo. Our data suggest that tissue elongation is an emergent property that arises from the collective regulation of graded, random cell motion rather than by the regulation of directionality of individual cellular movements. PMID:20613841

  1. Elongated nanostructures for radial junction solar cells

    NASA Astrophysics Data System (ADS)

    Kuang, Yinghuan; Di Vece, Marcel; Rath, Jatindra K.; van Dijk, Lourens; Schropp, Ruud E. I.

    2013-10-01

    In solar cell technology, the current trend is to thin down the active absorber layer. The main advantage of a thinner absorber is primarily the reduced consumption of material and energy during production. For thin film silicon (Si) technology, thinning down the absorber layer is of particular interest since both the device throughput of vacuum deposition systems and the stability of the devices are significantly enhanced. These features lead to lower cost per installed watt peak for solar cells, provided that the (stabilized) efficiency is the same as for thicker devices. However, merely thinning down inevitably leads to a reduced light absorption. Therefore, advanced light trapping schemes are crucial to increase the light path length. The use of elongated nanostructures is a promising method for advanced light trapping. The enhanced optical performance originates from orthogonalization of the light's travel path with respect to the direction of carrier collection due to the radial junction, an improved anti-reflection effect thanks to the three-dimensional geometric configuration and the multiple scattering between individual nanostructures. These advantages potentially allow for high efficiency at a significantly reduced quantity and even at a reduced material quality, of the semiconductor material. In this article, several types of elongated nanostructures with the high potential to improve the device performance are reviewed. First, we briefly introduce the conventional solar cells with emphasis on thin film technology, following the most commonly used fabrication techniques for creating nanostructures with a high aspect ratio. Subsequently, several representative applications of elongated nanostructures, such as Si nanowires in realistic photovoltaic (PV) devices, are reviewed. Finally, the scientific challenges and an outlook for nanostructured PV devices are presented.

  2. Elongated nanostructures for radial junction solar cells.

    PubMed

    Kuang, Yinghuan; Vece, Marcel Di; Rath, Jatindra K; Dijk, Lourens van; Schropp, Ruud E I

    2013-10-01

    In solar cell technology, the current trend is to thin down the active absorber layer. The main advantage of a thinner absorber is primarily the reduced consumption of material and energy during production. For thin film silicon (Si) technology, thinning down the absorber layer is of particular interest since both the device throughput of vacuum deposition systems and the stability of the devices are significantly enhanced. These features lead to lower cost per installed watt peak for solar cells, provided that the (stabilized) efficiency is the same as for thicker devices. However, merely thinning down inevitably leads to a reduced light absorption. Therefore, advanced light trapping schemes are crucial to increase the light path length. The use of elongated nanostructures is a promising method for advanced light trapping. The enhanced optical performance originates from orthogonalization of the light's travel path with respect to the direction of carrier collection due to the radial junction, an improved anti-reflection effect thanks to the three-dimensional geometric configuration and the multiple scattering between individual nanostructures. These advantages potentially allow for high efficiency at a significantly reduced quantity and even at a reduced material quality, of the semiconductor material. In this article, several types of elongated nanostructures with the high potential to improve the device performance are reviewed. First, we briefly introduce the conventional solar cells with emphasis on thin film technology, following the most commonly used fabrication techniques for creating nanostructures with a high aspect ratio. Subsequently, several representative applications of elongated nanostructures, such as Si nanowires in realistic photovoltaic (PV) devices, are reviewed. Finally, the scientific challenges and an outlook for nanostructured PV devices are presented.

  3. Molecular mechanism of viomycin inhibition of peptide elongation in bacteria.

    PubMed

    Holm, Mikael; Borg, Anneli; Ehrenberg, Måns; Sanyal, Suparna

    2016-01-26

    Viomycin is a tuberactinomycin antibiotic essential for treating multidrug-resistant tuberculosis. It inhibits bacterial protein synthesis by blocking elongation factor G (EF-G) catalyzed translocation of messenger RNA on the ribosome. Here we have clarified the molecular aspects of viomycin inhibition of the elongating ribosome using pre-steady-state kinetics. We found that the probability of ribosome inhibition by viomycin depends on competition between viomycin and EF-G for binding to the pretranslocation ribosome, and that stable viomycin binding requires an A-site bound tRNA. Once bound, viomycin stalls the ribosome in a pretranslocation state for a minimum of ∼ 45 s. This stalling time increases linearly with viomycin concentration. Viomycin inhibition also promotes futile cycles of GTP hydrolysis by EF-G. Finally, we have constructed a kinetic model for viomycin inhibition of EF-G catalyzed translocation, allowing for testable predictions of tuberactinomycin action in vivo and facilitating in-depth understanding of resistance development against this important class of antibiotics.

  4. Molecular mechanism of viomycin inhibition of peptide elongation in bacteria

    PubMed Central

    Holm, Mikael; Borg, Anneli; Ehrenberg, Måns; Sanyal, Suparna

    2016-01-01

    Viomycin is a tuberactinomycin antibiotic essential for treating multidrug-resistant tuberculosis. It inhibits bacterial protein synthesis by blocking elongation factor G (EF-G) catalyzed translocation of messenger RNA on the ribosome. Here we have clarified the molecular aspects of viomycin inhibition of the elongating ribosome using pre-steady-state kinetics. We found that the probability of ribosome inhibition by viomycin depends on competition between viomycin and EF-G for binding to the pretranslocation ribosome, and that stable viomycin binding requires an A-site bound tRNA. Once bound, viomycin stalls the ribosome in a pretranslocation state for a minimum of ∼45 s. This stalling time increases linearly with viomycin concentration. Viomycin inhibition also promotes futile cycles of GTP hydrolysis by EF-G. Finally, we have constructed a kinetic model for viomycin inhibition of EF-G catalyzed translocation, allowing for testable predictions of tuberactinomycin action in vivo and facilitating in-depth understanding of resistance development against this important class of antibiotics. PMID:26755601

  5. Characterization of pollutant dispersion near elongated ...

    EPA Pesticide Factsheets

    This paper presents a wind tunnel study of the effects of elongated rectangular buildings on the dispersion of pollutants from nearby stacks. The study examines the influence of source location, building aspect ratio, and wind direction on pollutant dispersion with the goal of developing improved algorithms within dispersion models. The paper also examines the current AERMOD/PRIME modeling capabilities compared to wind tunnel observations. Differences in the amount of plume material entrained in the wake region downwind of a building for various source locations and source heights are illustrated with vertical and lateral concentration profiles. These profiles were parameterized using the Gaussian equation and show the influence of building/source configurations on those parameters. When the building is oriented at 45° to the approach flow, for example, the effective plume height descends more rapidly than it does for a perpendicular building, enhancing the resulting surface concentrations in the wake region. Buildings at angles to the wind cause a cross-wind shift in the location of the plume resulting from a lateral mean flow established in the building wake. These and other effects that are not well represented in many dispersion models are important considerations when developing improved algorithms to estimate the location and magnitude of concentrations downwind of elongated buildings. The National Exposure Research Laboratory (NERL) Computational Exposur

  6. Nonlinear deformations of microcapsules in elongation flow

    NASA Astrophysics Data System (ADS)

    Deschamps, Julien; de Loubens, Clément; Boedec, Gwenn; Georgelin, Marc; Leonetti, Marc; Soft Matter; Biophysics Group Team

    2014-11-01

    Soft microcapsules are drops bounded by a thin elastic shell made of cross-linked proteins. They have numerous applications for drug delivery in bioengineering, pharmaceutics and medicine, where their mechanical stability and their dynamics under flow are crucial. They can also be used as red blood cells models. Here, we investigate the mechanical behaviour of microcapsules made of albumine in strong elongational flow, up to a stretching of 180% just before breaking. The set-up allows us to visualize the deformed shape in the two perpendicular main fields of view, to manage high capillary number and to manipulate soft microcapsules. The steady-state shape of a capsule in the planar elongational flow is non-axisymmetric. In each cross section, the shape is an ellipse but with different small axis which vary in opposite sense with the stretching. Whatever the degree of cross-linking and the size of the capsules, the deformations followed the same master-curve. Comparisons between numerical predictions and experimental results permit to conclude unambiguously that the more properly strain-energy model of membrane is the generalized Hooke model.

  7. Altering the interaction between σ70 and RNA polymerase generates complexes with distinct transcription-elongation properties

    PubMed Central

    Berghöfer-Hochheimer, Yvonne; Lu, Chi Zen; Gross, Carol A.

    2005-01-01

    We compare the elongation behavior of native Escherichia coli RNA polymerase holoenzyme assembled in vivo, holoenzyme reconstituted from σ70 and RNA polymerase in vitro, and holoenzyme with a specific alteration in the interface between σ70 and RNA polymerase. Elongating RNA polymerase from each holoenzyme has distinguishable properties, some of which cannot be explained by differential retention or rebinding of σ70 during elongation, or by differential presence of elongation factors. We suggest that interactions between RNA polymerase and σ70 may influence the ensemble of conformational states adopted by RNA polymerase during initiation. These states, in turn, may affect the conformational states adopted by the elongating enzyme, thereby physically and functionally imprinting RNA polymerase. PMID:15650048

  8. Induction of predominant tenogenic phenotype in human dermal fibroblasts via synergistic effect of TGF-β and elongated cell shape.

    PubMed

    Wang, Wenbo; Li, Jie; Wang, Keyun; Zhang, Zhiyong; Zhang, Wenjie; Zhou, Guangdong; Cao, Yilin; Ye, Mingliang; Zou, Hanfa; Liu, Wei

    2016-03-01

    Micropattern topography is widely investigated for its role in mediating stem cell differentiation, but remains unexplored for phenotype switch between mature cell types. This study investigated the potential of inducing tenogenic phenotype in human dermal fibroblasts (hDFs) by artificial elongation of cultured cells. Our results showed that a parallel microgrooved topography could convert spread hDFs into an elongated shape and induce a predominant tenogenic phenotype as the expression of biomarkers was significantly enhanced, such as scleraxis, tenomodulin, collagens I, III, VI, and decorin. It also enhanced the expression of transforming growth factor (TGF)-β1, but not α-smooth muscle actin. Elongated hDFs failed to induce other phenotypes, such as adiopogenic, chondrogenic, neurogenic, and myogenic lineages. By contrast, no tenogenic phenotype could be induced in elongated human chondrocytes, although chondrogenic phenotype was inhibited. Exogenous TGF-β1 could enhance the tenogenic phenotype in elongated hDFs at low dose (2 ng/ml), but promoted myofibroblast transdifferentiation of hDFs at high dose (10 ng/ml), regardless of cell shape. Elongated shape also resulted in decreased RhoA activity and increased Rho-associated protein kinase (ROCK) activity. Antagonizing TGF-β or inhibiting ROCK activity with Y27632 or depolymerizing actin with cytochalasin D could all significantly inhibit tenogenic phenotype induction, particularly in elongated hDFs. In conclusion, elongation of cultured dermal fibroblasts can induce a predominant tenogenic phenotype likely via synergistic effect of TGF-β and cytoskeletal signaling.

  9. Control of transcription elongation by GreA determines rate of gene expression in Streptococcus pneumoniae

    PubMed Central

    Yuzenkova, Yulia; Gamba, Pamela; Herber, Martijn; Attaiech, Laetitia; Shafeeq, Sulman; Kuipers, Oscar P.; Klumpp, Stefan; Zenkin, Nikolay; Veening, Jan-Willem

    2014-01-01

    Transcription by RNA polymerase may be interrupted by pauses caused by backtracking or misincorporation that can be resolved by the conserved bacterial Gre-factors. However, the consequences of such pausing in the living cell remain obscure. Here, we developed molecular biology and transcriptome sequencing tools in the human pathogen Streptococcus pneumoniae and provide evidence that transcription elongation is rate-limiting on highly expressed genes. Our results suggest that transcription elongation may be a highly regulated step of gene expression in S. pneumoniae. Regulation is accomplished via long-living elongation pauses and their resolution by elongation factor GreA. Interestingly, mathematical modeling indicates that long-living pauses cause queuing of RNA polymerases, which results in ‘transcription traffic jams’ on the gene and thus blocks its expression. Together, our results suggest that long-living pauses and RNA polymerase queues caused by them are a major problem on highly expressed genes and are detrimental for cell viability. The major and possibly sole function of GreA in S. pneumoniae is to prevent formation of backtracked elongation complexes. PMID:25190458

  10. Potential flow about elongated bodies of revolution

    NASA Technical Reports Server (NTRS)

    Kaplan, Carl

    1936-01-01

    This report presents a method of solving the problem of axial and transverse potential flows around arbitrary elongated bodies of revolution. The solutions of Laplace's equation for the velocity potentials of the axial and transverse flows, the system of coordinates being an elliptic one in a meridian plane, are given. The theory is applied to a body of revolution obtained from a symmetrical Joukowsky profile, a shape resembling an airship hull. The pressure distribution and the transverse-force distribution are calculated and serve as examples of the procedure to be followed in the case of an actual airship. A section on the determination of inertia coefficients is also included in which the validity of some earlier work is questioned.

  11. The Astoria Fan: An elongate type fan

    USGS Publications Warehouse

    Nelson, C.H.

    1984-01-01

    The Astoria Fan, a modern system, is located on a subducting oceanic crust and fills a north-south-trending trench along the Oregon continental margin. Well-developed channels cross the entire fan length; they display classic inner-fan leveed profiles but evolve into distributaries in the midfan area where the gradient decreases sharply. During periods of low sea level, inner- and middle-fan channels funnel sand to distal depositional sites in the outer-fan area where the sand/shale ratios are highest. This pattern of sand displacement and efficiency of transport appears to be characteristic of elongate fans fed by a major river and submarine canyon. ?? 1984 Springer-Verlag New York Inc.

  12. The Caenorhabditis elegans Elongator Complex Regulates Neuronal α-tubulin Acetylation

    PubMed Central

    Solinger, Jachen A.; Scorza, Francesco Berlanda; Marchesi, Stefano; Sauder, Ursula; Mitsushima, Dai; Capuani, Fabrizio; Stürzenbaum, Stephen R.; Cassata, Giuseppe

    2010-01-01

    Although acetylated α-tubulin is known to be a marker of stable microtubules in neurons, precise factors that regulate α-tubulin acetylation are, to date, largely unknown. Therefore, a genetic screen was employed in the nematode Caenorhabditis elegans that identified the Elongator complex as a possible regulator of α-tubulin acetylation. Detailed characterization of mutant animals revealed that the acetyltransferase activity of the Elongator is indeed required for correct acetylation of microtubules and for neuronal development. Moreover, the velocity of vesicles on microtubules was affected by mutations in Elongator. Elongator mutants also displayed defects in neurotransmitter levels. Furthermore, acetylation of α-tubulin was shown to act as a novel signal for the fine-tuning of microtubules dynamics by modulating α-tubulin turnover, which in turn affected neuronal shape. Given that mutations in the acetyltransferase subunit of the Elongator (Elp3) and in a scaffold subunit (Elp1) have previously been linked to human neurodegenerative diseases, namely Amyotrophic Lateral Sclerosis and Familial Dysautonomia respectively highlights the importance of this work and offers new insights to understand their etiology. PMID:20107598

  13. The Caenorhabditis elegans Elongator complex regulates neuronal alpha-tubulin acetylation.

    PubMed

    Solinger, Jachen A; Paolinelli, Roberta; Klöss, Holger; Scorza, Francesco Berlanda; Marchesi, Stefano; Sauder, Ursula; Mitsushima, Dai; Capuani, Fabrizio; Stürzenbaum, Stephen R; Cassata, Giuseppe

    2010-01-22

    Although acetylated alpha-tubulin is known to be a marker of stable microtubules in neurons, precise factors that regulate alpha-tubulin acetylation are, to date, largely unknown. Therefore, a genetic screen was employed in the nematode Caenorhabditis elegans that identified the Elongator complex as a possible regulator of alpha-tubulin acetylation. Detailed characterization of mutant animals revealed that the acetyltransferase activity of the Elongator is indeed required for correct acetylation of microtubules and for neuronal development. Moreover, the velocity of vesicles on microtubules was affected by mutations in Elongator. Elongator mutants also displayed defects in neurotransmitter levels. Furthermore, acetylation of alpha-tubulin was shown to act as a novel signal for the fine-tuning of microtubules dynamics by modulating alpha-tubulin turnover, which in turn affected neuronal shape. Given that mutations in the acetyltransferase subunit of the Elongator (Elp3) and in a scaffold subunit (Elp1) have previously been linked to human neurodegenerative diseases, namely Amyotrophic Lateral Sclerosis and Familial Dysautonomia respectively highlights the importance of this work and offers new insights to understand their etiology.

  14. Cotranslational response to proteotoxic stress by elongation pausing of ribosomes.

    PubMed

    Liu, Botao; Han, Yan; Qian, Shu-Bing

    2013-02-07

    Translational control permits cells to respond swiftly to a changing environment. Rapid attenuation of global protein synthesis under stress conditions has been largely ascribed to the inhibition of translation initiation. Here we report that intracellular proteotoxic stress reduces global protein synthesis by halting ribosomes on transcripts during elongation. Deep sequencing of ribosome-protected messenger RNA (mRNA) fragments reveals an early elongation pausing, roughly at the site where nascent polypeptide chains emerge from the ribosomal exit tunnel. Inhibiting endogenous chaperone molecules by a dominant-negative mutant or chemical inhibitors recapitulates the early elongation pausing, suggesting a dual role of molecular chaperones in facilitating polypeptide elongation and cotranslational folding. Our results further support the chaperone "trapping" mechanism in promoting the passage of nascent chains. Our study reveals that translating ribosomes fine tune the elongation rate by sensing the intracellular folding environment. The early elongation pausing represents a cotranslational stress response to maintain the intracellular protein homeostasis.

  15. Cell elongation is an adaptive response for clearing long chromatid arms from the cleavage plane.

    PubMed

    Kotadia, Shaila; Montembault, Emilie; Sullivan, William; Royou, Anne

    2012-11-26

    Chromosome segregation must be coordinated with cell cleavage to ensure correct transmission of the genome to daughter cells. Here we identify a novel mechanism by which Drosophila melanogaster neuronal stem cells coordinate sister chromatid segregation with cleavage furrow ingression. Cells adapted to a dramatic increase in chromatid arm length by transiently elongating during anaphase/telophase. The degree of cell elongation correlated with the length of the trailing chromatid arms and was concomitant with a slight increase in spindle length and an enlargement of the zone of cortical myosin distribution. Rho guanine-nucleotide exchange factor (Pebble)-depleted cells failed to elongate during segregation of long chromatids. As a result, Pebble-depleted adult flies exhibited morphological defects likely caused by cell death during development. These studies reveal a novel pathway linking trailing chromatid arms and cortical myosin that ensures the clearance of chromatids from the cleavage plane at the appropriate time during cytokinesis, thus preserving genome integrity.

  16. Runaway telomere elongation caused by telomerase RNA gene mutations.

    PubMed

    McEachern, M J; Blackburn, E H

    1995-08-03

    The ribonucleoprotein enzyme telomerase adds telomeric DNA onto chromosome ends and is normally regulated so that telomeric DNA lengths are kept within defined bounds. In the telomerase RNA gene from the yeast Kluyveromyces lactis, specific mutations that alter telomeric DNA sequences result in telomeres elongating to up to 100 times their normal length and impair cell growth. Some mutations cause immediate elongation whereas others behave like genetic time bombs, causing elongation only after a latent period of hundreds of generations.

  17. A coordinated codon-dependent regulation of translation by Elongator

    PubMed Central

    Bauer, Fanelie; Hermand, Damien

    2012-01-01

    More than a decade ago, the purification of the form of the RNA polymerase II (PolII) engaged in elongation led to the discovery of an associated, multi-subunit (Elp1-6) complex named “Elongator” by the Svejstrup lab. Although further evidence supported the original notion that Elongator is involved in transcription, Elongator lacked some of the expected features for a regulator of the elongating PolII. The discovery by the Byström lab, based on genetic dissection, that Elongator is pivotal for tRNA modifications, and that all the reported phenotypes of Elongator mutants are suppressed by the overexpression of two tRNAs added to the confusion. The increasing range of both potential substrates and biological processes regulated by Elongator in higher eukaryotes indicates that the major challenge of the field is to determine the biologically relevant function of Elongator. Our recent proteome-wide study in fission yeast supports a coordinated codon-dependent regulation of translation by Elongator. Here we provide additional analyses extending this hypothesis to budding yeast and worm. PMID:23165209

  18. Conditions for bubble elongation in cold ice-sheet ice

    USGS Publications Warehouse

    Alley, R.B.; Fitzpatrick, J.J.

    1999-01-01

    Highly elongated bubbles are sometimes observed in ice-sheet ice. Elongation is favored by rapid ice deformation, and opposed by diffusive processes. We use simple models to show that vapor transport dominates diffusion except possibly very close to the melting point, and that latent-heat effects are insignificant. Elongation is favored by larger bubbles at pore close-off, but is nearly independent of bubble compression below close-off. The simple presence of highly elongated bubbles indicates only that a critical ice-strain rate has been exceeded for significant time, and provides no information on possible disruption of stratigraphic continuity by ice deformation.

