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Sample records for rna cap structure

  1. Systematic identification of non-coding RNA 2,2,7-trimethylguanosine cap structures in Caenorhabditis elegans

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    Skogerbø Geir

    2007-09-01

    Full Text Available Abstract Background The 2,2,7-trimethylguanosine (TMG cap structure is an important functional characteristic of ncRNAs with critical cellular roles, such as some snRNAs. Here we used immunoprecipitation with both K121 and R1131 anti-TMG antibodies to systematically identify the TMG cap structures for all presently characterized ncRNAs in C. elegans. Results The two anti-TMG antibodies precipitated a similar group of the C. elegans ncRNAs. All snRNAs known to have a TMG cap structure were found in the precipitate, indicating that our identification system was efficient. Other ncRNA families related to splicing, such as SL RNAs and Sm Y RNAs, were also found in the precipitate, as were 7 C/D box snoRNAs. Further analysis showed that the SL RNAs and the Sm Y RNAs shared a very similar Sm binding site element (AAU4–5GGA, which sequence composition differed somewhat from those of other U snRNAs. There were also 16 ncRNAs without an Sm binding site element in the precipitate, suggesting that for these ncRNAs, TMG formation may occur independently of Sm proteins. Conclusion Our results showed that most ncRNAs predicted to be transcribed by RNA polymerase II had a TMG cap, while those predicted to be transcribed by RNA plymerase III or located in introns did not have a TMG cap structure. Compared to ncRNAs without a TMG cap, TMG-capped ncRNAs tended to have higher expression levels. Five functionally non-annotated ncRNAs also have a TMG cap structure, which might be helpful for identifying the cellular roles of these ncRNAs.

  2. Labeling of eukaryotic messenger RNA 5' terminus with phosphorus -32: use of tobacco acid pyrophosphatase for removal of cap structures

    International Nuclear Information System (INIS)

    Lockard, R.E.; Rieser, L.; Vournakis, J.N.

    1981-01-01

    In recent years, there has been a growing appreciation of the potential applications of 5'- 32 P-end-labeled mRNA, not only for screening recombinant clones and mapping gene structure, but also for revealing possible nucleotide sequence and structural signals within mRNA molecules themselves, which may be important for eukaryotic mRNA processing and turnover and for controlling differential rates of translational initiation. Three major problems, however, have retarded progress in this area, lack of methods for efficient and reproducible removal of m7G5ppp5'-cap structures, which maintain the integrity of an RNA molecule; inability to generate a sufficient amount of labeled mRNA, owing to the limited availability of most pure mRNA species; and the frequent problem of RNA degradation during in vitro end-labeling owing to RNAse contamination. The procedures presented here permit one to decap and label minute quantities of mRNA, effectively. Tobacco acid pyrophosphatase is relatively efficient in removing cap structures from even nanogram quantities of available mRNA, and enough radioactivity can be easily generated from minute amounts ofintact mRNA with very high-specific-activity [gamma- 32 P]ATP and the inhibition of ribonuclease contamination with diethylpyrocarbonate. These procedures can be modified and applied to almost any other type of RNA molecule as well. In Section III of this volume, we explore in detail how effectively 5'-end-labeled mRNA can be used not only for nucleotide sequence analysis, but also for mapping mRNA secondary structure

  3. Primary structure of the. cap alpha. -subunit of Na/sup +/, K/sup +/-ATPase. II. Isolation, reverse transcription, and cloning of messenger RNA

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    Petrukhin, K.E.; Broude, N.E.; Arsenyan, S.G.; Grishin, A.V.; Dzhandzhugazyan, K.N.; Modyanov, N.N.

    1986-10-01

    The messenger RNA coding the ..cap alpha..-subunit of Na/sup +/,K/sup +/-ATPase has been isolated from the outer medullary layer of porcine kidneys. The mRNA gives a specific hybridization band in the 25S-26S region with three oligonucleotide probes synthesized on the basis of information on the structure of three peptides isolated from a tryptic hydrolyzate of the ..cap alpha..-subunit of Na/sup +/,K/sup +/-ATPase. The translation of the mRNA in Xenopus laevis oocytes followed by immunochemical identification of the products of synthesis confirmed the presence of the mRNA of the ..cap alpha..-subunit of Na/sup +/,K/sup +/-ATPase in an enriched fraction of poly(A/sup +/)-RNA. This preparation has been used for the synthesis of cloning of double-stranded cDNA.

  4. Analysis of RNA binding by the dengue virus NS5 RNA capping enzyme.

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    Brittney R Henderson

    Full Text Available Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5' end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the K(D for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5' phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM. Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5' di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented.

  5. Structural changes of eIF4E upon binding to the mRNA 5' monomethylguanosine and trimethylguanosine Cap.

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    Rutkowska-Wlodarczyk, Izabela; Stepinski, Janusz; Dadlez, Michal; Darzynkiewicz, Edward; Stolarski, Ryszard; Niedzwiecka, Anna

    2008-03-04

    Recognition of the 5' cap by the eukaryotic initiation factor 4E (eIF4E) is the rate-limiting step in the ribosome recruitment to mRNAs. The regular cap consists of 7-monomethylguanosine (MMG) linked by a 5'-5' triphosphate bridge to the first transcribed nucleoside, while some primitive eukaryotes possess a N (2), N (2),7-trimethylguanosine (TMG) cap structure as a result of trans splicing. Mammalian eIF4E is highly specific to the MMG form of the cap in terms of association constants and thermodynamic driving force. We have investigated conformational changes of eIF4E induced by interaction with two cap analogues, 7-methyl-GTP and N (2), N (2),7-trimethyl-GTP. Hydrogen-deuterium exchange and electrospray mass spectrometry were applied to probe local dynamics of murine eIF4E in the apo and cap-bound forms. The data show that the cap binding induces long-range conformational changes in the protein, not only in the cap-binding pocket but also in a distant region of the 4E-BP/eIF4G binding site. Formation of the complex with 7-methyl-GTP makes the eIF4E structure more compact, while binding of N (2), N (2),7-trimethyl-GTP leads to higher solvent accessibility of the protein backbone in comparison with the apo form. The results suggest that the additional double methylation at the N (2)-amino group of the cap causes sterical effects upon binding to mammalian eIF4E which influence the overall solution dynamics of the protein, thus precluding formation of a tight complex.

  6. Enzymatic synthesis of RNAs capped with nucleotide analogues reveals the molecular basis for substrate selectivity of RNA capping enzyme: impacts on RNA metabolism.

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    Moheshwarnath Issur

    Full Text Available RNA cap binding proteins have evolved to specifically bind to the N7-methyl guanosine cap structure found at the 5' ends of eukaryotic mRNAs. The specificity of RNA capping enzymes towards GTP for the synthesis of this structure is therefore crucial for mRNA metabolism. The fact that ribavirin triphosphate was described as a substrate of a viral RNA capping enzyme, raised the possibility that RNAs capped with nucleotide analogues could be generated in cellulo. Owing to the fact that this prospect potentially has wide pharmacological implications, we decided to investigate whether the active site of the model Paramecium bursaria Chlorella virus-1 RNA capping enzyme was flexible enough to accommodate various purine analogues. Using this approach, we identified several key structural determinants at each step of the RNA capping reaction and generated RNAs harboring various different cap analogues. Moreover, we monitored the binding affinity of these novel capped RNAs to the eIF4E protein and evaluated their translational properties in cellulo. Overall, this study establishes a molecular rationale for the specific selection of GTP over other NTPs by RNA capping enzyme It also demonstrates that RNAs can be enzymatically capped with certain purine nucleotide analogs, and it also describes the impacts of modified RNA caps on specific steps involved in mRNA metabolism. For instance, our results indicate that the N7-methyl group of the classical N7-methyl guanosine cap is not always indispensable for binding to eIF4E and subsequently for translation when compensatory modifications are present on the capped residue. Overall, these findings have important implications for our understanding of the molecular determinants involved in both RNA capping and RNA metabolism.

  7. Immunoprecipitation of Tri-methylated Capped RNA.

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    Hayes, Karen E; Barr, Jamie A; Xie, Mingyi; Steitz, Joan A; Martinez, Ivan

    2018-02-05

    Cellular quiescence (also known as G 0 arrest) is characterized by reduced DNA replication, increased autophagy, and increased expression of cyclin-dependent kinase p27 Kip1 . Quiescence is essential for wound healing, organ regeneration, and preventing neoplasia. Previous findings indicate that microRNAs (miRNAs) play an important role in regulating cellular quiescence. Our recent publication demonstrated the existence of an alternative miRNA biogenesis pathway in primary human foreskin fibroblast (HFF) cells during quiescence. Indeed, we have identified a group of pri-miRNAs (whose mature miRNAs were found induced during quiescence) modified with a 2,2,7-trimethylguanosine (TMG)-cap by the trimethylguanosine synthase 1 (TGS1) protein and transported to the cytoplasm by the Exportin-1 (XPO1) protein. We used an antibody against (TMG)-caps (which does not cross-react with the (m 7 G)-caps that most pri-miRNAs or mRNAs contain [Luhrmann et al ., 1982]) to perform RNA immunoprecipitations from total RNA extracts of proliferating or quiescent HFFs. The novelty of this assay is the specific isolation of pri-miRNAs as well as other non-coding RNAs containing a TMG-cap modification.

  8. Characterization of a mimivirus RNA cap guanine-N2 methyltransferase.

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    Benarroch, Delphine; Qiu, Zhicheng R; Schwer, Beate; Shuman, Stewart

    2009-04-01

    A 2,2,7-trimethylguanosine (TMG) cap is a signature feature of eukaryal snRNAs, telomerase RNAs, and trans-spliced nematode mRNAs. TMG and 2,7-dimethylguanosine (DMG) caps are also present on mRNAs of two species of alphaviruses (positive strand RNA viruses of the Togaviridae family). It is presently not known how viral mRNAs might acquire a hypermethylated cap. Mimivirus, a giant DNA virus that infects amoeba, encodes many putative enzymes and proteins implicated in RNA transactions, including the synthesis and capping of viral mRNAs and the promotion of cap-dependent translation. Here we report the identification, purification, and characterization of a mimivirus cap-specific guanine-N2 methyltransferase (MimiTgs), a monomeric enzyme that catalyzes a single round of methyl transfer from AdoMet to an m(7)G cap substrate to form a DMG cap product. MimiTgs, is apparently unable to convert a DMG cap to a TMG cap, and is thereby distinguished from the structurally homologous yeast and human Tgs1 enzymes. Nonetheless, we show genetically that MimiTgs is a true ortholog of Saccharomyces cerevisiae Tgs1. Our results hint that DMG caps can satisfy many of the functions of TMG caps in vivo. We speculate that DMG capping of mimivirus mRNAs might favor viral protein synthesis in the infected host.

  9. 5'-Phospho-RNA Acceptor Specificity of GDP Polyribonucleotidyltransferase of Vesicular Stomatitis Virus in mRNA Capping.

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    Ogino, Minako; Ogino, Tomoaki

    2017-03-15

    The GDP polyribonucleotidyltransferase (PRNTase) domain of the multifunctional L protein of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus, catalyzes the transfer of 5'-phospho-RNA (pRNA) from 5'-triphospho-RNA (pppRNA) to GDP via a covalent enzyme-pRNA intermediate to generate a 5'-cap structure (GpppA). Here, using an improved oligo-RNA capping assay with the VSV L protein, we showed that the Michaelis constants for GDP and pppAACAG (VSV mRNA-start sequence) are 0.03 and 0.4 μM, respectively. A competition assay between GDP and GDP analogues in the GpppA formation and pRNA transfer assay using GDP analogues as pRNA acceptors indicated that the PRNTase domain recognizes the C-2-amino group, but not the C-6-oxo group, N-1-hydrogen, or N-7-nitrogen, of GDP for the cap formation. 2,6-Diaminopurine-riboside (DAP), 7-deazaguanosine (7-deaza-G), and 7-methylguanosine (m 7 G) diphosphates efficiently accepted pRNA, resulting in the formation of DAPpppA, 7-deaza-GpppA, and m 7 GpppA (cap 0), respectively. Furthermore, either the 2'- or 3'-hydroxyl group of GDP was found to be required for efficient pRNA transfer. A 5'-diphosphate form of antiviral ribavirin weakly inhibited the GpppA formation but did not act as a pRNA acceptor. These results indicate that the PRNTase domain has a unique guanosine-binding mode different from that of eukaryotic mRNA capping enzyme, guanylyltransferase. IMPORTANCE mRNAs of nonsegmented negative-strand (NNS) RNA viruses, such as VSV, possess a fully methylated cap structure, which is required for mRNA stability, efficient translation, and evasion of antiviral innate immunity in host cells. GDP polyribonucleotidyltransferase (PRNTase) is an unconventional mRNA capping enzyme of NNS RNA viruses that is distinct from the eukaryotic mRNA capping enzyme, guanylyltransferase. In this study, we studied the pRNA acceptor specificity of VSV PRNTase using various GDP analogues and identified chemical groups of GDP as

  10. 5′-Phospho-RNA Acceptor Specificity of GDP Polyribonucleotidyltransferase of Vesicular Stomatitis Virus in mRNA Capping

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    Ogino, Minako

    2017-01-01

    ABSTRACT The GDP polyribonucleotidyltransferase (PRNTase) domain of the multifunctional L protein of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus, catalyzes the transfer of 5′-phospho-RNA (pRNA) from 5′-triphospho-RNA (pppRNA) to GDP via a covalent enzyme-pRNA intermediate to generate a 5′-cap structure (GpppA). Here, using an improved oligo-RNA capping assay with the VSV L protein, we showed that the Michaelis constants for GDP and pppAACAG (VSV mRNA-start sequence) are 0.03 and 0.4 μM, respectively. A competition assay between GDP and GDP analogues in the GpppA formation and pRNA transfer assay using GDP analogues as pRNA acceptors indicated that the PRNTase domain recognizes the C-2-amino group, but not the C-6-oxo group, N-1-hydrogen, or N-7-nitrogen, of GDP for the cap formation. 2,6-Diaminopurine-riboside (DAP), 7-deazaguanosine (7-deaza-G), and 7-methylguanosine (m7G) diphosphates efficiently accepted pRNA, resulting in the formation of DAPpppA, 7-deaza-GpppA, and m7GpppA (cap 0), respectively. Furthermore, either the 2′- or 3′-hydroxyl group of GDP was found to be required for efficient pRNA transfer. A 5′-diphosphate form of antiviral ribavirin weakly inhibited the GpppA formation but did not act as a pRNA acceptor. These results indicate that the PRNTase domain has a unique guanosine-binding mode different from that of eukaryotic mRNA capping enzyme, guanylyltransferase. IMPORTANCE mRNAs of nonsegmented negative-strand (NNS) RNA viruses, such as VSV, possess a fully methylated cap structure, which is required for mRNA stability, efficient translation, and evasion of antiviral innate immunity in host cells. GDP polyribonucleotidyltransferase (PRNTase) is an unconventional mRNA capping enzyme of NNS RNA viruses that is distinct from the eukaryotic mRNA capping enzyme, guanylyltransferase. In this study, we studied the pRNA acceptor specificity of VSV PRNTase using various GDP analogues and identified chemical groups

  11. Cap-independent translation mechanism of red clover necrotic mosaic virus RNA2 differs from that of RNA1 and is linked to RNA replication.

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    Mizumoto, Hiroyuki; Iwakawa, Hiro-Oki; Kaido, Masanori; Mise, Kazuyuki; Okuno, Tetsuro

    2006-04-01

    The genome of Red clover necrotic mosaic virus (RCNMV) in the genus Dianthovirus is divided into two RNA molecules of RNA1 and RNA2, which have no cap structure at the 5' end and no poly(A) tail at the 3' end. The 3' untranslated region (3' UTR) of RCNMV RNA1 contains an essential RNA element (3'TE-DR1), which is required for cap-independent translation. In this study, we investigated a cap-independent translational mechanism of RNA2 using a firefly luciferase (Luc) gene expression assay system in cowpea protoplasts and a cell-free lysate (BYL) prepared from evacuolated tobacco BY2 protoplasts. We were unable to detect cis-acting RNA sequences in RNA2 that can replace the function of a cap structure, such as the 3'TE-DR1 of RNA1. However, the uncapped reporter RNA2, RNA2-Luc, in which the Luc open reading frame (ORF) was inserted between the 5' UTR and the movement protein ORF, was effectively translated in the presence of p27 and p88 in protoplasts in which RNA2-Luc was replicated. Time course experiments in protoplasts showed that the translational activity of RNA2-Luc did not reflect the amount of RNA2. Mutations in cis-acting RNA replication elements of RNA2 abolished the cap-independent translational activity of RNA2-Luc, suggesting that the translational activity of RNA2-Luc is coupled to RNA replication. Our results show that the translational mechanism differs between two segmented genomic RNAs of RCNMV. We present a model in which only RNA2 that is generated de novo through the viral RNA replication machinery functions as mRNA for translation.

  12. Lariat capping as a tool to manipulate the 5' end of individual yeast mRNA species in vivo

    DEFF Research Database (Denmark)

    Krogh, Nicolai; Pietschmann, Max; Schmid, Manfred

    2017-01-01

    The 5' cap structure of eukaryotic mRNA is critical for its processing, transport, translation, and stability. The many functions of the cap and the fact that most, if not all, mRNA carries the same type of cap makes it difficult to analyze cap function in vivo at individual steps of gene...... in the budding yeast Saccharomyces cerevisiae presumably without cofactors and that lariat capping occurs cotranscriptionally. The lariat-capped reporter mRNA is efficiently exported to the cytoplasm where it is found to be oligoadenylated and evenly distributed. Both the oligoadenylated form and a lariat......-capped mRNA with a templated poly(A) tail translates poorly, underlining the critical importance of the m(7)G cap in translation. Finally, the lariat-capped RNA exhibits a threefold longer half-life compared to its m(7)G-capped counterpart, consistent with a key role for the m(7)G cap in mRNA turnover. Our...

  13. The Rabies Virus L Protein Catalyzes mRNA Capping with GDP Polyribonucleotidyltransferase Activity

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    Minako Ogino

    2016-05-01

    Full Text Available The large (L protein of rabies virus (RABV plays multiple enzymatic roles in viral RNA synthesis and processing. However, none of its putative enzymatic activities have been directly demonstrated in vitro. In this study, we expressed and purified a recombinant form of the RABV L protein and verified its guanosine 5′-triphosphatase and GDP polyribonucleotidyltransferase (PRNTase activities, which are essential for viral mRNA cap formation by the unconventional mechanism. The RABV L protein capped 5′-triphosphorylated but not 5′-diphosphorylated RABV mRNA-start sequences, 5′-AACA(C/U, with GDP to generate the 5′-terminal cap structure G(5′ppp(5′A. The 5′-AAC sequence in the substrate RNAs was found to be strictly essential for RNA capping with the RABV L protein. Furthermore, site-directed mutagenesis showed that some conserved amino acid residues (G1112, T1170, W1201, H1241, R1242, F1285, and Q1286 in the PRNTase motifs A to E of the RABV L protein are required for cap formation. These findings suggest that the putative PRNTase domain in the RABV L protein catalyzes the rhabdovirus-specific capping reaction involving covalent catalysis of the pRNA transfer to GDP, thus offering this domain as a target for developing anti-viral agents.

  14. The Rabies Virus L Protein Catalyzes mRNA Capping with GDP Polyribonucleotidyltransferase Activity.

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    Ogino, Minako; Ito, Naoto; Sugiyama, Makoto; Ogino, Tomoaki

    2016-05-21

    The large (L) protein of rabies virus (RABV) plays multiple enzymatic roles in viral RNA synthesis and processing. However, none of its putative enzymatic activities have been directly demonstrated in vitro. In this study, we expressed and purified a recombinant form of the RABV L protein and verified its guanosine 5'-triphosphatase and GDP polyribonucleotidyltransferase (PRNTase) activities, which are essential for viral mRNA cap formation by the unconventional mechanism. The RABV L protein capped 5'-triphosphorylated but not 5'-diphosphorylated RABV mRNA-start sequences, 5'-AACA(C/U), with GDP to generate the 5'-terminal cap structure G(5')ppp(5')A. The 5'-AAC sequence in the substrate RNAs was found to be strictly essential for RNA capping with the RABV L protein. Furthermore, site-directed mutagenesis showed that some conserved amino acid residues (G1112, T1170, W1201, H1241, R1242, F1285, and Q1286) in the PRNTase motifs A to E of the RABV L protein are required for cap formation. These findings suggest that the putative PRNTase domain in the RABV L protein catalyzes the rhabdovirus-specific capping reaction involving covalent catalysis of the pRNA transfer to GDP, thus offering this domain as a target for developing anti-viral agents.

  15. RNA Thermodynamic Structural Entropy.

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    Garcia-Martin, Juan Antonio; Clote, Peter

    2015-01-01

    Conformational entropy for atomic-level, three dimensional biomolecules is known experimentally to play an important role in protein-ligand discrimination, yet reliable computation of entropy remains a difficult problem. Here we describe the first two accurate and efficient algorithms to compute the conformational entropy for RNA secondary structures, with respect to the Turner energy model, where free energy parameters are determined from UV absorption experiments. An algorithm to compute the derivational entropy for RNA secondary structures had previously been introduced, using stochastic context free grammars (SCFGs). However, the numerical value of derivational entropy depends heavily on the chosen context free grammar and on the training set used to estimate rule probabilities. Using data from the Rfam database, we determine that both of our thermodynamic methods, which agree in numerical value, are substantially faster than the SCFG method. Thermodynamic structural entropy is much smaller than derivational entropy, and the correlation between length-normalized thermodynamic entropy and derivational entropy is moderately weak to poor. In applications, we plot the structural entropy as a function of temperature for known thermoswitches, such as the repression of heat shock gene expression (ROSE) element, we determine that the correlation between hammerhead ribozyme cleavage activity and total free energy is improved by including an additional free energy term arising from conformational entropy, and we plot the structural entropy of windows of the HIV-1 genome. Our software RNAentropy can compute structural entropy for any user-specified temperature, and supports both the Turner'99 and Turner'04 energy parameters. It follows that RNAentropy is state-of-the-art software to compute RNA secondary structure conformational entropy. Source code is available at https://github.com/clotelab/RNAentropy/; a full web server is available at http

  16. RNA Thermodynamic Structural Entropy.

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    Juan Antonio Garcia-Martin

    Full Text Available Conformational entropy for atomic-level, three dimensional biomolecules is known experimentally to play an important role in protein-ligand discrimination, yet reliable computation of entropy remains a difficult problem. Here we describe the first two accurate and efficient algorithms to compute the conformational entropy for RNA secondary structures, with respect to the Turner energy model, where free energy parameters are determined from UV absorption experiments. An algorithm to compute the derivational entropy for RNA secondary structures had previously been introduced, using stochastic context free grammars (SCFGs. However, the numerical value of derivational entropy depends heavily on the chosen context free grammar and on the training set used to estimate rule probabilities. Using data from the Rfam database, we determine that both of our thermodynamic methods, which agree in numerical value, are substantially faster than the SCFG method. Thermodynamic structural entropy is much smaller than derivational entropy, and the correlation between length-normalized thermodynamic entropy and derivational entropy is moderately weak to poor. In applications, we plot the structural entropy as a function of temperature for known thermoswitches, such as the repression of heat shock gene expression (ROSE element, we determine that the correlation between hammerhead ribozyme cleavage activity and total free energy is improved by including an additional free energy term arising from conformational entropy, and we plot the structural entropy of windows of the HIV-1 genome. Our software RNAentropy can compute structural entropy for any user-specified temperature, and supports both the Turner'99 and Turner'04 energy parameters. It follows that RNAentropy is state-of-the-art software to compute RNA secondary structure conformational entropy. Source code is available at https://github.com/clotelab/RNAentropy/; a full web server is available at http

  17. Topology of RNA-RNA interaction structures

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    Andersen, Jørgen Ellegaard; Huang, Fenix Wenda; Penner, Robert

    2012-01-01

    Abstract The topological filtration of interacting RNA complexes is studied, and the role is analyzed of certain diagrams called irreducible shadows, which form suitable building blocks for more general structures. We prove that, for two interacting RNAs, called interaction structures, there exist...

  18. A Viral RNA Structural Element Alters Host Recognition of Nonself RNA

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    Hyde, J. L.; Gardner, C. L.; Kimura, T.; White, J. P.; Liu, G.; Trobaugh, D. W.; Huang, C.; Tonelli, M.; Paessler, S.; Takeda, K.; Klimstra, W. B.; Amarasinghe, G. K.; Diamond, M. S.

    2014-01-30

    Although interferon (IFN) signaling induces genes that limit viral infection, many pathogenic viruses overcome this host response. As an example, 2'-O methylation of the 5' cap of viral RNA subverts mammalian antiviral responses by evading restriction of Ifit1, an IFN-stimulated gene that regulates protein synthesis. However, alphaviruses replicate efficiently in cells expressing Ifit1 even though their genomic RNA has a 5' cap lacking 2'-O methylation. We show that pathogenic alphaviruses use secondary structural motifs within the 5' untranslated region (UTR) of their RNA to alter Ifit1 binding and function. Mutations within the 5'-UTR affecting RNA structural elements enabled restriction by or antagonism of Ifit1 in vitro and in vivo. These results identify an evasion mechanism by which viruses use RNA structural motifs to avoid immune restriction.

  19. Structural and Functional Diversity of Plant Virus 3'-Cap-Independent Translation Enhancers (3'-CITEs).

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    Truniger, Verónica; Miras, Manuel; Aranda, Miguel A

    2017-01-01

    Most of the positive-strand RNA plant viruses lack the 5'-cap and/or the poly(A)-tail that act synergistically to stimulate canonical translation of cellular mRNAs. However, they have RNA elements in the 5'- or 3'-untranslated regions of their RNAs that are required for their cap-independent translation. Cap-independent translation enhancers (CITEs) have been identified in the genomic 3'-end of viruses belonging to the family Tombusviridae and the genus Luteovirus . Seven classes of 3'-CITEs have been described to date based on their different RNA structures. They generally control the efficient formation of the translation initiation complex by varying mechanisms. Some 3'-CITEs bind eukaryotic translation initiation factors, others ribosomal subunits, bridging these to the 5'-end by different mechanisms, often long-distance RNA-RNA interactions. As previously proposed and recently found in one case in nature, 3'-CITEs are functionally independent elements that are transferable through recombination between viral genomes, leading to potential advantages for virus multiplication. In this review, the knowledge on 3'-CITEs and their functioning is updated. We also suggest that there is local structural conservation in the regions interacting with eIF4E of 3'-CITEs belonging to different classes.

  20. The human cap-binding complex is functionally connected to the nuclear RNA exosome

    DEFF Research Database (Denmark)

    Andersen, Peter Refsing; Domanski, Michal; Kristiansen, Maiken Søndergaard

    2013-01-01

    Nuclear processing and quality control of eukaryotic RNA is mediated by the RNA exosome, which is regulated by accessory factors. However, the mechanism of exosome recruitment to its ribonucleoprotein (RNP) targets remains poorly understood. Here we report a physical link between the human exosome...... and the cap-binding complex (CBC). The CBC associates with the ARS2 protein to form CBC-ARS2 (CBCA) and then further connects, together with the ZC3H18 protein, to the nuclear exosome targeting (NEXT) complex, thus forming CBC-NEXT (CBCN). RNA immunoprecipitation using CBCN factors as well as the analysis...

  1. Regulatory effects of cotranscriptional RNA structure formation and transitions.

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    Liu, Sheng-Rui; Hu, Chun-Gen; Zhang, Jin-Zhi

    2016-09-01

    RNAs, which play significant roles in many fundamental biological processes of life, fold into sophisticated and precise structures. RNA folding is a dynamic and intricate process, which conformation transition of coding and noncoding RNAs form the primary elements of genetic regulation. The cellular environment contains various intrinsic and extrinsic factors that potentially affect RNA folding in vivo, and experimental and theoretical evidence increasingly indicates that the highly flexible features of the RNA structure are affected by these factors, which include the flanking sequence context, physiochemical conditions, cis RNA-RNA interactions, and RNA interactions with other molecules. Furthermore, distinct RNA structures have been identified that govern almost all steps of biological processes in cells, including transcriptional activation and termination, transcriptional mutagenesis, 5'-capping, splicing, 3'-polyadenylation, mRNA export and localization, and translation. Here, we briefly summarize the dynamic and complex features of RNA folding along with a wide variety of intrinsic and extrinsic factors that affect RNA folding. We then provide several examples to elaborate RNA structure-mediated regulation at the transcriptional and posttranscriptional levels. Finally, we illustrate the regulatory roles of RNA structure and discuss advances pertaining to RNA structure in plants. WIREs RNA 2016, 7:562-574. doi: 10.1002/wrna.1350 For further resources related to this article, please visit the WIREs website. © 2016 Wiley Periodicals, Inc.

  2. On topological RNA interaction structures.

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    Qin, Jing; Reidys, Christian M

    2013-07-01

    Recently a folding algorithm of topological RNA pseudoknot structures was presented in Reidys et al. (2011). This algorithm folds single-stranded γ-structures, that is, RNA structures composed by distinct motifs of bounded topological genus. In this article, we set the theoretical foundations for the folding of the two backbone analogues of γ structures: the RNA γ-interaction structures. These are RNA-RNA interaction structures that are constructed by a finite number of building blocks over two backbones having genus at most γ. Combinatorial properties of γ-interaction structures are of practical interest since they have direct implications for the folding of topological interaction structures. We compute the generating function of γ-interaction structures and show that it is algebraic, which implies that the numbers of interaction structures can be computed recursively. We obtain simple asymptotic formulas for 0- and 1-interaction structures. The simplest class of interaction structures are the 0-interaction structures, which represent the two backbone analogues of secondary structures.

  3. Efficient RNA structure comparison algorithms.

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    Arslan, Abdullah N; Anandan, Jithendar; Fry, Eric; Monschke, Keith; Ganneboina, Nitin; Bowerman, Jason

    2017-12-01

    Recently proposed relative addressing-based ([Formula: see text]) RNA secondary structure representation has important features by which an RNA structure database can be stored into a suffix array. A fast substructure search algorithm has been proposed based on binary search on this suffix array. Using this substructure search algorithm, we present a fast algorithm that finds the largest common substructure of given multiple RNA structures in [Formula: see text] format. The multiple RNA structure comparison problem is NP-hard in its general formulation. We introduced a new problem for comparing multiple RNA structures. This problem has more strict similarity definition and objective, and we propose an algorithm that solves this problem efficiently. We also develop another comparison algorithm that iteratively calls this algorithm to locate nonoverlapping large common substructures in compared RNAs. With the new resulting tools, we improved the RNASSAC website (linked from http://faculty.tamuc.edu/aarslan ). This website now also includes two drawing tools: one specialized for preparing RNA substructures that can be used as input by the search tool, and another one for automatically drawing the entire RNA structure from a given structure sequence.

  4. A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement*

    Science.gov (United States)

    Chuang, Tzu-Wei; Lee, Kuo-Ming; Lou, Yuan-Chao; Lu, Chia-Chen; Tarn, Woan-Yuh

    2016-01-01

    Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis. PMID:26887951

  5. A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement.

    Science.gov (United States)

    Chuang, Tzu-Wei; Lee, Kuo-Ming; Lou, Yuan-Chao; Lu, Chia-Chen; Tarn, Woan-Yuh

    2016-04-15

    Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Marburg virus VP35 can both fully coat the backbone and cap the ends of dsRNA for interferon antagonism.

    Directory of Open Access Journals (Sweden)

    Shridhar Bale

    2012-09-01

    Full Text Available Filoviruses, including Marburg virus (MARV and Ebola virus (EBOV, cause fatal hemorrhagic fever in humans and non-human primates. All filoviruses encode a unique multi-functional protein termed VP35. The C-terminal double-stranded (dsRNA-binding domain (RBD of VP35 has been implicated in interferon antagonism and immune evasion. Crystal structures of the VP35 RBD from two ebolaviruses have previously demonstrated that the viral protein caps the ends of dsRNA. However, it is not yet understood how the expanses of dsRNA backbone, between the ends, are masked from immune surveillance during filovirus infection. Here, we report the crystal structure of MARV VP35 RBD bound to dsRNA. In the crystal structure, molecules of dsRNA stack end-to-end to form a pseudo-continuous oligonucleotide. This oligonucleotide is continuously and completely coated along its sugar-phosphate backbone by the MARV VP35 RBD. Analysis of dsRNA binding by dot-blot and isothermal titration calorimetry reveals that multiple copies of MARV VP35 RBD can indeed bind the dsRNA sugar-phosphate backbone in a cooperative manner in solution. Further, MARV VP35 RBD can also cap the ends of the dsRNA in solution, although this arrangement was not captured in crystals. Together, these studies suggest that MARV VP35 can both coat the backbone and cap the ends, and that for MARV, coating of the dsRNA backbone may be an essential mechanism by which dsRNA is masked from backbone-sensing immune surveillance molecules.

  7. Convection flow structure in the central polar cap

    Science.gov (United States)

    Bristow, W. A.

    2017-12-01

    A previous study of spatially averaged flow velocity in the central polar cap [Bristow et al., 2015] observed under steady IMF conditions found that it was extremely rare for the average to exceed 850 m/s (less than 0.2 % of the time). Anecdotally, however it is not uncommon to observe line-of-sight velocities in excess of 100 m/s in the McMurdo radar field of view directly over the magnetic pole. This discrepancy motivated this study, which examines the conditions under which high-velocity flows are observed at latitudes greater than 80° magnetic latitude. It was found that highly structured flows are common in the central polar cap, which leads to the flow within regions to have significant deviation from the average. In addition, the high-speed flow regions are usually directed away from the earth-sun line. No specific set of driving conditions was identified to be associated with high-speed flows. The study did conclude that 1)Polar cap velocities are generally highly structured. 2)Flow patterns typically illustrate narrow channels, vortical flow regions, and propagating features. 3) Persistent waves are a regular occurrence. 3)Features are observed to propagate from day side to night side, and from night side to day side.. 4)Convection often exhibits significant difference between the two hemispheres. And 5)About 10% of the time the velocity somewhere in the cap exceeds 1 Km/s The presentation will conclude with a discussion of the physical reasons for the flow structure. Bristow, W. A., E. Amata, J. Spaleta, and M. F. Marcucci (2015), Observations of the relationship between ionospheric central polar cap and dayside throat convection velocities, and solar wind/IMF driving, J. Geophys. Res. Space Physics, 120, doi:10.1002/2015JA021199.

  8. cap alpha. -spectra hyperfine structure resolution by silicon planar detectors

    Energy Technology Data Exchange (ETDEWEB)

    Eremin, V K; Verbitskaya, E M; Strokan, N B; Sukhanov, V L; Malyarenko, A M

    1986-10-01

    The lines with 13 keV step from the main one is ..cap alpha..-spectra of nuclei with an odd number of nucleons take place. Silicon planar detectors n-Si with the operation surface of 10 mm/sup 2/ are developed for resolution of this hyperfine structure. The mechanism of losses in detectors for short-range-path particles is analyzed. The results of measurements from detectors with 10 keV resolution are presented.

  9. RNA STRAND: The RNA Secondary Structure and Statistical Analysis Database

    Directory of Open Access Journals (Sweden)

    Andronescu Mirela

    2008-08-01

    Full Text Available Abstract Background The ability to access, search and analyse secondary structures of a large set of known RNA molecules is very important for deriving improved RNA energy models, for evaluating computational predictions of RNA secondary structures and for a better understanding of RNA folding. Currently there is no database that can easily provide these capabilities for almost all RNA molecules with known secondary structures. Results In this paper we describe RNA STRAND – the RNA secondary STRucture and statistical ANalysis Database, a curated database containing known secondary structures of any type and organism. Our new database provides a wide collection of known RNA secondary structures drawn from public databases, searchable and downloadable in a common format. Comprehensive statistical information on the secondary structures in our database is provided using the RNA Secondary Structure Analyser, a new tool we have developed to analyse RNA secondary structures. The information thus obtained is valuable for understanding to which extent and with which probability certain structural motifs can appear. We outline several ways in which the data provided in RNA STRAND can facilitate research on RNA structure, including the improvement of RNA energy models and evaluation of secondary structure prediction programs. In order to keep up-to-date with new RNA secondary structure experiments, we offer the necessary tools to add solved RNA secondary structures to our database and invite researchers to contribute to RNA STRAND. Conclusion RNA STRAND is a carefully assembled database of trusted RNA secondary structures, with easy on-line tools for searching, analyzing and downloading user selected entries, and is publicly available at http://www.rnasoft.ca/strand.

  10. Fatgraph models of RNA structure

    Directory of Open Access Journals (Sweden)

    Huang Fenix

    2017-01-01

    Full Text Available In this review paper we discuss fatgraphs as a conceptual framework for RNA structures. We discuss various notions of coarse-grained RNA structures and relate them to fatgraphs.We motivate and discuss the main intuition behind the fatgraph model and showcase its applicability to canonical as well as noncanonical base pairs. Recent discoveries regarding novel recursions of pseudoknotted (pk configurations as well as their translation into context-free grammars for pk-structures are discussed. This is shown to allow for extending the concept of partition functions of sequences w.r.t. a fixed structure having non-crossing arcs to pk-structures. We discuss minimum free energy folding of pk-structures and combine these above results outlining how to obtain an inverse folding algorithm for PK structures.

  11. Structure of a two-CAP-domain protein from the human hookworm parasite Necator americanus

    Energy Technology Data Exchange (ETDEWEB)

    Asojo, Oluwatoyin A., E-mail: oasojo@unmc.edu [Pathology and Microbiology Department, 986495 Nebraska Medical Center, Omaha, NE 68198-6495 (United States)

    2011-05-01

    The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite N. americanus refined to a resolution limit of 2.2 Å is presented. Major proteins secreted by the infective larval stage hookworms upon host entry include Ancylostoma secreted proteins (ASPs), which are characterized by one or two CAP (cysteine-rich secretory protein/antigen 5/pathogenesis related-1) domains. The CAP domain has been reported in diverse phylogenetically unrelated proteins, but has no confirmed function. The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite Necator americanus was refined to a resolution limit of 2.2 Å. The structure was solved by molecular replacement (MR) using Na-ASP-2, a one-CAP-domain ASP, as the search model. The correct MR solution could only be obtained by truncating the polyalanine model of Na-ASP-2 and removing several loops. The structure reveals two CAP domains linked by an extended loop. Overall, the carboxyl-terminal CAP domain is more similar to Na-ASP-2 than to the amino-terminal CAP domain. A large central cavity extends from the amino-terminal CAP domain to the carboxyl-terminal CAP domain, encompassing the putative CAP-binding cavity. The putative CAP-binding cavity is a characteristic cavity in the carboxyl-terminal CAP domain that contains a His and Glu pair. These residues are conserved in all single-CAP-domain proteins, but are absent in the amino-terminal CAP domain. The conserved His residues are oriented such that they appear to be capable of directly coordinating a zinc ion as observed for CAP proteins from reptile venoms. This first structure of a two-CAP-domain ASP can serve as a template for homology modeling of other two-CAP-domain proteins.

  12. Structure of a two-CAP-domain protein from the human hookworm parasite Necator americanus

    International Nuclear Information System (INIS)

    Asojo, Oluwatoyin A.

    2011-01-01

    The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite N. americanus refined to a resolution limit of 2.2 Å is presented. Major proteins secreted by the infective larval stage hookworms upon host entry include Ancylostoma secreted proteins (ASPs), which are characterized by one or two CAP (cysteine-rich secretory protein/antigen 5/pathogenesis related-1) domains. The CAP domain has been reported in diverse phylogenetically unrelated proteins, but has no confirmed function. The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite Necator americanus was refined to a resolution limit of 2.2 Å. The structure was solved by molecular replacement (MR) using Na-ASP-2, a one-CAP-domain ASP, as the search model. The correct MR solution could only be obtained by truncating the polyalanine model of Na-ASP-2 and removing several loops. The structure reveals two CAP domains linked by an extended loop. Overall, the carboxyl-terminal CAP domain is more similar to Na-ASP-2 than to the amino-terminal CAP domain. A large central cavity extends from the amino-terminal CAP domain to the carboxyl-terminal CAP domain, encompassing the putative CAP-binding cavity. The putative CAP-binding cavity is a characteristic cavity in the carboxyl-terminal CAP domain that contains a His and Glu pair. These residues are conserved in all single-CAP-domain proteins, but are absent in the amino-terminal CAP domain. The conserved His residues are oriented such that they appear to be capable of directly coordinating a zinc ion as observed for CAP proteins from reptile venoms. This first structure of a two-CAP-domain ASP can serve as a template for homology modeling of other two-CAP-domain proteins

  13. RNA-SSPT: RNA Secondary Structure Prediction Tools.

    Science.gov (United States)

    Ahmad, Freed; Mahboob, Shahid; Gulzar, Tahsin; Din, Salah U; Hanif, Tanzeela; Ahmad, Hifza; Afzal, Muhammad

    2013-01-01

    The prediction of RNA structure is useful for understanding evolution for both in silico and in vitro studies. Physical methods like NMR studies to predict RNA secondary structure are expensive and difficult. Computational RNA secondary structure prediction is easier. Comparative sequence analysis provides the best solution. But secondary structure prediction of a single RNA sequence is challenging. RNA-SSPT is a tool that computationally predicts secondary structure of a single RNA sequence. Most of the RNA secondary structure prediction tools do not allow pseudoknots in the structure or are unable to locate them. Nussinov dynamic programming algorithm has been implemented in RNA-SSPT. The current studies shows only energetically most favorable secondary structure is required and the algorithm modification is also available that produces base pairs to lower the total free energy of the secondary structure. For visualization of RNA secondary structure, NAVIEW in C language is used and modified in C# for tool requirement. RNA-SSPT is built in C# using Dot Net 2.0 in Microsoft Visual Studio 2005 Professional edition. The accuracy of RNA-SSPT is tested in terms of Sensitivity and Positive Predicted Value. It is a tool which serves both secondary structure prediction and secondary structure visualization purposes.

  14. Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to find them.

    Science.gov (United States)

    Leppek, Kathrin; Das, Rhiju; Barna, Maria

    2018-03-01

    RNA molecules can fold into intricate shapes that can provide an additional layer of control of gene expression beyond that of their sequence. In this Review, we discuss the current mechanistic understanding of structures in 5' untranslated regions (UTRs) of eukaryotic mRNAs and the emerging methodologies used to explore them. These structures may regulate cap-dependent translation initiation through helicase-mediated remodelling of RNA structures and higher-order RNA interactions, as well as cap-independent translation initiation through internal ribosome entry sites (IRESs), mRNA modifications and other specialized translation pathways. We discuss known 5' UTR RNA structures and how new structure probing technologies coupled with prospective validation, particularly compensatory mutagenesis, are likely to identify classes of structured RNA elements that shape post-transcriptional control of gene expression and the development of multicellular organisms.

  15. Primary and secondary structure of U8 small nuclear RNA

    International Nuclear Information System (INIS)

    Reddy, R.; Henning, D.; Busch, H.

    1985-01-01

    U8 small nuclear RNA is a new, capped, 140 nucleotides long RNA species found in Novikoff hepatoma cells. Its sequence is: m3GpppAmUmCGUCAGGA GGUUAAUCCU UACCUGUCCC UCCUUUCGGA GGGCAGAUAG AAAAUGAUGA UUGGAGCUUG CAUGAUCUGC UGAUUAUAGC AUUUCCGUGU AAUCAGGACC UGACAACAUC CUGAUUGCUU CUAUCUGAUUOH. This RNA is present in approximately 25,000 copies/cell, and it is enriched in nucleolar preparations. Like U1, U2, U4/U6, and U5 RNAs, U8 RNA was also present as a ribonucleoprotein associated with the Sm antigen. The rat U8 RNA was highly homologous (greater than 90%) to a recently characterized 5.4 S RNA from mouse cells infected with spleen focus-forming virus. In addition to the U8 RNA, three other U small nuclear RNAs were found in anti-Sm antibody immunoprecipitates from labeled rat and HeLa cells. Each of these contained a m3GpppAm cap structure; their apparent chain lengths were 60, 130, and 65 nucleotides. These U small nuclear RNAs are designated U7, U9, and U10 RNAs, respectively

  16. RNA Structural Alignments, Part I

    DEFF Research Database (Denmark)

    Havgaard, Jakob Hull; Gorodkin, Jan

    2014-01-01

    Simultaneous alignment and secondary structure prediction of RNA sequences is often referred to as "RNA structural alignment." A class of the methods for structural alignment is based on the principles proposed by Sankoff more than 25 years ago. The Sankoff algorithm simultaneously folds and aligns...... is so high that it took more than a decade before the first implementation of a Sankoff style algorithm was published. However, with the faster computers available today and the improved heuristics used in the implementations the Sankoff-based methods have become practical. This chapter describes...... the methods based on the Sankoff algorithm. All the practical implementations of the algorithm use heuristics to make them run in reasonable time and memory. These heuristics are also described in this chapter....

  17. MCT-1 protein interacts with the cap complex and modulates messenger RNA translational profiles

    DEFF Research Database (Denmark)

    Reinert, Line; Shi, B; Nandi, S

    2006-01-01

    MCT-1 is an oncogene that was initially identified in a human T cell lymphoma and has been shown to induce cell proliferation as well as activate survival-related pathways. MCT-1 contains the PUA domain, a recently described RNA-binding domain that is found in several tRNA and rRNA modification...... enzymes. Here, we established that MCT-1 protein interacts with the cap complex through its PUA domain and recruits the density-regulated protein (DENR/DRP), containing the SUI1 translation initiation domain. Through the use of microarray analysis on polysome-associated mRNAs, we showed that up......-regulation of MCT-1 was able to modulate the translation profiles of BCL2L2, TFDP1, MRE11A, cyclin D1, and E2F1 mRNAs, despite equivalent levels of mRNAs in the cytoplasm. Our data establish a role for MCT-1 in translational regulation, and support a linkage between translational control and oncogenesis....

  18. Modifying the 5'-Cap for Click Reactions of Eukaryotic mRNA and To Tune Translation Efficiency in Living Cells.

    Science.gov (United States)

    Holstein, Josephin M; Anhäuser, Lea; Rentmeister, Andrea

    2016-08-26

    The 5'-cap is a hallmark of eukaryotic mRNAs and plays fundamental roles in RNA metabolism, ranging from quality control to export and translation. Modifying the 5'-cap may thus enable modulation of the underlying processes and investigation or tuning of several biological functions. A straightforward approach is presented for the efficient production of a range of N7-modified caps based on the highly promiscuous methyltransferase Ecm1. We show that these, as well as N(2) -modified 5'-caps, can be used to tune translation of the respective mRNAs both in vitro and in cells. Appropriate modifications allow subsequent bioorthogonal chemistry, as demonstrated by intracellular live-cell labeling of a target mRNA. The efficient and versatile N7 manipulation of the mRNA cap makes mRNAs amenable to both modulation of their biological function and intracellular labeling, and represents a valuable addition to the chemical biology toolbox. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. The 3' untranslated region of tobacco necrosis virus RNA contains a barley yellow dwarf virus-like cap-independent translation element.

    Science.gov (United States)

    Shen, Ruizhong; Miller, W Allen

    2004-05-01

    RNAs of many viruses are translated efficiently in the absence of a 5' cap structure. The tobacco necrosis virus (TNV) genome is an uncapped, nonpolyadenylated RNA whose translation mechanism has not been well investigated. Computational analysis predicted a cap-independent translation element (TE) within the 3' untranslated region (3' UTR) of TNV RNA that resembles the TE of barley yellow dwarf virus (BYDV), a luteovirus. Here we report that such a TE does indeed exist in the 3' UTR of TNV strain D. Like the BYDV TE, the TNV TE (i) functions both in vitro and in vivo, (ii) requires additional sequence for cap-independent translation in vivo, (iii) has a similar secondary structure and the conserved sequence CGGAUCCUGGGAAACAGG, (iv) is inactivated by a four-base duplication in this conserved sequence, (v) can function in the 5' UTR, and (vi) when located in its natural 3' location, may form long-distance base pairing with the viral 5' UTR that is conserved and probably required. The TNV TE differs from the BYDV TE by having only three helical domains instead of four. Similar structures were found in all members of the Necrovirus genus of the Tombusviridae family, except satellite tobacco necrosis virus, which harbors a different 3' cap-independent translation domain. The presence of the BYDV-like TE in select genera of different families indicates that phylogenetic distribution of TEs does not follow standard viral taxonomic relationships. We propose a new class of cap-independent TE called BYDV-like TE.

  20. The 3′ Untranslated Region of Tobacco Necrosis Virus RNA Contains a Barley Yellow Dwarf Virus-Like Cap-Independent Translation Element

    Science.gov (United States)

    Shen, Ruizhong; Miller, W. Allen

    2004-01-01

    RNAs of many viruses are translated efficiently in the absence of a 5′ cap structure. The tobacco necrosis virus (TNV) genome is an uncapped, nonpolyadenylated RNA whose translation mechanism has not been well investigated. Computational analysis predicted a cap-independent translation element (TE) within the 3′ untranslated region (3′ UTR) of TNV RNA that resembles the TE of barley yellow dwarf virus (BYDV), a luteovirus. Here we report that such a TE does indeed exist in the 3′ UTR of TNV strain D. Like the BYDV TE, the TNV TE (i) functions both in vitro and in vivo, (ii) requires additional sequence for cap-independent translation in vivo, (iii) has a similar secondary structure and the conserved sequence CGGAUCCUGGGAAACAGG, (iv) is inactivated by a four-base duplication in this conserved sequence, (v) can function in the 5′ UTR, and (vi) when located in its natural 3′ location, may form long-distance base pairing with the viral 5′ UTR that is conserved and probably required. The TNV TE differs from the BYDV TE by having only three helical domains instead of four. Similar structures were found in all members of the Necrovirus genus of the Tombusviridae family, except satellite tobacco necrosis virus, which harbors a different 3′ cap-independent translation domain. The presence of the BYDV-like TE in select genera of different families indicates that phylogenetic distribution of TEs does not follow standard viral taxonomic relationships. We propose a new class of cap-independent TE called BYDV-like TE. PMID:15078948

  1. Structural and Functional Diversity of Plant Virus 3′-Cap-Independent Translation Enhancers (3′-CITEs

    Directory of Open Access Journals (Sweden)

    Verónica Truniger

    2017-11-01

    Full Text Available Most of the positive-strand RNA plant viruses lack the 5′-cap and/or the poly(A-tail that act synergistically to stimulate canonical translation of cellular mRNAs. However, they have RNA elements in the 5′- or 3′-untranslated regions of their RNAs that are required for their cap-independent translation. Cap-independent translation enhancers (CITEs have been identified in the genomic 3′-end of viruses belonging to the family Tombusviridae and the genus Luteovirus. Seven classes of 3′-CITEs have been described to date based on their different RNA structures. They generally control the efficient formation of the translation initiation complex by varying mechanisms. Some 3′-CITEs bind eukaryotic translation initiation factors, others ribosomal subunits, bridging these to the 5′-end by different mechanisms, often long-distance RNA–RNA interactions. As previously proposed and recently found in one case in nature, 3′-CITEs are functionally independent elements that are transferable through recombination between viral genomes, leading to potential advantages for virus multiplication. In this review, the knowledge on 3′-CITEs and their functioning is updated. We also suggest that there is local structural conservation in the regions interacting with eIF4E of 3′-CITEs belonging to different classes.

  2. Structural and Functional Diversity of Plant Virus 3′-Cap-Independent Translation Enhancers (3′-CITEs)

    Science.gov (United States)

    Truniger, Verónica; Miras, Manuel; Aranda, Miguel A.

    2017-01-01

    Most of the positive-strand RNA plant viruses lack the 5′-cap and/or the poly(A)-tail that act synergistically to stimulate canonical translation of cellular mRNAs. However, they have RNA elements in the 5′- or 3′-untranslated regions of their RNAs that are required for their cap-independent translation. Cap-independent translation enhancers (CITEs) have been identified in the genomic 3′-end of viruses belonging to the family Tombusviridae and the genus Luteovirus. Seven classes of 3′-CITEs have been described to date based on their different RNA structures. They generally control the efficient formation of the translation initiation complex by varying mechanisms. Some 3′-CITEs bind eukaryotic translation initiation factors, others ribosomal subunits, bridging these to the 5′-end by different mechanisms, often long-distance RNA–RNA interactions. As previously proposed and recently found in one case in nature, 3′-CITEs are functionally independent elements that are transferable through recombination between viral genomes, leading to potential advantages for virus multiplication. In this review, the knowledge on 3′-CITEs and their functioning is updated. We also suggest that there is local structural conservation in the regions interacting with eIF4E of 3′-CITEs belonging to different classes. PMID:29238357

  3. 5'-3' RNA-RNA interaction facilitates cap- and poly(A) tail-independent translation of tomato bushy stunt virus mrna: a potential common mechanism for tombusviridae.

    Science.gov (United States)

    Fabian, Marc R; White, K Andrew

    2004-07-09

    Tomato bushy stunt virus (TBSV) is the prototypical member of the genus Tombusvirus in the family Tombusviridae. The (+)-strand RNA genome of TBSV lacks both a 5' cap and a 3' poly(A) tail and instead contains a 3'-terminal RNA sequence that acts as a cap-independent translational enhancer (3' CITE). In this study, we have determined the RNA secondary structure of the translation-specific central segment of the 3' CITE, termed region 3.5 (R3.5). MFOLD structural modeling combined with solution structure mapping and comparative sequence analysis indicate that R3.5 adopts a branched structure that contains three major helices. Deletion and substitution studies revealed that two of these extended stem-loop (SL) structures are essential for 3' CITE activity in vivo. In particular, the terminal loop of one of these SLs, SL-B, was found to be critical for translation. Compensatory mutational analysis showed that SL-B functions by base pairing with another SL, SL3, in the 5' untranslated region of the TBSV genome. Thus, efficient translation of TBSV mRNA in vivo requires a 5'-3' RNA-RNA interaction that effectively circularizes the message. Similar types of interactions are also predicted to occur in TBSV subgenomic mRNAs between their 5' untranslated regions and the 3' CITE, and both genomic and subgenomic 5'-3' interactions are well conserved in all members of the genus Tombusvirus. In addition, a survey of other genera in Tombusviridae revealed the potential for similar 5'-3' RNA-RNA-based interactions in their viral mRNAs, suggesting that this mechanism extends throughout this large virus family.

  4. Structural basis of RNA folding and recognition in an AMP-RNA aptamer complex.

    Science.gov (United States)

    Jiang, F; Kumar, R A; Jones, R A; Patel, D J

    1996-07-11

    The catalytic properties of RNA and its well known role in gene expression and regulation are the consequence of its unique solution structures. Identification of the structural determinants of ligand recognition by RNA molecules is of fundamental importance for understanding the biological functions of RNA, as well as for the rational design of RNA Sequences with specific catalytic activities. Towards this latter end, Szostak et al. used in vitro selection techniques to isolate RNA sequences ('aptamers') containing a high-affinity binding site for ATP, the universal currency of cellular energy, and then used this motif to engineer ribozymes with polynucleotide kinase activity. Here we present the solution structure, as determined by multidimensional NMR spectroscopy and molecular dynamics calculations, of both uniformly and specifically 13C-, 15N-labelled 40-mer RNA containing the ATP-binding motif complexed with AMP. The aptamer adopts an L-shaped structure with two nearly orthogonal stems, each capped proximally by a G x G mismatch pair, binding the AMP ligand at their junction in a GNRA-like motif.

  5. The human Ago2 MC region does not contain an eIF4E-like mRNA cap binding motif

    Directory of Open Access Journals (Sweden)

    Grishin Nick V

    2009-01-01

    Full Text Available Abstract Background Argonaute (Ago proteins interact with small regulatory RNAs to mediate gene regulatory pathways. A recent report by Kiriakidou et al. 1 describes an MC sequence region identified in Ago2 that displays similarity to the cap-binding motif in translation initiation factor 4E (eIF4E. In a cap-bound eIF4E structure, two important aromatic residues of the motif stack on either side of a 7-methylguanosine 5'-triphosphate (m7Gppp base. The corresponding Ago2 aromatic residues (F450 and F505 were hypothesized to perform the same cap-binding function. However, the detected similarity between the MC sequence and the eIF4E cap-binding motif was questionable. Results A number of sequence-based and structure-based bioinformatics methods reveal the reported similarity between the Ago2 MC sequence region and the eIF4E cap-binding motif to be spurious. Alternatively, the MC sequence region is confidently assigned to the N-terminus of the Ago piwi module, within the mid domain of experimentally determined prokaryotic Ago structures. Confident mapping of the Ago2 MC sequence region to the piwi mid domain results in a homology-based structure model that positions the identified aromatic residues over 20 Å apart, with one of the aromatic side chains (F450 contributing instead to the hydrophobic core of the domain. Conclusion Correct functional prediction based on weak sequence similarity requires substantial evolutionary and structural support. The evolutionary context of the Ago mid domain suggested by multiple sequence alignment is limited to a conserved hydrophobicity profile required for the fold and a motif following the MC region that binds guide RNA. Mapping of the MC sequence to the mid domain structure reveals Ago2 aromatics that are incompatible with eIF4E-like mRNA cap-binding, yet display some limited local structure similarities that cause the chance sequence match to eIF4E. Reviewers This article was reviewed by Arcady Mushegian

  6. The mechanism of RNA 5 ' capping with NAD(+), NADH and desphospho-CoA

    Czech Academy of Sciences Publication Activity Database

    Bird, J.G.; Zhang, Y.; Tian, Y.; Panova, Natalya; Barvík, I.; Greene, L.; Liu, M.; Buckley, B.; Krásný, Libor; Lee, J.K.; Kaplan, C.D.; Ebright, R.H.; Nickels, B.E.

    2016-01-01

    Roč. 535, č. 7612 (2016), s. 444-447 ISSN 0028-0836 R&D Projects: GA ČR GA15-05228S Institutional support: RVO:61388971 Keywords : BACTERIAL RNA * STRUCTURAL BASIS * POLYMERASE-II Subject RIV: EE - Microbiology, Virology Impact factor: 40.137, year: 2016

  7. On RNA-RNA interaction structures of fixed topological genus.

    Science.gov (United States)

    Fu, Benjamin M M; Han, Hillary S W; Reidys, Christian M

    2015-04-01

    Interacting RNA complexes are studied via bicellular maps using a filtration via their topological genus. Our main result is a new bijection for RNA-RNA interaction structures and a linear time uniform sampling algorithm for RNA complexes of fixed topological genus. The bijection allows to either reduce the topological genus of a bicellular map directly, or to lose connectivity by decomposing the complex into a pair of single stranded RNA structures. Our main result is proved bijectively. It provides an explicit algorithm of how to rewire the corresponding complexes and an unambiguous decomposition grammar. Using the concept of genus induction, we construct bicellular maps of fixed topological genus g uniformly in linear time. We present various statistics on these topological RNA complexes and compare our findings with biological complexes. Furthermore we show how to construct loop-energy based complexes using our decomposition grammar. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Characteristics and Prediction of RNA Structure

    Directory of Open Access Journals (Sweden)

    Hengwu Li

    2014-01-01

    Full Text Available RNA secondary structures with pseudoknots are often predicted by minimizing free energy, which is NP-hard. Most RNAs fold during transcription from DNA into RNA through a hierarchical pathway wherein secondary structures form prior to tertiary structures. Real RNA secondary structures often have local instead of global optimization because of kinetic reasons. The performance of RNA structure prediction may be improved by considering dynamic and hierarchical folding mechanisms. This study is a novel report on RNA folding that accords with the golden mean characteristic based on the statistical analysis of the real RNA secondary structures of all 480 sequences from RNA STRAND, which are validated by NMR or X-ray. The length ratios of domains in these sequences are approximately 0.382L, 0.5L, 0.618L, and L, where L is the sequence length. These points are just the important golden sections of sequence. With this characteristic, an algorithm is designed to predict RNA hierarchical structures and simulate RNA folding by dynamically folding RNA structures according to the above golden section points. The sensitivity and number of predicted pseudoknots of our algorithm are better than those of the Mfold, HotKnots, McQfold, ProbKnot, and Lhw-Zhu algorithms. Experimental results reflect the folding rules of RNA from a new angle that is close to natural folding.