  19. Venus - A Large Elongated Caldera 'Sacajawea Patera

    NASA Technical Reports Server (NTRS)

    1991-01-01

    This Magellan image reveals Sacajawea Patera, a large, elongate caldera located in Western Ishtar Terra on the smooth plateau of Lakshmi Planum. The image is centered at 64.5 degrees North latitude and 337 degrees East longitude. It is approximately 420 kilometers (252 miles) wide at the base. Sacajawea is a depression approximately 1-2 kilometers (0.6-1.2 miles) deep and 120 x 215 kilometers (74 x 133 miles) in diameter; it is elongate in a southwest-northeast direction. The depression is bounded by a zone of circumferential curvilinear structures interpreted to be graben and fault scarps. These structures are spaced 0.5-4 kilometers (0.3-2.5 miles) apart, are 0.6-4.0 kilometers (0.4-2.5 miles) in width and up to 100 kilometers (62 miles) in length. Extending up to approximately 140 kilometers (87 miles) in length from the southeast of the patera is a system of linear structures thought to represent a flanking rift zone along which the lateral injection and eruption of magma may have occurred. A shield edifice 12 kilometers (7 miles) in diameter with a prominent central pit lies along the trend of one of these features. The impact crater Zlata, approximately 6 kilometers (4 miles) in diameter is located within the zone of graben to the northwest of the patera. Few flow features are observed in association with Sacajawea, possibly due to age and state of degradation of the flows. Mottled bright deposits 4-20 kilometers (2.5-12 miles) in width are located near the periphery and in the center of the patera floor within local topographic lows. Diffuse patches of dark material approximately 40 kilometers (25 miles) in width are observed southwest of the patera, superposed on portions of the surrounding graben. The formation of Sacajawea is thought to be related to the drainage and collapse of a large magma chamber. Gravitational relaxation may have caused the resultant caldera to sag, producing the numerous faults and graben that circumscribe the patera. Regions of

  20. Glycoproteome of Elongating Cotton Fiber Cells*

    PubMed Central

    Kumar, Saravanan; Kumar, Krishan; Pandey, Pankaj; Rajamani, Vijayalakshmi; Padmalatha, Kethireddy Venkata; Dhandapani, Gurusamy; Kanakachari, Mogilicherla; Leelavathi, Sadhu; Kumar, Polumetla Ananda; Reddy, Vanga Siva

    2013-01-01

    Cotton ovule epidermal cell differentiation into long fibers primarily depends on wall-oriented processes such as loosening, elongation, remodeling, and maturation. Such processes are governed by cell wall bound structural proteins and interacting carbohydrate active enzymes. Glycosylation plays a major role in the structural, functional, and localization aspects of the cell wall and extracellular destined proteins. Elucidating the glycoproteome of fiber cells would reflect its wall composition as well as compartmental requirement, which must be system specific. Following complementary proteomic approaches, we have identified 334 unique proteins comprising structural and regulatory families. Glycopeptide-based enrichment followed by deglycosylation with PNGase F and A revealed 92 unique peptides containing 106 formerly N-linked glycosylated sites from 67 unique proteins. Our results showed that structural proteins like arabinogalactans and carbohydrate active enzymes were relatively more abundant and showed stage- and isoform-specific expression patterns in the differentiating fiber cell. Furthermore, our data also revealed the presence of heterogeneous and novel forms of structural and regulatory glycoproteins. Comparative analysis with other plant glycoproteomes highlighted the unique composition of the fiber glycoproteome. The present study provides the first insight into the identity, abundance, diversity, and composition of the glycoproteome within single celled cotton fibers. The elucidated composition also indirectly provides clues about unicellular compartmental requirements underlying single cell differentiation. PMID:24019148

  1. Emerging brain morphologies from axonal elongation

    PubMed Central

    Holland, Maria A.; Miller, Kyle E.; Kuhl, Ellen

    2015-01-01

    Understanding the characteristic morphology of our brain remains a challenging, yet important task in human evolution, developmental biology, and neurosciences. Mathematical modeling shapes our understanding of cortical folding and provides functional relations between cortical wavelength, thickness, and stiffness. Yet, current mathematical models are phenomenologically isotropic and typically predict non-physiological, periodic folding patterns. Here we establish a mechanistic model for cortical folding, in which macroscopic changes in white matter volume are a natural consequence of microscopic axonal growth. To calibrate our model, we consult axon elongation experiments in chick sensory neurons. We demonstrate that a single parameter, the axonal growth rate, explains a wide variety of in vitro conditions including immediate axonal thinning and gradual thickness restoration. We embed our axonal growth model into a continuum model for brain development using axonal orientation distributions motivated by diffusion spectrum imaging. Our simulations suggest that white matter anisotropy - as an emergent property from directional axonal growth - intrinsically induces symmetry breaking, and predicts more physiological, less regular morphologies with regionally varying gyral wavelengths and sulcal depths. Mechanistic modeling of brain development could establish valuable relationships between brain connectivity, brain anatomy, and brain function. PMID:25824370

  2. Genome-Wide and Experimental Resolution of Relative Translation Elongation Speed at Individual Gene Level in Human Cells

    PubMed Central

    Gu, Wei; Cui, Yizhi; Zhong, Jiayong; Jin, Jingjie; He, Qing-Yu; Wang, Tong; Zhang, Gong

    2016-01-01

    In the process of translation, ribosomes first assemble on mRNAs (translation initiation) and then translate along the mRNA (elongation) to synthesize proteins. Elongation pausing is deemed highly relevant to co-translational folding of nascent peptides and the functionality of protein products, which positioned the evaluation of elongation speed as one of the central questions in the field of translational control. By integrating three types of RNA-seq methods, we experimentally and computationally resolved elongation speed, with our proposed elongation velocity index (EVI), a relative measure at individual gene level and under physiological condition in human cells. We successfully distinguished slow-translating genes from the background translatome. We demonstrated that low-EVI genes encoded more stable proteins. We further identified cell-specific slow-translating codons, which might serve as a causal factor of elongation deceleration. As an example for the biological relevance, we showed that the relatively slow-translating genes tended to be associated with the maintenance of malignant phenotypes per pathway analyses. In conclusion, EVI opens a new view to understand why human cells tend to avoid simultaneously speeding up translation initiation and decelerating elongation, and the possible cancer relevance of translating low-EVI genes to gain better protein quality. PMID:26926465

  3. Yeast and Human RNA Polymerase II Elongation Complexes: Evidence for Functional Differences and Postinitiation Recruitment of Factors†

    PubMed Central

    Pardee, Timothy S.; Ghazy, Mohamed A.; Ponticelli, Alfred S.

    2003-01-01

    Immobilized DNA templates, glycerol gradient centrifugation, and native gel analysis were utilized to isolate and compare functional RNA polymerase II (RNAPII) elongation complexes from Saccharomyces cerevisiae and human cell nuclear extracts. Yeast elongation complexes blocked by incorporation of 3′-O-methyl-GTP into the nascent transcript exhibited a sedimentation coefficient of 35S, were less tightly associated to the template than their human counterparts, and displayed no detectable 3′-5′ exonuclease activity on the associated transcript. In contrast, blocked human elongation complexes were more tightly bound to the template, and multiple forms were identified, with the largest exhibiting a sedimentation coefficient of 60S. Analysis of the associated transcripts revealed that a subset of the human elongation complexes exhibited strong 3′-5′ exonuclease activity. Although isolated human preinitiation complexes were competent for efficient transcription, their ability to generate 60S elongation complexes was strikingly impaired. These findings demonstrate functional and size differences between S. cerevisiae and human RNAPII elongation complexes and support the view that the formation of mature elongation complexes involves recruitment of nuclear factors after the initiation of transcription. PMID:12684381

  4. Positive grid corrosion elongation analysis using CAE with corrosion deformation transformed into thermal phenomenon

    NASA Astrophysics Data System (ADS)

    Mukaitani, Ichiroh; Hayashi, Koji; Shimoura, Ichiro; Takemasa, Arihiko; Takahashi, Isamu; Tsubakino, Harushige

    Valve-regulated lead-acid (VRLA) batteries have been commercially available for more than 20 years and have been enthusiastically embraced by users of uninterruptible power supplies (UPS) because of the anticipated reduction in installation and operating costs, smaller footprint and fewer environmental concerns. In Japan, communication networks are demanding reduced costs and longer life from their batteries. Among the factors limiting the life of VRLA batteries, the corrosion of positive grid material has been proven to cause elongation of the plates, loss of electrical contact and shorter lifetime. The content of Sn is also a key factor and addition of Sn in the grid alloy results in better performance in creep resistance, tensile strength and corrosion resistance [R. David Prenagaman, The Battery Man, vol. 39, September 1997, p. 16. I. Mukaitani, T. Sakamoto, T. Kikuoka, Y. Yamaguchi, H. Tsubakino, Proceedings of the 40th Battery Symposium in Japan, 1999, p. 99]. A key point is what the ratio of Sn to Ca should be, since too much Sn may lead to even worse elongation of the plates [I. Mukaitani, T. Sakamoto, T. Kikuoka, Y. Yamaguchi, H. Tsubakino, Proceedings of the 40th Battery Symposium in Japan, 1999, p. 99]. We have determined that microstructure control with a composition of lead-calcium-tin (Pb-Ca-Sn) alloy is optimal for better performance of the plates [I. Mukaitani, T. Sakamoto, T. Kikuoka, Y. Yamaguchi, H. Tsubakino, Proceedings of the 40th Battery Symposium in Japan, 1999, p. 99]. We developed a "simulation of current collector corrosion elongation" which is a technique of estimating corrosion elongation from the current collector design [I. Mukaitani, K. Hayashi, I. Shimoura, H. Takabayashi, M. Terada, A. Takemasa, I. Takahashi, K. Okamoto, Proceedings of the 44th Battery Symposium in Japan, 2003, p. 652]. Corrosion elongation occurs as the corrosion material layer grows out of the current collector metal. We resolved this problem using generally CAD

  5. Ballooning mode stability of elongated high-beta tokamaks

    NASA Astrophysics Data System (ADS)

    Mauel, Michael E.

    1987-12-01

    The variational principle derived by Choe and Freidberg [Phys. Fluids 29, 1766 (1986)] and used to estimate the geometry of high-beta tokamak equilibria is extended to include elongation. Ballooning mode stability is then investigated, illustrating the influence of elongation on local and global stability.

  6. An elongation method for large systems toward bio-systems.

    PubMed

    Aoki, Yuriko; Gu, Feng Long

    2012-06-07

    The elongation method, proposed in the early 1990s, originally for theoretical synthesis of aperiodic polymers, has been reviewed. The details of derivation of the localization scheme adopted by the elongation method are described along with the elongation processes. The reliability and efficiency of the elongation method have been proven by applying it to various models of bio-systems, such as gramicidin A, collagen, DNA, etc. By means of orbital shift, the elongation method has been successfully applied to delocalized π-conjugated systems. The so-called orbital shift works in such a way that during the elongation process, some strongly delocalized frozen orbitals are assigned as active orbitals and joined with the interaction of the attacking monomer. By this treatment, it has been demonstrated that the total energies and non-linear optical properties determined by the elongation method are more accurate even for bio-systems and delocalized systems like fused porphyrin wires. The elongation method has been further developed for treating any three-dimensional (3D) systems and its applicability is confirmed by applying it to entangled insulin models whose terminal is capped by both neutral and zwitterionic sequences.

  7. Blastocyst Elongation, Trophoblastic Differentiation and Embryonic Pattern Formation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The molecular basis behind elongation and concomitant gastrulation in ungulates that occurs during pre-implantation is still poorly understood. In-depth transcriptome analysis of the elongating porcine conceptus at specific stages has demonstrated that protein synthesis, protein trafficking, cell g...

  8. Halogenated auxins affect microtubules and root elongation in Lactuca sativa

    NASA Technical Reports Server (NTRS)

    Zhang, N.; Hasenstein, K. H.

    2000-01-01

    We studied the effect of 4,4,4-trifluoro-3-(indole-3-)butyric acid (TFIBA), a recently described root growth stimulator, and 5,6-dichloro-indole-3-acetic acid (DCIAA) on growth and microtubule (MT) organization in roots of Lactuca sativa L. DCIAA and indole-3-butyric acid (IBA) inhibited root elongation and depolymerized MTs in the cortex of the elongation zone, inhibited the elongation of stele cells, and promoted xylem maturation. Both auxins caused the plane of cell division to shift from anticlinal to periclinal. In contrast, TFIBA (100 micromolar) promoted elongation of primary roots by 40% and stimulated the elongation of lateral roots, even in the presence of IBA, the microtubular inhibitors oryzalin and taxol, or the auxin transport inhibitor naphthylphthalamic acid. However, TFIBA inhibited the formation of lateral root primordia. Immunostaining showed that TFIBA stabilized MTs orientation perpendicular to the root axis, doubled the cortical cell length, but delayed xylem maturation. The data indicate that the auxin-induced inhibition of elongation and swelling of roots results from reoriented phragmoplasts, the destabilization of MTs in elongating cells, and promotion of vessel formation. In contrast, TFIBA induced promotion of root elongation by enhancing cell length, prolonging transverse MT orientation, delaying cell and xylem maturation.

  9. Isolated Horner Syndrome From an Elongated Styloid Process (Eagle Syndrome).

    PubMed

    Chang, Caitlin A; Lin, Tony; Fung, Kevin; Sharma, Manas; Fraser, J Alexander

    2015-12-01

    Eagle syndrome occurs when an elongated styloid process causes otolaryngological or neurological symptoms or signs. We report a patient who had an isolated asymptomatic Horner syndrome that resulted from a pinned internal carotid artery being dynamically injured by an elongated styloid process during chiropractic neck manipulation. There was no evidence of arterial dissection.

  10. Induction of Coleoptile Elongation by Carbon Dioxide 1

    PubMed Central

    Evans, Michael L.; Ray, Peter M.; Reinhold, Leonora

    1971-01-01

    The ability of CO2 to induce elongation of Avena sativa coleoptile segments was examined with the use of a high resolution growth-recording device. CO2-saturated water causes an 8- to 16-fold promotion in the rate of elongation within 1 minute. This elongation is insensitive to a variety of metabolic inhibitors that suppress auxin-induced elongation, and the CO2 effect cannot be prevented by pretreatment with these inhibitors. Buffers of pH 3 to 4 also stimulate elongation quickly, and it seems that at least a major part of the action of CO2 depends upon its ability to reduce pH. The rate of elongation of auxin-promoted segments can be further enhanced by treatment with CO2 but not vice versa. The response to CO2 can be inhibited by mannitol at osmotic concentrations that inhibit normal growth, by calcium, and by brief pretreatment with heavy water (D2O). The elongation rate that results from CO2 treatment is sensitive to temperature, but the induction by CO2 itself appears to be almost temperature-independent. Elongation following treatment with CO2 may be a physical flow phenomenon, essentially independent of immediate biochemical participation, which occurs when wall polymer interactions that normally restrict strain in the cell wall are weakened or broken by CO2 in a manner that in effect substitutes for the role of metabolism in normal auxin-inducible cell enlargement. PMID:16657618

  11. The Effects of Microgravity on Seated Height (Spinal Elongation)

    NASA Technical Reports Server (NTRS)

    Young, K. S.; Rajulu, S.

    2011-01-01

    ABSTRACT Many physiological factors, such as spinal elongation, fluid shifts, bone atrophy, and muscle loss, occur during an exposure to a microgravity environment. Spinal elongation is just one of the factors that can also affect the safety and performance of a crewmember while in space. Spinal elongation occurs due to the lack of gravity/compression on the spinal column. This allows for the straightening of the natural spinal curve. There is a possible fluid shift in the inter-vertebral disks that may also result in changes in height. This study aims at collecting the overall change in seated height for crewmembers exposed to a microgravity environment. During previous Programs, Apollo-Soyuz Test Project (ASTP) and Skylab, spinal elongation data was collected from a small number of subjects in a standing posture but were limited in scope. Data from these studies indicated a quick increase in stature during the first few days of weightlessness, after which stature growth reached a plateau resulting in up to a 3% increase of the original measurement [1-5]. However, this data was collected only for crewmembers in standing posture and not in a seated posture. Seated height may have a different effect than standing height due to a change in posture as well as due to a compounded effect of wearing restraints and a potential compression of the gluteal area. Seated height was deemed as a critical measurement in the design of the Constellation Program s (CxP) Crew Exploration Vehicle (CEV), called Orion which is now the point-of-departure vehicle for the Multi-Purpose Crew Vehicle (MPCV) Program; therefore a better understanding of the effects of microgravity on seated height is necessary. Potential changes in seated height that may not have impacted crew accommodation in previous Programs will have significant effects on crew accommodation due to the layout of seats in the Orion.. The current and existing configuration is such that the four crewmembers are stacked two by

  12. 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.

  13. Red Light-inhibited Mesocotyl Elongation in Maize Seedlings

    PubMed Central

    Vanderhoef, Larry N.; Briggs, Winslow R.

    1978-01-01

    Red light-inhibited mesocotyl elongation, which occurs in intact Zea mays L. seedlings, was studied in excised segments which included the coleoptile (or parts therefrom) and apical centimeter of the mesocotyl. Experiments took into account, first, the ability of the segments to regenerate auxin supply sites, and, second, that auxin uptake can be greatly reduced if there is no cut surface, apical to the elongating cells, to act as a port of entry. In all cases, auxin completely reversed the inhibition of elongation by light. The results support the hypothesis that light regulates mesocotyl elongation by controlling auxin supply from the coleoptile. Sucrose concentration had no effect on auxin reversal of light-inhibited elongation, but relatively high concentrations of gibberellic acid (10 μm) could substitute for auxin in this system. PMID:16660331

  14. High n ballooning modes in highly elongated tokamaks

    SciTech Connect

    An, C.H.; Bateman, G.

    1980-02-01

    An analytic study of stability against high n ballooning modes in highly elongated axisymmetric plasmas is presented and compared with computational results. From the equation for the marginal pressure gradient, it is found that the local shear plays an important role on the stability of elongated and shifted plasma, and that high elongation deteriorates the stability by decreasing the stabilizing effects of field line bending and local shear. The net contribution of the local shear to stability decreases with elongation and shift for strongly ballooning modes (eigenfunctions strongly localized near the outer edge of the toroidal flux surfaces) but increases for interchange modes (eigenfunctions more uniform along the flux surfaces). The computational study of high n ballooning modes in a highly elongated plasma reveals that lowering the aspect ratio and broadening the pressure profile enhance the marginal beta for ..beta../sub p/ less than unity but severely reduce the marginal beta for ..beta../sub p/ larger than unity.

  15. Sequence-dependent elongation dynamics on macrolide-bound ribosomes.

    PubMed

    Johansson, Magnus; Chen, Jin; Tsai, Albert; Kornberg, Guy; Puglisi, Joseph D

    2014-06-12

    The traditional view of macrolide antibiotics as plugs inside the ribosomal nascent peptide exit tunnel (NPET) has lately been challenged in favor of a more complex, heterogeneous mechanism, where drug-peptide interactions determine the fate of a translating ribosome. To investigate these highly dynamic processes, we applied single-molecule tracking of elongating ribosomes during inhibition of elongation by erythromycin of several nascent chains, including ErmCL and H-NS, which were shown to be, respectively, sensitive and resistant to erythromycin. Peptide sequence-specific changes were observed in translation elongation dynamics in the presence of a macrolide-obstructed NPET. Elongation rates were not severely inhibited in general by the presence of the drug; instead, stalls or pauses were observed as abrupt events. The dynamic pathways of nascent-chain-dependent elongation pausing in the presence of macrolides determine the fate of the translating ribosome stalling or readthrough.

  16. [New treatment for peripheral nerve defects: nerve elongation].

    PubMed

    Kou, Y H; Jiang, B G

    2016-10-18

    Peripheral nerve defects are still a major challenge in clinical practice, and the most commonly used method of treatment for peripheral nerve defects is nerve transplantation, which has certain limitations and shortcomings, so new repair methods and techniques are needed. The peripheral nerve is elongated in limb lengthening surgery without injury, from which we got inspirations and proposed a new method to repair peripheral nerve defects: peripheral nerve elongation. The peripheral nerve could beelongated by a certain percent, but the physiological change and the maximum elongation range were still unknown. This study discussed the endurance, the physiological and pathological change of peripheral nerve elongation in detail, and got a lot of useful data. First, we developed peripheral nerve extender which could match the slow and even extension of peripheral nerve. Then, our animal experiment result confirmed that the peripheral nerve had better endurance for chronic elongation than that of acute elongation and cleared the extensibility of peripheral nerve and the range of repair for peripheral nerve defects. Our result also revealed the histological basis and changed the rule for pathological physiology of peripheral nerve elongation: the most important structure foundation of peripheral nerve elongation was Fontana band, which was the coiling of nerve fibers under the epineurium, so peripheral nerve could be stretched for 8.5%-10.0% without injury because of the Fontana band. We confirmed that peripheral nerve extending technology could have the same repair effect as traditional nerve transplantation through animal experiments. Finally, we compared the clinical outcomes between nerve elongation and performance of the conventional method in the repair of short-distance transection injuries in human elbows, and the post-operative follow-up results demonstrated that early neurological function recovery was better in the nerve elongation group than in the

  17. Selenotrisulfide inhibits initiation by RNA polymerase II but not elongation

    SciTech Connect

    Frenkel, G.D.; Falvey, D.

    1989-03-01

    We previously reported that RNA polymerase II (purified from wheat germ) is inhibited by selenotrisulfides, the products of the reaction of selenite with sulfhydryl compounds. We have now found that the initiation stage of the reaction is inhibited by selenotrisulfide but the elongation stage of the reaction is not. The actual start of the RNA chain is not inhibited by the selenotrisulfide, but rather the formation of the enzyme-DNA binary complex. Selenotrisulfide has a similar differential effect on initiation and elongation by RNA polymerase II from HeLa cells; in contrast, with E. coli RNA polymerase, it inhibits elongation as well.

  18. Phytochrome Control of Maize Coleoptile Section Elongation 1

    PubMed Central

    Warner, Timothy J.; Ross, James D.; Coombs, James

    1981-01-01

    A rapid loss of far red light (FR) reversibility of red-light (R) stimulated elongation of maize coleoptile sections was observed. Reversal was not possible when the interval between R and FR treatment was greater than 45 seconds. Most of the R-stimulated elongation occurred during the first 15 hours after irradiation. Exogenous gibberellic acid did not alter the time course of R/FR reversibility loss. These results are interpreted as indicating independence of R- and gibberellic acid-stimulated elongation in this system. PMID:16661674

  19. A pollen-specific RALF from tomato that regulates pollen tube elongation.

    PubMed

    Covey, Paul A; Subbaiah, Chalivendra C; Parsons, Ronald L; Pearce, Gregory; Lay, Fung T; Anderson, Marilyn A; Ryan, Clarence A; Bedinger, Patricia A

    2010-06-01

    Rapid Alkalinization Factors (RALFs) are plant peptides that rapidly increase the pH of plant suspension cell culture medium and inhibit root growth. A pollen-specific tomato (Solanum lycopersicum) RALF (SlPRALF) has been identified. The SlPRALF gene encodes a preproprotein that appears to be processed and released from the pollen tube as an active peptide. A synthetic SlPRALF peptide based on the putative active peptide did not affect pollen hydration or viability but inhibited the elongation of normal pollen tubes in an in vitro growth system. Inhibitory effects of SlPRALF were detectable at concentrations as low as 10 nm, and complete inhibition was observed at 1 mum peptide. At least 10-fold higher levels of alkSlPRALF, which lacks disulfide bonds, were required to see similar effects. A greater effect of peptide was observed in low-pH-buffered medium. Inhibition of pollen tube elongation was reversible if peptide was removed within 15 min of exposure. Addition of 100 nm SlPRALF to actively growing pollen tubes inhibited further elongation until tubes were 40 to 60 mum in length, after which pollen tubes became resistant to the peptide. The onset of resistance correlated with the timing of the exit of the male germ unit from the pollen grain into the tube. Thus, exogenous SlPRALF acts as a negative regulator of pollen tube elongation within a specific developmental window.

  20. Phosphatidylinositol phosphate 5-kinase genes respond to phosphate deficiency for root hair elongation in Arabidopsis thaliana.

    PubMed

    Wada, Yukika; Kusano, Hiroaki; Tsuge, Tomohiko; Aoyama, Takashi

    2015-02-01

    Plants drastically alter their root system architecture to adapt to different underground growth conditions. During phosphate (Pi) deficiency, most plants including Arabidopsis thaliana enhance the development of lateral roots and root hairs, resulting in bushy and hairy roots. To elucidate the signal pathway specific for the root hair elongation response to Pi deficiency, we investigated the expression of type-B phosphatidylinositol phosphate 5-kinase (PIP5K) genes, as a quantitative factor for root hair elongation in Arabidopsis. At young seedling stages, the PIP5K3 and PIP5K4 genes responded to Pi deficiency in steady-state transcript levels via PHR1-binding sequences (P1BSs) in their upstream regions. Both pip5k3 and pip5k4 single mutants, which exhibit short-root-hair phenotypes, remained responsive to Pi deficiency for root hair elongation; however the pip5k3pip5k4 double mutant exhibited shorter root hairs than the single mutants, and lost responsiveness to Pi deficiency at young seedling stages. In the tactical complementation line in which modified PIP5K3 and PIP5K4 genes with base substitutions in their P1BSs were co-introduced into the double mutant, root hairs of young seedlings had normal lengths under Pi-sufficient conditions, but were not responsive to Pi deficiency. From these results, we conclude that a Pi-deficiency signal is transferred to the pathway for root hair elongation via the PIP5K genes.

  1. Mechanical modelling quantifies the functional importance of outer tissue layers during root elongation and bending

    PubMed Central

    Dyson, Rosemary J; Vizcay-Barrena, Gema; Band, Leah R; Fernandes, Anwesha N; French, Andrew P; Fozard, John A; Hodgman, T Charlie; Kenobi, Kim; Pridmore, Tony P; Stout, Michael; Wells, Darren M; Wilson, Michael H; Bennett, Malcolm J; Jensen, Oliver E

    2014-01-01

    Root elongation and bending require the coordinated expansion of multiple cells of different types. These processes are regulated by the action of hormones that can target distinct cell layers. We use a mathematical model to characterise the influence of the biomechanical properties of individual cell walls on the properties of the whole tissue. Taking a simple constitutive model at the cell scale which characterises cell walls via yield and extensibility parameters, we derive the analogous tissue-level model to describe elongation and bending. To accurately parameterise the model, we take detailed measurements of cell turgor, cell geometries and wall thicknesses. The model demonstrates how cell properties and shapes contribute to tissue-level extensibility and yield. Exploiting the highly organised structure of the elongation zone (EZ) of the Arabidopsis root, we quantify the contributions of different cell layers, using the measured parameters. We show how distributions of material and geometric properties across the root cross-section contribute to the generation of curvature, and relate the angle of a gravitropic bend to the magnitude and duration of asymmetric wall softening. We quantify the geometric factors which lead to the predominant contribution of the outer cell files in driving root elongation and bending. PMID:24641449

  2. The Ssu72 Phosphatase Mediates the RNA Polymerase II Initiation-Elongation Transition*

    PubMed Central

    Rosado-Lugo, Jesús D.; Hampsey, Michael

    2014-01-01

    Transitions between the different stages of the RNAPII transcription cycle involve the recruitment and exchange of factors, including mRNA capping enzymes, elongation factors, splicing factors, 3′-end-processing complexes, and termination factors. These transitions are coordinated by the dynamic phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNAPII (Rpb1). The CTD is composed of reiterated heptapeptide repeats (Y1S2P3T4S5P6S7) that undergo phosphorylation and dephosphorylation as RNAPII transitions through the transcription cycle. An essential phosphatase in this process is Ssu72, which exhibits catalytic specificity for Ser(P)5 and Ser(P)7. Ssu72 is unique in that it is specific for Ser(P)5 in one orientation of the CTD and for Ser(P)7 when bound in the opposite orientation. Moreover, Ssu72 interacts with components of the initiation machinery and affects start site selection yet is an integral component of the CPF 3′-end-processing complex. Here we provide a comprehensive view of the effects of Ssu72 with respect to its Ser(P)5 phosphatase activity. We demonstrate that Ssu72 dephosphorylates Ser(P)5 at the initiation-elongation transition. Furthermore, Ssu72 indirectly affects the levels of Ser(P)2 during the elongation stage of transcription but does so independent of its catalytic activity. PMID:25339178

  3. Arabidopsis thaliana root elongation growth is sensitive to lunisolar tidal acceleration and may also be weakly correlated with geomagnetic variations

    PubMed Central

    Barlow, Peter W.; Fisahn, Joachim; Yazdanbakhsh, Nima; Moraes, Thiago A.; Khabarova, Olga V.; Gallep, Cristiano M.

    2013-01-01

    Background Correlative evidence suggests a relationship between the lunisolar tidal acceleration and the elongation rate of arabidopsis roots grown under free-running conditions of constant low light. Methods Seedlings of Arabidopsis thaliana were grown in a controlled-climate chamber maintained at a constant temperature and subjected to continuous low-level illumination from fluorescent tubes, conditions that approximate to a ‘free-running’ state in which most of the abiotic factors that entrain root growth rates are excluded. Elongation of evenly spaced, vertical primary roots was recorded continuously over periods of up to 14 d using high temporal- and spatial-resolution video imaging and were analysed in conjunction with geophysical variables. Key Results and Conclusions The results confirm the lunisolar tidal/root elongation relationship. Also presented are relationships between the hourly elongation rates and the contemporaneous variations in geomagnetic activity, as evaluated from the disturbance storm time and ap indices. On the basis of time series of root elongation rates that extend over ≥4 d and recorded at different seasons of the year, a provisional conclusion is that root elongation responds to variation in the lunisolar force and also appears to adjust in accordance with variations in the geomagnetic field. Thus, both lunisolar tidal acceleration and the geomagnetic field should be considered as modulators of root growth rate, alongside other, stronger and more well-known abiotic environmental regulators, and perhaps unexplored factors such as air ions. Major changes in atmospheric pressure are not considered to be a factor contributing to oscillations of root elongation rate. PMID:23532042

  4. 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

  5. A role for Chk1 in blocking transcriptional elongation of p21 RNA during the S-phase checkpoint

    PubMed Central

    Beckerman, Rachel; Donner, Aaron J.; Mattia, Melissa; Peart, Melissa J.; Manley, James L.; Espinosa, Joaquin M.; Prives, Carol

    2009-01-01

    We reported previously that when cells are arrested in S phase, a subset of p53 target genes fails to be strongly induced despite the presence of high levels of p53. When DNA replication is inhibited, reduced p21 mRNA accumulation is correlated with a marked reduction in transcription elongation. Here we show that ablation of the protein kinase Chk1 rescues the p21 transcription elongation defect when cells are blocked in S phase, as measured by increases in both p21 mRNA levels and the presence of the elongating form of RNA polymerase II (RNAPII) toward the 3′ end of the p21 gene. Recruitment of specific elongation and 3′ processing factors (DSIF, CstF-64, and CPSF-100) is also restored. While additional components of the RNAPII transcriptional machinery, such as TFIIB and CDK7, are recruited more extensively to the p21 locus after DNA damage than after replication stress, their recruitment is not enhanced by ablation of Chk1. Significantly, ablating Chk2, a kinase closely related in substrate specificity to Chk1, does not rescue p21 mRNA levels during S-phase arrest. Thus, Chk1 has a direct and selective role in the elongation block to p21 observed during S-phase arrest. These findings demonstrate for the first time a link between the replication checkpoint mediated by ATR/Chk1 and the transcription elongation/3′ processing machinery. PMID:19487575

  6. Analysis of changes in relative elemental growth rate patterns in the elongation zone of Arabidopsis roots upon gravistimulation

    NASA Technical Reports Server (NTRS)

    Mullen, J. L.; Ishikawa, H.; Evans, M. L.

    1998-01-01

    Although Arabidopsis is an important system for studying root physiology, the localized growth patterns of its roots have not been well defined, particularly during tropic responses. In order to characterize growth rate profiles along the apex of primary roots of Arabidopsis thaliana (L.) Heynh (ecotype Columbia) we applied small charcoal particles to the root surface and analyzed their displacement during growth using an automated video digitizer system with custom software for tracking the markers. When growing vertically, the maximum elongation rate occurred 481 +/- 50 microns back from the extreme tip of the root (tip of root cap), and the elongation zone extended back to 912 +/- 137 microns. The distal elongation zone (DEZ) has previously been described as the apical region of the elongation zone in which the relative elemental growth rate (REGR) is < or = 30% of the peak rate in the central elongation zone. By this definition, our data indicate that the basal limit of the DEZ was located 248 +/- 30 microns from the root tip. However, after gravistimulation, the growth patterns of the root changed. Within the first hour of graviresponse, the basal limit of the DEZ and the position of peak REGR shifted apically on the upper flank of the root. This was due to a combination of increased growth in the DEZ and growth inhibition in the central elongation zone. On the lower flank, the basal limit of the DEZ shifted basipetally as the REGR decreased. These factors set up the gradient of growth rate across the root, which drives curvature.

  7. Amplitude-dependent contraction/elongation of nonlinear Lamb waves

    NASA Astrophysics Data System (ADS)

    Packo, Pawel; Staszewski, Wieslaw J.; Uhl, Tadeusz; Leamy, Michael J.

    2016-04-01

    Nonlinear elastic guided waves find application in various disciplines of science and engineering, such as non- destructive testing and structural health monitoring. Recent recognition and quantification of their amplitude- dependent changes in spectral properties has contributed to the development of new monitoring concepts for mechanical structures. The focus of this work is to investigate and predict amplitude-dependent shifts in Lamb wave dispersion curves. The theory for frequency/wavenumber shifts for plate waves, based on a Lindstedt-Poincaré perturbation approach, was presented by the authors in previous years. Equivalently, spectral properties changes can be seen as wavelength contraction/elongation. Within the proposed framework, the wavelength of a Lamb wave depends on several factors; e.g., wave amplitude and second-, third- and fourth-order elastic constants, and others. Various types of nonlinear effects are considered in presented studies. Sensitivity studies for model parameters, i.e. higher-order elastic constants, are performed to quantify their influence on Lamb wave frequency/wavenumber shifting, and to identify the key parameters governing wavelength tuning.

  8. The Emerging Role of Forces in Axonal Elongation

    PubMed Central

    Suter, Daniel M.; Miller, Kyle E.

    2011-01-01

    An understanding of how axons elongate is needed to develop rational strategies to treat neurological diseases and nerve injury. Growth cone-mediated neuronal elongation is currently viewed as occurring through cytoskeletal dynamics involving the polymerization of actin and tubulin subunits at the tip of the axon. However, recent work suggests that axons and growth cones also generate forces (through cytoskeletal dynamics, kinesin, dynein, and myosin), forces induce axonal elongation, and axons lengthen by stretching. This review highlights results from various model systems (Drosophila, Aplysia, Xenopus, chicken, mouse, rat, and PC12 cells), supporting a role for forces, bulk microtubule movements, and intercalated mass addition in the process of axonal elongation. We think that a satisfying answer to the question, “How do axons grow?” will come by integrating the best aspects of biophysics, genetics, and cell biology. PMID:21527310

  9. Arabidopsis NF-YCs Mediate the Light-Controlled Hypocotyl Elongation via Modulating Histone Acetylation.

    PubMed

    Tang, Yang; Liu, Xuncheng; Liu, Xu; Li, Yuge; Wu, Keqiang; Hou, Xingliang

    2017-02-13

    Light is a crucial environmental signal that promotes photomorphogenesis, the developmental process with a series of light-dependent alterations for plants to adapt various external challenges. Chromatin modification has been proposed to be involved in such light-mediated growth, but the underlying mechanism is still elusive. In this study, we identified four Arabidopsis thaliana Nuclear Factor-YC homologs, NF-YC1, NF-YC3, NF-YC4, and NF-YC9 (NF-YCs), which function redundantly as repressors of light-controlled hypocotyl elongation via histone deacetylation. Obvious etiolation phenotypes are observed in NF-YCs loss-of-function mutant seedlings grown under light conditions, including significant elongated hypocotyls and fewer opened cotyledons. We found that NF-YCs interact with histone deacetylase HDA15 in the light, co-target the promoters of a set of hypocotyl elongation-related genes, and modulate the levels of histone H4 acetylation on the associated chromatins, thus repressing gene expression. In contrast, NF-YC-HDA15 complex is dismissed from the target genes in the dark, resulting in increased level of H4 acetylation and consequent etiolated growth. Further analyses revealed that transcriptional repression activity of NF-YCs on the light-controlled hypocotyl elongation partially depends on the deacetylation activity of HDA15, and loss of HDA15 function could rescue the short-hypocotyl phenotype of NF-YCs overexpression plants. Taken together, our results indicate that NF-YC1, NF-YC3, NF-YC4, and NF-YC9 function as transcriptional co-repressors by interacting with HDA15 to inhibit hypocotyl elongation in photomorphogenesis during the early seedling stage. Our findings highlight that NF-YCs can modulate plant development in response to environmental cues via epigenetic regulation.

  10. TEFM is a potent stimulator of mitochondrial transcription elongation in vitro

    PubMed Central

    Posse, Viktor; Shahzad, Saba; Falkenberg, Maria; Hällberg, B. Martin; Gustafsson, Claes M.

    2015-01-01

    A single-subunit RNA polymerase, POLRMT, transcribes the mitochondrial genome in human cells. Recently, a factor termed as the mitochondrial transcription elongation factor, TEFM, was shown to stimulate transcription elongation in vivo, but its effect in vitro was relatively modest. In the current work, we have isolated active TEFM in recombinant form and used a reconstituted in vitro transcription system to characterize its activities. We show that TEFM strongly promotes POLRMT processivity as it dramatically stimulates the formation of longer transcripts. TEFM also abolishes premature transcription termination at conserved sequence block II, an event that has been linked to primer formation during initiation of mtDNA synthesis. We show that POLRMT pauses at a wide range of sites in a given DNA sequence. In the absence of TEFM, this leads to termination; however, the presence of TEFM abolishes this effect and aids POLRMT in continuation of transcription. Further, we show that TEFM substantially increases the POLRMT affinity to an elongation-like DNA:RNA template. In combination with previously published in vivo observations, our data establish TEFM as an essential component of the mitochondrial transcription machinery. PMID:25690892

  11. Numerical experiments with flows of elongated granules

    NASA Technical Reports Server (NTRS)

    Elrod, Harold G.; Brewe, David E.

    1992-01-01

    Theory and numerical results are given for a program simulating two dimensional granular flow (1) between two infinite, counter-moving, parallel, roughened walls, and (2) for an infinitely wide slider. Each granule is simulated by a central repulsive force field ratcheted with force restitution factor to introduce dissipation. Transmission of angular momentum between particles occurs via Coulomb friction. The effect of granular hardness is explored. Gaps from 7 to 28 particle diameters are investigated, with solid fractions ranging from 0.2 to 0.9. Among features observed are: slip flow at boundaries, coagulation at high densities, and gross fluctuation in surface stress. A videotape has been prepared to demonstrate the foregoing effects.

  12. The elongation rate of RNA polymerase determines the fate of transcribed nucleosomes

    PubMed Central

    Bintu, Lacramioara; Kopaczynska, Marta; Hodges, Courtney; Lubkowska, Lucyna; Kashlev, Mikhail; Bustamante, Carlos

    2011-01-01

    Upon transcription, histones can either detach from DNA or transfer behind the polymerase through a process believed to involve template looping. The details governing nucleosomal fate during transcription are not well understood. Our atomic force microscopy images of RNA polymerase II-nucleosome complexes confirm the presence of looped transcriptional intermediates and provide mechanistic insight into the histone-transfer process via the distribution of transcribed nucleosome positions. Significantly, we find that a fraction of the transcribed nucleosomes are remodeled to hexasomes, and that this fraction depends on the transcription elongation rate. A simple model involving the kinetic competition between transcription elongation, histone transfer, and histone-histone dissociation quantitatively rationalizes our observations and unifies results obtained with other polymerases. Factors affecting the relative magnitude of these processes provide the physical basis for nucleosomal fate during transcription and, therefore, for the regulation of gene expression. PMID:22081017

  13. An Integrated Approach Reveals Regulatory Controls on Bacterial Translation Elongation

    PubMed Central

    Subramaniam, Arvind R.; Zid, Brian M.; O’Shea, Erin K.

    2014-01-01

    Summary Ribosomes elongate at a non-uniform rate during translation. Theoretical models and experiments disagree on the in vivo determinants of elongation rate and the mechanism by which elongation rate affects protein levels. To resolve this conflict, we measured transcriptome-wide ribosome occupancy under multiple conditions and used it to formulate a whole-cell model of translation in E. coli. Our model predicts that elongation rates at most codons during nutrient-rich growth are not limited by the intracellular concentrations of aminoacyl-tRNAs. However, elongation pausing during starvation for single amino acids is highly sensitive to the kinetics of tRNA aminoacylation. We further show that translation abortion upon pausing accounts for the observed ribosome occupancy along mRNAs during starvation. Abortion reduces global protein synthesis, but it enhances the translation of a subset of mRNAs. These results suggest a regulatory role for aminoacylation and abortion during stress, and our study provides an experimentally-constrained framework for modeling translation. PMID:25416955

  14. Protein Synthesis in E. coli: Dependence of Codon-Specific Elongation on tRNA Concentration and Codon Usage.

    PubMed

    Rudorf, Sophia; Lipowsky, Reinhard

    2015-01-01

    To synthesize a protein, a ribosome moves along a messenger RNA (mRNA), reads it codon by codon, and takes up the corresponding ternary complexes which consist of aminoacylated transfer RNAs (aa-tRNAs), elongation factor Tu (EF-Tu), and GTP. During this process of translation elongation, the ribosome proceeds with a codon-specific rate. Here, we present a general theoretical framework to calculate codon-specific elongation rates and error frequencies based on tRNA concentrations and codon usages. Our theory takes three important aspects of in-vivo translation elongation into account. First, non-cognate, near-cognate and cognate ternary complexes compete for the binding sites on the ribosomes. Second, the corresponding binding rates are determined by the concentrations of free ternary complexes, which must be distinguished from the total tRNA concentrations as measured in vivo. Third, for each tRNA species, the difference between total tRNA and ternary complex concentration depends on the codon usages of the corresponding cognate and near-cognate codons. Furthermore, we apply our theory to two alternative pathways for tRNA release from the ribosomal E site and show how the mechanism of tRNA release influences the concentrations of free ternary complexes and thus the codon-specific elongation rates. Using a recently introduced method to determine kinetic rates of in-vivo translation from in-vitro data, we compute elongation rates for all codons in Escherichia coli. We show that for some tRNA species only a few tRNA molecules are part of ternary complexes and, thus, available for the translating ribosomes. In addition, we find that codon-specific elongation rates strongly depend on the overall codon usage in the cell, which could be altered experimentally by overexpression of individual genes.

  15. Elongate summit calderas as Neogene paleostress indicators in Antarctica

    USGS Publications Warehouse

    Paulsen, T.S.; Wilson, T.J.

    2007-01-01

    The orientations and ages of elongate summit calderas on major polygenetic volcanoes were compiled to document Miocene to Pleistocene Sh (minimum horizontal stress) directions on the western and northern flanks of the West Antarctic rift system. Miocene to Pleistocene summit calderas along the western Ross Sea show relatively consistent ENE long axis trends, which are at a high angle to the Transantarctic Mountain Front and parallel to the N77ºE Sh direction at Cape Roberts. The elongation directions of many Miocene to Pleistocene summit calderas in Marie Byrd Land parallel the alignment of polygenetic volcanoes in which they occur, except several Pleistocene calderas with consistent NNE to NE trends. The overall pattern of elongate calderas in Marie Byrd Land is probably due to a combination of structurally controlled orientations and regional stress fields in which Sh is oriented NNE to NE at a moderate to high angle to the trace of the West Antarctic rift system.

  16. An Elongated Tetrakaidecahedron Model for Open-Celled Foams

    NASA Technical Reports Server (NTRS)

    Sullivan, Roy M.; Ghosn, Louis J.; Lerch, Bradley A.

    2007-01-01

    A micro-mechanics model for non-isotropic, open-celled foams is developed using an elongated tetrakaidecahedron (Kelvin model) as the repeating unit cell. The micro-mechanics model employs an elongated Kelvin model geometry which is more general than that employed by previous authors. Assuming the cell edges possess axial and bending rigidity, the mechanics of deformation of the elongated tetrakaidecahedron lead to a set of equations for the Young's modulus, Poisson's ratio and strength of the foam in the principal material directions. These equations are written as a function of the cell edge lengths and cross-section properties, the inclination angle and the strength and stiffness of the solid material. The model is applied to predict the strength and stiffness of several polymeric foams. Good agreement is observed between the model results and the experimental measurements.

  17. Break-induced replication and recombinational telomere elongation in yeast.

    PubMed

    McEachern, Michael J; Haber, James E

    2006-01-01

    When a telomere becomes unprotected or if only one end of a chromosomal double-strand break succeeds in recombining with a template sequence, DNA can be repaired by a recombination-dependent DNA replication process termed break-induced replication (BIR). In budding yeasts, there are two BIR pathways, one dependent on the Rad51 recombinase protein and one Rad51 independent; these two repair processes lead to different types of survivors in cells lacking the telomerase enzyme that is required for normal telomere maintenance. Recombination at telomeres is triggered by either excessive telomere shortening or disruptions in the function of telomere-binding proteins. Telomere elongation by BIR appears to often occur through a "roll and spread" mechanism. In this process, a telomeric circle produced by recombination at a dysfunctional telomere acts as a template for a rolling circle BIR event to form an elongated telomere. Additional BIR events can then copy the elongated sequence to all other telomeres.

  18. Elongation effects on the Therac 6 linear accelerator.

    PubMed

    Dawson, D J

    1978-01-01

    The effects of field elongation on the central-axis output at dmax and on the depth-dose curves for the 6-MV x rays from the Therac 6 linear accelerator are considered. These parameters are independent of the collimator angle for both square and elongated fields. The exchange of collimator pairs results in significant output differences but has a negligible effect on the depth-dose curves. Both the central-axis output at dmax and the depth-dose curves agree satisfactorily with the predictions of the equivalent-square technique. An alternative method of greater accuracy is also indicated for the prediction of central-axis outputs at dmax for elongated fields.

  19. Non-targeted metabolomic evaluation of the uterine milieu during the transitional period of embryo elongation in the pig

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alterations in the signaling of critical molecular factors within the uterine milieu lead to deficiencies in embryo elongation. The objective of this study was to identify metabolites within the uterine environment that are present as porcine embryos transition between spherical, ovoid, and tubular ...

  20. NTR1 is required for transcription elongation checkpoints at alternative exons in Arabidopsis

    PubMed Central

    Dolata, Jakub; Guo, Yanwu; Kołowerzo, Agnieszka; Smoliński, Dariusz; Brzyżek, Grzegorz; Jarmołowski, Artur; Świeżewski, Szymon

    2015-01-01

    The interconnection between transcription and splicing is a subject of intense study. We report that Arabidopsis homologue of spliceosome disassembly factor NTR1 is required for correct expression and splicing of DOG1, a regulator of seed dormancy. Global splicing analysis in atntr1 mutants revealed a bias for downstream 5′ and 3′ splice site selection and an enhanced rate of exon skipping. A local reduction in PolII occupancy at misspliced exons and introns in atntr1 mutants suggests that directionality in splice site selection is a manifestation of fast PolII elongation kinetics. In agreement with this model, we found AtNTR1 to bind target genes and co-localise with PolII. A minigene analysis further confirmed that strong alternative splice sites constitute an AtNTR1-dependent transcriptional roadblock. Plants deficient in PolII endonucleolytic cleavage showed opposite effects for splice site choice and PolII occupancy compared to atntr1 mutants, and inhibition of PolII elongation or endonucleolytic cleavage in atntr1 mutant resulted in partial reversal of splicing defects. We propose that AtNTR1 is part of a transcription elongation checkpoint at alternative exons in Arabidopsis. PMID:25568310

  1. A conserved PUF/Ago/eEF1A complex attenuates translation elongation

    PubMed Central

    Friend, Kyle; Campbell, Zachary T.; Cooke, Amy; Kroll-Conner, Peggy; Wickens, Marvin P.; Kimble, Judith

    2011-01-01

    SUMMARY PUF (Pumilio/FBF) RNA-binding proteins and Argonaute (Ago) miRNA-binding proteins regulate mRNAs post-transcriptionally, each acting through similar yet distinct mechanisms. Here, we report that PUF and Ago proteins can also function together in a complex with a core translation elongation factor, eEF1A, to repress translation elongation. Both nematode and mammalian PUF/Ago/eEF1A complexes were identified, using co-immunoprecipitation and recombinant protein assays. Nematode CSR-1 (Ago) promotes repression of FBF (PUF) target mRNAs in in vivo assays, and the FBF-1/CSR-1 heterodimer inhibits EFT-3 (eEF1A) GTPase activity in vitro. Mammalian PUM2/Ago/eEF1A inhibits translation of nonadenylated and polyadenylated reporter mRNAs in vitro. This repression occurs after translation initiation and leads to ribosome accumulation within the open reading frame, roughly at the site where the nascent polypeptide emerges from the ribosomal exit tunnel. Together, these data suggest that a conserved PUF/Ago/eEF1A complex attenuates translation elongation. PMID:22231398

  2. A SnapShot of Ubiquitin Chain Elongation

    PubMed Central

    Kovacev, Jordan; Wu, Kenneth; Spratt, Donald E.; Chong, Robert A.; Lee, Chan; Nayak, Jaladhi; Shaw, Gary S.; Pan, Zhen-Qiang

    2014-01-01

    We have explored the mechanisms of polyubiquitin chain assembly with reconstituted ubiquitination of IκBα and β-catenin by the Skp1-cullin 1-βTrCP F-box protein (SCFβTrCP) E3 ubiquitin (Ub) ligase complex. Competition experiments revealed that SCFβTrCP formed a complex with IκBα and that the Nedd8 modified E3-substrate platform engaged in dynamic interactions with the Cdc34 E2 Ub conjugating enzyme for chain elongation. Using “elongation intermediates” containing β-catenin linked with Ub chains of defined length, it was observed that a Lys-48-Ub chain of a length greater than four, but not its Lys-63 linkage counterparts, slowed the rate of additional Ub conjugation. Thus, the Ub chain length and linkage impact kinetic rates of chain elongation. Given that Lys-48-tetra-Ub is packed into compact conformations due to extensive intrachain interactions between Ub subunits, this topology may limit the accessibility of SCFβTrCP/Cdc34 to the distal Ub Lys-48 and result in slowed elongation. PMID:24464578

  3. FtsZ-Dependent Elongation of a Coccoid Bacterium

    PubMed Central

    Pereira, Ana R.; Hsin, Jen; Król, Ewa; Tavares, Andreia C.; Flores, Pierre; Hoiczyk, Egbert; Ng, Natalie; Dajkovic, Alex; Brun, Yves V.; VanNieuwenhze, Michael S.; Roemer, Terry; Carballido-Lopez, Rut; Huang, Kerwyn Casey

    2016-01-01

    ABSTRACT A mechanistic understanding of the determination and maintenance of the simplest bacterial cell shape, a sphere, remains elusive compared with that of more complex shapes. Cocci seem to lack a dedicated elongation machinery, and a spherical shape has been considered an evolutionary dead-end morphology, as a transition from a spherical to a rod-like shape has never been observed in bacteria. Here we show that a Staphylococcus aureus mutant (M5) expressing the ftsZG193D allele exhibits elongated cells. Molecular dynamics simulations and in vitro studies indicate that FtsZG193D filaments are more twisted and shorter than wild-type filaments. In vivo, M5 cell wall deposition is initiated asymmetrically, only on one side of the cell, and progresses into a helical pattern rather than into a constricting ring as in wild-type cells. This helical pattern of wall insertion leads to elongation, as in rod-shaped cells. Thus, structural flexibility of FtsZ filaments can result in an FtsZ-dependent mechanism for generating elongated cells from cocci. PMID:27601570

  4. Evidence for parallel elongated structures in the mesosphere

    NASA Technical Reports Server (NTRS)

    Adams, G. W.; Brosnahan, J. W.; Walden, D. C.