  9. Simulation studies on structural and thermal properties of alkane thiol capped gold nanoparticles.

    Science.gov (United States)

    Devi, J Meena

    2017-06-01

    The structural and thermal properties of the passivated gold nanoparticles were explored employing molecular dynamics simulation for the different surface coverage densities of the self-assembled monolayer (SAM) of alkane thiol. The structural properties of the monolayer protected gold nanoparticles such us overall shape, organization and conformation of the capping alkane thiol chains were found to be influenced by the capping density. The structural order of the thiol capped gold nanoparticles enhances with the increase in the surface coverage density. The specific heat capacity of the alkane thiol capped gold nanoparticles was found to increase linearly with the thiol coverage density. This may be attributed to the enhancement in the lattice vibrational energy. The present simulation results suggest, that the structural and thermal properties of the alkane thiol capped gold nanoparticles may be modified by the suitable selection of the SAM coverage density. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Inverse folding of RNA pseudoknot structures

    Directory of Open Access Journals (Sweden)

    Li Linda YM

    2010-06-01

    Full Text Available Abstract Background RNA exhibits a variety of structural configurations. Here we consider a structure to be tantamount to the noncrossing Watson-Crick and G-U-base pairings (secondary structure and additional cross-serial base pairs. These interactions are called pseudoknots and are observed across the whole spectrum of RNA functionalities. In the context of studying natural RNA structures, searching for new ribozymes and designing artificial RNA, it is of interest to find RNA sequences folding into a specific structure and to analyze their induced neutral networks. Since the established inverse folding algorithms, RNAinverse, RNA-SSD as well as INFO-RNA are limited to RNA secondary structures, we present in this paper the inverse folding algorithm Inv which can deal with 3-noncrossing, canonical pseudoknot structures. Results In this paper we present the inverse folding algorithm Inv. We give a detailed analysis of Inv, including pseudocodes. We show that Inv allows to design in particular 3-noncrossing nonplanar RNA pseudoknot 3-noncrossing RNA structures-a class which is difficult to construct via dynamic programming routines. Inv is freely available at http://www.combinatorics.cn/cbpc/inv.html. Conclusions The algorithm Inv extends inverse folding capabilities to RNA pseudoknot structures. In comparison with RNAinverse it uses new ideas, for instance by considering sets of competing structures. As a result, Inv is not only able to find novel sequences even for RNA secondary structures, it does so in the context of competing structures that potentially exhibit cross-serial interactions.

  11. Exploring RNA structure by integrative molecular modelling

    DEFF Research Database (Denmark)

    Masquida, Benoît; Beckert, Bertrand; Jossinet, Fabrice

    2010-01-01

    RNA molecular modelling is adequate to rapidly tackle the structure of RNA molecules. With new structured RNAs constituting a central class of cellular regulators discovered every year, the need for swift and reliable modelling methods is more crucial than ever. The pragmatic method based...... on interactive all-atom molecular modelling relies on the observation that specific structural motifs are recurrently found in RNA sequences. Once identified by a combination of comparative sequence analysis and biochemical data, the motifs composing the secondary structure of a given RNA can be extruded...

  12. Predicting RNA Structure Using Mutual Information

    DEFF Research Database (Denmark)

    Freyhult, E.; Moulton, V.; Gardner, P. P.

    2005-01-01

    , to display and predict conserved RNA secondary structure (including pseudoknots) from an alignment. Results: We show that MIfold can be used to predict simple pseudoknots, and that the performance can be adjusted to make it either more sensitive or more selective. We also demonstrate that the overall...... package. Conclusion: MIfold provides a useful supplementary tool to programs such as RNA Structure Logo, RNAalifold and COVE, and should be useful for automatically generating structural predictions for databases such as Rfam. Availability: MIfold is freely available from http......Background: With the ever-increasing number of sequenced RNAs and the establishment of new RNA databases, such as the Comparative RNA Web Site and Rfam, there is a growing need for accurately and automatically predicting RNA structures from multiple alignments. Since RNA secondary structure...

  13. Functional 5′ UTR mRNA structures in eukaryotic translation regulation and how to find them

    Science.gov (United States)

    Leppek, Kathrin; Das, Rhiju; Barna, Maria

    2017-01-01

    RNA molecules can fold into intricate shapes that can provide an additional layer of control of gene expression beyond that of their sequence. In this Review, we discuss the current mechanistic understanding of structures in 5′ untranslated regions (UTRs) of eukaryotic mRNAs and the emerging methodologies used to explore them. These structures may regulate cap-dependent translation initiation through helicase-mediated remodelling of RNA structures and higher-order RNA interactions, as well as cap-independent translation initiation through internal ribosome entry sites (IRESs), mRNA modifications and other specialized translation pathways. We discuss known 5′ UTR RNA structures and how new structure probing technologies coupled with prospective validation, particularly compensatory mutagenesis, are likely to identify classes of structured RNA elements that shape post-transcriptional control of gene expression and the development of multicellular organisms. PMID:29165424

  14. Four RNA families with functional transient structures.

    Science.gov (United States)

    Zhu, Jing Yun A; Meyer, Irmtraud M

    2015-01-01

    Protein-coding and non-coding RNA transcripts perform a wide variety of cellular functions in diverse organisms. Several of their functional roles are expressed and modulated via RNA structure. A given transcript, however, can have more than a single functional RNA structure throughout its life, a fact which has been previously overlooked. Transient RNA structures, for example, are only present during specific time intervals and cellular conditions. We here introduce four RNA families with transient RNA structures that play distinct and diverse functional roles. Moreover, we show that these transient RNA structures are structurally well-defined and evolutionarily conserved. Since Rfam annotates one structure for each family, there is either no annotation for these transient structures or no such family. Thus, our alignments either significantly update and extend the existing Rfam families or introduce a new RNA family to Rfam. For each of the four RNA families, we compile a multiple-sequence alignment based on experimentally verified transient and dominant (dominant in terms of either the thermodynamic stability and/or attention received so far) RNA secondary structures using a combination of automated search via covariance model and manual curation. The first alignment is the Trp operon leader which regulates the operon transcription in response to tryptophan abundance through alternative structures. The second alignment is the HDV ribozyme which we extend to the 5' flanking sequence. This flanking sequence is involved in the regulation of the transcript's self-cleavage activity. The third alignment is the 5' UTR of the maturation protein from Levivirus which contains a transient structure that temporarily postpones the formation of the final inhibitory structure to allow translation of maturation protein. The fourth and last alignment is the SAM riboswitch which regulates the downstream gene expression by assuming alternative structures upon binding of SAM. All

  15. Small catalytic RNA: Structure, function and application

    Energy Technology Data Exchange (ETDEWEB)

    Monforte, Joseph Albert [Univ. of California, Berkeley, CA (United States)

    1991-04-01

    We have utilized a combination of photochemical cross-linking techniques and site-directed mutagenesis to obtain secondary and tertiary structure information for the self-cleaving, self-ligating subsequence of RNA from the negative strand of Satellite Tobacco Ringspot Virus. We have found that the helical regions fold about a hinge to promoting four different possible tertiary interactions, creating a molecular of similar shape to a paperclip. A model suggesting that the ``paperclip`` and ``hammerhead`` RNAs share a similar three dimensional structure is proposed. We have used a self-cleaving RNA molecule related to a subsequence of plant viroids, a ``hammerhead,`` to study the length-dependent folding of RNA produced during transcription by RNA polymerase. We have used this method to determine the length of RNA sequestered within elongating E. coli and T7 RNA polymerase complexes. The data show that for E. coli RNA polymerase 121±s are sequestered within the ternary complex, which is consistent with the presence of an RNA-DNA hybrid within the transcription bubble, as proposed by others. The result for T7 RNA polymerase differs from E. coli RNA polymerase, with only 10{plus_minus}1 nucleotides sequestered within the ternary complex, setting a new upper limit for the minimum RNA-DNA required for a stable elongating complex. Comparisons between E. coli and T7 RNA polymerase are made. The relevance of the results to models or transcription termination, abortive initiation, and initiation to elongation mode transitions are discussed.

  16. Alternate capping mechanisms for transcription of spring viremia of carp virus: evidence for independent mRNA initiation.

    Science.gov (United States)

    Gupta, K C; Roy, P

    1980-01-01

    Two alternate mechanisms of mRNA capping for spring viremia of carp virus have been observed. Under normal reaction conditions, a ppG residue of the capping GTP is transferred to a pA moiety of the 5' termini of mRNA transcripts. However, in reaction conditions where GppNHp is used instead of GTP, an alternate capping mechanism occurs whereby a pG residue of the capping GTP is transferred to a ppA moiety of the transcripts. The first mechanism is identical to that described previously for vesicular stomatitis virus (G. Abraham, D. P. Rhodes, and A. K. Banerjee, Nature [London] 255:37-40, 1975; A. K. Banerjee, S. A. Moyer, and D. P. Rhodes, Virology 61:547-558, 1974), and thus appears to be a conserved function during the evolution of rhabdoviruses. The alternate mechanism of capping indicates not only that capping can take place by two procedures, but also that the substrate termini have di- or triphosphate 5' ends, indicating that they are probably independently initiated. An analog of ATP, AppNHp, has been found to completely inhibit the initiation of transcription by spring viremia of carp virus, suggesting that a cleavage between the beta and gamma phosphates of ATP is essential for the initiation of transcription. However, in the presence of GppNHp, uncapped (ppAp and pppAp), capped (GpppAp), and capped methylated (m7GpppAmpAp and GpppAmpAp) transcripts are detected. Size analyses of oligodeoxythymidylic acid-cellulose-bound transcripts resolved by formamide gel electrophoresis demonstrated that full-size mRNA transcripts are synthesized as well as larger RNA species. The presence of GppNHp and S-adenosylhomocysteine in reaction mixtures did not have any effect on the type of unmethylated transcription products. Our results favor a transcription model postulated previously (D. H. L. Bishop, in H. Fraenkel-Conrat and R. R. Wagner, ed., Comprehensive Virology, vol. 10, Plenum Press, New York, 1977; D. H. L. Bishop and A. Flamand, in D. C. Burke and W. C. Russell

  17. How to find the optimal partner--studies of snurportin 1 interactions with U snRNA 5' TMG-cap analogues containing modified 2-amino group of 7-methylguanosine.

    Science.gov (United States)

    Piecyk, Karolina; Niedzwiecka, Anna; Ferenc-Mrozek, Aleksandra; Lukaszewicz, Maciej; Darzynkiewicz, Edward; Jankowska-Anyszka, Marzena

    2015-08-01

    Snurportin 1 is an adaptor protein that mediates the active nuclear import of uridine-rich small nuclear RNAs (U snRNA) by the importin-β receptor pathway. Its cellular activity influences the overall transport yield of small ribonucleoprotein complexes containing N(2),N(2),7-trimethylguanosine (TMG) capped U snRNA. So far little is still known about structural requirements related to molecular recognition of the trimethylguanosine moiety by snurportin in solution. Since these interactions are of a great biomedical importance, we synthesized a series of new 7-methylguanosine cap analogues with extended substituents at the exocyclic 2-amino group to gain a deeper insight into how the TMG-cap is adapted into the snurportin cap-binding pocket. Prepared chemical tools were applied in binding assays using emission spectroscopy. Surprisingly, our results revealed strict selectivity of snurportin towards the TMG-cap structure that relied mainly on its structural stiffness and compactness. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Facilitating RNA structure prediction with microarrays.

    Science.gov (United States)

    Kierzek, Elzbieta; Kierzek, Ryszard; Turner, Douglas H; Catrina, Irina E

    2006-01-17

    Determining RNA secondary structure is important for understanding structure-function relationships and identifying potential drug targets. This paper reports the use of microarrays with heptamer 2'-O-methyl oligoribonucleotides to probe the secondary structure of an RNA and thereby improve the prediction of that secondary structure. When experimental constraints from hybridization results are added to a free-energy minimization algorithm, the prediction of the secondary structure of Escherichia coli 5S rRNA improves from 27 to 92% of the known canonical base pairs. Optimization of buffer conditions for hybridization and application of 2'-O-methyl-2-thiouridine to enhance binding and improve discrimination between AU and GU pairs are also described. The results suggest that probing RNA with oligonucleotide microarrays can facilitate determination of secondary structure.

  19. Genome-scale characterization of RNA tertiary structures and their functional impact by RNA solvent accessibility prediction.

    Science.gov (United States)

    Yang, Yuedong; Li, Xiaomei; Zhao, Huiying; Zhan, Jian; Wang, Jihua; Zhou, Yaoqi

    2017-01-01

    As most RNA structures are elusive to structure determination, obtaining solvent accessible surface areas (ASAs) of nucleotides in an RNA structure is an important first step to characterize potential functional sites and core structural regions. Here, we developed RNAsnap, the first machine-learning method trained on protein-bound RNA structures for solvent accessibility prediction. Built on sequence profiles from multiple sequence alignment (RNAsnap-prof), the method provided robust prediction in fivefold cross-validation and an independent test (Pearson correlation coefficients, r, between predicted and actual ASA values are 0.66 and 0.63, respectively). Application of the method to 6178 mRNAs revealed its positive correlation to mRNA accessibility by dimethyl sulphate (DMS) experimentally measured in vivo (r = 0.37) but not in vitro (r = 0.07), despite the lack of training on mRNAs and the fact that DMS accessibility is only an approximation to solvent accessibility. We further found strong association across coding and noncoding regions between predicted solvent accessibility of the mutation site of a single nucleotide variant (SNV) and the frequency of that variant in the population for 2.2 million SNVs obtained in the 1000 Genomes Project. Moreover, mapping solvent accessibility of RNAs to the human genome indicated that introns, 5' cap of 5' and 3' cap of 3' untranslated regions, are more solvent accessible, consistent with their respective functional roles. These results support conformational selections as the mechanism for the formation of RNA-protein complexes and highlight the utility of genome-scale characterization of RNA tertiary structures by RNAsnap. The server and its stand-alone downloadable version are available at http://sparks-lab.org. © 2016 Yang et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  20. New windows into retroviral RNA structures.

    Science.gov (United States)

    Jayaraman, Dhivya; Kenyon, Julia Claire

    2018-01-25

    The multiple roles of both viral and cellular RNAs have become increasingly apparent in recent years, and techniques to model them have become significantly more powerful, enabling faster and more accurate visualization of RNA structures. Techniques such as SHAPE (selective 2'OH acylation analysed by primer extension) have revolutionized the field, and have been used to examine RNAs belonging to many and diverse retroviruses. Secondary structure probing reagents such as these have been aided by the development of faster methods of analysis either via capillary or next-generation sequencing, allowing the analysis of entire genomes, and of retroviral RNA structures within virions. Techniques to model the three-dimensional structures of these large RNAs have also recently developed. The flexibility of retroviral RNAs, both structural and functional, is clear from the results of these new experimental techniques. Retroviral RNA structures and structural changes control many stages of the lifecycle, and both the RNA structures themselves and their interactions with ligands are potential new drug targets. In addition, our growing understanding of retroviral RNA structures is aiding our knowledge of cellular RNA form and function.

  1. The structural effects of cap and trade climate policy

    International Nuclear Information System (INIS)

    Goettle, Richard J.; Fawcett, Allen A.

    2009-01-01

    The Inter-temporal General Equilibrium Model (IGEM) explores the cost to the U.S. economy of increasingly more stringent cap and trade regimes. The economy-wide losses are small with energy, agriculture, chemicals, high tech manufacturing and trade being most affected. The availability of lower cost offsets substantially reduces these economic losses. The economy becomes less capital but more labor intensive. Household welfare losses are smaller for full consumption (goods, services and leisure). A more inelastic trade-off between consumption and leisure dramatically reduces policy costs as do more favorable revenue recycling options. Induced technical change yields a small, measurable reduction in policy costs. (author)

  2. MicroRNA-target binding structures mimic microRNA duplex structures in humans.

    Directory of Open Access Journals (Sweden)

    Xi Chen

    Full Text Available Traditionally, researchers match a microRNA guide strand to mRNA sequences using sequence comparisons to predict its potential target genes. However, many of the predictions can be false positives due to limitations in sequence comparison alone. In this work, we consider the association of two related RNA structures that share a common guide strand: the microRNA duplex and the microRNA-target binding structure. We have analyzed thousands of such structure pairs and found many of them share high structural similarity. Therefore, we conclude that when predicting microRNA target genes, considering just the microRNA guide strand matches to gene sequences may not be sufficient--the microRNA duplex structure formed by the guide strand and its companion passenger strand must also be considered. We have developed software to translate RNA binding structure into encoded representations, and we have also created novel automatic comparison methods utilizing such encoded representations to determine RNA structure similarity. Our software and methods can be utilized in the other RNA secondary structure comparisons as well.

  3. T-Cap Pull-Off and Bending Behavior for Stitched Structure

    Science.gov (United States)

    Lovejoy, Andrew E.; Leone, Frank A., Jr.

    2016-01-01

    The Pultruded Rod Stitched Efficient Unitized Structure (PRSEUS) is a structural concept that was developed by The Boeing Company to address the complex structural design aspects associated with a pressurized hybrid wing body aircraft configuration. An important design feature required for assembly is the integrally stitched T-cap, which provides connectivity of the corner (orthogonal) joint between adjacent panels. A series of tests were conducted on T-cap test articles, with and without a rod stiffener penetrating the T-cap web, under tension (pull-off) and bending loads. Three designs were tested, including the baseline design used in largescale test articles. The baseline had only the manufacturing stitch row adjacent to the fillet at the base of the T-cap web. Two new designs added stitching rows to the T-cap web at either 0.5- or 1.0-inch spacing along the height of the web. Testing was conducted at NASA Langley Research Center to determine the behavior of the T-cap region resulting from the applied loading. Results show that stitching arrests the initial delamination failures so that the maximum strength capability exceeds the load at which the initial delaminations develop. However, it was seen that the added web stitching had very little effect on the initial delamination failure load, but actually decreased the initial delamination failure load for tension loading of test articles without a stiffener passing through the web. Additionally, the added web stitching only increased the maximum load capability by between 1% and 12.5%. The presence of the stiffener, however, did increase the initial and maximum loads for both tension and bending loading as compared to the stringerless baseline design. Based on the results of the few samples tested, the additional stitching in the T-cap web showed little advantage over the baseline design in terms of structural failure at the T-cap web/skin junction for the current test articles.

  4. Small catalytic RNA: Structure, function and application

    Energy Technology Data Exchange (ETDEWEB)

    Monforte, J.A.

    1991-04-01

    We have utilized a combination of photochemical cross-linking techniques and site-directed mutagenesis to obtain secondary and tertiary structure information for the self-cleaving, self-ligating subsequence of RNA from the negative strand of Satellite Tobacco Ringspot Virus. We have found that the helical regions fold about a hinge to promoting four different possible tertiary interactions, creating a molecular of similar shape to a paperclip. A model suggesting that the paperclip'' and hammerhead'' RNAs share a similar three dimensional structure is proposed. We have used a self-cleaving RNA molecule related to a subsequence of plant viroids, a hammerhead,'' to study the length-dependent folding of RNA produced during transcription by RNA polymerase. We have used this method to determine the length of RNA sequestered within elongating E. coli and T7 RNA polymerase complexes. The data show that for E. coli RNA polymerase 12{plus minus}1 nucleotides are sequestered within the ternary complex, which is consistent with the presence of an RNA-DNA hybrid within the transcription bubble, as proposed by others. The result for T7 RNA polymerase differs from E. coli RNA polymerase, with only 10{plus minus}1 nucleotides sequestered within the ternary complex, setting a new upper limit for the minimum RNA-DNA required for a stable elongating complex. Comparisons between E. coli and T7 RNA polymerase are made. The relevance of the results to models or transcription termination, abortive initiation, and initiation to elongation mode transitions are discussed.

  5. Origin of interfacial perpendicular magnetic anisotropy in MgO/CoFe/metallic capping layer structures.

    Science.gov (United States)

    Peng, Shouzhong; Wang, Mengxing; Yang, Hongxin; Zeng, Lang; Nan, Jiang; Zhou, Jiaqi; Zhang, Youguang; Hallal, Ali; Chshiev, Mairbek; Wang, Kang L; Zhang, Qianfan; Zhao, Weisheng

    2015-12-11

    Spin-transfer-torque magnetic random access memory (STT-MRAM) attracts extensive attentions due to its non-volatility, high density and low power consumption. The core device in STT-MRAM is CoFeB/MgO-based magnetic tunnel junction (MTJ), which possesses a high tunnel magnetoresistance ratio as well as a large value of perpendicular magnetic anisotropy (PMA). It has been experimentally proven that a capping layer coating on CoFeB layer is essential to obtain a strong PMA. However, the physical mechanism of such effect remains unclear. In this paper, we investigate the origin of the PMA in MgO/CoFe/metallic capping layer structures by using a first-principles computation scheme. The trend of PMA variation with different capping materials agrees well with experimental results. We find that interfacial PMA in the three-layer structures comes from both the MgO/CoFe and CoFe/capping layer interfaces, which can be analyzed separately. Furthermore, the PMAs in the CoFe/capping layer interfaces are analyzed through resolving the magnetic anisotropy energy by layer and orbital. The variation of PMA with different capping materials is attributed to the different hybridizations of both d and p orbitals via spin-orbit coupling. This work can significantly benefit the research and development of nanoscale STT-MRAM.

  6. RNA secondary structure prediction using soft computing.

    Science.gov (United States)

    Ray, Shubhra Sankar; Pal, Sankar K

    2013-01-01

    Prediction of RNA structure is invaluable in creating new drugs and understanding genetic diseases. Several deterministic algorithms and soft computing-based techniques have been developed for more than a decade to determine the structure from a known RNA sequence. Soft computing gained importance with the need to get approximate solutions for RNA sequences by considering the issues related with kinetic effects, cotranscriptional folding, and estimation of certain energy parameters. A brief description of some of the soft computing-based techniques, developed for RNA secondary structure prediction, is presented along with their relevance. The basic concepts of RNA and its different structural elements like helix, bulge, hairpin loop, internal loop, and multiloop are described. These are followed by different methodologies, employing genetic algorithms, artificial neural networks, and fuzzy logic. The role of various metaheuristics, like simulated annealing, particle swarm optimization, ant colony optimization, and tabu search is also discussed. A relative comparison among different techniques, in predicting 12 known RNA secondary structures, is presented, as an example. Future challenging issues are then mentioned.

  7. Human cytomegalovirus TRS1 protein associates with the 7-methylguanosine mRNA cap and facilitates translation.

    Science.gov (United States)

    Ziehr, Benjamin; Lenarcic, Erik; Vincent, Heather A; Cecil, Chad; Garcia, Benjamin; Shenk, Thomas; Moorman, Nathaniel J

    2015-06-01

    Viruses rely on the host translation machinery for the synthesis of viral proteins. Human cells have evolved sensors that recognize viral RNAs and inhibit mRNA translation in order to limit virus replication. Understanding how viruses manipulate the host translation machinery to gain access to ribosomes and disable the antiviral response is therefore a critical aspect of the host/pathogen interface. In this study, we used a proteomics approach to identify human cytomegalovirus (HCMV) proteins that might contribute to viral mRNA translation. The HCMV TRS1 protein (pTRS1) associated with the 7-methylguanosine mRNA cap, increased the total level of protein synthesis, and colocalized with mRNAs undergoing translation initiation during infection. pTRS1 stimulated translation of a nonviral reporter gene and increased the translation of a reporter containing an HCMV 5' untranslated region (5'UTR) to a greater extent. The preferential effect of pTRS1 on translation of an mRNA containing a viral 5'UTR required the pTRS1 RNA and double-stranded RNA-dependent protein kinase (PKR)-binding domains, and was likely the result of PKR inhibition. However, pTRS1 also stimulated the total level of protein synthesis and translation directed by an HCMV 5'UTR in cells lacking PKR. Thus our results demonstrate that pTRS1 stimulates translation through both PKR-dependent and PKR-independent mechanisms. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Accelerated probabilistic inference of RNA structure evolution

    Directory of Open Access Journals (Sweden)

    Holmes Ian

    2005-03-01

    Full Text Available Abstract Background Pairwise stochastic context-free grammars (Pair SCFGs are powerful tools for evolutionary analysis of RNA, including simultaneous RNA sequence alignment and secondary structure prediction, but the associated algorithms are intensive in both CPU and memory usage. The same problem is faced by other RNA alignment-and-folding algorithms based on Sankoff's 1985 algorithm. It is therefore desirable to constrain such algorithms, by pre-processing the sequences and using this first pass to limit the range of structures and/or alignments that can be considered. Results We demonstrate how flexible classes of constraint can be imposed, greatly reducing the computational costs while maintaining a high quality of structural homology prediction. Any score-attributed context-free grammar (e.g. energy-based scoring schemes, or conditionally normalized Pair SCFGs is amenable to this treatment. It is now possible to combine independent structural and alignment constraints of unprecedented general flexibility in Pair SCFG alignment algorithms. We outline several applications to the bioinformatics of RNA sequence and structure, including Waterman-Eggert N-best alignments and progressive multiple alignment. We evaluate the performance of the algorithm on test examples from the RFAM database. Conclusion A program, Stemloc, that implements these algorithms for efficient RNA sequence alignment and structure prediction is available under the GNU General Public License.

  9. Retroviral RNA Dimerization: From Structure to Functions

    Directory of Open Access Journals (Sweden)

    Noé Dubois

    2018-03-01

    Full Text Available The genome of the retroviruses is a dimer composed by two homologous copies of genomic RNA (gRNA molecules of positive polarity. The dimerization process allows two gRNA molecules to be non-covalently linked together through intermolecular base-pairing. This step is critical for the viral life cycle and is highly conserved among retroviruses with the exception of spumaretroviruses. Furthermore, packaging of two gRNA copies into viral particles presents an important evolutionary advantage for immune system evasion and drug resistance. Recent studies reported RNA switches models regulating not only gRNA dimerization, but also translation and packaging, and a spatio-temporal characterization of viral gRNA dimerization within cells are now at hand. This review summarizes our current understanding on the structural features of the dimerization signals for a variety of retroviruses (HIVs, MLV, RSV, BLV, MMTV, MPMV…, the mechanisms of RNA dimer formation and functional implications in the retroviral cycle.

  10. Structural, spectroscopic and biological investigation of copper oxides nanoparticles with various capping agents

    Energy Technology Data Exchange (ETDEWEB)

    Nowak, A., E-mail: ana.maria.nowak@gmail.com [A. Chelkowski Institute of Physics, University of Silesia, Katowice (Poland); Szade, J.; Talik, E.; Ratuszna, A. [A. Chelkowski Institute of Physics, University of Silesia, Katowice (Poland); Ostafin, M. [Agricultural University of Cracow, Department of Microbiology, Krakow (Poland); Peszke, J. [A. Chelkowski Institute of Physics, University of Silesia, Katowice (Poland)

    2014-06-01

    Powder composed of copper oxides nanoparticles with various capping agents has been synthesized and characterized with the use of X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and X-ray diffraction (XRD). Polyvinyl alcohol (PVA), glycol propylene, glycerin and glycerin plus ammonia were used as capping agents. The scanning electron microscopy (SEM) studies showed that nanoparticles form agglomerates with the size from 80 to 120 nm while particles size determined from the XRD experiment was in the range from 7 to 21 nm. XPS and XRD experiments revealed that depending on capping and reducing agents used in the synthesis nanoparticles are composed of Cu{sub 2}O, CuO or a mixture of them. The biological activity test performed for a selected sample where the capping agent was glycerin plus ammonia has shown promising killing/inhibiting behavior, very effective especially for Gram negatives bacteria. - Highlights: • We obtained copper oxide nanoparticles in a powder form. • Several capping agents were tested. • Structural and chemical tests showed that the main component were Cu{sub 2}O and CuO. • The size of nanoparticles was in the range 7–21 nm. • Nanoparticles with glycerin and ammonia capping agent showed good antibacterial properties.

  11. The art of editing RNA structural alignments

    DEFF Research Database (Denmark)

    Andersen, Ebbe Sloth

    2014-01-01

    Manual editing of RNA structural alignments may be considered more art than science, since it still requires an expert biologist to take multiple levels of information into account and be slightly creative when constructing high-quality alignments. Even though the task is rather tedious, it is re......Manual editing of RNA structural alignments may be considered more art than science, since it still requires an expert biologist to take multiple levels of information into account and be slightly creative when constructing high-quality alignments. Even though the task is rather tedious...

  12. An essential role for trimethylguanosine RNA caps in Saccharomyces cerevisiae meiosis and their requirement for splicing of SAE3 and PCH2 meiotic pre-mRNAs.

    Science.gov (United States)

    Qiu, Zhicheng R; Shuman, Stewart; Schwer, Beate

    2011-07-01

    Tgs1 is the enzyme that converts m(7)G RNA caps to the 2,2,7-trimethylguanosine (TMG) caps characteristic of spliceosomal snRNAs. Fungi grow vegetatively without TMG caps, thereby raising the question of what cellular transactions, if any, are TMG cap-dependent. Here, we report that Saccharomyces cerevisiae Tgs1 methyltransferase activity is essential for meiosis. tgs1Δ cells are specifically defective in splicing PCH2 and SAE3 meiotic pre-mRNAs. The TMG requirement for SAE3 splicing is alleviated by two intron mutations: a UAUUAAC to UACUAAC change that restores a consensus branchpoint and disruption of a stem-loop encompassing the branchpoint. The TMG requirement for PCH2 splicing is alleviated by a CACUAAC to UACUAAC change restoring a consensus branchpoint and by shortening the PCH2 5' exon. Placing the SAE3 and PCH2 introns within a HIS3 reporter confers Tgs1-dependent histidine prototrophy, signifying that the respective introns are portable determinants of TMG-dependent gene expression. Analysis of in vitro splicing in extracts of TGS1 versus tgs1Δ cells showed that SAE3 intron removal was enfeebled without TMG caps, whereas splicing of ACT1 was unaffected. Our findings illuminate a new mode of tunable splicing, a reliance on TMG caps for an essential developmental RNA transaction, and three genetically distinct meiotic splicing regulons in budding yeast.

  13. Rational design of avian metapneumovirus live attenuated vaccines by inhibiting viral messenger RNA cap methyltransferase

    Science.gov (United States)

    Avian metapneumovirus (aMPV), also known as avian pneumovirus or turkey rhinotracheitis, is a non-segmented negative-sense RNA virus belonging to the family of Paramyxoviridae, the subfamily Pneumovirinae, and the genus Metapneumovirus. aMPV is the causative agent of respiratory tract infection and ...

  14. The performance of multi-factor term structure models for pricing and hedging caps and swaptions

    NARCIS (Netherlands)

    Driessen, J.J.A.G.; Klaassen, P.; Melenberg, B.

    2000-01-01

    In this paper we empirically compare a wide range of different term structure models when it comes to the pricing and, in particular, hedging of caps and swaptions. We analyze the influence of the number of factors on the hedging and pricing results, and investigate which type of data "interest rate

  15. The Performance of Multi-Factor Term Structure Models for Pricing and Hedging Caps and Swaptions

    NARCIS (Netherlands)

    Driessen, J.J.A.G.; Klaassen, P.; Melenberg, B.

    2000-01-01

    In this paper we empirically compare different term structure models when it comes to the pricing and hedging of caps and swaptions.We analyze the influence of the number of factors on the pricing and hedging results, and investigate which type of data -interest rate data or derivative price data-

  16. Synthesis, effect of capping agents, structural, optical and photoluminescence properties of ZnO nanoparticles

    International Nuclear Information System (INIS)

    Singh, A.K.; Viswanath, V.; Janu, V.C.

    2009-01-01

    Zinc oxide nanoparticles were synthesized using chemical method in alcohol base. During synthesis three capping agents, i.e. triethanolamine (TEA), oleic acid and thioglycerol, were used and the effect of concentrations was analyzed for their effectiveness in limiting the particle growth. Thermal stability of ZnO nanoparticles prepared using TEA, oleic acid and thioglycerol capping agents, was studied using thermogravimetric analyzer (TGA). ZnO nanoparticles capped with TEA showed maximum weight loss. X-ray diffraction (XRD) and scanning electron microscopy (SEM) were used for structural and morphological characterization of ZnO nanoparticles. Particle size was evaluated using effective mass approximation method from UV-vis spectroscopy and Scherrer's formula from XRD patterns. XRD analysis revealed single crystal ZnO nanoparticles of size 12-20 nm in case of TEA capping. TEA, oleic acid and thioglycerol capped synthesized ZnO nanoparticles were investigated at room temperature photoluminescence for three excitation wavelengths i.e. 304, 322 and 325 nm, showing strong peaks at about 471 nm when excited at 322 and 325 nm whereas strong peak was observed at 411 for 304 nm excitation.

  17. Prolonged reorganization of thiol-capped Au nanoparticles layered structures

    Directory of Open Access Journals (Sweden)

    Sarathi Kundu

    2013-09-01

    Full Text Available Prolonged reorganization behaviour of mono-, di-, tri- and multi-layer films of Au nanoparticles prepared by Langmuir-Blodgett method on hydrophobic Si(001 substrates have been studied by using X-ray scattering techniques. Out-of-plane study shows that although at the initial stage the reorganization occurs through the compaction of the films keeping the layered structure unchanged but finally all layered structures modify to monolayer structure. Due to this reorganization the Au density increases within the nanometer thick films. In-plane study shows that inside the reorganized films Au nanoparticles are distributed randomly and the particle size modifies as the metallic core of Au nanoparticles coalesces.

  18. The structure of hookworm platelet inhibitor (HPI), a CAP superfamily member from Ancylostoma caninum.

    Science.gov (United States)

    Ma, Dongying; Francischetti, Ivo M B; Ribeiro, Jose M C; Andersen, John F

    2015-06-01

    Secreted protein components of hookworm species include a number of representatives of the cysteine-rich/antigen 5/pathogenesis-related 1 (CAP) protein family known as Ancylostoma-secreted proteins (ASPs). Some of these have been considered as candidate antigens for the development of vaccines against hookworms. The functions of most CAP superfamily members are poorly understood, but one form, the hookworm platelet inhibitor (HPI), has been isolated as a putative antagonist of the platelet integrins αIIbβ3 and α2β1. Here, the crystal structure of HPI is described and its structural features are examined in relation to its possible function. The HPI structure is similar to those of other ASPs and shows incomplete conservation of the sequence motifs CAP1 and CAP2 that are considered to be diagnostic of CAP superfamily members. The asymmetric unit of the HPI crystal contains a dimer with an extensive interaction interface, but chromatographic measurements indicate that it is primarily monomeric in solution. In the dimeric structure, the putative active-site cleft areas from both monomers are united into a single negatively charged depression. A potential Lys-Gly-Asp disintegrin-like motif was identified in the sequence of HPI, but is not positioned at the apex of a tight turn, making it unlikely that it interacts with the integrin. Recombinant HPI produced in Escherichia coli was found not to inhibit the adhesion of human platelets to collagen or fibrinogen, despite having a native structure as shown by X-ray diffraction. This result corroborates previous analyses of recombinant HPI and suggests that it might require post-translational modification or have a different biological function.

  19. Solving the RNA polymerase I structural puzzle

    Energy Technology Data Exchange (ETDEWEB)

    Moreno-Morcillo, María [European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg (Germany); Taylor, Nicholas M. I. [Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid (Spain); Gruene, Tim [Georg-August-University, Tammannstrasse 4, 37077 Göttingen (Germany); Legrand, Pierre [SOLEIL Synchrotron, L’Orme de Merisiers, Saint Aubin, Gif-sur-Yvette (France); Rashid, Umar J. [European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg (Germany); Ruiz, Federico M. [Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid (Spain); Steuerwald, Ulrich; Müller, Christoph W. [European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg (Germany); Fernández-Tornero, Carlos, E-mail: cftornero@cib.csic.es [Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid (Spain); European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg (Germany)

    2014-10-01

    Details of the RNA polymerase I crystal structure determination provide a framework for solution of the structures of other multi-subunit complexes. Simple crystallographic experiments are described to extract relevant biological information such as the location of the enzyme active site. Knowing the structure of multi-subunit complexes is critical to understand basic cellular functions. However, when crystals of these complexes can be obtained they rarely diffract beyond 3 Å resolution, which complicates X-ray structure determination and refinement. The crystal structure of RNA polymerase I, an essential cellular machine that synthesizes the precursor of ribosomal RNA in the nucleolus of eukaryotic cells, has recently been solved. Here, the crucial steps that were undertaken to build the atomic model of this multi-subunit enzyme are reported, emphasizing how simple crystallographic experiments can be used to extract relevant biological information. In particular, this report discusses the combination of poor molecular replacement and experimental phases, the application of multi-crystal averaging and the use of anomalous scatterers as sequence markers to guide tracing and to locate the active site. The methods outlined here will likely serve as a reference for future structural determination of large complexes at low resolution.

  20. Protein Structure and the Sequential Structure of mRNA

    DEFF Research Database (Denmark)

    Brunak, Søren; Engelbrecht, Jacob

    1996-01-01

    entries in the Brookhaven Protein Data Bank produced 719 protein chains with matching mRNA sequence, amino acid sequence, and secondary structure assignment, By neural network analysis, we found strong signals in mRNA sequence regions surrounding helices and sheets, These signals do not originate from......A direct comparison of experimentally determined protein structures and their corresponding protein coding mRNA sequences has been performed, We examine whether real world data support the hypothesis that clusters of rare codons correlate with the location of structural units in the resulting...... protein, The degeneracy of the genetic code allows for a biased selection of codons which may control the translational rate of the ribosome, and may thus in vivo have a catalyzing effect on the folding of the polypeptide chain, A complete search for GenBank nucleotide sequences coding for structural...

  1. Structural basis for the regulation mechanism of the tyrosine kinase CapB from Staphylococcus aureus

    DEFF Research Database (Denmark)

    Olivares-Illana, Vanesa; Meyer, Philippe; Bechet, Emmanuelle

    2008-01-01

    Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly...... understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus...... be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high...

  2. RNA2DMut: a web tool for the design and analysis of RNA structure mutations.

    Science.gov (United States)

    Moss, Walter N

    2018-03-01

    With the widespread application of high-throughput sequencing, novel RNA sequences are being discovered at an astonishing rate. The analysis of function, however, lags behind. In both the cis - and trans -regulatory functions of RNA, secondary structure (2D base-pairing) plays essential regulatory roles. In order to test RNA function, it is essential to be able to design and analyze mutations that can affect structure. This was the motivation for the creation of the RNA2DMut web tool. With RNA2DMut, users can enter in RNA sequences to analyze, constrain mutations to specific residues, or limit changes to purines/pyrimidines. The sequence is analyzed at each base to determine the effect of every possible point mutation on 2D structure. The metrics used in RNA2DMut rely on the calculation of the Boltzmann structure ensemble and do not require a robust 2D model of RNA structure for designing mutations. This tool can facilitate a wide array of uses involving RNA: for example, in designing and evaluating mutants for biological assays, interrogating RNA-protein interactions, identifying key regions to alter in SELEX experiments, and improving RNA folding and crystallization properties for structural biology. Additional tools are available to help users introduce other mutations (e.g., indels and substitutions) and evaluate their effects on RNA structure. Example calculations are shown for five RNAs that require 2D structure for their function: the MALAT1 mascRNA, an influenza virus splicing regulatory motif, the EBER2 viral noncoding RNA, the Xist lncRNA repA region, and human Y RNA 5. RNA2DMut can be accessed at https://rna2dmut.bb.iastate.edu/. © 2018 Moss; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  3. The tRNA-like structure of Turnip yellow mosaic virus RNA is a 3'-translational enhancer

    International Nuclear Information System (INIS)

    Matsuda, Daiki; Dreher, Theo W.

    2004-01-01

    Many positive stand RNA viral genomes lack the poly(A) tail that is characteristic of cellular mRNAs and that promotes translation in cis. The 3' untranslated regions (UTRs) of such genomes are expected to provide similar translation-enhancing properties as a poly(A) tail, yet the great variety of 3' sequences suggests that this is accomplished in a range of ways. We have identified a translational enhancer present in the 3' UTR of Turnip yellow mosaic virus (TYMV) RNA using luciferase reporter RNAs with generic 5' sequences transfected into plant cells. The 3' terminal 109 nucleotides comprising the tRNA-like structure (TLS) and an upstream pseudoknot (UPSK) act in synergy with a 5'-cap to enhance translation, with a minor contribution in stabilizing the RNA. Maximum enhancement requires that the RNA be capable of aminoacylation, but either the native valine or engineered methionine is acceptable. Mutations that decrease the affinity for translation elongation factor eEF1A (but also diminish aminoacylation efficiency) strongly decrease translational enhancement, suggesting that eEF1A is mechanistically involved. The UPSK seems to act as an important, though nonspecific, spacer element ensuring proper presentation of a functional TLS. Our studies have uncovered a novel type of translational enhancer and a new role for a plant viral TLS

  4. Synthesis, Structural and Optical Properties of TOPO and HDA Capped Cadmium Sulphide Nanocrystals, and the Effect of Capping Ligand Concentration

    Directory of Open Access Journals (Sweden)

    Damian C. Onwudiwe

    2015-01-01

    Full Text Available The thermal decomposition of bis(N,N-diallyldithiocarbamatoCd(II in a “one-pot” synthesis in tri-n-octylphosphine oxide (TOPO and hexadecylamine (HDA afforded CdS (TOPO-CdS and HDA-CdS of varying optical properties and morphologies. The influence of the ratio of the precursor concentration to the capping molecule, as a factor affecting the morphology and size of the nanoparticles, was investigated. The particles varied in shape from spheres to rods and show quantum size effects in their optical spectra with clear differences in the photoluminescence (PL spectra. The PL spectrum of the HDA capped CdS nanoparticles has an emission maximum centred at 468, 472, and 484 nm for the precursor to HDA concentration ratio of 1 : 10, 1 : 15, and 1 : 20, respectively, while the TOPO capped nanoparticles show emission peaks at 483, 494, and 498 nm at the same concentration ratio. Powdered X-ray diffraction (p-XRD shows the nanoparticles to be hexagonal. The crystallinity of the nanoparticles was evident from high resolution transmission electron microscopy (HRTEM which gave well-defined images of particles with clear lattice fringes.

  5. Structural imprints in vivo decode RNA regulatory mechanisms.

    Science.gov (United States)

    Spitale, Robert C; Flynn, Ryan A; Zhang, Qiangfeng Cliff; Crisalli, Pete; Lee, Byron; Jung, Jong-Wha; Kuchelmeister, Hannes Y; Batista, Pedro J; Torre, Eduardo A; Kool, Eric T; Chang, Howard Y

    2015-03-26

    Visualizing the physical basis for molecular behaviour inside living cells is a great challenge for biology. RNAs are central to biological regulation, and the ability of RNA to adopt specific structures intimately controls every step of the gene expression program. However, our understanding of physiological RNA structures is limited; current in vivo RNA structure profiles include only two of the four nucleotides that make up RNA. Here we present a novel biochemical approach, in vivo click selective 2'-hydroxyl acylation and profiling experiment (icSHAPE), which enables the first global view, to our knowledge, of RNA secondary structures in living cells for all four bases. icSHAPE of the mouse embryonic stem cell transcriptome versus purified RNA folded in vitro shows that the structural dynamics of RNA in the cellular environment distinguish different classes of RNAs and regulatory elements. Structural signatures at translational start sites and ribosome pause sites are conserved from in vitro conditions, suggesting that these RNA elements are programmed by sequence. In contrast, focal structural rearrangements in vivo reveal precise interfaces of RNA with RNA-binding proteins or RNA-modification sites that are consistent with atomic-resolution structural data. Such dynamic structural footprints enable accurate prediction of RNA-protein interactions and N(6)-methyladenosine (m(6)A) modification genome wide. These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression.

  6. Structural Chemistry of Human RNA Methyltransferases.

    Science.gov (United States)

    Schapira, Matthieu

    2016-03-18

    RNA methyltransferases (RNMTs) play important roles in RNA stability, splicing, and epigenetic mechanisms. They constitute a promising target class that is underexplored by the medicinal chemistry community. Information of relevance to drug design can be extracted from the rich structural coverage of human RNMTs. In this work, the structural chemistry of this protein family is analyzed in depth. Unlike most methyltransferases, RNMTs generally feature a substrate-binding site that is largely open on the cofactor-binding pocket, favoring the design of bisubstrate inhibitors. Substrate purine or pyrimidines are often sandwiched between hydrophobic walls that can accommodate planar ring systems. When the substrate base is laying on a shallow surface, a 5' flanking base is sometimes anchored in a druggable cavity. The cofactor-binding site is structurally more diverse than in protein methyltransferases and more druggable in SPOUT than in Rossman-fold enzymes. Finally, conformational plasticity observed both at the substrate and cofactor binding sites may be a challenge for structure-based drug design. The landscape drawn here may inform ongoing efforts toward the discovery of the first human RNMT inhibitors.

  7. RStrucFam: a web server to associate structure and cognate RNA for RNA-binding proteins from sequence information.

    Science.gov (United States)

    Ghosh, Pritha; Mathew, Oommen K; Sowdhamini, Ramanathan

    2016-10-07

    RNA-binding proteins (RBPs) interact with their cognate RNA(s) to form large biomolecular assemblies. They are versatile in their functionality and are involved in a myriad of processes inside the cell. RBPs with similar structural features and common biological functions are grouped together into families and superfamilies. It will be useful to obtain an early understanding and association of RNA-binding property of sequences of gene products. Here, we report a web server, RStrucFam, to predict the structure, type of cognate RNA(s) and function(s) of proteins, where possible, from mere sequence information. The web server employs Hidden Markov Model scan (hmmscan) to enable association to a back-end database of structural and sequence families. The database (HMMRBP) comprises of 437 HMMs of RBP families of known structure that have been generated using structure-based sequence alignments and 746 sequence-centric RBP family HMMs. The input protein sequence is associated with structural or sequence domain families, if structure or sequence signatures exist. In case of association of the protein with a family of known structures, output features like, multiple structure-based sequence alignment (MSSA) of the query with all others members of that family is provided. Further, cognate RNA partner(s) for that protein, Gene Ontology (GO) annotations, if any and a homology model of the protein can be obtained. The users can also browse through the database for details pertaining to each family, protein or RNA and their related information based on keyword search or RNA motif search. RStrucFam is a web server that exploits structurally conserved features of RBPs, derived from known family members and imprinted in mathematical profiles, to predict putative RBPs from sequence information. Proteins that fail to associate with such structure-centric families are further queried against the sequence-centric RBP family HMMs in the HMMRBP database. Further, all other essential

  8. Structural and luminescent properties of Fe3+ doped PVA capped CdTe nanoparticles

    Directory of Open Access Journals (Sweden)

    Ravindranadh K.

    2017-07-01

    Full Text Available During recent decades, magnetic and semiconductor nanoparticles have attracted significant attention of scientists in various fields of engineering, physics, chemistry, biology and medicine. Fe3+ doped PVA capped CdTe nanoparticles were prepared by co-precipitation method and characterized by powder X-ray diffraction, SEM, TEM, FT-IR, optical, EPR and PL techniques to collect the information about the crystal structure, coordination/local site symmetry of doped Fe3+ ions in the host lattice and the luminescent properties of prepared sample. Powder XRD data revealed that the crystal structure belongs to a cubic system and its lattice cell parameters were evaluated. The average crystallite size was estimated to be 8 nm. The morphology of prepared samples was analyzed by using SEM and TEM investigations. Functional groups of the prepared sample were observed in FT-IR spectra. Optical absorption and EPR studies have shown that on doping, Fe3+ ions enter the host lattice in octahedral site symmetry. PL studies of Fe3+ doped PVA capped CdTe nanoparticles revealed UV and blue emission bands. CIE chromaticity coordinates were also calculated from the emission spectrum of Fe3+ doped PVA capped CdTe nanoparticles.

  9. RNA.

    Science.gov (United States)

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  10. Automated and fast building of three-dimensional RNA structures.

    Science.gov (United States)

    Zhao, Yunjie; Huang, Yangyu; Gong, Zhou; Wang, Yanjie; Man, Jianfen; Xiao, Yi

    2012-01-01

    Building tertiary structures of non-coding RNA is required to understand their functions and design new molecules. Current algorithms of RNA tertiary structure prediction give satisfactory accuracy only for small size and simple topology and many of them need manual manipulation. Here, we present an automated and fast program, 3dRNA, for RNA tertiary structure prediction with reasonable accuracy for RNAs of larger size and complex topology.

  11. RNA structure alignment by a unit-vector approach.

    Science.gov (United States)

    Capriotti, Emidio; Marti-Renom, Marc A

    2008-08-15

    The recent discovery of tiny RNA molecules such as microRNAs and small interfering RNA are transforming the view of RNA as a simple information transfer molecule. Similar to proteins, the native three-dimensional structure of RNA determines its biological activity. Therefore, classifying the current structural space is paramount for functionally annotating RNA molecules. The increasing numbers of RNA structures deposited in the PDB requires more accurate, automatic and benchmarked methods for RNA structure comparison. In this article, we introduce a new algorithm for RNA structure alignment based on a unit-vector approach. The algorithm has been implemented in the SARA program, which results in RNA structure pairwise alignments and their statistical significance. The SARA program has been implemented to be of general applicability even when no secondary structure can be calculated from the RNA structures. A benchmark against the ARTS program using a set of 1275 non-redundant pairwise structure alignments results in inverted approximately 6% extra alignments with at least 50% structurally superposed nucleotides and base pairs. A first attempt to perform RNA automatic functional annotation based on structure alignments indicates that SARA can correctly assign the deepest SCOR classification to >60% of the query structures. The SARA program is freely available through a World Wide Web server http://sgu.bioinfo.cipf.es/services/SARA/. Supplementary data are available at Bioinformatics online.

  12. Supramolecular Self-Assembly of Histidine-Capped-Dialkoxy-Anthracene: A Visible Light Triggered Platform for facile siRNA Delivery

    KAUST Repository

    Patil, Sachin

    2016-06-29

    Supramolecular self-assembly of histidine-capped-dialkoxy-anthracene (HDA) results in the formation of light responsive nanostructures.Single-crystal X-ray diffraction analysis of HDA shows two types of hydrogen bonding. The first hydrogen bond is established between the imidazole moieties while the second involves the oxygen atom of one amide group and the hydrogen atom of a second amide group. When protonated in acidic aqueous media, HDA successfully complexes siRNA yielding spherical nanostructures. This biocompatible platform controllably delivers siRNA with high efficacy upon visible light irradiation leading up to 90% of gene silencing in live cells.

  13. The crystal structure of tRNA

    Indian Academy of Sciences (India)

    Madhu

    of yeast alanine tRNA by Robert Holley's group at Cornell. University ... decode nonsense codons) with John Smith and Brenner. However, my ... tRNA from 10 g of unfractionated tRNA. ... tRNA crystals were, in fact, protein (Hendrikson et al.

  14. Probing of RNA structures in a positive sense RNA virus reveals selection pressures for structural elements

    Science.gov (United States)

    Watters, Kyle E; Choudhary, Krishna; Aviran, Sharon; Perry, Keith L

    2018-01-01

    Abstract In single stranded (+)-sense RNA viruses, RNA structural elements (SEs) play essential roles in the infection process from replication to encapsidation. Using selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) and covariation analysis, we explore the structural features of the third genome segment of cucumber mosaic virus (CMV), RNA3 (2216 nt), both in vitro and in plant cell lysates. Comparing SHAPE-Seq and covariation analysis results revealed multiple SEs in the coat protein open reading frame and 3′ untranslated region. Four of these SEs were mutated and serially passaged in Nicotiana tabacum plants to identify biologically selected changes to the original mutated sequences. After passaging, loop mutants showed partial reversion to their wild-type sequence and SEs that were structurally disrupted by mutations were restored to wild-type-like structures via synonymous mutations in planta. These results support the existence and selection of virus open reading frame SEs in the host organism and provide a framework for further studies on the role of RNA structure in viral infection. Additionally, this work demonstrates the applicability of high-throughput chemical probing in plant cell lysates and presents a new method for calculating SHAPE reactivities from overlapping reverse transcriptase priming sites. PMID:29294088

  15. A folding algorithm for extended RNA secondary structures.

    Science.gov (United States)

    Höner zu Siederdissen, Christian; Bernhart, Stephan H; Stadler, Peter F; Hofacker, Ivo L

    2011-07-01

    RNA secondary structure contains many non-canonical base pairs of different pair families. Successful prediction of these structural features leads to improved secondary structures with applications in tertiary structure prediction and simultaneous folding and alignment. We present a theoretical model capturing both RNA pair families and extended secondary structure motifs with shared nucleotides using 2-diagrams. We accompany this model with a number of programs for parameter optimization and structure prediction. All sources (optimization routines, RNA folding, RNA evaluation, extended secondary structure visualization) are published under the GPLv3 and available at www.tbi.univie.ac.at/software/rnawolf/.

  16. Cervical Cap

    Science.gov (United States)

    ... Videos for Educators Search English Español The Cervical Cap KidsHealth / For Teens / The Cervical Cap What's in ... Call the Doctor? Print What Is a Cervical Cap? A cervical cap is a small cup made ...

  17. Structural and functional characterization of the CAP domain of pathogen-related yeast 1 (Pry1) protein

    Science.gov (United States)

    Darwiche, Rabih; Kelleher, Alan; Hudspeth, Elissa M.; Schneiter, Roger; Asojo, Oluwatoyin A.

    2016-06-01

    The production, crystal structure, and functional characterization of the C-terminal cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) domain of pathogen-related yeast protein-1 (Pry1) from Saccharomyces cerevisiae is presented. The CAP domain of Pry1 (Pry1CAP) is functional in vivo as its expression restores cholesterol export to yeast mutants lacking endogenous Pry1 and Pry2. Recombinant Pry1CAP forms dimers in solution, is sufficient for in vitro cholesterol binding, and has comparable binding properties as full-length Pry1. Two crystal structures of Pry1CAP are reported, one with Mg2+ coordinated to the conserved CAP tetrad (His208, Glu215, Glu233 and His250) in spacegroup I41 and the other without divalent cations in spacegroup P6122. The latter structure contains four 1,4-dioxane molecules from the crystallization solution, one of which sits in the cholesterol binding site. Both structures reveal that the divalent cation and cholesterol binding sites are connected upon dimerization, providing a structural basis for the observed Mg2+-dependent sterol binding by Pry1.

  18. Structural plasticity of the N-terminal capping helix of the TPR domain of kinesin light chain.

    Directory of Open Access Journals (Sweden)

    The Quyen Nguyen

    Full Text Available Kinesin1 plays a major role in neuronal transport by recruiting many different cargos through its kinesin light chain (KLC. Various structurally unrelated cargos interact with the conserved tetratricopeptide repeat (TPR domain of KLC. The N-terminal capping helix of the TPR domain exhibits an atypical sequence and structural features that may contribute to the versatility of the TPR domain to bind different cargos. We determined crystal structures of the TPR domain of both KLC1 and KLC2 encompassing the N-terminal capping helix and show that this helix exhibits two distinct and defined orientations relative to the rest of the TPR domain. Such a difference in orientation gives rise, at the N-terminal part of the groove, to the formation of one hydrophobic pocket, as well as to electrostatic variations at the groove surface. We present a comprehensive structural analysis of available KLC1/2-TPR domain structures that highlights that ligand binding into the groove can be specific of one or the other N-terminal capping helix orientations. Further, structural analysis reveals that the N-terminal capping helix is always involved in crystal packing contacts, especially in a TPR1:TPR1' contact which highlights its propensity to be a protein-protein interaction site. Together, these results underline that the structural plasticity of the N-terminal capping helix might represent a structural determinant for TPR domain structural versatility in cargo binding.

  19. Reconstruction of ancestral RNA sequences under multiple structural constraints

    OpenAIRE

    Tremblay-Savard, Olivier; Reinharz, Vladimir; Waldisp?hl, J?r?me

    2016-01-01

    Background Secondary structures form the scaffold of multiple sequence alignment of non-coding RNA (ncRNA) families. An accurate reconstruction of ancestral ncRNAs must use this structural signal. However, the inference of ancestors of a single ncRNA family with a single consensus structure may bias the results towards sequences with high affinity to this structure, which are far from the true ancestors. Methods In this paper, we introduce achARNement, a maximum parsimony approach that, given...

  20. High-throughput determination of RNA structure by proximity ligation.

    Science.gov (United States)

    Ramani, Vijay; Qiu, Ruolan; Shendure, Jay

    2015-09-01

    We present an unbiased method to globally resolve RNA structures through pairwise contact measurements between interacting regions. RNA proximity ligation (RPL) uses proximity ligation of native RNA followed by deep sequencing to yield chimeric reads with ligation junctions in the vicinity of structurally proximate bases. We apply RPL in both baker's yeast (Saccharomyces cerevisiae) and human cells and generate contact probability maps for ribosomal and other abundant RNAs, including yeast snoRNAs, the RNA subunit of the signal recognition particle and the yeast U2 spliceosomal RNA homolog. RPL measurements correlate with established secondary structures for these RNA molecules, including stem-loop structures and long-range pseudoknots. We anticipate that RPL will complement the current repertoire of computational and experimental approaches in enabling the high-throughput determination of secondary and tertiary RNA structures.

  1. High performance EUV multilayer structures insensitive to capping layer optical parameters.

    Science.gov (United States)

    Pelizzo, Maria Guglielmina; Suman, Michele; Monaco, Gianni; Nicolosi, Piergiorgio; Windt, David L

    2008-09-15

    We have designed and tested a-periodic multilayer structures containing protective capping layers in order to obtain improved stability with respect to any possible changes of the capping layer optical properties (due to oxidation and contamination, for example)-while simultaneously maximizing the EUV reflection efficiency for specific applications, and in particular for EUV lithography. Such coatings may be particularly useful in EUV lithographic apparatus, because they provide both high integrated photon flux and higher stability to the harsh operating environment, which can affect seriously the performance of the multilayer-coated projector system optics. In this work, an evolutive algorithm has been developed in order to design these a-periodic structures, which have been proven to have also the property of stable performance with respect to random layer thickness errors that might occur during coating deposition. Prototypes have been fabricated, and tested with EUV and X-ray reflectometry, and secondary electron spectroscopy. The experimental results clearly show improved performance of our new a-periodic coatings design compared with standard periodic multilayer structures.

  2. Structural basis for the regulation mechanism of the tyrosine kinase CapB from Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Vanesa Olivares-Illana

    2008-06-01

    Full Text Available Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function.

  3. Azimuthal Structure of the Sand Erg that Encircles the North Polar Water-Ice Cap

    Science.gov (United States)

    Teodoro, L. A.; Elphic, R. C.; Eke, V. R.; Feldman, W. C.; Maurice, S.; Pathare, A.

    2011-12-01

    The sand erg that completely encircles the perennial water-ice cap that covers the Martian north geographic pole displays considerable azimuthal structure as seen in visible and near-IR images. Much of this structure is associated with the terminations of the many steep troughs that cut spiral the approximately 3 km thick polar ice cap. Other contributions come from the katabatic winds that spill over steep-sided edges of the cap, such as what bounds the largest set of dunes that comprise Olympia Undae. During the spring and summer months when these winds initiate from the higher altitudes that contain sublimating CO2 ice, which is very cold and dry, heat adiabatically when they compress as they lose altitude. These winds should then remove H2O moisture from the uppermost layer of the sand dunes that are directly in their path. Two likely locations where this desiccation may occur preferentially is at the termination of Chasma Boreale and the ice cap at Olympia Undae. We will search for this effect by sharpening the spatial structure of the epithermal neutron counting rates measured at northern high latitudes using the Mars Odyssey Neutron Spectrometer (MONS). The epithermal range of neutron energies is nearly uniquely sensitive to the hydrogen content of surface soils, which should likely be in the form of H2O/OH molecules/radicals. We therefore convert epithermal counting rates in terms of Water-Equivalent-Hydrogen, WEH. However, MONS counting-rate data have a FWHM of ~550 km., which is sufficiently broad to prevent a close association of WEH variability with images of geological features. In this study, we reduce spurious features in the instrument smeared neutron counting rates through deconvolution. We choose the PIXON numerical deconvolution technique for this purpose. This technique uses a statistical approach (Pina 2001, Eke 2001), which is capable of removing spurious features in the data in the presence of noise. We have previously carried out a detailed

  4. The 5'-poly(A leader of poxvirus mRNA confers a translational advantage that can be achieved in cells with impaired cap-dependent translation.