    1983-01-01

    The physical cause of partial reflection from the mesosphere is of interest. Data are presented from an image-forming radar at Brighton, Colorado, that suggest that some of the radar scattering is caused by parallel elongated structures lying almost directly overhead. Possible physical sources for such structures include gravity waves and roll vortices.

  5. Molecular development of the mid-stage elongating cotton fiber

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cotton fiber is one of the leading natural textile fibers and is the leading value added crop in the USA. The annual business revenue from the cotton industry exceeds $120 billion. The growth of the cotton fiber is divided into four unique, yet overlapping stages; initiation, elongation, secondary w...

  6. Collisional diffusion in toroidal plasmas with elongation and triangularity

    SciTech Connect

    Martin, P.; Castro, E.; Haines, M. G.

    2007-05-15

    Collisional diffusion is analyzed for plasma tokamaks with different ellipticities and triangularities. Improved nonlinear equations for the families of magnetic surfaces are used here. Dimensionless average velocities are calculated as a function of the inductive electric field, elongation, triangularity, and Shafranov shift. Confinement has been found to depend significantly on triangularity.

  7. Quantitative regulation of FLC via coordinated transcriptional initiation and elongation

    PubMed Central

    Wu, Zhe; Ietswaart, Robert; Liu, Fuquan; Yang, Hongchun; Howard, Martin; Dean, Caroline

    2016-01-01

    The basis of quantitative regulation of gene expression is still poorly understood. In Arabidopsis thaliana, quantitative variation in expression of FLOWERING LOCUS C (FLC) influences the timing of flowering. In ambient temperatures, FLC expression is quantitatively modulated by a chromatin silencing mechanism involving alternative polyadenylation of antisense transcripts. Investigation of this mechanism unexpectedly showed that RNA polymerase II (Pol II) occupancy changes at FLC did not reflect RNA fold changes. Mathematical modeling of these transcriptional dynamics predicted a tight coordination of transcriptional initiation and elongation. This prediction was validated by detailed measurements of total and chromatin-bound FLC intronic RNA, a methodology appropriate for analyzing elongation rate changes in a range of organisms. Transcription initiation was found to vary ∼25-fold with elongation rate varying ∼8- to 12-fold. Premature sense transcript termination contributed very little to expression differences. This quantitative variation in transcription was coincident with variation in H3K36me3 and H3K4me2 over the FLC gene body. We propose different chromatin states coordinately influence transcriptional initiation and elongation rates and that this coordination is likely to be a general feature of quantitative gene regulation in a chromatin context. PMID:26699513

  8. Widespread regulation of translation by elongation pausing in heat shock.

    PubMed

    Shalgi, Reut; Hurt, Jessica A; Krykbaeva, Irina; Taipale, Mikko; Lindquist, Susan; Burge, Christopher B

    2013-02-07

    Global repression of protein synthesis is a hallmark of the cellular stress response and has been attributed primarily to inhibition of translation initiation, although this mechanism may not always explain the full extent of repression. Here, using ribosome footprinting, we show that 2 hr of severe heat stress triggers global pausing of translation elongation at around codon 65 on most mRNAs in both mouse and human cells. The genome-wide nature of the phenomenon, its location, and features of protein N termini suggested the involvement of ribosome-associated chaperones. After severe heat shock, Hsp70's interactions with the translational machinery were markedly altered and its association with ribosomes was reduced. Pretreatment with mild heat stress or overexpression of Hsp70 protected cells from heat shock-induced elongation pausing, while inhibition of Hsp70 activity triggered elongation pausing without heat stress. Our findings suggest that regulation of translation elongation in general, and by chaperones in particular, represents a major component of cellular stress responses.

  9. Binary asteroid population. 3. Secondary rotations and elongations

    NASA Astrophysics Data System (ADS)

    Pravec, P.; Scheirich, P.; Kušnirák, P.; Hornoch, K.; Galád, A.; Naidu, S. P.; Pray, D. P.; Világi, J.; Gajdoš, Š.; Kornoš, L.; Krugly, Yu. N.; Cooney, W. R.; Gross, J.; Terrell, D.; Gaftonyuk, N.; Pollock, J.; Husárik, M.; Chiorny, V.; Stephens, R. D.; Durkee, R.; Reddy, V.; Dyvig, R.; Vraštil, J.; Žižka, J.; Mottola, S.; Hellmich, S.; Oey, J.; Benishek, V.; Kryszczyńska, A.; Higgins, D.; Ries, J.; Marchis, F.; Baek, M.; Macomber, B.; Inasaridze, R.; Kvaratskhelia, O.; Ayvazian, V.; Rumyantsev, V.; Masi, G.; Colas, F.; Lecacheux, J.; Montaigut, R.; Leroy, A.; Brown, P.; Krzeminski, Z.; Molotov, I.; Reichart, D.; Haislip, J.; LaCluyze, A.

    2016-03-01

    We collected data on rotations and elongations of 46 secondaries of binary and triple systems among near-Earth, Mars-crossing and small main belt asteroids. 24 were found or are strongly suspected to be synchronous (in 1:1 spin-orbit resonance), and the other 22, generally on more distant and/or eccentric orbits, were found or are suggested to have asynchronous rotations. For 18 of the synchronous secondaries, we constrained their librational angles, finding that their long axes pointed to within 20° of the primary on most epochs. The observed anti-correlation of secondary synchroneity with orbital eccentricity and the limited librational angles agree with the theories by Ćuk and Nesvorný (Ćuk, M., Nesvorný, D. [2010]. Icarus 207, 732-743) and Naidu and Margot (Naidu, S.P., Margot, J.-L. [2015]. Astron. J. 149, 80). A reason for the asynchronous secondaries being on wider orbits than synchronous ones may be longer tidal circularization time scales at larger semi-major axes. The asynchronous secondaries show relatively fast spins; their rotation periods are typically < 10 h. An intriguing observation is a paucity of chaotic secondary rotations; with an exception of (35107) 1991 VH, the secondary rotations are single-periodic with no signs of chaotic rotation and their periods are constant on timescales from weeks to years. The secondary equatorial elongations show an upper limit of a2 /b2 ∼ 1.5 . The lack of synchronous secondaries with greater elongations appears consistent, considering uncertainties of the axis ratio estimates, with the theory by Ćuk and Nesvorný that predicts large regions of chaotic rotation in the phase space for a2 /b2 ≳√{ 2 } . Alternatively, secondaries may not form or stay very elongated in gravitational (tidal) field of the primary. It could be due to the secondary fission mechanism suggested by Jacobson and Scheeres (Jacobson, S.A., Scheeres, D.J. [2011]. Icarus 214, 161-178), as its efficiency is correlated with the

  10. Stimulation of root elongation and curvature by calcium.

    PubMed

    Takahashi, H; Scott, T K; Suge, H

    1992-01-01

    Ca2+ has been proposed to mediate inhibition of root elongation. However, exogenous Ca2+ at 10 or 20 millimolar, applied directly to the root cap, significantly stimulated root elongation in pea (Pisum sativum L.) and corn (Zea mays L.) seedlings. Furthermore, Ca2+ at 1 to 20 millimolar, applied unilaterally to the caps of Alaska pea roots, caused root curvature away from the Ca2+ source, which was caused by an acceleration of elongation growth on the convex side (Ca2+ side) of the roots. Roots of an agravitropic pea mutant, ageotropum, responded to a greater extent. Roots of Merit and Silver Queen corn also responded to Ca2+ in similar ways but required a higher Ca2+ concentration than that of pea roots. Roots of all other cultivars tested (additional four cultivars of pea and one of corn) curved away from the unilateral Ca2+ source as well. The Ca(2+)-stimulated curvature was substantially enhanced by light. A Ca2+ ionophore, A23187, at 20 micromolar or abscisic acid at 0.1 to 100 micromolar partially substituted for the light effect and enhanced the Ca(2+)-stimulated curvature in the dark. Unilateral application of Ca2+ to the elongation zone of intact roots or to the cut end of detipped roots caused either no curvature or very slight curvature toward the Ca2+. Thus, Ca2+ action on root elongation differs depending on its site of application. The stimulatory action of Ca2+ may involve an elevation of cytoplasmic Ca2+ in root cap cells and may partipate in root tropisms.

  11. Adenylate cyclase regulates elongation of mammalian primary cilia

    SciTech Connect

    Ou, Young; Ruan, Yibing; Cheng, Min; Moser, Joanna J.; Rattner, Jerome B.; Hoorn, Frans A. van der

    2009-10-01

    The primary cilium is a non-motile microtubule-based structure that shares many similarities with the structures of flagella and motile cilia. It is well known that the length of flagella is under stringent control, but it is not known whether this is true for primary cilia. In this study, we found that the length of primary cilia in fibroblast-like synoviocytes, either in log phase culture or in quiescent state, was confined within a range. However, when lithium was added to the culture to a final concentration of 100 mM, primary cilia of synoviocytes grew beyond this range, elongating to a length that was on average approximately 3 times the length of untreated cilia. Lithium is a drug approved for treating bipolar disorder. We dissected the molecular targets of this drug, and observed that inhibition of adenylate cyclase III (ACIII) by specific inhibitors mimicked the effects of lithium on primary cilium elongation. Inhibition of GSK-3{beta} by four different inhibitors did not induce primary cilia elongation. ACIII was found in primary cilia of a variety of cell types, and lithium treatment of these cell types led to their cilium elongation. Further, we demonstrate that different cell types displayed distinct sensitivities to the lithium treatment. However, in all cases examined primary cilia elongated as a result of lithium treatment. In particular, two neuronal cell types, rat PC-12 adrenal medulla cells and human astrocytes, developed long primary cilia when lithium was used at or close to the therapeutic relevant concentration (1-2 mM). These results suggest that the length of primary cilia is controlled, at least in part, by the ACIII-cAMP signaling pathway.

  12. Stimulation of root elongation and curvature by calcium

    NASA Technical Reports Server (NTRS)

    Takahashi, H.; Scott, T. K.; Suge, H.

    1992-01-01

    Ca2+ has been proposed to mediate inhibition of root elongation. However, exogenous Ca2+ at 10 or 20 millimolar, applied directly to the root cap, significantly stimulated root elongation in pea (Pisum sativum L.) and corn (Zea mays L.) seedlings. Furthermore, Ca2+ at 1 to 20 millimolar, applied unilaterally to the caps of Alaska pea roots, caused root curvature away from the Ca2+ source, which was caused by an acceleration of elongation growth on the convex side (Ca2+ side) of the roots. Roots of an agravitropic pea mutant, ageotropum, responded to a greater extent. Roots of Merit and Silver Queen corn also responded to Ca2+ in similar ways but required a higher Ca2+ concentration than that of pea roots. Roots of all other cultivars tested (additional four cultivars of pea and one of corn) curved away from the unilateral Ca2+ source as well. The Ca(2+)-stimulated curvature was substantially enhanced by light. A Ca2+ ionophore, A23187, at 20 micromolar or abscisic acid at 0.1 to 100 micromolar partially substituted for the light effect and enhanced the Ca(2+)-stimulated curvature in the dark. Unilateral application of Ca2+ to the elongation zone of intact roots or to the cut end of detipped roots caused either no curvature or very slight curvature toward the Ca2+. Thus, Ca2+ action on root elongation differs depending on its site of application. The stimulatory action of Ca2+ may involve an elevation of cytoplasmic Ca2+ in root cap cells and may partipate in root tropisms.

  13. Hypusine-containing protein eIF5A promotes translation elongation.

    PubMed

    Saini, Preeti; Eyler, Daniel E; Green, Rachel; Dever, Thomas E

    2009-05-07

    Translation elongation factors facilitate protein synthesis by the ribosome. Previous studies identified two universally conserved translation elongation factors, EF-Tu in bacteria (known as eEF1A in eukaryotes) and EF-G (eEF2), which deliver aminoacyl-tRNAs to the ribosome and promote ribosomal translocation, respectively. The factor eIF5A (encoded by HYP2 and ANB1 in Saccharomyces cerevisiae), the sole protein in eukaryotes and archaea to contain the unusual amino acid hypusine (N(epsilon)-(4-amino-2-hydroxybutyl)lysine), was originally identified based on its ability to stimulate the yield (endpoint) of methionyl-puromycin synthesis-a model assay for first peptide bond synthesis thought to report on certain aspects of translation initiation. Hypusine is required for eIF5A to associate with ribosomes and to stimulate methionyl-puromycin synthesis. Because eIF5A did not stimulate earlier steps of translation initiation, and depletion of eIF5A in yeast only modestly impaired protein synthesis, it was proposed that eIF5A function was limited to stimulating synthesis of the first peptide bond or that eIF5A functioned on only a subset of cellular messenger RNAs. However, the precise cellular role of eIF5A is unknown, and the protein has also been linked to mRNA decay, including the nonsense-mediated mRNA decay pathway, and to nucleocytoplasmic transport. Here we use molecular genetic and biochemical studies to show that eIF5A promotes translation elongation. Depletion or inactivation of eIF5A in the yeast S. cerevisiae resulted in the accumulation of polysomes and an increase in ribosomal transit times. Addition of recombinant eIF5A from yeast, but not a derivative lacking hypusine, enhanced the rate of tripeptide synthesis in vitro. Moreover, inactivation of eIF5A mimicked the effects of the eEF2 inhibitor sordarin, indicating that eIF5A might function together with eEF2 to promote ribosomal translocation. Because eIF5A is a structural homologue of the bacterial

  14. Regulation of Stalk Elongation by Phosphate in Caulobacter crescentus

    PubMed Central

    Gonin, Madeleine; Quardokus, Ellen M.; O'Donnol, Danielle; Maddock, Janine; Brun, Yves V.

    2000-01-01

    In Caulobacter crescentus, stalk biosynthesis is regulated by cell cycle cues and by extracellular phosphate concentration. Phosphate-starved cells undergo dramatic stalk elongation to produce stalks as much as 30 times as long as those of cells growing in phosphate-rich medium. To identify genes involved in the control of stalk elongation, transposon mutants were isolated that exhibited a long-stalk phenotype irrespective of extracellular phosphate concentration. The disrupted genes were identified as homologues of the high-affinity phosphate transport genes pstSCAB of Escherichia coli. In E. coli, pst mutants have a constitutively expressed phosphate (Pho) regulon. To determine if stalk elongation is regulated by the Pho regulon, the Caulobacter phoB gene that encodes the transcriptional activator of the Pho regulon was cloned and mutated. While phoB was not required for stalk synthesis or for the cell cycle timing of stalk synthesis initiation, it was required for stalk elongation in response to phosphate starvation. Both pstS and phoB mutants were deficient in phosphate transport. When a phoB mutant was grown with limiting phosphate concentrations, stalks only increased in length by an average of 1.4-fold compared to the average 9-fold increase in stalk length of wild-type cells grown in the same medium. Thus, the phenotypes of phoB and pst mutants were opposite. phoB mutants were unable to elongate stalks during phosphate starvation, whereas pst mutants made long stalks in both high- and low-phosphate media. Analysis of double pst phoB mutants indicated that the long-stalk phenotype of pst mutants was dependent on phoB. In addition, analysis of a pstS-lacZ transcriptional fusion showed that pstS transcription is dependent on phoB. These results suggest that the signal transduction pathway that stimulates stalk elongation in response to phosphate starvation is mediated by the Pst proteins and the response regulator PhoB. PMID:10629178

  15. DNA sequencing by synthesis based on elongation delay detection

    NASA Astrophysics Data System (ADS)

    Manturov, Alexey O.; Grigoryev, Anton V.

    2015-03-01

    The one of most important problem in modern genetics, biology and medicine is determination of the primary nucleotide sequence of the DNA of living organisms (DNA sequencing). This paper describes the label-free DNA sequencing approach, based on the observation of a discrete dynamics of DNA sequence elongation phase. The proposed DNA sequencing principle are studied by numerical simulation. The numerical model for proposed label-free DNA sequencing approach is based on a cellular automaton, which can simulate the elongation stage (growth of DNA strands) and dynamics of nucleotides incorporation to rising DNA strand. The estimates for number of copied DNA sequences for required probability of nucleotide incorporation event detection and correct DNA sequence determination was obtained. The proposed approach can be applied at all known DNA sequencing devices with "sequencing by synthesis" principle of operation.

  16. Elongated Dihydrogen versus Compressed Dihydride in Osmium Complexes.

    PubMed

    Eguillor, Beatriz; Esteruelas, Miguel A; Lezáun, Virginia; Oliván, Montserrat; Oñate, Enrique

    2017-01-31

    Small modifications on the co-ligands of complexes containing two coordinated hydrogen atoms can determine the elongated dihydrogen versus compressed dihydride nature of these species and therefore their chemical behavior. 2,6-diphenylpyridine favors the formation of the osmium(IV) cation [OsH2 (C6 H4 pyPh)(PiPr3 )2 ](+) , whereas 2-phenoxy-6-phenylpyridine, which contains an oxygen atom between the heterocycle and one of the phenyl groups, stabilizes the osmium(II) elongated dihydrogen species [Os(C6 H4 pyOPh)(η(2) -H2 )(PiPr3 )2 ](+) . In contrast to the latter, the former shows a marked tendency to undergo reductive elimination of the heterocycle.

  17. Critical dynamics of vesicle stretching transition in elongational flow.

    PubMed

    Kantsler, Vasiliy; Segre, Enrico; Steinberg, Victor

    2008-07-25

    We present results on the stretching of single tubular vesicles in an elongation flow toward dumbbell shapes, and on their relaxation. A critical strain rate epsilonc exists; for strain rates epsilonelongation flow.

  18. Elongated solid electrolyte cell configurations and flexible connections therefor

    DOEpatents

    Reichner, P.

    1989-10-17

    A flexible, high temperature, solid oxide electrolyte electrochemical cell stack configuration is made, comprising a plurality of flattened, elongated, connected cell combinations, each cell combination containing an interior electrode having a top surface and a plurality of interior gas feed conduits, through its axial length, electrolyte contacting the interior electrode and exterior electrode contacting electrolyte, where a major portion of the air electrode top surface is covered by interconnection material, and where each cell has at least one axially elongated, electronically conductive, flexible, porous, metal fiber felt material in electronic connection with the air electrode through contact with a major portion of the interconnection material, the metal fiber felt being effective as a shock absorbent body between the cells. 4 figs.

  19. Elongated solid electrolyte cell configurations and flexible connections therefor

    DOEpatents

    Reichner, Philip

    1989-01-01

    A flexible, high temperature, solid oxide electrolyte electrochemical cell stack configuration is made, comprising a plurality of flattened, elongated, connected cell combinations 1, each cell combination containing an interior electrode 2 having a top surface and a plurality of interior gas feed conduits 3, through its axial length, electrolyte 5 contacting the interior electrode and exterior electrode 8 contacting electrolyte, where a major portion of the air electrode top surface 7 is covered by interconnection material 6, and where each cell has at least one axially elongated, electronically conductive, flexible, porous, metal fiber felt material 9 in electronic connection with the air electrode 2 through contact with a major portion of the interconnection material 6, the metal fiber felt being effective as a shock absorbent body between the cells.

  20. Investigation of Impact Load Absorption through Suspension Line Elongation

    DTIC Science & Technology

    1952-12-01

    16 1. Charts . . ’ . . . .. . . 16 2. glong~tion Ratin of Li; Goups . . . 163. Graphs . .. .. .. .. .. .. .. ... . 16 SECTION IV - DISCUSSION OF...Tester . ...................... z14 Figure 16 . Frazier Air Porosity Tester in Use ....... 215 Figure 17. 30 ft., Extended Skirt CsnoW’ in Deployment Bag...line than on canopies strung with high elongation line. WMADR 5&~5T 1 CONCLUSIONS; 15. Nylon is superior to fortisan in shock absorbing capacity. 16

  1. Neuroprotective copper bis(thiosemicarbazonato) complexes promote neurite elongation.

    PubMed

    Bica, Laura; Liddell, Jeffrey R; Donnelly, Paul S; Duncan, Clare; Caragounis, Aphrodite; Volitakis, Irene; Paterson, Brett M; Cappai, Roberto; Grubman, Alexandra; Camakaris, James; Crouch, Peter J; White, Anthony R

    2014-01-01

    Abnormal biometal homeostasis is a central feature of many neurodegenerative disorders including Alzheimer's disease (AD), Parkinson's disease (PD), and motor neuron disease. Recent studies have shown that metal complexing compounds behaving as ionophores such as clioquinol and PBT2 have robust therapeutic activity in animal models of neurodegenerative disease; however, the mechanism of neuroprotective action remains unclear. These neuroprotective or neurogenerative processes may be related to the delivery or redistribution of biometals, such as copper and zinc, by metal ionophores. To investigate this further, we examined the effect of the bis(thiosemicarbazonato)-copper complex, Cu(II)(gtsm) on neuritogenesis and neurite elongation (neurogenerative outcomes) in PC12 neuronal-related cultures. We found that Cu(II)(gtsm) induced robust neurite elongation in PC12 cells when delivered at concentrations of 25 or 50 nM overnight. Analogous effects were observed with an alternative copper bis(thiosemicarbazonato) complex, Cu(II)(atsm), but at a higher concentration. Induction of neurite elongation by Cu(II)(gtsm) was restricted to neurites within the length range of 75-99 µm with a 2.3-fold increase in numbers of neurites in this length range with 50 nM Cu(II)(gtsm) treatment. The mechanism of neurogenerative action was investigated and revealed that Cu(II)(gtsm) inhibited cellular phosphatase activity. Treatment of cultures with 5 nM FK506 (calcineurin phosphatase inhibitor) resulted in analogous elongation of neurites compared to 50 nM Cu(II)(gtsm), suggesting a potential link between Cu(II)(gtsm)-mediated phosphatase inhibition and neurogenerative outcomes.

  2. Directional ultrasonic backscattering in polycrystals with elongated grains

    NASA Astrophysics Data System (ADS)

    Lobkis, O. I.; Yang, L.; Li, J.; Rokhlin, S. I.

    2012-05-01

    An analytical solution for a three dimensional integral representation of the backscattering (BS) coefficient in polycrystals with elongated grains is obtained. The theory was applied to evaluation of experimental data in Ti alloy with duplex microstructure, which consists of micro-textured regions (MTR) and smaller crystallites. Experiment shows that for microstructure characterization there is significant advantage in using the directional ratios of backscattering coefficients instead their absolute values for data analysis.

  3. Solitary waves in elongated clouds of strongly interacting bosons

    SciTech Connect

    Oegren, M.; Kavoulakis, G.M.; Jackson, A.D.

    2005-08-15

    We examine the propagation of solitary waves in elongated clouds of trapped bosonic atoms as the confinement, the strength of the interatomic interaction, and the atom density are varied. We identify three different physical regimes and develop a general formalism that allows us to interpolate between them. Finally we pay special attention to the transition to the Tonks-Girardeau limit of strongly interacting bosons.