    Directory of Open Access Journals (Sweden)

    Pragyesh Dhungel

    2017-08-01

    Full Text Available The poly(A leader at the 5'-untranslated region (5'-UTR is an unusually striking feature of all poxvirus mRNAs transcribed after viral DNA replication (post-replicative mRNAs. These poly(A leaders are non-templated and of heterogeneous lengths; and their function during poxvirus infection remains a long-standing question. Here, we discovered that a 5'-poly(A leader conferred a selective translational advantage to mRNA in poxvirus-infected cells. A constitutive and uninterrupted 5'-poly(A leader with 12 residues was optimal. Because the most frequent lengths of the 5'-poly(A leaders are 8-12 residues, the result suggests that the poly(A leader has been evolutionarily optimized to boost poxvirus protein production. A 5'-poly(A leader also could increase protein production in the bacteriophage T7 promoter-based expression system of vaccinia virus, the prototypic member of poxviruses. Interestingly, although vaccinia virus post-replicative mRNAs do have 5'- methylated guanosine caps and can use cap-dependent translation, in vaccinia virus-infected cells, mRNA with a 5'-poly(A leader could also be efficiently translated in cells with impaired cap-dependent translation. However, the translation was not mediated through an internal ribosome entry site (IRES. These results point to a fundamental mechanism poxvirus uses to efficiently translate its post-replicative mRNAs.

  5. Distributional effects and structural change induced by various CAP Pillar 1 proposals; the case of the Czech Republic

    OpenAIRE

    Ratinger, Tomas; Foltyn, Ivan; Jelinek, Ladislav; Kristkova, Zuzana

    2012-01-01

    This paper deals with the potential effects of the CAP pillar 1 on farm incomes and structural changes. It uses a dynamic Computable General Equilibrium model and a specific analysis on distributional effects. The effect of payments ceiling in the current CAP 2020 proposal with subtracting labour costs will bring only insignificant payment reduction for most farmers except large extensive beef breeders whose direct payments will drop by 13% on average. However, if the condition on labour cost...

  6. Optimization of a Hot Structure Aeroshell and Nose Cap for Mars Atmospheric Entry

    Science.gov (United States)

    Langston, Sarah L.; Lang, Christapher G.; Samareh, Jamshid A.; Daryabeigi, Kamran

    2016-01-01

    The National Aeronautics and Space Administration (NASA) is preparing to send humans beyond Low Earth Orbit and eventually to the surface of Mars. As part of the Evolvable Mars Campaign, different vehicle configurations are being designed and considered for delivering large payloads to the surface of Mars. Weight and packing volume are driving factors in the vehicle design, and the thermal protection system (TPS) for planetary entry is a technology area which can offer potential weight and volume savings. The feasibility and potential benefits of a ceramic matrix composite hot structure concept for different vehicle configurations are explored in this paper, including the nose cap for a Hypersonic Inflatable Aerodynamic Decelerator (HIAD) and an aeroshell for a mid lift-to-drag (Mid L/D) concept. The TPS of a planetary entry vehicle is a critical component required to survive the severe aerodynamic heating environment during atmospheric en- try. The current state-of-the-art is an ablative material to protect the vehicle from the heat load. The ablator is bonded to an underlying structure, which carries the mechanical loads associated with entry. The alternative hot structure design utilizes an advanced carbon-carbon material system on the outer surface of the vehicle, which is exposed to the severe heating and acts as a load carrying structure. The preliminary design using the hot structure concept and the ablative concept is determined for the spherical nose cap of the HIAD entry vehicle and the aeroshell of the Mid L/D entry vehicle. The results of the study indicate that the use of hot structures for both vehicle concepts leads to a feasible design with potential weight and volume savings benefits over current state-of-the-art TPS technology that could enable future missions.

  7. Interfacial structures in confined cap-turbulent and churn-turbulent flows

    International Nuclear Information System (INIS)

    Sun Xiaodong; Kim, Seungjin; Cheng Ling; Ishii, Mamoru; Beus, Stephen G.

    2004-01-01

    The objective of the present work is to study and model the interfacial structure development of air-water two-phase flow in a confined flow passage. Experiments of a total of 13 flow conditions in cap-turbulent and churn-turbulent flow regimes are carried out in a vertical air-water upward two-phase flow experimental loop with a test section of 200 mm in width and 10 mm in gap. Miniaturized four-sensor conductivity probes are used to measure local two-phase parameters at three different elevations for each flow condition. Bubble characteristics captured by the probes are categorized into two groups in view of the two-group interfacial area transport equation, i.e., spherical/distorted bubbles as Group 1 and cap/churn-turbulent bubbles as Group 2. The acquired local parameters are time-averaged void fraction, interfacial velocity, bubble number frequency, interfacial area concentration, and bubble Sauter mean diameter for each group of bubbles. Also, the line-averaged and area-averaged data are presented and discussed in detail. The comparisons of these parameters at different elevations demonstrate the development of interfacial structures along the flow direction due to bubble interactions and the hydrodynamic effects. Furthermore, these data can serve as one part of the experimental data for investigation of the interfacial area transport in a confined two-phase flow

  8. Visualization of RNA structure models within the Integrative Genomics Viewer.

    Science.gov (United States)

    Busan, Steven; Weeks, Kevin M

    2017-07-01

    Analyses of the interrelationships between RNA structure and function are increasingly important components of genomic studies. The SHAPE-MaP strategy enables accurate RNA structure probing and realistic structure modeling of kilobase-length noncoding RNAs and mRNAs. Existing tools for visualizing RNA structure models are not suitable for efficient analysis of long, structurally heterogeneous RNAs. In addition, structure models are often advantageously interpreted in the context of other experimental data and gene annotation information, for which few tools currently exist. We have developed a module within the widely used and well supported open-source Integrative Genomics Viewer (IGV) that allows visualization of SHAPE and other chemical probing data, including raw reactivities, data-driven structural entropies, and data-constrained base-pair secondary structure models, in context with linear genomic data tracks. We illustrate the usefulness of visualizing RNA structure in the IGV by exploring structure models for a large viral RNA genome, comparing bacterial mRNA structure in cells with its structure under cell- and protein-free conditions, and comparing a noncoding RNA structure modeled using SHAPE data with a base-pairing model inferred through sequence covariation analysis. © 2017 Busan and Weeks; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  9. Rtools: a web server for various secondary structural analyses on single RNA sequences.

    Science.gov (United States)

    Hamada, Michiaki; Ono, Yukiteru; Kiryu, Hisanori; Sato, Kengo; Kato, Yuki; Fukunaga, Tsukasa; Mori, Ryota; Asai, Kiyoshi

    2016-07-08

    The secondary structures, as well as the nucleotide sequences, are the important features of RNA molecules to characterize their functions. According to the thermodynamic model, however, the probability of any secondary structure is very small. As a consequence, any tool to predict the secondary structures of RNAs has limited accuracy. On the other hand, there are a few tools to compensate the imperfect predictions by calculating and visualizing the secondary structural information from RNA sequences. It is desirable to obtain the rich information from those tools through a friendly interface. We implemented a web server of the tools to predict secondary structures and to calculate various structural features based on the energy models of secondary structures. By just giving an RNA sequence to the web server, the user can get the different types of solutions of the secondary structures, the marginal probabilities such as base-paring probabilities, loop probabilities and accessibilities of the local bases, the energy changes by arbitrary base mutations as well as the measures for validations of the predicted secondary structures. The web server is available at http://rtools.cbrc.jp, which integrates software tools, CentroidFold, CentroidHomfold, IPKnot, CapR, Raccess, Rchange and RintD. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Rapid NMR screening of RNA secondary structure and binding

    International Nuclear Information System (INIS)

    Helmling, Christina; Keyhani, Sara; Sochor, Florian; Fürtig, Boris; Hengesbach, Martin; Schwalbe, Harald

    2015-01-01

    Determination of RNA secondary structures by NMR spectroscopy is a useful tool e.g. to elucidate RNA folding space or functional aspects of regulatory RNA elements. However, current approaches of RNA synthesis and preparation are usually time-consuming and do not provide analysis with single nucleotide precision when applied for a large number of different RNA sequences. Here, we significantly improve the yield and 3′ end homogeneity of RNA preparation by in vitro transcription. Further, by establishing a native purification procedure with increased throughput, we provide a shortcut to study several RNA constructs simultaneously. We show that this approach yields μmol quantities of RNA with purities comparable to PAGE purification, while avoiding denaturation of the RNA

  11. Rapid NMR screening of RNA secondary structure and binding

    Energy Technology Data Exchange (ETDEWEB)

    Helmling, Christina; Keyhani, Sara; Sochor, Florian; Fürtig, Boris; Hengesbach, Martin; Schwalbe, Harald, E-mail: schwalbe@nmr.uni-frankfurt.de [Johann Wolfgang Goethe-Universität, Institut für Organische Chemie und Chemische Biologie, Center for Biomolecular Magnetic Resonance (BMRZ) (Germany)

    2015-09-15

    Determination of RNA secondary structures by NMR spectroscopy is a useful tool e.g. to elucidate RNA folding space or functional aspects of regulatory RNA elements. However, current approaches of RNA synthesis and preparation are usually time-consuming and do not provide analysis with single nucleotide precision when applied for a large number of different RNA sequences. Here, we significantly improve the yield and 3′ end homogeneity of RNA preparation by in vitro transcription. Further, by establishing a native purification procedure with increased throughput, we provide a shortcut to study several RNA constructs simultaneously. We show that this approach yields μmol quantities of RNA with purities comparable to PAGE purification, while avoiding denaturation of the RNA.

  12. ncRNA consensus secondary structure derivation using grammar strings.

    Science.gov (United States)

    Achawanantakun, Rujira; Sun, Yanni; Takyar, Seyedeh Shohreh

    2011-04-01

    Many noncoding RNAs (ncRNAs) function through both their sequences and secondary structures. Thus, secondary structure derivation is an important issue in today's RNA research. The state-of-the-art structure annotation tools are based on comparative analysis, which derives consensus structure of homologous ncRNAs. Despite promising results from existing ncRNA aligning and consensus structure derivation tools, there is a need for more efficient and accurate ncRNA secondary structure modeling and alignment methods. In this work, we introduce a consensus structure derivation approach based on grammar string, a novel ncRNA secondary structure representation that encodes an ncRNA's sequence and secondary structure in the parameter space of a context-free grammar (CFG) and a full RNA grammar including pseudoknots. Being a string defined on a special alphabet constructed from a grammar, grammar string converts ncRNA alignment into sequence alignment. We derive consensus secondary structures from hundreds of ncRNA families from BraliBase 2.1 and 25 families containing pseudoknots using grammar string alignment. Our experiments have shown that grammar string-based structure derivation competes favorably in consensus structure quality with Murlet and RNASampler. Source code and experimental data are available at http://www.cse.msu.edu/~yannisun/grammar-string.

  13. On the importance of cotranscriptional RNA structure formation

    Science.gov (United States)

    Lai, Daniel; Proctor, Jeff R.; Meyer, Irmtraud M.

    2013-01-01

    The expression of genes, both coding and noncoding, can be significantly influenced by RNA structural features of their corresponding transcripts. There is by now mounting experimental and some theoretical evidence that structure formation in vivo starts during transcription and that this cotranscriptional folding determines the functional RNA structural features that are being formed. Several decades of research in bioinformatics have resulted in a wide range of computational methods for predicting RNA secondary structures. Almost all state-of-the-art methods in terms of prediction accuracy, however, completely ignore the process of structure formation and focus exclusively on the final RNA structure. This review hopes to bridge this gap. We summarize the existing evidence for cotranscriptional folding and then review the different, currently used strategies for RNA secondary-structure prediction. Finally, we propose a range of ideas on how state-of-the-art methods could be potentially improved by explicitly capturing the process of cotranscriptional structure formation. PMID:24131802

  14. Computer-Aided Design of RNA Origami Structures.

    Science.gov (United States)

    Sparvath, Steffen L; Geary, Cody W; Andersen, Ebbe S

    2017-01-01

    RNA nanostructures can be used as scaffolds to organize, combine, and control molecular functionalities, with great potential for applications in nanomedicine and synthetic biology. The single-stranded RNA origami method allows RNA nanostructures to be folded as they are transcribed by the RNA polymerase. RNA origami structures provide a stable framework that can be decorated with functional RNA elements such as riboswitches, ribozymes, interaction sites, and aptamers for binding small molecules or protein targets. The rich library of RNA structural and functional elements combined with the possibility to attach proteins through aptamer-based binding creates virtually limitless possibilities for constructing advanced RNA-based nanodevices.In this chapter we provide a detailed protocol for the single-stranded RNA origami design method using a simple 2-helix tall structure as an example. The first step involves 3D modeling of a double-crossover between two RNA double helices, followed by decoration with tertiary motifs. The second step deals with the construction of a 2D blueprint describing the secondary structure and sequence constraints that serves as the input for computer programs. In the third step, computer programs are used to design RNA sequences that are compatible with the structure, and the resulting outputs are evaluated and converted into DNA sequences to order.

  15. Advances in RNA Structure Determination | Center for Cancer Research

    Science.gov (United States)

    The recent years have witnessed a revolution in the field of RNA structure and function. Until recently the main contribution of RNA in cellular and disease functions was considered to be a role defined by the central dogma, namely DNA codes for mRNAs, which in turn encode for proteins, a notion facilitated by non-coding ribosomal RNA and tRNA. It was also assumed at the time

  16. Solving RNA's structural secrets: interaction with antibodies and crystal structure of a nuclease resistant RNA

    International Nuclear Information System (INIS)

    Wallace, S.T.

    1998-10-01

    This Ph.D. thesis concerns the structural characterization of RNA. The work is split into two sections: 1) in vitro selection and characterization of RNAs which bind antibiotics and 2) crystal structure of a nuclease resistant RNA molecule used in antisense applications. Understanding antibiotic-RNA interactions is crucial in aiding rational drug design. We were interested in studying antibiotic interactions with RNAs small enough to characterize at the molecular and possibly at the atomic level. In order to do so, we previously performed in vitro selection to find small RNAs which bind to the peptide antibiotic viomycin and the aminoglycoside antibiotic streptomycin. The characterization of the viomycin-binding RNAs revealed the necessity of a pseudoknot-structure in order to interact with the antibiotic. The RNAs which were selected to interact with streptomycin require the presence of magnesium to bind the antibiotic. One of the RNAs, upon interacting with streptomycin undergoes a significant conformational change spanning the entire RNA sequence needed to bind the antibiotic. In a quest to design oligodeoxynucleotides (ODNs) which are able to specifically bid and inactivate the mRNA of a gene, it is necessary to fulfill two criteria: 1) increase binding affinity between the ODN and the target RNA and 2) increase the ODN's resistance to nuclease degradation. An ODN with an aminopropyl modification at the 2' position of its ribose has emerged as the most successful candidate at fulfilling both criteria. It is the most nuclease resistant modification known to date. We were interested in explaining how this modification is able to circumvent degradation by nucleases. A dodecamer containing a single 2'-O-aminopropyl modified nucleotide was crystallized and the structure was solved to a resolution of 1.6 A. In an attempt to explain the nuclease resistance, the crystal coordinates were modeled into the active exonuclease site of DNA polymerase I. We propose the

  17. RNA folding: structure prediction, folding kinetics and ion electrostatics.

    Science.gov (United States)

    Tan, Zhijie; Zhang, Wenbing; Shi, Yazhou; Wang, Fenghua

    2015-01-01

    Beyond the "traditional" functions such as gene storage, transport and protein synthesis, recent discoveries reveal that RNAs have important "new" biological functions including the RNA silence and gene regulation of riboswitch. Such functions of noncoding RNAs are strongly coupled to the RNA structures and proper structure change, which naturally leads to the RNA folding problem including structure prediction and folding kinetics. Due to the polyanionic nature of RNAs, RNA folding structure, stability and kinetics are strongly coupled to the ion condition of solution. The main focus of this chapter is to review the recent progress in the three major aspects in RNA folding problem: structure prediction, folding kinetics and ion electrostatics. This chapter will introduce both the recent experimental and theoretical progress, while emphasize the theoretical modelling on the three aspects in RNA folding.

  18. RNA 3D modules in genome-wide predictions of RNA 2D structure

    DEFF Research Database (Denmark)

    Theis, Corinna; Zirbel, Craig L; Zu Siederdissen, Christian Höner

    2015-01-01

    . These modules can, for example, occur inside structural elements which in RNA 2D predictions appear as internal loops. Hence one question is if the use of such RNA 3D information can improve the prediction accuracy of RNA secondary structure at a genome-wide level. Here, we use RNAz in combination with 3D......Recent experimental and computational progress has revealed a large potential for RNA structure in the genome. This has been driven by computational strategies that exploit multiple genomes of related organisms to identify common sequences and secondary structures. However, these computational...... approaches have two main challenges: they are computationally expensive and they have a relatively high false discovery rate (FDR). Simultaneously, RNA 3D structure analysis has revealed modules composed of non-canonical base pairs which occur in non-homologous positions, apparently by independent evolution...

  19. Capping layer growth rate and the optical and structural properties of GaAsSbN-capped InAs/GaAs quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Ulloa, J. M., E-mail: jmulloa@isom.upm.es; Utrilla, A. D.; Guzman, A.; Hierro, A. [Institute for Systems based on Optoelectronics and Microtechnology (ISOM) and Dpto. Ingeniería Electrónica, Universidad Politecnica de Madrid, Ciudad Universitaria s/n, 28040 Madrid (Spain); Reyes, D. F.; Ben, T.; González, D. [Departamento de Ciencia de los Materiales e IM y QI, Universidad de Cádiz, 11510 Puerto Real (Cádiz) (Spain)

    2014-10-07

    Changing the growth rate during the heteroepitaxial capping of InAs/GaAs quantum dots (QDs) with a 5 nm-thick GaAsSbN capping layer (CL) strongly modifies the QD structural and optical properties. A size and shape transition from taller pyramids to flatter lens-shaped QDs is observed when the CL growth rate is decreased from 1.5 to 0.5 ML/s. This indicates that the QD dissolution processes taking place during capping can be controlled to some extent by the GaAsSbN CL growth rate, with high growth rates allowing a complete preservation of the QDs. However, the dissolution processes are shown to have a leveling effect on the QD height, giving rise to a narrower size distribution for lower growth rates. Contrary to what could be expected, these effects are opposite to the strong blue-shift and improvement of the photoluminescence (PL) observed for higher growth rates. Nevertheless, the PL results can be understood in terms of the strong impact of the growth rate on the Sb and N incorporation into the CL, which results in lower Sb and N contents at higher growth rates. Besides the QD-CL band offsets and QD strain, the different CL composition alters the band alignment of the system, which can be transformed to type-II at low growth rates. These results show the key role of the alloyed CL growth parameters on the resulting QD properties and demonstrate an intricate correlation between the PL spectra and the sample morphology in complex QD-CL structures.

  20. Characterization of cap binding proteins associated with the nucleus

    International Nuclear Information System (INIS)

    Patzelt, E.

    1986-04-01

    Eucaryotic mRNAs a carry 7-methylguanosine triphosphate residue (called cap structure) at their 5' terminus. The cap plays an important role in RNA recognition. Cap binding proteins (CBP) of HeLa cells were identified by photoaffinity labelling using the cap analogue γ-( 32 P)-(4-(benzoyl-phenyl)methylamido)-7-methylguanosine-5'-triphosphate (BP-m 7 GTP). Photoreaction of this cap analogue with HeLa cell initiation factors resulted in specific labelling of two polypeptides of Msub(r) 37000 and 26000. The latter was also labelled in crude initiation factors prepared from reticulocytes and is identical to the cap binding protein CBP I previously identified. These cap binding proteins were also affinity labelled in poliovirus infected cell extracts. Photoaffinity reaction with BP-m 7 GTP of whole HeLa cell homogenate showed three additional polypeptides with Msub(r) 120000, 89000 and 80000. These cap binding proteins were found to be associated with the nucleus and are therefore referred to as nuclear cap binding proteins, i.e. NCBP 1, NCBP 2 and NCBP 3. They were also present in splicing extracts. Photoaffinity labelling in these nuclear extracts was differentially inhibited by various cap analogues and capped mRNAs. Affinity chromatography on immobilized globin mRNA led to a partial separation of the three nuclear cap binding proteins. Chromatography on m 7 GTP-Sepharose resulted in a specific binding of NCBP 3. The different behaviour of the cap binding proteins suggests that they are functionally distinct and that they might be involved in different processes requiring cap recognition. (Author)

  1. A Method to Predict the Structure and Stability of RNA/RNA Complexes.

    Science.gov (United States)

    Xu, Xiaojun; Chen, Shi-Jie

    2016-01-01

    RNA/RNA interactions are essential for genomic RNA dimerization and regulation of gene expression. Intermolecular loop-loop base pairing is a widespread and functionally important tertiary structure motif in RNA machinery. However, computational prediction of intermolecular loop-loop base pairing is challenged by the entropy and free energy calculation due to the conformational constraint and the intermolecular interactions. In this chapter, we describe a recently developed statistical mechanics-based method for the prediction of RNA/RNA complex structures and stabilities. The method is based on the virtual bond RNA folding model (Vfold). The main emphasis in the method is placed on the evaluation of the entropy and free energy for the loops, especially tertiary kissing loops. The method also uses recursive partition function calculations and two-step screening algorithm for large, complicated structures of RNA/RNA complexes. As case studies, we use the HIV-1 Mal dimer and the siRNA/HIV-1 mutant (T4) to illustrate the method.

  2. Global Analysis of RNA Secondary Structure in Two Metazoans

    Directory of Open Access Journals (Sweden)

    Fan Li

    2012-01-01

    Full Text Available The secondary structure of RNA is necessary for its maturation, regulation, processing, and function. However, the global influence of RNA folding in eukaryotes is still unclear. Here, we use a high-throughput, sequencing-based, structure-mapping approach to identify the paired (double-stranded RNA [dsRNA] and unpaired (single-stranded RNA [ssRNA] components of the Drosophila melanogaster and Caenorhabditis elegans transcriptomes, which allows us to identify conserved features of RNA secondary structure in metazoans. From this analysis, we find that ssRNAs and dsRNAs are significantly correlated with specific epigenetic modifications. Additionally, we find key structural patterns across protein-coding transcripts that indicate that RNA folding demarcates regions of protein translation and likely affects microRNA-mediated regulation of mRNAs in animals. Finally, we identify and characterize 546 mRNAs whose folding pattern is significantly correlated between these metazoans, suggesting that their structure has some function. Overall, our findings provide a global assessment of RNA folding in animals.

  3. Functional RNA structures throughout the Hepatitis C Virus genome.

    Science.gov (United States)

    Adams, Rebecca L; Pirakitikulr, Nathan; Pyle, Anna Marie

    2017-06-01

    The single-stranded Hepatitis C Virus (HCV) genome adopts a set of elaborate RNA structures that are involved in every stage of the viral lifecycle. Recent advances in chemical probing, sequencing, and structural biology have facilitated analysis of RNA folding on a genome-wide scale, revealing novel structures and networks of interactions. These studies have underscored the active role played by RNA in every function of HCV and they open the door to new types of RNA-targeted therapeutics. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Deregulation of cap-dependent mRNA translation increases tumour radiosensitivity through reduction of the hypoxic fraction

    NARCIS (Netherlands)

    Rouschop, K.M.; Dubois, L.; Schaaf, M.B.; Beucken, T. van den; Lieuwes, N.; Keulers, T.G.; Savelkouls, K.G.; Bussink, J.; Kogel, A.J. van der; Koritzinsky, M.; Wouters, B.G.

    2011-01-01

    BACKGROUND AND PURPOSE: Tumour hypoxia is an important limiting factor in the successful treatment of cancer. Adaptation to hypoxia includes inhibition of mTOR, causing scavenging of eukaryotic initiation factor 4E (eIF4E), the rate-limiting factor for cap-dependent translation. The aim of this

  5. Rational design of human metapneumovirus live attenuated vaccine candidates by inhibiting viral mRNA cap methyltransferase.

    Science.gov (United States)

    Zhang, Yu; Wei, Yongwei; Zhang, Xiaodong; Cai, Hui; Niewiesk, Stefan; Li, Jianrong

    2014-10-01

    The paramyxoviruses human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), and human parainfluenza virus type 3 (hPIV3) are responsible for the majority of pediatric respiratory diseases and inflict significant economic loss, health care costs, and emotional burdens. Despite major efforts, there are no vaccines available for these viruses. The conserved region VI (CR VI) of the large (L) polymerase proteins of paramyxoviruses catalyzes methyltransferase (MTase) activities that typically methylate viral mRNAs at positions guanine N-7 (G-N-7) and ribose 2'-O. In this study, we generated a panel of recombinant hMPVs carrying mutations in the S-adenosylmethionine (SAM) binding site in CR VI of L protein. These recombinant viruses were specifically defective in ribose 2'-O methylation but not G-N-7 methylation and were genetically stable and highly attenuated in cell culture and viral replication in the upper and lower respiratory tracts of cotton rats. Importantly, vaccination of cotton rats with these recombinant hMPVs (rhMPVs) with defective MTases triggered a high level of neutralizing antibody, and the rats were completely protected from challenge with wild-type rhMPV. Collectively, our results indicate that (i) amino acid residues in the SAM binding site in the hMPV L protein are essential for 2'-O methylation and (ii) inhibition of mRNA cap MTase can serve as a novel target to rationally design live attenuated vaccines for hMPV and perhaps other paramyxoviruses, such as hRSV and hPIV3. Human paramyxoviruses, including hRSV, hMPV, and hPIV3, cause the majority of acute upper and lower respiratory tract infections in humans, particularly in infants, children, the elderly, and immunocompromised individuals. Currently, there is no licensed vaccine available. A formalin-inactivated vaccine is not suitable for these viruses because it causes enhanced lung damage upon reinfection with the same virus. A live attenuated vaccine is the most promising

  6. Computational RNA secondary structure design: empirical complexity and improved methods

    Directory of Open Access Journals (Sweden)

    Condon Anne

    2007-01-01

    Full Text Available Abstract Background We investigate the empirical complexity of the RNA secondary structure design problem, that is, the scaling of the typical difficulty of the design task for various classes of RNA structures as the size of the target structure is increased. The purpose of this work is to understand better the factors that make RNA structures hard to design for existing, high-performance algorithms. Such understanding provides the basis for improving the performance of one of the best algorithms for this problem, RNA-SSD, and for characterising its limitations. Results To gain insights into the practical complexity of the problem, we present a scaling analysis on random and biologically motivated structures using an improved version of the RNA-SSD algorithm, and also the RNAinverse algorithm from the Vienna package. Since primary structure constraints are relevant for designing RNA structures, we also investigate the correlation between the number and the location of the primary structure constraints when designing structures and the performance of the RNA-SSD algorithm. The scaling analysis on random and biologically motivated structures supports the hypothesis that the running time of both algorithms scales polynomially with the size of the structure. We also found that the algorithms are in general faster when constraints are placed only on paired bases in the structure. Furthermore, we prove that, according to the standard thermodynamic model, for some structures that the RNA-SSD algorithm was unable to design, there exists no sequence whose minimum free energy structure is the target structure. Conclusion Our analysis helps to better understand the strengths and limitations of both the RNA-SSD and RNAinverse algorithms, and suggests ways in which the performance of these algorithms can be further improved.

  7. Cervical Cap

    Science.gov (United States)

    ... giving birth vaginally, which means the cervical cap may not fit as well. Inconsistent or incorrect use of the cervical cap increases your risk of pregnancy. For example, you may get pregnant when using the cervical cap if: ...

  8. A Two-Way Street: Regulatory Interplay between RNA Polymerase and Nascent RNA Structure.

    Science.gov (United States)

    Zhang, Jinwei; Landick, Robert

    2016-04-01

    The vectorial (5'-to-3' at varying velocity) synthesis of RNA by cellular RNA polymerases (RNAPs) creates a rugged kinetic landscape, demarcated by frequent, sometimes long-lived, pauses. In addition to myriad gene-regulatory roles, these pauses temporally and spatially program the co-transcriptional, hierarchical folding of biologically active RNAs. Conversely, these RNA structures, which form inside or near the RNA exit channel, interact with the polymerase and adjacent protein factors to influence RNA synthesis by modulating pausing, termination, antitermination, and slippage. Here, we review the evolutionary origin, mechanistic underpinnings, and regulatory consequences of this interplay between RNAP and nascent RNA structure. We categorize and rationalize the extensive linkage between the transcriptional machinery and its product, and provide a framework for future studies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. On infrared spectroscopic analysis of transfer RNA secondary structure

    Energy Technology Data Exchange (ETDEWEB)

    Semenov, M A; Starikov, E B

    1987-07-14

    Various techniques of IR spectroscopy in the 1550-1750 cm/sup -1/ region employed to analyse the tRNA secondary structure are discussed and a novel improved method is proposed. The main novel features of this method are the approximation of tRNA helical region spectra by catalogue carbonyl absorption bands and approximation of tRNA nonhelical region spectra by those of homopolyribonucleotides. The IR spectra of tRNA/sub yeast//sup phe/ and tRNA/sub E.coli//sup fmet/ in the carbonyl vibration region are explained on the basis of calculated transition moment coupling.

  10. Structure of Escherichia coli Hfq bound to polyriboadenylate RNA

    DEFF Research Database (Denmark)

    Link, Todd M; Valentin-Hansen, Poul; Brennan, Richard G

    2009-01-01

    (A) RNA, A(15). The structure reveals a unique RNA binding mechanism. Unlike uridine-containing sequences, which bind to the "proximal" face, the poly(A) tract binds to the "distal" face of Hfq using 6 tripartite binding motifs. Each motif consists of an adenosine specificity site (A site), which......Hfq is a small, highly abundant hexameric protein that is found in many bacteria and plays a critical role in mRNA expression and RNA stability. As an "RNA chaperone," Hfq binds AU-rich sequences and facilitates the trans annealing of small RNAs (sRNAs) to their target mRNAs, typically resulting...... in the down-regulation of gene expression. Hfq also plays a key role in bacterial RNA decay by binding tightly to polyadenylate [poly(A)] tracts. The structural mechanism by which Hfq recognizes and binds poly(A) is unknown. Here, we report the crystal structure of Escherichia coli Hfq bound to the poly...

  11. RNAstructure: software for RNA secondary structure prediction and analysis.

    Science.gov (United States)

    Reuter, Jessica S; Mathews, David H

    2010-03-15

    To understand an RNA sequence's mechanism of action, the structure must be known. Furthermore, target RNA structure is an important consideration in the design of small interfering RNAs and antisense DNA oligonucleotides. RNA secondary structure prediction, using thermodynamics, can be used to develop hypotheses about the structure of an RNA sequence. RNAstructure is a software package for RNA secondary structure prediction and analysis. It uses thermodynamics and utilizes the most recent set of nearest neighbor parameters from the Turner group. It includes methods for secondary structure prediction (using several algorithms), prediction of base pair probabilities, bimolecular structure prediction, and prediction of a structure common to two sequences. This contribution describes new extensions to the package, including a library of C++ classes for incorporation into other programs, a user-friendly graphical user interface written in JAVA, and new Unix-style text interfaces. The original graphical user interface for Microsoft Windows is still maintained. The extensions to RNAstructure serve to make RNA secondary structure prediction user-friendly. The package is available for download from the Mathews lab homepage at http://rna.urmc.rochester.edu/RNAstructure.html.

  12. Evolutionary rate variation and RNA secondary structure prediction

    DEFF Research Database (Denmark)

    Knudsen, B.; Andersen, E.S.; Damgaard, C.

    2004-01-01

    Predicting RNA secondary structure using evolutionary history can be carried out by using an alignment of related RNA sequences with conserved structure. Accurately determining evolutionary substitution rates for base pairs and single stranded nucleotides is a concern for methods based on this type...... by applying rates derived from tRNA and rRNA to the prediction of the much more rapidly evolving 5'-region of HIV-1. We find that the HIV-1 prediction is in agreement with experimental data, even though the relative evolutionary rate between A and G is significantly increased, both in stem and loop regions...

  13. RNA-Pareto: interactive analysis of Pareto-optimal RNA sequence-structure alignments.

    Science.gov (United States)

    Schnattinger, Thomas; Schöning, Uwe; Marchfelder, Anita; Kestler, Hans A

    2013-12-01

    Incorporating secondary structure information into the alignment process improves the quality of RNA sequence alignments. Instead of using fixed weighting parameters, sequence and structure components can be treated as different objectives and optimized simultaneously. The result is not a single, but a Pareto-set of equally optimal solutions, which all represent different possible weighting parameters. We now provide the interactive graphical software tool RNA-Pareto, which allows a direct inspection of all feasible results to the pairwise RNA sequence-structure alignment problem and greatly facilitates the exploration of the optimal solution set.

  14. Free energy minimization to predict RNA secondary structures and computational RNA design.

    Science.gov (United States)

    Churkin, Alexander; Weinbrand, Lina; Barash, Danny

    2015-01-01

    Determining the RNA secondary structure from sequence data by computational predictions is a long-standing problem. Its solution has been approached in two distinctive ways. If a multiple sequence alignment of a collection of homologous sequences is available, the comparative method uses phylogeny to determine conserved base pairs that are more likely to form as a result of billions of years of evolution than by chance. In the case of single sequences, recursive algorithms that compute free energy structures by using empirically derived energy parameters have been developed. This latter approach of RNA folding prediction by energy minimization is widely used to predict RNA secondary structure from sequence. For a significant number of RNA molecules, the secondary structure of the RNA molecule is indicative of its function and its computational prediction by minimizing its free energy is important for its functional analysis. A general method for free energy minimization to predict RNA secondary structures is dynamic programming, although other optimization methods have been developed as well along with empirically derived energy parameters. In this chapter, we introduce and illustrate by examples the approach of free energy minimization to predict RNA secondary structures.

  15. BEAM web server: a tool for structural RNA motif discovery.

    Science.gov (United States)

    Pietrosanto, Marco; Adinolfi, Marta; Casula, Riccardo; Ausiello, Gabriele; Ferrè, Fabrizio; Helmer-Citterich, Manuela

    2018-03-15

    RNA structural motif finding is a relevant problem that becomes computationally hard when working on high-throughput data (e.g. eCLIP, PAR-CLIP), often represented by thousands of RNA molecules. Currently, the BEAM server is the only web tool capable to handle tens of thousands of RNA in input with a motif discovery procedure that is only limited by the current secondary structure prediction accuracies. The recently developed method BEAM (BEAr Motifs finder) can analyze tens of thousands of RNA molecules and identify RNA secondary structure motifs associated to a measure of their statistical significance. BEAM is extremely fast thanks to the BEAR encoding that transforms each RNA secondary structure in a string of characters. BEAM also exploits the evolutionary knowledge contained in a substitution matrix of secondary structure elements, extracted from the RFAM database of families of homologous RNAs. The BEAM web server has been designed to streamline data pre-processing by automatically handling folding and encoding of RNA sequences, giving users a choice for the preferred folding program. The server provides an intuitive and informative results page with the list of secondary structure motifs identified, the logo of each motif, its significance, graphic representation and information about its position in the RNA molecules sharing it. The web server is freely available at http://beam.uniroma2.it/ and it is implemented in NodeJS and Python with all major browsers supported. marco.pietrosanto@uniroma2.it. Supplementary data are available at Bioinformatics online.

  16. A comprehensive comparison of comparative RNA structure prediction approaches

    DEFF Research Database (Denmark)

    Gardner, P. P.; Giegerich, R.

    2004-01-01

    -finding and multiple-sequence-alignment algorithms. Results Here we evaluate a number of RNA folding algorithms using reliable RNA data-sets and compare their relative performance. Conclusions We conclude that comparative data can enhance structure prediction but structure-prediction-algorithms vary widely in terms......Background An increasing number of researchers have released novel RNA structure analysis and prediction algorithms for comparative approaches to structure prediction. Yet, independent benchmarking of these algorithms is rarely performed as is now common practice for protein-folding, gene...

  17. CompaRNA: a server for continuous benchmarking of automated methods for RNA secondary structure prediction

    Science.gov (United States)

    Puton, Tomasz; Kozlowski, Lukasz P.; Rother, Kristian M.; Bujnicki, Janusz M.

    2013-01-01

    We present a continuous benchmarking approach for the assessment of RNA secondary structure prediction methods implemented in the CompaRNA web server. As of 3 October 2012, the performance of 28 single-sequence and 13 comparative methods has been evaluated on RNA sequences/structures released weekly by the Protein Data Bank. We also provide a static benchmark generated on RNA 2D structures derived from the RNAstrand database. Benchmarks on both data sets offer insight into the relative performance of RNA secondary structure prediction methods on RNAs of different size and with respect to different types of structure. According to our tests, on the average, the most accurate predictions obtained by a comparative approach are generated by CentroidAlifold, MXScarna, RNAalifold and TurboFold. On the average, the most accurate predictions obtained by single-sequence analyses are generated by CentroidFold, ContextFold and IPknot. The best comparative methods typically outperform the best single-sequence methods if an alignment of homologous RNA sequences is available. This article presents the results of our benchmarks as of 3 October 2012, whereas the rankings presented online are continuously updated. We will gladly include new prediction methods and new measures of accuracy in the new editions of CompaRNA benchmarks. PMID:23435231

  18. CompaRNA: a server for continuous benchmarking of automated methods for RNA secondary structure prediction.

    Science.gov (United States)

    Puton, Tomasz; Kozlowski, Lukasz P; Rother, Kristian M; Bujnicki, Janusz M

    2013-04-01

    We present a continuous benchmarking approach for the assessment of RNA secondary structure prediction methods implemented in the CompaRNA web server. As of 3 October 2012, the performance of 28 single-sequence and 13 comparative methods has been evaluated on RNA sequences/structures released weekly by the Protein Data Bank. We also provide a static benchmark generated on RNA 2D structures derived from the RNAstrand database. Benchmarks on both data sets offer insight into the relative performance of RNA secondary structure prediction methods on RNAs of different size and with respect to different types of structure. According to our tests, on the average, the most accurate predictions obtained by a comparative approach are generated by CentroidAlifold, MXScarna, RNAalifold and TurboFold. On the average, the most accurate predictions obtained by single-sequence analyses are generated by CentroidFold, ContextFold and IPknot. The best comparative methods typically outperform the best single-sequence methods if an alignment of homologous RNA sequences is available. This article presents the results of our benchmarks as of 3 October 2012, whereas the rankings presented online are continuously updated. We will gladly include new prediction methods and new measures of accuracy in the new editions of CompaRNA benchmarks.

  19. RNA secondary structures of the bacteriophage phi6 packaging regions.

    OpenAIRE

    Pirttimaa, M J; Bamford, D H

    2000-01-01

    Bacteriophage phi6 genome consists of three segments of double-stranded RNA. During maturation, single-stranded copies of these segments are packaged into preformed polymerase complex particles. Only phi6 RNA is packaged, and each particle contains only one copy of each segment. An in vitro packaging and replication assay has been developed for phi6, and the packaging signals (pac sites) have been mapped to the 5' ends of the RNA segments. In this study, we propose secondary structure models ...

  20. Structure of an RNA dimer of a regulatory element from human thymidylate synthase mRNA

    OpenAIRE

    Dibrov, Sergey; McLean, Jaime; Hermann, Thomas

    2011-01-01

    An oligonucleotide representing a regulatory element of human thymidylate synthase mRNA has been crystallized as a dimer. The structure of the asymmetric dimer has been determined at 1.97 Å resolution.

  1. Approaches to link RNA secondary structures with splicing regulation

    DEFF Research Database (Denmark)

    Plass, Mireya; Eyras, Eduardo

    2014-01-01

    In higher eukaryotes, alternative splicing is usually regulated by protein factors, which bind to the pre-mRNA and affect the recognition of splicing signals. There is recent evidence that the secondary structure of the pre-mRNA may also play an important role in this process, either by facilitat...... describes the steps in the analysis of the secondary structure of the pre-mRNA and its possible relation to splicing. As a working example, we use the case of yeast and the problem of the recognition of the 3' splice site (3'ss).......In higher eukaryotes, alternative splicing is usually regulated by protein factors, which bind to the pre-mRNA and affect the recognition of splicing signals. There is recent evidence that the secondary structure of the pre-mRNA may also play an important role in this process, either...

  2. Automated identification of RNA 3D modules with discriminative power in RNA structural alignments

    DEFF Research Database (Denmark)

    Theis, Corinna; Höner zu Siederdissen, Christian; Hofacker, Ivo L.

    2013-01-01

    Recent progress in predicting RNA structure is moving towards filling the 'gap' in 2D RNA structure prediction where, for example, predicted internal loops often form non-canonical base pairs. This is increasingly recognized with the steady increase of known RNA 3D modules. There is a general...... comparative evidence. Subsequently, the modules, initially represented by a graph, are turned into models for the RMDetect program, which allows to test their discriminative power using real and randomized Rfam alignments. An initial extraction of 22495 3D modules in all PDB files results in 977 internal loop...

  3. High-Throughput Sequencing Based Methods of RNA Structure Investigation

    DEFF Research Database (Denmark)

    Kielpinski, Lukasz Jan

    In this thesis we describe the development of four related methods for RNA structure probing that utilize massive parallel sequencing. Using them, we were able to gather structural data for multiple, long molecules simultaneously. First, we have established an easy to follow experimental...... and computational protocol for detecting the reverse transcription termination sites (RTTS-Seq). This protocol was subsequently applied to hydroxyl radical footprinting of three dimensional RNA structures to give a probing signal that correlates well with the RNA backbone solvent accessibility. Moreover, we applied...

  4. MultiSETTER: web server for multiple RNA structure comparison.

    Science.gov (United States)

    Čech, Petr; Hoksza, David; Svozil, Daniel

    2015-08-12

    Understanding the architecture and function of RNA molecules requires methods for comparing and analyzing their tertiary and quaternary structures. While structural superposition of short RNAs is achievable in a reasonable time, large structures represent much bigger challenge. Therefore, we have developed a fast and accurate algorithm for RNA pairwise structure superposition called SETTER and implemented it in the SETTER web server. However, though biological relationships can be inferred by a pairwise structure alignment, key features preserved by evolution can be identified only from a multiple structure alignment. Thus, we extended the SETTER algorithm to the alignment of multiple RNA structures and developed the MultiSETTER algorithm. In this paper, we present the updated version of the SETTER web server that implements a user friendly interface to the MultiSETTER algorithm. The server accepts RNA structures either as the list of PDB IDs or as user-defined PDB files. After the superposition is computed, structures are visualized in 3D and several reports and statistics are generated. To the best of our knowledge, the MultiSETTER web server is the first publicly available tool for a multiple RNA structure alignment. The MultiSETTER server offers the visual inspection of an alignment in 3D space which may reveal structural and functional relationships not captured by other multiple alignment methods based either on a sequence or on secondary structure motifs.

  5. A combinatorial enumeration problem of RNA secondary structures

    African Journals Online (AJOL)

    use

    2011-12-21

    Dec 21, 2011 ... interesting combinatorial questions (Chen et al., 2005;. Liu, 2006; Schmitt and Waterman 1994; Stein and. Waterman 1978). The research on the enumeration of. RNA secondary structures becomes one of the hot topics in Computational Molecular Biology. An RNA molecule is described by its sequences of.

  6. Atomic Structure and Biochemical Characterization of an RNA Endonuclease in the N Terminus of Andes Virus L Protein.

    Directory of Open Access Journals (Sweden)

    Yaiza Fernández-García

    2016-06-01

    Full Text Available Andes virus (ANDV is a human-pathogenic hantavirus. Hantaviruses presumably initiate their mRNA synthesis by using cap structures derived from host cell mRNAs, a mechanism called cap-snatching. A signature for a cap-snatching endonuclease is present in the N terminus of hantavirus L proteins. In this study, we aimed to solve the atomic structure of the ANDV endonuclease and characterize its biochemical features. However, the wild-type protein was refractory to expression in Escherichia coli, presumably due to toxic enzyme activity. To circumvent this problem, we introduced attenuating mutations in the domain that were previously shown to enhance L protein expression in mammalian cells. Using this approach, 13 mutant proteins encompassing ANDV L protein residues 1-200 were successfully expressed and purified. Protein stability and nuclease activity of the mutants was analyzed and the crystal structure of one mutant was solved to a resolution of 2.4 Å. Shape in solution was determined by small angle X-ray scattering. The ANDV endonuclease showed structural similarities to related enzymes of orthobunya-, arena-, and orthomyxoviruses, but also differences such as elongated shape and positively charged patches surrounding the active site. The enzyme was dependent on manganese, which is bound to the active site, most efficiently cleaved single-stranded RNA substrates, did not cleave DNA, and could be inhibited by known endonuclease inhibitors. The atomic structure in conjunction with stability and activity data for the 13 mutant enzymes facilitated inference of structure-function relationships in the protein. In conclusion, we solved the structure of a hantavirus cap-snatching endonuclease, elucidated its catalytic properties, and present a highly active mutant form, which allows for inhibitor screening.

  7. Computing the Partition Function for Kinetically Trapped RNA Secondary Structures

    Science.gov (United States)

    Lorenz, William A.; Clote, Peter

    2011-01-01

    An RNA secondary structure is locally optimal if there is no lower energy structure that can be obtained by the addition or removal of a single base pair, where energy is defined according to the widely accepted Turner nearest neighbor model. Locally optimal structures form kinetic traps, since any evolution away from a locally optimal structure must involve energetically unfavorable folding steps. Here, we present a novel, efficient algorithm to compute the partition function over all locally optimal secondary structures of a given RNA sequence. Our software, RNAlocopt runs in time and space. Additionally, RNAlocopt samples a user-specified number of structures from the Boltzmann subensemble of all locally optimal structures. We apply RNAlocopt to show that (1) the number of locally optimal structures is far fewer than the total number of structures – indeed, the number of locally optimal structures approximately equal to the square root of the number of all structures, (2) the structural diversity of this subensemble may be either similar to or quite different from the structural diversity of the entire Boltzmann ensemble, a situation that depends on the type of input RNA, (3) the (modified) maximum expected accuracy structure, computed by taking into account base pairing frequencies of locally optimal structures, is a more accurate prediction of the native structure than other current thermodynamics-based methods. The software RNAlocopt constitutes a technical breakthrough in our study of the folding landscape for RNA secondary structures. For the first time, locally optimal structures (kinetic traps in the Turner energy model) can be rapidly generated for long RNA sequences, previously impossible with methods that involved exhaustive enumeration. Use of locally optimal structure leads to state-of-the-art secondary structure prediction, as benchmarked against methods involving the computation of minimum free energy and of maximum expected accuracy. Web server

  8. Computing the partition function for kinetically trapped RNA secondary structures.

    Directory of Open Access Journals (Sweden)

    William A Lorenz

    Full Text Available An RNA secondary structure is locally optimal if there is no lower energy structure that can be obtained by the addition or removal of a single base pair, where energy is defined according to the widely accepted Turner nearest neighbor model. Locally optimal structures form kinetic traps, since any evolution away from a locally optimal structure must involve energetically unfavorable folding steps. Here, we present a novel, efficient algorithm to compute the partition function over all locally optimal secondary structures of a given RNA sequence. Our software, RNAlocopt runs in O(n3 time and O(n2 space. Additionally, RNAlocopt samples a user-specified number of structures from the Boltzmann subensemble of all locally optimal structures. We apply RNAlocopt to show that (1 the number of locally optimal structures is far fewer than the total number of structures--indeed, the number of locally optimal structures approximately equal to the square root of the number of all structures, (2 the structural diversity of this subensemble may be either similar to or quite different from the structural diversity of the entire Boltzmann ensemble, a situation that depends on the type of input RNA, (3 the (modified maximum expected accuracy structure, computed by taking into account base pairing frequencies of locally optimal structures, is a more accurate prediction of the native structure than other current thermodynamics-based methods. The software RNAlocopt constitutes a technical breakthrough in our study of the folding landscape for RNA secondary structures. For the first time, locally optimal structures (kinetic traps in the Turner energy model can be rapidly generated for long RNA sequences, previously impossible with methods that involved exhaustive enumeration. Use of locally optimal structure leads to state-of-the-art secondary structure prediction, as benchmarked against methods involving the computation of minimum free energy and of maximum expected

  9. Ensemble-based prediction of RNA secondary structures.

    Science.gov (United States)

    Aghaeepour, Nima; Hoos, Holger H

    2013-04-24

    Accurate structure prediction methods play an important role for the understanding of RNA function. Energy-based, pseudoknot-free secondary structure prediction is one of the most widely used and versatile approaches, and improved methods for this task have received much attention over the past five years. Despite the impressive progress that as been achieved in this area, existing evaluations of the prediction accuracy achieved by various algorithms do not provide a comprehensive, statistically sound assessment. Furthermore, while there is increasing evidence that no prediction algorithm consistently outperforms all others, no work has been done to exploit the complementary strengths of multiple approaches. In this work, we present two contributions to the area of RNA secondary structure prediction. Firstly, we use state-of-the-art, resampling-based statistical methods together with a previously published and increasingly widely used dataset of high-quality RNA structures to conduct a comprehensive evaluation of existing RNA secondary structure prediction procedures. The results from this evaluation clarify the performance relationship between ten well-known existing energy-based pseudoknot-free RNA secondary structure prediction methods and clearly demonstrate the progress that has been achieved in recent years. Secondly, we introduce AveRNA, a generic and powerful method for combining a set of existing secondary structure prediction procedures into an ensemble-based method that achieves significantly higher prediction accuracies than obtained from any of its component procedures. Our new, ensemble-based method, AveRNA, improves the state of the art for energy-based, pseudoknot-free RNA secondary structure prediction by exploiting the complementary strengths of multiple existing prediction procedures, as demonstrated using a state-of-the-art statistical resampling approach. In addition, AveRNA allows an intuitive and effective control of the trade-off between

  10. DCJ-RNA - double cut and join for RNA secondary structures.

    Science.gov (United States)

    Badr, Ghada H; Al-Aqel, Haifa A

    2017-10-16

    Genome rearrangements are essential processes for evolution and are responsible for existing varieties of genome architectures. Many studies have been conducted to obtain an algorithm that identifies the minimum number of inversions that are necessary to transform one genome into another; this allows for genome sequence representation in polynomial time. Studies have not been conducted on the topic of rearranging a genome when it is represented as a secondary structure. Unlike sequences, the secondary structure preserves the functionality of the genome. Sequences can be different, but they all share the same structure and, therefore, the same functionality. This paper proposes a double cut and join for RNA secondary structures (DCJ-RNA) algorithm. This algorithm allows for the description of evolutionary scenarios that are based on secondary structures rather than sequences. The main aim of this paper is to suggest an efficient algorithm that can help researchers compare two ribonucleic acid (RNA) secondary structures based on rearrangement operations. The results, which are based on real datasets, show that the algorithm is able to count the minimum number of rearrangement operations, as well as to report an optimum scenario that can increase the similarity between the two structures. The algorithm calculates the distance between structures and reports a scenario based on the minimum rearrangement operations required to make the given structure similar to the other. DCJ-RNA can also be used to measure the distance between the two structures. This can help identify the common functionalities between different species.

  11. A method for rapid similarity analysis of RNA secondary structures

    Directory of Open Access Journals (Sweden)

    Liu Na

    2006-11-01

    Full Text Available Abstract Background Owing to the rapid expansion of RNA structure databases in recent years, efficient methods for structure comparison are in demand for function prediction and evolutionary analysis. Usually, the similarity of RNA secondary structures is evaluated based on tree models and dynamic programming algorithms. We present here a new method for the similarity analysis of RNA secondary structures. Results Three sets of real data have been used as input for the example applications. Set I includes the structures from 5S rRNAs. Set II includes the secondary structures from RNase P and RNase MRP. Set III includes the structures from 16S rRNAs. Reasonable phylogenetic trees are derived for these three sets of data by using our method. Moreover, our program runs faster as compared to some existing ones. Conclusion The famous Lempel-Ziv algorithm can efficiently extract the information on repeated patterns encoded in RNA secondary structures and makes our method an alternative to analyze the similarity of RNA secondary structures. This method will also be useful to researchers who are interested in evolutionary analysis.

  12. Polar cap electric field structures with a northward interplanetary magnetic field

    International Nuclear Information System (INIS)

    Burke, W.J.; Kelley, M.C.; Sagalyn, R.C.; Smiddy, M.; Lai, S.T.

    1979-01-01

    Polar cap electric fields patterns are presented from times when the S3-2 Satellite was near the dawn-dusk meridian and IMF data were available. With B/sub z/> or =0.7γ, two characteristic types of electric field patterns were measured in the polar cap. In the sunlit polar cap the convection pattern usually consisted of four cells. Two of the cells were confined to the polar cap with sunward convection in the central portion of the cap. The other pair of cells were marked by anti-sunward flow along the flanks of the polar cap and by sunward flow in the auroral oval. These observations are interpreted in terms of a model for magnetic merging at the poleward wall of the dayside polar cusp. The sunward flow in the auroral zone is not predicted by the magnetic model and may be due to a viscous interaction between the solar wind and and magnetosphere. The second type, which was observed in some of the summer hemisphere passes and all of the winter ones, was characterized by an electric field pattern which was very turbulent, and may be related to inhomogeneous merging

  13. Topological classification and enumeration of RNA structures by genus

    DEFF Research Database (Denmark)

    Andersen, Joergen Ellegard; Penner, Robert C.; Reidys, Christian

    2013-01-01

    To an RNA pseudoknot structure is naturally associated a topological surface, which has its associated genus, and structures can thus be classified by the genus. Based on earlier work of Harer-Zagier, we compute the generating function for the number of those structures of fixed genus and minimum...

  14. HD-RNAS: An automated hierarchical database of RNA structures

    Directory of Open Access Journals (Sweden)

    Shubhra Sankar eRay

    2012-04-01

    Full Text Available One of the important goals of most biological investigations is to classify and organize the experimental findings so that they are readily useful for deriving generalized rules. Although there is a huge amount of information on RNA structures in PDB, there are redundant files, ambiguous synthetic sequences etc. Moreover, a systematic hierarchical organization, reflecting RNA classification, is missing in PDB. In this investigation, we have classified all the available RNA crystal structures from PDB through a programmatic approach. Hence, it would be now a simple assignment to regularly update the classification as and when new structures are released. The classification can further determine (i a non-redundant set of RNA structures and (ii if available, a set of structures of identical sequence and function, which can highlight structural polymorphism, ligand-induced conformational alterations etc. Presently, we have classified the available structures (2095 PDB entries having RNA chain longer than 9 nucleotides solved by X-ray crystallography or NMR spectroscopy into nine functional classes. The structures of same function and same source are mostly seen to be similar with subtle differences depending on their functional complexation. The web-server is available online at http://www.saha.ac.in/biop/www/HD-RNAS.html and is updated regularly.

  15. RNA secondary structures of the bacteriophage phi6 packaging regions.

    Science.gov (United States)

    Pirttimaa, M J; Bamford, D H

    2000-06-01

    Bacteriophage phi6 genome consists of three segments of double-stranded RNA. During maturation, single-stranded copies of these segments are packaged into preformed polymerase complex particles. Only phi6 RNA is packaged, and each particle contains only one copy of each segment. An in vitro packaging and replication assay has been developed for phi6, and the packaging signals (pac sites) have been mapped to the 5' ends of the RNA segments. In this study, we propose secondary structure models for the pac sites of phi6 single-stranded RNA segments. Our models accommodate data from structure-specific chemical modifications, free energy minimizations, and phylogenetic comparisons. Previously reported pac site deletion studies are also discussed. Each pac site possesses a unique architecture, that, however, contains common structural elements.

  16. Chemical Synthesis of a 5'-Terminal TMG-Capped Triribonucleotide m(3)(2,2,7)G(5)(')pppAmpUmpA of U1 RNA.

    Science.gov (United States)

    Sekine, Mitsuo; Kadokura, Michinori; Satoh, Takahiko; Seio, Kohji; Wada, Takeshi; Fischer, Utz; Sumpter, Vicki; Lührmann, Reinhard

    1996-06-26

    The 5'-terminal TMG-capped triribonucleotide, m(3)(2,2,7)G(5)(')pppAmpUmpA, has been synthesized by condensation of an appropriately protected triribonucleotide derivative of ppAmpUmpA with a new TMG-capping reagent. During this total synthesis, it was found that the regioselective 2'-O-methylation of 3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-N-(4-monomethoxytrityl)adenosine was achieved by use of MeI/Ag(2)O without affecting the base moiety. A new route to 2-N,2-N-dimethylguanosine from guanosine via a three-step reaction has also been developed by reductive methylation using paraformaldehyde and sodium cyanoborohydride. These key intermediates were used as starting materials for the construction of a fully protected derivative of pAmpUmpA and a TMG-capping reagent of Im-pm(3)(2,2,7)G. The target TMG-capped tetramer, m(3)(2,2,7)G(5)(')pppAmpUmpA, was synthesized by condensation of a partially protected triribonucleotide 5'-terminal diphosphate species, ppA(MMTr)mpUmpA, with Im-pm(3)(2,2,7)G followed by treatment with 80% acetic acid. The structure of m(3)(2,2,7)G(5)(')pppAmpUmpA was characterized by (1)H and (31)P NMR spectroscopy as well as enzymatic assay using snake venom phosphodiesterase, calf intestinal phosphatase, and nuclease P1.

  17. Characterization of the PB2 Cap Binding Domain Accelerates Inhibitor Design

    Directory of Open Access Journals (Sweden)

    Amanda E. Constantinides

    2018-01-01

    Full Text Available X-ray crystallographic structural determinations of the PB2 cap binding domain (PB2cap have improved the conformational characterization of the RNA-dependent RNA polymerase machinery (PA, PB2, and PB1 of the influenza virus. Geometrically, the catalytic PB1 subunit resembles the palm of a human hand. PA lies near the thumb region, and PB2 lies near the finger region. PB2 binds the cap moiety in the pre-mRNA of the host cell, while the endonuclease of PA cleaves the pre-mRNA 10–13 nucleotides downstream. The truncated RNA piece performs as a primer for PB1 to synthesize the viral mRNA. Precisely targeting PB2cap with a small molecule inhibitor will halt viral proliferation via interference of the cap-snatching activity. Wild-type and mutant PB2cap from A/California/07/2009 H1N1 were expressed in Escherichia coli, purified by nickel affinity and size exclusion chromatography, crystallized, and subjected to X-ray diffraction experiments. The crystal of mutant PB2cap liganded with m7GTP was prepared by co-crystallization. Structures were solved by the molecular replacement method, refined, and deposited in the Protein Data Bank (PDB. Structural determination and comparative analyses of these structures revealed the functions of Glu361, Lys376, His357, Phe404, Phe323, Lys339, His432, Asn429, Gln406, and Met401 in PB2cap, and the dissociation of the influenza A PB2cap C-terminal subdomain (residues 446–479 upon ligand binding. Understanding the role of these residues will aid in the ultimate development of a small-molecule inhibitor that binds both Influenza A and B virus PB2cap.

  18. Effect of capping agents: Structural, optical and biological properties of ZnO nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Javed, Rabia [Department of Biotechnology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320 (Pakistan); Usman, Muhammad, E-mail: uk_phy@yahoo.com [Department of Physics, Faculty of Natural Sciences, Quaid-i-Azam University, Islamabad 45320 (Pakistan); Department of Physics, School of Science and Engineering, Lahore University of Management Sciences, Lahore 54729 (Pakistan); Tabassum, Saira; Zia, Muhammad [Department of Biotechnology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320 (Pakistan)

    2016-11-15

    Highlights: • ZnO nanoparticles have been effectively capped with polyethylene glycol (PEG) and polyvinyl pyrrolidone (PVP) shown by the data of XRD, FTIR and UV–visible spectroscopy. • Reduction in size occurred from 34 nm to 26 nm due to capping agent and band gap energy increases with the decrease in the particle size. • Antibacterial activity against Gram-positive bacteria is greater than the Gram-negative bacteria. • All biological assays reveal highest activities in capped ZnO nanoparticles as compared to the uncapped ZnO nanoparticles. • Highest antibacterial activity has been exhibited by ZnO-PVP while highest antioxidant and antidiabetic activities have been conferred by ZnO- PEG. - Abstract: Different biological activities of capped and uncapped ZnO nanoparticles were investigated, and the effects of potential capping agents on these biological activities were studied. ZnO nanoparticles were synthesized and capped by polyethylene glycol (PEG) and polyvinyl pyrrolidone (PVP) using a simple chemical method of co-precipitation. Characterization by X-ray diffraction (XRD), Fourier transform Infrared spectroscopy (FTIR) and UV–vis spectroscopy confirmed the crystallinity, size, functional group, and band gap of synthesized nanoparticles. Reduction in size occurred from 34 nm to 26 nm due to surfactant. Results of all biological activities indicated significantly higher values in capped as compared to uncapped nanoparticles. Antibacterial activity against Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), Escherichia coli (ATCC15224), and Acetobacter was obtained. This activity was more prominent against Gram-positive bacteria, and ZnO-PVP nanoparticles elucidated highest antibacterial activity (zone of inhibition 17 mm) against Gram-positive, Bacillus subtilis species. Antioxidant activities including total flavonoid content, total phenolic content, total antioxidant capacity, total reducing power and %age inhibition of DPPH, and

  19. Effect of capping agents: Structural, optical and biological properties of ZnO nanoparticles

    International Nuclear Information System (INIS)

    Javed, Rabia; Usman, Muhammad; Tabassum, Saira; Zia, Muhammad

    2016-01-01

    Highlights: • ZnO nanoparticles have been effectively capped with polyethylene glycol (PEG) and polyvinyl pyrrolidone (PVP) shown by the data of XRD, FTIR and UV–visible spectroscopy. • Reduction in size occurred from 34 nm to 26 nm due to capping agent and band gap energy increases with the decrease in the particle size. • Antibacterial activity against Gram-positive bacteria is greater than the Gram-negative bacteria. • All biological assays reveal highest activities in capped ZnO nanoparticles as compared to the uncapped ZnO nanoparticles. • Highest antibacterial activity has been exhibited by ZnO-PVP while highest antioxidant and antidiabetic activities have been conferred by ZnO- PEG. - Abstract: Different biological activities of capped and uncapped ZnO nanoparticles were investigated, and the effects of potential capping agents on these biological activities were studied. ZnO nanoparticles were synthesized and capped by polyethylene glycol (PEG) and polyvinyl pyrrolidone (PVP) using a simple chemical method of co-precipitation. Characterization by X-ray diffraction (XRD), Fourier transform Infrared spectroscopy (FTIR) and UV–vis spectroscopy confirmed the crystallinity, size, functional group, and band gap of synthesized nanoparticles. Reduction in size occurred from 34 nm to 26 nm due to surfactant. Results of all biological activities indicated significantly higher values in capped as compared to uncapped nanoparticles. Antibacterial activity against Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633), Escherichia coli (ATCC15224), and Acetobacter was obtained. This activity was more prominent against Gram-positive bacteria, and ZnO-PVP nanoparticles elucidated highest antibacterial activity (zone of inhibition 17 mm) against Gram-positive, Bacillus subtilis species. Antioxidant activities including total flavonoid content, total phenolic content, total antioxidant capacity, total reducing power and %age inhibition of DPPH, and

  20. Heart structure-specific transcriptomic atlas reveals conserved microRNA-mRNA interactions.

    Science.gov (United States)

    Vacchi-Suzzi, Caterina; Hahne, Florian; Scheubel, Philippe; Marcellin, Magali; Dubost, Valerie; Westphal, Magdalena; Boeglen, Catherine; Büchmann-Møller, Stine; Cheung, Ming Sin; Cordier, André; De Benedetto, Christopher; Deurinck, Mark; Frei, Moritz; Moulin, Pierre; Oakeley, Edward; Grenet, Olivier; Grevot, Armelle; Stull, Robert; Theil, Diethilde; Moggs, Jonathan G; Marrer, Estelle; Couttet, Philippe

    2013-01-01

    MicroRNAs are short non-coding RNAs that regulate gene expression at the post-transcriptional level and play key roles in heart development and cardiovascular diseases. Here, we have characterized the expression and distribution of microRNAs across eight cardiac structures (left and right ventricles, apex, papillary muscle, septum, left and right atrium and valves) in rat, Beagle dog and cynomolgus monkey using microRNA sequencing. Conserved microRNA signatures enriched in specific heart structures across these species were identified for cardiac valve (miR-let-7c, miR-125b, miR-127, miR-199a-3p, miR-204, miR-320, miR-99b, miR-328 and miR-744) and myocardium (miR-1, miR-133b, miR-133a, miR-208b, miR-30e, miR-499-5p, miR-30e*). The relative abundance of myocardium-enriched (miR-1) and valve-enriched (miR-125b-5p and miR-204) microRNAs was confirmed using in situ hybridization. MicroRNA-mRNA interactions potentially relevant for cardiac functions were explored using anti-correlation expression analysis and microRNA target prediction algorithms. Interactions between miR-1/Timp3, miR-125b/Rbm24, miR-204/Tgfbr2 and miR-208b/Csnk2a2 were identified and experimentally investigated in human pulmonary smooth muscle cells and luciferase reporter assays. In conclusion, we have generated a high-resolution heart structure-specific mRNA/microRNA expression atlas for three mammalian species that provides a novel resource for investigating novel microRNA regulatory circuits involved in cardiac molecular physiopathology.

  1. Heart structure-specific transcriptomic atlas reveals conserved microRNA-mRNA interactions.

    Directory of Open Access Journals (Sweden)

    Caterina Vacchi-Suzzi

    Full Text Available MicroRNAs are short non-coding RNAs that regulate gene expression at the post-transcriptional level and play key roles in heart development and cardiovascular diseases. Here, we have characterized the expression and distribution of microRNAs across eight cardiac structures (left and right ventricles, apex, papillary muscle, septum, left and right atrium and valves in rat, Beagle dog and cynomolgus monkey using microRNA sequencing. Conserved microRNA signatures enriched in specific heart structures across these species were identified for cardiac valve (miR-let-7c, miR-125b, miR-127, miR-199a-3p, miR-204, miR-320, miR-99b, miR-328 and miR-744 and myocardium (miR-1, miR-133b, miR-133a, miR-208b, miR-30e, miR-499-5p, miR-30e*. The relative abundance of myocardium-enriched (miR-1 and valve-enriched (miR-125b-5p and miR-204 microRNAs was confirmed using in situ hybridization. MicroRNA-mRNA interactions potentially relevant for cardiac functions were explored using anti-correlation expression analysis and microRNA target prediction algorithms. Interactions between miR-1/Timp3, miR-125b/Rbm24, miR-204/Tgfbr2 and miR-208b/Csnk2a2 were identified and experimentally investigated in human pulmonary smooth muscle cells and luciferase reporter assays. In conclusion, we have generated a high-resolution heart structure-specific mRNA/microRNA expression atlas for three mammalian species that provides a novel resource for investigating novel microRNA regulatory circuits involved in cardiac molecular physiopathology.

  2. RNA secondary structure image - fRNAdb | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us fRNAdb RNA secondary structure image Data detail Data name RNA secondary structure image DOI... 10.18908/lsdba.nbdc00452-005 Description of data contents RNA secondary structure images - png.zip: RNA secondary structure image...s (PNG) - pdf.zip: RNA secondary structure images (PDF) - thumbnail.zip: Thumbnails of... RNA secondary structure images Data file File name: RNA_secondary_structure_image... File URL: ftp://ftp.biosciencedbc.jp/archive/frnadb/LATEST/RNA_secondary_structure_image File size: 9.6 GB

  3. The 2’-O-ribose methyltransferase for cap 1 of spliced leader RNA and U1 small nuclear RNA in Trypanosoma brucei

    Czech Academy of Sciences Publication Activity Database

    Zamudio, J. R.; Mittra, B.; Foldynová-Trantírková, Silvie; Zeiner, G. M.; Lukeš, Julius; Bujnicki, J. M.; Sturm, N. R.; Campbell, D. A.

    2007-01-01

    Roč. 27, č. 17 (2007), s. 6084-6092 ISSN 0270-7306 R&D Projects: GA MŠk 2B06129; GA MŠk LC07032 Institutional research plan: CEZ:AV0Z60220518 Keywords : methylation * Trypanosoma brucei * methyltransferase * RNA interference Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.420, year: 2007

  4. Structure of noncoding RNA is a determinant of function of RNA binding proteins in transcriptional regulation

    Directory of Open Access Journals (Sweden)

    Oyoshi Takanori

    2012-01-01

    Full Text Available Abstract The majority of the noncoding regions of mammalian genomes have been found to be transcribed to generate noncoding RNAs (ncRNAs, resulting in intense interest in their biological roles. During the past decade, numerous ncRNAs and aptamers have been identified as regulators of transcription. 6S RNA, first described as a ncRNA in E. coli, mimics an open promoter structure, which has a large bulge with two hairpin/stalk structures that regulate transcription through interactions with RNA polymerase. B2 RNA, which has stem-loops and unstructured single-stranded regions, represses transcription of mRNA in response to various stresses, including heat shock in mouse cells. The interaction of TLS (translocated in liposarcoma with CBP/p300 was induced by ncRNAs that bind to TLS, and this in turn results in inhibition of CBP/p300 histone acetyltransferase (HAT activity in human cells. Transcription regulator EWS (Ewing's sarcoma, which is highly related to TLS, and TLS specifically bind to G-quadruplex structures in vitro. The carboxy terminus containing the Arg-Gly-Gly (RGG repeat domains in these proteins are necessary for cis-repression of transcription activation and HAT activity by the N-terminal glutamine-rich domain. Especially, the RGG domain in the carboxy terminus of EWS is important for the G-quadruplex specific binding. Together, these data suggest that functions of EWS and TLS are modulated by specific structures of ncRNAs.

  5. Reconstruction of ancestral RNA sequences under multiple structural constraints

    Directory of Open Access Journals (Sweden)

    Olivier Tremblay-Savard

    2016-11-01

    Full Text Available Abstract Background Secondary structures form the scaffold of multiple sequence alignment of non-coding RNA (ncRNA families. An accurate reconstruction of ancestral ncRNAs must use this structural signal. However, the inference of ancestors of a single ncRNA family with a single consensus structure may bias the results towards sequences with high affinity to this structure, which are far from the true ancestors. Methods In this paper, we introduce achARNement, a maximum parsimony approach that, given two alignments of homologous ncRNA families with consensus secondary structures and a phylogenetic tree, simultaneously calculates ancestral RNA sequences for these two families. Results We test our methodology on simulated data sets, and show that achARNement outperforms classical maximum parsimony approaches in terms of accuracy, but also reduces by several orders of magnitude the number of candidate sequences. To conclude this study, we apply our algorithms on the Glm clan and the FinP-traJ clan from the Rfam database. Conclusions Our results show that our methods reconstruct small sets of high-quality candidate ancestors with better agreement to the two target structures than with classical approaches. Our program is freely available at: http://csb.cs.mcgill.ca/acharnement .

  6. Reconstruction of ancestral RNA sequences under multiple structural constraints.

    Science.gov (United States)

    Tremblay-Savard, Olivier; Reinharz, Vladimir; Waldispühl, Jérôme

    2016-11-11

    Secondary structures form the scaffold of multiple sequence alignment of non-coding RNA (ncRNA) families. An accurate reconstruction of ancestral ncRNAs must use this structural signal. However, the inference of ancestors of a single ncRNA family with a single consensus structure may bias the results towards sequences with high affinity to this structure, which are far from the true ancestors. In this paper, we introduce achARNement, a maximum parsimony approach that, given two alignments of homologous ncRNA families with consensus secondary structures and a phylogenetic tree, simultaneously calculates ancestral RNA sequences for these two families. We test our methodology on simulated data sets, and show that achARNement outperforms classical maximum parsimony approaches in terms of accuracy, but also reduces by several orders of magnitude the number of candidate sequences. To conclude this study, we apply our algorithms on the Glm clan and the FinP-traJ clan from the Rfam database. Our results show that our methods reconstruct small sets of high-quality candidate ancestors with better agreement to the two target structures than with classical approaches. Our program is freely available at: http://csb.cs.mcgill.ca/acharnement .

  7. Strategies for measuring evolutionary conservation of RNA secondary structures

    Directory of Open Access Journals (Sweden)

    Hofacker Ivo L

    2008-02-01

    Full Text Available Abstract Background Evolutionary conservation of RNA secondary structure is a typical feature of many functional non-coding RNAs. Since almost all of the available methods used for prediction and annotation of non-coding RNA genes rely on this evolutionary signature, accurate measures for structural conservation are essential. Results We systematically assessed the ability of various measures to detect conserved RNA structures in multiple sequence alignments. We tested three existing and eight novel strategies that are based on metrics of folding energies, metrics of single optimal structure predictions, and metrics of structure ensembles. We find that the folding energy based SCI score used in the RNAz program and a simple base-pair distance metric are by far the most accurate. The use of more complex metrics like for example tree editing does not improve performance. A variant of the SCI performed particularly well on highly conserved alignments and is thus a viable alternative when only little evolutionary information is available. Surprisingly, ensemble based methods that, in principle, could benefit from the additional information contained in sub-optimal structures, perform particularly poorly. As a general trend, we observed that methods that include a consensus structure prediction outperformed equivalent methods that only consider pairwise comparisons. Conclusion Structural conservation can be measured accurately with relatively simple and intuitive metrics. They have the potential to form the basis of future RNA gene finders, that face new challenges like finding lineage specific structures or detecting mis-aligned sequences.

  8. Analysis of the modified optical properties and band structure of GaAs1-xSbx-capped InAs/GaAs quantum dots

    NARCIS (Netherlands)

    Ulloa, J.M.; Llorens, J.M.; Moral, del M.; Bozkurt, M.; Koenraad, P.M.; Hierro, A.

    2012-01-01

    The origin of the modified optical properties of InAs/GaAs quantum dots (QD) capped with a thin GaAs1-xSbx layer is analyzed in terms of the band structure. To do so, the size, shape, and composition of the QDs and capping layer are determined through cross-sectional scanning tunnelling microscopy

  9. tRNA-like structure regulates translation of Brome mosaic virus RNA.

    Science.gov (United States)

    Barends, Sharief; Rudinger-Thirion, Joëlle; Florentz, Catherine; Giegé, Richard; Pleij, Cornelis W A; Kraal, Barend

    2004-04-01

    For various groups of plant viruses, the genomic RNAs end with a tRNA-like structure (TLS) instead of the 3' poly(A) tail of common mRNAs. The actual function of these TLSs has long been enigmatic. Recently, however, it became clear that for turnip yellow mosaic virus, a tymovirus, the valylated TLS(TYMV) of the single genomic RNA functions as a bait for host ribosomes and directs them to the internal initiation site of translation (with N-terminal valine) of the second open reading frame for the polyprotein. This discovery prompted us to investigate whether the much larger TLSs of a different genus of viruses have a comparable function in translation. Brome mosaic virus (BMV), a bromovirus, has a tripartite RNA genome with a subgenomic RNA4 for coat protein expression. All four RNAs carry a highly conserved and bulky 3' TLS(BMV) (about 200 nucleotides) with determinants for tyrosylation. We discovered TLS(BMV)-catalyzed self-tyrosylation of the tyrosyl-tRNA synthetase but could not clearly detect tyrosine incorporation into any virus-encoded protein. We established that BMV proteins do not need TLS(BMV) tyrosylation for their initiation. However, disruption of the TLSs strongly reduced the translation of genomic RNA1, RNA2, and less strongly, RNA3, whereas coat protein expression from RNA4 remained unaffected. This aberrant translation could be partially restored by providing the TLS(BMV) in trans. Intriguingly, a subdomain of the TLS(BMV) could even almost fully restore translation to the original pattern. We discuss here a model with a central and dominant role for the TLS(BMV) during the BMV infection cycle.

  10. Structure of the radio emission from the NGC 1579/LkH. cap alpha. 101 region

    Energy Technology Data Exchange (ETDEWEB)

    Brown, R L [National Radio Astronomy Observatory, Charlottesville, Va. (USA); Broderick, J J; Knapp, G R

    1976-06-01

    Radio-frequency observations at 3.7 and 11 cm of the NGC 1579/LkH..cap alpha..101 region show that the radio emission arises in a compact, < 1'' core concentric with a more extended approximately 1' emission region. At these wavelengths the compact component is optically thick, with a spectrum increasing as ..nu.., whereas the extended region is optically thin and contributes at least 80 per cent of the total flux density. LkH..cap alpha..101 appears to be the source of excitation for all of the radio emission; this result, together with the total infrared luminosity, suggests that an appropriate spectral classification for LkH..cap alpha..101 is B1 IIe.

  11. Deregulation of cap-dependent mRNA translation increases tumour radiosensitivity through reduction of the hypoxic fraction

    International Nuclear Information System (INIS)

    Rouschop, Kasper M.A.; Dubois, Ludwig; Schaaf, Marco B.E.; Beucken, Twan van den; Lieuwes, Natasja; Keulers, Tom G.H.; Savelkouls, Kim G.M.; Bussink, Johan; Kogel, Albert J. van der; Koritzinsky, Marianne; Wouters, Bradly G.

    2011-01-01

    Background and purpose: Tumour hypoxia is an important limiting factor in the successful treatment of cancer. Adaptation to hypoxia includes inhibition of mTOR, causing scavenging of eukaryotic initiation factor 4E (eIF4E), the rate-limiting factor for cap-dependent translation. The aim of this study was to determine the effect of preventing mTOR-dependent translation inhibition on hypoxic cell survival and tumour sensitivity towards irradiation. Material and methods: The effect of eIF4E-overexpression on cell proliferation, hypoxia-tolerance, and radiation sensitivity was assessed using isogenic, inducible U373 and HCT116 cells. Results: We found that eIF4E-overexpression significantly enhanced proliferation of cells under normal conditions, but not during hypoxia, caused by increased cell death during hypoxia. Furthermore, eIF4E-overexpression stimulated overall rates of tumour growth, but resulted in selective loss of hypoxic cells in established tumours and increased levels of necrosis. This markedly increased overall tumour sensitivity to irradiation. Conclusions: Our results demonstrate that hypoxia induced inhibition of translational control through regulation of eIF4E is an important mediator of hypoxia tolerance and radioresistance of tumours. These data also demonstrate that deregulation of metabolic pathways such as mTOR can influence the proliferation and survival of tumour cells experiencing metabolic stress in opposite ways of nutrient replete cells.

  12. RNA structure and scalar coupling constants

    Energy Technology Data Exchange (ETDEWEB)

    Tinoco, I. Jr.; Cai, Z.; Hines, J.V.; Landry, S.M.; SantaLucia, J. Jr.; Shen, L.X.; Varani, G. [Univ. of California, Berkeley, CA (United States)

    1994-12-01

    Signs and magnitudes of scalar coupling constants-spin-spin splittings-comprise a very large amount of data that can be used to establish the conformations of RNA molecules. Proton-proton and proton-phosphorus splittings have been used the most, but the availability of {sup 13}C-and {sup 15}N-labeled molecules allow many more coupling constants to be used for determining conformation. We will systematically consider the torsion angles that characterize a nucleotide unit and the coupling constants that depend on the values of these torsion angles. Karplus-type equations have been established relating many three-bond coupling constants to torsion angles. However, one- and two-bond coupling constants can also depend on conformation. Serianni and coworkers measured carbon-proton coupling constants in ribonucleosides and have calculated their values as a function of conformation. The signs of two-bond coupling can be very useful because it is easier to measure a sign than an accurate magnitude.

  13. Structure of Drosophila Oskar reveals a novel RNA binding protein

    Science.gov (United States)

    Yang, Na; Yu, Zhenyu; Hu, Menglong; Wang, Mingzhu; Lehmann, Ruth; Xu, Rui-Ming

    2015-01-01

    Oskar (Osk) protein plays critical roles during Drosophila germ cell development, yet its functions in germ-line formation and body patterning remain poorly understood. This situation contrasts sharply with the vast knowledge about the function and mechanism of osk mRNA localization. Osk is predicted to have an N-terminal LOTUS domain (Osk-N), which has been suggested to bind RNA, and a C-terminal hydrolase-like domain (Osk-C) of unknown function. Here, we report the crystal structures of Osk-N and Osk-C. Osk-N shows a homodimer of winged-helix–fold modules, but without detectable RNA-binding activity. Osk-C has a lipase-fold structure but lacks critical catalytic residues at the putative active site. Surprisingly, we found that Osk-C binds the 3′UTRs of osk and nanos mRNA in vitro. Mutational studies identified a region of Osk-C important for mRNA binding. These results suggest possible functions of Osk in the regulation of stability, regulation of translation, and localization of relevant mRNAs through direct interaction with their 3′UTRs, and provide structural insights into a novel protein–RNA interaction motif involving a hydrolase-related domain. PMID:26324911

  14. RNA secondary structure diagrams for very large molecules: RNAfdl

    DEFF Research Database (Denmark)

    Hecker, Nikolai; Wiegels, Tim; Torda, Andrew E.

    2013-01-01

    There are many programs that can read the secondary structure of an RNA molecule and draw a diagram, but hardly any that can cope with 10 3 bases. RNAfdl is slow but capable of producing intersection-free diagrams for ribosome-sized structures, has a graphical user interface for adjustments...

  15. Nucleosome Positioning and NDR Structure at RNA Polymerase III Promoters

    DEFF Research Database (Denmark)

    Helbo, Alexandra Søgaard; Lay, Fides D; Jones, Peter A

    2017-01-01

    Chromatin is structurally involved in the transcriptional regulation of all genes. While the nucleosome positioning at RNA polymerase II (pol II) promoters has been extensively studied, less is known about the chromatin structure at pol III promoters in human cells. We use a high...

  16. Experimental and numerical investigation of the cap-shock structure in over expanded thrust-optimized nozzles

    Energy Technology Data Exchange (ETDEWEB)

    Reijasse, P.; Bouvier, F.; Servel, P.

    2002-07-01

    This paper deals with the aerodynamics of an over-expanded nozzle, when the internal parabolic contour of the nozzle extension is highly thrust-optimized in terms of specific impulse-to-weight ratio. This optimization leads to an internal focusing shock issuing from a little downstream from the throat, even when the nozzle is running at nearly vacuum conditions. When such a nozzle is over-expanded, the focusing shock thus interferes with the over-expansion shock, and it forms from this shock interference a particular shock system, named 'cap-shock' because of the cap-like luminous shape seen in the over-expanded plumes of some real engines. Navier-Stokes calcinations performed in Europe had permitted to numerically analyze such a flow pattern, and they have revealed notably a recirculation bubble on the centerline downstream of the Mach disk, which had never been measured yet. A test campaign characterizing the flow separation in over-expanded sub-scale nozzles has been performed in the R2Ch blowdown wind tunnel of the Onera Chalais-Meudon center. Schlieren photographs of the exhaust jet have authorized a detailed description of the cap-shock pattern. Two-components Laser Doppler Velocimetry measurements have confirmed the existence of a recirculation bubble surrounded by an annular supersonic jet and has given its size. In addition to the calculations and the Schlieren interpretative sketches, these first quantitative experimental characterization of the cap-shock structure permit to state a physical description of the cap-shock induced flow field in the thrust-optimized nozzles. (authors)

  17. Structural, spectroscopic and anti-microbial inspection of PEG capped ZnO nanoparticles for biomedical applications

    Science.gov (United States)

    Meshram, J. V.; Koli, V. B.; Kumbhar, S. G.; Borde, L. C.; Phadatare, M. R.; Pawar, S. H.

    2018-04-01

    Zinc oxide (ZnO) nanoparticles (NPs) have a wide range of biomedical applications. Present study demonstrates the new methodology in sol-gel technology for synthesizing Polyethylene glycol (PEG) capped ZnO NPs and its size effect on anti-microbial activity. The reaction time was increased from 1 h to 5 h for the synthesis of ZnO NPs at 130 °C. The size of PEG capped ZnO NPs is increased from 10 to 84 nm by increasing the reaction upto 5 h. The x-ray diffraction studies and transmission electron microscopy analysis reveals the phase purity and hexagonal wurtzite crystal structure with uniform PEG capping on the surface of ZnO NPs. UV–visible spectroscopy exhibits the peak at 366 nm which is attributed to ZnO NPs. No adverse effect is observed in case of absorbance spectroscopy. Further, Fourier transforms infrared spectroscopy and thermo gravimetric analysis depicts the adsorption of PEG molecules on the ZnO NPs surface. The anti-microbial activities for both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria were studied by optical density (OD) mesurement. The remarkable anti-microbial activity was observed for PEG capped ZnO NPs synthesized at 1 h reaction time showing higher activity in comparison with that synthesized from 2 h to 5 h reaction time. The microbial growth was found to be inhibited after 10 h OD measurement for both the bacteria. The anti-microbial activity may be attributed to the generation of ROS and H2O2. However, these generated species plays a vital role in inhibition of microbial growth. Hence, PEG capped ZnO NPs has promising biomedical applications.

  18. Age structure and mortality of walleyes in Kansas reservoirs: Use of mortality caps to establish realistic management objectives

    Science.gov (United States)

    Quist, M.C.; Stephen, J.L.; Guy, C.S.; Schultz, R.D.

    2004-01-01

    Age structure, total annual mortality, and mortality caps (maximum mortality thresholds established by managers) were investigated for walleye Sander vitreus (formerly Stizostedion vitreum) populations sampled from eight Kansas reservoirs during 1991-1999. We assessed age structure by examining the relative frequency of different ages in the population; total annual mortality of age-2 and older walleyes was estimated by use of a weighted catch curve. To evaluate the utility of mortality caps, we modeled threshold values of mortality by varying growth rates and management objectives. Estimated mortality thresholds were then compared with observed growth and mortality rates. The maximum age of walleyes varied from 5 to 11 years across reservoirs. Age structure was dominated (???72%) by walleyes age 3 and younger in all reservoirs, corresponding to ages that were not yet vulnerable to harvest. Total annual mortality rates varied from 40.7% to 59.5% across reservoirs and averaged 51.1% overall (SE = 2.3). Analysis of mortality caps indicated that a management objective of 500 mm for the mean length of walleyes harvested by anglers was realistic for all reservoirs with a 457-mm minimum length limit but not for those with a 381-mm minimum length limit. For a 500-mm mean length objective to be realized for reservoirs with a 381-mm length limit, managers must either reduce mortality rates (e.g., through restrictive harvest regulations) or increase growth of walleyes. When the assumed objective was to maintain the mean length of harvested walleyes at current levels, the observed annual mortality rates were below the mortality cap for all reservoirs except one. Mortality caps also provided insight on management objectives expressed in terms of proportional stock density (PSD). Results indicated that a PSD objective of 20-40 was realistic for most reservoirs. This study provides important walleye mortality information that can be used for monitoring or for inclusion into

  19. Use of tiling array data and RNA secondary structure predictions to identify noncoding RNA genes

    DEFF Research Database (Denmark)

    Weile, Christian; Gardner, Paul P; Hedegaard, Mads M

    2007-01-01

    neuroblastoma cell line SK-N-AS. Using this strategy, we identify thousands of human candidate RNA genes. To further verify the expression of these genes, we focused on candidate genes that had a stable hairpin structures or a high level of covariance. Using northern blotting, we verify the expression of 2 out...

  20. Disease-associated mutations that alter the RNA structural ensemble.

    Directory of Open Access Journals (Sweden)

    Matthew Halvorsen

    2010-08-01

    Full Text Available Genome-wide association studies (GWAS often identify disease-associated mutations in intergenic and non-coding regions of the genome. Given the high percentage of the human genome that is transcribed, we postulate that for some observed associations the disease phenotype is caused by a structural rearrangement in a regulatory region of the RNA transcript. To identify such mutations, we have performed a genome-wide analysis of all known disease-associated Single Nucleotide Polymorphisms (SNPs from the Human Gene Mutation Database (HGMD that map to the untranslated regions (UTRs of a gene. Rather than using minimum free energy approaches (e.g. mFold, we use a partition function calculation that takes into consideration the ensemble of possible RNA conformations for a given sequence. We identified in the human genome disease-associated SNPs that significantly alter the global conformation of the UTR to which they map. For six disease-states (Hyperferritinemia Cataract Syndrome, beta-Thalassemia, Cartilage-Hair Hypoplasia, Retinoblastoma, Chronic Obstructive Pulmonary Disease (COPD, and Hypertension, we identified multiple SNPs in UTRs that alter the mRNA structural ensemble of the associated genes. Using a Boltzmann sampling procedure for sub-optimal RNA structures, we are able to characterize and visualize the nature of the conformational changes induced by the disease-associated mutations in the structural ensemble. We observe in several cases (specifically the 5' UTRs of FTL and RB1 SNP-induced conformational changes analogous to those observed in bacterial regulatory Riboswitches when specific ligands bind. We propose that the UTR and SNP combinations we identify constitute a "RiboSNitch," that is a regulatory RNA in which a specific SNP has a structural consequence that results in a disease phenotype. Our SNPfold algorithm can help identify RiboSNitches by leveraging GWAS data and an analysis of the mRNA structural ensemble.

  1. Structural analysis of closure cap barriers: A pre-test study for the Bentonite Mat Demonstration Project

    International Nuclear Information System (INIS)

    Gong, Chung; Pelfrey, J.R.

    1993-01-01

    The Bentonite Mat Demonstration Project (BMDP) is a field demonstration study to determine the construction/installation requirements, permeability, and subsidence performance characteristics of a composite barrier. The composite barrier will consist of on-site sandy-clay blanketed by a bentonite mat and a flexible High Density Polyethylene (HDPE) liner (also called flexible membrane liner). Construction of one control test pad and three bentonite test pads are planned. The control test pad will be used to establish baseline data. Underneath the composite clay cap is a four feet thick loose sand layer in which cavities will be created by evacuation of sand. The present work provides a mathematical model for the BMDP. The mathematical model will be used to simulate the mechanical and structural responses of the composite clay cap during the testing processes. Based upon engineering experience and technical references, a set of nominal soil parameters have been selected

  2. Network Properties of the Ensemble of RNA Structures

    Science.gov (United States)

    Clote, Peter; Bayegan, Amir

    2015-01-01

    We describe the first dynamic programming algorithm that computes the expected degree for the network, or graph G = (V, E) of all secondary structures of a given RNA sequence a = a 1, …, a n. Here, the nodes V correspond to all secondary structures of a, while an edge exists between nodes s, t if the secondary structure t can be obtained from s by adding, removing or shifting a base pair. Since secondary structure kinetics programs implement the Gillespie algorithm, which simulates a random walk on the network of secondary structures, the expected network degree may provide a better understanding of kinetics of RNA folding when allowing defect diffusion, helix zippering, and related conformation transformations. We determine the correlation between expected network degree, contact order, conformational entropy, and expected number of native contacts for a benchmarking dataset of RNAs. Source code is available at http://bioinformatics.bc.edu/clotelab/RNAexpNumNbors. PMID:26488894

  3. Landscape and variation of RNA secondary structure across the human transcriptome.

    OpenAIRE

    Wan, Y; Qu, K; Zhang, QC; Flynn, RA; Manor, O; Ouyang, Z; Zhang, J; Spitale, RC; Snyder, MP; Segal, E; Chang, HY

    2014-01-01

    In parallel to the genetic code for protein synthesis, a second layer of information is embedded in all RNA transcripts in the form of RNA structure. RNA structure influences practically every step in the gene expression program. However, the nature of most RNA structures or effects of sequence variation on structure are not known. Here we report the initial landscape and variation of RNA secondary structures (RSSs) in a human family trio (mother, father and their child). This provides a comp...

  4. Investigation of RNA structure in satellite panicum mosaic virus

    International Nuclear Information System (INIS)

    Makino, D.L.; Day, J.; Larson, S.B.; McPherson, A.

    2006-01-01

    Three new crystal forms of satellite panicum mosaic virus (SPMV) were grown and their structures solved from X-ray diffraction data using molecular replacement techniques. The crystals were grown under conditions of pH and ionic strength that were appreciably different then those used for the original structure determination. In rhombohedral crystals grown at pH 8.5 and low ionic strength PEG 3350 solutions, Fourier syntheses revealed segments, ten amino acid residues long, of amino-terminal polypeptides not previously seen, as well as masses of electron density within concavities on the interior of the capsid, which appeared in the neighborhoods of icosahedral five- and threefold axes. The densities were compatible with secondary structural domains of RNA, and they included a segment of double helical RNA of about four to five base pairs oriented, at least approximately, along the fivefold axes. The distribution of RNA observed for SPMV appears to be distinctly different than the encapsidated nucleic acid conformation previously suggested for another satellite virus, satellite tobacco mosaic virus. This study further shows that analysis of viruses in crystals grown under different chemical conditions may reveal additional information regarding the structure of encapsidated RNA

  5. A combinatorial enumeration problem of RNA secondary structures

    African Journals Online (AJOL)

    use

    2011-12-21

    Dec 21, 2011 ... connection between Discrete Mathematics and Compu- tational Molecular Biology (Chen et al, 2005; Hofacker et ... in Computational Molecular Biology. An RNA molecule is described by its sequences of bases ... Here, a mathematical definition of secondary structure is given (Stein and Waterman 1978).

  6. lncRNA Structure: Message to the Heart.

    Science.gov (United States)

    Fazal, Furqan M; Chang, Howard Y

    2016-10-06

    In this issue, Xue et al. (2016) describe the secondary structure of the heart-specific long non-coding RNA Braveheart, leading to the discovery of a short, asymmetric G-rich loop that controls cardiac lineage commitment by interacting with the transcription factor CNBP. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Accurate SHAPE-directed RNA secondary structure modeling, including pseudoknots.

    Science.gov (United States)

    Hajdin, Christine E; Bellaousov, Stanislav; Huggins, Wayne; Leonard, Christopher W; Mathews, David H; Weeks, Kevin M

    2013-04-02

    A pseudoknot forms in an RNA when nucleotides in a loop pair with a region outside the helices that close the loop. Pseudoknots occur relatively rarely in RNA but are highly overrepresented in functionally critical motifs in large catalytic RNAs, in riboswitches, and in regulatory elements of viruses. Pseudoknots are usually excluded from RNA structure prediction algorithms. When included, these pairings are difficult to model accurately, especially in large RNAs, because allowing this structure dramatically increases the number of possible incorrect folds and because it is difficult to search the fold space for an optimal structure. We have developed a concise secondary structure modeling approach that combines SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) experimental chemical probing information and a simple, but robust, energy model for the entropic cost of single pseudoknot formation. Structures are predicted with iterative refinement, using a dynamic programming algorithm. This melded experimental and thermodynamic energy function predicted the secondary structures and the pseudoknots for a set of 21 challenging RNAs of known structure ranging in size from 34 to 530 nt. On average, 93% of known base pairs were predicted, and all pseudoknots in well-folded RNAs were identified.

  8. Selective small-molecule inhibition of an RNA structural element

    Energy Technology Data Exchange (ETDEWEB)

    Howe, John A.; Wang, Hao; Fischmann, Thierry O.; Balibar, Carl J.; Xiao, Li; Galgoci, Andrew M.; Malinverni, Juliana C.; Mayhood, Todd; Villafania, Artjohn; Nahvi, Ali; Murgolo, Nicholas; Barbieri, Christopher M.; Mann, Paul A.; Carr, Donna; Xia, Ellen; Zuck, Paul; Riley, Dan; Painter, Ronald E.; Walker, Scott S.; Sherborne, Brad; de Jesus, Reynalda; Pan, Weidong; Plotkin, Michael A.; Wu, Jin; Rindgen, Diane; Cummings, John; Garlisi, Charles G.; Zhang, Rumin; Sheth, Payal R.; Gill, Charles J.; Tang, Haifeng; Roemer , Terry (Merck)

    2015-09-30

    Riboswitches are non-coding RNA structures located in messenger RNAs that bind endogenous ligands, such as a specific metabolite or ion, to regulate gene expression. As such, riboswitches serve as a novel, yet largely unexploited, class of emerging drug targets. Demonstrating this potential, however, has proven difficult and is restricted to structurally similar antimetabolites and semi-synthetic analogues of their cognate ligand, thus greatly restricting the chemical space and selectivity sought for such inhibitors. Here we report the discovery and characterization of ribocil, a highly selective chemical modulator of bacterial riboflavin riboswitches, which was identified in a phenotypic screen and acts as a structurally distinct synthetic mimic of the natural ligand, flavin mononucleotide, to repress riboswitch-mediated ribB gene expression and inhibit bacterial cell growth. Our findings indicate that non-coding RNA structural elements may be more broadly targeted by synthetic small molecules than previously expected.

  9. Secondary structure of 5S RNA: NMR experiments on RNA molecules partially labeled with Nitrogen-15

    International Nuclear Information System (INIS)

    Gewirth, D.T.; Abo, S.R.; Leontis, N.B.; Moore, P.B.

    1987-01-01

    A method has been found for reassembling fragment 1 of Escherichia coli 5S RNA from mixtures containing strand III (bases 69-87) and the complex consisting of strand II (bases 89-120) and strand IV (bases 1-11). The reassembled molecule is identical with unreconstituted fragment 1. With this technique, fragment 1 molecules have been constructed 15 N-labeled either in strand III or in the strand II-strand IV complex. Spectroscopic data obtained with these partially labeled molecules show that the terminal helix of 5S RNA includes the GU and GC base pairs at positions 9 and 10 which the standard model for 5S secondary structure predicts but that these base pairs are unstable both in the fragment and in native 5S RNA. The data also assign three resonances to the helix V region of the molecule (bases 70-77 and 99-106). None of these resonances has a normal chemical shift even though two of them correspond to AU or GU base pairs in the standard model. The implications of these findings for the authors understanding of the structure of 5S RNA and its complex with ribosomal protein L25 are discussed

  10. Secondary structural entropy in RNA switch (Riboswitch) identification.

    Science.gov (United States)

    Manzourolajdad, Amirhossein; Arnold, Jonathan

    2015-04-28

    RNA regulatory elements play a significant role in gene regulation. Riboswitches, a widespread group of regulatory RNAs, are vital components of many bacterial genomes. These regulatory elements generally function by forming a ligand-induced alternative fold that controls access to ribosome binding sites or other regulatory sites in RNA. Riboswitch-mediated mechanisms are ubiquitous across bacterial genomes. A typical class of riboswitch has its own unique structural and biological complexity, making de novo riboswitch identification a formidable task. Traditionally, riboswitches have been identified through comparative genomics based on sequence and structural homology. The limitations of structural-homology-based approaches, coupled with the assumption that there is a great diversity of undiscovered riboswitches, suggests the need for alternative methods for riboswitch identification, possibly based on features intrinsic to their structure. As of yet, no such reliable method has been proposed. We used structural entropy of riboswitch sequences as a measure of their secondary structural dynamics. Entropy values of a diverse set of riboswitches were compared to that of their mutants, their dinucleotide shuffles, and their reverse complement sequences under different stochastic context-free grammar folding models. Significance of our results was evaluated by comparison to other approaches, such as the base-pairing entropy and energy landscapes dynamics. Classifiers based on structural entropy optimized via sequence and structural features were devised as riboswitch identifiers and tested on Bacillus subtilis, Escherichia coli, and Synechococcus elongatus as an exploration of structural entropy based approaches. The unusually long untranslated region of the cotH in Bacillus subtilis, as well as upstream regions of certain genes, such as the sucC genes were associated with significant structural entropy values in genome-wide examinations. Various tests show that there

  11. Quantitative DMS mapping for automated RNA secondary structure inference

    OpenAIRE

    Cordero, Pablo; Kladwang, Wipapat; VanLang, Christopher C.; Das, Rhiju

    2012-01-01

    For decades, dimethyl sulfate (DMS) mapping has informed manual modeling of RNA structure in vitro and in vivo. Here, we incorporate DMS data into automated secondary structure inference using a pseudo-energy framework developed for 2'-OH acylation (SHAPE) mapping. On six non-coding RNAs with crystallographic models, DMS- guided modeling achieves overall false negative and false discovery rates of 9.5% and 11.6%, comparable or better than SHAPE-guided modeling; and non-parametric bootstrappin...

  12. Integrating chemical footprinting data into RNA secondary structure prediction.

    Directory of Open Access Journals (Sweden)

    Kourosh Zarringhalam

    Full Text Available Chemical and enzymatic footprinting experiments, such as shape (selective 2'-hydroxyl acylation analyzed by primer extension, yield important information about RNA secondary structure. Indeed, since the [Formula: see text]-hydroxyl is reactive at flexible (loop regions, but unreactive at base-paired regions, shape yields quantitative data about which RNA nucleotides are base-paired. Recently, low error rates in secondary structure prediction have been reported for three RNAs of moderate size, by including base stacking pseudo-energy terms derived from shape data into the computation of minimum free energy secondary structure. Here, we describe a novel method, RNAsc (RNA soft constraints, which includes pseudo-energy terms for each nucleotide position, rather than only for base stacking positions. We prove that RNAsc is self-consistent, in the sense that the nucleotide-specific probabilities of being unpaired in the low energy Boltzmann ensemble always become more closely correlated with the input shape data after application of RNAsc. From this mathematical perspective, the secondary structure predicted by RNAsc should be 'correct', in as much as the shape data is 'correct'. We benchmark RNAsc against the previously mentioned method for eight RNAs, for which both shape data and native structures are known, to find the same accuracy in 7 out of 8 cases, and an improvement of 25% in one case. Furthermore, we present what appears to be the first direct comparison of shape data and in-line probing data, by comparing yeast asp-tRNA shape data from the literature with data from in-line probing experiments we have recently performed. With respect to several criteria, we find that shape data appear to be more robust than in-line probing data, at least in the case of asp-tRNA.

  13. Stochastic sampling of the RNA structural alignment space.

    Science.gov (United States)

    Harmanci, Arif Ozgun; Sharma, Gaurav; Mathews, David H

    2009-07-01

    A novel method is presented for predicting the common secondary structures and alignment of two homologous RNA sequences by sampling the 'structural alignment' space, i.e. the joint space of their alignments and common secondary structures. The structural alignment space is sampled according to a pseudo-Boltzmann distribution based on a pseudo-free energy change that combines base pairing probabilities from a thermodynamic model and alignment probabilities from a hidden Markov model. By virtue of the implicit comparative analysis between the two sequences, the method offers an improvement over single sequence sampling of the Boltzmann ensemble. A cluster analysis shows that the samples obtained from joint sampling of the structural alignment space cluster more closely than samples generated by the single sequence method. On average, the representative (centroid) structure and alignment of the most populated cluster in the sample of structures and alignments generated by joint sampling are more accurate than single sequence sampling and alignment based on sequence alone, respectively. The 'best' centroid structure that is closest to the known structure among all the centroids is, on average, more accurate than structure predictions of other methods. Additionally, cluster analysis identifies, on average, a few clusters, whose centroids can be presented as alternative candidates. The source code for the proposed method can be downloaded at http://rna.urmc.rochester.edu.

  14. Crowdsourcing RNA structural alignments with an online computer game.

    Science.gov (United States)

    Waldispühl, Jérôme; Kam, Arthur; Gardner, Paul P

    2015-01-01

    The annotation and classification of ncRNAs is essential to decipher molecular mechanisms of gene regulation in normal and disease states. A database such as Rfam maintains alignments, consensus secondary structures, and corresponding annotations for RNA families. Its primary purpose is the automated, accurate annotation of non-coding RNAs in genomic sequences. However, the alignment of RNAs is computationally challenging, and the data stored in this database are often subject to improvements. Here, we design and evaluate Ribo, a human-computing game that aims to improve the accuracy of RNA alignments already stored in Rfam. We demonstrate the potential of our techniques and discuss the feasibility of large scale collaborative annotation and classification of RNA families.

  15. Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9).

    Science.gov (United States)

    Fu, Qinqin; Yuan, Y Adam

    2013-03-01

    Intensive research interest has focused on small RNA-processing machinery and the RNA-induced silencing complex (RISC), key cellular machines in RNAi pathways. However, the structural mechanism regarding RISC assembly, the primary step linking small RNA processing and RNA-mediated gene silencing, is largely unknown. Human RNA helicase A (DHX9) was reported to function as an RISC-loading factor, and such function is mediated mainly by its dsRNA-binding domains (dsRBDs). Here, we report the crystal structures of human RNA helicase A (RHA) dsRBD1 and dsRBD2 domains in complex with dsRNAs, respectively. Structural analysis not only reveals higher siRNA duplex-binding affinity displayed by dsRBD1, but also identifies a crystallographic dsRBD1 pair of physiological significance in cooperatively recognizing dsRNAs. Structural observations are further validated by isothermal titration calorimetric (ITC) assay. Moreover, co-immunoprecipitation (co-IP) assay coupled with mutagenesis demonstrated that both dsRBDs are required for RISC association, and such association is mediated by dsRNA. Hence, our structural and functional efforts have revealed a potential working model for siRNA recognition by RHA tandem dsRBDs, and together they provide direct structural insights into RISC assembly facilitated by RHA.

  16. Investigations on structural and optical properties of starch capped ZnS nanoparticles synthesized by microwave irradiation method

    Science.gov (United States)

    Lalithadevi, B.; Mohan Rao, K.; Ramananda, D.

    2018-05-01

    Following a green synthesis method, zinc sulfide (ZnS) nanoparticles were prepared by chemical co-precipitation technique using starch as capping agent. Microwave irradiation was used as heating source. X-ray diffraction studies indicated that nanopowders obtained were polycrystalline possessing ZnS simple cubic structure. Transmission electron microscopic studies indicated that starch limits the agglomeration by steric stabilization. Interaction between ZnS and starch was confirmed by Fourier transform infrared spectroscopy as well as Raman scattering studies. Quantum size effects were observed in optical absorption studies while quenching of defect states on nanoparticles was improved with increase in starch addition as indicated by photoluminescence spectra.

  17. Simulation study of HEMT structures with HfO2 cap layer for mitigating inverse piezoelectric effect related device failures

    Directory of Open Access Journals (Sweden)

    Deepthi Nagulapally

    2015-01-01

    Full Text Available The Inverse Piezoelectric Effect (IPE is thought to contribute to possible device failure of GaN High Electron Mobility Transistors (HEMTs. Here we focus on a simulation study to probe the possible mitigation of the IPE by reducing the internal electric fields and related elastic energy through the use of high-k materials. Inclusion of a HfO2 “cap layer” above the AlGaN barrier particularly with a partial mesa structure is shown to have potential advantages. Simulations reveal even greater reductions in the internal electric fields by using “field plates” in concert with high-k oxides.

  18. Random generation of RNA secondary structures according to native distributions

    Directory of Open Access Journals (Sweden)

    Nebel Markus E

    2011-10-01

    Full Text Available Abstract Background Random biological sequences are a topic of great interest in genome analysis since, according to a powerful paradigm, they represent the background noise from which the actual biological information must differentiate. Accordingly, the generation of random sequences has been investigated for a long time. Similarly, random object of a more complicated structure like RNA molecules or proteins are of interest. Results In this article, we present a new general framework for deriving algorithms for the non-uniform random generation of combinatorial objects according to the encoding and probability distribution implied by a stochastic context-free grammar. Briefly, the framework extends on the well-known recursive method for (uniform random generation and uses the popular framework of admissible specifications of combinatorial classes, introducing weighted combinatorial classes to allow for the non-uniform generation by means of unranking. This framework is used to derive an algorithm for the generation of RNA secondary structures of a given fixed size. We address the random generation of these structures according to a realistic distribution obtained from real-life data by using a very detailed context-free grammar (that models the class of RNA secondary structures by distinguishing between all known motifs in RNA structure. Compared to well-known sampling approaches used in several structure prediction tools (such as SFold ours has two major advantages: Firstly, after a preprocessing step in time O(n2 for the computation of all weighted class sizes needed, with our approach a set of m random secondary structures of a given structure size n can be computed in worst-case time complexity Om⋅n⋅ log(n while other algorithms typically have a runtime in O(m⋅n2. Secondly, our approach works with integer arithmetic only which is faster and saves us from all the discomforting details of using floating point arithmetic with

  19. Crystal structures of two eukaryotic nucleases involved in RNA metabolism

    DEFF Research Database (Denmark)

    Jonstrup, Anette Thyssen; Midtgaard, Søren Fuglsang; Van, Lan Bich

    RNA serves a number of functions in the cell: mRNAs are the carriers of information between gene and protein, tRNAs and rRNAs are involved in the synthesis of proteins, whereas a number of additional RNA species are responsible for other functions in the cell. The quality of the different RNAs...... RNAs. We have solved the structures of two nucleases involved in 3'-5' degradation of RNA; the S. pombe Pop2p and the S. cerevisiae Rrp6p. Pop2p is part of the main cytoplasmatic deadenylation complex in yeast, which also contains the nuclease Ccr4p. Deadenylation, where the poly(A)-tail is removed...... specific transcripts. Here, we present the crystal structure of the S. pombe Pop2p protein to 1.4 Å resolution. The high resolution structure provides a clear picture of the active site architecture. Structural alignment of single nucleotides and poly(A)-oligonucleotides from earlier co-crystal structures...

  20. Drifting field-aligned density structures in the night-side polar cap

    Czech Academy of Sciences Publication Activity Database

    Santolík, Ondřej; Persoon, A. M.; Gurnett, D. A.; Décréau, P. M. E.; Pickett, J. S.; Maršálek, O.; Maksimovic, M.; Cornilleau-Wehrlin, N.

    2005-01-01

    Roč. 32, - (2005), L06106-1 ISSN 0094-8276 R&D Projects: GA ČR(CZ) GA202/03/0832; GA MŠk ME 650; GA MŠk 1P05ME811 Grant - others: NASA (US) NAG5-9974; NASA (US) NNG04GB98G; NSF(US) 0307319; ESA PECS(XE) 98025 Institutional research plan: CEZ:AV0Z30420517 Keywords : Magnetospheric Physics * Plasma convection * Plasma waves and instabilities * Polar cap phenomena * Magnetospheric configuration and dynamics Subject RIV: BL - Plasma and Gas Discharge Physics Impact factor: 2.491, year: 2005

  1. A Structural Overview of RNA-Dependent RNA Polymerases from the Flaviviridae Family

    Directory of Open Access Journals (Sweden)

    Jiqin Wu

    2015-06-01

    Full Text Available RNA-dependent RNA polymerases (RdRPs from the Flaviviridae family are representatives of viral polymerases that carry out RNA synthesis through a de novo initiation mechanism. They share a ≈ 600-residue polymerase core that displays a canonical viral RdRP architecture resembling an encircled right hand with palm, fingers, and thumb domains surrounding the active site. Polymerase catalytic motifs A–E in the palm and motifs F/G in the fingers are shared by all viral RdRPs with sequence and/or structural conservations regardless of the mechanism of initiation. Different from RdRPs carrying out primer-dependent initiation, Flaviviridae and other de novo RdRPs utilize a priming element often integrated in the thumb domain to facilitate primer-independent initiation. Upon the transition to the elongation phase, this priming element needs to undergo currently unresolved conformational rearrangements to accommodate the growth of the template-product RNA duplex. In the genera of Flavivirus and Pestivirus, the polymerase module in the C-terminal part of the RdRP protein may be regulated in cis by the N-terminal region of the same polypeptide. Either being a methyltransferase in Flavivirus or a functionally unclarified module in Pestivirus, this region could play auxiliary roles for the canonical folding and/or the catalysis of the polymerase, through defined intra-molecular interactions.

  2. Comparative structural analysis of human DEAD-box RNA helicases.

    Directory of Open Access Journals (Sweden)

    Patrick Schütz

    2010-09-01

    Full Text Available DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.

  3. Comparative structural analysis of human DEAD-box RNA helicases.

    Science.gov (United States)

    Schütz, Patrick; Karlberg, Tobias; van den Berg, Susanne; Collins, Ruairi; Lehtiö, Lari; Högbom, Martin; Holmberg-Schiavone, Lovisa; Tempel, Wolfram; Park, Hee-Won; Hammarström, Martin; Moche, Martin; Thorsell, Ann-Gerd; Schüler, Herwig

    2010-09-30

    DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.

  4. A phase transition in energy-filtered RNA secondary structures

    DEFF Research Database (Denmark)

    Han, Hillary Siwei; reidys, Christian

    2012-01-01

    In this paper we study the effect of energy parameters on minimum free energy (mfe) RNA secondary structures. Employing a simplified combinatorial energy model, that is only dependent on the diagram representation and that is not sequence specific, we prove the following dichotomy result. Mfe...... this phase transition from a discrete limit to a central limit distribution and subsequently put our result into the context of quantifying the effect of sparsification of the folding of these respective mfe-structures. We show that the sparsification of realistic mfe-structures leads to a constant time...

  5. Fine structure of the magnetic spectrum of. cap alpha. -rays of ionium

    Energy Technology Data Exchange (ETDEWEB)

    Rosenblum, S; Valadares, M; Vial, J

    1948-11-22

    Using as source of Io--Th an electrolytic deposit on Pt made in 1945 (27% Io), the authors studied the magnetic spectrum of the ..cap alpha..-radiation of Io from long-exposure photographs (4 to 21 days) submitted to a microscopic counting. From a simultaneously obtained ..cap alpha..-spectrum of ThC a ratio was obtained which permitted the assigning of a value of 4682 +- 10 keV to the energy of the most rapid group of Io. This group is separated from another, of a similar order of intensity, by 69 keV. A still slower group seems to exist at 170 keV from the first one (cf Ward, Proc Cambridge Phil Soc 35 322(1939)). There are indications that the groups are of a complex nature, which points to the possible existence either of a soft gamma spectrum or of a complicated beta spectrum. The presence of Ra accumulated since 1945 is visible on the spectra obtained; in a few years it will be possible to determine directly, by the method used, the half life of Ra.

  6. Structural, optical, XPS and magnetic properties of Zn particles capped by ZnO nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Morozov, Iu.G., E-mail: yugmor@hotmail.com [Institute of Structural Macrokinetics and Materials Science, Russian Academy of Sciences, Academician Osipyan Street 8, Chernogolovka, Moscow Region 142432 (Russian Federation); Belousova, O.V. [Institute of Structural Macrokinetics and Materials Science, Russian Academy of Sciences, Academician Osipyan Street 8, Chernogolovka, Moscow Region 142432 (Russian Federation); Ortega, D., E-mail: daniel.ortega@imdea.org [Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA-Nanociencia), Cantoblanco 28049, Madrid (Spain); Mafina, M.-K., E-mail: m.k.mafina@qmul.ac.uk [School of Engineering and Materials Science, Queen Mary University of London, Mile End, Eng, 231, London E1 4NS (United Kingdom); Kuznetcov, M.V., E-mail: maxim1968@mail.ru [Department of Chemistry, Materials Chemistry Research Centre, University College London, 20 Gordon Street, London WC1H 0AJ (United Kingdom)

    2015-06-05

    Highlights: • Levitation-jet aerosol synthesis of Zn particles capped by ZnO nanoparticles (NPs). • TEM, XRD, UV–vis, FT-IR, Raman, XPS and magnetic characterization of the NPs. • Correlation between unit-cell volume of crystal lattice and maximum magnetization. - Abstract: Spherical zinc particles ranging from 42 to 760 nm in average size and capped with plate-like zinc oxide particles of 10–30 nm in sizes have been prepared by levitation-jet aerosol synthesis through condensation of zinc vapor in an inert/oxidizer gas flow. The nanoparticles have been characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), BET measurements, ultra violet visible (UV–vis) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, Raman spectroscopy, X-ray electron spectroscopy (XPS), superconducting quantum interference device (SQUID), and vibrating-sample magnetometer (VSM). Magnetic and XRD data indicate that the observed ferromagnetic ordering related to the changes in unit-cell volume of Zn in the Zn/ZnO interface of the nanoparticles. These results are in good correlation with the optical measurements data.

  7. NoFold: RNA structure clustering without folding or alignment.

    Science.gov (United States)

    Middleton, Sarah A; Kim, Junhyong

    2014-11-01

    Structures that recur across multiple different transcripts, called structure motifs, often perform a similar function-for example, recruiting a specific RNA-binding protein that then regulates translation, splicing, or subcellular localization. Identifying common motifs between coregulated transcripts may therefore yield significant insight into their binding partners and mechanism of regulation. However, as most methods for clustering structures are based on folding individual sequences or doing many pairwise alignments, this results in a tradeoff between speed and accuracy that can be problematic for large-scale data sets. Here we describe a novel method for comparing and characterizing RNA secondary structures that does not require folding or pairwise alignment of the input sequences. Our method uses the idea of constructing a distance function between two objects by their respective distances to a collection of empirical examples or models, which in our case consists of 1973 Rfam family covariance models. Using this as a basis for measuring structural similarity, we developed a clustering pipeline called NoFold to automatically identify and annotate structure motifs within large sequence data sets. We demonstrate that NoFold can simultaneously identify multiple structure motifs with an average sensitivity of 0.80 and precision of 0.98 and generally exceeds the performance of existing methods. We also perform a cross-validation analysis of the entire set of Rfam families, achieving an average sensitivity of 0.57. We apply NoFold to identify motifs enriched in dendritically localized transcripts and report 213 enriched motifs, including both known and novel structures. © 2014 Middleton and Kim; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  8. Problems connected with the use of oligonucleotide probes with a high degree of degeneracy. Identification of mRNA and of cDNA clones corresponding to the gene of the. cap alpha. -subunit of Na/sup +/, K/sup +/-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Petrukhin, K.E.; Grishin, A.V.; Arsenyan, S.G.; Broude, N.E.; Grinkevich, V.A.; Filippova, L.Yu.; Severtsova, I.V.; Modyanov, N.N.

    1986-10-01

    To identify and search for nucleotide sequences containing the structural part of the gene of the ..cap alpha..-subunit of Na/sup +/, K/sup +/-ATPase, 17-membered oligonucleotide probes corresponding to the peptide Lys-Asp-Ala-Phe-Gln-Asn have been synthesized. It has been shown that, with a 64-fold degeneracyd, the 17-membered probe is suitable only for the identification of a specific sequence in mRNA. To search for clones containing cDNA fragments, preliminary fractionation of the probes with the aid of HPLC or the resynthesis of groups of oligonucleotides with a lower degeneracy is necessary.

  9. Development of Interfacial Structure in a Confined Air-Water Cap-Turbulent and Churn-Turbulent Flow

    International Nuclear Information System (INIS)

    Sun, X.; Kim, S.; Cheng, L.; Ishii, M.; Beus, S.G.

    2001-01-01

    The objective of the present work is to study and model the interfacial structure development of air-water two-phase flow in a confined test section. Experiments of a total of 9 flow conditions in a cap-turbulent and churn-turbulent flow regimes are carried out in a vertical air-water upward two-phase flow experimental loop with a test section of 20-cm in width and 1-cm in gap. The miniaturized four-sensor conductivity probes are used to measure local two-phase parameters at three different elevations for each flow condition. The bubbles captured by the probes are categorized into two groups in view of the two-group interfacial area transport equation, i.e., spherical/distorted bubbles as Group 1 and cap/churn-turbulent bubbles as Group 2. The acquired parameters are time-averaged local void fraction, interfacial velocity, bubble number frequency, interfacial area concentration, and bubble Sauter mean diameter for both groups of bubbles. Also, the line-averaged and area-averaged data are presented and discussed. The comparisons of these parameters at different elevations demonstrate the development of interfacial structure along the flow direction due to bubble interactions

  10. Development of Interfacial Structure in a Confined Air-Water Cap-Turbulent and Churn-Turbulent Flow

    International Nuclear Information System (INIS)

    Xiaodong Sun; Seungjin Kim; Ling Cheng; Mamoru Ishii; Beus, Stephen G.