  4. Clamped-filament elongation model for actin-based motors.

    PubMed Central

    Dickinson, Richard B; Purich, Daniel L

    2002-01-01

    Although actin-based motility drives cell crawling and intracellular locomotion of organelles and certain pathogens, the underlying mechanism of force generation remains a mystery. Recent experiments demonstrated that Listeria exhibit episodes of 5.4-nm stepwise motion corresponding to the periodicity of the actin filament subunits, and extremely small positional fluctuations during the intermittent pauses [S. C. Kuo and J. L. McGrath. 2000. Nature. 407:1026-1029]. These findings suggest that motile bacteria remain firmly bound to actin filament ends as they elongate, a behavior that appears to rule out previous models for actin-based motility. We propose and analyze a new mechanochemical model (called the "Lock, Load & Fire" mechanism) for force generation by means of affinity-modulated, clamped-filament elongation. During the locking step, the filament's terminal ATP-containing subunit binds tightly to a clamp situated on the surface of a motile object; in the loading step, actin.ATP monomer(s) bind to the filament end, an event that triggers the firing step, wherein ATP hydrolysis on the clamped subunit attenuates the filament's affinity for the clamp. This last step initiates translocation of the new ATP-containing terminus to the clamp, whereupon another cycle begins anew. This model explains how surface-tethered filaments can grow while exerting flexural or tensile force on the motile surface. Moreover, stochastic simulations of the model reproduce the signature motions of Listeria. This elongation motor, which we term actoclampin, exploits actin's intrinsic ATPase activity to provide a simple, high-fidelity enzymatic reaction cycle for force production that does not require elongating filaments to dissociate from the motile surface. This mechanism may operate whenever actin polymerization is called upon to generate the forces that drive cell crawling or intracellular organelle motility. PMID:11806905

  5. Neuroprotective Copper Bis(thiosemicarbazonato) Complexes Promote Neurite Elongation

    PubMed Central

    Bica, Laura; Liddell, Jeffrey R.; Donnelly, Paul S.; Duncan, Clare; Caragounis, Aphrodite; Volitakis, Irene; Paterson, Brett M.; Cappai, Roberto; Grubman, Alexandra; Camakaris, James; Crouch, Peter J.; White, Anthony R.

    2014-01-01

    Abnormal biometal homeostasis is a central feature of many neurodegenerative disorders including Alzheimer's disease (AD), Parkinson's disease (PD), and motor neuron disease. Recent studies have shown that metal complexing compounds behaving as ionophores such as clioquinol and PBT2 have robust therapeutic activity in animal models of neurodegenerative disease; however, the mechanism of neuroprotective action remains unclear. These neuroprotective or neurogenerative processes may be related to the delivery or redistribution of biometals, such as copper and zinc, by metal ionophores. To investigate this further, we examined the effect of the bis(thiosemicarbazonato)-copper complex, CuII(gtsm) on neuritogenesis and neurite elongation (neurogenerative outcomes) in PC12 neuronal-related cultures. We found that CuII(gtsm) induced robust neurite elongation in PC12 cells when delivered at concentrations of 25 or 50 nM overnight. Analogous effects were observed with an alternative copper bis(thiosemicarbazonato) complex, CuII(atsm), but at a higher concentration. Induction of neurite elongation by CuII(gtsm) was restricted to neurites within the length range of 75–99 µm with a 2.3-fold increase in numbers of neurites in this length range with 50 nM CuII(gtsm) treatment. The mechanism of neurogenerative action was investigated and revealed that CuII(gtsm) inhibited cellular phosphatase activity. Treatment of cultures with 5 nM FK506 (calcineurin phosphatase inhibitor) resulted in analogous elongation of neurites compared to 50 nM CuII(gtsm), suggesting a potential link between CuII(gtsm)-mediated phosphatase inhibition and neurogenerative outcomes. PMID:24587210

  6. A review of elongation of os calcis for flat feet.

    PubMed

    Phillips, G E

    1983-01-01

    Between 1959 and 1974 the late Dillwyn Evans treated severe symptomatic flat feet by elongating the os calcis. The long-term follow-up of 20 of these patients with a total of 23 feet is presented 7 to 20 years after the operation. At review 17 of the 23 feet showed very good or good results and it was concluded that this is a useful procedure for severe cases of flat feet which appears to stand the test of time.

  7. Photo-oxidation of LDPE: Effects on elongational viscosity

    NASA Astrophysics Data System (ADS)

    Rolón-Garrido, Víctor H.; Wagner, Manfred H.

    2013-04-01

    Sheets of low-density polyethylene (LDPE) were photo-oxidatively treated at room temperature, and subsequently characterized rheologically in the melt state by shear and uniaxial extensional experiments. For photo-oxidation, a xenon lamp was used to irradiate the samples for times between 1 day and 6 weeks. Linear-viscoelastic characterization was performed in a temperature range of 130 to 220°C to obtain the master curve at 170°C, the reference temperature at which the elongational viscosities were measured. Linear viscoelasticity is increasingly affected by increasing photo-oxidation due to crosslinking of LDPE, as corroborated by an increasing gel fraction as determined by a solvent extraction method. The elongational measurements reveal a strong enhancement of strain hardening until a saturation level is achieved. The elongational data are analyzed in the frame work of two constitutive equations, the rubber-like liquid and the molecular stress function models. Within the experimental window, time-deformation separability is confirmed for all samples, independent of the degree of photo-oxidation.

  8. Role of dipole elongation in orientationally ordered liquids

    NASA Astrophysics Data System (ADS)

    Wei, Dong-Qing; Gao, Lin; Zhang, Jiao; Yan, Li-Wei; Hu, Jin-He; Chen, Lang; Gong, Zi-Zheng; Guo, Yong-Xin; Han, Yu

    2011-06-01

    A recent study shows that the dipole elongation in the extended dipole model plays a significant role in the phase transitions of liquid crystal phases. In this paper, molecular dynamics (MD) simulations were performed for the dipole model with different distances between the two charges keeping the total dipole moment the same. The potential energy consists of the Lennard-Jones potential and the site-site electrostatic contribution of partial charges. Detailed analyses were made with respect to the average order parameters and as functions of density along with other thermodynamic properties. When the reduced dipole elongations are 0.16 and 0.32, respectively, it is shown that the chainlike structures in the low density regime, liquid phases with columnar and smectic orders, and solid phases are formed; the phase with nematic order is not present anymore. At 0.64, the phases with antiferroelectric order were favored. The transition is found at the reduced elongation 0.55. It shows that the phase transitions are quite sensitive to the molecular charge distribution; this simple system could exhibit rather rich phase behaviors, which represents a significant advance in identifying molecular and state parameters of the future ferroelectric liquids.

  9. Mechanism of gibberellin-dependent stem elongation in peas

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.; Sovonick-Dunford, S. A.

    1989-01-01

    Stem elongation in peas (Pisum sativum L.) is under partial control by gibberellins, yet the mechanism of such control is uncertain. In this study, we examined the cellular and physical properties that govern stem elongation, to determine how gibberellins influence pea stem growth. Stem elongation of etiolated seedlings was retarded with uniconozol, a gibberellin synthesis inhibitor, and the growth retardation was reversed by exogenous gibberellin. Using the pressure probe and vapor pressure osmometry, we found little effect of uniconozol and gibberellin on cell turgor pressure or osmotic pressure. In contrast, these treatments had major effects on in vivo stress relaxation, measured by turgor relaxation and pressure-block techniques. Uniconozol-treated plants exhibited reduced wall relaxation (both initial rate and total amount). The results show that growth retardation is effected via a reduction in the wall yield coefficient and an increase in the yield threshold. These effects were largely reversed by exogenous gibberellin. When we measured the mechanical characteristics of the wall by stress/strain (Instron) analysis, we found only minor effects of uniconozol and gibberellin on the plastic compliance. This observation indicates that these agents did not alter wall expansion through effects on the mechanical (viscoelastic) properties of the wall. Our results suggest that wall expansion in peas is better viewed as a chemorheological, rather than a viscoelastic, process.

  10. Thin blanket designs for the Elongated Tokamak Commercial Reactor

    SciTech Connect

    Wong, C.P.C.; Bourque, R.F.; Cheng, E.T.; Creedon, R.L.; Schultz, K.R.

    1986-11-01

    The Elongated Tokamak (ET)* is an innovative concept that uses as highly elongated plasma (plasma height-to-width ratio of 6-10) to allow high plasma current and high toroidal betas. ET has the potential for the development of small-size, high-power density, low-cost fusion reactors using normal conducting coils. The elongated plasma shape is achieved by use of a continuous stack of PF coils parallel to the plasma surface on both inbound and outbound sides. To achieve plasma stability, these coil stacks must be located no further than one plasma minor radius from the plasma edge, greatly restricting the space available for blankets. In order to assess the potential of a small reactor, the authors evaluated and designed blankets 30 to 40 cm thick. Three different thin blanket designs were found to be acceptable: FLiBe self-cooled, helium-cooled lithium, and helium-cooled 17Li83Pb blanket designs. A lithium-cooled integrated blanket-coil design (BLITZ-coil) was also found to be suitable for the ET commercial reactors.

  11. ACL reconstruction: in vivo measurement of patellar tendon graft elongation.

    PubMed

    Berruto, M; Howe, J G; Beynnon, B D; Johnson, R J; Nichols, C E; Pope, M H

    1991-06-01

    The implantation of a free autogenous patellar tendon graft is the surgical technique that currently offers the best results in anterior cruciate ligament reconstruction. However, numerous aspects regarding both technique and postoperative rehabilitation can still be improved. The aim of this study was to measure the elongation of the patellar tendon in vivo in the operating room after reconstructive surgery, subjecting the knee to normal strain such as passive mobilization or anterior displacement of the tibia. Three volunteers were studied. Our results were different from those reported in a previous study conducted in vivo on a normal anterior cruciate ligament (ACL). In spite of the isometric position of the tendon, passive mobilization provoked a progressive increase in the elongation of the graft within each cycle of flexion-extension and between one cycle and the next. This also occurred during the Lachman test. These findings suggest that the graft undergoes a process of tensile adjustment when it is first put under strain. Continued elongation once this process appears stabilized raises doubts as to the reliability of isometric measuring devices.

  12. A triple helix-loop-helix/basic helix-loop-helix cascade controls cell elongation downstream of multiple hormonal and environmental signaling pathways in Arabidopsis.

    PubMed

    Bai, Ming-Yi; Fan, Min; Oh, Eunkyoo; Wang, Zhi-Yong

    2012-12-01

    Environmental and endogenous signals, including light, temperature, brassinosteroid (BR), and gibberellin (GA), regulate cell elongation largely by influencing the expression of the paclobutrazol-resistant (PRE) family helix-loop-helix (HLH) factors, which promote cell elongation by interacting antagonistically with another HLH factor, IBH1. However, the molecular mechanism by which PREs and IBH1 regulate gene expression has remained unknown. Here, we show that IBH1 interacts with and inhibits a DNA binding basic helix-loop-helix (bHLH) protein, HBI1, in Arabidopsis thaliana. Overexpression of HBI1 increased hypocotyl and petiole elongation, whereas dominant inactivation of HBI1 and its homologs caused a dwarf phenotype, indicating that HBI1 is a positive regulator of cell elongation. In vitro and in vivo experiments showed that HBI1 directly bound to the promoters and activated two EXPANSIN genes encoding cell wall-loosening enzymes; HBI1's DNA binding and transcriptional activities were inhibited by IBH1, but the inhibitory effects of IBH1 were abolished by PRE1. The results indicate that PREs activate the DNA binding bHLH factor HBI1 by sequestering its inhibitor IBH1. Altering each of the three factors affected plant sensitivities to BR, GA, temperature, and light. Our study demonstrates that PREs, IBH1, and HBI1 form a chain of antagonistic switches that regulates cell elongation downstream of multiple external and endogenous signals.

  13. Localized recruitment of a chromatin-remodeling activity by an activator in vivo drives transcriptional elongation

    PubMed Central

    Corey, Laura L.; Weirich, Christine S.; Benjamin, Ivor J.; Kingston, Robert E.

    2003-01-01

    To understand the role of chromatin-remodeling activities in transcription, it is necessary to understand how they interact with transcriptional activators in vivo to regulate the different steps of transcription. Human heat shock factor 1 (HSF1) stimulates both transcriptional initiation and elongation. We replaced mouse HSF1 in fibroblasts with wild-type and mutant human HSF1 constructs and characterized regulation of an endogenous mouse hsp70 gene. A mutation that diminished transcriptional initiation led to twofold reductions in hsp70 mRNA induction and recruitment of a SWI/SNF remodeling complex. In contrast, a mutation that diminished transcriptional elongation abolished induction of full-length mRNA, SWI/SNF recruitment, and chromatin remodeling, but minimally impaired initiation from the hsp70 promoter. Another remodeling factor, SNF2h, is constitutively present at the promoter irrespective of the genotype of HSF1. These data suggest that localized recruitment of SWI/SNF drives a specialized remodeling reaction necessary for the production of full-length hsp70 mRNA. PMID:12782657

  14. Telomere elongation (Tel), a new mutation in Drosophila melanogaster that produces long telomeres.

    PubMed Central

    Siriaco, Giorgia M; Cenci, Giovanni; Haoudi, Abdelali; Champion, Larry E; Zhou, Chun; Gatti, Maurizio; Mason, James M

    2002-01-01

    In most eukaryotes telomeres are extended by telomerase. Drosophila melanogaster, however, lacks telomerase, and telomere-specific non-LTR retrotransposons, HeT-A and TART, transpose specifically to chromosome ends. A Drosophila strain, Gaiano, that has long telomeres has been identified. We extracted the major Gaiano chromosomes into an Oregon-R genetic background and examined the resulting stocks after 60 generations. In situ hybridization using HeT-A and TART sequences showed that, in stocks carrying either the X or the second chromosome from Gaiano, only the Gaiano-derived chromosomes display long telomeres. However, in stocks carrying the Gaiano third chromosome, all telomeres are substantially elongated, indicating that the Gaiano chromosome 3 carries a factor that increases HeT-A and TART addition to the telomeres. We show that this factor, termed Telomere elongation (Tel), is dominant and localizes as a single unit to 69 on the genetic map. The long telomeres tend to associate with each other in both polytene and mitotic cells. These associations depend on telomere length rather than the presence of Tel. Associations between metaphase chromosomes are resolved during anaphase, suggesting that they are mediated by either proteinaceous links or DNA hydrogen bonding, rather than covalent DNA-DNA bonds. PMID:11805059

  15. Ethylene Regulates the Arabidopsis Microtubule-Associated Protein WAVE-DAMPENED2-LIKE5 in Etiolated Hypocotyl Elongation1[OPEN

    PubMed Central

    Sun, Jingbo; Ma, Qianqian; Mao, Tonglin

    2015-01-01

    The phytohormone ethylene plays crucial roles in the negative regulation of plant etiolated hypocotyl elongation. The microtubule cytoskeleton also participates in hypocotyl cell growth. However, it remains unclear if ethylene signaling-mediated etiolated hypocotyl elongation involves the microtubule cytoskeleton. In this study, we functionally identified the previously uncharacterized microtubule-associated protein WAVE-DAMPENED2-LIKE5 (WDL5) as a microtubule-stabilizing protein that plays a positive role in ethylene-regulated etiolated hypocotyl cell elongation in Arabidopsis (Arabidopsis thaliana). ETHYLENE-INSENSITIVE3, a key transcription factor in the ethylene signaling pathway, directly targets and up-regulates WDL5. Etiolated hypocotyls from a WDL5 loss-of-function mutant (wdl5-1) were more insensitive to 1-aminocyclopropane-1-carboxylic acid treatment than the wild type. Decreasing WDL5 expression partially rescued the shorter etiolated hypocotyl phenotype in the ethylene overproduction mutant eto1-1. Reorganization of cortical microtubules in etiolated hypocotyl cells from the wdl5-1 mutant was less sensitive to 1-aminocyclopropane-1-carboxylic acid treatment. These findings indicate that WDL5 is an important participant in ethylene signaling inhibition of etiolated hypocotyl growth. This study reveals a mechanism involved in the ethylene regulation of microtubules through WDL5 to inhibit etiolated hypocotyl cell elongation. PMID:26134166

  16. Myosin IIIa boosts elongation of stereocilia by transporting espin 1 to the plus ends of actin filaments.

    PubMed

    Salles, Felipe T; Merritt, Raymond C; Manor, Uri; Dougherty, Gerard W; Sousa, Aurea D; Moore, Judy E; Yengo, Christopher M; Dosé, Andréa C; Kachar, Bechara

    2009-04-01

    Two proteins implicated in inherited deafness, myosin IIIa, a plus-end-directed motor, and espin, an actin-bundling protein containing the actin-monomer-binding motif WH2, have been shown to influence the length of mechanosensory stereocilia. Here we report that espin 1, an ankyrin repeat-containing isoform of espin, colocalizes with myosin IIIa at stereocilia tips and interacts with a unique conserved domain of myosin IIIa. We show that combined overexpression of these proteins causes greater elongation of stereocilia, compared with overexpression of either myosin IIIa alone or espin 1 alone. When these two proteins were co-expressed in the fibroblast-like COS-7 cell line they induced a tenfold elongation of filopodia. This extraordinary filopodia elongation results from the transport of espin 1 to the plus ends of F-actin by myosin IIIa and depends on espin 1 WH2 activity. This study provides the basis for understanding the role of myosin IIIa and espin 1 in regulating stereocilia length, and presents a physiological example where myosins can boost elongation of actin protrusions by transporting actin regulatory factors to the plus ends of actin filaments.

  17. Is the Glass Half Full or Half Empty? How to Reverse the Effect of Glass Elongation on the Volume Poured

    PubMed Central

    Caljouw, Simone R.; van Wijck, Ruud

    2014-01-01

    To reduce the volume of drinks and the risk of overconsumption, health professionals recommend the use of tall skinny instead of short wide glasses. Yet the results of the present study contradict this health advice. Participants who generously filled up a glass with lemonade served 9% more in tall narrow compared with short wide glasses (p<0.05). In addition, when pouring a small amount (i.e., a shot), participants poured 3% more in a short wide than in a tall narrow glass (p<0.05). Elongation may bias the perceived volume that is poured but also the perceived volume of the free space in the glass. We hypothesised that shifting attention from the bottom to the brim of the glass when filling it close to capacity might reverse the glass elongation effect on the quantity poured. This hypothesis was tested, by investigating two pouring tasks that differed in the required focus of attention. When the instruction was to match a reference volume, participants poured more liquid in the short wide compared with the tall narrow glass (p<0.05). The effect of glass elongation on poured volume was the opposite when the instruction was to leave space in the glasses for the reference volume. It seems likely that task and individual factors affect the pourer's viewing strategy and thus may determine the direction of the glass elongation effect on the volume poured. PMID:25343252

  18. Ethylene Regulates the Arabidopsis Microtubule-Associated Protein WAVE-DAMPENED2-LIKE5 in Etiolated Hypocotyl Elongation.

    PubMed

    Sun, Jingbo; Ma, Qianqian; Mao, Tonglin

    2015-09-01

    The phytohormone ethylene plays crucial roles in the negative regulation of plant etiolated hypocotyl elongation. The microtubule cytoskeleton also participates in hypocotyl cell growth. However, it remains unclear if ethylene signaling-mediated etiolated hypocotyl elongation involves the microtubule cytoskeleton. In this study, we functionally identified the previously uncharacterized microtubule-associated protein WAVE-DAMPENED2-LIKE5 (WDL5) as a microtubule-stabilizing protein that plays a positive role in ethylene-regulated etiolated hypocotyl cell elongation in Arabidopsis (Arabidopsis thaliana). ETHYLENE-INSENSITIVE3, a key transcription factor in the ethylene signaling pathway, directly targets and up-regulates WDL5. Etiolated hypocotyls from a WDL5 loss-of-function mutant (wdl5-1) were more insensitive to 1-aminocyclopropane-1-carboxylic acid treatment than the wild type. Decreasing WDL5 expression partially rescued the shorter etiolated hypocotyl phenotype in the ethylene overproduction mutant eto1-1. Reorganization of cortical microtubules in etiolated hypocotyl cells from the wdl5-1 mutant was less sensitive to 1-aminocyclopropane-1-carboxylic acid treatment. These findings indicate that WDL5 is an important participant in ethylene signaling inhibition of etiolated hypocotyl growth. This study reveals a mechanism involved in the ethylene regulation of microtubules through WDL5 to inhibit etiolated hypocotyl cell elongation.

  19. Relative Mesothelioma Potencies for Unregulated Respirable Elongated Mineral and Synthetic Particles

    EPA Science Inventory

    For decades uncertainties and contradictions have surrounded the issue of whether exposures to respirable elongated mineral and synthetic particles (REMPs and RESPs) present health risks such as those recognized for exposures to elongated asbestiform mineral particles from the fi...

  20. Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation.

    PubMed

    Vallerga, María Belén; Mansilla, Sabrina F; Federico, María Belén; Bertolin, Agustina P; Gottifredi, Vanesa

    2015-12-01

    After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UV-damaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates.

  1. Gibberellin-Regulation and Genetic Variations in Leaf Elongation for Tall Fescue in Association with Differential Gene Expression Controlling Cell Expansion

    PubMed Central

    Xu, Qian; Krishnan, Sanalkumar; Merewitz, Emily; Xu, Jichen; Huang, Bingru

    2016-01-01

    Leaf elongation rate (LER) is an important factor controlling plant growth and productivity. The objective of this study was to determine whether genetic variation in LER for a fast-growing (‘K-31’), and a dwarf cultivar (‘Bonsai’) of tall fescue (Festuca arundinacea) and gibberellic acid (GA) regulation of LER were associated with differential expression of cell-expansion genes. Plants were treated with GA3, trinexapac-ethyl (TE) (GA inhibitor), or water (untreated control) in a hydroponic system. LER of ‘K-31’ was 63% greater than that of ‘Bonsai’, which corresponded with 32% higher endogenous GA4 content in leaf and greater cell elongation and production rates under the untreated control condition. Exogenous application of GA3 significantly enhanced LER while TE treatment inhibited leaf elongation due to GA3-stimulation or TE-inhibition of cell elongation and production rate in leaves for both cultivars. Real-time quantitative polymerase chain reaction analysis revealed that three α-expansins, one β-expansin, and three xyloglucan endotransglycosylase (XET) genes were associated with GA-stimulation of leaf elongation, of which, the differential expression of EXPA4 and EXPA7 was related to the genotypic variation in LER of two cultivars. Those differentially-expressed expansin and XET genes could play major roles in genetic variation and GA-regulated leaf elongation in tall fescue. PMID:27457585

  2. Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation

    PubMed Central

    Vallerga, María Belén; Mansilla, Sabrina F.; Federico, María Belén; Bertolin, Agustina P.; Gottifredi, Vanesa

    2015-01-01

    After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UV-damaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates. PMID:26627254

  3. Response to Interrupted Hyperopia after Restraint of Axial Elongation in Tree Shrews

    PubMed Central

    Siegwart, John T.; Norton, Thomas T.

    2012-01-01

    Purpose To determine if early restraint of axial elongation in response to plus lenses increases the subsequent response to interrupted hyperopia. Methods The normal, interrupted hyperopia group (n=5) had normal visual exposure until 24 days of visual experience (VE). Then, from 24 to 45 days of VE, the animals wore binocular −4 D lenses which shifted the refractive state of the eyes in the direction of hyperopia. Interrupted hyperopia was produced by removing the lenses for 2 hours per day. The early restraint, interrupted hyperopia group (n=5) wore binocular +4 D lenses continuously from 11 to 24 days of VE, becoming emmetropic with the lenses in place and hyperopic when they were removed. Then, from 24 to 45 days of VE, the lenses were removed 22 hours per day and replaced for 2 hours per day. This created the same initial regimen of interrupted hyperopia as in the normal, interrupted hyperopia group. A plus-lens control group wore binocular +4 D lenses (n=5) continuously from 11 to 45 Days of VE to assess the stability of the refractive compensation. Results In the normal, interrupted hyperopia animals, 2 hours of relief from the imposed hyperopia was sufficient to prevent myopia development. In the early restraint, interrupted hyperopia animals, 2 hours of relief from the hyperopia did not prevent myopia development; the eyes became myopic while wearing the lens. The control animals compensated for the +4 D lenses and maintained a stable with-the-lens emmetropia through 45 days of VE, demonstrating that the myopic shift in the early-restraint group was due to the interrupted hyperopia. Conclusions Compensation for plus lenses, involving slowed axial elongation, increases the response to subsequent interrupted hyperopia. Similar to previous reports of an eye-size factor in elongated eyes, these data provide evidence for an eye-size mechanism operating, in this case, in eyes that have restrained their axial length. PMID:23314128

  4. Metabolic and Physical Control of Cell Elongation Rate

    PubMed Central

    Green, P. B.; Erickson, R. O.; Buggy, J.

    1971-01-01

    Several levels of control of elongation rate are revealed through the detailed study of responses of the Nitella internode to abrupt shifts in turgor. The immediate response, which apparently reflects the physical state of the cell, is approximately described by the equation r = (P — Y)m where r is rate, P is pressure, Y is the wall's yielding threshold, and m is related to the wall's apparent fluidity (reciprocal viscosity). Because P and Y are in the range 5 to 6 atmospheres, and (P — Y) is roughly 0.2 atmosphere, elongation rate is initially extremely sensitive to changes in P. A small step-down in turgor (0.7 atmosphere) stops growth, and a similar rise greatly accelerates it. These initial responses are, however, soon (15 minutes) compensated by changes in Y. An apparent metabolism-dependent reaction (azide-sensitive) lowers Y; strain hardening (azide-insensitive) raises it. These two opposing processes, acting on Y, serve as a governor on (P — Y), tending to maintain it at a given value despite changes in P. This ability to compensate is itself a function of turgor. Turgor step-downs are less and less well compensated, leading to lower rate, as turgor falls from 5 atmospheres to about 2 atmospheres where growth appears not to resume. This is the lowest attainable yield value, Y1. The turgor dependency of compensation reflects a turgor requirement of the Y-lowering (“wall-softening”) process. Thus the relation between steady state, rs, and turgor is an indirect one, derived from time-dependent alterations of the cell wall. This relationship superficially resembles the instantaneously valid one in that, roughly, rs = (P — Y1)ms. Y1 and ms, however, have much lower values than Y and m. The duality of the elongation rate versus turgor relation and the prominent role of Y in regulating rate are the major features of growth control in Nitella. PMID:16657635

  5. Elongated optical bottle beams generated by composite binary axicons

    NASA Astrophysics Data System (ADS)

    Porfirev, A. P.; Skidanov, R. V.

    2016-04-01

    We provide analytical, numerical and experimental study of the possibility of forming elongated optical bottle beams (OBBs) using composite binary phase axicons. In this case, the OBB is generated by the superposition of Bessel beams in the near-field region on the axicon. To generate the OBB experimentally, we utilized a spatial light modulator. The experimental results are qualitatively consistent with the results of numerical simulations performed using Fresnel transform. Such type of optical trap can be applied in many applications of microbiology, micromechanics and meteorology to manipulate micro- and nanoobjects in liquid or gaseous medium.

  6. Longitudinal domain wall formation in elongated assemblies of ferromagnetic nanoparticles.

    PubMed

    Varón, Miriam; Beleggia, Marco; Jordanovic, Jelena; Schiøtz, Jakob; Kasama, Takeshi; Puntes, Victor F; Frandsen, Cathrine

    2015-09-29

    Through evaporation of dense colloids of ferromagnetic ~13 nm ε-Co particles onto carbon substrates, anisotropic magnetic dipolar interactions can support formation of elongated particle structures with aggregate thicknesses of 100-400 nm and lengths of up to some hundred microns. Lorenz microscopy and electron holography reveal collective magnetic ordering in these structures. However, in contrast to continuous ferromagnetic thin films of comparable dimensions, domain walls appear preferentially as longitudinal, i.e., oriented parallel to the long axis of the nanoparticle assemblies. We explain this unusual domain structure as the result of dipolar interactions and shape anisotropy, in the absence of inter-particle exchange coupling.

  7. Controlled laser production of elongated articles from particulates

    DOEpatents

    Dixon, Raymond D.; Lewis, Gary K.; Milewski, John O.

    2002-01-01

    It has been discovered that wires and small diameter rods can be produced using laser deposition technology in a novel way. An elongated article such as a wire or rod is constructed by melting and depositing particulate material into a deposition zone which has been designed to yield the desired article shape and dimensions. The article is withdrawn from the deposition zone as it is formed, thus enabling formation of the article in a continuous process. Alternatively, the deposition zone is moved along any of numerous deposition paths away from the article being formed.

  8. Connectedness Percolation of Elongated Hard Particles in an External Field

    NASA Astrophysics Data System (ADS)

    Otten, Ronald H. J.; van der Schoot, Paul

    2012-02-01

    A theory is presented of how orienting fields and steric interactions conspire against the formation of a percolating network of, in some sense, connected elongated colloidal particles in fluid dispersions. We find that the network that forms above a critical loading breaks up again at higher loadings due to interaction-induced enhancement of the particle alignment. Upon approach of the percolation threshold, the cluster dimensions diverge with the same critical exponent parallel and perpendicular to the field direction, implying that connectedness percolation is not in the universality class of directed percolation.

  9. Modeling cell elongation during germ band retraction: cell autonomy versus applied anisotropic stress

    PubMed Central

    Lynch, Holley E.; Veldhuis, Jim; Brodland, G. Wayne; Hutson, M. Shane

    2014-01-01

    The morphogenetic process of germ band retraction in Drosophila embryos involves coordinated movements of two epithelial tissues – germ band and amnioserosa. The germ band shortens along its rostral-caudal or head-to-tail axis, widens along its perpendicular dorsal-ventral axis, and uncurls from an initial ‘U’ shape. The amnioserosa mechanically assists this process by pulling on the crook of the U-shaped germ band. The amnioserosa may also provide biochemical signals that drive germ band cells to change shape in a mechanically autonomous fashion. Here, we use a finite-element model to investigate how these two contributions reshape the germ band. We do so by modeling the response to laser-induced wounds in each of the germ band’s spatially distinct segments (T1-T3, A1-A9) during the middle of retraction when segments T1-A3 form the ventral arm of the ‘U’, A4-A7 form its crook, and A8-A9 complete the dorsal arm. We explore these responses under a range of externally applied stresses and internal anisotropy of cell edge tensions – akin to a planar cell polarity that can drive elongation of cells in a direction parallel to the minimum edge tension – and identify regions of parameter space (edge-tension anisotropy versus stress anisotropy) that best match previous experiments for each germ band segment. All but three germ band segments are best fit when the applied stress anisotropy and the edge-tension anisotropy work against one another – i.e., when the isolated effects would elongate cells in perpendicular directions. Segments in the crook of the germ band (A4-A7) have cells that elongate in the direction of maximum external stress, i.e., external stress anisotropy is dominant. In most other segments, the dominant factor is internal edge-tension anisotropy. These results are consistent with models in which the amnioserosa pulls on the crook of the germ band to mechanically assist retraction. In addition, they suggest a mechanical cue for edge

  10. Regulation of elongation phase of mRNA translation in diabetic nephropathy: amelioration by rapamycin.

    PubMed

    Sataranatarajan, Kavithalakshmi; Mariappan, Meenalakshmi M; Lee, Myung Ja; Feliers, Denis; Choudhury, Goutam Ghosh; Barnes, Jeffrey L; Kasinath, Balakuntalam S

    2007-12-01

    High glucose and high insulin, pathogenic factors in type 2 diabetes, induce rapid synthesis of the matrix protein laminin-beta1 in renal proximal tubular epithelial cells by stimulation of initiation phase of mRNA translation. We investigated if elongation phase of translation also contributes to high glucose and high insulin induction of laminin-beta1 synthesis in proximal tubular epithelial cells. High glucose or high insulin rapidly increased activating Thr56 dephosphorylation of eEF2 and inactivating Ser366 phosphorylation of eEF2 kinase, events that facilitate elongation. Studies with inhibitors showed that PI3 kinase-Akt-mTOR-p70S6 kinase pathway controlled changes in phosphorylation of eEF2 and eEF2 kinase induced by high glucose or high insulin. Renal cortical homogenates from db/db mice in early stage of type 2 diabetes showed decrease in eEF2 phosphorylation and increment in eEF2 kinase phosphorylation in association with renal hypertrophy and glomerular and tubular increase in laminin-beta1 content. Rapamycin, an inhibitor of mTOR, abolished diabetes-induced changes in phosphorylation of eEF2, eEF2 kinase, and p70S6 kinase and ameliorated renal hypertrophy and laminin-beta1 protein content, without affecting hyperglycemia. These data show that mTOR is an attractive target for amelioration of diabetes-induced renal injury.

  11. Progesterone receptor induces bcl-x expression through intragenic binding sites favoring RNA polymerase II elongation

    PubMed Central

    Bertucci, Paola Y.; Nacht, A. Silvina; Alló, Mariano; Rocha-Viegas, Luciana; Ballaré, Cecilia; Soronellas, Daniel; Castellano, Giancarlo; Zaurin, Roser; Kornblihtt, Alberto R.; Beato, Miguel; Vicent, Guillermo P.; Pecci, Adali

    2013-01-01

    Steroid receptors were classically described for regulating transcription by binding to target gene promoters. However, genome-wide studies reveal that steroid receptors-binding sites are mainly located at intragenic regions. To determine the role of these sites, we examined the effect of progestins on the transcription of the bcl-x gene, where only intragenic progesterone receptor-binding sites (PRbs) were identified. We found that in response to hormone treatment, the PR is recruited to these sites along with two histone acetyltransferases CREB-binding protein (CBP) and GCN5, leading to an increase in histone H3 and H4 acetylation and to the binding of the SWI/SNF complex. Concomitant, a more relaxed chromatin was detected along bcl-x gene mainly in the regions surrounding the intragenic PRbs. PR also mediated the recruitment of the positive elongation factor pTEFb, favoring RNA polymerase II (Pol II) elongation activity. Together these events promoted the re-distribution of the active Pol II toward the 3′-end of the gene and a decrease in the ratio between proximal and distal transcription. These results suggest a novel mechanism by which PR regulates gene expression by facilitating the proper passage of the polymerase along hormone-dependent genes. PMID:23640331

  12. RBPJ maintains brain tumor–initiating cells through CDK9-mediated transcriptional elongation

    PubMed Central

    Xie, Qi; Wu, Qiulian; Kim, Leo; Miller, Tyler E.; Liau, Brian B.; Mack, Stephen C.; Yang, Kailin; Factor, Daniel C.; Fang, Xiaoguang; Huang, Zhi; Zhou, Wenchao; Alazem, Kareem; Wang, Xiuxing; Bernstein, Bradley E.; Bao, Shideng; Rich, Jeremy N.

    2016-01-01

    Glioblastomas co-opt stem cell regulatory pathways to maintain brain tumor–initiating cells (BTICs), also known as cancer stem cells. NOTCH signaling has been a molecular target in BTICs, but NOTCH antagonists have demonstrated limited efficacy in clinical trials. Recombining binding protein suppressor of hairless (RBPJ) is considered a central transcriptional mediator of NOTCH activity. Here, we report that pharmacologic NOTCH inhibitors were less effective than targeting RBPJ in suppressing tumor growth. While NOTCH inhibitors decreased canonical NOTCH gene expression, RBPJ regulated a distinct profile of genes critical to BTIC stemness and cell cycle progression. RBPJ was preferentially expressed by BTICs and required for BTIC self-renewal and tumor growth. MYC, a key BTIC regulator, bound the RBPJ promoter and treatment with a bromodomain and extraterminal domain (BET) family bromodomain inhibitor decreased MYC and RBPJ expression. Proteomic studies demonstrated that RBPJ binds CDK9, a component of positive transcription elongation factor b (P-TEFb), to target gene promoters, enhancing transcriptional elongation. Collectively, RBPJ links MYC and transcriptional control through CDK9, providing potential nodes of fragility for therapeutic intervention, potentially distinct from NOTCH. PMID:27322055

  13. RBPJ maintains brain tumor-initiating cells through CDK9-mediated transcriptional elongation.

    PubMed

    Xie, Qi; Wu, Qiulian; Kim, Leo; Miller, Tyler E; Liau, Brian B; Mack, Stephen C; Yang, Kailin; Factor, Daniel C; Fang, Xiaoguang; Huang, Zhi; Zhou, Wenchao; Alazem, Kareem; Wang, Xiuxing; Bernstein, Bradley E; Bao, Shideng; Rich, Jeremy N

    2016-07-01

    Glioblastomas co-opt stem cell regulatory pathways to maintain brain tumor-initiating cells (BTICs), also known as cancer stem cells. NOTCH signaling has been a molecular target in BTICs, but NOTCH antagonists have demonstrated limited efficacy in clinical trials. Recombining binding protein suppressor of hairless (RBPJ) is considered a central transcriptional mediator of NOTCH activity. Here, we report that pharmacologic NOTCH inhibitors were less effective than targeting RBPJ in suppressing tumor growth. While NOTCH inhibitors decreased canonical NOTCH gene expression, RBPJ regulated a distinct profile of genes critical to BTIC stemness and cell cycle progression. RBPJ was preferentially expressed by BTICs and required for BTIC self-renewal and tumor growth. MYC, a key BTIC regulator, bound the RBPJ promoter and treatment with a bromodomain and extraterminal domain (BET) family bromodomain inhibitor decreased MYC and RBPJ expression. Proteomic studies demonstrated that RBPJ binds CDK9, a component of positive transcription elongation factor b (P-TEFb), to target gene promoters, enhancing transcriptional elongation. Collectively, RBPJ links MYC and transcriptional control through CDK9, providing potential nodes of fragility for therapeutic intervention, potentially distinct from NOTCH.

  14. Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells.

    PubMed

    Min, Irene M; Waterfall, Joshua J; Core, Leighton J; Munroe, Robert J; Schimenti, John; Lis, John T

    2011-04-01

    Transitions between pluripotent stem cells and differentiated cells are executed by key transcription regulators. Comparative measurements of RNA polymerase distribution over the genome's primary transcription units in different cell states can identify the genes and steps in the transcription cycle that are regulated during such transitions. To identify the complete transcriptional profiles of RNA polymerases with high sensitivity and resolution, as well as the critical regulated steps upon which regulatory factors act, we used genome-wide nuclear run-on (GRO-seq) to map the density and orientation of transcriptionally engaged RNA polymerases in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). In both cell types, progression of a promoter-proximal, paused RNA polymerase II (Pol II) into productive elongation is a rate-limiting step in transcription of ∼40% of mRNA-encoding genes. Importantly, quantitative comparisons between cell types reveal that transcription is controlled frequently at paused Pol II's entry into elongation. Furthermore, "bivalent" ESC genes (exhibiting both active and repressive histone modifications) bound by Polycomb group complexes PRC1 (Polycomb-repressive complex 1) and PRC2 show dramatically reduced levels of paused Pol II at promoters relative to an average gene. In contrast, bivalent promoters bound by only PRC2 allow Pol II pausing, but it is confined to extremely 5' proximal regions. Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation.

  15. The Effects of a Valgus Collapse Knee Position on In Vivo ACL Elongation

    PubMed Central

    Utturkar, G. M.; Irribarra, L. A.; Taylor, K. A.; Spritzer, C. E.; Taylor, D. C.; Garrett, W. E.; DeFrate, Louis E.

    2013-01-01

    There are conflicting data regarding what motions increase ACL injury risk. More specifically, the mechanical role of valgus collapse positions during ACL injury remains controversial. Our objective was to evaluate ACL elongation in a model that mimics knee movements thought to occur during ACL injury. Eight healthy male subjects were imaged using MR and biplanar fluoroscopy to measure the in vivo elongation of the ACL and its functional bundles during three static knee positions: full extension, 30° of flexion, and a position intended to mimic a valgus collapse position described in the literature. For this study, the valgus collapse position consisted of 30° of knee flexion, internal rotation of the hip, and 10° of external tibial rotation. ACL length decreased significantly from full extension (30.2 ± 2.6 mm) to 30° of flexion (27.1 ± 2.2 mm). ACL length further decreased in the valgus collapse position (25.6 ± 2.4 mm). Both functional bundles of the ACL followed similar trends with regards to decreases in length in each of the three positions. Since strain would follow patterns of ACL length, landing on an extended knee may be a more relevant risk factor for ACL injuries than the valgus collapse position in males. Future studies should evaluate the effects of dynamic motion patterns on in vivo ACL strains. PMID:22855117

  16. IGF-1 Induces GHRH Neuronal Axon Elongation during Early Postnatal Life in Mice

    PubMed Central

    Clemessy, Maud; Heurtier, Victor; Ledent, Tatiana; Robinson, Iain C.; Mollard, Patrice; Epelbaum, Jacques; Meaney, Michael J.; Garel, Sonia; Le Bouc, Yves; Kappeler, Laurent

    2017-01-01

    Nutrition during the perinatal period programs body growth. Growth hormone (GH) secretion from the pituitary regulates body growth and is controlled by Growth Hormone Releasing Hormone (GHRH) neurons located in the arcuate nucleus of the hypothalamus. We observed that dietary restriction during the early postnatal period (i.e. lactation) in mice influences postnatal growth by permanently altering the development of the somatotropic axis in the pituitary gland. This alteration may be due to a lack of GHRH signaling during this critical developmental period. Indeed, underfed pups showed decreased insulin-like growth factor I (IGF-I) plasma levels, which are associated with lower innervation of the median eminence by GHRH axons at 10 days of age relative to normally fed pups. IGF-I preferentially stimulated axon elongation of GHRH neurons in in vitro arcuate explant cultures from 7 day-old normally fed pups. This IGF-I stimulating effect was selective since other arcuate neurons visualized concomitantly by neurofilament labeling, or AgRP immunochemistry, did not significantly respond to IGF-I stimulation. Moreover, GHRH neurons in explants from age-matched underfed pups lost the capacity to respond to IGF-I stimulation. Molecular analyses indicated that nutritional restriction was associated with impaired activation of AKT. These results highlight a role for IGF-I in axon elongation that appears to be cell selective and participates in the complex cellular mechanisms that link underfeeding during the early postnatal period with programming of the growth trajectory. PMID:28076448

  17. Defective telomere elongation and hematopoiesis from telomerase-mutant aplastic anemia iPSCs

    PubMed Central

    Winkler, Thomas; Hong, So Gun; Decker, Jake E.; Morgan, Mary J.; Wu, Chuanfeng; Hughes, William M.; Yang, Yanqin; Wangsa, Danny; Padilla-Nash, Hesed M.; Ried, Thomas; Young, Neal S.; Dunbar, Cynthia E.; Calado, Rodrigo T.

    2013-01-01

    Critically short telomeres activate p53-mediated apoptosis, resulting in organ failure and leading to malignant transformation. Mutations in genes responsible for telomere maintenance are linked to a number of human diseases. We derived induced pluripotent stem cells (iPSCs) from 4 patients with aplastic anemia or hypocellular bone marrow carrying heterozygous mutations in the telomerase reverse transcriptase (TERT) or the telomerase RNA component (TERC) telomerase genes. Both mutant and control iPSCs upregulated TERT and TERC expression compared with parental fibroblasts, but mutant iPSCs elongated telomeres at a lower rate compared with healthy iPSCs, and the deficit correlated with the mutations’ impact on telomerase activity. There was no evidence for alternative lengthening of telomere (ALT) pathway activation. Elongation varied among iPSC clones derived from the same patient and among clones from siblings harboring identical mutations. Clonal heterogeneity was linked to genetic and environmental factors, but was not influenced by residual expression of reprogramming transgenes. Hypoxia increased telomere extension in both mutant and normal iPSCs. Additionally, telomerase-mutant iPSCs showed defective hematopoietic differentiation in vitro, mirroring the clinical phenotype observed in patients and demonstrating that human telomere diseases can be modeled utilizing iPSCs. Our data support the necessity of studying multiple clones when using iPSCs to model disease. PMID:23585473

  18. The effects of a valgus collapse knee position on in vivo ACL elongation.

    PubMed

    Utturkar, G M; Irribarra, L A; Taylor, K A; Spritzer, C E; Taylor, D C; Garrett, W E; Defrate, Louis E

    2013-01-01

    There are conflicting data regarding what motions increase ACL injury risk. More specifically, the mechanical role of valgus collapse positions during ACL injury remains controversial. Our objective was to evaluate ACL elongation in a model that mimics knee movements thought to occur during ACL injury. Eight healthy male subjects were imaged using MR and biplanar fluoroscopy to measure the in vivo elongation of the ACL and its functional bundles during three static knee positions: full extension, 30° of flexion, and a position intended to mimic a valgus collapse position described in the literature. For this study, the valgus collapse position consisted of 30° of knee flexion, internal rotation of the hip, and 10° of external tibial rotation. ACL length decreased significantly from full extension (30.2 ± 2.6 mm) to 30° of flexion (27.1 ± 2.2 mm). ACL length further decreased in the valgus collapse position (25.6 ± 2.4 mm). Both functional bundles of the ACL followed similar trends with regards to decreases in length in each of the three positions. Since strain would follow patterns of ACL length, landing on an extended knee may be a more relevant risk factor for ACL injuries than the valgus collapse position in males. Future studies should evaluate the effects of dynamic motion patterns on in vivo ACL strains.

  19. Effect of temperature on plant elongation and cell wall extensibility.

    PubMed

    Pietruszka, M; Lewicka, S

    2007-03-01

    Lockhart equation was derived for explaining plant cell expansion where both cell wall extension and water uptake must occur concomitantly. Its fundamental contribution was to express turgor pressure explicitly in terms of osmosis and wall mechanics. Here we present a new equation in which pressure is determined by temperature. It also accounts for the role of osmosis and consequently the role of water uptake in growing cell. By adopting literature data, we also attempt to report theoretically the close relation between plant elongation and cell wall extensibility. This is accomplished by the modified equation of growth solved for various temperatures in case of two different species. The results enable to interpret empirical data in terms of our model and fully confirm its applicability to the investigation of the problem of plant cell extensibility in function of environmental temperature. Moreover, by separating elastic effects from growth process we specified the characteristic temperature common for both processes which corresponds to the resonance energy of biochemical reactions as well as to the rapid softening of the elastic modes toward the high temperature end where we encountered viscoelastic and/or plastic behavior as dominating. By introducing analytical formulae connected with growth and elastic properties of the cell wall, we conclude with the statement how these both processes contribute quantitatively to the resonance-like shape of the elongation curve. In addition, the tension versus temperature "phase diagram" for a living plant cell is presented.

  20. Ancestral role of caudal genes in axis elongation and segmentation.

    PubMed

    Copf, Tijana; Schröder, Reinhard; Averof, Michalis

    2004-12-21

    caudal (cad/Cdx) genes are essential for the formation of posterior structures in Drosophila, Caenorhabditis elegans, and vertebrates. In contrast to Drosophila, the majority of arthropods generate their segments sequentially from a posteriorly located growth zone, a process known as short-germ development. caudal homologues are expressed in the growth zone of diverse short-germ arthropods, but until now their functional role in these animals had not been studied. Here, we use RNA interference to examine the function of caudal genes in two short-germ arthropods, the crustacean Artemia franciscana and the beetle Tribolium castaneum. We show that, in both species, caudal is required for the formation of most body segments. In animals with reduced levels of caudal expression, axis elongation stops, resulting in severe truncations that remove most trunk segments. We also show that caudal function is required for the early phases of segmentation and Hox gene expression. The observed phenotypes suggest that in arthropods caudal had an ancestral role in axis elongation and segmentation, and was required for the formation of most body segments. Similarities to the function of vertebrate Cdx genes in the presomitic mesoderm, from which somites are generated, indicate that this role may also predate the origin of the Bilateria.