    2002-01-01

    The objective of the present work is to study and model the interfacial structure development of air-water two-phase flow in a confined test section. Experiments of a total of 9 flow conditions in cap-turbulent and churn-turbulent flow regimes are carried out in a vertical air-water upward two-phase flow experimental loop with a test section of 200-mm in width and 10-mm in gap. Miniaturized four-sensor conductivity probes are used to measure local two-phase parameters at three different elevations for each flow condition. The bubbles captured by the probes are categorized into two groups in view of the two-group interfacial area transport equation, i.e., spherical/distorted bubbles as Group 1 and cap/churn-turbulent bubbles as Group 2. The acquired parameters are time-averaged local void fraction, interfacial velocity, bubble number frequency, interfacial area concentration, and bubble Sauter mean diameter for both groups of bubbles. Also, the line-averaged and area-averaged data are presented and discussed. The comparisons of these parameters at different elevations demonstrate the development of interfacial structure along the flow direction due to bubble interactions. (authors)

  11. Evolution of Tertiary Structure of Viral RNA Dependent Polymerases

    Czech Academy of Sciences Publication Activity Database

    Černý, Jiří; Černá, B.; Valdés, James J.; Grubhoffer, Libor; Růžek, Daniel

    2014-01-01

    Roč. 9, č. 5 (2014), e96070 E-ISSN 1932-6203 R&D Projects: GA ČR GAP502/11/2116; GA ČR GAP302/12/2490; GA MŠk(CZ) EE2.3.30.0032 Institutional support: RVO:60077344 Keywords : Q-BETA replicase * C virus RNA * crystal structure Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.234, year: 2014

  12. Structural basis of genomic RNA (gRNA) dimerization and packaging determinants of mouse mammary tumor virus (MMTV).

    Science.gov (United States)

    Aktar, Suriya J; Vivet-Boudou, Valérie; Ali, Lizna M; Jabeen, Ayesha; Kalloush, Rawan M; Richer, Delphine; Mustafa, Farah; Marquet, Roland; Rizvi, Tahir A

    2014-11-14

    One of the hallmarks of retroviral life cycle is the efficient and specific packaging of two copies of retroviral gRNA in the form of a non-covalent RNA dimer by the assembling virions. It is becoming increasingly clear that the process of dimerization is closely linked with gRNA packaging, and in some retroviruses, the latter depends on the former. Earlier mutational analysis of the 5' end of the MMTV genome indicated that MMTV gRNA packaging determinants comprise sequences both within the 5' untranslated region (5' UTR) and the beginning of gag. The RNA secondary structure of MMTV gRNA packaging sequences was elucidated employing selective 2'hydroxyl acylation analyzed by primer extension (SHAPE). SHAPE analyses revealed the presence of a U5/Gag long-range interaction (U5/Gag LRI), not predicted by minimum free-energy structure predictions that potentially stabilizes the global structure of this region. Structure conservation along with base-pair covariations between different strains of MMTV further supported the SHAPE-validated model. The 5' region of the MMTV gRNA contains multiple palindromic (pal) sequences that could initiate intermolecular interaction during RNA dimerization. In vitro RNA dimerization, SHAPE analysis, and structure prediction approaches on a series of pal mutants revealed that MMTV RNA utilizes a palindromic point of contact to initiate intermolecular interactions between two gRNAs, leading to dimerization. This contact point resides within pal II (5' CGGCCG 3') at the 5' UTR and contains a canonical "GC" dyad and therefore likely constitutes the MMTV RNA dimerization initiation site (DIS). Further analyses of these pal mutants employing in vivo genetic approaches indicate that pal II, as well as pal sequences located in the primer binding site (PBS) are both required for efficient MMTV gRNA packaging. Employing structural prediction, biochemical, and genetic approaches, we show that pal II functions as a primary point of contact between

  13. A probabilistic model for the evolution of RNA structure

    Directory of Open Access Journals (Sweden)

    Holmes Ian

    2004-10-01

    Full Text Available Abstract Background For the purposes of finding and aligning noncoding RNA gene- and cis-regulatory elements in multiple-genome datasets, it is useful to be able to derive multi-sequence stochastic grammars (and hence multiple alignment algorithms systematically, starting from hypotheses about the various kinds of random mutation event and their rates. Results Here, we consider a highly simplified evolutionary model for RNA, called "The TKF91 Structure Tree" (following Thorne, Kishino and Felsenstein's 1991 model of sequence evolution with indels, which we have implemented for pairwise alignment as proof of principle for such an approach. The model, its strengths and its weaknesses are discussed with reference to four examples of functional ncRNA sequences: a riboswitch (guanine, a zipcode (nanos, a splicing factor (U4 and a ribozyme (RNase P. As shown by our visualisations of posterior probability matrices, the selected examples illustrate three different signatures of natural selection that are highly characteristic of ncRNA: (i co-ordinated basepair substitutions, (ii co-ordinated basepair indels and (iii whole-stem indels. Conclusions Although all three types of mutation "event" are built into our model, events of type (i and (ii are found to be better modeled than events of type (iii. Nevertheless, we hypothesise from the model's performance on pairwise alignments that it would form an adequate basis for a prototype multiple alignment and genefinding tool.

  14. SimRNA: a coarse-grained method for RNA folding simulations and 3D structure prediction.

    Science.gov (United States)

    Boniecki, Michal J; Lach, Grzegorz; Dawson, Wayne K; Tomala, Konrad; Lukasz, Pawel; Soltysinski, Tomasz; Rother, Kristian M; Bujnicki, Janusz M

    2016-04-20

    RNA molecules play fundamental roles in cellular processes. Their function and interactions with other biomolecules are dependent on the ability to form complex three-dimensional (3D) structures. However, experimental determination of RNA 3D structures is laborious and challenging, and therefore, the majority of known RNAs remain structurally uncharacterized. Here, we present SimRNA: a new method for computational RNA 3D structure prediction, which uses a coarse-grained representation, relies on the Monte Carlo method for sampling the conformational space, and employs a statistical potential to approximate the energy and identify conformations that correspond to biologically relevant structures. SimRNA can fold RNA molecules using only sequence information, and, on established test sequences, it recapitulates secondary structure with high accuracy, including correct prediction of pseudoknots. For modeling of complex 3D structures, it can use additional restraints, derived from experimental or computational analyses, including information about secondary structure and/or long-range contacts. SimRNA also can be used to analyze conformational landscapes and identify potential alternative structures. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Structural and functional basis for RNA cleavage by Ire1

    Directory of Open Access Journals (Sweden)

    Stroud Robert M

    2011-07-01

    Full Text Available Abstract Background The unfolded protein response (UPR controls the protein folding capacity of the endoplasmic reticulum (ER. Central to this signaling pathway is the ER-resident bifunctional transmembrane kinase/endoribonuclease Ire1. The endoribonuclease (RNase domain of Ire1 initiates a non-conventional mRNA splicing reaction, leading to the production of a transcription factor that controls UPR target genes. The mRNA splicing reaction is an obligatory step of Ire1 signaling, yet its mechanism has remained poorly understood due to the absence of substrate-bound crystal structures of Ire1, the lack of structural similarity between Ire1 and other RNases, and a scarcity of quantitative enzymological data. Here, we experimentally define the active site of Ire1 RNase and quantitatively evaluate the contribution of the key active site residues to catalysis. Results This analysis and two new crystal structures suggest that Ire1 RNase uses histidine H1061 and tyrosine Y1043 as the general acid-general base pair contributing ≥ 7.6 kcal/mol and 1.4 kcal/mol to transition state stabilization, respectively, and asparagine N1057 and arginine R1056 for coordination of the scissile phosphate. Investigation of the stem-loop recognition revealed that additionally to the stem-loops derived from the classic Ire1 substrates HAC1 and Xbp1 mRNA, Ire1 can site-specifically and rapidly cleave anticodon stem-loop (ASL of unmodified tRNAPhe, extending known substrate specificity of Ire1 RNase. Conclusions Our data define the catalytic center of Ire1 RNase and suggest a mechanism of RNA cleavage: each RNase monomer apparently contains a separate catalytic apparatus for RNA cleavage, whereas two RNase subunits contribute to RNA stem-loop docking. Conservation of the key residues among Ire1 homologues suggests that the mechanism elucidated here for yeast Ire1 applies to Ire1 in metazoan cells, and to the only known Ire1 homologue RNase L.

  16. Structural basis for m7G recognition and 2'-O-methyl discrimination in capped RNAs by the innate immune receptor RIG-I

    Energy Technology Data Exchange (ETDEWEB)

    Devarkar, Swapnil C.; Wang, Chen; Miller, Matthew T.; Ramanathan, Anand; Jiang, Fuguo; Khan, Abdul G.; Patel, Smita S.; Marcotrigiano, Joseph

    2016-01-05

    The cytosolic innate immune receptor Retinoic Acid Inducible Gene-I (RIG-I) is the principal detector of pathogenic RNAs carrying a 5'-triphosphate (5'ppp). Self RNAs like mRNAs evade recognition by RIG-I due to posttranscriptional modifications like 5'-end capping with 7-methyl guanosine (m7G) and 2'-O-methylation of 5'-end nucleotides. Viruses have also evolved mechanisms to mimic these modifications, which in part is believed to aid in immune evasion. Currently, it is unclear how these modifications modulate RIG-I recognition. This paper provides structural and mechanistic insights into the roles of the m7G cap and 2'-O-methylation in RIG-I evasion. We show that RIG-I accommodates the m7G base while maintaining the 5'ppp contacts and can recognize Cap-0 RNAs but not Cap-1.

  17. Fine-tuning structural RNA alignments in the twilight zone

    Directory of Open Access Journals (Sweden)

    Schirmer Stefanie

    2010-04-01

    Full Text Available Abstract Background A widely used method to find conserved secondary structure in RNA is to first construct a multiple sequence alignment, and then fold the alignment, optimizing a score based on thermodynamics and covariance. This method works best around 75% sequence similarity. However, in a "twilight zone" below 55% similarity, the sequence alignment tends to obscure the covariance signal used in the second phase. Therefore, while the overall shape of the consensus structure may still be found, the degree of conservation cannot be estimated reliably. Results Based on a combination of available methods, we present a method named planACstar for improving structure conservation in structural alignments in the twilight zone. After constructing a consensus structure by alignment folding, planACstar abandons the original sequence alignment, refolds the sequences individually, but consistent with the consensus, aligns the structures, irrespective of sequence, by a pure structure alignment method, and derives an improved sequence alignment from the alignment of structures, to be re-submitted to alignment folding, etc.. This circle may be iterated as long as structural conservation improves, but normally, one step suffices. Conclusions Employing the tools ClustalW, RNAalifold, and RNAforester, we find that for sequences with 30-55% sequence identity, structural conservation can be improved by 10% on average, with a large variation, measured in terms of RNAalifold's own criterion, the structure conservation index.

  18. Prediction of RNA secondary structure using generalized centroid estimators.

    Science.gov (United States)

    Hamada, Michiaki; Kiryu, Hisanori; Sato, Kengo; Mituyama, Toutai; Asai, Kiyoshi

    2009-02-15

    Recent studies have shown that the methods for predicting secondary structures of RNAs on the basis of posterior decoding of the base-pairing probabilities has an advantage with respect to prediction accuracy over the conventionally utilized minimum free energy methods. However, there is room for improvement in the objective functions presented in previous studies, which are maximized in the posterior decoding with respect to the accuracy measures for secondary structures. We propose novel estimators which improve the accuracy of secondary structure prediction of RNAs. The proposed estimators maximize an objective function which is the weighted sum of the expected number of the true positives and that of the true negatives of the base pairs. The proposed estimators are also improved versions of the ones used in previous works, namely CONTRAfold for secondary structure prediction from a single RNA sequence and McCaskill-MEA for common secondary structure prediction from multiple alignments of RNA sequences. We clarify the relations between the proposed estimators and the estimators presented in previous works, and theoretically show that the previous estimators include additional unnecessary terms in the evaluation measures with respect to the accuracy. Furthermore, computational experiments confirm the theoretical analysis by indicating improvement in the empirical accuracy. The proposed estimators represent extensions of the centroid estimators proposed in Ding et al. and Carvalho and Lawrence, and are applicable to a wide variety of problems in bioinformatics. Supporting information and the CentroidFold software are available online at: http://www.ncrna.org/software/centroidfold/.

  19. Cradle Cap (For Parents)

    Science.gov (United States)

    ... Safe Videos for Educators Search English Español Cradle Cap (Infantile Seborrheic Dermatitis) KidsHealth / For Parents / Cradle Cap ( ... many babies develop called cradle cap. About Cradle Cap Cradle cap is the common term for seborrheic ...

  20. Structural basis underlying CAC RNA recognition by the RRM domain of dimeric RNA-binding protein RBPMS

    Energy Technology Data Exchange (ETDEWEB)

    Teplova, Marianna; Farazi, Thalia A.; Tuschl, Thomas; Patel, Dinshaw J.

    2015-09-08

    Abstract

    RNA-binding protein with multiple splicing (designated RBPMS) is a higher vertebrate mRNA-binding protein containing a single RNA recognition motif (RRM). RBPMS has been shown to be involved in mRNA transport, localization and stability, with key roles in axon guidance, smooth muscle plasticity, as well as regulation of cancer cell proliferation and migration. We report on structure-function studies of the RRM domain of RBPMS bound to a CAC-containing single-stranded RNA. These results provide insights into potential topologies of complexes formed by the RBPMS RRM domain and the tandem CAC repeat binding sites as detected by photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation. These studies establish that the RRM domain of RBPMS forms a symmetrical dimer in the free state, with each monomer binding sequence-specifically to all three nucleotides of a CAC segment in the RNA bound state. Structure-guided mutations within the dimerization and RNA-binding interfaces of RBPMS RRM on RNA complex formation resulted in both disruption of dimerization and a decrease in RNA-binding affinity as observed by size exclusion chromatography and isothermal titration calorimetry. As anticipated from biochemical binding studies, over-expression of dimerization or RNA-binding mutants of Flag-HA-tagged RBPMS were no longer able to track with stress granules in HEK293 cells, thereby documenting the deleterious effects of such mutationsin vivo.

  1. The identification and functional annotation of RNA structures conserved in vertebrates

    DEFF Research Database (Denmark)

    Seemann, Ernst Stefan; Mirza, Aashiq Hussain; Hansen, Claus

    2017-01-01

    Structured elements of RNA molecules are essential in, e.g., RNA stabilization, localization and protein interaction, and their conservation across species suggests a common functional role. We computationally screened vertebrate genomes for Conserved RNA Structures (CRSs), leveraging structure-b......-structured counterparts. Our findings of transcribed uncharacterized regulatory regions that contain CRSs support their RNA-mediated functionality.......Structured elements of RNA molecules are essential in, e.g., RNA stabilization, localization and protein interaction, and their conservation across species suggests a common functional role. We computationally screened vertebrate genomes for Conserved RNA Structures (CRSs), leveraging structure......-based, rather than sequence-based, alignments. After careful correction for sequence identity and GC content, we predict ~516k human genomic regions containing CRSs. We find that a substantial fraction of human-mouse CRS regions (i) co-localize consistently with binding sites of the same RNA binding proteins...

  2. RNA Secondary Structure Prediction by Using Discrete Mathematics: An Interdisciplinary Research Experience for Undergraduate Students

    Science.gov (United States)

    Ellington, Roni; Wachira, James; Nkwanta, Asamoah

    2010-01-01

    The focus of this Research Experience for Undergraduates (REU) project was on RNA secondary structure prediction by using a lattice walk approach. The lattice walk approach is a combinatorial and computational biology method used to enumerate possible secondary structures and predict RNA secondary structure from RNA sequences. The method uses…

  3. ConStruct: Improved construction of RNA consensus structures

    Directory of Open Access Journals (Sweden)

    Steger Gerhard

    2008-04-01

    Full Text Available Abstract Background Aligning homologous non-coding RNAs (ncRNAs correctly in terms of sequence and structure is an unresolved problem, due to both mathematical complexity and imperfect scoring functions. High quality alignments, however, are a prerequisite for most consensus structure prediction approaches, homology searches, and tools for phylogeny inference. Automatically created ncRNA alignments often need manual corrections, yet this manual refinement is tedious and error-prone. Results We present an extended version of CONSTRUCT, a semi-automatic, graphical tool suitable for creating RNA alignments correct in terms of both consensus sequence and consensus structure. To this purpose CONSTRUCT combines sequence alignment, thermodynamic data and various measures of covariation. One important feature is that the user is guided during the alignment correction step by a consensus dotplot, which displays all thermodynamically optimal base pairs and the corresponding covariation. Once the initial alignment is corrected, optimal and suboptimal secondary structures as well as tertiary interaction can be predicted. We demonstrate CONSTRUCT's ability to guide the user in correcting an initial alignment, and show an example for optimal secondary consensus structure prediction on very hard to align SECIS elements. Moreover we use CONSTRUCT to predict tertiary interactions from sequences of the internal ribosome entry site of CrP-like viruses. In addition we show that alignments specifically designed for benchmarking can be easily be optimized using CONSTRUCT, although they share very little sequence identity. Conclusion CONSTRUCT's graphical interface allows for an easy alignment correction based on and guided by predicted and known structural constraints. It combines several algorithms for prediction of secondary consensus structure and even tertiary interactions. The CONSTRUCT package can be downloaded from the URL listed in the Availability and

  4. Improved measurements of RNA structure conservation with generalized centroid estimators

    Directory of Open Access Journals (Sweden)

    Yohei eOkada

    2011-08-01

    Full Text Available Identification of non-protein-coding RNAs (ncRNAs in genomes is acrucial task for not only molecular cell biology but alsobioinformatics. Secondary structures of ncRNAs are employed as a keyfeature of ncRNA analysis since biological functions of ncRNAs aredeeply related to their secondary structures. Although the minimumfree energy (MFE structure of an RNA sequence is regarded as the moststable structure, MFE alone could not be an appropriate measure foridentifying ncRNAs since the free energy is heavily biased by thenucleotide composition. Therefore, instead of MFE itself, severalalternative measures for identifying ncRNAs have been proposed such asthe structure conservation index (SCI and the base pair distance(BPD, both of which employ MFE structures. However, thesemeasurements are unfortunately not suitable for identifying ncRNAs insome cases including the genome-wide search and incur high falsediscovery rate. In this study, we propose improved measurements basedon SCI and BPD, applying generalized centroid estimators toincorporate the robustness against low quality multiple alignments.Our experiments show that our proposed methods achieve higher accuracythan the original SCI and BPD for not only human-curated structuralalignments but also low quality alignments produced by CLUSTALW. Furthermore, the centroid-based SCI on CLUSTAL W alignments is moreaccurate than or comparable with that of the original SCI onstructural alignments generated with RAF, a high quality structuralaligner, for which two-fold expensive computational time is requiredon average. We conclude that our methods are more suitable forgenome-wide alignments which are of low quality from the point of viewon secondary structures than the original SCI and BPD.

  5. Supramolecular Self-Assembly of Histidine-Capped-Dialkoxy-Anthracene: A Visible Light Triggered Platform for facile siRNA Delivery

    KAUST Repository

    Patil, Sachin; Moosa, Basem; Alsaiari, Shahad; Alamoudi, Kholod; Alshamsan, Aws; Almailk, Abdulaziz; Adil, Karim; Eddaoudi, Mohamed; Khashab, Niveen M.

    2016-01-01

    Supramolecular self-assembly of histidine-capped-dialkoxy-anthracene (HDA) results in the formation of light responsive nanostructures.Single-crystal X-ray diffraction analysis of HDA shows two types of hydrogen bonding. The first hydrogen bond

  6. Crystal Structure of Serine Racemase that Produces Neurotransmitter font-variant:small-caps">d-Serine for Stimulation of the NMDA Receptor

    Science.gov (United States)

    Goto, Masaru

    font-variant:small-caps">d-Serine is an endogenous coagonist for the N-methyl-font-variant:small-caps">d-aspartate receptor and is involved in excitatory neurotransmission in the brain. Mammalian pyridoxal 5’-phosphate-dependent serine racemase, which is localized in the mammalian brain, catalyzes the racemization of font-variant:small-caps">l-serine to yield font-variant:small-caps">d-serine and vice versa. We have determined the structures of three forms of the mammalian enzyme homolog from Schizosaccharomyces pombe. Lys57 and Ser82 located on the protein and solvent sides, respectively, with respect to the cofactor plane, are acid-base catalysts that shuttle protons to the substrate. The modified enzyme, which has a unique lysino-font-variant:small-caps">d-alanyl residue at the active site, also binds the substrate serine in the active site, suggesting that the lysino-font-variant:small-caps">d-alanyl residue acts as a catalytic base in the same manner as Lys57 of the wild type enzyme.

  7. DNA structure in human RNA polymerase II promoters

    DEFF Research Database (Denmark)

    Pedersen, Anders Gorm; Baldi, Pierre; Chauvin, Yves

    1998-01-01

    with a very low level of sequence similarity. The sequences, which include both TATA-containing and TATA-less promoters, are aligned by hidden Markov models. Using three different models of sequence-derived DNA bendability, the aligned promoters display a common structural profile with bendability being low...... protein in a manner reminiscent of DNA in a nucleosome. This notion is further supported by the finding that the periodic bendability is caused mainly by the complementary triplet pairs CAG/CTG and GGC/GCC, which previously have been found to correlate with nucleosome positioning. We present models where......The fact that DNA three-dimensional structure is important for transcriptional regulation begs the question of whether eukaryotic promoters contain general structural features independently of what genes they control. We present an analysis of a large set of human RNA polymerase II promoters...

  8. Primary structure, gene organization and polypeptide expression of poliovirus RNA

    Energy Technology Data Exchange (ETDEWEB)

    Kitamura, N. (State Univ. of New York, Stony Brook); Semler, B.L.; Rothberg, P.G.

    1981-06-18

    The primary structure of the poliovirus genome has been determined. The RNA molecule is 7433 nucleotides long, polyadenylated at the 3' terminus, and covalently linked to a small protein (VPg) at the 5' terminus. An open reading frame of 2207 consecutive triplets spans over 89% of the nucleotide sequence and codes for the viral polyprotein NCVPOO. Twelve viral polypeptides have been mapped by amino acid sequence analysis and were found to be proteolytic cleavage products of the polyprotein, cleavages occurring predominantly at Gln-Gly pairs.

  9. Landscape and variation of RNA secondary structure across the human transcriptome.

    Science.gov (United States)

    Wan, Yue; Qu, Kun; Zhang, Qiangfeng Cliff; Flynn, Ryan A; Manor, Ohad; Ouyang, Zhengqing; Zhang, Jiajing; Spitale, Robert C; Snyder, Michael P; Segal, Eran; Chang, Howard Y

    2014-01-30

    In parallel to the genetic code for protein synthesis, a second layer of information is embedded in all RNA transcripts in the form of RNA structure. RNA structure influences practically every step in the gene expression program. However, the nature of most RNA structures or effects of sequence variation on structure are not known. Here we report the initial landscape and variation of RNA secondary structures (RSSs) in a human family trio (mother, father and their child). This provides a comprehensive RSS map of human coding and non-coding RNAs. We identify unique RSS signatures that demarcate open reading frames and splicing junctions, and define authentic microRNA-binding sites. Comparison of native deproteinized RNA isolated from cells versus refolded purified RNA suggests that the majority of the RSS information is encoded within RNA sequence. Over 1,900 transcribed single nucleotide variants (approximately 15% of all transcribed single nucleotide variants) alter local RNA structure. We discover simple sequence and spacing rules that determine the ability of point mutations to impact RSSs. Selective depletion of 'riboSNitches' versus structurally synonymous variants at precise locations suggests selection for specific RNA shapes at thousands of sites, including 3' untranslated regions, binding sites of microRNAs and RNA-binding proteins genome-wide. These results highlight the potentially broad contribution of RNA structure and its variation to gene regulation.

  10. Statistical properties of thermodynamically predicted RNA secondary structures in viral genomes

    Science.gov (United States)

    Spanò, M.; Lillo, F.; Miccichè, S.; Mantegna, R. N.

    2008-10-01

    By performing a comprehensive study on 1832 segments of 1212 complete genomes of viruses, we show that in viral genomes the hairpin structures of thermodynamically predicted RNA secondary structures are more abundant than expected under a simple random null hypothesis. The detected hairpin structures of RNA secondary structures are present both in coding and in noncoding regions for the four groups of viruses categorized as dsDNA, dsRNA, ssDNA and ssRNA. For all groups, hairpin structures of RNA secondary structures are detected more frequently than expected for a random null hypothesis in noncoding rather than in coding regions. However, potential RNA secondary structures are also present in coding regions of dsDNA group. In fact, we detect evolutionary conserved RNA secondary structures in conserved coding and noncoding regions of a large set of complete genomes of dsDNA herpesviruses.

  11. Death cap

    DEFF Research Database (Denmark)

    Rudbæk, Torsten R; Kofoed, Pernille Bouteloup; Bove, Jeppe

    2014-01-01

    Death cap (Amanita phalloides) is commonly found and is one of the five most toxic fungi in Denmark. Toxicity is due to amatoxin, and poisoning is a serious medical condition, causing organ failure with potential fatal outcome. Acknowledgement and clarification of exposure, symptomatic and focused...

  12. Modelling the structure of a ceRNA-theoretical, bipartite microRNA-mRNA interaction network regulating intestinal epithelial cellular pathways using R programming.

    Science.gov (United States)

    Robinson, J M; Henderson, W A

    2018-01-12

    We report a method using functional-molecular databases and network modelling to identify hypothetical mRNA-miRNA interaction networks regulating intestinal epithelial barrier function. The model forms a data-analysis component of our cell culture experiments, which produce RNA expression data from Nanostring Technologies nCounter ® system. The epithelial tight-junction (TJ) and actin cytoskeleton interact as molecular components of the intestinal epithelial barrier. Upstream regulation of TJ-cytoskeleton interaction is effected by the Rac/Rock/Rho signaling pathway and other associated pathways which may be activated or suppressed by extracellular signaling from growth factors, hormones, and immune receptors. Pathway activations affect epithelial homeostasis, contributing to degradation of the epithelial barrier associated with osmotic dysregulation, inflammation, and tumor development. The complexity underlying miRNA-mRNA interaction networks represents a roadblock for prediction and validation of competing-endogenous RNA network function. We developed a network model to identify hypothetical co-regulatory motifs in a miRNA-mRNA interaction network related to epithelial function. A mRNA-miRNA interaction list was generated using KEGG and miRWalk2.0 databases. R-code was developed to quantify and visualize inherent network structures. We identified a sub-network with a high number of shared, targeting miRNAs, of genes associated with cellular proliferation and cancer, including c-MYC and Cyclin D.

  13. Atomic-resolution structure of the CAP-Gly domain of dynactin on polymeric microtubules determined by magic angle spinning NMR spectroscopy.

    Science.gov (United States)

    Yan, Si; Guo, Changmiao; Hou, Guangjin; Zhang, Huilan; Lu, Xingyu; Williams, John Charles; Polenova, Tatyana

    2015-11-24

    Microtubules and their associated proteins perform a broad array of essential physiological functions, including mitosis, polarization and differentiation, cell migration, and vesicle and organelle transport. As such, they have been extensively studied at multiple levels of resolution (e.g., from structural biology to cell biology). Despite these efforts, there remain significant gaps in our knowledge concerning how microtubule-binding proteins bind to microtubules, how dynamics connect different conformational states, and how these interactions and dynamics affect cellular processes. Structures of microtubule-associated proteins assembled on polymeric microtubules are not known at atomic resolution. Here, we report a structure of the cytoskeleton-associated protein glycine-rich (CAP-Gly) domain of dynactin motor on polymeric microtubules, solved by magic angle spinning NMR spectroscopy. We present the intermolecular interface of CAP-Gly with microtubules, derived by recording direct dipolar contacts between CAP-Gly and tubulin using double rotational echo double resonance (dREDOR)-filtered experiments. Our results indicate that the structure adopted by CAP-Gly varies, particularly around its loop regions, permitting its interaction with multiple binding partners and with the microtubules. To our knowledge, this study reports the first atomic-resolution structure of a microtubule-associated protein on polymeric microtubules. Our approach lays the foundation for atomic-resolution structural analysis of other microtubule-associated motors.

  14. Effects of GaN capping on the structural and the optical properties of InN nanostructures grown by using MOCVD

    International Nuclear Information System (INIS)

    Sun, Yuanping; Cho, Yonghoon; Wang, Hui; Wang, Lili; Zhang, Shuming; Yang, Hui

    2010-01-01

    InN nanostructures with and without GaN capping layers were grown by using metal-organic chemical vapor deposition. Morphological, structural, and optical properties were systematically studied by using atomic force microscopy, X-ray diffraction (XRD) and temperature-dependent photoluminescence (PL). XRD results show that an InGaN structure is formed for the sample with a GaN capping layer, which will reduce the quality and the IR PL emission of the InN. The lower emission peak at ∼0.7 eV was theoretically fitted and assigned as the band edge emission of InN. Temperature-dependent PL shows a good quantum efficiency for the sample without a GaN capping layers; this corresponds to a lower density of dislocations and a small activation energy.

  15. A Comparative Taxonomy of Parallel Algorithms for RNA Secondary Structure Prediction

    Science.gov (United States)

    Al-Khatib, Ra’ed M.; Abdullah, Rosni; Rashid, Nur’Aini Abdul

    2010-01-01

    RNA molecules have been discovered playing crucial roles in numerous biological and medical procedures and processes. RNA structures determination have become a major problem in the biology context. Recently, computer scientists have empowered the biologists with RNA secondary structures that ease an understanding of the RNA functions and roles. Detecting RNA secondary structure is an NP-hard problem, especially in pseudoknotted RNA structures. The detection process is also time-consuming; as a result, an alternative approach such as using parallel architectures is a desirable option. The main goal in this paper is to do an intensive investigation of parallel methods used in the literature to solve the demanding issues, related to the RNA secondary structure prediction methods. Then, we introduce a new taxonomy for the parallel RNA folding methods. Based on this proposed taxonomy, a systematic and scientific comparison is performed among these existing methods. PMID:20458364

  16. Crystallization and X-ray crystallographic analysis of the cap-binding domain of influenza A virus H1N1 polymerase subunit PB2

    International Nuclear Information System (INIS)

    Liu, Yong; Meng, Geng; Luo, Ming; Zheng, Xiaofeng

    2013-01-01

    Substrate-free cap-binding domain of influenza A virus H1N1 polymerase subunit PB2 has been crystallized to show the structural details and clarify whether obvious conformational changes exist between the substrate-free and substrate-bound cap-binding domain. PB2 is one of the subunits of the influenza virus heterotrimeric polymerase. By its cap-binding domain (PB2 cap ), PB2 captures the 5′ cap of the host pre-mRNA to generate a capped 5′ oligonucleotide primer for virus transcription. The crystal structure of influenza A virus H3N2 PB2 cap with bound cap analogue m 7 GTP has been reported previously. To show the substrate-free structural details of PB2 cap and clarify whether obvious conformational changes exist between the substrate-free and substrate-bound cap-binding domain, we have successfully obtained the crystal of substrate-free H1N1 PB2 cap . The crystal of H1N1 PB2 cap diffracted to a high resolution of 1.32 Å. The crystal symmetry belongs to space group P1 with unit-cell parameters a = 29.49, b = 37.04, c = 38.33 Å, α = 71.10, β = 69.84, γ = 75.85°. There is one molecule in the asymmetric unit

  17. A semi-supervised learning approach for RNA secondary structure prediction.

    Science.gov (United States)

    Yonemoto, Haruka; Asai, Kiyoshi; Hamada, Michiaki

    2015-08-01

    RNA secondary structure prediction is a key technology in RNA bioinformatics. Most algorithms for RNA secondary structure prediction use probabilistic models, in which the model parameters are trained with reliable RNA secondary structures. Because of the difficulty of determining RNA secondary structures by experimental procedures, such as NMR or X-ray crystal structural analyses, there are still many RNA sequences that could be useful for training whose secondary structures have not been experimentally determined. In this paper, we introduce a novel semi-supervised learning approach for training parameters in a probabilistic model of RNA secondary structures in which we employ not only RNA sequences with annotated secondary structures but also ones with unknown secondary structures. Our model is based on a hybrid of generative (stochastic context-free grammars) and discriminative models (conditional random fields) that has been successfully applied to natural language processing. Computational experiments indicate that the accuracy of secondary structure prediction is improved by incorporating RNA sequences with unknown secondary structures into training. To our knowledge, this is the first study of a semi-supervised learning approach for RNA secondary structure prediction. This technique will be useful when the number of reliable structures is limited. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. WhiteCap System, structural analysis reports. Progress report, April 1995--June 1995

    Energy Technology Data Exchange (ETDEWEB)

    Chai, Y.H.; Romstad, K.M. [Univ. of California, Davis, CA (United States)

    1995-04-27

    The authors have completed an engineering investigation of the CoolRoof System as applicable to typical building construction in the Sacramento Valley. The purpose of this investigation is to provide structural engineering recommendations regarding structural modifications to typical building construction required by CoolRoof. This report presents the results of our investigation.

  19. Fast flexible modeling of RNA structure using internal coordinates.

    Science.gov (United States)

    Flores, Samuel Coulbourn; Sherman, Michael A; Bruns, Christopher M; Eastman, Peter; Altman, Russ Biagio

    2011-01-01

    Modeling the structure and dynamics of large macromolecules remains a critical challenge. Molecular dynamics (MD) simulations are expensive because they model every atom independently, and are difficult to combine with experimentally derived knowledge. Assembly of molecules using fragments from libraries relies on the database of known structures and thus may not work for novel motifs. Coarse-grained modeling methods have yielded good results on large molecules but can suffer from difficulties in creating more detailed full atomic realizations. There is therefore a need for molecular modeling algorithms that remain chemically accurate and economical for large molecules, do not rely on fragment libraries, and can incorporate experimental information. RNABuilder works in the internal coordinate space of dihedral angles and thus has time requirements proportional to the number of moving parts rather than the number of atoms. It provides accurate physics-based response to applied forces, but also allows user-specified forces for incorporating experimental information. A particular strength of RNABuilder is that all Leontis-Westhof basepairs can be specified as primitives by the user to be satisfied during model construction. We apply RNABuilder to predict the structure of an RNA molecule with 160 bases from its secondary structure, as well as experimental information. Our model matches the known structure to 10.2 Angstroms RMSD and has low computational expense.

  20. Impact of target mRNA structure on siRNA silencing efficiency: A large-scale study.

    Science.gov (United States)

    Gredell, Joseph A; Berger, Angela K; Walton, S Patrick

    2008-07-01

    The selection of active siRNAs is generally based on identifying siRNAs with certain sequence and structural properties. However, the efficiency of RNA interference has also been shown to depend on the structure of the target mRNA, primarily through studies using exogenous transcripts with well-defined secondary structures in the vicinity of the target sequence. While these studies provide a means for examining the impact of target sequence and structure independently, the predicted secondary structures for these transcripts are often not reflective of structures that form in full-length, native mRNAs where interactions can occur between relatively remote segments of the mRNAs. Here, using a combination of experimental results and analysis of a large dataset, we demonstrate that the accessibility of certain local target structures on the mRNA is an important determinant in the gene silencing ability of siRNAs. siRNAs targeting the enhanced green fluorescent protein were chosen using a minimal siRNA selection algorithm followed by classification based on the predicted minimum free energy structures of the target transcripts. Transfection into HeLa and HepG2 cells revealed that siRNAs targeting regions of the mRNA predicted to have unpaired 5'- and 3'-ends resulted in greater gene silencing than regions predicted to have other types of secondary structure. These results were confirmed by analysis of gene silencing data from previously published siRNAs, which showed that mRNA target regions unpaired at either the 5'-end or 3'-end were silenced, on average, approximately 10% more strongly than target regions unpaired in the center or primarily paired throughout. We found this effect to be independent of the structure of the siRNA guide strand. Taken together, these results suggest minimal requirements for nucleation of hybridization between the siRNA guide strand and mRNA and that both mRNA and guide strand structure should be considered when choosing candidate si

  1. Simultaneous characterization of cellular RNA structure and function with in-cell SHAPE-Seq.

    Science.gov (United States)

    Watters, Kyle E; Abbott, Timothy R; Lucks, Julius B

    2016-01-29

    Many non-coding RNAs form structures that interact with cellular machinery to control gene expression. A central goal of molecular and synthetic biology is to uncover design principles linking RNA structure to function to understand and engineer this relationship. Here we report a simple, high-throughput method called in-cell SHAPE-Seq that combines in-cell probing of RNA structure with a measurement of gene expression to simultaneously characterize RNA structure and function in bacterial cells. We use in-cell SHAPE-Seq to study the structure-function relationship of two RNA mechanisms that regulate translation in Escherichia coli. We find that nucleotides that participate in RNA-RNA interactions are highly accessible when their binding partner is absent and that changes in RNA structure due to RNA-RNA interactions can be quantitatively correlated to changes in gene expression. We also characterize the cellular structures of three endogenously expressed non-coding RNAs: 5S rRNA, RNase P and the btuB riboswitch. Finally, a comparison between in-cell and in vitro folded RNA structures revealed remarkable similarities for synthetic RNAs, but significant differences for RNAs that participate in complex cellular interactions. Thus, in-cell SHAPE-Seq represents an easily approachable tool for biologists and engineers to uncover relationships between sequence, structure and function of RNAs in the cell. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Apical cap

    International Nuclear Information System (INIS)

    McLoud, T.C.; Isler, R.J.; Novelline, R.A.; Putman, C.E.; Simeone, J.; Stark, P.

    1981-01-01

    Apical caps, either unilateral or bilateral, are a common feature of advancing age and are usually the result of subpleural scarring unassociated with other diseases. Pancoast (superior sulcus) tumors are a well recognized cause of unilateral asymmetric apical density. Other lesions arising in the lung, pleura, or extrapleural space may produce unilateral or bilateral apical caps. These include: (1) inflammatory: tuberculosis and extrapleural abscesses extending from the neck; (2) post radiation fibrosis after mantle therapy for Hodgkin disease or supraclavicular radiation in the treatment of breast carcinoma; (3) neoplasm: lymphoma extending from the neck or mediastinum, superior sulcus bronchogenic carcinoma, and metastases; (4) traumatic: extrapleural dissection of blood from a ruptured aorta, fractures of the ribs or spine, or hemorrhage due to subclavian line placement; (5) vascular: coarctation of the aorta with dilated collaterals over the apex, fistula between the subclavian artery and vein; and (6) miscellaneous: mediastinal lipomatosis with subcostal fat extending over the apices

  3. RNA three-way junctions can act as flexible RNA structural elements in large RNA molecules: a molecular simulation analysis

    Czech Academy of Sciences Publication Activity Database

    Beššeová, Ivana; Réblová, Kamila; Leontis, N.B.; Šponer, Jiří

    2009-01-01

    Roč. 26, č. 6 (2009), s. 830-831 ISSN 0739-1102. [The 17th Conversation . 16.06.2009-20.06.2009, Albany] Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : RNA three-way junctions * RNA Subject RIV: BO - Biophysics

  4. RNA-dependent RNA polymerase of hepatitis C virus binds to its coding region RNA stem-loop structure, 5BSL3.2, and its negative strand.

    Science.gov (United States)

    Kanamori, Hiroshi; Yuhashi, Kazuhito; Ohnishi, Shin; Koike, Kazuhiko; Kodama, Tatsuhiko

    2010-05-01

    The hepatitis C virus NS5B RNA-dependent RNA polymerase (RdRp) is a key enzyme involved in viral replication. Interaction between NS5B RdRp and the viral RNA sequence is likely to be an important step in viral RNA replication. The C-terminal half of the NS5B-coding sequence, which contains the important cis-acting replication element, has been identified as an NS5B-binding sequence. In the present study, we confirm the specific binding of NS5B to one of the RNA stem-loop structures in the region, 5BSL3.2. In addition, we show that NS5B binds to the complementary strand of 5BSL3.2 (5BSL3.2N). The bulge structure of 5BSL3.2N was shown to be indispensable for tight binding to NS5B. In vitro RdRp activity was inhibited by 5BSL3.2N, indicating the importance of the RNA element in the polymerization by RdRp. These results suggest the involvement of the RNA stem-loop structure of the negative strand in the replication process.

  5. An efficient algorithm for planar drawing of RNA structures with pseudoknots of any type.

    Science.gov (United States)

    Byun, Yanga; Han, Kyungsook

    2016-06-01

    An RNA pseudoknot is a tertiary structural element in which bases of a loop pair with complementary bases are outside the loop. A drawing of RNA secondary structures is a tree, but a drawing of RNA pseudoknots is a graph that has an inner cycle within a pseudoknot and possibly outer cycles formed between the pseudoknot and other structural elements. Visualizing a large-scale RNA structure with pseudoknots as a planar drawing is challenging because a planar drawing of an RNA structure requires both pseudoknots and an entire structure enclosing the pseudoknots to be embedded into a plane without overlapping or crossing. This paper presents an efficient heuristic algorithm for visualizing a pseudoknotted RNA structure as a planar drawing. The algorithm consists of several parts for finding crossing stems and page mapping the stems, for the layout of stem-loops and pseudoknots, and for overlap detection between structural elements and resolving it. Unlike previous algorithms, our algorithm generates a planar drawing for a large RNA structure with pseudoknots of any type and provides a bracket view of the structure. It generates a compact and aesthetic structure graph for a large pseudoknotted RNA structure in O([Formula: see text]) time, where n is the number of stems of the RNA structure.

  6. If the cap fits, wear it: an overview of telomeric structures over evolution.

    Science.gov (United States)

    Fulcher, Nick; Derboven, Elisa; Valuchova, Sona; Riha, Karel

    2014-03-01

    Genome organization into linear chromosomes likely represents an important evolutionary innovation that has permitted the development of the sexual life cycle; this process has consequently advanced nuclear expansion and increased complexity of eukaryotic genomes. Chromosome linearity, however, poses a major challenge to the internal cellular machinery. The need to efficiently recognize and repair DNA double-strand breaks that occur as a consequence of DNA damage presents a constant threat to native chromosome ends known as telomeres. In this review, we present a comparative survey of various solutions to the end protection problem, maintaining an emphasis on DNA structure. This begins with telomeric structures derived from a subset of prokaryotes, mitochondria, and viruses, and will progress into the typical telomere structure exhibited by higher organisms containing TTAGG-like tandem sequences. We next examine non-canonical telomeres from Drosophila melanogaster, which comprise arrays of retrotransposons. Finally, we discuss telomeric structures in evolution and possible switches between canonical and non-canonical solutions to chromosome end protection.

  7. Sequence-structure relationships in RNA loops: establishing the basis for loop homology modeling.

    Science.gov (United States)

    Schudoma, Christian; May, Patrick; Nikiforova, Viktoria; Walther, Dirk

    2010-01-01

    The specific function of RNA molecules frequently resides in their seemingly unstructured loop regions. We performed a systematic analysis of RNA loops extracted from experimentally determined three-dimensional structures of RNA molecules. A comprehensive loop-structure data set was created and organized into distinct clusters based on structural and sequence similarity. We detected clear evidence of the hallmark of homology present in the sequence-structure relationships in loops. Loops differing by structures. Thus, our results support the application of homology modeling for RNA loop model building. We established a threshold that may guide the sequence divergence-based selection of template structures for RNA loop homology modeling. Of all possible sequences that are, under the assumption of isosteric relationships, theoretically compatible with actual sequences observed in RNA structures, only a small fraction is contained in the Rfam database of RNA sequences and classes implying that the actual RNA loop space may consist of a limited number of unique loop structures and conserved sequences. The loop-structure data sets are made available via an online database, RLooM. RLooM also offers functionalities for the modeling of RNA loop structures in support of RNA engineering and design efforts.

  8. Structural explanation for the role of Mn2+ in the activity of phi6 RNA-dependent RNA polymerase.

    Science.gov (United States)

    Poranen, Minna M; Salgado, Paula S; Koivunen, Minni R L; Wright, Sam; Bamford, Dennis H; Stuart, David I; Grimes, Jonathan M

    2008-11-01

    The biological role of manganese (Mn(2+)) has been a long-standing puzzle, since at low concentrations it activates several polymerases whilst at higher concentrations it inhibits. Viral RNA polymerases possess a common architecture, reminiscent of a closed right hand. The RNA-dependent RNA polymerase (RdRp) of bacteriophage 6 is one of the best understood examples of this important class of polymerases. We have probed the role of Mn(2+) by biochemical, biophysical and structural analyses of the wild-type enzyme and of a mutant form with an altered Mn(2+)-binding site (E491 to Q). The E491Q mutant has much reduced affinity for Mn(2+), reduced RNA binding and a compromised elongation rate. Loss of Mn(2+) binding structurally stabilizes the enzyme. These data and a re-examination of the structures of other viral RNA polymerases clarify the role of manganese in the activation of polymerization: Mn(2+) coordination of a catalytic aspartate is necessary to allow the active site to properly engage with the triphosphates of the incoming NTPs. The structural flexibility caused by Mn(2+) is also important for the enzyme dynamics, explaining the requirement for manganese throughout RNA polymerization.

  9. Tetrameric structure of the flagellar cap protein FliD from Serratia marcescens.

    Science.gov (United States)

    Cho, So Yeon; Song, Wan Seok; Hong, Ho Jeong; Lee, Geun-Shik; Kang, Seung Goo; Ko, Hyun-Jeong; Kim, Pyeung-Hyeun; Yoon, Sung-Il

    2017-07-15

    Bacterial motility is provided by the flagellum. FliD is located at the distal end of the flagellum and plays a key role in the insertion of each flagellin protein at the growing tip of the flagellar filament. Because FliD functions as an oligomer, the determination of the oligomeric state of FliD is critical to understanding the molecular mechanism of FliD-mediated flagellar growth. FliD has been shown to adopt a pentameric or a hexameric structure depending on the bacterial species. Here, we report another distinct oligomeric form of FliD based on structural and biochemical studies. The crystal structures of the D2 and D3 domains of Serratia marcescens FliD (smFliD) were determined in two crystal forms and together revealed that smFliD assembles into a tetrameric architecture that resembles a four-pointed star plate. smFliD tetramerization was also confirmed in solution by cross-linking experiments. Although smFliD oligomerizes in a head-to-tail orientation using a common primary binding interface between the D2 and D3' domains (the prime denotes the second subunit in the oligomer) similarly to other FliD orthologs, the smFliD tetramer diverges to present a unique secondary D2-D2' binding interface. Our structure-based comparative analysis of FliD suggests that bacteria have developed diverse species-specific oligomeric forms of FliD that range from tetramers to hexamers for flagellar growth. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Temporal Translational Control by a Metastable RNA Structure

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Franch, Thomas; Gerdes, Kenn

    2001-01-01

    Programmed cell death by the hok/sok locus of plasmid R1 relies on a complex translational control mechanism. The highly stable hok mRNA is activated by 3'-end exonucleolytical processing. Removal of the mRNA 3' end releases a 5'-end sequence that triggers refolding of the mRNA. The refolded hok m......-transcriptional control mechanism....

  11. RNA processing and ribonucleoprotein assembly studied in vivo by RNA transfection

    International Nuclear Information System (INIS)

    Kleinschmidt, A.M.; Pederson, T.

    1990-01-01

    The authors present a method for studying RNA processing and ribonucleoprotein assembly in vivo, by using RNA synthesized in vitro. SP6-transcribed 32 P-labeled U2 small nuclear RNA precursor molecules were introduced into cultured human 293 cells by calcium phosphate-mediated uptake, as in standard DNA transfection experiments. RNase protection mapping demonstrated that the introduced pre-U2 RNA underwent accurate 3' end processing. The introduced U2 RNA was assembled into ribonucleoprotein particles that reacted with an antibody specific for proteins known to be associated with the U2 small nuclear ribonucleoprotein particle. The 3' end-processed, ribonucleoprotein-assembled U2 RNA accumulated in the nuclear fraction. When pre-U2 RNA with a 7-methylguanosine group at the 5' end was introduced into cells, it underwent conversion to a 2,2,7-trimethylguanosine cap structure, a characteristic feature of the U-small nuclear RNAs. Pre-U2 RNA introduced with an adenosine cap (Ap-ppG) also underwent processing, small nuclear ribonucleoprotein assembly, and nuclear accumulation, establishing that a methylated guanosine cap structure is not required for these steps in U2 small nuclear ribonucleprotein biosynthesis. Beyond its demonstrated usefulness in the study of small nuclear ribonucleoprotein biosynthesis, RNA transfection may be of general applicability to the investigation of eukaryotic RNA processing in vivo and may also offer opportunities for introducing therapeutically targeted RNAs (ribozymes or antisense RNA) into cells

  12. Non-destructive determination of ultra-thin GaN cap layer thickness in AlGaN/GaN HEMT structure by angle resolved x-ray photoelectron spectroscopy (ARXPS)

    Science.gov (United States)

    Goyal, Anshu; Yadav, Brajesh S.; Raman, R.; Kapoor, Ashok K.

    2018-02-01

    Angle resolved X-ray photoelectron spectroscopy (ARXPS) and secondary ion mass spectrometry (SIMS) investigations have been carried out to characterize the GaN cap layer in AlGaN/GaN HEMT structure. The paper discusses the qualitative (presence or absence of a cap layer) and quantitative (cap layer thickness) characterization of cap layer in HEMT structure non-destructively using ARXPS measurements in conjunction with the theoretical modeling. Further the relative sensitive factor (RSF=σ/Ga σAl ) for Ga to Al ratio was estimated to be 0.963 and was used in the quantification of GaN cap layer thickness. Our results show that Al/Ga intensity ratio varies with the emission angle in the presence of GaN cap layer and otherwise remains constant. Also, the modeling of this intensity ratio gives its thickness. The finding of ARXPS was also substantiated by SIMS depth profiling studies.

  13. Non-destructive determination of ultra-thin GaN cap layer thickness in AlGaN/GaN HEMT structure by angle resolved x-ray photoelectron spectroscopy (ARXPS

    Directory of Open Access Journals (Sweden)

    Anshu Goyal

    2018-02-01

    Full Text Available Angle resolved X-ray photoelectron spectroscopy (ARXPS and secondary ion mass spectrometry (SIMS investigations have been carried out to characterize the GaN cap layer in AlGaN/GaN HEMT structure. The paper discusses the qualitative (presence or absence of a cap layer and quantitative (cap layer thickness characterization of cap layer in HEMT structure non-destructively using ARXPS measurements in conjunction with the theoretical modeling. Further the relative sensitive factor (RSF=σGaσAl for Ga to Al ratio was estimated to be 0.963 and was used in the quantification of GaN cap layer thickness. Our results show that Al/Ga intensity ratio varies with the emission angle in the presence of GaN cap layer and otherwise remains constant. Also, the modeling of this intensity ratio gives its thickness. The finding of ARXPS was also substantiated by SIMS depth profiling studies.

  14. Vfold: a web server for RNA structure and folding thermodynamics prediction.

    Science.gov (United States)

    Xu, Xiaojun; Zhao, Peinan; Chen, Shi-Jie

    2014-01-01

    The ever increasing discovery of non-coding RNAs leads to unprecedented demand for the accurate modeling of RNA folding, including the predictions of two-dimensional (base pair) and three-dimensional all-atom structures and folding stabilities. Accurate modeling of RNA structure and stability has far-reaching impact on our understanding of RNA functions in human health and our ability to design RNA-based therapeutic strategies. The Vfold server offers a web interface to predict (a) RNA two-dimensional structure from the nucleotide sequence, (b) three-dimensional structure from the two-dimensional structure and the sequence, and (c) folding thermodynamics (heat capacity melting curve) from the sequence. To predict the two-dimensional structure (base pairs), the server generates an ensemble of structures, including loop structures with the different intra-loop mismatches, and evaluates the free energies using the experimental parameters for the base stacks and the loop entropy parameters given by a coarse-grained RNA folding model (the Vfold model) for the loops. To predict the three-dimensional structure, the server assembles the motif scaffolds using structure templates extracted from the known PDB structures and refines the structure using all-atom energy minimization. The Vfold-based web server provides a user friendly tool for the prediction of RNA structure and stability. The web server and the source codes are freely accessible for public use at "http://rna.physics.missouri.edu".

  15. Evolution of the RNase P RNA structural domain in Leptospira spp

    NARCIS (Netherlands)

    Ravishankar, Vigneshwaran; Ahmed, Ahmed; Sivagnanam, Ulaganathan; Muthuraman, Krishnaraja; Karthikaichamy, Anbarasu; Wilson, Herald A.; Devendran, Ajay; Hartskeerl, Rudy A.; Raj, Stephen M. L.

    2014-01-01

    We have employed the RNase P RNA (RPR) gene, which is present as single copy in chromosome I of Leptospira spp. to investigate the phylogeny of structural domains present in the RNA subunit of the tRNA processing enzyme, RNase P. RPR gene sequences of 150 strains derived from NCBI database along

  16. RNA structural constraints in the evolution of the influenza A virus genome NP segment

    NARCIS (Netherlands)

    A.P. Gultyaev (Alexander); A. Tsyganov-Bodounov (Anton); M.I. Spronken (Monique); S. Van Der Kooij (Sander); R.A.M. Fouchier (Ron); R.C.L. Olsthoorn (René)

    2014-01-01

    textabstractConserved RNA secondary structures were predicted in the nucleoprotein (NP) segment of the influenza A virus genome using comparative sequence and structure analysis. A number of structural elements exhibiting nucleotide covariations were identified over the whole segment length,

  17. Non-Watson Crick base pairs might stabilize RNA structural motifs in ...

    Indian Academy of Sciences (India)

    Watson Crick base pairs, internal loops and pseudoknots have been the highlighting feature of recent structural determination of RNAs. The recent crystal structure of group-I introns has demonstrated that these might constitute RNA structural ...

  18. Poliovirus RNA synthesis in vitro: structural elements and antibody inhibition

    International Nuclear Information System (INIS)

    Semler, B.L.; Hanecak, R.; Dorner, L.F.; Anderson, C.W.; Wimmer, E.

    1983-01-01

    The poliovirus RNA polymerase complex has been analyzed by immunoautoradiography using antibody probes derived from purified replicase (P3) region viral polypeptides. Antibody preparations made against the polio RNA polymerase, P3-4b, detected a previously unreported cellular protein that copurifies with the RNA polymerase. An IgG fraction purified from rabbit antiserum to polypeptide P3-2, a precursor fo the RNA polymerase, specifically inhibits poliovirus RNA synthesis in vitro. The authors have also immunoprecipitated a 60,000-dalton protein (P3-4a) with antiserum to protein P3-4b and have determined the precise genomic map position of this protein by automated Edman degradation. Protein P3-4a originates by cleavage of the RNA polymerase precursor at a glutamine-glucine amino acid pair not previously reported to be a viral cleavage site

  19. An RNA-binding compound that stabilizes the HIV-1 gRNA packaging signal structure and specifically blocks HIV-1 RNA encapsidation.

    Science.gov (United States)

    Ingemarsdotter, Carin K; Zeng, Jingwei; Long, Ziqi; Lever, Andrew M L; Kenyon, Julia C

    2018-03-14

    NSC260594, a quinolinium derivative from the NCI diversity set II compound library, was previously identified in a target-based assay as an inhibitor of the interaction between the HIV-1 (ψ) stem-loop 3 (SL3) RNA and Gag. This compound was shown to exhibit potent antiviral activity. Here, the effects of this compound on individual stages of the viral lifecycle were examined by qRT-PCR, ELISA and Western blot, to see if its actions were specific to the viral packaging stage. The structural effects of NSC260594 binding to the HIV-1 gRNA were also examined by SHAPE and dimerization assays. Treatment of cells with NSC260594 did not reduce the number of integration events of incoming virus, and treatment of virus producing cells did not affect the level of intracellular Gag protein or viral particle release as determined by immunoblot. However, NSC260594 reduced the incorporation of gRNA into virions by up to 82%, without affecting levels of gRNA inside the cell. This reduction in packaging correlated closely with the reduction in infectivity of the released viral particles. To establish the structural effects of NSC260594 on the HIV-1 gRNA, we performed SHAPE analyses to pinpoint RNA structural changes. NSC260594 had a stabilizing effect on the wild type RNA that was not confined to SL3, but that was propagated across the structure. A packaging mutant lacking SL3 did not show this effect. NSC260594 acts as a specific inhibitor of HIV-1 RNA packaging. No other viral functions are affected. Its action involves preventing the interaction of Gag with SL3 by stabilizing this small RNA stem-loop which then leads to stabilization of the global packaging signal region (psi or ψ). This confirms data, previously only shown in analyses of isolated SL3 oligonucleotides, that SL3 is structurally labile in the presence of Gag and that this is critical for the complete psi region to be able to adopt different conformations. Since replication is otherwise unaffected by NSC260594

  20. Structure and assembly of the essential RNA ring component of a viral DNA packaging motor.

    Science.gov (United States)

    Ding, Fang; Lu, Changrui; Zhao, Wei; Rajashankar, Kanagalaghatta R; Anderson, Dwight L; Jardine, Paul J; Grimes, Shelley; Ke, Ailong

    2011-05-03

    Prohead RNA (pRNA) is an essential component in the assembly and operation of the powerful bacteriophage 29 DNA packaging motor. The pRNA forms a multimeric ring via intermolecular base-pairing interactions between protomers that serves to guide the assembly of the ring ATPase that drives DNA packaging. Here we report the quaternary structure of this rare multimeric RNA at 3.5 Å resolution, crystallized as tetrameric rings. Strong quaternary interactions and the inherent flexibility helped rationalize how free pRNA is able to adopt multiple oligomerization states in solution. These characteristics also allowed excellent fitting of the crystallographic pRNA protomers into previous prohead/pRNA cryo-EM reconstructions, supporting the presence of a pentameric, but not hexameric, pRNA ring in the context of the DNA packaging motor. The pentameric pRNA ring anchors itself directly to the phage prohead by interacting specifically with the fivefold symmetric capsid structures that surround the head-tail connector portal. From these contacts, five RNA superhelices project from the pRNA ring, where they serve as scaffolds for binding and assembly of the ring ATPase, and possibly mediate communication between motor components. Construction of structure-based designer pRNAs with little sequence similarity to the wild-type pRNA were shown to fully support the packaging of 29 DNA.

  1. RNA secondary structure prediction with pseudoknots: Contribution of algorithm versus energy model.

    Science.gov (United States)

    Jabbari, Hosna; Wark, Ian; Montemagno, Carlo

    2018-01-01

    RNA is a biopolymer with various applications inside the cell and in biotechnology. Structure of an RNA molecule mainly determines its function and is essential to guide nanostructure design. Since experimental structure determination is time-consuming and expensive, accurate computational prediction of RNA structure is of great importance. Prediction of RNA secondary structure is relatively simpler than its tertiary structure and provides information about its tertiary structure, therefore, RNA secondary structure prediction has received attention in the past decades. Numerous methods with different folding approaches have been developed for RNA secondary structure prediction. While methods for prediction of RNA pseudoknot-free structure (structures with no crossing base pairs) have greatly improved in terms of their accuracy, methods for prediction of RNA pseudoknotted secondary structure (structures with crossing base pairs) still have room for improvement. A long-standing question for improving the prediction accuracy of RNA pseudoknotted secondary structure is whether to focus on the prediction algorithm or the underlying energy model, as there is a trade-off on computational cost of the prediction algorithm versus the generality of the method. The aim of this work is to argue when comparing different methods for RNA pseudoknotted structure prediction, the combination of algorithm and energy model should be considered and a method should not be considered superior or inferior to others if they do not use the same scoring model. We demonstrate that while the folding approach is important in structure prediction, it is not the only important factor in prediction accuracy of a given method as the underlying energy model is also as of great value. Therefore we encourage researchers to pay particular attention in comparing methods with different energy models.

  2. The identification and functional annotation of RNA structures conserved in vertebrates

    DEFF Research Database (Denmark)

    Seemann, Ernst Stefan; Mirza, Aashiq Hussain; Hansen, Claus

    2017-01-01

    Structured elements of RNA molecules are essential in, e.g., RNA stabilization, localization and protein interaction, and their conservation across species suggests a common functional role. We computationally screened vertebrate genomes for Conserved RNA Structures (CRSs), leveraging structure...... (RBPs) or (ii) are transcribed in corresponding tissues. Additionally, a CaptureSeq experiment revealed expression of many of our CRS regions in human fetal brain, including 662 novel ones. For selected human and mouse candidate pairs, qRT-PCR and in vitro RNA structure probing supported both shared...... expression and shared structure despite low abundance and low sequence identity. About 30k CRS regions are located near coding or long non-coding RNA genes or within enhancers. Structured (CRS overlapping) enhancer RNAs and extended 3' ends have significantly increased expression levels over their non-structured...