  1. The Many Faces of Elongator in Neurodevelopment and Disease

    PubMed Central

    Kojic, Marija; Wainwright, Brandon

    2016-01-01

    Development of the nervous system requires a variety of cellular activities, such as proliferation, migration, axonal outgrowth and guidance and synapse formation during the differentiation of neural precursors into mature neurons. Malfunction of these highly regulated and coordinated events results in various neurological diseases. The Elongator complex is a multi-subunit complex highly conserved in eukaryotes whose function has been implicated in the majority of cellular activities underlying neurodevelopment. These activities include cell motility, actin cytoskeleton organization, exocytosis, polarized secretion, intracellular trafficking and the maintenance of neural function. Several studies have associated mutations in Elongator subunits with the neurological disorders familial dysautonomia (FD), intellectual disability (ID), amyotrophic lateral sclerosis (ALS) and rolandic epilepsy (RE). Here, we review the various cellular activities assigned to this complex and discuss the implications for neural development and disease. Further research in this area has the potential to generate new diagnostic tools, better prevention strategies and more effective treatment options for a wide variety of neurological disorders. PMID:27847465

  2. Cooperativity of peptidoglycan synthases active in bacterial cell elongation.

    PubMed

    Banzhaf, Manuel; van den Berg van Saparoea, Bart; Terrak, Mohammed; Fraipont, Claudine; Egan, Alexander; Philippe, Jules; Zapun, André; Breukink, Eefjan; Nguyen-Distèche, Martine; den Blaauwen, Tanneke; Vollmer, Waldemar

    2012-07-01

    Growth of the bacterial cell wall peptidoglycan sacculus requires the co-ordinated activities of peptidoglycan synthases, hydrolases and cell morphogenesis proteins, but the details of these interactions are largely unknown. We now show that the Escherichia coli peptidoglycan glycosyltrasferase-transpeptidase PBP1A interacts with the cell elongation-specific transpeptidase PBP2 in vitro and in the cell. Cells lacking PBP1A are thinner and initiate cell division later in the cell cycle. PBP1A localizes mainly to the cylindrical wall of the cell, supporting its role in cell elongation. Our in vitro peptidoglycan synthesis assays provide novel insights into the cooperativity of peptidoglycan synthases with different activities. PBP2 stimulates the glycosyltransferase activity of PBP1A, and PBP1A and PBP2 cooperate to attach newly synthesized peptidoglycan to sacculi. PBP2 has peptidoglycan transpeptidase activity in the presence of active PBP1A. Our data also provide a possible explanation for the depletion of lipid II precursors in penicillin-treated cells.

  3. Device for measuring hole elongation in a bolted joint

    NASA Technical Reports Server (NTRS)

    Wichorek, Gregory R. (Inventor)

    1987-01-01

    A device to determine the operable failure mode of mechanically fastened lightweight composite joints by measuring the hole elongation of a bolted joint is disclosed. The double-lap joint test apparatus comprises a stud, a test specimen having a hole, two load transfer plates, and linear displacement measuring instruments. The test specimen is sandwiched between the two load transfer plates and clamped together with the stud. Spacer washers are placed between the test specimen and each load transfer plate to provide a known, controllable area for the determination of clamping forces around the hole of the specimen attributable to bolt torque. The spacer washers also provide a gap for the mounting of reference angles on each side of the test specimen. Under tensile loading, elongation of the hole of the test specimen causes the stud to move away from the reference angles. This displacement is measured by the voltage output of two linear displacement measuring instruments that are attached to the stud and remain in contact with the reference angles throughout the tensile loading. The present invention obviates previous problems in obtaining specimen deformation measurements by monitoring the reference angles to the test specimen and the linear displacement measuring instruments to the stud.

  4. The interplay of stiffness and force anisotropies drives embryo elongation

    PubMed Central

    Vuong-Brender, Thanh Thi Kim; Ben Amar, Martine; Pontabry, Julien; Labouesse, Michel

    2017-01-01

    The morphogenesis of tissues, like the deformation of an object, results from the interplay between their material properties and the mechanical forces exerted on them. The importance of mechanical forces in influencing cell behaviour is widely recognized, whereas the importance of tissue material properties, in particular stiffness, has received much less attention. Using Caenorhabditis elegans as a model, we examine how both aspects contribute to embryonic elongation. Measuring the opening shape of the epidermal actin cortex after laser nano-ablation, we assess the spatiotemporal changes of actomyosin-dependent force and stiffness along the antero-posterior and dorso-ventral axis. Experimental data and analytical modelling show that myosin-II-dependent force anisotropy within the lateral epidermis, and stiffness anisotropy within the fiber-reinforced dorso-ventral epidermis are critical in driving embryonic elongation. Together, our results establish a quantitative link between cortical tension, material properties and morphogenesis of an entire embryo. DOI: http://dx.doi.org/10.7554/eLife.23866.001 PMID:28181905

  5. Initiation and elongation of lateral roots in Lactuca sativa

    NASA Technical Reports Server (NTRS)

    Zhang, N.; Hasenstein, K. H.

    1999-01-01

    Lactuca sativa cv. Baijianye seedlings do not normally produce lateral roots, but removal of the root tip or application of auxin, especially indole-butyric acid, triggered the formation of lateral roots. Primordia initiated within 9 h and were fully developed after 24 h by activating the pericycle cells opposite the xylem pole. The pericycle cells divided asymmetrically into short and long cells. The short cells divided further to form primordia. The effect of root tip removal and auxin application was reversed by 6-benzylaminopurine at concentrations >10(-8) M. The cytokinin oxidase inhibitor N1-(2chloro4pyridyl)-N2-phenylurea also suppressed auxin-induced lateral rooting. The elongation of primary roots was promoted by L-alpha-(2-aminoethoxyvinyl) glycine and silver ions, but only the latter enhanced elongation of lateral roots. The data indicate that the induction of lateral roots is controlled by basipetally moving cytokinin and acropetally moving auxin. Lateral roots appear to not produce ethylene.

  6. Application of an Elongated Kelvin Model to Space Shuttle Foams

    NASA Technical Reports Server (NTRS)

    Sullivan, Roy M.; Ghosn, Louis J.; Lerch, Bradley A.

    2009-01-01

    The space shuttle foams are rigid closed-cell polyurethane foams. The two foams used most-extensively oil space shuttle external tank are BX-265 and NCFL4-124. Because of the foaming and rising process, the foam microstructures are elongated in the rise direction. As a result, these two foams exhibit a nonisotropic mechanical behavior. A detailed microstructural characterization of the two foams is presented. Key features of the foam cells are described and the average cell dimensions in the two foams are summarized. Experimental studies are also conducted to measure the room temperature mechanical response of the two foams in the two principal material directions (parallel to the rise and perpendicular to the rise). The measured elastic modulus, proportional limit stress, ultimate tensile strength, and Poisson's ratios are reported. The generalized elongated Kelvin foam model previously developed by the authors is reviewed and the equations which result from this model are summarized. Using the measured microstructural dimensions and the measured stiffness ratio, the foam tensile strength ratio and Poisson's ratios are predicted for both foams and are compared with the experimental data. The predicted tensile strength ratio is in close agreement with the measured strength ratio for both BX-265 and NCFI24-124. The comparison between the predicted Poisson's ratios and the measured values is not as favorable.

  7. Mode-converted ultrasonic scattering in polycrystals with elongated grains.

    PubMed

    Arguelles, Andrea P; Kube, Christopher M; Hu, Ping; Turner, Joseph A

    2016-09-01

    Elastic wave scattering is used to study polycrystalline media for a wide range of applications. Received signals, which include scattering from the randomly oriented grains comprising the polycrystal, contain information from which useful microstructural parameters may often be inferred. Recently, a mode-converted diffuse ultrasonic scattering model was developed for evaluating the scattered response of a transverse wave from an incident longitudinal wave in a polycrystalline medium containing equiaxed single-phase grains with cubic elastic symmetry. In this article, that theoretical mode-converted scattering model is modified to account for grain elongation within the sample. The model shows the dependence on scattering angle relative to the grain axis orientation. Experimental measurements were performed on a sample of 7475-T7351 aluminum using a pitch-catch transducer configuration. The results show that the mode-converted scattering can be used to determine the dimensions of the elongated grains. The average grain shape determined from the experimental measurements is compared with dimensions extracted from electron backscatter diffraction, an electron imaging technique. The results suggest that mode-converted diffuse ultrasonic scattering has the potential to quantify detailed information about grain microstructure.

  8. Elongation of primed DNA templates by eukaryotic DNA polymerases.

    PubMed Central

    Ikeda, J E; Longiaru, M; Horwitz, M S; Hurwitz, J

    1980-01-01

    The combined action of DNA polymerase alpha and DNA polymerase beta leads to the synthesis of full-length linear DNA strands with phi X174 DNA templates containing an RNA primer. The reaction can be carried out in two stages. In the first stage, DNA polymerase alpha catalyzes the synthesis of a chain that averaged 230 deoxynucleotides long and was covalently linked to the RNA primer. In the second stage, DNA polymerase beta elongates the DNA strand covalently attached to the RNA primer to full length. With DNA primers, DNA polymerase alpha catalyzes only limited deoxynucleotide addition whereas DNA polymerase beta alone elongates DNA primed templates to full length. DNA polymerase beta can also stimulate the synthesis of adenovirus DNA in vitro in the presence of a cytosol extract from adenovirus-infected cells. In all of these systems, dNMP incorporation catalyzed by DNA polymerase beta was sensitive to N-ethylmaleimide; however, this polymerase activity was resistant to N-ethylmaleimide with poly(rA) x (dT) as the primer template. Images PMID:6160581

  9. Structural basis for transcription elongation by bacterial RNA polymerase.

    PubMed

    Vassylyev, Dmitry G; Vassylyeva, Marina N; Perederina, Anna; Tahirov, Tahir H; Artsimovitch, Irina

    2007-07-12

    The RNA polymerase elongation complex (EC) is both highly stable and processive, rapidly extending RNA chains for thousands of nucleotides. Understanding the mechanisms of elongation and its regulation requires detailed information about the structural organization of the EC. Here we report the 2.5-A resolution structure of the Thermus thermophilus EC; the structure reveals the post-translocated intermediate with the DNA template in the active site available for pairing with the substrate. DNA strand separation occurs one position downstream of the active site, implying that only one substrate at a time can specifically bind to the EC. The upstream edge of the RNA/DNA hybrid stacks on the beta'-subunit 'lid' loop, whereas the first displaced RNA base is trapped within a protein pocket, suggesting a mechanism for RNA displacement. The RNA is threaded through the RNA exit channel, where it adopts a conformation mimicking that of a single strand within a double helix, providing insight into a mechanism for hairpin-dependent pausing and termination.

  10. Strain and elongation of the human semitendinosus muscle - tendon unit.

    PubMed

    Kellis, Eleftherios; Patsika, Glykeria; Karagiannidis, Evaggelos

    2013-12-01

    The semitendinosus (ST) consists of a long distal tendon and it is divided in two parts by a tendinous inscription (TI). The purpose of this study was to quantify strain and elongation of the TI and the distal tendon of ST. Fourteen subjects performed ramp isometric contractions of the knee flexors at 0°, 45° and 90° of knee flexion. Two ultrasound probes were used to visualize the displacement of the distal tendon and selected points across the TI and aponeuroses. Three-way analysis of variance designs indicated that: (a) strain and elongation of the ST distal muscle-tendon junction were higher than that of the aponeurosis - TI junction points (p < 0.05) (b) the long arm of the TI reach strain of 49.86 ± 7.77% which was significantly (p < 0.05) higher than that displayed by the short arm (28.35 ± 0.59%) (c) Strain of tendinous and TI-aponeuroses segments significantly increased from 90° to 0° of knee flexion while the inverse was observed for the TI arm length (p < 0.05). (d) Tendon strain was significantly higher than strain of the TI-aponeuroses segments at 45° and 90° of knee flexion while the opposite was observed at 0° of knee flexion. The arrangement of TI along ST length results in differential local strains, indicating that the mechanical properties of the ST muscle are affected by tendon, aponeuroses and tendinous inscription interactions.

  11. Gibberellin biosynthesis and signal transduction is essential for internode elongation in deepwater rice

    PubMed Central

    Ayano, Madoka; Kani, Takahiro; Kojima, Mikiko; Sakakibara, Hitoshi; Kitaoka, Takuya; Kuroha, Takeshi; Angeles-Shim, Rosalyn B; Kitano, Hidemi; Nagai, Keisuke; Ashikari, Motoyuki

    2014-01-01

    Under flooded conditions, the leaves and internodes of deepwater rice can elongate above the water surface to capture oxygen and prevent drowning. Our previous studies showed that three major quantitative trait loci (QTL) regulate deepwater-dependent internode elongation in deepwater rice. In this study, we investigated the age-dependent internode elongation in deepwater rice. We also investigated the relationship between deepwater-dependent internode elongation and the phytohormone gibberellin (GA) by physiological and genetic approach using a QTL pyramiding line (NIL-1 + 3 + 12). Deepwater rice did not show internode elongation before the sixth leaf stage under deepwater condition. Additionally, deepwater-dependent internode elongation occurred on the sixth and seventh internodes during the sixth leaf stage. These results indicate that deepwater rice could not start internode elongation until the sixth leaf stage. Ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS) method for the phytohormone contents showed a deepwater-dependent GA1 and GA4 accumulation in deepwater rice. Additionally, a GA inhibitor abolished deepwater-dependent internode elongation in deepwater rice. On the contrary, GA feeding mimicked internode elongation under ordinary growth conditions. However, mutations in GA biosynthesis and signal transduction genes blocked deepwater-dependent internode elongation. These data suggested that GA biosynthesis and signal transduction are essential for deepwater-dependent internode elongation in deepwater rice. Deepwater rice obtained the ability for rapid internode elongation to avoid drowning and adapt to flooded condition. How does it regulate internode elongation? Using both physiological and genetic approach, this paper shows that the plant hormone, gibberellin (GA) regulates internode elongation. PMID:24891164

  12. CLASH: THE ENHANCED LENSING EFFICIENCY OF THE HIGHLY ELONGATED MERGING CLUSTER MACS J0416.1-2403

    SciTech Connect

    Zitrin, A.; Bartelmann, M.; Carrasco, M.; Meneghetti, M.; Umetsu, K.; Broadhurst, T.; Bradley, L.; Coe, D.; Koekemoer, A. M.; Postman, M.; Ford, H.; Medezinski, E.; Kelson, D.; Moustakas, J.; Moustakas, L. A.; Nonino, M.; Rosati, P.; Seidel, G.; Seitz, S.; and others

    2013-01-10

    We perform a strong lensing analysis of the merging galaxy cluster MACS J0416.1-2403 (M0416; z = 0.42) in recent CLASH/HST observations. We identify 70 new multiple images and candidates of 23 background sources in the range 0.7 {approx}< z{sub phot} {approx}< 6.14 including two probable high-redshift dropouts, revealing a highly elongated lens with axis ratio {approx_equal}5:1, and a major axis of {approx}100'' (z{sub s} {approx} 2). Compared to other well-studied clusters, M0416 shows an enhanced lensing efficiency. Although the critical area is not particularly large ({approx_equal} 0.6 {open_square}'; z{sub s} {approx} 2), the number of multiple images, per critical area, is anomalously high. We calculate that the observed elongation boosts the number of multiple images, per critical area, by a factor of {approx}2.5 Multiplication-Sign , due to the increased ratio of the caustic area relative to the critical area. Additionally, we find that the observed separation between the two main mass components enlarges the critical area by a factor of {approx}2. These geometrical effects can account for the high number (density) of multiple images observed. We find in numerical simulations that only {approx}4% of the clusters (with M{sub vir} {>=} 6 Multiplication-Sign 10{sup 14} h {sup -1} M{sub Sun }) exhibit critical curves as elongated as in M0416.

  13. Radiostereometric Evaluation of Tendon Elongation After Distal Biceps Repair

    PubMed Central

    Marshall, Nathan E.; Keller, Robert A.; Okoroha, Kelechi; Guest, John Michael; Yu, Charles; Muh, Stephanie; Moutzouros, Vasilios

    2016-01-01

    Background: Operative repair of distal biceps tendon ruptures has shown successful outcomes. However, little is known about the amount of tendon or repair site lengthening after repair. Purpose/Hypothesis: The purpose of this study was to evaluate distal biceps tendon repair via intratendinous radiostereometric analysis to analyze tendon lengthening at different time intervals of healing. The hypothesis was that there is significant lengthening after repair. Study Design: Case series; Level of evidence, 4. Methods: Eleven patients with distal biceps ruptures requiring operative repair were recruited. During repair, two 2-mm tantalum beads with laser-etched holes were sutured to the distal biceps tendon. Beads were evaluated via computed tomography scans immediately postoperatively and at 16 weeks. Radiographs were obtained at time 0 and then at 4, 8, and 16 weeks postoperatively. Measurements were made using the button-to-bead and bead-to-bead distances to assess repair site elongation as well as tendon elongation over time. After final follow-up, patients filled out the Disabilities of the Arm, Shoulder, and Hand (DASH) questionnaire and underwent ultrasound to confirm the integrity of the tendon. Results: Ten patients had complete ruptures, with 1 having a partial rupture that underwent completion of the tear and subsequent repair. All patients showed statistically significant lengthening after surgery. The mean amount of tendon lengthening after surgery was 22.8 mm (range, 11.2-30.9 mm; P < .05), and the repair site lengthened a mean 17.0 mm (range, 9.6-30.6 mm; P < .05) from surgery to final follow-up. The greatest change in lengthening was noted between time 0 and week 4 (mean, 11.3 mm; P < .05), with the least amount of lengthening between weeks 8 and 16 (mean, 2.6 mm; P < .05). The mean DASH score was 11.2. Final ultrasound evaluations found all tendons to be in continuity. Conclusion: All patients undergoing distal biceps tendon repair have significant

  14. Longitudinal domain wall formation in elongated assemblies of ferromagnetic nanoparticles

    PubMed Central

    Varón, Miriam; Beleggia, Marco; Jordanovic, Jelena; Schiøtz, Jakob; Kasama, Takeshi; Puntes, Victor F.; Frandsen, Cathrine

    2015-01-01

    Through evaporation of dense colloids of ferromagnetic ~13 nm ε-Co particles onto carbon substrates, anisotropic magnetic dipolar interactions can support formation of elongated particle structures with aggregate thicknesses of 100–400 nm and lengths of up to some hundred microns. Lorenz microscopy and electron holography reveal collective magnetic ordering in these structures. However, in contrast to continuous ferromagnetic thin films of comparable dimensions, domain walls appear preferentially as longitudinal, i.e., oriented parallel to the long axis of the nanoparticle assemblies. We explain this unusual domain structure as the result of dipolar interactions and shape anisotropy, in the absence of inter-particle exchange coupling. PMID:26416297

  15. Intermediate electrostatic field for the generalized elongation method.

    PubMed

    Liu, Kai; Korchowiec, Jacek; Aoki, Yuriko

    2015-05-18

    An intermediate electrostatic field is introduced to improve the accuracy of fragment-based quantum-chemical computational methods by including long-range polarizations of biomolecules. The point charge distribution of the intermediate field is generated by a charge sensitivity analysis that is parameterized for five different population analyses, namely, atoms-in-molecules, Hirshfeld, Mulliken, natural orbital, and Voronoi population analysis. Two model systems are chosen to demonstrate the performance of the generalized elongation method (ELG) combined with the intermediate electrostatic field. The calculations are performed for the STO-3G, 6-31G, and 6-31G(d) basis sets and compared with reference Hartree-Fock calculations. It is shown that the error in the total energy is reduced by one order of magnitude, independently of the population analyses used. This demonstrates the importance of long-range polarization in electronic-structure calculations by fragmentation techniques.

  16. The barberplaid illusion: plaid motion is biased by elongated apertures

    NASA Technical Reports Server (NTRS)

    Beutter, B. R.; Mulligan, J. B.; Stone, L. S.

    1996-01-01

    The perceived direction of motion of plaids windowed by elongated spatial Gaussians is biased toward the window's long axis. The bias increases as the relative angle between the plaid motion and the long axis of the window increases, peaks at a relative angle of approximately 45 deg, and then decreases. The bias increases as the window is made narrower (at fixed height) and decreases as the component spatial frequency increases (at fixed aperture size). We examine several models of human motion processing (cross-correlation, motion-energy, intersection-of-constraints, and vector-sum), and show that none of these standard models can predict our data. We conclude that spatial integration of motion signals plays a crucial role in plaid motion perception and that current models must be explicitly expanded to include such spatial interactions.

  17. Reconstruction of recurrent diaphragmatic eventration with an elongated polytetrafluoroethylene sheet

    PubMed Central

    Ikeda, Masaki; Sonobe, Makoto; Bando, Toru; Date, Hiroshi

    2013-01-01

    We report the case of a 31-year old woman with recurrence of left diaphragmatic eventration 3 years after a previous surgery for this condition. At the initial occurrence, she had experienced dyspnoea on exercise and subsequently underwent laparoscopic plication of the diaphragm with an endo-stapler at a local hospital. Immediately after the operation, the diaphragm was torn and the intestine entered the thorax. Therefore, plication involving sewing was performed. Then, 3 years later, the patient again experienced dyspnoea and was diagnosed as having recurrence of left diaphragmatic eventration. Observation under thoracoscopy revealed that the centre of the left diaphragm was thin but not torn. We reconstructed the left diaphragm with an elongated polytetrafluoroethylene sheet on the naïve diaphragm. The patient was discharged from our hospital 5 days after surgery. Her respiratory function improved and she has not experienced recurrence. PMID:23644727

  18. Reconstruction of recurrent diaphragmatic eventration with an elongated polytetrafluoroethylene sheet.

    PubMed

    Ikeda, Masaki; Sonobe, Makoto; Bando, Toru; Date, Hiroshi

    2013-08-01

    We report the case of a 31-year old woman with recurrence of left diaphragmatic eventration 3 years after a previous surgery for this condition. At the initial occurrence, she had experienced dyspnoea on exercise and subsequently underwent laparoscopic plication of the diaphragm with an endo-stapler at a local hospital. Immediately after the operation, the diaphragm was torn and the intestine entered the thorax. Therefore, plication involving sewing was performed. Then, 3 years later, the patient again experienced dyspnoea and was diagnosed as having recurrence of left diaphragmatic eventration. Observation under thoracoscopy revealed that the centre of the left diaphragm was thin but not torn. We reconstructed the left diaphragm with an elongated polytetrafluoroethylene sheet on the naïve diaphragm. The patient was discharged from our hospital 5 days after surgery. Her respiratory function improved and she has not experienced recurrence.

  19. Content-Based Retrieval of Medical Images with Elongated Structures

    NASA Astrophysics Data System (ADS)

    Machado, Alexei Manso Correa; Teixeira, Christiano Augusto Caldas

    In this paper we propose a set of methods to describe, register and retrieve images of elongated structures from a database based on their shape content. Registration is performed based on an elastic matching algorithm that jointly takes into account the gross shape of the structure and the shape of its boundary, resulting in anatomically consistent deformations. The method determines a medial axis that represents the full extent of the structure with no branches. Discriminative anatomic features are computed from the results of registration and used as variables in a content-based image retrieval system. A case study on the morphology of the corpus callosum in the chromosome 22q11.2 deletion syndrome illustrates the effectiveness of the method and corroborates the hypothesis that retrieval systems may also act as knowledge discovery tools.

  20. Laser diffraction particle sizing: Instrument probe volume relocation and elongation

    NASA Technical Reports Server (NTRS)

    Anderson, Robert C.; Buchele, Donald R.; Hovenac, Edward A.; Lock, James A.

    1990-01-01

    The effective probe volume of laser diffraction particle sizing instruments depends on many instrument parameters. In particular the probe volume axial boundaries and its location along laser beam are essentially defined by the onset of a vignetting effect where light scattered at large angles from small particles misses the transform lens. This vignetting effect results in a probe volume that must be inconveniently close to the lens in order to detect smaller diameter particles (less than 100 micrometers). With the addition of an appropriately designed Keplerian telescope, the probe volume may be relocated and elongated. The theory of operation of this supplemental optical system is described. Design considerations for these supplemental optical systems are described, including recommendations for lens specifications, assembly and use. An image transfer system is described which has been designed for use on a Malvern 2600HSD instrument. Experimental validation of this image transfer system is described.