  3. Innate immune restriction and antagonism of viral RNA lacking 2'-O methylation

    Energy Technology Data Exchange (ETDEWEB)

    Hyde, Jennifer L. [Departments of Medicine, Washington University School of Medicine, St Louis., MO 63110 (United States); Diamond, Michael S., E-mail: diamond@borcim.wustl.edu [Departments of Medicine, Washington University School of Medicine, St Louis., MO 63110 (United States); Molecular Microbiology, Washington University School of Medicine, St Louis., MO 63110 (United States); Pathology & Immunology, Washington University School of Medicine, St Louis., MO 63110 (United States); The Center for Human Immunology and Immunotherapy Programs, Washington University School of Medicine, St Louis., MO 63110 (United States)

    2015-05-15

    N-7 and 2′-O methylation of host cell mRNA occurs in the nucleus and results in the generation of cap structures (cap 0, m{sup 7}GpppN; cap 1, m{sup 7}GpppNm) that control gene expression by modulating nuclear export, splicing, turnover, and protein synthesis. Remarkably, RNA cap modification also contributes to mammalian cell host defense as viral RNA lacking 2′-O methylation is sensed and inhibited by IFIT1, an interferon (IFN) stimulated gene (ISG). Accordingly, pathogenic viruses that replicate in the cytoplasm have evolved mechanisms to circumvent IFIT1 restriction and facilitate infection of mammalian cells. These include: (a) generating cap 1 structures on their RNA through cap-snatching or virally-encoded 2′-O methyltransferases, (b) using cap-independent means of translation, or (c) using RNA secondary structural motifs to antagonize IFIT1 binding. This review will discuss new insights as to how specific modifications at the 5′-end of viral RNA modulate host pathogen recognition responses to promote infection and disease.

  4. An efficient genetic algorithm for structural RNA pairwise alignment and its application to non-coding RNA discovery in yeast

    Directory of Open Access Journals (Sweden)

    Taneda Akito

    2008-12-01

    Full Text Available Abstract Background Aligning RNA sequences with low sequence identity has been a challenging problem since such a computation essentially needs an algorithm with high complexities for taking structural conservation into account. Although many sophisticated algorithms for the purpose have been proposed to date, further improvement in efficiency is necessary to accelerate its large-scale applications including non-coding RNA (ncRNA discovery. Results We developed a new genetic algorithm, Cofolga2, for simultaneously computing pairwise RNA sequence alignment and consensus folding, and benchmarked it using BRAliBase 2.1. The benchmark results showed that our new algorithm is accurate and efficient in both time and memory usage. Then, combining with the originally trained SVM, we applied the new algorithm to novel ncRNA discovery where we compared S. cerevisiae genome with six related genomes in a pairwise manner. By focusing our search to the relatively short regions (50 bp to 2,000 bp sandwiched by conserved sequences, we successfully predict 714 intergenic and 1,311 sense or antisense ncRNA candidates, which were found in the pairwise alignments with stable consensus secondary structure and low sequence identity (≤ 50%. By comparing with the previous predictions, we found that > 92% of the candidates is novel candidates. The estimated rate of false positives in the predicted candidates is 51%. Twenty-five percent of the intergenic candidates has supports for expression in cell, i.e. their genomic positions overlap those of the experimentally determined transcripts in literature. By manual inspection of the results, moreover, we obtained four multiple alignments with low sequence identity which reveal consensus structures shared by three species/sequences. Conclusion The present method gives an efficient tool complementary to sequence-alignment-based ncRNA finders.

  5. RNA FRABASE 2.0: an advanced web-accessible database with the capacity to search the three-dimensional fragments within RNA structures

    Directory of Open Access Journals (Sweden)

    Wasik Szymon

    2010-05-01

    Full Text Available Abstract Background Recent discoveries concerning novel functions of RNA, such as RNA interference, have contributed towards the growing importance of the field. In this respect, a deeper knowledge of complex three-dimensional RNA structures is essential to understand their new biological functions. A number of bioinformatic tools have been proposed to explore two major structural databases (PDB, NDB in order to analyze various aspects of RNA tertiary structures. One of these tools is RNA FRABASE 1.0, the first web-accessible database with an engine for automatic search of 3D fragments within PDB-derived RNA structures. This search is based upon the user-defined RNA secondary structure pattern. In this paper, we present and discuss RNA FRABASE 2.0. This second version of the system represents a major extension of this tool in terms of providing new data and a wide spectrum of novel functionalities. An intuitionally operated web server platform enables very fast user-tailored search of three-dimensional RNA fragments, their multi-parameter conformational analysis and visualization. Description RNA FRABASE 2.0 has stored information on 1565 PDB-deposited RNA structures, including all NMR models. The RNA FRABASE 2.0 search engine algorithms operate on the database of the RNA sequences and the new library of RNA secondary structures, coded in the dot-bracket format extended to hold multi-stranded structures and to cover residues whose coordinates are missing in the PDB files. The library of RNA secondary structures (and their graphics is made available. A high level of efficiency of the 3D search has been achieved by introducing novel tools to formulate advanced searching patterns and to screen highly populated tertiary structure elements. RNA FRABASE 2.0 also stores data and conformational parameters in order to provide "on the spot" structural filters to explore the three-dimensional RNA structures. An instant visualization of the 3D RNA

  6. General enumeration of RNA secondary structures based on new ...

    African Journals Online (AJOL)

    akpobome

    coding, transferring and retrieving genetic information, and in directing cell metabolism. The nucleic acid includes DNA and RNA molecule. RNA molecule is a single-stranded nucleic acid of four different kinds of nucleotides. The four nucleotides only differ by one part, called bases. Hence, one usually identifies nucleotides.

  7. GARN: Sampling RNA 3D Structure Space with Game Theory and Knowledge-Based Scoring Strategies.

    Science.gov (United States)

    Boudard, Mélanie; Bernauer, Julie; Barth, Dominique; Cohen, Johanne; Denise, Alain

    2015-01-01

    Cellular processes involve large numbers of RNA molecules. The functions of these RNA molecules and their binding to molecular machines are highly dependent on their 3D structures. One of the key challenges in RNA structure prediction and modeling is predicting the spatial arrangement of the various structural elements of RNA. As RNA folding is generally hierarchical, methods involving coarse-grained models hold great promise for this purpose. We present here a novel coarse-grained method for sampling, based on game theory and knowledge-based potentials. This strategy, GARN (Game Algorithm for RNa sampling), is often much faster than previously described techniques and generates large sets of solutions closely resembling the native structure. GARN is thus a suitable starting point for the molecular modeling of large RNAs, particularly those with experimental constraints. GARN is available from: http://garn.lri.fr/.

  8. An automated procedure for covariation-based detection of RNA structure

    International Nuclear Information System (INIS)

    Winker, S.; Overbeek, R.; Woese, C.R.; Olsen, G.J.; Pfluger, N.

    1989-12-01

    This paper summarizes our investigations into the computational detection of secondary and tertiary structure of ribosomal RNA. We have developed a new automated procedure that not only identifies potential bondings of secondary and tertiary structure, but also provides the covariation evidence that supports the proposed bondings, and any counter-evidence that can be detected in the known sequences. A small number of previously unknown bondings have been detected in individual RNA molecules (16S rRNA and 7S RNA) through the use of our automated procedure. Currently, we are systematically studying mitochondrial rRNA. Our goal is to detect tertiary structure within 16S rRNA and quaternary structure between 16S and 23S rRNA. Our ultimate hope is that automated covariation analysis will contribute significantly to a refined picture of ribosome structure. Our colleagues in biology have begun experiments to test certain hypotheses suggested by an examination of our program's output. These experiments involve sequencing key portions of the 23S ribosomal RNA for species in which the known 16S ribosomal RNA exhibits variation (from the dominant pattern) at the site of a proposed bonding. The hope is that the 23S ribosomal RNA of these species will exhibit corresponding complementary variation or generalized covariation. 24 refs

  9. An automated procedure for covariation-based detection of RNA structure

    Energy Technology Data Exchange (ETDEWEB)

    Winker, S.; Overbeek, R.; Woese, C.R.; Olsen, G.J.; Pfluger, N.

    1989-12-01

    This paper summarizes our investigations into the computational detection of secondary and tertiary structure of ribosomal RNA. We have developed a new automated procedure that not only identifies potential bondings of secondary and tertiary structure, but also provides the covariation evidence that supports the proposed bondings, and any counter-evidence that can be detected in the known sequences. A small number of previously unknown bondings have been detected in individual RNA molecules (16S rRNA and 7S RNA) through the use of our automated procedure. Currently, we are systematically studying mitochondrial rRNA. Our goal is to detect tertiary structure within 16S rRNA and quaternary structure between 16S and 23S rRNA. Our ultimate hope is that automated covariation analysis will contribute significantly to a refined picture of ribosome structure. Our colleagues in biology have begun experiments to test certain hypotheses suggested by an examination of our program's output. These experiments involve sequencing key portions of the 23S ribosomal RNA for species in which the known 16S ribosomal RNA exhibits variation (from the dominant pattern) at the site of a proposed bonding. The hope is that the 23S ribosomal RNA of these species will exhibit corresponding complementary variation or generalized covariation. 24 refs.

  10. Mapping RNA Structure In Vitro with SHAPE Chemistry and Next-Generation Sequencing (SHAPE-Seq).

    Science.gov (United States)

    Watters, Kyle E; Lucks, Julius B

    2016-01-01

    Mapping RNA structure with selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry has proven to be a versatile method for characterizing RNA structure in a variety of contexts. SHAPE reagents covalently modify RNAs in a structure-dependent manner to create adducts at the 2'-OH group of the ribose backbone at nucleotides that are structurally flexible. The positions of these adducts are detected using reverse transcriptase (RT) primer extension, which stops one nucleotide before the modification, to create a pool of cDNAs whose lengths reflect the location of SHAPE modification. Quantification of the cDNA pools is used to estimate the "reactivity" of each nucleotide in an RNA molecule to the SHAPE reagent. High reactivities indicate nucleotides that are structurally flexible, while low reactivities indicate nucleotides that are inflexible. These SHAPE reactivities can then be used to infer RNA structures by restraining RNA structure prediction algorithms. Here, we provide a state-of-the-art protocol describing how to perform in vitro RNA structure probing with SHAPE chemistry using next-generation sequencing to quantify cDNA pools and estimate reactivities (SHAPE-Seq). The use of next-generation sequencing allows for higher throughput, more consistent data analysis, and multiplexing capabilities. The technique described herein, SHAPE-Seq v2.0, uses a universal reverse transcription priming site that is ligated to the RNA after SHAPE modification. The introduced priming site allows for the structural analysis of an RNA independent of its sequence.

  11. Investigation of RNA Structure by High-Throughput SHAPE-Based Probing Methods

    DEFF Research Database (Denmark)

    Poulsen, Line Dahl

    of highthroughput SHAPE-based approaches to investigate RNA structure based on novel SHAPE reagents that permit selection of full-length cDNAs. The SHAPE Selection (SHAPES) method is applied to the foot-and-mouth disease virus (FMDV) plus strand RNA genome, and the data is used to construct a genome-wide structural...... that they are functional. The SHAPES method is further applied to the hepatitis C virus (HCV), where the data is used to refine known and predicted structures. Over the past years, the interest of studying RNA structure in their native environment has been increased, and to allow studying RNA structure inside living cells...... using the SHAPE Selection approach, I introduce a biotinylated probing reagent. This chemical can cross cell membranes and reacts with RNA inside the cells, allowing the structural conformations to be studied in the context of physiological relevant conditions in living cells. The methods and results...

  12. Downregulation of viral RNA translation by hepatitis C virus non-structural protein NS5A requires the poly(U/UC) sequence in the 3' UTR.

    Science.gov (United States)

    Hoffman, Brett; Li, Zhubing; Liu, Qiang

    2015-08-01

    Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is essential for viral replication; however, its effect on HCV RNA translation remains controversial partially due to the use of reporters lacking the 3' UTR, where NS5A binds to the poly(U/UC) sequence. We investigated the role of NS5A in HCV translation using a monocistronic RNA containing a Renilla luciferase gene flanked by the HCV UTRs. We found that NS5A downregulated viral RNA translation in a dose-dependent manner. This downregulation required both the 5' and 3' UTRs of HCV because substitution of either sequence with the 5' and 3' UTRs of enterovirus 71 or a cap structure at the 5' end eliminated the effects of NS5A on translation. Translation of the HCV genomic RNA was also downregulated by NS5A. The inhibition of HCV translation by NS5A required the poly(U/UC) sequence in the 3' UTR as NS5A did not affect translation when it was deleted. In addition, we showed that, whilst the amphipathic α-helix of NS5A has no effect on viral translation, the three domains of NS5A can inhibit translation independently, also dependent on the presence of the poly(U/UC) sequence in the 3' UTR. These results suggested that NS5A downregulated HCV RNA translation through a mechanism involving the poly(U/UC) sequence in the 3' UTR.

  13. Structural Studies of RNA Helicases Involved in Eukaryotic Pre-mRNA Splicing, Ribosome Biogenesis, and Translation Initiation

    DEFF Research Database (Denmark)

    He, Yangzi

    and ligates the neighbouring exons to generate mature mRNAs. Prp43 is an RNA helicase of the DEAH/RHA family. In yeast, once mRNAs are released, Prp43 catalyzes the disassembly of spliceosomes. The 18S, 5.8S and 25S rRNAs are transcribed as a single polycistronic transcript—the 35S pre......-rRNA. It is nucleolytically cleaved and chemically modified to generate mature rRNAs, which assemble with ribosomal proteins to form the ribosome. Prp43 is required for the processing of the 18S rRNA. Using X-ray crystallography, I determined a high resolution structure of Prp43 bound to ADP, the first structure of a DEAH....../RHA helicase. It defined the conserved structural features of all DEAH/RHA helicases, and unveiled a novel nucleotide binding site. Additionally a preliminary low resolution structure of a ternary complex comprising Prp43, a non-hydrolyzable ATP analogue, and a single-stranded RNA, was obtained. The ribosome...

  14. The nematode eukaryotic translation initiation factor 4E/G complex works with a trans-spliced leader stem-loop to enable efficient translation of trimethylguanosine-capped RNAs.

    Science.gov (United States)

    Wallace, Adam; Filbin, Megan E; Veo, Bethany; McFarland, Craig; Stepinski, Janusz; Jankowska-Anyszka, Marzena; Darzynkiewicz, Edward; Davis, Richard E

    2010-04-01

    Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5' end through the eIF4F initiation complex binding to the 5' m(7)G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5' end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m(7)G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5' untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs.

  15. The Nematode Eukaryotic Translation Initiation Factor 4E/G Complex Works with a trans-Spliced Leader Stem-Loop To Enable Efficient Translation of Trimethylguanosine-Capped RNAs ▿ †

    Science.gov (United States)

    Wallace, Adam; Filbin, Megan E.; Veo, Bethany; McFarland, Craig; Stepinski, Janusz; Jankowska-Anyszka, Marzena; Darzynkiewicz, Edward; Davis, Richard E.

    2010-01-01

    Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5′ end through the eIF4F initiation complex binding to the 5′ m7G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5′ end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m7G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5′ untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs. PMID:20154140

  16. Differential accumulation of nif structural gene mRNA in Azotobacter vinelandii.

    Science.gov (United States)

    Hamilton, Trinity L; Jacobson, Marty; Ludwig, Marcus; Boyd, Eric S; Bryant, Donald A; Dean, Dennis R; Peters, John W

    2011-09-01

    Northern analysis was employed to investigate mRNA produced by mutant strains of Azotobacter vinelandii with defined deletions in the nif structural genes and in the intergenic noncoding regions. The results indicate that intergenic RNA secondary structures effect the differential accumulation of transcripts, supporting the high Fe protein-to-MoFe protein ratio required for optimal diazotrophic growth.

  17. Kinetic analysis of the effects of target structure on siRNA efficiency

    Science.gov (United States)

    Chen, Jiawen; Zhang, Wenbing

    2012-12-01

    RNAi efficiency for target cleavage and protein expression is related to the target structure. Considering the RNA-induced silencing complex (RISC) as a multiple turnover enzyme, we investigated the effect of target mRNA structure on siRNA efficiency with kinetic analysis. The 4-step model was used to study the target cleavage kinetic process: hybridization nucleation at an accessible target site, RISC-mRNA hybrid elongation along with mRNA target structure melting, target cleavage, and enzyme reactivation. At this model, the terms accounting for the target accessibility, stability, and the seed and the nucleation site effects are all included. The results are in good agreement with that of experiments which show different arguments about the structure effects on siRNA efficiency. It shows that the siRNA efficiency is influenced by the integrated factors of target's accessibility, stability, and the seed effects. To study the off-target effects, a simple model of one siRNA binding to two mRNA targets was designed. By using this model, the possibility for diminishing the off-target effects by the concentration of siRNA was discussed.

  18. R2R - software to speed the depiction of aesthetic consensus RNA secondary structures

    Science.gov (United States)

    2011-01-01

    Background With continuing identification of novel structured noncoding RNAs, there is an increasing need to create schematic diagrams showing the consensus features of these molecules. RNA structural diagrams are typically made either with general-purpose drawing programs like Adobe Illustrator, or with automated or interactive programs specific to RNA. Unfortunately, the use of applications like Illustrator is extremely time consuming, while existing RNA-specific programs produce figures that are useful, but usually not of the same aesthetic quality as those produced at great cost in Illustrator. Additionally, most existing RNA-specific applications are designed for drawing single RNA molecules, not consensus diagrams. Results We created R2R, a computer program that facilitates the generation of aesthetic and readable drawings of RNA consensus diagrams in a fraction of the time required with general-purpose drawing programs. Since the inference of a consensus RNA structure typically requires a multiple-sequence alignment, the R2R user annotates the alignment with commands directing the layout and annotation of the RNA. R2R creates SVG or PDF output that can be imported into Adobe Illustrator, Inkscape or CorelDRAW. R2R can be used to create consensus sequence and secondary structure models for novel RNA structures or to revise models when new representatives for known RNA classes become available. Although R2R does not currently have a graphical user interface, it has proven useful in our efforts to create 100 schematic models of distinct noncoding RNA classes. Conclusions R2R makes it possible to obtain high-quality drawings of the consensus sequence and structural models of many diverse RNA structures with a more practical amount of effort. R2R software is available at http://breaker.research.yale.edu/R2R and as an Additional file. PMID:21205310

  19. R2R--software to speed the depiction of aesthetic consensus RNA secondary structures.

    Science.gov (United States)

    Weinberg, Zasha; Breaker, Ronald R

    2011-01-04

    With continuing identification of novel structured noncoding RNAs, there is an increasing need to create schematic diagrams showing the consensus features of these molecules. RNA structural diagrams are typically made either with general-purpose drawing programs like Adobe Illustrator, or with automated or interactive programs specific to RNA. Unfortunately, the use of applications like Illustrator is extremely time consuming, while existing RNA-specific programs produce figures that are useful, but usually not of the same aesthetic quality as those produced at great cost in Illustrator. Additionally, most existing RNA-specific applications are designed for drawing single RNA molecules, not consensus diagrams. We created R2R, a computer program that facilitates the generation of aesthetic and readable drawings of RNA consensus diagrams in a fraction of the time required with general-purpose drawing programs. Since the inference of a consensus RNA structure typically requires a multiple-sequence alignment, the R2R user annotates the alignment with commands directing the layout and annotation of the RNA. R2R creates SVG or PDF output that can be imported into Adobe Illustrator, Inkscape or CorelDRAW. R2R can be used to create consensus sequence and secondary structure models for novel RNA structures or to revise models when new representatives for known RNA classes become available. Although R2R does not currently have a graphical user interface, it has proven useful in our efforts to create 100 schematic models of distinct noncoding RNA classes. R2R makes it possible to obtain high-quality drawings of the consensus sequence and structural models of many diverse RNA structures with a more practical amount of effort. R2R software is available at http://breaker.research.yale.edu/R2R and as an Additional file.

  20. R2R - software to speed the depiction of aesthetic consensus RNA secondary structures

    Directory of Open Access Journals (Sweden)

    Weinberg Zasha

    2011-01-01

    Full Text Available Abstract Background With continuing identification of novel structured noncoding RNAs, there is an increasing need to create schematic diagrams showing the consensus features of these molecules. RNA structural diagrams are typically made either with general-purpose drawing programs like Adobe Illustrator, or with automated or interactive programs specific to RNA. Unfortunately, the use of applications like Illustrator is extremely time consuming, while existing RNA-specific programs produce figures that are useful, but usually not of the same aesthetic quality as those produced at great cost in Illustrator. Additionally, most existing RNA-specific applications are designed for drawing single RNA molecules, not consensus diagrams. Results We created R2R, a computer program that facilitates the generation of aesthetic and readable drawings of RNA consensus diagrams in a fraction of the time required with general-purpose drawing programs. Since the inference of a consensus RNA structure typically requires a multiple-sequence alignment, the R2R user annotates the alignment with commands directing the layout and annotation of the RNA. R2R creates SVG or PDF output that can be imported into Adobe Illustrator, Inkscape or CorelDRAW. R2R can be used to create consensus sequence and secondary structure models for novel RNA structures or to revise models when new representatives for known RNA classes become available. Although R2R does not currently have a graphical user interface, it has proven useful in our efforts to create 100 schematic models of distinct noncoding RNA classes. Conclusions R2R makes it possible to obtain high-quality drawings of the consensus sequence and structural models of many diverse RNA structures with a more practical amount of effort. R2R software is available at http://breaker.research.yale.edu/R2R and as an Additional file.

  1. The chemical structure of DNA sequence signals for RNA transcription

    Science.gov (United States)

    George, D. G.; Dayhoff, M. O.

    1982-01-01

    The proposed recognition sites for RNA transcription for E. coli NRA polymerase, bacteriophage T7 RNA polymerase, and eukaryotic RNA polymerase Pol II are evaluated in the light of the requirements for efficient recognition. It is shown that although there is good experimental evidence that specific nucleic acid sequence patterns are involved in transcriptional regulation in bacteria and bacterial viruses, among the sequences now available, only in the case of the promoters recognized by bacteriophage T7 polymerase does it seem likely that the pattern is sufficient. It is concluded that the eukaryotic pattern that is investigated is not restrictive enough to serve as a recognition site.

  2. Crystal-Structure-Guided Design of Self-Assembling RNA Nanotriangles.

    Science.gov (United States)

    Boerneke, Mark A; Dibrov, Sergey M; Hermann, Thomas

    2016-03-14

    RNA nanotechnology uses RNA structural motifs to build nanosized architectures that assemble through selective base-pair interactions. Herein, we report the crystal-structure-guided design of highly stable RNA nanotriangles that self-assemble cooperatively from short oligonucleotides. The crystal structure of an 81 nucleotide nanotriangle determined at 2.6 Å resolution reveals the so-far smallest circularly closed nanoobject made entirely of double-stranded RNA. The assembly of the nanotriangle architecture involved RNA corner motifs that were derived from ligand-responsive RNA switches, which offer the opportunity to control self-assembly and dissociation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Probing RNA native conformational ensembles with structural constraints

    DEFF Research Database (Denmark)

    Fonseca, Rasmus; van den Bedem, Henry; Bernauer, Julie

    2016-01-01

    substates, which are difficult to characterize experimentally and computationally. Here, we present an innovative, entirely kinematic computational procedure to efficiently explore the native ensemble of RNA molecules. Our procedure projects degrees of freedom onto a subspace of conformation space defined...

  4. Structure and Dynamics of the tRNA-like Structure Domain of Brome Mosaic Virus

    Science.gov (United States)

    Vieweger, Mario; Nesbitt, David

    2014-03-01

    Conformational switching is widely accepted as regulatory mechanism in gene expression in bacterial systems. More recently, similar regulation mechanisms are emerging for viral systems. One of the most abundant and best studied systems is the tRNA-like structure domain that is found in a number of plant viruses across eight genera. In this work, the folding dynamics of the tRNA-like structure domain of Brome Mosaic Virus are investigated using single-molecule Fluorescence Resonance Energy Transfer techniques. In particular, Burst fluorescence is applied to observe metal-ion induced folding in freely diffusing RNA constructs resembling the 3'-terminal 169nt of BMV RNA3. Histograms of EFRET probabilities reveal a complex equilibrium of three distinct populations. A step-wise kinetic model for TLS folding is developed in accord with the evolution of conformational populations and structural information in the literature. In this mechanism, formation of functional TLS domains from unfolded RNAs requires two consecutive steps; 1) hybridization of a long-range stem interaction followed by 2) formation of a 3' pseudoknot. This three-state equilibrium is well described by step-wise dissociation constants K1(328(30) μM) and K2(1092(183) μM) for [Mg2+] and K1(74(6) mM) and K2(243(52) mM) for [Na+]-induced folding. The kinetic model is validated by oligo competition with the STEM interaction. Implications of this conformational folding mechanism are discussed in regards to regulation of virus replication.

  5. RNACompress: Grammar-based compression and informational complexity measurement of RNA secondary structure

    Directory of Open Access Journals (Sweden)

    Chen Chun

    2008-03-01

    Full Text Available Abstract Background With the rapid emergence of RNA databases and newly identified non-coding RNAs, an efficient compression algorithm for RNA sequence and structural information is needed for the storage and analysis of such data. Although several algorithms for compressing DNA sequences have been proposed, none of them are suitable for the compression of RNA sequences with their secondary structures simultaneously. This kind of compression not only facilitates the maintenance of RNA data, but also supplies a novel way to measure the informational complexity of RNA structural data, raising the possibility of studying the relationship between the functional activities of RNA structures and their complexities, as well as various structural properties of RNA based on compression. Results RNACompress employs an efficient grammar-based model to compress RNA sequences and their secondary structures. The main goals of this algorithm are two fold: (1 present a robust and effective way for RNA structural data compression; (2 design a suitable model to represent RNA secondary structure as well as derive the informational complexity of the structural data based on compression. Our extensive tests have shown that RNACompress achieves a universally better compression ratio compared with other sequence-specific or common text-specific compression algorithms, such as Gencompress, winrar and gzip. Moreover, a test of the activities of distinct GTP-binding RNAs (aptamers compared with their structural complexity shows that our defined informational complexity can be used to describe how complexity varies with activity. These results lead to an objective means of comparing the functional properties of heteropolymers from the information perspective. Conclusion A universal algorithm for the compression of RNA secondary structure as well as the evaluation of its informational complexity is discussed in this paper. We have developed RNACompress, as a useful tool

  6. Rclick: a web server for comparison of RNA 3D structures.

    Science.gov (United States)

    Nguyen, Minh N; Verma, Chandra

    2015-03-15

    RNA molecules play important roles in key biological processes in the cell and are becoming attractive for developing therapeutic applications. Since the function of RNA depends on its structure and dynamics, comparing and classifying the RNA 3D structures is of crucial importance to molecular biology. In this study, we have developed Rclick, a web server that is capable of superimposing RNA 3D structures by using clique matching and 3D least-squares fitting. Our server Rclick has been benchmarked and compared with other popular servers and methods for RNA structural alignments. In most cases, Rclick alignments were better in terms of structure overlap. Our server also recognizes conformational changes between structures. For this purpose, the server produces complementary alignments to maximize the extent of detectable similarity. Various examples showcase the utility of our web server for comparison of RNA, RNA-protein complexes and RNA-ligand structures. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  7. The modeled structure of the RNA dependent RNA polymerase of GBV-C Virus suggests a role for motif E in Flaviviridae RNA polymerases

    Directory of Open Access Journals (Sweden)

    Dutartre Hélène

    2005-10-01

    Full Text Available Abstract Background The Flaviviridae virus family includes major human and animal pathogens. The RNA dependent RNA polymerase (RdRp plays a central role in the replication process, and thus is a validated target for antiviral drugs. Despite the increasing structural and enzymatic characterization of viral RdRps, detailed molecular replication mechanisms remain unclear. The hepatitis C virus (HCV is a major human pathogen difficult to study in cultured cells. The bovine viral diarrhea virus (BVDV is often used as a surrogate model to screen antiviral drugs against HCV. The structure of BVDV RdRp has been recently published. It presents several differences relative to HCV RdRp. These differences raise questions about the relevance of BVDV as a surrogate model, and cast novel interest on the "GB" virus C (GBV-C. Indeed, GBV-C is genetically closer to HCV than BVDV, and can lead to productive infection of cultured cells. There is no structural data for the GBV-C RdRp yet. Results We show in this study that the GBV-C RdRp is closest to the HCV RdRp. We report a 3D model of the GBV-C RdRp, developed using sequence-to-structure threading and comparative modeling based on the atomic coordinates of the HCV RdRp structure. Analysis of the predicted structural features in the phylogenetic context of the RNA polymerase family allows rationalizing most of the experimental data available. Both available structures and our model are explored to examine the catalytic cleft, allosteric and substrate binding sites. Conclusion Computational methods were used to infer evolutionary relationships and to predict the structure of a viral RNA polymerase. Docking a GTP molecule into the structure allows defining a GTP binding pocket in the GBV-C RdRp, such as that of BVDV. The resulting model suggests a new proposition for the mechanism of RNA synthesis, and may prove useful to design new experiments to implement our knowledge on the initiation mechanism of RNA

  8. Structural and functional characterisation of Aichi virus RNA dependent RNA polymerase

    Czech Academy of Sciences Publication Activity Database

    Dubánková, Anna; Humpolíčková, Jana; Šilhán, Jan; Bäumlová, Adriana; Chalupská, Dominika; Klíma, Martin; Bouřa, Evžen

    2017-01-01

    Roč. 15, č. 1 (2017), s. 7-8 ISSN 2336-7202. [Mezioborové setkání mladých biologů, biochemiků a chemiků /17./. 30.05.2017-01.06.2017, Milovy] Institutional support: RVO:61388963 Keywords : Aichi virus * RNA replication Subject RIV: CE - Biochemistry

  9. Influence of microgravity on root-cap regeneration and the structure of columella cells in Zea mays

    Science.gov (United States)

    Moore, R.; McClelen, C. E.; Fondren, W. M.; Wang, C. L.

    1987-01-01

    We launched imbibed seeds and seedlings of Zea mays into outer space aboard the space shuttle Columbia to determine the influence of microgravity on 1) root-cap regeneration, and 2) the distribution of amyloplasts and endoplasmic reticulum (ER) in the putative statocytes (i.e., columella cells) of roots. Decapped roots grown on Earth completely regenerated their caps within 4.8 days after decapping, while those grown in microgravity did not regenerate caps. In Earth-grown seedlings, the ER was localized primarily along the periphery of columella cells, and amyloplasts sedimented in response to gravity to the lower sides of the cells. Seeds germinated on Earth and subsequently launched into outer space had a distribution of ER in columella cells similar to that of Earth-grown controls, but amyloplasts were distributed throughout the cells. Seeds germinated in outer space were characterized by the presence of spherical and ellipsoidal masses of ER and randomly distributed amyloplasts in their columella cells. These results indicate that 1) gravity is necessary for regeneration of the root cap, 2) columella cells can maintain their characteristic distribution of ER in microgravity only if they are exposed previously to gravity, and 3) gravity is necessary to distribute the ER in columella cells of this cultivar of Z. mays.

  10. Structure-Function Model for Kissing Loop Interactions That Initiate Dimerization of Ty1 RNA

    Directory of Open Access Journals (Sweden)

    Eric R. Gamache

    2017-04-01

    Full Text Available The genomic RNA of the retrotransposon Ty1 is packaged as a dimer into virus-like particles. The 5′ terminus of Ty1 RNA harbors cis-acting sequences required for translation initiation, packaging and initiation of reverse transcription (TIPIRT. To identify RNA motifs involved in dimerization and packaging, a structural model of the TIPIRT domain in vitro was developed from single-nucleotide resolution RNA structural data. In general agreement with previous models, the first 326 nucleotides of Ty1 RNA form a pseudoknot with a 7-bp stem (S1, a 1-nucleotide interhelical loop and an 8-bp stem (S2 that delineate two long, structured loops. Nucleotide substitutions that disrupt either pseudoknot stem greatly reduced helper-Ty1-mediated retrotransposition of a mini-Ty1, but only mutations in S2 destabilized mini-Ty1 RNA in cis and helper-Ty1 RNA in trans. Nested in different loops of the pseudoknot are two hairpins with complementary 7-nucleotide motifs at their apices. Nucleotide substitutions in either motif also reduced retrotransposition and destabilized mini- and helper-Ty1 RNA. Compensatory mutations that restore base-pairing in the S2 stem or between the hairpins rescued retrotransposition and RNA stability in cis and trans. These data inform a model whereby a Ty1 RNA kissing complex with two intermolecular kissing-loop interactions initiates dimerization and packaging.

  11. JNSViewer-A JavaScript-based Nucleotide Sequence Viewer for DNA/RNA secondary structures.

    Science.gov (United States)

    Shi, Jieming; Li, Xi; Dong, Min; Graham, Mitchell; Yadav, Nehul; Liang, Chun

    2017-01-01

    Many tools are available for visualizing RNA or DNA secondary structures, but there is scarce implementation in JavaScript that provides seamless integration with the increasingly popular web computational platforms. We have developed JNSViewer, a highly interactive web service, which is bundled with several popular tools for DNA/RNA secondary structure prediction and can provide precise and interactive correspondence among nucleotides, dot-bracket data, secondary structure graphs, and genic annotations. In JNSViewer, users can perform RNA secondary structure predictions with different programs and settings, add customized genic annotations in GFF format to structure graphs, search for specific linear motifs, and extract relevant structure graphs of sub-sequences. JNSViewer also allows users to choose a transcript or specific segment of Arabidopsis thaliana genome sequences and predict the corresponding secondary structure. Popular genome browsers (i.e., JBrowse and BrowserGenome) were integrated into JNSViewer to provide powerful visualizations of chromosomal locations, genic annotations, and secondary structures. In addition, we used StructureFold with default settings to predict some RNA structures for Arabidopsis by incorporating in vivo high-throughput RNA structure profiling data and stored the results in our web server, which might be a useful resource for RNA secondary structure studies in plants. JNSViewer is available at http://bioinfolab.miamioh.edu/jnsviewer/index.html.

  12. JNSViewer—A JavaScript-based Nucleotide Sequence Viewer for DNA/RNA secondary structures

    Science.gov (United States)

    Dong, Min; Graham, Mitchell; Yadav, Nehul

    2017-01-01

    Many tools are available for visualizing RNA or DNA secondary structures, but there is scarce implementation in JavaScript that provides seamless integration with the increasingly popular web computational platforms. We have developed JNSViewer, a highly interactive web service, which is bundled with several popular tools for DNA/RNA secondary structure prediction and can provide precise and interactive correspondence among nucleotides, dot-bracket data, secondary structure graphs, and genic annotations. In JNSViewer, users can perform RNA secondary structure predictions with different programs and settings, add customized genic annotations in GFF format to structure graphs, search for specific linear motifs, and extract relevant structure graphs of sub-sequences. JNSViewer also allows users to choose a transcript or specific segment of Arabidopsis thaliana genome sequences and predict the corresponding secondary structure. Popular genome browsers (i.e., JBrowse and BrowserGenome) were integrated into JNSViewer to provide powerful visualizations of chromosomal locations, genic annotations, and secondary structures. In addition, we used StructureFold with default settings to predict some RNA structures for Arabidopsis by incorporating in vivo high-throughput RNA structure profiling data and stored the results in our web server, which might be a useful resource for RNA secondary structure studies in plants. JNSViewer is available at http://bioinfolab.miamioh.edu/jnsviewer/index.html. PMID:28582416

  13. JNSViewer-A JavaScript-based Nucleotide Sequence Viewer for DNA/RNA secondary structures.

    Directory of Open Access Journals (Sweden)

    Jieming Shi

    Full Text Available Many tools are available for visualizing RNA or DNA secondary structures, but there is scarce implementation in JavaScript that provides seamless integration with the increasingly popular web computational platforms. We have developed JNSViewer, a highly interactive web service, which is bundled with several popular tools for DNA/RNA secondary structure prediction and can provide precise and interactive correspondence among nucleotides, dot-bracket data, secondary structure graphs, and genic annotations. In JNSViewer, users can perform RNA secondary structure predictions with different programs and settings, add customized genic annotations in GFF format to structure graphs, search for specific linear motifs, and extract relevant structure graphs of sub-sequences. JNSViewer also allows users to choose a transcript or specific segment of Arabidopsis thaliana genome sequences and predict the corresponding secondary structure. Popular genome browsers (i.e., JBrowse and BrowserGenome were integrated into JNSViewer to provide powerful visualizations of chromosomal locations, genic annotations, and secondary structures. In addition, we used StructureFold with default settings to predict some RNA structures for Arabidopsis by incorporating in vivo high-throughput RNA structure profiling data and stored the results in our web server, which might be a useful resource for RNA secondary structure studies in plants. JNSViewer is available at http://bioinfolab.miamioh.edu/jnsviewer/index.html.

  14. Computational strategies for the automated design of RNA nanoscale structures from building blocks using NanoTiler.

    Science.gov (United States)

    Bindewald, Eckart; Grunewald, Calvin; Boyle, Brett; O'Connor, Mary; Shapiro, Bruce A

    2008-10-01

    One approach to designing RNA nanoscale structures is to use known RNA structural motifs such as junctions, kissing loops or bulges and to construct a molecular model by connecting these building blocks with helical struts. We previously developed an algorithm for detecting internal loops, junctions and kissing loops in RNA structures. Here we present algorithms for automating or assisting many of the steps that are involved in creating RNA structures from building blocks: (1) assembling building blocks into nanostructures using either a combinatorial search or constraint satisfaction; (2) optimizing RNA 3D ring structures to improve ring closure; (3) sequence optimisation; (4) creating a unique non-degenerate RNA topology descriptor. This effectively creates a computational pipeline for generating molecular models of RNA nanostructures and more specifically RNA ring structures with optimized sequences from RNA building blocks. We show several examples of how the algorithms can be utilized to generate RNA tecto-shapes.

  15. Computational strategies for the automated design of RNA nanoscale structures from building blocks using NanoTiler☆

    Science.gov (United States)

    Bindewald, Eckart; Grunewald, Calvin; Boyle, Brett; O’Connor, Mary; Shapiro, Bruce A.

    2013-01-01

    One approach to designing RNA nanoscale structures is to use known RNA structural motifs such as junctions, kissing loops or bulges and to construct a molecular model by connecting these building blocks with helical struts. We previously developed an algorithm for detecting internal loops, junctions and kissing loops in RNA structures. Here we present algorithms for automating or assisting many of the steps that are involved in creating RNA structures from building blocks: (1) assembling building blocks into nanostructures using either a combinatorial search or constraint satisfaction; (2) optimizing RNA 3D ring structures to improve ring closure; (3) sequence optimisation; (4) creating a unique non-degenerate RNA topology descriptor. This effectively creates a computational pipeline for generating molecular models of RNA nanostructures and more specifically RNA ring structures with optimized sequences from RNA building blocks. We show several examples of how the algorithms can be utilized to generate RNA tecto-shapes. PMID:18838281

  16. MCTBI: a web server for predicting metal ion effects in RNA structures.

    Science.gov (United States)

    Sun, Li-Zhen; Zhang, Jing-Xiang; Chen, Shi-Jie

    2017-08-01

    Metal ions play critical roles in RNA structure and function. However, web servers and software packages for predicting ion effects in RNA structures are notably scarce. Furthermore, the existing web servers and software packages mainly neglect ion correlation and fluctuation effects, which are potentially important for RNAs. We here report a new web server, the MCTBI server (http://rna.physics.missouri.edu/MCTBI), for the prediction of ion effects for RNA structures. This server is based on the recently developed MCTBI, a model that can account for ion correlation and fluctuation effects for nucleic acid structures and can provide improved predictions for the effects of metal ions, especially for multivalent ions such as Mg 2+ effects, as shown by extensive theory-experiment test results. The MCTBI web server predicts metal ion binding fractions, the most probable bound ion distribution, the electrostatic free energy of the system, and the free energy components. The results provide mechanistic insights into the role of metal ions in RNA structure formation and folding stability, which is important for understanding RNA functions and the rational design of RNA structures. © 2017 Sun et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  17. Structure of Hepatitis C Virus Polymerase in Complex with Primer-Template RNA

    Energy Technology Data Exchange (ETDEWEB)

    Mosley, Ralph T.; Edwards, Thomas E.; Murakami, Eisuke; Lam, Angela M.; Grice, Rena L.; Du, Jinfa; Sofia, Michael J.; Furman, Philip A.; Otto, Michael J. (Pharmasset); (Emerald)

    2012-08-01

    The replication of the hepatitis C viral (HCV) genome is accomplished by the NS5B RNA-dependent RNA polymerase (RdRp), for which mechanistic understanding and structure-guided drug design efforts have been hampered by its propensity to crystallize in a closed, polymerization-incompetent state. The removal of an autoinhibitory {beta}-hairpin loop from genotype 2a HCV NS5B increases de novo RNA synthesis by >100-fold, promotes RNA binding, and facilitated the determination of the first crystallographic structures of HCV polymerase in complex with RNA primer-template pairs. These crystal structures demonstrate the structural realignment required for primer-template recognition and elongation, provide new insights into HCV RNA synthesis at the molecular level, and may prove useful in the structure-based design of novel antiviral compounds. Additionally, our approach for obtaining the RNA primer-template-bound structure of HCV polymerase may be generally applicable to solving RNA-bound complexes for other viral RdRps that contain similar regulatory {beta}-hairpin loops, including bovine viral diarrhea virus, dengue virus, and West Nile virus.

  18. A comparative method for finding and folding RNA secondary structures within protein-coding regions

    DEFF Research Database (Denmark)

    Pedersen, Jakob Skou; Meyer, Irmtraud Margret; Forsberg, Roald

    2004-01-01

    that RNA-DECODER's parameters can be automatically trained to successfully fold known secondary structures within the HCV genome. We scan the genomes of HCV and polio virus for conserved secondary-structure elements, and analyze performance as a function of available evolutionary information. On known...... secondary structures, RNA-DECODER shows a sensitivity similar to the programs MFOLD, PFOLD and RNAALIFOLD. When scanning the entire genomes of HCV and polio virus for structure elements, RNA-DECODER's results indicate a markedly higher specificity than MFOLD, PFOLD and RNAALIFOLD....

  19. De Novo Discovery of Structured ncRNA Motifs in Genomic Sequences

    DEFF Research Database (Denmark)

    Ruzzo, Walter L; Gorodkin, Jan

    2014-01-01

    De novo discovery of "motifs" capturing the commonalities among related noncoding ncRNA structured RNAs is among the most difficult problems in computational biology. This chapter outlines the challenges presented by this problem, together with some approaches towards solving them, with an emphas...... on an approach based on the CMfinder CMfinder program as a case study. Applications to genomic screens for novel de novo structured ncRNA ncRNA s, including structured RNA elements in untranslated portions of protein-coding genes, are presented.......De novo discovery of "motifs" capturing the commonalities among related noncoding ncRNA structured RNAs is among the most difficult problems in computational biology. This chapter outlines the challenges presented by this problem, together with some approaches towards solving them, with an emphasis...

  20. TRANSAT-- method for detecting the conserved helices of functional RNA structures, including transient, pseudo-knotted and alternative structures.

    Science.gov (United States)

    Wiebe, Nicholas J P; Meyer, Irmtraud M

    2010-06-24

    The prediction of functional RNA structures has attracted increased interest, as it allows us to study the potential functional roles of many genes. RNA structure prediction methods, however, assume that there is a unique functional RNA structure and also do not predict functional features required for in vivo folding. In order to understand how functional RNA structures form in vivo, we require sophisticated experiments or reliable prediction methods. So far, there exist only a few, experimentally validated transient RNA structures. On the computational side, there exist several computer programs which aim to predict the co-transcriptional folding pathway in vivo, but these make a range of simplifying assumptions and do not capture all features known to influence RNA folding in vivo. We want to investigate if evolutionarily related RNA genes fold in a similar way in vivo. To this end, we have developed a new computational method, Transat, which detects conserved helices of high statistical significance. We introduce the method, present a comprehensive performance evaluation and show that Transat is able to predict the structural features of known reference structures including pseudo-knotted ones as well as those of known alternative structural configurations. Transat can also identify unstructured sub-sequences bound by other molecules and provides evidence for new helices which may define folding pathways, supporting the notion that homologous RNA sequence not only assume a similar reference RNA structure, but also fold similarly. Finally, we show that the structural features predicted by Transat differ from those assuming thermodynamic equilibrium. Unlike the existing methods for predicting folding pathways, our method works in a comparative way. This has the disadvantage of not being able to predict features as function of time, but has the considerable advantage of highlighting conserved features and of not requiring a detailed knowledge of the cellular

  1. Structure of Lipid Nanoparticles Containing siRNA or mRNA by Dynamic Nuclear Polarization-Enhanced NMR Spectroscopy.

    Science.gov (United States)

    Viger-Gravel, Jasmine; Schantz, Anna; Pinon, Arthur C; Rossini, Aaron J; Schantz, Staffan; Emsley, Lyndon

    2018-02-22

    Here, we show how dynamic nuclear polarization (DNP) NMR spectroscopy experiments permit the atomic level structural characterization of loaded and empty lipid nanoparticles (LNPs). The LNPs used here were synthesized by the microfluidic mixing technique and are composed of ionizable cationic lipid (DLin-MC3-DMA), a phospholipid (distearoylphosphatidylcholine, DSPC), cholesterol, and poly(ethylene glycol) (PEG) (dimyristoyl phosphatidyl ethanolamine (DMPE)-PEG 2000), as well as encapsulated cargoes that are either phosphorothioated siRNA (50 or 100%) or mRNA. We show that LNPs form physically stable complexes with bioactive drug siRNA for a period of 94 days. Relayed DNP experiments are performed to study 1 H- 1 H spin diffusion and to determine the spatial location of the various components of the LNP by studying the average enhancement factors as a function of polarization time. We observe a striking feature of LNPs in the presence and in the absence of encapsulating siRNA or mRNA by comparing our experimental results to numerical spin-diffusion modeling. We observe that LNPs form a layered structure, and we detect that DSPC and DMPE-PEG 2000 lipids form a surface rich layer in the presence (or absence) of the cargoes and that the cholesterol and ionizable cationic lipid are embedded in the core. Furthermore, relayed DNP 31 P solid-state NMR experiments allow the location of the cargo encapsulated in the LNPs to be determined. On the basis of the results, we propose a new structural model for the LNPs that features a homogeneous core with a tendency for layering of DSPC and DMPE-PEG at the surface.

  2. Evolving stochastic context-free grammars for RNA secondary structure prediction

    DEFF Research Database (Denmark)

    Anderson, James WJ; Tataru, Paula Cristina; Stains, Joe

    2012-01-01

    Background Stochastic Context-Free Grammars (SCFGs) were applied successfully to RNA secondary structure prediction in the early 90s, and used in combination with comparative methods in the late 90s. The set of SCFGs potentially useful for RNA secondary structure prediction is very large, but a few...... to structure prediction as has been previously suggested. Results These search techniques were applied to predict RNA secondary structure on a maximal data set and revealed new and interesting grammars, though none are dramatically better than classic grammars. In general, results showed that many grammars...... with quite different structure could have very similar predictive ability. Many ambiguous grammars were found which were at least as effective as the best current unambiguous grammars. Conclusions Overall the method of evolving SCFGs for RNA secondary structure prediction proved effective in finding many...

  3. CentroidFold: a web server for RNA secondary structure prediction

    OpenAIRE

    Sato, Kengo; Hamada, Michiaki; Asai, Kiyoshi; Mituyama, Toutai

    2009-01-01

    The CentroidFold web server (http://www.ncrna.org/centroidfold/) is a web application for RNA secondary structure prediction powered by one of the most accurate prediction engine. The server accepts two kinds of sequence data: a single RNA sequence and a multiple alignment of RNA sequences. It responses with a prediction result shown as a popular base-pair notation and a graph representation. PDF version of the graph representation is also available. For a multiple alignment sequence, the ser...

  4. RNA secondary structure prediction by using discrete mathematics: an interdisciplinary research experience for undergraduate students.

    Science.gov (United States)

    Ellington, Roni; Wachira, James; Nkwanta, Asamoah

    2010-01-01

    The focus of this Research Experience for Undergraduates (REU) project was on RNA secondary structure prediction by using a lattice walk approach. The lattice walk approach is a combinatorial and computational biology method used to enumerate possible secondary structures and predict RNA secondary structure from RNA sequences. The method uses discrete mathematical techniques and identifies specified base pairs as parameters. The goal of the REU was to introduce upper-level undergraduate students to the principles and challenges of interdisciplinary research in molecular biology and discrete mathematics. At the beginning of the project, students from the biology and mathematics departments of a mid-sized university received instruction on the role of secondary structure in the function of eukaryotic RNAs and RNA viruses, RNA related to combinatorics, and the National Center for Biotechnology Information resources. The student research projects focused on RNA secondary structure prediction on a regulatory region of the yellow fever virus RNA genome and on an untranslated region of an mRNA of a gene associated with the neurological disorder epilepsy. At the end of the project, the REU students gave poster and oral presentations, and they submitted written final project reports to the program director. The outcome of the REU was that the students gained transferable knowledge and skills in bioinformatics and an awareness of the applications of discrete mathematics to biological research problems.

  5. RNA Secondary Structure Prediction by Using Discrete Mathematics: An Interdisciplinary Research Experience for Undergraduate Students

    Science.gov (United States)

    Ellington, Roni; Wachira, James

    2010-01-01

    The focus of this Research Experience for Undergraduates (REU) project was on RNA secondary structure prediction by using a lattice walk approach. The lattice walk approach is a combinatorial and computational biology method used to enumerate possible secondary structures and predict RNA secondary structure from RNA sequences. The method uses discrete mathematical techniques and identifies specified base pairs as parameters. The goal of the REU was to introduce upper-level undergraduate students to the principles and challenges of interdisciplinary research in molecular biology and discrete mathematics. At the beginning of the project, students from the biology and mathematics departments of a mid-sized university received instruction on the role of secondary structure in the function of eukaryotic RNAs and RNA viruses, RNA related to combinatorics, and the National Center for Biotechnology Information resources. The student research projects focused on RNA secondary structure prediction on a regulatory region of the yellow fever virus RNA genome and on an untranslated region of an mRNA of a gene associated with the neurological disorder epilepsy. At the end of the project, the REU students gave poster and oral presentations, and they submitted written final project reports to the program director. The outcome of the REU was that the students gained transferable knowledge and skills in bioinformatics and an awareness of the applications of discrete mathematics to biological research problems. PMID:20810968

  6. Structural insights into RNA processing by the human RISC-loading complex.

    Science.gov (United States)

    Wang, Hong-Wei; Noland, Cameron; Siridechadilok, Bunpote; Taylor, David W; Ma, Enbo; Felderer, Karin; Doudna, Jennifer A; Nogales, Eva

    2009-11-01

    Targeted gene silencing by RNA interference (RNAi) requires loading of a short guide RNA (small interfering RNA (siRNA) or microRNA (miRNA)) onto an Argonaute protein to form the functional center of an RNA-induced silencing complex (RISC). In humans, Argonaute2 (AGO2) assembles with the guide RNA-generating enzyme Dicer and the RNA-binding protein TRBP to form a RISC-loading complex (RLC), which is necessary for efficient transfer of nascent siRNAs and miRNAs from Dicer to AGO2. Here, using single-particle EM analysis, we show that human Dicer has an L-shaped structure. The RLC Dicer's N-terminal DExH/D domain, located in a short 'base branch', interacts with TRBP, whereas its C-terminal catalytic domains in the main body are proximal to AGO2. A model generated by docking the available atomic structures of Dicer and Argonaute homologs into the RLC reconstruction suggests a mechanism for siRNA transfer from Dicer to AGO2.

  7. Structural plasticity of Barley yellow dwarf virus-like cap-independent translation elements in four genera of plant viral RNAs.

    Science.gov (United States)

    Wang, Zhaohui; Kraft, Jelena J; Hui, Alice Y; Miller, W Allen

    2010-06-20

    The 3' untranslated regions (UTRs) of many plant viral RNAs contain cap-independent translation elements (3' CITEs). Among the 3' CITEs, the Barley yellow dwarf virus (BYDV)-like translation elements (BTEs) form a structurally variable and widely distributed group. Viruses in three genera were known to harbor 3' BTEs, defined by the presence of a 17-nt consensus sequence. To understand BTE function, knowledge of phylogenetically conserved structure is essential, yet the secondary structure has been determined only for the BYDV BTE. Here we show that Rose spring dwarf-associated luteovirus, and two viruses in a fourth genus, Umbravirus, contain functional BTEs, despite deviating in the 17nt consensus sequence. Structure probing by selective 2'-hydroxyl acylation and primer extension (SHAPE) revealed conserved and highly variable structures in BTEs in all four genera. We conclude that BTEs tolerate striking evolutionary plasticity in structure, while retaining the ability to stimulate cap-independent translation. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  8. Visualizing RNA Secondary Structure Base Pair Binding Probabilities using Nested Concave Hulls

    OpenAIRE

    Sansen , Joris; Bourqui , Romain; Thebault , Patricia; Allali , Julien; Auber , David

    2015-01-01

    International audience; The challenge 1 of the BIOVIS 2015 design contest consists in designing an intuitive visual depiction of base pairs binding probabilities for secondary structure of ncRNA. Our representation depicts the potential nucleotide pairs binding using nested concave hulls over the computed MFE ncRNA secondary structure. Thus, it allows to identify regions with a high level of uncertainty in the MFE computation and the structures which seem to match to reality.

  9. The Globular State of the Single-Stranded RNA: Effect of the Secondary Structure Rearrangements

    Science.gov (United States)

    Grigoryan, Zareh A.; Karapetian, Armen T.

    2015-01-01

    The mutual influence of the slow rearrangements of secondary structure and fast collapse of the long single-stranded RNA (ssRNA) in approximation of coarse-grained model is studied with analytic calculations. It is assumed that the characteristic time of the secondary structure rearrangement is much longer than that for the formation of the tertiary structure. A nonequilibrium phase transition of the 2nd order has been observed. PMID:26345143

  10. The Globular State of the Single-Stranded RNA: Effect of the Secondary Structure Rearrangements

    Directory of Open Access Journals (Sweden)

    Zareh A. Grigoryan

    2015-01-01

    Full Text Available The mutual influence of the slow rearrangements of secondary structure and fast collapse of the long single-stranded RNA (ssRNA in approximation of coarse-grained model is studied with analytic calculations. It is assumed that the characteristic time of the secondary structure rearrangement is much longer than that for the formation of the tertiary structure. A nonequilibrium phase transition of the 2nd order has been observed.

  11. Crystal structure analysis reveals functional flexibility in the selenocysteine-specific tRNA from mouse.

    Directory of Open Access Journals (Sweden)

    Oleg M Ganichkin

    Full Text Available Selenocysteine tRNAs (tRNA(Sec exhibit a number of unique identity elements that are recognized specifically by proteins of the selenocysteine biosynthetic pathways and decoding machineries. Presently, these identity elements and the mechanisms by which they are interpreted by tRNA(Sec-interacting factors are incompletely understood.We applied rational mutagenesis to obtain well diffracting crystals of murine tRNA(Sec. tRNA(Sec lacking the single-stranded 3'-acceptor end ((ΔGCCARNA(Sec yielded a crystal structure at 2.0 Å resolution. The global structure of (ΔGCCARNA(Sec resembles the structure of human tRNA(Sec determined at 3.1 Å resolution. Structural comparisons revealed flexible regions in tRNA(Sec used for induced fit binding to selenophosphate synthetase. Water molecules located in the present structure were involved in the stabilization of two alternative conformations of the anticodon stem-loop. Modeling of a 2'-O-methylated ribose at position U34 of the anticodon loop as found in a sub-population of tRNA(Secin vivo showed how this modification favors an anticodon loop conformation that is functional during decoding on the ribosome. Soaking of crystals in Mn(2+-containing buffer revealed eight potential divalent metal ion binding sites but the located metal ions did not significantly stabilize specific structural features of tRNA(Sec.We provide the most highly resolved structure of a tRNA(Sec molecule to date and assessed the influence of water molecules and metal ions on the molecule's conformation and dynamics. Our results suggest how conformational changes of tRNA(Sec support its interaction with proteins.

  12. An image processing approach to computing distances between RNA secondary structures dot plots

    Directory of Open Access Journals (Sweden)

    Sapiro Guillermo

    2009-02-01

    Full Text Available Abstract Background Computing the distance between two RNA secondary structures can contribute in understanding the functional relationship between them. When used repeatedly, such a procedure may lead to finding a query RNA structure of interest in a database of structures. Several methods are available for computing distances between RNAs represented as strings or graphs, but none utilize the RNA representation with dot plots. Since dot plots are essentially digital images, there is a clear motivation to devise an algorithm for computing the distance between dot plots based on image processing methods. Results We have developed a new metric dubbed 'DoPloCompare', which compares two RNA structures. The method is based on comparing dot plot diagrams that represent the secondary structures. When analyzing two diagrams and motivated by image processing, the distance is based on a combination of histogram correlations and a geometrical distance measure. We introduce, describe, and illustrate the procedure by two applications that utilize this metric on RNA sequences. The first application is the RNA design problem, where the goal is to find the nucleotide sequence for a given secondary structure. Examples where our proposed distance measure outperforms others are given. The second application locates peculiar point mutations that induce significant structural alternations relative to the wild type predicted secondary structure. The approach reported in the past to solve this problem was tested on several RNA sequences with known secondary structures to affirm their prediction, as well as on a data set of ribosomal pieces. These pieces were computationally cut from a ribosome for which an experimentally derived secondary structure is available, and on each piece the prediction conveys similarity to the experimental result. Our newly proposed distance measure shows benefit in this problem as well when compared to standard methods used for assessing

  13. In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features.

    Science.gov (United States)

    Ding, Yiliang; Tang, Yin; Kwok, Chun Kit; Zhang, Yu; Bevilacqua, Philip C; Assmann, Sarah M

    2014-01-30

    RNA structure has critical roles in processes ranging from ligand sensing to the regulation of translation, polyadenylation and splicing. However, a lack of genome-wide in vivo RNA structural data has limited our understanding of how RNA structure regulates gene expression in living cells. Here we present a high-throughput, genome-wide in vivo RNA structure probing method, structure-seq, in which dimethyl sulphate methylation of unprotected adenines and cytosines is identified by next-generation sequencing. Application of this method to Arabidopsis thaliana seedlings yielded the first in vivo genome-wide RNA structure map at nucleotide resolution for any organism, with quantitative structural information across more than 10,000 transcripts. Our analysis reveals a three-nucleotide periodic repeat pattern in the structure of coding regions, as well as a less-structured region immediately upstream of the start codon, and shows that these features are strongly correlated with translation efficiency. We also find patterns of strong and weak secondary structure at sites of alternative polyadenylation, as well as strong secondary structure at 5' splice sites that correlates with unspliced events. Notably, in vivo structures of messenger RNAs annotated for stress responses are poorly predicted in silico, whereas mRNA structures of genes related to cell function maintenance are well predicted. Global comparison of several structural features between these two categories shows that the mRNAs associated with stress responses tend to have more single-strandedness, longer maximal loop length and higher free energy per nucleotide, features that may allow these RNAs to undergo conformational changes in response to environmental conditions. Structure-seq allows the RNA structurome and its biological roles to be interrogated on a genome-wide scale and should be applicable to any organism.

  14. Web-Beagle: a web server for the alignment of RNA secondary structures.

    Science.gov (United States)

    Mattei, Eugenio; Pietrosanto, Marco; Ferrè, Fabrizio; Helmer-Citterich, Manuela

    2015-07-01

    Web-Beagle (http://beagle.bio.uniroma2.it) is a web server for the pairwise global or local alignment of RNA secondary structures. The server exploits a new encoding for RNA secondary structure and a substitution matrix of RNA structural elements to perform RNA structural alignments. The web server allows the user to compute up to 10 000 alignments in a single run, taking as input sets of RNA sequences and structures or primary sequences alone. In the latter case, the server computes the secondary structure prediction for the RNAs on-the-fly using RNAfold (free energy minimization). The user can also compare a set of input RNAs to one of five pre-compiled RNA datasets including lncRNAs and 3' UTRs. All types of comparison produce in output the pairwise alignments along with structural similarity and statistical significance measures for each resulting alignment. A graphical color-coded representation of the alignments allows the user to easily identify structural similarities between RNAs. Web-Beagle can be used for finding structurally related regions in two or more RNAs, for the identification of homologous regions or for functional annotation. Benchmark tests show that Web-Beagle has lower computational complexity, running time and better performances than other available methods. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. RNA-TVcurve: a Web server for RNA secondary structure comparison based on a multi-scale similarity of its triple vector curve representation.

    Science.gov (United States)

    Li, Ying; Shi, Xiaohu; Liang, Yanchun; Xie, Juan; Zhang, Yu; Ma, Qin

    2017-01-21

    RNAs have been found to carry diverse functionalities in nature. Inferring the similarity between two given RNAs is a fundamental step to understand and interpret their functional relationship. The majority of functional RNAs show conserved secondary structures, rather than sequence conservation. Those algorithms relying on sequence-based features usually have limitations in their prediction performance. Hence, integrating RNA structure features is very critical for RNA analysis. Existing algorithms mainly fall into two categories: alignment-based and alignment-free. The alignment-free algorithms of RNA comparison usually have lower time complexity than alignment-based algorithms. An alignment-free RNA comparison algorithm was proposed, in which novel numerical representations RNA-TVcurve (triple vector curve representation) of RNA sequence and corresponding secondary structure features are provided. Then a multi-scale similarity score of two given RNAs was designed based on wavelet decomposition of their numerical representation. In support of RNA mutation and phylogenetic analysis, a web server (RNA-TVcurve) was designed based on this alignment-free RNA comparison algorithm. It provides three functional modules: 1) visualization of numerical representation of RNA secondary structure; 2) detection of single-point mutation based on secondary structure; and 3) comparison of pairwise and multiple RNA secondary structures. The inputs of the web server require RNA primary sequences, while corresponding secondary structures are optional. For the primary sequences alone, the web server can compute the secondary structures using free energy minimization algorithm in terms of RNAfold tool from Vienna RNA package. RNA-TVcurve is the first integrated web server, based on an alignment-free method, to deliver a suite of RNA analysis functions, including visualization, mutation analysis and multiple RNAs structure comparison. The comparison results with two popular RNA

  16. The identification and functional annotation of RNA structures conserved in vertebrates.

    Science.gov (United States)

    Seemann, Stefan E; Mirza, Aashiq H; Hansen, Claus; Bang-Berthelsen, Claus H; Garde, Christian; Christensen-Dalsgaard, Mikkel; Torarinsson, Elfar; Yao, Zizhen; Workman, Christopher T; Pociot, Flemming; Nielsen, Henrik; Tommerup, Niels; Ruzzo, Walter L; Gorodkin, Jan

    2017-08-01

    Structured elements of RNA molecules are essential in, e.g., RNA stabilization, localization, and protein interaction, and their conservation across species suggests a common functional role. We computationally screened vertebrate genomes for conserved RNA structures (CRSs), leveraging structure-based, rather than sequence-based, alignments. After careful correction for sequence identity and GC content, we predict ∼516,000 human genomic regions containing CRSs. We find that a substantial fraction of human-mouse CRS regions (1) colocalize consistently with binding sites of the same RNA binding proteins (RBPs) or (2) are transcribed in corresponding tissues. Additionally, a CaptureSeq experiment revealed expression of many of our CRS regions in human fetal brain, including 662 novel ones. For selected human and mouse candidate pairs, qRT-PCR and in vitro RNA structure probing supported both shared expression and shared structure despite low abundance and low sequence identity. About 30,000 CRS regions are located near coding or long noncoding RNA genes or within enhancers. Structured (CRS overlapping) enhancer RNAs and extended 3' ends have significantly increased expression levels over their nonstructured counterparts. Our findings of transcribed uncharacterized regulatory regions that contain CRSs support their RNA-mediated functionality. © 2017 Seemann et al.; Published by Cold Spring Harbor Laboratory Press.

  17. Structural Analysis of Monomeric RNA-Dependent Polymerases: Evolutionary and Therapeutic Implications.

    Directory of Open Access Journals (Sweden)

    Rodrigo Jácome

    Full Text Available The crystal structures of monomeric RNA-dependent RNA polymerases and reverse transcriptases of more than 20 different viruses are available in the Protein Data Bank. They all share the characteristic right-hand shape of DNA- and RNA polymerases formed by the fingers, palm and thumb subdomains, and, in many cases, "fingertips" that extend from the fingers towards the thumb subdomain, giving the viral enzyme a closed right-hand appearance. Six conserved structural motifs that contain key residues for the proper functioning of the enzyme have been identified in all these RNA-dependent polymerases. These enzymes share a two divalent metal-ion mechanism of polymerization in which two conserved aspartate residues coordinate the interactions with the metal ions to catalyze the nucleotidyl transfer reaction. The recent availability of crystal structures of polymerases of the Orthomyxoviridae and Bunyaviridae families allowed us to make pairwise comparisons of the tertiary structures of polymerases belonging to the four main RNA viral groups, which has led to a phylogenetic tree in which single-stranded negative RNA viral polymerases have been included for the first time. This has also allowed us to use a homology-based structural prediction approach to develop a general three-dimensional model of the Ebola virus RNA-dependent RNA polymerase. Our model includes several of the conserved structural motifs and residues described in other viral RNA-dependent RNA polymerases that define the catalytic and highly conserved palm subdomain, as well as portions of the fingers and thumb subdomains. The results presented here help to understand the current use and apparent success of antivirals, i.e. Brincidofovir, Lamivudine and Favipiravir, originally aimed at other types of polymerases, to counteract the Ebola virus infection.

  18. Integrated structural biology to unravel molecular mechanisms of protein-RNA recognition.

    Science.gov (United States)

    Schlundt, Andreas; Tants, Jan-Niklas; Sattler, Michael

    2017-04-15

    Recent advances in RNA sequencing technologies have greatly expanded our knowledge of the RNA landscape in cells, often with spatiotemporal resolution. These techniques identified many new (often non-coding) RNA molecules. Large-scale studies have also discovered novel RNA binding proteins (RBPs), which exhibit single or multiple RNA binding domains (RBDs) for recognition of specific sequence or structured motifs in RNA. Starting from these large-scale approaches it is crucial to unravel the molecular principles of protein-RNA recognition in ribonucleoprotein complexes (RNPs) to understand the underlying mechanisms of gene regulation. Structural biology and biophysical studies at highest possible resolution are key to elucidate molecular mechanisms of RNA recognition by RBPs and how conformational dynamics, weak interactions and cooperative binding contribute to the formation of specific, context-dependent RNPs. While large compact RNPs can be well studied by X-ray crystallography and cryo-EM, analysis of dynamics and weak interaction necessitates the use of solution methods to capture these properties. Here, we illustrate methods to study the structure and conformational dynamics of protein-RNA complexes in solution starting from the identification of interaction partners in a given RNP. Biophysical and biochemical techniques support the characterization of a protein-RNA complex and identify regions relevant in structural analysis. Nuclear magnetic resonance (NMR) is a powerful tool to gain information on folding, stability and dynamics of RNAs and characterize RNPs in solution. It provides crucial information that is complementary to the static pictures derived from other techniques. NMR can be readily combined with other solution techniques, such as small angle X-ray and/or neutron scattering (SAXS/SANS), electron paramagnetic resonance (EPR), and Förster resonance energy transfer (FRET), which provide information about overall shapes, internal domain

  19. The PETfold and PETcofold web servers for intra- and intermolecular structures of multiple RNA sequences

    DEFF Research Database (Denmark)

    Seemann, Ernst Stefan; Menzel, Karl Peter; Backofen, Rolf

    2011-01-01

    gene. We present web servers to analyze multiple RNA sequences for common RNA structure and for RNA interaction sites. The web servers are based on the recent PET (Probabilistic Evolutionary and Thermodynamic) models PETfold and PETcofold, but add user friendly features ranging from a graphical layer...... to interactive usage of the predictors. Additionally, the web servers provide direct access to annotated RNA alignments, such as the Rfam 10.0 database and multiple alignments of 16 vertebrate genomes with human. The web servers are freely available at: http://rth.dk/resources/petfold/...

  20. A novel knowledge-based potential for RNA 3D structure evaluation

    Science.gov (United States)

    Yang, Yi; Gu, Qi; Zhang, Ben-Gong; Shi, Ya-Zhou; Shao, Zhi-Gang

    2018-03-01

    Ribonucleic acids (RNAs) play a vital role in biology, and knowledge of their three-dimensional (3D) structure is required to understand their biological functions. Recently structural prediction methods have been developed to address this issue, but a series of RNA 3D structures are generally predicted by most existing methods. Therefore, the evaluation of the predicted structures is generally indispensable. Although several methods have been proposed to assess RNA 3D structures, the existing methods are not precise enough. In this work, a new all-atom knowledge-based potential is developed for more accurately evaluating RNA 3D structures. The potential not only includes local and nonlocal interactions but also fully considers the specificity of each RNA by introducing a retraining mechanism. Based on extensive test sets generated from independent methods, the proposed potential correctly distinguished the native state and ranked near-native conformations to effectively select the best. Furthermore, the proposed potential precisely captured RNA structural features such as base-stacking and base-pairing. Comparisons with existing potential methods show that the proposed potential is very reliable and accurate in RNA 3D structure evaluation. Project supported by the National Science Foundation of China (Grants Nos. 11605125, 11105054, 11274124, and 11401448).

  1. Study of RNA structures with a connection to random matrix theory

    International Nuclear Information System (INIS)

    Bhadola, Pradeep; Deo, Nivedita

    2015-01-01

    This manuscript investigates the level of complexity and thermodynamic properties of the real RNA structures and compares the properties with the random RNA sequences. A discussion on the similarities of thermodynamical properties of the real structures with the non linear random matrix model of RNA folding is presented. The structural information contained in the PDB file is exploited to get the base pairing information. The complexity of an RNA structure is defined by a topological quantity called genus which is calculated from the base pairing information. Thermodynamic analysis of the real structures is done numerically. The real structures have a minimum free energy which is very small compared to the randomly generated sequences of the same length. This analysis suggests that there are specific patterns in the structures which are preserved during the evolution of the sequences and certain sequences are discarded by the evolutionary process. Further analyzing the sequences of a fixed length reveal that the RNA structures exist in ensembles i.e. although all the sequences in the ensemble have different series of nucleotides (sequence) they fold into structures that have the same pairs of hydrogen bonding as well as the same minimum free energy. The specific heat of the RNA molecule is numerically estimated at different lengths. The specific heat curve with temperature shows a bump and for some RNA, a double peak behavior is observed. The same behavior is seen in the study of the random matrix model with non linear interaction of RNA folding. The bump in the non linear matrix model can be controlled by the change in the interaction strength.

  2. Correlation of RNA secondary structure statistics with thermodynamic stability and applications to folding.

    Science.gov (United States)

    Wu, Johnny C; Gardner, David P; Ozer, Stuart; Gutell, Robin R; Ren, Pengyu

    2009-08-28

    The accurate prediction of the secondary and tertiary structure of an RNA with different folding algorithms is dependent on several factors, including the energy functions. However, an RNA higher-order structure cannot be predicted accurately from its sequence based on a limited set of energy parameters. The inter- and intramolecular forces between this RNA and other small molecules and macromolecules, in addition to other factors in the cell such as pH, ionic strength, and temperature, influence the complex dynamics associated with transition of a single stranded RNA to its secondary and tertiary structure. Since all of the factors that affect the formation of an RNAs 3D structure cannot be determined experimentally, statistically derived potential energy has been used in the prediction of protein structure. In the current work, we evaluate the statistical free energy of various secondary structure motifs, including base-pair stacks, hairpin loops, and internal loops, using their statistical frequency obtained from the comparative analysis of more than 50,000 RNA sequences stored in the RNA Comparative Analysis Database (rCAD) at the Comparative RNA Web (CRW) Site. Statistical energy was computed from the structural statistics for several datasets. While the statistical energy for a base-pair stack correlates with experimentally derived free energy values, suggesting a Boltzmann-like distribution, variation is observed between different molecules and their location on the phylogenetic tree of life. Our statistical energy values calculated for several structural elements were utilized in the Mfold RNA-folding algorithm. The combined statistical energy values for base-pair stacks, hairpins and internal loop flanks result in a significant improvement in the accuracy of secondary structure prediction; the hairpin flanks contribute the most.

  3. Structure, dynamics and RNA binding of the multi-domain splicing factor TIA-1

    Science.gov (United States)

    Wang, Iren; Hennig, Janosch; Jagtap, Pravin Kumar Ankush; Sonntag, Miriam; Valcárcel, Juan; Sattler, Michael

    2014-01-01

    Alternative pre-messenger ribonucleic acid (pre-mRNA) splicing is an essential process in eukaryotic gene regulation. The T-cell intracellular antigen-1 (TIA-1) is an apoptosis-promoting factor that modulates alternative splicing of transcripts, including the pre-mRNA encoding the membrane receptor Fas. TIA-1 is a multi-domain ribonucleic acid (RNA) binding protein that recognizes poly-uridine tract RNA sequences to facilitate 5′ splice site recognition by the U1 small nuclear ribonucleoprotein (snRNP). Here, we characterize the RNA interaction and conformational dynamics of TIA-1 by nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC) and small angle X-ray scattering (SAXS). Our NMR-derived solution structure of TIA-1 RRM2–RRM3 (RRM2,3) reveals that RRM2 adopts a canonical RNA recognition motif (RRM) fold, while RRM3 is preceded by an non-canonical helix α0. NMR and SAXS data show that all three RRMs are largely independent structural modules in the absence of RNA, while RNA binding induces a compact arrangement. RRM2,3 binds to pyrimidine-rich FAS pre-mRNA or poly-uridine (U9) RNA with nanomolar affinities. RRM1 has little intrinsic RNA binding affinity and does not strongly contribute to RNA binding in the context of RRM1,2,3. Our data unravel the role of binding avidity and the contributions of the TIA-1 RRMs for recognition of pyrimidine-rich RNAs. PMID:24682828

  4. Advancing viral RNA structure prediction: measuring the thermodynamics of pyrimidine-rich internal loops.

    Science.gov (United States)

    Phan, Andy; Mailey, Katherine; Saeki, Jessica; Gu, Xiaobo; Schroeder, Susan J

    2017-05-01

    Accurate thermodynamic parameters improve RNA structure predictions and thus accelerate understanding of RNA function and the identification of RNA drug binding sites. Many viral RNA structures, such as internal ribosome entry sites, have internal loops and bulges that are potential drug target sites. Current models used to predict internal loops are biased toward small, symmetric purine loops, and thus poorly predict asymmetric, pyrimidine-rich loops with >6 nucleotides (nt) that occur frequently in viral RNA. This article presents new thermodynamic data for 40 pyrimidine loops, many of which can form UU or protonated CC base pairs. Uracil and protonated cytosine base pairs stabilize asymmetric internal loops. Accurate prediction rules are presented that account for all thermodynamic measurements of RNA asymmetric internal loops. New loop initiation terms for loops with >6 nt are presented that do not follow previous assumptions that increasing asymmetry destabilizes loops. Since the last 2004 update, 126 new loops with asymmetry or sizes greater than 2 × 2 have been measured. These new measurements significantly deepen and diversify the thermodynamic database for RNA. These results will help better predict internal loops that are larger, pyrimidine-rich, and occur within viral structures such as internal ribosome entry sites. © 2017 Phan et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  5. The crystal structure and RNA-binding of an orthomyxovirus nucleoprotein.

    Directory of Open Access Journals (Sweden)

    Wenjie Zheng

    2013-09-01

    Full Text Available Genome packaging for viruses with segmented genomes is often a complex problem. This is particularly true for influenza viruses and other orthomyxoviruses, whose genome consists of multiple negative-sense RNAs encapsidated as ribonucleoprotein (RNP complexes. To better understand the structural features of orthomyxovirus RNPs that allow them to be packaged, we determined the crystal structure of the nucleoprotein (NP of a fish orthomyxovirus, the infectious salmon anemia virus (ISAV (genus Isavirus. As the major protein component of the RNPs, ISAV-NP possesses a bi-lobular structure similar to the influenza virus NP. Because both RNA-free and RNA-bound ISAV NP forms stable dimers in solution, we were able to measure the NP RNA binding affinity as well as the stoichiometry using recombinant proteins and synthetic oligos. Our RNA binding analysis revealed that each ISAV-NP binds ~12 nts of RNA, shorter than the 24-28 nts originally estimated for the influenza A virus NP based on population average. The 12-nt stoichiometry was further confirmed by results from electron microscopy and dynamic light scattering. Considering that RNPs of ISAV and the influenza viruses have similar morphologies and dimensions, our findings suggest that NP-free RNA may exist on orthomyxovirus RNPs, and selective RNP packaging may be accomplished through direct RNA-RNA interactions.

  6. Exact calculation of loop formation probability identifies folding motifs in RNA secondary structures

    Science.gov (United States)

    Sloma, Michael F.; Mathews, David H.

    2016-01-01

    RNA secondary structure prediction is widely used to analyze RNA sequences. In an RNA partition function calculation, free energy nearest neighbor parameters are used in a dynamic programming algorithm to estimate statistical properties of the secondary structure ensemble. Previously, partition functions have largely been used to estimate the probability that a given pair of nucleotides form a base pair, the conditional stacking probability, the accessibility to binding of a continuous stretch of nucleotides, or a representative sample of RNA structures. Here it is demonstrated that an RNA partition function can also be used to calculate the exact probability of formation of hairpin loops, internal loops, bulge loops, or multibranch loops at a given position. This calculation can also be used to estimate the probability of formation of specific helices. Benchmarking on a set of RNA sequences with known secondary structures indicated that loops that were calculated to be more probable were more likely to be present in the known structure than less probable loops. Furthermore, highly probable loops are more likely to be in the known structure than the set of loops predicted in the lowest free energy structures. PMID:27852924

  7. Capturing alternative secondary structures of RNA by decomposition of base-pairing probabilities.

    Science.gov (United States)

    Hagio, Taichi; Sakuraba, Shun; Iwakiri, Junichi; Mori, Ryota; Asai, Kiyoshi

    2018-02-19

    It is known that functional RNAs often switch their functions by forming different secondary structures. Popular tools for RNA secondary structures prediction, however, predict the single 'best' structures, and do not produce alternative structures. There are bioinformatics tools to predict suboptimal structures, but it is difficult to detect which alternative secondary structures are essential. We proposed a new computational method to detect essential alternative secondary structures from RNA sequences by decomposing the base-pairing probability matrix. The decomposition is calculated by a newly implemented software tool, RintW, which efficiently computes the base-pairing probability distributions over the Hamming distance from arbitrary reference secondary structures. The proposed approach has been demonstrated on ROSE element RNA thermometer sequence and Lysine RNA ribo-switch, showing that the proposed approach captures conformational changes in secondary structures. We have shown that alternative secondary structures are captured by decomposing base-paring probabilities over Hamming distance. Source code is available from http://www.ncRNA.org/RintW .

  8. URS DataBase: universe of RNA structures and their motifs.

    Science.gov (United States)

    Baulin, Eugene; Yacovlev, Victor; Khachko, Denis; Spirin, Sergei; Roytberg, Mikhail

    2016-01-01

    The Universe of RNA Structures DataBase (URSDB) stores information obtained from all RNA-containing PDB entries (2935 entries in October 2015). The content of the database is updated regularly. The database consists of 51 tables containing indexed data on various elements of the RNA structures. The database provides a web interface allowing user to select a subset of structures with desired features and to obtain various statistical data for a selected subset of structures or for all structures. In particular, one can easily obtain statistics on geometric parameters of base pairs, on structural motifs (stems, loops, etc.) or on different types of pseudoknots. The user can also view and get information on an individual structure or its selected parts, e.g. RNA-protein hydrogen bonds. URSDB employs a new original definition of loops in RNA structures. That definition fits both pseudoknot-free and pseudoknotted secondary structures and coincides with the classical definition in case of pseudoknot-free structures. To our knowledge, URSDB is the first database supporting searches based on topological classification of pseudoknots and on extended loop classification.Database URL: http://server3.lpm.org.ru/urs/. © The Author(s) 2016. Published by Oxford University Press.

  9. Structural organization of the transfer RNA operon I of Vibrio cholerae

    Indian Academy of Sciences (India)

    Unknown

    [Ghatak A, Majumdar A and Ghosh R K 2005 Structural organization of the transfer RNA operon I of Vibrio cholerae: Differences ..... clonal relationship are of utmost importance. ... rately derived from environmental, nontoxigenic, non-O1.

  10. Determination of low-energy structures of a small RNA hairpin using ...

    Indian Academy of Sciences (India)

    2012-06-25

    Jun 25, 2012 ... such as water clusters, Argon clusters and different biomo- lecules. ... Keywords. Energy landscape; Monte Carlo; optimization; RNA; tertiary structure .... tui large ribosomal subunit (Residues 310–321, PDB ID: 1JJ2).

  11. Quantitation of base substitutions in eukaryotic 5S rRNA: selection for the maintenance of RNA secondary structure.

    Science.gov (United States)

    Curtiss, W C; Vournakis, J N

    1984-01-01

    Eukaryotic 5S rRNA sequences from 34 diverse species were compared by the following method: (1) The sequences were aligned; (2) the positions of substitutions were located by comparison of all possible pairs of sequences; (3) the substitution sites were mapped to an assumed general base pairing model; and (4) the R-Y model of base stacking was used to study stacking pattern relationships in the structure. An analysis of the sequence and structure variability in each region of the molecule is presented. It was found that the degree of base substitution varies over a wide range, from absolute conservation to occurrence of over 90% of the possible observable substitutions. The substitutions are located primarily in stem regions of the 5S rRNA secondary structure. More than 88% of the substitutions in helical regions maintain base pairing. The disruptive substitutions are primarily located at the edges of helical regions, resulting in shortening of the helical regions and lengthening of the adjacent nonpaired regions. Base stacking patterns determined by the R-Y model are mapped onto the general secondary structure. Intrastrand and interstrand stacking could stabilize alternative coaxial structures and limit the conformational flexibility of nonpaired regions. Two short contiguous regions are 100% conserved in all species. This may reflect evolutionary constraints imposed at the DNA level by the requirement for binding of a 5S gene transcription initiation factor during gene expression.

  12. Simulation study of HEMT structures with HfO{sub 2} cap layer for mitigating inverse piezoelectric effect related device failures

    Energy Technology Data Exchange (ETDEWEB)

    Nagulapally, Deepthi; Joshi, Ravi P., E-mail: rjoshi@odu.edu [Department of Electrical and Computer Engineering, Old Dominion University, Norfolk, VA 23529-0246 (United States); Pradhan, Aswini [Department of Engineering and Center for Materials Research, Norfolk State University, 700 Park Avenue, Norfolk, VA 23504 (United States)

    2015-01-15

    The Inverse Piezoelectric Effect (IPE) is thought to contribute to possible device failure of GaN High Electron Mobility Transistors (HEMTs). Here we focus on a simulation study to probe the possible mitigation of the IPE by reducing the internal electric fields and related elastic energy through the use of high-k materials. Inclusion of a HfO{sub 2} “cap layer” above the AlGaN barrier particularly with a partial mesa structure is shown to have potential advantages. Simulations reveal even greater reductions in the internal electric fields by using “field plates” in concert with high-k oxides.

  13. Influence of an Fe cap layer on the structural and magnetic properties of Fe49Pt51/Fe bi-layers

    International Nuclear Information System (INIS)

    Chao-Yang, Duan; Bin, Ma; Zong-Zhi, Zhang; Qing-Yuan, Jin; Fu-Lin, Wei

    2009-01-01

    The influences of an Fe cap layer on the structural and magnetic properties of FePt/Fe bi-layers are investigated. Compared with single FePt alloy films, a thin Fe layer can affect the crystalline orientation and improve the chemical ordering of L1 0 FePt films. Moreover, the coercivity increases when a thin Fe layer covers the FePt layer. Beyond a critical thickness, however, the Fe cover layer quickens the magnetization reversal of Fe 49 Pt 51 /Fe bi-layers by their exchange coupling

  14. In vitro synthesis of minus-strand RNA by an isolated cereal yellow dwarf virus RNA-dependent RNA polymerase requires VPg and a stem-loop structure at the 3' end of the virus RNA.

    Science.gov (United States)

    Osman, Toba A M; Coutts, Robert H A; Buck, Kenneth W

    2006-11-01

    Cereal yellow dwarf virus (CYDV) RNA has a 5'-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, being able to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, Red clover necrotic mosaic virus, Cucumber mosaic virus, and Tobacco mosaic virus. RNA synthesis catalyzed by the RdRp required a 3'-terminal GU sequence and the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3' terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge in the stem, and the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3' end of the template. The data are consistent with a model in which the RdRp binds to the stem-loop structure which positions the active site to recognize the 3'-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg.

  15. The FOLDALIGN web server for pairwise structural RNA alignment and mutual motif search

    DEFF Research Database (Denmark)

    Havgaard, Jakob Hull; Lyngsø, Rune B.; Gorodkin, Jan

    2005-01-01

    FOLDALIGN is a Sankoff-based algorithm for making structural alignments of RNA sequences. Here, we present a web server for making pairwise alignments between two RNA sequences, using the recently updated version of FOLDALIGN. The server can be used to scan two sequences for a common structural RNA...... motif of limited size, or the entire sequences can be aligned locally or globally. The web server offers a graphical interface, which makes it simple to make alignments and manually browse the results. the web server can be accessed at http://foldalign.kvl.dk...

  16. Unzippers, Resolvers and Sensors: A Structural and Functional Biochemistry Tale of RNA Helicases

    Directory of Open Access Journals (Sweden)

    Ana Lúcia Leitão

    2015-01-01

    Full Text Available The centrality of RNA within the biological world is an irrefutable fact that currently attracts increasing attention from the scientific community. The panoply of functional RNAs requires the existence of specific biological caretakers, RNA helicases, devoted to maintain the proper folding of those molecules, resolving unstable structures. However, evolution has taken advantage of the specific position and characteristics of RNA helicases to develop new functions for these proteins, which are at the interface of the basic processes for transference of information from DNA to proteins. RNA helicases are involved in many biologically relevant processes, not only as RNA chaperones, but also as signal transducers, scaffolds of molecular complexes, and regulatory elements. Structural biology studies during the last decade, founded in X-ray crystallography, have characterized in detail several RNA-helicases. This comprehensive review summarizes the structural knowledge accumulated in the last two decades within this family of proteins, with special emphasis on the structure-function relationships of the most widely-studied families of RNA helicases: the DEAD-box, RIG-I-like and viral NS3 classes.

  17. Structural Analysis of ‘key’ Interactions in Functional RNA Molecules

    KAUST Repository

    Chawla, Mohit

    2018-04-01

    The main objective of the thesis is to carry out structural bioinformatics study along with usage of advanced quantum chemical methods to look at the structural stability and energetics of RNA building blocks. The main focus of the work described here lies on understanding the reasons behind the intrinsic stability of key interactions in nucleic acids. Crystal structures of RNA molecules exhibit fascinating variety of non-covalent interactions, which play an important role in maintaining the three dimensional structures. An accurate atomic level description of these interactions in the structural building blocks of RNA is a key to understand the structure-function relationship in these molecules. An effort has been made to link the conclusions drawn from quantum chemical computations on RNA base pairs in wide biochemical context of their occurrence in RNA structures. The initial attention was on the impact of natural and non-natural modifications of the nucleic acid bases on the structure and stability of base pairs that they are involved in. In the remaining sections we cover other molecular interactions shaping nucleic acids, as the interaction between ribose and the bases, and the fluoride sensing riboswitch system in order to investigate structure and dynamics of nucleic acids at the atomic level and to gain insight into the physical chemistry behind.

  18. Structural Analysis of ‘key’ Interactions in Functional RNA Molecules

    KAUST Repository

    Chawla, Mohit

    2018-01-01

    The main objective of the thesis is to carry out structural bioinformatics study along with usage of advanced quantum chemical methods to look at the structural stability and energetics of RNA building blocks. The main focus of the work described here lies on understanding the reasons behind the intrinsic stability of key interactions in nucleic acids. Crystal structures of RNA molecules exhibit fascinating variety of non-covalent interactions, which play an important role in maintaining the three dimensional structures. An accurate atomic level description of these interactions in the structural building blocks of RNA is a key to understand the structure-function relationship in these molecules. An effort has been made to link the conclusions drawn from quantum chemical computations on RNA base pairs in wide biochemical context of their occurrence in RNA structures. The initial attention was on the impact of natural and non-natural modifications of the nucleic acid bases on the structure and stability of base pairs that they are involved in. In the remaining sections we cover other molecular interactions shaping nucleic acids, as the interaction between ribose and the bases, and the fluoride sensing riboswitch system in order to investigate structure and dynamics of nucleic acids at the atomic level and to gain insight into the physical chemistry behind.

  19. Thermodynamic heuristics with case-based reasoning: combined insights for RNA pseudoknot secondary structure.

    Science.gov (United States)

    Al-Khatib, Ra'ed M; Rashid, Nur'Aini Abdul; Abdullah, Rosni

    2011-08-01

    The secondary structure of RNA pseudoknots has been extensively inferred and scrutinized by computational approaches. Experimental methods for determining RNA structure are time consuming and tedious; therefore, predictive computational approaches are required. Predicting the most accurate and energy-stable pseudoknot RNA secondary structure has been proven to be an NP-hard problem. In this paper, a new RNA folding approach, termed MSeeker, is presented; it includes KnotSeeker (a heuristic method) and Mfold (a thermodynamic algorithm). The global optimization of this thermodynamic heuristic approach was further enhanced by using a case-based reasoning technique as a local optimization method. MSeeker is a proposed algorithm for predicting RNA pseudoknot structure from individual sequences, especially long ones. This research demonstrates that MSeeker improves the sensitivity and specificity of existing RNA pseudoknot structure predictions. The performance and structural results from this proposed method were evaluated against seven other state-of-the-art pseudoknot prediction methods. The MSeeker method had better sensitivity than the DotKnot, FlexStem, HotKnots, pknotsRG, ILM, NUPACK and pknotsRE methods, with 79% of the predicted pseudoknot base-pairs being correct.

  20. The ins and outs of lncRNA structure: How, why and what comes next?

    Science.gov (United States)

    Blythe, Amanda J; Fox, Archa H; Bond, Charles S

    2016-01-01

    The field of structural biology has the unique advantage of being able to provide a comprehensive picture of biological mechanisms at the molecular and atomic level. Long noncoding RNAs (lncRNAs) represent the new frontier in the molecular biology of complex organisms yet remain the least characterised of all the classes of RNA. Thousands of new lncRNAs are being reported each year yet very little structural data exists for this rapidly expanding field. The length of lncRNAs ranges from 200 nt to over 100 kb in length and they generally exhibit low cellular abundance. Therefore, obtaining sufficient quantities of lncRNA to use for structural analysis is challenging. However, as technologies develop structures of lncRNAs are starting to emerge providing important information regarding their mechanism of action. Here we review the current methods used to determine the structure of lncRNA and lncRNA:protein complexes and describe the significant contribution structural biology has and will make to the field of lncRNA research. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Full-length RNA structure prediction of the HIV-1 genome reveals a conserved core domain

    DEFF Research Database (Denmark)

    Sükösd, Zsuzsanna; Andersen, Ebbe Sloth; Seemann, Ernst Stefan

    2015-01-01

    of the HIV-1 genome is highly variable in most regions, with a limited number of stable and conserved RNA secondary structures. Most interesting, a set of long distance interactions form a core organizing structure (COS) that organize the genome into three major structural domains. Despite overlapping...

  2. Structural Basis for dsRNA Recognition by NS1 Protein of Influenza A Virus

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, A.; Wong, S; Yuan, Y

    2009-01-01

    Influenza A viruses are important human pathogens causing periodic pandemic threats. Nonstructural protein 1 (NS1) protein of influenza A virus (NS1A) shields the virus against host defense. Here, we report the crystal structure of NS1A RNA-binding domain (RBD) bound to a double-stranded RNA (dsRNA) at 1.7A. NS1A RBD forms a homodimer to recognize the major groove of A-form dsRNA in a length-independent mode by its conserved concave surface formed by dimeric anti-parallel alpha-helices. dsRNA is anchored by a pair of invariable arginines (Arg38) from both monomers by extensive hydrogen bonds. In accordance with the structural observation, isothermal titration calorimetry assay shows that the unique Arg38-Arg38 pair and two Arg35-Arg46 pairs are crucial for dsRNA binding, and that Ser42 and Thr49 are also important for dsRNA binding. Agrobacterium co-infiltration assay further supports that the unique Arg38 pair plays important roles in dsRNA binding in vivo.

  3. Structure of the second RRM domain of Nrd1, a fission yeast MAPK target RNA binding protein, and implication for its RNA recognition and regulation

    Energy Technology Data Exchange (ETDEWEB)

    Kobayashi, Ayaho; Kanaba, Teppei [Graduate School of Science and Engineering, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji 192-0397 (Japan); Satoh, Ryosuke [Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku 141-0021, Tokyo (Japan); Fujiwara, Toshinobu [Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku 141-0021, Tokyo (Japan); Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku,Nagoya 467-8603 (Japan); Ito, Yutaka [Graduate School of Science and Engineering, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji 192-0397 (Japan); Sugiura, Reiko [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka 577-8502 (Japan); Mishima, Masaki, E-mail: mishima-masaki@tmu.ac.jp [Graduate School of Science and Engineering, Tokyo Metropolitan University, Minamiosawa 1-1, Hachioji 192-0397 (Japan)

    2013-07-19

    Highlights: •Solution structure of the second RRM of Nrd1 was determined. •RNA binding site of the second RRM was estimated. •Regulatory mechanism of RNA binding by phosphorylation is discussed. -- Abstract: Negative regulator of differentiation 1 (Nrd1) is known as a negative regulator of sexual differentiation in fission yeast. Recently, it has been revealed that Nrd1 also regulates cytokinesis, in which physical separation of the cell is achieved by a contractile ring comprising many proteins including actin and myosin. Cdc4, a myosin II light chain, is known to be required for cytokinesis. Nrd1 binds and stabilizes Cdc4 mRNA, and thereby suppressing the cytokinesis defects of the cdc4 mutants. Interestingly, Pmk1 MAPK phosphorylates Nrd1, resulting in markedly reduced RNA binding activity. Furthermore, Nrd1 localizes to stress granules in response to various stresses, and Pmk1 phosphorylation enhances the localization. Nrd1 consists of four RRM domains, although the mechanism by which Pmk1 regulates the RNA binding activity of Nrd1 is unknown. In an effort to delineate the relationship between Nrd1 structure and function, we prepared each RNA binding domain of Nrd1 and examined RNA binding to chemically synthesized oligo RNA using NMR. The structure of the second RRM domain of Nrd1 was determined and the RNA binding site on the second RRM domain was mapped by NMR. A plausible mechanism pertaining to the regulation of RNA binding activity by phosphorylation is also discussed.

  4. Structure of the second RRM domain of Nrd1, a fission yeast MAPK target RNA binding protein, and implication for its RNA recognition and regulation

    International Nuclear Information System (INIS)

    Kobayashi, Ayaho; Kanaba, Teppei; Satoh, Ryosuke; Fujiwara, Toshinobu; Ito, Yutaka; Sugiura, Reiko; Mishima, Masaki

    2013-01-01

    Highlights: •Solution structure of the second RRM of Nrd1 was determined. •RNA binding site of the second RRM was estimated. •Regulatory mechanism of RNA binding by phosphorylation is discussed. -- Abstract: Negative regulator of differentiation 1 (Nrd1) is known as a negative regulator of sexual differentiation in fission yeast. Recently, it has been revealed that Nrd1 also regulates cytokinesis, in which physical separation of the cell is achieved by a contractile ring comprising many proteins including actin and myosin. Cdc4, a myosin II light chain, is known to be required for cytokinesis. Nrd1 binds and stabilizes Cdc4 mRNA, and thereby suppressing the cytokinesis defects of the cdc4 mutants. Interestingly, Pmk1 MAPK phosphorylates Nrd1, resulting in markedly reduced RNA binding activity. Furthermore, Nrd1 localizes to stress granules in response to various stresses, and Pmk1 phosphorylation enhances the localization. Nrd1 consists of four RRM domains, although the mechanism by which Pmk1 regulates the RNA binding activity of Nrd1 is unknown. In an effort to delineate the relationship between Nrd1 structure and function, we prepared each RNA binding domain of Nrd1 and examined RNA binding to chemically synthesized oligo RNA using NMR. The structure of the second RRM domain of Nrd1 was determined and the RNA binding site on the second RRM domain was mapped by NMR. A plausible mechanism pertaining to the regulation of RNA binding activity by phosphorylation is also discussed

  5. Terminal structures of West Nile virus genomic RNA and their interactions with viral NS5 protein

    International Nuclear Information System (INIS)

    Dong Hongping; Zhang Bo; Shi Peiyong

    2008-01-01

    Genome cyclization is essential for flavivirus replication. We used RNases to probe the structures formed by the 5'-terminal 190 nucleotides and the 3'-terminal 111 nucleotides of the West Nile virus (WNV) genomic RNA. When analyzed individually, the two RNAs adopt stem-loop structures as predicted by the thermodynamic-folding program. However, when mixed together, the two RNAs form a duplex that is mediated through base-pairings of two sets of RNA elements (5'CS/3'CSI and 5'UAR/3'UAR). Formation of the RNA duplex facilitates a conformational change that leaves the 3'-terminal nucleotides of the genome (position - 8 to - 16) to be single-stranded. Viral NS5 binds specifically to the 5'-terminal stem-loop (SL1) of the genomic RNA. The 5'SL1 RNA structure is essential for WNV replication. The study has provided further evidence to suggest that flavivirus genome cyclization and NS5/5'SL1 RNA interaction facilitate NS5 binding to the 3' end of the genome for the initiation of viral minus-strand RNA synthesis

  6. RNA-Seq-Based Transcript Structure Analysis with TrBorderExt.

    Science.gov (United States)

    Wang, Yejun; Sun, Ming-An; White, Aaron P

    2018-01-01

    RNA-Seq has become a routine strategy for genome-wide gene expression comparisons in bacteria. Despite lower resolution in transcript border parsing compared with dRNA-Seq, TSS-EMOTE, Cappable-seq, Term-seq, and others, directional RNA-Seq still illustrates its advantages: low cost, quantification and transcript border analysis with a medium resolution (±10-20 nt). To facilitate mining of directional RNA-Seq datasets especially with respect to transcript structure analysis, we developed a tool, TrBorderExt, which can parse transcript start sites and termination sites accurately in bacteria. A detailed protocol is described in this chapter for how to use the software package step by step to identify bacterial transcript borders from raw RNA-Seq data. The package was developed with Perl and R programming languages, and is accessible freely through the website: http://www.szu-bioinf.org/TrBorderExt .

  7. Sparse RNA folding revisited: space-efficient minimum free energy structure prediction.

    Science.gov (United States)

    Will, Sebastian; Jabbari, Hosna

    2016-01-01

    RNA secondary structure prediction by energy minimization is the central computational tool for the analysis of structural non-coding RNAs and their interactions. Sparsification has been successfully applied to improve the time efficiency of various structure prediction algorithms while guaranteeing the same result; however, for many such folding problems, space efficiency is of even greater concern, particularly for long RNA sequences. So far, space-efficient sparsified RNA folding with fold reconstruction was solved only for simple base-pair-based pseudo-energy models. Here, we revisit the problem of space-efficient free energy minimization. Whereas the space-efficient minimization of the free energy has been sketched before, the reconstruction of the optimum structure has not even been discussed. We show that this reconstruction is not possible in trivial extension of the method for simple energy models. Then, we present the time- and space-efficient sparsified free energy minimization algorithm SparseMFEFold that guarantees MFE structure prediction. In particular, this novel algorithm provides efficient fold reconstruction based on dynamically garbage-collected trace arrows. The complexity of our algorithm depends on two parameters, the number of candidates Z and the number of trace arrows T; both are bounded by [Formula: see text], but are typically much smaller. The time complexity of RNA folding is reduced from [Formula: see text] to [Formula: see text]; the space complexity, from [Formula: see text] to [Formula: see text]. Our empirical results show more than 80 % space savings over RNAfold [Vienna RNA package] on the long RNAs from the RNA STRAND database (≥2500 bases). The presented technique is intentionally generalizable to complex prediction algorithms; due to their high space demands, algorithms like pseudoknot prediction and RNA-RNA-interaction prediction are expected to profit even stronger than "standard" MFE folding. SparseMFEFold is free

  8. Tree decomposition based fast search of RNA structures including pseudoknots in genomes.

    Science.gov (United States)

    Song, Yinglei; Liu, Chunmei; Malmberg, Russell; Pan, Fangfang; Cai, Liming

    2005-01-01

    Searching genomes for RNA secondary structure with computational methods has become an important approach to the annotation of non-coding RNAs. However, due to the lack of efficient algorithms for accurate RNA structure-sequence alignment, computer programs capable of fast and effectively searching genomes for RNA secondary structures have not been available. In this paper, a novel RNA structure profiling model is introduced based on the notion of a conformational graph to specify the consensus structure of an RNA family. Tree decomposition yields a small tree width t for such conformation graphs (e.g., t = 2 for stem loops and only a slight increase for pseudo-knots). Within this modelling framework, the optimal alignment of a sequence to the structure model corresponds to finding a maximum valued isomorphic subgraph and consequently can be accomplished through dynamic programming on the tree decomposition of the conformational graph in time O(k(t)N(2)), where k is a small parameter; and N is the size of the projiled RNA structure. Experiments show that the application of the alignment algorithm to search in genomes yields the same search accuracy as methods based on a Covariance model with a significant reduction in computation time. In particular; very accurate searches of tmRNAs in bacteria genomes and of telomerase RNAs in yeast genomes can be accomplished in days, as opposed to months required by other methods. The tree decomposition based searching tool is free upon request and can be downloaded at our site h t t p ://w.uga.edu/RNA-informatics/software/index.php.

  9. Identification of RNA species in the RNA-toxin complex and structure of the complex in Clostridium botulinum type E.

    Science.gov (United States)

    Kitamura, Masaru

    2002-02-15

    Clostridium botulinum type E toxin was isolated in the form of a complex with RNA(s) from bacterial cells. Characterization of the complexed RNA remains to be elucidated. The RNA is identified here as ribosomal RNA (rRNA) having 23S and 16S components. The RNA-toxin complexes were found to be made up of three types with different molecular sizes. The three types of RNA-toxin complex are toxin bound to both the 23S and 16S rRNA, toxin bound to the 16S rRNA and a small amount of 23S rRNA, and toxin bound only to the 16S rRNA. ©2002 Elsevier Science (USA).

  10. The cervical cap (image)

    Science.gov (United States)

    The cervical cap is a flexible rubber cup-like device that is filled with spermicide and self-inserted over the cervix ... left in place several hours after intercourse. The cap is a prescribed device fitted by a health ...

  11. Structure optimization and preliminary bioactivity evaluation of N-hydroxybenzamide-based HDAC inhibitors with Y-shaped cap.

    Science.gov (United States)

    Yu, Chenggong; He, Feng; Qu, Ying; Zhang, Qiuqiong; Lv, Jiahui; Zhang, Xiangna; Xu, Ana; Miao, Pannan; Wu, Jingde

    2018-05-01

    Histone deacetylase inhibitors (HDACIs) are effective small molecules in the treatment of human cancers. In our continuing efforts to develop novel N-hydroxyterephthalamide-based HDACIs, herein we report the design and development of a new class of N-hydroxybenzamide-based HDACIs. In this new class of analogs, we inserted an ethylene moiety in the linker and used indole as a part of the Y-shaped cap group. Biological characterization identified compounds 4o, 4p, 4q and 4t to show improved HDAC inhibition, while no isoform selectivity for HDACs was observed. These compounds also exhibited improved anti-proliferative activity against multiple cancer cell lines when compared to their parent compound and positive control SAHA. Copyright © 2018 Elsevier Ltd. All rights reserved.

  12. On the Structure and Adjustment of Inversion-Capped Neutral Atmospheric Boundary-Layer Flows: Large-Eddy Simulation Study

    DEFF Research Database (Denmark)

    Pedersen, Jesper Grønnegaard; Gryning, Sven-Erik; Kelly, Mark C.

    2014-01-01

    A range of large-eddy simulations, with differing free atmosphere stratification and zero or slightly positive surface heat flux, is investigated to improve understanding of the neutral and near-neutral, inversion-capped, horizontally homogeneous, barotropic atmospheric boundary layer with emphasis...... on the upper region. We find that an adjustment time of at least 16 h is needed for the simulated flow to reach a quasi-steady state. The boundary layer continues to grow, but at a slow rate that changes little after 8 h of simulation time. A common feature of the neutral simulations is the development...... of a super-geostrophic jet near the top of the boundary layer. The analytical wind-shear models included do not account for such a jet, and the best agreement with simulated wind shear is seen in cases with weak stratification above the boundary layer. Increasing the surface heat flux decreases the magnitude...

  13. Revisiting magnetism of capped Au and ZnO nanoparticles: Surface band structure and atomic orbital with giant magnetic moment

    Energy Technology Data Exchange (ETDEWEB)

    Hernando, Antonio; Crespo, Patricia [Instituto de Magnetismo Aplicado, UCM-CSIC-ADIF, Las Rozas. P.O. Box 155, 28230 Madrid (Spain); Dept. Fisica de Materiales, Universidad Complutense, Madrid (Spain); Garcia, Miguel Angel [Instituto de Ceramica y Vidrio, CSIC, C/ Kelsen, 5, Madrid 28049 (Spain); Coey, Michael [Trinity College Dublin, Dublin (Ireland); Ayuela, Andres; Echenique, Pedro Miguel [Centro de Fisica de Materiales, CFM-MPC CSIC-UPV/EHU, Donostia International Physics Center (DIPC), 20018 San Sebastian (Spain); Departamento de Fisica de Materiales, Fac. de Quimicas, Universidad del Pais Vasco UPV-EHU, 20018 San Sebastian (Spain)

    2011-10-15

    In this article we review the exotic magnetism of nanoparticles (NPs) formed by substances that are not magnetic in bulk as described with generality in Section 1. In particular, the intrinsic character of the magnetism observed on capped Au and ZnO NPs is analysed. X-ray magnetic circular dichroism (XMCD) analysis has shown that the magnetic moments are intrinsic and lie in the Au and Zn atoms, respectively, as analysed in Section 2, where the general theoretical ideas are also revisited. Since impurity atoms bonded to the surface act as donor or acceptor of electrons that occupy the surface states, the anomalous magnetic response is analysed in terms of the surface band in Section 3. Finally, Section 4 summarizes our last theoretical proposal. (Copyright copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  14. RNAmutants: a web server to explore the mutational landscape of RNA secondary structures

    Science.gov (United States)

    Waldispühl, Jerome; Devadas, Srinivas; Berger, Bonnie; Clote, Peter

    2009-01-01

    The history and mechanism of molecular evolution in DNA have been greatly elucidated by contributions from genetics, probability theory and bioinformatics—indeed, mathematical developments such as Kimura's neutral theory, Kingman's coalescent theory and efficient software such as BLAST, ClustalW, Phylip, etc., provide the foundation for modern population genetics. In contrast to DNA, the function of most noncoding RNA depends on tertiary structure, experimentally known to be largely determined by secondary structure, for which dynamic programming can efficiently compute the minimum free energy secondary structure. For this reason, understanding the effect of pointwise mutations in RNA secondary structure could reveal fundamental properties of structural RNA molecules and improve our understanding of molecular evolution of RNA. The web server RNAmutants provides several efficient tools to compute the ensemble of low-energy secondary structures for all k-mutants of a given RNA sequence, where k is bounded by a user-specified upper bound. As we have previously shown, these tools can be used to predict putative deleterious mutations and to analyze regulatory sequences from the hepatitis C and human immunodeficiency genomes. Web server is available at http://bioinformatics.bc.edu/clotelab/RNAmutants/, and downloadable binaries at http://rnamutants.csail.mit.edu/. PMID:19531740

  15. Structural Insights into RNA Recognition by the Alternate-Splicing Regulator CUG-Binding Protein 1

    Energy Technology Data Exchange (ETDEWEB)

    M Teplova; J Song; H Gaw; A Teplov; D Patel

    2011-12-31

    CUG-binding protein 1 (CUGBP1) regulates multiple aspects of nuclear and cytoplasmic mRNA processing, with implications for onset of myotonic dystrophy. CUGBP1 harbors three RRM domains and preferentially targets UGU-rich mRNA elements. We describe crystal structures of CUGBP1 RRM1 and tandem RRM1/2 domains bound to RNAs containing tandem UGU(U/G) elements. Both RRM1 in RRM1-RNA and RRM2 in RRM1/2-RNA complexes use similar principles to target UGU(U/G) elements, with recognition mediated by face-to-edge stacking and water-mediated hydrogen-bonding networks. The UG step adopts a left-handed Z-RNA conformation, with the syn guanine recognized through Hoogsteen edge-protein backbone hydrogen-bonding interactions. NMR studies on the RRM1/2-RNA complex establish that both RRM domains target tandem UGUU motifs in solution, whereas filter-binding assays identify a preference for recognition of GU over AU or GC steps. We discuss the implications of CUGBP1-mediated targeting and sequestration of UGU(U/G) elements on pre-mRNA alternative-splicing regulation, translational regulation, and mRNA decay.

  16. PRince: a web server for structural and physicochemical analysis of protein-RNA interface.

    Science.gov (United States)

    Barik, Amita; Mishra, Abhishek; Bahadur, Ranjit Prasad

    2012-07-01

    We have developed a web server, PRince, which analyzes the structural features and physicochemical properties of the protein-RNA interface. Users need to submit a PDB file containing the atomic coordinates of both the protein and the RNA molecules in complex form (in '.pdb' format). They should also mention the chain identifiers of interacting protein and RNA molecules. The size of the protein-RNA interface is estimated by measuring the solvent accessible surface area buried in contact. For a given protein-RNA complex, PRince calculates structural, physicochemical and hydration properties of the interacting surfaces. All these parameters generated by the server are presented in a tabular format. The interacting surfaces can also be visualized with software plug-in like Jmol. In addition, the output files containing the list of the atomic coordinates of the interacting protein, RNA and interface water molecules can be downloaded. The parameters generated by PRince are novel, and users can correlate them with the experimentally determined biophysical and biochemical parameters for better understanding the specificity of the protein-RNA recognition process. This server will be continuously upgraded to include more parameters. PRince is publicly accessible and free for use. Available at http://www.facweb.iitkgp.ernet.in/~rbahadur/prince/home.html.

  17. nRC: non-coding RNA Classifier based on structural features.

    Science.gov (United States)

    Fiannaca, Antonino; La Rosa, Massimo; La Paglia, Laura; Rizzo, Riccardo; Urso, Alfonso

    2017-01-01

    Non-coding RNA (ncRNA) are small non-coding sequences involved in gene expression regulation of many biological processes and diseases. The recent discovery of a large set of different ncRNAs with biologically relevant roles has opened the way to develop methods able to discriminate between the different ncRNA classes. Moreover, the lack of knowledge about the complete mechanisms in regulative processes, together with the development of high-throughput technologies, has required the help of bioinformatics tools in addressing biologists and clinicians with a deeper comprehension of the functional roles of ncRNAs. In this work, we introduce a new ncRNA classification tool, nRC (non-coding RNA Classifier). Our approach is based on features extraction from the ncRNA secondary structure together with a supervised classification algorithm implementing a deep learning architecture based on convolutional neural networks. We tested our approach for the classification of 13 different ncRNA classes. We obtained classification scores, using the most common statistical measures. In particular, we reach an accuracy and sensitivity score of about 74%. The proposed method outperforms other similar classification methods based on secondary structure features and machine learning algorithms, including the RNAcon tool that, to date, is the reference classifier. nRC tool is freely available as a docker image at https://hub.docker.com/r/tblab/nrc/. The source code of nRC tool is also available at https://github.com/IcarPA-TBlab/nrc.

  18. Structure and Dynamics of RNA Repeat Expansions That Cause Huntington's Disease and Myotonic Dystrophy Type 1.

    Science.gov (United States)

    Chen, Jonathan L; VanEtten, Damian M; Fountain, Matthew A; Yildirim, Ilyas; Disney, Matthew D

    2017-07-11

    RNA repeat expansions cause a host of incurable, genetically defined diseases. The most common class of RNA repeats consists of trinucleotide repeats. These long, repeating transcripts fold into hairpins containing 1 × 1 internal loops that can mediate disease via a variety of mechanism(s) in which RNA is the central player. Two of these disorders are Huntington's disease and myotonic dystrophy type 1, which are caused by r(CAG) and r(CUG) repeats, respectively. We report the structures of two RNA constructs containing three copies of a r(CAG) [r(3×CAG)] or r(CUG) [r(3×CUG)] motif that were modeled with nuclear magnetic resonance spectroscopy and simulated annealing with restrained molecular dynamics. The 1 × 1 internal loops of r(3×CAG) are stabilized by one-hydrogen bond (cis Watson-Crick/Watson-Crick) AA pairs, while those of r(3×CUG) prefer one- or two-hydrogen bond (cis Watson-Crick/Watson-Crick) UU pairs. Assigned chemical shifts for the residues depended on the identity of neighbors or next nearest neighbors. Additional insights into the dynamics of these RNA constructs were gained by molecular dynamics simulations and a discrete path sampling method. Results indicate that the global structures of the RNA are A-form and that the loop regions are dynamic. The results will be useful for understanding the dynamic trajectory of these RNA repeats but also may aid in the development of therapeutics.

  19. GC content around splice sites affects splicing through pre-mRNA secondary structures

    Directory of Open Access Journals (Sweden)

    Chen Liang

    2011-01-01

    Full Text Available Abstract Background Alternative splicing increases protein diversity by generating multiple transcript isoforms from a single gene through different combinations of exons or through different selections of splice sites. It has been reported that RNA secondary structures are involved in alternative splicing. Here we perform a genomic study of RNA secondary structures around splice sites in humans (Homo sapiens, mice (Mus musculus, fruit flies (Drosophila melanogaster, and nematodes (Caenorhabditis elegans to further investigate this phenomenon. Results We observe that GC content around splice sites is closely associated with the splice site usage in multiple species. RNA secondary structure is the possible explanation, because the structural stability difference among alternative splice sites, constitutive splice sites, and skipped splice sites can be explained by the GC content difference. Alternative splice sites tend to be GC-enriched and exhibit more stable RNA secondary structures in all of the considered species. In humans and mice, splice sites of first exons and long exons tend to be GC-enriched and hence form more stable structures, indicating the special role of RNA secondary structures in promoter proximal splicing events and the splicing of long exons. In addition, GC-enriched exon-intron junctions tend to be overrepresented in tissue-specific alternative splice sites, indicating the functional consequence of the GC effect. Compared with regions far from splice sites and decoy splice sites, real splice sites are GC-enriched. We also found that the GC-content effect is much stronger than the nucleotide-order effect to form stable secondary structures. Conclusion All of these results indicate that GC content is related to splice site usage and it may mediate the splicing process through RNA secondary structures.

  20. New families of human regulatory RNA structures identified by comparative analysis of vertebrate genomes.

    Science.gov (United States)

    Parker, Brian J; Moltke, Ida; Roth, Adam; Washietl, Stefan; Wen, Jiayu; Kellis, Manolis; Breaker, Ronald; Pedersen, Jakob Skou

    2011-11-01

    Regulatory RNA structures are often members of families with multiple paralogous instances across the genome. Family members share functional and structural properties, which allow them to be studied as a whole, facilitating both bioinformatic and experimental characterization. We have developed a comparative method, EvoFam, for genome-wide identification of families of regulatory RNA structures, based on primary sequence and secondary structure similarity. We apply EvoFam to a 41-way genomic vertebrate alignment. Genome-wide, we identify 220 human, high-confidence families outside protein-coding regions comprising 725 individual structures, including 48 families with known structural RNA elements. Known families identified include both noncoding RNAs, e.g., miRNAs and the recently identified MALAT1/MEN β lincRNA family; and cis-regulatory structures, e.g., iron-responsive elements. We also identify tens of new families supported by strong evolutionary evidence and other statistical evidence, such as GO term enrichments. For some of these, detailed analysis has led to the formulation of specific functional hypotheses. Examples include two hypothesized auto-regulatory feedback mechanisms: one involving six long hairpins in the 3'-UTR of MAT2A, a key metabolic gene that produces the primary human methyl donor S-adenosylmethionine; the other involving a tRNA-like structure in the intron of the tRNA maturation gene POP1. We experimentally validate the predicted MAT2A structures. Finally, we identify potential new regulatory networks, including large families of short hairpins enriched in immunity-related genes, e.g., TNF, FOS, and CTLA4, which include known transcript destabilizing elements. Our findings exemplify the diversity of post-transcriptional regulation and provide a resource for further characterization of new regulatory mechanisms and families of noncoding RNAs.

  1. Cradle Cap: Treatment

    Science.gov (United States)

    Cradle cap Treatment Cradle cap usually doesn't require medical treatment. It clears up on its own within a few months. In the meantime, wash ... tips can help you control and manage cradle cap. Gently rub your baby's scalp with your fingers ...

  2. Initiation of translation in bacteria by a structured eukaryotic IRES RNA.

    Science.gov (United States)

    Colussi, Timothy M; Costantino, David A; Zhu, Jianyu; Donohue, John Paul; Korostelev, Andrei A; Jaafar, Zane A; Plank, Terra-Dawn M; Noller, Harry F; Kieft, Jeffrey S

    2015-03-05

    The central dogma of gene expression (DNA to RNA to protein) is universal, but in different domains of life there are fundamental mechanistic differences within this pathway. For example, the canonical molecular signals used to initiate protein synthesis in bacteria and eukaryotes are mutually exclusive. However, the core structures and conformational dynamics of ribosomes that are responsible for the translation steps that take place after initiation are ancient and conserved across the domains of life. We wanted to explore whether an undiscovered RNA-based signal might be able to use these conserved features, bypassing mechanisms specific to each domain of life, and initiate protein synthesis in both bacteria and eukaryotes. Although structured internal ribosome entry site (IRES) RNAs can manipulate ribosomes to initiate translation in eukaryotic cells, an analogous RNA structure-based mechanism has not been observed in bacteria. Here we report our discovery that a eukaryotic viral IRES can initiate translation in live bacteria. We solved the crystal structure of this IRES bound to a bacterial ribosome to 3.8 Å resolution, revealing that despite differences between bacterial and eukaryotic ribosomes this IRES binds directly to both and occupies the space normally used by transfer RNAs. Initiation in both bacteria and eukaryotes depends on the structure of the IRES RNA, but in bacteria this RNA uses a different mechanism that includes a form of ribosome repositioning after initial recruitment. This IRES RNA bridges billions of years of evolutionary divergence and provides an example of an RNA structure-based translation initiation signal capable of operating in two domains of life.

  3. NMR-study of dynamic structural transtions in RNA-molecules

    OpenAIRE

    Fürtig, Boris

    2007-01-01

    The following thesis is concerned with the elucidation of structural changes of RNA molecules during the time course of dynamic processes that are commonly denoted as folding reactions. In contrast to the field of protein folding, the concept of RNA folding comprises not only folding reactions itself but also refolding- or conformational switching- and assembly processes (see chapter III). The method in this thesis to monitor these diverse processes is high resolution liquid-state NMR spectro...

  4. An evolutionary model for protein-coding regions with conserved RNA structure

    DEFF Research Database (Denmark)

    Pedersen, Jakob Skou; Forsberg, Roald; Meyer, Irmtraud Margret

    2004-01-01

    in the RNA structure. The overlap of these fundamental dependencies is sufficient to cause "contagious" context dependencies which cascade across many nucleotide sites. Such large-scale dependencies challenge the use of traditional phylogenetic models in evolutionary inference because they explicitly assume...... components of traditional phylogenetic models. We applied this to a data set of full-genome sequences from the hepatitis C virus where five RNA structures are mapped within the coding region. This allowed us to partition the effects of selection on different structural elements and to test various hypotheses......Here we present a model of nucleotide substitution in protein-coding regions that also encode the formation of conserved RNA structures. In such regions, apparent evolutionary context dependencies exist, both between nucleotides occupying the same codon and between nucleotides forming a base pair...

  5. Structures of two exonucleases involved in controlled RNA turnover in yeast

    DEFF Research Database (Denmark)

    Jonstrup, Anette Thyssen; Midtgaard, Søren Fuglsang; Van, Lan Bich

    divalent cations. The Pop2p structure reveals that the ability of this enzyme to degrade poly(A)/(U)/(C), but not poly(G) may be determined by structural hindrance of interaction with this specific nucleotide. In Rrp6p, mutations known to confer specific RNA degradation phenotypes in yeast nuclei can now...... rid of aberrant RNAs. Here we describe the structures of two 3'-5' exonucleases involved in controlled RNA decay in yeast, Pop2p and Rrp6p. Rrp6p is associated with the nuclear exosome where it participates in the degradation of improperly processed precursor mRNAs and trimming of stable RNAs [1]. Pop...