  1. Gibberellin-Stimulation of Rhizome Elongation and Differential GA-Responsive Proteomic Changes in Two Grass Species

    PubMed Central

    Ma, Xiqing; Huang, Bingru

    2016-01-01

    Rapid and extensive rhizome development is a desirable trait for perennial grass growth and adaptation to environmental stresses. The objective of this study was to determine proteomic changes and associated metabolic pathways of gibberellin (GA) -regulation of rhizome elongation in two perennial grass species differing in rhizome development. Plants of a short-rhizome bunch-type tall fescue (TF; Festuca arundinacea; ‘BR’) and an extensive rhizomatous Kentucky bluegrass (KB; Poa pratensis; ‘Baron’) were treated with 10 μM GA3 in hydroponic culture in growth chambers. The average rhizome length in KB was significantly longer than that in TF regardless of GA3 treatment, and increased significantly with GA3 treatment, to a greater extent than that in TF. Comparative proteomic analysis using two-dimensional electrophoresis and mass spectrometry was performed to further investigate proteins and associated metabolic pathways imparting increased rhizome elongation by GA. A total of 37 and 38 differentially expressed proteins in response to GA3 treatment were identified in TF and KB plants, respectively, which were mainly involved in photosynthesis, energy and amino acid metabolism, protein synthesis, defense and cell development processes. Accelerated rhizome elongation in KB by GA could be mainly associated with the increased abundance of proteins involved in energy metabolism (glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase, and ATP synthase), amino acid metabolism (S-adenosylmethionine and adenosylhomocysteinase), protein synthesis (HSP90, elongation factor Tu and eukaryotic translation initiation factor 5A), cell-wall development (cell dividion cycle protein, alpha tubulin-2A and actin), and signal transduction (calreticulin). These proteins could be used as candidate proteins for further analysis of molecular mechanisms controlling rhizome growth. PMID:27446135

  2. Contraction/elongation behavior of cation-modified polyacrylonitrile fibers

    NASA Astrophysics Data System (ADS)

    Kim, Kwang J.; Caligiuri, Jerusha; Choe, Kiyoug; Shahinpoor, Mohsen

    2003-07-01

    In this paper, the authors present the contraction/elongation behavior of cation-modified Polyacrylonitrile (PAN) fibers, which identifies the fibers to be effectively used as biomimetic actuators and artifical muscles. The research was intiated by realizing that the contraction/elongation behavior of PAN is governed by the diffusional processes of ions/solvents interaction. The PAN fibers were suitably annealed, cross-linked and hydrolyzed to become "active". The cation-modified process was performed using KOH, NaOH, and LiOH, respectively, for the boiling and alkaline-soaking mediums. It was found that the PAN fibers, regardless of whether being activated in KOH, NaOH, or LiOH, increased from their initial length after being activated and soaked in distilled water. Lengths then decreased after the fibers were soaked in the bases. Fibers treated with LiOH had the largest increase in length following immersion in distilled water. Fibers soaked in any of the three mediums generally had the same decrease in length following immersion in the alkaline solutions, as also occurred following immersion in HCl. Especially noticeable with the fibers treated with LiOH was that greater displacement in the lengths occurred using the 2 N solutions. It is our general notion that the Osmotic pressure of free ions plays an import role on the properties of PAN. However, the observation that Li+ treated PAN fibers exhibit the largest contraction/expansion capability compared to Na+ or K+ treated PANs, can raise another important issue, i.e. "hydration". Realizing that the Osmotic pressure of electrolyte systems in weakly dependent upon the types of ions, it is highly likely that the "hydration" phenomena of free ions within the PAN network plays a key role on its deformation properties. It should be noted that PAN fibers have the capability of changing their effective longitudinal strain more than 100% and have comparable strength to human muscle.

  3. Elongational rheology and cohesive fracture of photo-oxidated LDPE

    SciTech Connect

    Rolón-Garrido, Víctor H. Wagner, Manfred H.

    2014-01-15

    It was found recently that low-density polyethylene (LDPE) samples with different degrees of photo-oxidation represent an interesting system to study the transition from ductile to cohesive fracture and the aspects of the cohesive rupture in elongational flow. Sheets of LDPE were subjected to photo-oxidation in the presence of air using a xenon lamp to irradiate the samples for times between 1 day and 6 weeks. Characterisation methods included Fourier transform infrared spectroscopy, solvent extraction method, and rheology in shear and uniaxial extensional flows. Linear viscoelasticity was increasingly affected by increasing photo-oxidation due to crosslinking of LDPE, as corroborated by the carbonyl index, acid and aldehydes groups, and gel fraction. The molecular stress function model was used to quantify the experimental data, and the nonlinear model parameter β was found to be correlated with the gel content. The uniaxial data showed that the transition from ductile to cohesive fracture was shifted to lower elongational rates, the higher the gel content was. From 2 weeks photo-oxidation onwards, cohesive rupture occurred at every strain rate investigated. The true strain and true stress at cohesive fracture as well as the energy density applied to the sample up to fracture were analyzed. At low gel content, rupture was mainly determined by the melt fraction while at high gel content, rupture occurred predominantly in the gel structure. The strain at break was found to be independent of strain rate, contrary to the stress at break and the energy density. Thus, the true strain and not the stress at break or the energy density was found to be the relevant physical quantity to describe cohesive fracture behavior of photo-oxidated LDPE. The equilibrium modulus of the gel structures was correlated with the true strain at rupture. The stiffer the gel structure, the lower was the deformation tolerated before the sample breaks.

  4. Prevalence and pattern of the elongated styloid process among geriatric patients in Saudi Arabia

    PubMed Central

    AlZarea, Bader K

    2017-01-01

    Objective The objectives of this study were to assess the elongation of the styloid process on digital panoramic radiographs and to evaluate the prevalence of the elongation according to age, sex and types. Patients and methods Digital panoramic radiographs of 198 geriatric edentulous patients were analyzed. The radiographic length of the styloid process was measured on both sides using the measurement toolbars on the accompanying analysis software. For statistical analysis, Student’s t-test, chi-square test and analysis of variance test were used. Results The elongated styloid process was seen in 87 of the 198 patients. It was observed that as age increased, elongation of the styloid process increased with a male predominance. Type I elongation was more common than other types of elongation. Bilateral elongation was most commonly found than unilateral elongation, and both types of elongation were frequently seen in males compared with females. Conclusion The prevalence of the elongated styloid process in the present study was higher in comparison to the other reports from edentulous patients.

  5. Dilution of individual microtubules observed in real time in vitro: evidence that cap size is small and independent of elongation rate

    PubMed Central

    1991-01-01

    Although the mechanism of microtubule dynamic instability is thought to involve the hydrolysis of tubulin-bound GTP, the mechanism of GTP hydrolysis and the basis of microtubule stability are controversial. Video microscopy of individual microtubules and dilution protocols were used to examine the size and lifetime of the stabilizing cap. Purified porcine brain tubulin (7-23 microM) was assembled at 37 degrees C onto both ends of isolated sea urchin axoneme fragments in a miniature flow cell to give a 10-fold variation in elongation rate. The tubulin concentration in the region of microtubule growth could be diluted rapidly (by 84% within 3 s of the onset of dilution). Upon perfusion with buffer containing no tubulin, microtubules experienced a catastrophe (conversion from elongation to rapid shortening) within 4-6 s on average after dilution to 16% of the initial concentration, independent of the predilution rate of elongation and length. Based on extrapolation of catastrophe frequency to zero tubulin concentration, the estimated lifetime of the stable cap after infinite dilution was less than 3-4 s for plus and minus ends, much shorter than the approximately 200 s observed at steady state (Walker, R. A., E. T. O'Brien, N. K. Pryer, M. Soboeiro, W. A. Voter, H. P. Erickson, and E. D. Salmon. 1988. J. Cell Biol. 107:1437-1448.). We conclude that during elongation, both plus and minus ends are stabilized by a short region (approximately 200 dimers or less) and that the size of the stable cap is independent of 10-fold variation in elongation rate. These results eliminate models of dynamic instability which predict extensive "build- up" stabilizing caps and support models which constrain the cap to the elongating tip. We propose that the cell may take advantage of such an assembly mechanism by using "catastrophe factors" that can promote frequent catastrophe even at high elongation rates by transiently binding to microtubule ends and briefly inhibiting GTP

  6. Coping with asymmetry: How infants and adults walk with one elongated leg

    PubMed Central

    Cole, Whitney G.; Gill, Simone V.; Vereijken, Beatrix; Adolph, Karen E.

    2014-01-01

    The stability of a system affects how it will handle a perturbation: The system may compensate for the perturbation or not. This study examined how 14-month-old infants—notoriously unstable walkers—and adults cope with a perturbation to walking. We attached a platform to one of participants' shoes, forcing them to walk with one elongated leg. At first, the platform shoe caused both age groups to slow down and limp, and caused infants to misstep and fall. But after a few trials, infants altered their gait to compensate for the platform shoe whereas adults did not; infants recovered symmetrical gait whereas adults continued to limp. Apparently, adult walking was stable enough to cope with the perturbation, but infants risked falling if they did not compensate. Compensation depends on the interplay of multiple factors: The availability of a compensatory response, the cost of compensation, and the stability of the system being perturbed. PMID:24857934

  7. Identification of Elongated Primary Cilia with Impaired Mechanotransduction in Idiopathic Scoliosis Patients

    PubMed Central

    Oliazadeh, Niaz; Gorman, Kristen F.; Eveleigh, Robert; Bourque, Guillaume; Moreau, Alain

    2017-01-01

    The primary cilium is an outward projecting antenna-like organelle with an important role in bone mechanotransduction. The capacity to sense mechanical stimuli can affect important cellular and molecular aspects of bone tissue. Idiopathic scoliosis (IS) is a complex pediatric disease of unknown cause, defined by abnormal spinal curvatures. We demonstrate significant elongation of primary cilia in IS patient bone cells. In response to mechanical stimulation, these IS cells differentially express osteogenic factors, mechanosensitive genes, and signaling genes. Considering that numerous ciliary genes are associated with a scoliosis phenotype, among ciliopathies and knockout animal models, we expected IS patients to have an accumulation of rare variants in ciliary genes. Instead, our SKAT-O analysis of whole exomes showed an enrichment among IS patients for rare variants in genes with a role in cellular mechanotransduction. Our data indicates defective cilia in IS bone cells, which may be linked to heterogeneous gene variants pertaining to cellular mechanotransduction. PMID:28290481

  8. Three-dimensional structure of photosystem II from Thermosynechococcus elongates in complex with terbutryn

    SciTech Connect

    Gabdulkhakov, A. G. Dontsova, M. V.; Saenger, W.

    2011-11-15

    Photosystem II is a key component of the photosynthetic pathway producing oxygen at the thylakoid membrane of cyanobacteria, green algae, and plants. The three-dimensional structure of photosystem II from the cyanobacterium Thermosynechococcus elongates in a complex with herbicide terbutryn (a photosynthesis inhibitor) was determined for the first time by X-ray diffraction and refined at 3.2 Angstrom-Sign resolution (R{sub factor} = 26.9%, R{sub free} = 29.9%, rmsd for bond lengths is 0.013 Angstrom-Sign , and rmsd for bond angles is 2.2 Degree-Sign ). The terbutryn molecule was located in the binding pocket of the mobile plastoquinone. The atomic coordinates of the refined structure of photosystem II in a complex with terbutryn were deposited in the Protein Data Bank.

  9. Defective Dendrite Elongation but Normal Fertility in Mice Lacking the Rho-Like GTPase Activator Dbl

    PubMed Central

    Hirsch, Emilio; Pozzato, Michela; Vercelli, Alessandro; Barberis, Laura; Azzolino, Ornella; Russo, Chiara; Vanni, Cristina; Silengo, Lorenzo; Eva, Alessandra; Altruda, Fiorella

    2002-01-01

    Dbl is the prototype of a large family of GDP-GTP exchange factors for small GTPases of the Rho family. In vitro, Dbl is known to activate Rho and Cdc42 and to induce a transformed phenotype. Dbl is specifically expressed in brain and gonads, but its in vivo functions are largely unknown. To assess its role in neurogenesis and gametogenesis, targeted deletion of the murine Dbl gene was accomplished in embryonic stem cells. Dbl-null mice are viable and did not show either decreased reproductive performances or obvious neurological defects. Histological analysis of mutant testis showed normal morphology and unaltered proliferation and survival of spermatogonia. Dbl-null brains indicated a correct disposition of the major neural structures. Analysis of cortical stratification indicated that Dbl is not crucial for neuronal migration. However, in distinct populations of Dbl-null cortical pyramidal neurons, the length of dendrites was significantly reduced, suggesting a role for Dbl in dendrite elongation. PMID:11940671

  10. Mitotic Transcriptional Activation: Clearance of Actively Engaged Pol II via Transcriptional Elongation Control in Mitosis.

    PubMed

    Liang, Kaiwei; Woodfin, Ashley R; Slaughter, Brian D; Unruh, Jay R; Box, Andrew C; Rickels, Ryan A; Gao, Xin; Haug, Jeffrey S; Jaspersen, Sue L; Shilatifard, Ali

    2015-11-05

    Although it is established that some general transcription factors are inactivated at mitosis, many details of mitotic transcription inhibition (MTI) and its underlying mechanisms are largely unknown. We have identified mitotic transcriptional activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MTI. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MTI whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division.

  11. The Acid Growth Theory of auxin-induced cell elongation is alive and well

    NASA Technical Reports Server (NTRS)

    Rayle, D. L.; Cleland, R. E.

    1992-01-01

    Plant cells elongate irreversibly only when load-bearing bonds in the walls are cleaved. Auxin causes the elongation of stem and coleoptile cells by promoting wall loosening via cleavage of these bonds. This process may be coupled with the intercalation of new cell wall polymers. Because the primary site of auxin action appears to be the plasma membrane or some intracellular site, and wall loosening is extracellular, there must be communication between the protoplast and the wall. Some "wall-loosening factor" must be exported from auxin-impacted cells, which sets into motion the wall loosening events. About 20 years ago, it was suggested that the wall-loosening factor is hydrogen ions. This idea and subsequent supporting data gave rise to the Acid Growth Theory, which states that when exposed to auxin, susceptible cells excrete protons into the wall (apoplast) at an enhanced rate, resulting in a decrease in apoplastic pH. The lowered wall pH then activates wall-loosening processes, the precise nature of which is unknown. Because exogenous acid causes a transient (1-4 h) increase in growth rate, auxin must also mediate events in addition to wall acidification for growth to continue for an extended period of time. These events may include osmoregulation, cell wall synthesis, and maintenance of the capacity of walls to undergo acid-induced wall loosening. At present, we do not know if these phenomena are tightly coupled to wall acidification or if they are the products of multiple independent signal transduction pathways.

  12. Elongation kinetics of polyglutamine peptide fibrils: a quartz crystal microbalance with dissipation study.

    PubMed

    Walters, Robert H; Jacobson, Kurt H; Pedersen, Joel A; Murphy, Regina M

    2012-08-10

    Abnormally expanded polyglutamine domains in proteins are associated with several neurodegenerative diseases, including Huntington's disease. Expansion of the polyglutamine (polyQ) domain facilitates aggregation of the affected protein, and several studies directly link aggregation to neurotoxicity. Studies of synthetic polyQ peptides have contributed substantially to our understanding of the mechanism of aggregation. In this report, polyQ fibrils were immobilized onto a sensor, and their elongation by polyQ peptides of various length and conformation was examined using quartz crystal microbalance with dissipation monitoring (QCM-D). The rate of elongation increased as the peptide length increased from 8 to 24 glutamines (Q8, Q20, and Q24). Monomer conformation affected elongation rates: insertion of a β-turn template d-Pro-Gly in the center of the peptide increased elongation rates several-fold, while insertion of Pro-Pro dramatically slowed elongation. Dissipation measurements of the QCM-D provided qualitative information about mechanical properties of the elongating fibrils. These data showed clear differences in the characteristics of the elongating aggregates, depending on the specific identity of the associating polyQ peptide. Elongation rates were sensitive to the pH and ionic strength of the buffer. Comparison of QCM-D data with those obtained by optical waveguide lightmode spectroscopy revealed that very little water was associated with the elongation of fibrils by the peptide containing d-Pro-Gly, but a significant amount of water was associated when the fibrils were elongated by Q20. Together, the data indicate that elongation of polyQ fibrils can occur without full consolidation to the fibril structure, resulting in variations to the aggregate structure during elongation.

  13. The dual functions of WLIM1a in cell elongation and secondary wall formation in developing cotton fibers.

    PubMed

    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

    2013-11-01

    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.

  14. Elongation and Termination Reactions of Protein Synthesis on Maize Root Tip Polyribosomes Studied in a Homologous Cell-Free System 1

    PubMed Central

    Webster, Cecelia; Kim, Chang-Yub; Roberts, Justin K. M.

    1991-01-01

    We show that the control of gene expression at the level of elongation and termination of protein synthesis can be observed in vitro. Free cytoplasmic polyribosomes were isolated from maize (Zea mays) root tips, and translated in root tip extracts that had been fractionated with ammonium sulfate to contain elongation factors, and be depleted in initiation factors. The root tip extract performs elongation and termination reactions as efficiently as wheat germ extracts. The translation products of the maize system are the same as made in vivo. The dependence of these in vitro elongation and termination reactions on pH was determined. Total protein synthesis in this system exhibits an optimum at pH ∼7.5. However, the pH dependence of rates of synthesis of individual proteins is not at all uniform; many polyribosomes become stalled when translated at low pH. These data were compared with the elongation and termination capacity of polyribosomes isolated from oxygenated and hypoxic root tips (tissue having, respectively, high and low cytoplasmic pH values). We observed an inverse relationship between the relative abundance of many specific translatable mRNAs in polyribosomes of hypoxic root tips, and the relative rates of elongation and termination reactions on the different mRNAs at low pH in vitro. These results suggest that changes in intracellular pH in hypoxic root tips can be sensed directly by the translational machinery and thereby selectively modulate gene expression. ImagesFigure 2Figure 4Figure 5Figure 6Figure 7 PMID:16668202

  15. Elongator function in tRNA wobble uridine modification is conserved between yeast and plants

    PubMed Central

    Mehlgarten, Constance; Jablonowski, Daniel; Wrackmeyer, Uta; Tschitschmann, Susan; Sondermann, David; Jäger, Gunilla; Gong, Zhizhong; Byström, Anders S; Schaffrath, Raffael; Breunig, Karin D

    2010-01-01

    Based on studies in yeast and mammalian cells the Elongator complex has been implicated in functions as diverse as histone acetylation, polarized protein trafficking and tRNA modification. Here we show that Arabidopsis mutants lacking the Elongator subunit AtELP3/ELO3 have a defect in tRNA wobble uridine modification. Moreover, we demonstrate that yeast elp3 and elp1 mutants expressing the respective Arabidopsis Elongator homologues AtELP3/ELO3 and AtELP1/ELO2 assemble integer Elongator complexes indicating a high degree of structural conservation. Surprisingly, in vivo complementation studies based on Elongator-dependent tRNA nonsense suppression and zymocin tRNase toxin assays indicated that while AtELP1 rescued defects of a yeast elp1 mutant, the most conserved Elongator gene AtELP3, failed to complement an elp3 mutant. This lack of complementation is due to incompatibility with yeast ELP1 as coexpression of both plant genes in an elp1 elp3 yeast mutant restored Elongator's tRNA modification function in vivo. Similarly, AtELP1, not ScELP1 also supported partial complementation by yeast–plant Elp3 hybrids suggesting that AtElp1 has less stringent sequence requirements for Elp3 than ScElp1. We conclude that yeast and plant Elongator share tRNA modification roles and propose that this function might be conserved in Elongator from all eukaryotic kingdoms of life. PMID:20398216

  16. Micro- and Nanoscale Capacitors that Incorporate an Array of Conductive Elements Having Elongated Bodies

    NASA Technical Reports Server (NTRS)

    Manohara, Harish (Inventor); Del Castillo, Linda Y. (Inventor); Mojarradi, Mohammed M. (Inventor)

    2016-01-01

    Systems and methods in accordance with embodiments of the invention implement micro- and nanoscale capacitors that incorporate a conductive element that conforms to the shape of an array elongated bodies. In one embodiment, a capacitor that incorporates a conductive element that conforms to the shape of an array of elongated bodies includes: a first conductive element that conforms to the shape of an array of elongated bodies; a second conductive element that conforms to the shape of an array of elongated bodies; and a dielectric material disposed in between the first conductive element and the second conductive element, and thereby physically separates them.

  17. Fitness consequences of natural variation in flooding-induced shoot elongation in Rumex palustris.

    PubMed

    Chen, Xin; Visser, Eric J W; de Kroon, Hans; Pierik, Ronald; Voesenek, Laurentius A C J; Huber, Heidrun

    2011-04-01

    • Plants can respond to their environment by morphological plasticity. Generally, the potential benefits of adaptive plastic responses are beyond doubt under predictable environmental changes. However, the net benefits may be less straightforward when plants encounter temporal stresses, such as flooding in river flood plains. • Here, we tested whether the balance of costs and benefits associated with flooding-induced shoot elongation depends on the flooding regime, by subjecting Rumex palustris plants with different elongation capacity to submergence of different frequency and duration. • Our results showed that reaching the surface by shoot elongation is associated with fitness benefits, as under less frequent, but longer, flooding episodes plants emerging above the floodwater had greater biomass production than plants that were kept below the surface. As we predicted, slow-elongating plants had clear advantages over fast-elongating ones if submergence was frequent but of short duration, indicating that elongation also incurs costs. • Our data suggest that high costs select for weak plasticity under frequent environmental change. In contrast to our predictions, however, fast-elongating plants did not have an overall advantage over slow-elongating plants when floods lasted longer. This indicates that the delicate balance between benefits and costs of flooding-induced elongation depends on the specific characteristics of the flooding regime.

  18. Application of an Elongated Kelvin Model to Space Shuttle Foams

    NASA Technical Reports Server (NTRS)

    Sullivan, Roy M.; Ghosn, Louis J.; Lerch, Bradley A.

    2008-01-01

    Spray-on foam insulation is applied to the exterior of the Space Shuttle s External Tank to limit propellant boil-off and to prevent ice formation. The Space Shuttle foams are rigid closed-cell polyurethane foams. The two foams used most extensively on the Space Shuttle External Tank are BX-265 and NCFI24-124. Since the catastrophic loss of the Space Shuttle Columbia, numerous studies have been conducted to mitigate the likelihood and the severity of foam shedding during the Shuttle s ascent to space. Due to the foaming and rising process, the foam microstructures are elongated in the rise direction. As a result, these two foams exhibit a non-isotropic mechanical behavior. In this paper, a detailed microstructural characterization of the two foams is presented. The key features of the foam cells are summarized and the average cell dimensions in the two foams are compared. Experimental studies to measure the room temperature mechanical response of the two foams in the two principal material directions (parallel to the rise and perpendicular to the rise) are also reported. The measured elastic modulus, proportional limit stress, ultimate tensile stress and the Poisson s ratios for the two foams are compared. The generalized elongated Kelvin foam model previously developed by the authors is reviewed and the equations which result from this model are presented. The resulting equations show that the ratio of the elastic modulus in the rise direction to that in the perpendicular-to-rise direction as well as the ratio of the strengths in the two material directions is only a function of the microstructural dimensions. Using the measured microstructural dimensions and the measured stiffness ratio, the foam tensile strength ratio and Poisson s ratios are predicted for both foams. The predicted tensile strength ratio is in close agreement with the measured strength ratios for both BX-265 and NCFI24-124. The comparison between the predicted Poisson s ratios and the measured

  19. Suppression of translucent elongated structures: applications in chest radiography.

    PubMed

    Hogeweg, Laurens; Sanchez, Clara I; van Ginneken, Bram

    2013-11-01

    Projection images, such as those routinely acquired in radiological practice, are difficult to analyze because multiple 3-D structures superimpose at a single point in the 2-D image. Removal of particular superimposed structures may improve interpretation of these images, both by humans and by computers. This work therefore presents a general method to isolate and suppress structures in 2-D projection images. The focus is on elongated structures, which allows an intensity model of a structure of interest to be extracted using local information only. The model is created from profiles sampled perpendicular to the structure. Profiles containing other structures are detected and removed to reduce the influence on the model. Subspace filtering, using blind source separation techniques, is applied to separate the structure to be suppressed from other structures. By subtracting the modeled structure from the original image a structure suppressed image is created. The method is evaluated in four experiments. In the first experiment ribs are suppressed in 20 artificial radiographs simulated from 3-D lung computed tomography (CT) images. The proposed method with blind source separation and outlier detection shows superior suppression of ribs in simulated radiographs, compared to a simplified approach without these techniques. Additionally, the ability of three observers to discriminate between patches containing ribs and containing no ribs, as measured by the area under the rece