  6. Homology Modeling and Analysis of Structure Predictions of the Bovine Rhinitis B Virus RNA Dependent RNA Polymerase (RdRp

    Directory of Open Access Journals (Sweden)

    Devendra K. Rai

    2012-07-01

    Full Text Available Bovine Rhinitis B Virus (BRBV is a picornavirus responsible for mild respiratory infection of cattle. It is probably the least characterized among the aphthoviruses. BRBV is the closest relative known to Foot and Mouth Disease virus (FMDV with a ~43% identical polyprotein sequence and as much as 67% identical sequence for the RNA dependent RNA polymerase (RdRp, which is also known as 3D polymerase (3Dpol. In the present study we carried out phylogenetic analysis, structure based sequence alignment and prediction of three-dimensional structure of BRBV 3Dpol using a combination of different computational tools. Model structures of BRBV 3Dpol were verified for their stereochemical quality and accuracy. The BRBV 3Dpol structure predicted by SWISS-MODEL exhibited highest scores in terms of stereochemical quality and accuracy, which were in the range of 2Å resolution crystal structures. The active site, nucleic acid binding site and overall structure were observed to be in agreement with the crystal structure of unliganded as well as template/primer (T/P, nucleotide tri-phosphate (NTP and pyrophosphate (PPi bound FMDV 3Dpol (PDB, 1U09 and 2E9Z. The closest proximity of BRBV and FMDV 3Dpol as compared to human rhinovirus type 16 (HRV-16 and rabbit hemorrhagic disease virus (RHDV 3Dpols is also substantiated by phylogeny analysis and root-mean square deviation (RMSD between C-α traces of the polymerase structures. The absence of positively charged α-helix at C terminal, significant differences in non-covalent interactions especially salt bridges and CH-pi interactions around T/P channel of BRBV 3Dpol compared to FMDV 3Dpol, indicate that despite a very high homology to FMDV 3Dpol, BRBV 3Dpol may adopt a different mechanism for handling its substrates and adapting to physiological requirements. Our findings will be valuable in the

  7. Prediction of RNA secondary structures: from theory to models and real molecules

    International Nuclear Information System (INIS)

    Schuster, Peter

    2006-01-01

    RNA secondary structures are derived from RNA sequences, which are strings built form the natural four letter nucleotide alphabet, {AUGC}. These coarse-grained structures, in turn, are tantamount to constrained strings over a three letter alphabet. Hence, the secondary structures are discrete objects and the number of sequences always exceeds the number of structures. The sequences built from two letter alphabets form perfect structures when the nucleotides can form a base pair, as is the case with {GC} or {AU}, but the relation between the sequences and structures differs strongly from the four letter alphabet. A comprehensive theory of RNA structure is presented, which is based on the concepts of sequence space and shape space, being a space of structures. It sets the stage for modelling processes in ensembles of RNA molecules like evolutionary optimization or kinetic folding as dynamical phenomena guided by mappings between the two spaces. The number of minimum free energy (mfe) structures is always smaller than the number of sequences, even for two letter alphabets. Folding of RNA molecules into mfe energy structures constitutes a non-invertible mapping from sequence space onto shape space. The preimage of a structure in sequence space is defined as its neutral network. Similarly the set of suboptimal structures is the preimage of a sequence in shape space. This set represents the conformation space of a given sequence. The evolutionary optimization of structures in populations is a process taking place in sequence space, whereas kinetic folding occurs in molecular ensembles that optimize free energy in conformation space. Efficient folding algorithms based on dynamic programming are available for the prediction of secondary structures for given sequences. The inverse problem, the computation of sequences for predefined structures, is an important tool for the design of RNA molecules with tailored properties. Simultaneous folding or cofolding of two or more RNA

  8. 3dRPC: a web server for 3D RNA-protein structure prediction.

    Science.gov (United States)

    Huang, Yangyu; Li, Haotian; Xiao, Yi

    2018-04-01

    RNA-protein interactions occur in many biological processes. To understand the mechanism of these interactions one needs to know three-dimensional (3D) structures of RNA-protein complexes. 3dRPC is an algorithm for prediction of 3D RNA-protein complex structures and consists of a docking algorithm RPDOCK and a scoring function 3dRPC-Score. RPDOCK is used to sample possible complex conformations of an RNA and a protein by calculating the geometric and electrostatic complementarities and stacking interactions at the RNA-protein interface according to the features of atom packing of the interface. 3dRPC-Score is a knowledge-based potential that uses the conformations of nucleotide-amino-acid pairs as statistical variables and that is used to choose the near-native complex-conformations obtained from the docking method above. Recently, we built a web server for 3dRPC. The users can easily use 3dRPC without installing it locally. RNA and protein structures in PDB (Protein Data Bank) format are the only needed input files. It can also incorporate the information of interface residues or residue-pairs obtained from experiments or theoretical predictions to improve the prediction. The address of 3dRPC web server is http://biophy.hust.edu.cn/3dRPC. yxiao@hust.edu.cn.

  9. Hydration sites of unpaired RNA bases: a statistical analysis of the PDB structures

    Directory of Open Access Journals (Sweden)

    Carugo Oliviero

    2011-10-01

    Full Text Available Abstract Background Hydration is crucial for RNA structure and function. X-ray crystallography is the most commonly used method to determine RNA structures and hydration and, therefore, statistical surveys are based on crystallographic results, the number of which is quickly increasing. Results A statistical analysis of the water molecule distribution in high-resolution X-ray structures of unpaired RNA nucleotides showed that: different bases have the same penchant to be surrounded by water molecules; clusters of water molecules indicate possible hydration sites, which, in some cases, match those of the major and minor grooves of RNA and DNA double helices; complex hydrogen bond networks characterize the solvation of the nucleotides, resulting in a significant rigidity of the base and its surrounding water molecules. Interestingly, the hydration sites around unpaired RNA bases do not match, in general, the positions that are occupied by the second nucleotide when the base-pair is formed. Conclusions The hydration sites around unpaired RNA bases were found. They do not replicate the atom positions of complementary bases in the Watson-Crick pairs.

  10. New families of human regulatory RNA structures identified by comparative analysis of vertebrate genomes

    DEFF Research Database (Denmark)

    Parker, Brian John; Moltke, Ida; Roth, Adam

    2011-01-01

    a comparative method, EvoFam, for genome-wide identification of families of regulatory RNA structures, based on primary sequence and secondary structure similarity. We apply EvoFam to a 41-way genomic vertebrate alignment. Genome-wide, we identify 220 human, high-confidence families outside protein...

  11. DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo.

    Science.gov (United States)

    Zubradt, Meghan; Gupta, Paromita; Persad, Sitara; Lambowitz, Alan M; Weissman, Jonathan S; Rouskin, Silvi

    2017-01-01

    Coupling of structure-specific in vivo chemical modification to next-generation sequencing is transforming RNA secondary structure studies in living cells. The dominant strategy for detecting in vivo chemical modifications uses reverse transcriptase truncation products, which introduce biases and necessitate population-average assessments of RNA structure. Here we present dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-MaPseq), which encodes DMS modifications as mismatches using a thermostable group II intron reverse transcriptase. DMS-MaPseq yields a high signal-to-noise ratio, can report multiple structural features per molecule, and allows both genome-wide studies and focused in vivo investigations of even low-abundance RNAs. We apply DMS-MaPseq for the first analysis of RNA structure within an animal tissue and to identify a functional structure involved in noncanonical translation initiation. Additionally, we use DMS-MaPseq to compare the in vivo structure of pre-mRNAs with their mature isoforms. These applications illustrate DMS-MaPseq's capacity to dramatically expand in vivo analysis of RNA structure.

  12. Bi-objective integer programming for RNA secondary structure prediction with pseudoknots.

    Science.gov (United States)

    Legendre, Audrey; Angel, Eric; Tahi, Fariza

    2018-01-15

    RNA structure prediction is an important field in bioinformatics, and numerous methods and tools have been proposed. Pseudoknots are specific motifs of RNA secondary structures that are difficult to predict. Almost all existing methods are based on a single model and return one solution, often missing the real structure. An alternative approach would be to combine different models and return a (small) set of solutions, maximizing its quality and diversity in order to increase the probability that it contains the real structure. We propose here an original method for predicting RNA secondary structures with pseudoknots, based on integer programming. We developed a generic bi-objective integer programming algorithm allowing to return optimal and sub-optimal solutions optimizing simultaneously two models. This algorithm was then applied to the combination of two known models of RNA secondary structure prediction, namely MEA and MFE. The resulting tool, called BiokoP, is compared with the other methods in the literature. The results show that the best solution (structure with the highest F 1 -score) is, in most cases, given by BiokoP. Moreover, the results of BiokoP are homogeneous, regardless of the pseudoknot type or the presence or not of pseudoknots. Indeed, the F 1 -scores are always higher than 70% for any number of solutions returned. The results obtained by BiokoP show that combining the MEA and the MFE models, as well as returning several optimal and several sub-optimal solutions, allow to improve the prediction of secondary structures. One perspective of our work is to combine better mono-criterion models, in particular to combine a model based on the comparative approach with the MEA and the MFE models. This leads to develop in the future a new multi-objective algorithm to combine more than two models. BiokoP is available on the EvryRNA platform: https://EvryRNA.ibisc.univ-evry.fr .

  13. The 5S rRNA loop E: chemical probing and phylogenetic data versus crystal structure.

    Science.gov (United States)

    Leontis, N B; Westhof, E

    1998-09-01

    A significant fraction of the bases in a folded, structured RNA molecule participate in noncanonical base pairing interactions, often in the context of internal loops or multi-helix junction loops. The appearance of each new high-resolution RNA structure provides welcome data to guide efforts to understand and predict RNA 3D structure, especially when the RNA in question is a functionally conserved molecule. The recent publication of the crystal structure of the "Loop E" region of bacterial 5S ribosomal RNA is such an event [Correll CC, Freeborn B, Moore PB, Steitz TA, 1997, Cell 91:705-712]. In addition to providing more examples of already established noncanonical base pairs, such as purine-purine sheared pairings, trans-Hoogsteen UA, and GU wobble pairs, the structure provides the first high-resolution views of two new purine-purine pairings and a new GU pairing. The goal of the present analysis is to expand the capabilities of both chemical probing and phylogenetic analysis to predict with greater accuracy the structures of RNA molecules. First, in light of existing chemical probing data, we investigate what lessons could be learned regarding the interpretation of this widely used method of RNA structure probing. Then we analyze the 3D structure with reference to molecular phylogeny data (assuming conservation of function) to discover what alternative base pairings are geometrically compatible with the structure. The comparisons between previous modeling efforts and crystal structures show that the intricate involvements of ions and water molecules in the maintenance of non-Watson-Crick pairs render the process of correctly identifying the interacting sites in such pairs treacherous, except in cases of trans-Hoogsteen A/U or sheared A/G pairs for the adenine N1 site. The phylogenetic analysis identifies A/A, A/C, A/U and C/A, C/C, and C/U pairings isosteric with sheared A/G, as well as A/A and A/C pairings isosteric with both G/U and G/G bifurcated pairings

  14. Ebola virus RNA editing depends on the primary editing site sequence and an upstream secondary structure.

    Directory of Open Access Journals (Sweden)

    Masfique Mehedi

    Full Text Available Ebolavirus (EBOV, the causative agent of a severe hemorrhagic fever and a biosafety level 4 pathogen, increases its genome coding capacity by producing multiple transcripts encoding for structural and nonstructural glycoproteins from a single gene. This is achieved through RNA editing, during which non-template adenosine residues are incorporated into the EBOV mRNAs at an editing site encoding for 7 adenosine residues. However, the mechanism of EBOV RNA editing is currently not understood. In this study, we report for the first time that minigenomes containing the glycoprotein gene editing site can undergo RNA editing, thereby eliminating the requirement for a biosafety level 4 laboratory to study EBOV RNA editing. Using a newly developed dual-reporter minigenome, we have characterized the mechanism of EBOV RNA editing, and have identified cis-acting sequences that are required for editing, located between 9 nt upstream and 9 nt downstream of the editing site. Moreover, we show that a secondary structure in the upstream cis-acting sequence plays an important role in RNA editing. EBOV RNA editing is glycoprotein gene-specific, as a stretch encoding for 7 adenosine residues located in the viral polymerase gene did not serve as an editing site, most likely due to an absence of the necessary cis-acting sequences. Finally, the EBOV protein VP30 was identified as a trans-acting factor for RNA editing, constituting a novel function for this protein. Overall, our results provide novel insights into the RNA editing mechanism of EBOV, further understanding of which might result in novel intervention strategies against this viral pathogen.

  15. Ebola virus RNA editing depends on the primary editing site sequence and an upstream secondary structure.

    Science.gov (United States)

    Mehedi, Masfique; Hoenen, Thomas; Robertson, Shelly; Ricklefs, Stacy; Dolan, Michael A; Taylor, Travis; Falzarano, Darryl; Ebihara, Hideki; Porcella, Stephen F; Feldmann, Heinz

    2013-01-01

    Ebolavirus (EBOV), the causative agent of a severe hemorrhagic fever and a biosafety level 4 pathogen, increases its genome coding capacity by producing multiple transcripts encoding for structural and nonstructural glycoproteins from a single gene. This is achieved through RNA editing, during which non-template adenosine residues are incorporated into the EBOV mRNAs at an editing site encoding for 7 adenosine residues. However, the mechanism of EBOV RNA editing is currently not understood. In this study, we report for the first time that minigenomes containing the glycoprotein gene editing site can undergo RNA editing, thereby eliminating the requirement for a biosafety level 4 laboratory to study EBOV RNA editing. Using a newly developed dual-reporter minigenome, we have characterized the mechanism of EBOV RNA editing, and have identified cis-acting sequences that are required for editing, located between 9 nt upstream and 9 nt downstream of the editing site. Moreover, we show that a secondary structure in the upstream cis-acting sequence plays an important role in RNA editing. EBOV RNA editing is glycoprotein gene-specific, as a stretch encoding for 7 adenosine residues located in the viral polymerase gene did not serve as an editing site, most likely due to an absence of the necessary cis-acting sequences. Finally, the EBOV protein VP30 was identified as a trans-acting factor for RNA editing, constituting a novel function for this protein. Overall, our results provide novel insights into the RNA editing mechanism of EBOV, further understanding of which might result in novel intervention strategies against this viral pathogen.

  16. RNA Structural Dynamics As Captured by Molecular Simulations: A Comprehensive Overview

    Science.gov (United States)

    2018-01-01

    With both catalytic and genetic functions, ribonucleic acid (RNA) is perhaps the most pluripotent chemical species in molecular biology, and its functions are intimately linked to its structure and dynamics. Computer simulations, and in particular atomistic molecular dynamics (MD), allow structural dynamics of biomolecular systems to be investigated with unprecedented temporal and spatial resolution. We here provide a comprehensive overview of the fast-developing field of MD simulations of RNA molecules. We begin with an in-depth, evaluatory coverage of the most fundamental methodological challenges that set the basis for the future development of the field, in particular, the current developments and inherent physical limitations of the atomistic force fields and the recent advances in a broad spectrum of enhanced sampling methods. We also survey the closely related field of coarse-grained modeling of RNA systems. After dealing with the methodological aspects, we provide an exhaustive overview of the available RNA simulation literature, ranging from studies of the smallest RNA oligonucleotides to investigations of the entire ribosome. Our review encompasses tetranucleotides, tetraloops, a number of small RNA motifs, A-helix RNA, kissing-loop complexes, the TAR RNA element, the decoding center and other important regions of the ribosome, as well as assorted others systems. Extended sections are devoted to RNA–ion interactions, ribozymes, riboswitches, and protein/RNA complexes. Our overview is written for as broad of an audience as possible, aiming to provide a much-needed interdisciplinary bridge between computation and experiment, together with a perspective on the future of the field. PMID:29297679

  17. Translation of the flavivirus kunjin NS3 gene in cis but not its RNA sequence or secondary structure is essential for efficient RNA packaging.

    Science.gov (United States)

    Pijlman, Gorben P; Kondratieva, Natasha; Khromykh, Alexander A

    2006-11-01

    Our previous studies using trans-complementation analysis of Kunjin virus (KUN) full-length cDNA clones harboring in-frame deletions in the NS3 gene demonstrated the inability of these defective complemented RNAs to be packaged into virus particles (W. J. Liu, P. L. Sedlak, N. Kondratieva, and A. A. Khromykh, J. Virol. 76:10766-10775). In this study we aimed to establish whether this requirement for NS3 in RNA packaging is determined by the secondary RNA structure of the NS3 gene or by the essential role of the translated NS3 gene product. Multiple silent mutations of three computer-predicted stable RNA structures in the NS3 coding region of KUN replicon RNA aimed at disrupting RNA secondary structure without affecting amino acid sequence did not affect RNA replication and packaging into virus-like particles in the packaging cell line, thus demonstrating that the predicted conserved RNA structures in the NS3 gene do not play a role in RNA replication and/or packaging. In contrast, double frameshift mutations in the NS3 coding region of full-length KUN RNA, producing scrambled NS3 protein but retaining secondary RNA structure, resulted in the loss of ability of these defective RNAs to be packaged into virus particles in complementation experiments in KUN replicon-expressing cells. Furthermore, the more robust complementation-packaging system based on established stable cell lines producing large amounts of complemented replicating NS3-deficient replicon RNAs and infection with KUN virus to provide structural proteins also failed to detect any secreted virus-like particles containing packaged NS3-deficient replicon RNAs. These results have now firmly established the requirement of KUN NS3 protein translated in cis for genome packaging into virus particles.

  18. The ribosome structure controls and directs mRNA entry, translocation and exit dynamics

    International Nuclear Information System (INIS)

    Kurkcuoglu, Ozge; Doruker, Pemra; Jernigan, Robert L; Sen, Taner Z; Kloczkowski, Andrzej

    2008-01-01

    The protein-synthesizing ribosome undergoes large motions to effect the translocation of tRNAs and mRNA; here, the domain motions of this system are explored with a coarse-grained elastic network model using normal mode analysis. Crystal structures are used to construct various model systems of the 70S complex with/without tRNA, elongation factor Tu and the ribosomal proteins. Computed motions reveal the well-known ratchet-like rotational motion of the large subunits, as well as the head rotation of the small subunit and the high flexibility of the L1 and L7/L12 stalks, even in the absence of ribosomal proteins. This result indicates that these experimentally observed motions during translocation are inherently controlled by the ribosomal shape and only partially dependent upon GTP hydrolysis. Normal mode analysis further reveals the mobility of A- and P-tRNAs to increase in the absence of the E-tRNA. In addition, the dynamics of the E-tRNA is affected by the absence of the ribosomal protein L1. The mRNA in the entrance tunnel interacts directly with helicase proteins S3 and S4, which constrain the mRNA in a clamp-like fashion, as well as with protein S5, which likely orients the mRNA to ensure correct translation. The ribosomal proteins S7, S11 and S18 may also be involved in assuring translation fidelity by constraining the mRNA at the exit site of the channel. The mRNA also interacts with the 16S 3' end forming the Shine–Dalgarno complex at the initiation step; the 3' end may act as a 'hook' to reel in the mRNA to facilitate its exit

  19. Mathematical and Biological Modelling of RNA Secondary Structure and Its Effects on Gene Expression

    Directory of Open Access Journals (Sweden)

    T. A. Hughes

    2006-01-01

    Full Text Available Secondary structures within the 5′ untranslated regions of messenger RNAs can have profound effects on the efficiency of translation of their messages and thereby on gene expression. Consequently they can act as important regulatory motifs in both physiological and pathological settings. Current approaches to predicting the secondary structure of these RNA sequences find the structure with the global-minimum free energy. However, since RNA folds progressively from the 5′ end when synthesised or released from the translational machinery, this may not be the most probable structure. We discuss secondary structure prediction based on local-minimisation of free energy with thermodynamic fluctuations as nucleotides are added to the 3′ end and show that these can result in different secondary structures. We also discuss approaches for studying the extent of the translational inhibition specified by structures within the 5′ untranslated region.

  20. An empirical strategy to detect bacterial transcript structure from directional RNA-seq transcriptome data.

    Science.gov (United States)

    Wang, Yejun; MacKenzie, Keith D; White, Aaron P

    2015-05-07

    As sequencing costs are being lowered continuously, RNA-seq has gradually been adopted as the first choice for comparative transcriptome studies with bacteria. Unlike microarrays, RNA-seq can directly detect cDNA derived from mRNA transcripts at a single nucleotide resolution. Not only does this allow researchers to determine the absolute expression level of genes, but it also conveys information about transcript structure. Few automatic software tools have yet been established to investigate large-scale RNA-seq data for bacterial transcript structure analysis. In this study, 54 directional RNA-seq libraries from Salmonella serovar Typhimurium (S. Typhimurium) 14028s were examined for potential relationships between read mapping patterns and transcript structure. We developed an empirical method, combined with statistical tests, to automatically detect key transcript features, including transcriptional start sites (TSSs), transcriptional termination sites (TTSs) and operon organization. Using our method, we obtained 2,764 TSSs and 1,467 TTSs for 1331 and 844 different genes, respectively. Identification of TSSs facilitated further discrimination of 215 putative sigma 38 regulons and 863 potential sigma 70 regulons. Combining the TSSs and TTSs with intergenic distance and co-expression information, we comprehensively annotated the operon organization in S. Typhimurium 14028s. Our results show that directional RNA-seq can be used to detect transcriptional borders at an acceptable resolution of ±10-20 nucleotides. Technical limitations of the RNA-seq procedure may prevent single nucleotide resolution. The automatic transcript border detection methods, statistical models and operon organization pipeline that we have described could be widely applied to RNA-seq studies in other bacteria. Furthermore, the TSSs, TTSs, operons, promoters and unstranslated regions that we have defined for S. Typhimurium 14028s may constitute valuable resources that can be used for

  1. Microtubule dynamics: Caps, catastrophes, and coupled hydrolysis

    DEFF Research Database (Denmark)

    Flyvbjerg, H.; Holy, T.E.; Leibler, S.

    1996-01-01

    An effective theory is formulated for the dynamics of the guanosine triphosphate (GTP) cap believed to stabilize growing microtubules. The theory provides a ''coarse-grained'' description of the cap's dynamics. ''Microscopic'' details, such as the microtubule lattice structure and the fate of its...

  2. Synthesis of 4-thiouridine, 6-thioinosine, and 6-thioguanosine 3',5'-O-bisphosphates as donor molecules for RNA ligation and their application to the synthesis of photoactivatable TMG-capped U1 snRNA fragments.

    Science.gov (United States)

    Kadokura, M; Wada, T; Seio, K; Sekine, M

    2000-08-25

    4-Thiouridine, 6-thioguanosine, and 6-thioinosine 3',5'-bisphosphates (9, 20, and 28) were synthesized in good yields by considerably improved methods. In the former two compounds, uridine and 2-N-phenylacetylguanosine were converted via transient O-trimethylsilylation to the corresponding 4- and 6-O-benzenesulfonyl intermediates (2 and 13), which, in turn, were allowed to react with 2-cyanoethanethiol in the presence of N-methylpyrrolidine to give 4-thiouridine (3) and 2-N-phenylacetyl-6-thioguanosine derivatives (14), respectively. In situ dimethoxytritylation of these thionucleoside derivatives gave the 5'-masked products 4 and 15 in high overall yields from 1 and 11. 6-S-(2-Cyanoethyl)-5'-O-(4,4'-dimethoxytrityl)-6-thioinosine (23) was synthesized via substitution of the 5'-O-tritylated 6-chloropurine riboside derivative 22 with 2-cyanoethanethiol. These S-(2-cyanoethyl)thionucleosides were converted to the 2'-O-(tert-butyldimethylsilyl)ribonucleoside 3'-phosphoramidite derivatives 7, 18, and 26 or 3',5'-bisphosphate derivatives 8, 19, and 27. Treatment of 8, 19, and 27 with DBU gave thionucleoside 3',5'-bisphosphate derivatives 9, 20, and 28, which were found to be substrates of T4 RNA ligase. These thionucleoside 3',5'-bisphosphates were examined as donors for ligation with m3(2,2,7) G5'pppAmUmA, i.e., the 5'-terminal tetranucleotide fragment of U1 snRNA, The 4-thiouridine 3',5'-bisphosphate derivative 9 was found to serve as the most active substrate of T4 RNA ligase with a reaction efficiency of 96%.

  3. General enumeration of RNA secondary structures based on new ...

    African Journals Online (AJOL)

    Crick base pairs between AU and GC. Based on the new representation, this paper also computes the number of various types of constrained secondary structures taking the minimum stack length 1 and minimum size m for each bonding loop as ...

  4. A 3'-end structure in RNA2 of a crinivirus is essential for viral RNA synthesis and contributes to replication-associated translation activity.

    Science.gov (United States)

    Mongkolsiriwattana, Chawin; Zhou, Jaclyn S; Ng, James C K

    2016-10-03

    The terminal ends in the genome of RNA viruses contain features that regulate viral replication and/or translation. We have identified a Y-shaped structure (YSS) in the 3' terminal regions of the bipartite genome of Lettuce chlorosis virus (LCV), a member in the genus Crinivirus (family Closteroviridae). The YSS is the first in this family of viruses to be determined using Selective 2'-Hydroxyl Acylation Analyzed by Primer Extension (SHAPE). Using luciferase constructs/replicons, in vivo and in vitro assays showed that the 5' and YSS-containing 3' terminal regions of LCV RNA1 supported translation activity. In contrast, similar regions from LCV RNA2, including those upstream of the YSS, did not. LCV RNA2 mutants with nucleotide deletions or replacements that affected the YSS were replication deficient. In addition, the YSS of LCV RNA1 and RNA2 were interchangeable without affecting viral RNA synthesis. Translation and significant replication were observed for specific LCV RNA2 replicons only in the presence of LCV RNA1, but both processes were impaired when the YSS and/or its upstream region were incomplete or altered. These results are evidence that the YSS is essential to the viral replication machinery, and contributes to replication enhancement and replication-associated translation activity in the RNA2 replicons.

  5. Nuclei of aged myofibres undergo structural and functional changes suggesting impairment in RNA processing

    Directory of Open Access Journals (Sweden)

    C Pellicciari

    2009-06-01

    Full Text Available Advancing adult age is associated with a progressive decrease in skeletal muscle mass, strength and quality known as sarcopenia. The mechanisms underlying age-related skeletal muscle wasting and weakness are manifold and still remain to be fully elucidated. Despite the increasing evidence that the progress of muscle diseases leading to muscle atrophy/dystrophy may be related to defective RNA processing, no data on the morpho-functional features of skeletal muscle nuclei in sarcopenia are available at present. In this view, we have investigated, by combining morphometry and immunocytochemistry at light and electron microscopy, the fine structure of myonuclei as well as the distribution and amount of RNA processing factors in skeletal myofibres of biceps brachii and quadriceps femoris from adult and old rats. Results demonstrate that the myonuclei of aged type II fibres show an increased amount of condensed chromatin and lower amounts of phosphorylated polymerase II and DNA/RNA hybrid molecules, clearly indicating a decrease in pre-mRNA transcription rate compared to adult animals. In addition, myonuclei of aged fibres show decreased amounts of nucleoplasmic splicing factors and an accumulation of cleavage factors, polyadenilated RNA and perichromatin granules, suggesting a reduction in the processing and transport rate of premRNA. During ageing, it seems therefore that in rat myonuclei the entire production chain of mRNA, from synthesis to cytoplasmic export, is less efficient. This failure likely contributes to the reduced responsiveness of muscle cells to anabolic stimuli in the elderly.

  6. Structural insights into mechanisms of the small RNA methyltransferase HEN1

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Ying; Ji, Lijuan; Huang, Qichen; Vassylyev, Dmitry G.; Chen, Xuemei; Ma, Jin-Biao; (UAB); (UCR)

    2010-02-22

    RNA silencing is a conserved regulatory mechanism in fungi, plants and animals that regulates gene expression and defence against viruses and transgenes. Small silencing RNAs of {approx}20-30 nucleotides and their associated effector proteins, the Argonaute family proteins, are the central components in RNA silencing. A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2'-O-methylation on the 3' terminal nucleotide. A conserved S-adenosyl-L-methionine-dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification. Here we report the 3.1 {angstrom} crystal structure of full-length HEN1 from Arabidopsis in complex with a 22-nucleotide small RNA duplex and cofactor product S-adenosyl-L-homocysteine. Highly cooperative recognition of the small RNA substrate by multiple RNA binding domains and the methyltransferase domain in HEN1 measures the length of the RNA duplex and determines the substrate specificity. Metal ion coordination by both 2' and 3' hydroxyls on the 3'-terminal nucleotide and four invariant residues in the active site of the methyltransferase domain suggests a novel Mg{sup 2+}-dependent 2'-O-methylation mechanism.

  7. Structure determination of an 11-subunit exosome in complex with RNA by molecular replacement

    International Nuclear Information System (INIS)

    Makino, Debora Lika; Conti, Elena

    2013-01-01

    The crystallographic steps towards the structure determination of a complete eukaryotic exosome complex bound to RNA are presented. Phasing of this 11-protein subunit complex was carried out via molecular replacement. The RNA exosome is an evolutionarily conserved multi-protein complex involved in the 3′ degradation of a variety of RNA transcripts. In the nucleus, the exosome participates in the maturation of structured RNAs, in the surveillance of pre-mRNAs and in the decay of a variety of noncoding transcripts. In the cytoplasm, the exosome degrades mRNAs in constitutive and regulated turnover pathways. Several structures of subcomplexes of eukaryotic exosomes or related prokaryotic exosome-like complexes are known, but how the complete assembly is organized to fulfil processive RNA degradation has been unclear. An atomic snapshot of a Saccharomyces cerevisiae 420 kDa exosome complex bound to an RNA substrate in the pre-cleavage state of a hydrolytic reaction has been determined. Here, the crystallographic steps towards the structural elucidation, which was carried out by molecular replacement, are presented

  8. Structure of the prolyl-tRNA synthetase from the eukaryotic pathogen Giardia lamblia

    Energy Technology Data Exchange (ETDEWEB)

    Larson, Eric T.; Kim, Jessica E.; Napuli, Alberto J.; Verlinde, Christophe L. M. J.; Fan, Erkang; Zucker, Frank H.; Van Voorhis, Wesley C.; Buckner, Frederick S.; Hol, Wim G. J.; Merritt, Ethan A., E-mail: merritt@u.washington.edu [Medical Structural Genomics of Pathogenic Protozoa, (United States); University of Washington, Seattle, WA 98195 (United States)

    2012-09-01

    The structure of Giardia prolyl-tRNA synthetase cocrystallized with proline and ATP shows evidence for half-of-the-sites activity, leading to a corresponding mixture of reaction substrates and product (prolyl-AMP) in the two active sites of the dimer. The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolution crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine.

  9. Rational Modular RNA Engineering Based on In Vivo Profiling of Structural Accessibility.

    Science.gov (United States)

    Leistra, Abigail N; Amador, Paul; Buvanendiran, Aishwarya; Moon-Walker, Alex; Contreras, Lydia M

    2017-12-15

    Bacterial small RNAs (sRNAs) have been established as powerful parts for controlling gene expression. However, development and application of engineered sRNAs has primarily focused on regulating novel synthetic targets. In this work, we demonstrate a rational modular RNA engineering approach that uses in vivo structural accessibility measurements to tune the regulatory activity of a multisubstrate sRNA for differential control of its native target network. Employing the CsrB global sRNA regulator as a model system, we use published in vivo structural accessibility data to infer the contribution of its local structures (substructures) to function and select a subset for engineering. We then modularly recombine the selected substructures, differentially representing those of presumed high or low functional contribution, to build a library of 21 CsrB variants. Using fluorescent translational reporter assays, we demonstrate that the CsrB variants achieve a 5-fold gradient of control of well-characterized Csr network targets. Interestingly, results suggest that less conserved local structures within long, multisubstrate sRNAs may represent better targets for rational engineering than their well-conserved counterparts. Lastly, mapping the impact of sRNA variants on a signature Csr network phenotype indicates the potential of this approach for tuning the activity of global sRNA regulators in the context of metabolic engineering applications.

  10. Trans-acting RNAs as molecular probes for monitoring time-dependent structural change of an RNA complex adapting two structures.

    Science.gov (United States)

    Maeda, Yuri; Furuta, Hiroyuki; Ikawa, Yoshiya

    2011-03-01

    As dynamic structural changes are pivotal for the functions of some classes of RNA molecule, it is important to develop methods to monitor structural changes in RNA in a time-dependent manner without chemical modification. Based on previous reports that trans-acting RNAs can be used as probes for analysis and control of 3D structures of target RNAs, we applied this method to monitor time-dependent structural changes in RNA. We designed and performed a proof-of-principle study using a simple model RNA complex that adopts two different structures as a target. The time-dependent structural changes in the target RNA were successfully monitored using two trans-acting RNAs, which stably form a ternary complex with the bimolecular target RNA and act as a catalyst to join two RNA fragments of the target complex, respectively. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  11. Self containment, a property of modular RNA structures, distinguishes microRNAs.

    Directory of Open Access Journals (Sweden)

    Miler T Lee

    2008-08-01

    Full Text Available RNA molecules will tend to adopt a folded conformation through the pairing of bases on a single strand; the resulting so-called secondary structure is critical to the function of many types of RNA. The secondary structure of a particular substring of functional RNA may depend on its surrounding sequence. Yet, some RNAs such as microRNAs retain their specific structures during biogenesis, which involves extraction of the substructure from a larger structural context, while other functional RNAs may be composed of a fusion of independent substructures. Such observations raise the question of whether particular functional RNA substructures may be selected for invariance of secondary structure to their surrounding nucleotide context. We define the property of self containment to be the tendency for an RNA sequence to robustly adopt the same optimal secondary structure regardless of whether it exists in isolation or is a substring of a longer sequence of arbitrary nucleotide content. We measured degree of self containment using a scoring method we call the self-containment index and found that miRNA stem loops exhibit high self containment, consistent with the requirement for structural invariance imposed by the miRNA biogenesis pathway, while most other structured RNAs do not. Further analysis revealed a trend toward higher self containment among clustered and conserved miRNAs, suggesting that high self containment may be a characteristic of novel miRNAs acquiring new genomic contexts. We found that miRNAs display significantly enhanced self containment compared to other functional RNAs, but we also found a trend toward natural selection for self containment in most functional RNA classes. We suggest that self containment arises out of selection for robustness against perturbations, invariance during biogenesis, and modular composition of structural function. Analysis of self containment will be important for both annotation and design of functional

  12. Base pairing and structural insights into the 5-formylcytosine in RNA duplex

    Science.gov (United States)

    Wang, Rui; Luo, Zhipu; He, Kaizhang; Delaney, Michael O.; Chen, Doris; Sheng, Jia

    2016-01-01

    Abstract 5-Formylcytidine (f5C), a previously discovered natural nucleotide in the mitochondrial tRNA of many species including human, has been recently detected as the oxidative product of 5-methylcytidine (m5C) through 5-hydroxymethylcytidine (hm5C) in total RNA of mammalian cells. The discovery indicated that these cytosine derivatives in RNA might also play important epigenetic roles similar as in DNA, which has been intensively investigated in the past few years. In this paper, we studied the base pairing specificity of f5C in different RNA duplex contexts. We found that the 5-formyl group could increase duplex thermal stability and enhance base pairing specificity. We present three high-resolution crystal structures of an octamer RNA duplex [5′-GUA(f5C)GUAC-3′]2 that have been solved under three crystallization conditions with different buffers and pH values. Our results showed that the 5-formyl group is located in the same plane as the cytosine base and forms an intra-residue hydrogen bond with the amino group in the N4 position. In addition, this modification increases the base stacking between the f5C and the neighboring bases while not causing significant global and local structure perturbations. This work provides insights into the effects of 5-formylcytosine on RNA duplex. PMID:27079978

  13. Annotating the protein-RNA interaction sites in proteins using evolutionary information and protein backbone structure.

    Science.gov (United States)

    Li, Tao; Li, Qian-Zhong

    2012-11-07

    RNA-protein interactions play important roles in various biological processes. The precise detection of RNA-protein interaction sites is very important for understanding essential biological processes and annotating the function of the proteins. In this study, based on various features from amino acid sequence and structure, including evolutionary information, solvent accessible surface area and torsion angles (φ, ψ) in the backbone structure of the polypeptide chain, a computational method for predicting RNA-binding sites in proteins is proposed. When the method is applied to predict RNA-binding sites in three datasets: RBP86 containing 86 protein chains, RBP107 containing 107 proteins chains and RBP109 containing 109 proteins chains, better sensitivities and specificities are obtained compared to previously published methods in five-fold cross-validation tests. In order to make further examination for the efficiency of our method, the RBP107 dataset is used as training set, RBP86 and RBP109 datasets are used as the independent test sets. In addition, as examples of our prediction, RNA-binding sites in a few proteins are presented. The annotated results are consistent with the PDB annotation. These results show that our method is useful for annotating RNA binding sites of novel proteins.

  14. Structure of E. coli 16S RNA elucidated by psoralen crosslinking

    International Nuclear Information System (INIS)

    Thompson, J.F.; Hearst, J.E.

    1983-01-01

    E. coli 16S RNA in solution was photoreacted with hydroxymethyltrimethylpsoralen and long wave ultraviolet light. Positions of crosslinks were determined to high resolution by partially digesting the RNA with T 1 RNase, separating the crosslinked fragments by two-dimensional gel electrophoresis, reversing the crosslink, and sequencing the separated fragments. This method yielded the locations of crosslinks to +/-15 nucleotides. Even finer placement has been made on the basis of our knowledge of psoralen reactivity. Thirteen unique crosslinks were mapped. Seven crosslinks confirmed regions of secondary structure which had been predicted in published phylogenetic models, three crosslinks discriminated between phylogenetic models, and three proved the existence of new structures. The new structures were all long-range interactions which appear to be in dynamic equilibrium with local secondary structure. Because this technique yields direct information about the secondary structure of large RNAs, it should prove invaluable in studying the structure of other RNAs of all sizes

  15. Rich RNA Structure Landscapes Revealed by Mutate-and-Map Analysis.

    Directory of Open Access Journals (Sweden)

    Pablo Cordero

    2015-11-01

    Full Text Available Landscapes exhibiting multiple secondary structures arise in natural RNA molecules that modulate gene expression, protein synthesis, and viral infection [corrected]. We report herein that high-throughput chemical experiments can isolate an RNA's multiple alternative secondary structures as they are stabilized by systematic mutagenesis (mutate-and-map, M2 and that a computational algorithm, REEFFIT, enables unbiased reconstruction of these states' structures and populations. In an in silico benchmark on non-coding RNAs with complex landscapes, M2-REEFFIT recovers 95% of RNA helices present with at least 25% population while maintaining a low false discovery rate (10% and conservative error estimates. In experimental benchmarks, M2-REEFFIT recovers the structure landscapes of a 35-nt MedLoop hairpin, a 110-nt 16S rRNA four-way junction with an excited state, a 25-nt bistable hairpin, and a 112-nt three-state adenine riboswitch with its expression platform, molecules whose characterization previously required expert mutational analysis and specialized NMR or chemical mapping experiments. With this validation, M2-REEFFIT enabled tests of whether artificial RNA sequences might exhibit complex landscapes in the absence of explicit design. An artificial flavin mononucleotide riboswitch and a randomly generated RNA sequence are found to interconvert between three or more states, including structures for which there was no design, but that could be stabilized through mutations. These results highlight the likely pervasiveness of rich landscapes with multiple secondary structures in both natural and artificial RNAs and demonstrate an automated chemical/computational route for their empirical characterization.

  16. Defining the structural requirements for a helix in 23 S ribosomal RNA that confers erythromycin resistance

    DEFF Research Database (Denmark)

    Douthwaite, S; Powers, T; Lee, J Y

    1989-01-01

    The helix spanning nucleotides 1198 to 1247 (helix 1200-1250) in Escherichia coli 23 S ribosomal RNA (rRNA) is functionally important in protein synthesis, and deletions in this region confer erythromycin resistance. In order to define the structural requirements for resistance, we have dissected...... deletion mutants show a sensitive phenotype. Deletions that extend into the base-pairing between GCC1208 and GGU1240 result in non-functional 23 S RNAs, which consequently do not confer resistance. A number of phylogenetically conserved nucleotides have been shown to be non-essential for 23 S RNA function....... However, removal of either these or non-conserved nucleotides from helix 1200-1250 measurably reduces the efficiency of 23 S RNA in forming functional ribosomes. We have used chemical probing and a modified primer extension method to investigate erythromycin binding to wild-type and resistant ribosomes...

  17. The Structure of the RNA m5C Methyltransferase YebU from Escherichia coli Reveals a C-terminal RNA-recruiting PUA Domain

    DEFF Research Database (Denmark)

    Hallberg, B. Martin; Ericsson, Ulrika B.; Johnson, Kenneth A

    2006-01-01

    potential that differ from other RNA-MTase structures, suggesting that YebU interacts with its RNA target in a different manner. Docking of YebU onto the 30 S subunit indicates that the PUA and MTase domains make several contacts with 16 S rRNA as well as with the ribosomal protein S12. The ribosomal...... protein interactions would explain why the assembled 30 S subunit, and not naked 16 S rRNA, is the preferred substrate for YebU....... by X-ray crystallography, and we present a molecular model for how YebU specifically recognizes, binds and methylates its ribosomal substrate. The YebU protein has an N-terminal SAM-binding catalytic domain with structural similarity to the equivalent domains in several other m(5)C RNA MTases including...

  18. Structural profiles of human miRNA families from pairwise clustering

    DEFF Research Database (Denmark)

    Kaczkowski, Bogumil; Þórarinsson, Elfar; Reiche, Kristin

    2009-01-01

    secondary structure already predicted, little is known about the patterns of structural conservation among pre-miRNAs. We address this issue by clustering the human pre-miRNA sequences based on pairwise, sequence and secondary structure alignment using FOLDALIGN, followed by global multiple alignment...... of obtained clusters by WAR. As a result, the common secondary structure was successfully determined for four FOLDALIGN clusters: the RF00027 structural family of the Rfam database and three clusters with previously undescribed consensus structures. Availability: http://genome.ku.dk/resources/mirclust...

  19. Modeling the structure of RNA molecules with small-angle X-ray scattering data.

    Directory of Open Access Journals (Sweden)

    Michal Jan Gajda

    Full Text Available We propose a novel fragment assembly method for low-resolution modeling of RNA and show how it may be used along with small-angle X-ray solution scattering (SAXS data to model low-resolution structures of particles having as many as 12 independent secondary structure elements. We assessed this model-building procedure by using both artificial data on a previously proposed benchmark and publicly available data. With the artificial data, SAXS-guided models show better similarity to native structures than ROSETTA decoys. The publicly available data showed that SAXS-guided models can be used to reinterpret RNA structures previously deposited in the Protein Data Bank. Our approach allows for fast and efficient building of de novo models of RNA using approximate secondary structures that can be readily obtained from existing bioinformatic approaches. We also offer a rigorous assessment of the resolving power of SAXS in the case of small RNA structures, along with a small multimetric benchmark of the proposed method.

  20. RNA-Binding Proteins in Plant Immunity

    Directory of Open Access Journals (Sweden)

    Virginia Woloshen

    2011-01-01

    Full Text Available Plant defence responses against pathogen infection are crucial to plant survival. The high degree of regulation of plant immunity occurs both transcriptionally and posttranscriptionally. Once transcribed, target gene RNA must be processed prior to translation. This includes polyadenylation, 5′capping, editing, splicing, and mRNA export. RNA-binding proteins (RBPs have been implicated at each level of RNA processing. Previous research has primarily focused on structural RNA-binding proteins of yeast and mammals; however, more recent work has characterized a number of plant RBPs and revealed their roles in plant immune responses. This paper provides an update on the known functions of RBPs in plant immune response regulation. Future in-depth analysis of RBPs and other related players will unveil the sophisticated regulatory mechanisms of RNA processing during plant immune responses.

  1. NOBAI: a web server for character coding of geometrical and statistical features in RNA structure

    Science.gov (United States)

    Knudsen, Vegeir; Caetano-Anollés, Gustavo

    2008-01-01

    The Numeration of Objects in Biology: Alignment Inferences (NOBAI) web server provides a web interface to the applications in the NOBAI software package. This software codes topological and thermodynamic information related to the secondary structure of RNA molecules as multi-state phylogenetic characters, builds character matrices directly in NEXUS format and provides sequence randomization options. The web server is an effective tool that facilitates the search for evolutionary history embedded in the structure of functional RNA molecules. The NOBAI web server is accessible at ‘http://www.manet.uiuc.edu/nobai/nobai.php’. This web site is free and open to all users and there is no login requirement. PMID:18448469

  2. Evolution of the RNase P RNA structural domain in Leptospira spp.

    Science.gov (United States)

    Ravishankar, Vigneshwaran; Ahmed, Ahmed; Sivagnanam, Ulaganathan; Muthuraman, Krishnaraja; Karthikaichamy, Anbarasu; Wilson, Herald A; Devendran, Ajay; Hartskeerl, Rudy A; Raj, Stephen M L

    2014-12-01

    We have employed the RNase P RNA (RPR) gene, which is present as single copy in chromosome I of Leptospira spp. to investigate the phylogeny of structural domains present in the RNA subunit of the tRNA processing enzyme, RNase P. RPR gene sequences of 150 strains derived from NCBI database along with sequences determined from 8 reference strains were examined to fathom strain specific structural differences present in leptospiral RPR. Sequence variations in the RPR gene impacted on the configuration of loops, stems and bulges found in the RPR highlighting species and strain specific structural motifs. In vitro transcribed leptospiral RPR ribozymes are demonstrated to process pre-tRNA into mature tRNA in consonance with the positioning of Leptospira in the taxonomic domain of bacteria. RPR sequence datasets used to construct a phylogenetic tree exemplified the segregation of strains into their respective lineages with a (re)speciation of strain SH 9 to Leptospira borgpetersenii, strains Fiocruz LV 3954 and Fiocruz LV 4135 to Leptospira santarosai, strain CBC 613 to Leptospira kirschneri and strain HAI 1536 to Leptospira noguchii. Furthermore, it allowed characterization of an isolate P2653, presumptively characterized as either serovar Hebdomadis, Kremastos or Longnan to Leptospira weilii, serovar Longnan. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  3. A quantitative analysis of secondary RNA structure using domination based parameters on trees

    Directory of Open Access Journals (Sweden)

    Zou Yue

    2006-03-01

    Full Text Available Abstract Background It has become increasingly apparent that a comprehensive database of RNA motifs is essential in order to achieve new goals in genomic and proteomic research. Secondary RNA structures have frequently been represented by various modeling methods as graph-theoretic trees. Using graph theory as a modeling tool allows the vast resources of graphical invariants to be utilized to numerically identify secondary RNA motifs. The domination number of a graph is a graphical invariant that is sensitive to even a slight change in the structure of a tree. The invariants selected in this study are variations of the domination number of a graph. These graphical invariants are partitioned into two classes, and we define two parameters based on each of these classes. These parameters are calculated for all small order trees and a statistical analysis of the resulting data is conducted to determine if the values of these parameters can be utilized to identify which trees of orders seven and eight are RNA-like in structure. Results The statistical analysis shows that the domination based parameters correctly distinguish between the trees that represent native structures and those that are not likely candidates to represent RNA. Some of the trees previously identified as candidate structures are found to be "very" RNA like, while others are not, thereby refining the space of structures likely to be found as representing secondary RNA structure. Conclusion Search algorithms are available that mine nucleotide sequence databases. However, the number of motifs identified can be quite large, making a further search for similar motif computationally difficult. Much of the work in the bioinformatics arena is toward the development of better algorithms to address the computational problem. This work, on the other hand, uses mathematical descriptors to more clearly characterize the RNA motifs and thereby reduce the corresponding search space. These

  4. Thermodynamics and kinetics of RNA tertiary structure formation in the junctionless hairpin ribozyme.

    Science.gov (United States)

    White, Neil A; Hoogstraten, Charles G

    2017-09-01

    The hairpin ribozyme consists of two RNA internal loops that interact to form the catalytically active structure. This docking transition is a rare example of intermolecular formation of RNA tertiary structure without coupling to helix annealing. We have used temperature-dependent surface plasmon resonance (SPR) to characterize the thermodynamics and kinetics of RNA tertiary structure formation for the junctionless form of the ribozyme, in which loops A and B reside on separate molecules. We find docking to be strongly enthalpy-driven and to be accompanied by substantial activation barriers for association and dissociation, consistent with the structural reorganization of both internal loops upon complex formation. Comparisons with the parallel analysis of a ribozyme variant carrying a 2'-O-methyl modification at the self-cleavage site and with published data in other systems reveal a surprising diversity of thermodynamic signatures, emphasizing the delicate balance of contributions to the free energy of formation of RNA tertiary structure. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. RNA is an integral component of chromatin that contributes to its structural organization.

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    Antonio Rodríguez-Campos

    Full Text Available Chromatin structure is influenced by multiples factors, such as pH, temperature, nature and concentration of counterions, post-translational modifications of histones and binding of structural non-histone proteins. RNA is also known to contribute to the regulation of chromatin structure as chromatin-induced gene silencing was shown to depend on the RNAi machinery in S. pombe, plants and Drosophila. Moreover, both in Drosophila and mammals, dosage compensation requires the contribution of specific non-coding RNAs. However, whether RNA itself plays a direct structural role in chromatin is not known. Here, we report results that indicate a general structural role for RNA in eukaryotic chromatin. RNA is found associated to purified chromatin prepared from chicken liver, or cultured Drosophila S2 cells, and treatment with RNase A alters the structural properties of chromatin. Our results indicate that chromatin-associated RNAs, which account for 2%-5% of total chromatin-associated nucleic acids, are polyA(- and show a size similar to that of the DNA contained in the corresponding chromatin fragments. Chromatin-associated RNA(s are not likely to correspond to nascent transcripts as they are also found bound to chromatin when cells are treated with alpha-amanitin. After treatment with RNase A, chromatin fragments of molecular weight >3.000 bp of DNA showed reduced sedimentation through sucrose gradients and increased sensitivity to micrococcal nuclease digestion. This structural transition, which is observed both at euchromatic and heterochromatic regions, proceeds without loss of histone H1 or any significant change in core-histone composition and integrity.

  6. Crystal Structure of the N-Terminal RNA Recognition Motif of mRNA Decay Regulator AUF1

    Directory of Open Access Journals (Sweden)

    Young Jun Choi

    2016-01-01

    Full Text Available AU-rich element binding/degradation factor 1 (AUF1 plays a role in destabilizing mRNAs by forming complexes with AU-rich elements (ARE in the 3′-untranslated regions. Multiple AUF1-ARE complexes regulate the translation of encoded products related to the cell cycle, apoptosis, and inflammation. AUF1 contains two tandem RNA recognition motifs (RRM and a Gln- (Q- rich domain in their C-terminal region. To observe how the two RRMs are involved in recognizing ARE, we obtained the AUF1-p37 protein covering the two RRMs. However, only N-terminal RRM (RRM1 was crystallized and its structure was determined at 1.7 Å resolution. It appears that the RRM1 and RRM2 separated before crystallization. To demonstrate which factors affect the separate RRM1-2, we performed limited proteolysis using trypsin. The results indicated that the intact proteins were cleaved by unknown proteases that were associated with them prior to crystallization. In comparison with each of the monomers, the conformations of the β2-β3 loops were highly variable. Furthermore, a comparison with the RRM1-2 structures of HuR and hnRNP A1 revealed that a dimer of RRM1 could be one of the possible conformations of RRM1-2. Our data may provide a guidance for further structural investigations of AUF1 tandem RRM repeat and its mode of ARE binding.

  7. Extracellular plasma RNA from colon cancer patients is confined in a vesicle-like structure and is mRNA-enriched

    Science.gov (United States)

    García, José Miguel; García, Vanesa; Peña, Cristina; Domínguez, Gemma; Silva, Javier; Diaz, Raquel; Espinosa, Pablo; Citores, Maria Jesús; Collado, Manuel; Bonilla, Félix

    2008-01-01

    Little is yet known about the origin and protective mechanism of free nucleic acids in plasma. We investigated the possibility of these free nucleic acids being particle associated. Plasma samples from colon cancer patients and cell culture media were subjected to various antibody incubations, ultracentrifugation, and RNA extraction protocols for total RNA, epithelial RNA, and mRNA. Flow cytometry using a Ber-EP4 antibody and confocal laser microscopy after staining with propidium iodide were also performed. mRNA levels of the LISCH7 and SDHA genes were determined in cells and in culture media. Ber-EP4 antibody and polystyrene beads coated with oligo dT sequences were employed. We observed that, after incubation, total RNA and mRNA were always detected after membrane digestion, and that epithelial RNA was detected before this procedure. In ultracentrifugation, mRNA was caught in the supernatant only if a former lysis mediated or in the pellet if there was no previous digestion. Flow cytometry determinations showed that antibody-coated microbeads keep acellular structures bearing epithelial antigens apart. Confocal laser microscopy made 1- to 2-μm-diameter particles perceptible in the vicinity of magnetic polystyrene beads. Relevant differences were observed between mRNA of cells and culture media, as there was a considerable difference in LISCH7 mRNA levels between HT29 and IMR90 cell co-cultures and their culture media. Our results support the view that extracellular RNA found in plasma from cancer patients circulates in association with or is protected in a multiparticle complex, and that an active release mechanism by tumor cells may be a possible origin. PMID:18456845

  8. Viral replication. Structural basis for RNA replication by the hepatitis C virus polymerase.

    Science.gov (United States)

    Appleby, Todd C; Perry, Jason K; Murakami, Eisuke; Barauskas, Ona; Feng, Joy; Cho, Aesop; Fox, David; Wetmore, Diana R; McGrath, Mary E; Ray, Adrian S; Sofia, Michael J; Swaminathan, S; Edwards, Thomas E

    2015-02-13

    Nucleotide analog inhibitors have shown clinical success in the treatment of hepatitis C virus (HCV) infection, despite an incomplete mechanistic understanding of NS5B, the viral RNA-dependent RNA polymerase. Here we study the details of HCV RNA replication by determining crystal structures of stalled polymerase ternary complexes with enzymes, RNA templates, RNA primers, incoming nucleotides, and catalytic metal ions during both primed initiation and elongation of RNA synthesis. Our analysis revealed that highly conserved active-site residues in NS5B position the primer for in-line attack on the incoming nucleotide. A β loop and a C-terminal membrane-anchoring linker occlude the active-site cavity in the apo state, retract in the primed initiation assembly to enforce replication of the HCV genome from the 3' terminus, and vacate the active-site cavity during elongation. We investigated the incorporation of nucleotide analog inhibitors, including the clinically active metabolite formed by sofosbuvir, to elucidate key molecular interactions in the active site. Copyright © 2015, American Association for the Advancement of Science.

  9. The suboptimal structures find the optimal RNAs: homology search for bacterial non-coding RNAsusing suboptimal RNA structures

    Czech Academy of Sciences Publication Activity Database

    Pánek, Josef; Krásný, Libor; Bobek, Jan; Ježková, E.; Korelusová, Jana; Vohradský, Jiří

    -, - (2010), s. 1-9 ISSN 1362-4962 R&D Projects: GA MŠk 2B06065; GA ČR GA303/09/0475; GA ČR GA310/07/1009 Institutional research plan: CEZ:AV0Z50200510 Keywords : ncRNAs * RNA structures Subject RIV: EE - Microbiology, Virology

  10. Structure of a conserved hypothetical protein SA1388 from S. aureus reveals a capped hexameric toroid with two PII domain lids and a dinuclear metal center

    Directory of Open Access Journals (Sweden)

    Leybourne Matthew

    2006-12-01

    Full Text Available Abstract Background The protein encoded by the SA1388 gene from Staphylococcus aureus was chosen for structure determination to elucidate its domain organization and confirm our earlier remote homology based prediction that it housed a nitrogen regulatory PII protein-like domain. SA1388 was predicted to contain a central PII-like domain and two flanking regions, which together belong to the NIF3-like protein family. Proteins like SA1388 remain a poorly studied group and their structural characterization could guide future investigations aimed at understanding their function. Results The structure of SA1388 has been solved to 2.0Å resolution by single wavelength anomalous dispersion phasing method using selenium anomalous signals. It reveals a canonical NIF3-like fold containing two domains with a PII-like domain inserted in the middle of the polypeptide. The N and C terminal halves of the NIF3-like domains are involved in dimerization, while the PII domain forms trimeric contacts with symmetry related monomers. Overall, the NIF3-like domains of SA1388 are organized as a hexameric toroid similar to its homologs, E. coli ybgI and the hypothetical protein SP1609 from Streptococcus pneumoniae. The openings on either side of the toroid are partially covered by trimeric "lids" formed by the PII domains. The junction of the two NIF3 domains has two zinc ions bound at what appears to be a histidine rich active site. A well-defined electron density corresponding to an endogenously bound ligand of unknown identity is observed in close proximity to the metal site. Conclusion SA1388 is the third member of the NIF3-like family of proteins to be structurally characterized, the other two also being hypothetical proteins of unknown function. The structure of SA1388 confirms our earlier prediction that the inserted domain that separates the two NIF3 domains adopts a PII-like fold and reveals an overall capped toroidal arrangement for the protein hexamer. The

  11. Finding the most significant common sequence and structure motifs in a set of RNA sequences

    DEFF Research Database (Denmark)

    Gorodkin, Jan; Heyer, L.J.; Stormo, G.D.

    1997-01-01

    We present a computational scheme to locally align a collection of RNA sequences using sequence and structure constraints, In addition, the method searches for the resulting alignments with the most significant common motifs, among all possible collections, The first part utilizes a simplified...

  12. Bioinformatical approaches to RNA structure prediction & Sequencing of an ancient human genome

    DEFF Research Database (Denmark)

    Lindgreen, Stinus

    Stinus Lindgreen has been working in two different fields during his Ph.D. The first part has been focused on computational approaches to predict the structure of non-coding RNA molecules at the base pairing level. This has resulted in the analysis of various measures of the base pairing potentia...

  13. SHAPE selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data

    DEFF Research Database (Denmark)

    Poulsen, Line Dahl; Kielpinski, Lukasz Jan; Salama, Sofie R

    2015-01-01

    transcriptase. Here, we introduce a SHAPE Selection (SHAPES) reagent, N-propanone isatoic anhydride (NPIA), which retains the ability of SHAPE reagents to accurately probe RNA structure, but also allows covalent coupling between the SHAPES reagent and a biotin molecule. We demonstrate that SHAPES...

  14. A possible contribution of mRNA secondary structure to translation initiation efficiency in Lactococcus lactis

    NARCIS (Netherlands)

    Guchte, Maarten van de; Lende, Ted van der; Kok, Jan; Venema, Gerard

    1991-01-01

    Gene expression signals derived from Lactococcus lactis were linked to lacZ-fused genes with different 5'-nucleotide sequences. Computer predictions of mRNA secondary structure were combined with lacZ expression studies to direct base-substitutions that could possibly influence gene expression.

  15. Structure of Pseudoknot PK26 Shows 3D Domain Swapping in an RNA

    Science.gov (United States)

    Lietzke, Susan E; Barnes, Cindy L.

    1998-01-01

    3D domain swapping provides a facile pathway for the evolution of oligomeric proteins and allosteric mechanisms and a means for using monomer-oligomer equilibria to regulate biological activity. The term "3D domain swapping" describes the exchange of identical domains between two protein monomers to create an oligomer. 3D domain swapping has, so far, only been recognized in proteins. In this study, the structure of the pseudoknot PK26 is reported and it is a clear example of 3D domain swapping in RNA. PK26 was chosen for study because RNA pseudoknots are required structures in several biological processes and they arise frequently in in vitro selection experiments directed against protein targets. PK26 specifically inhibits HIV-1 reverse transcriptase with nanomolar affinity. We have now determined the 3.1 A resolution crystal structure of PK26 and find that it forms a 3D domain swapped dimer. PK26 shows extensive base pairing between and within strands. Formation of the dimer requires the linker region between the pseudoknot folds to adopt a unique conformation that allows a base within a helical stem to skip one base in the stacking register. Rearrangement of the linker would permit a monomeric pseudoknot to form. This structure shows how RNA can use 3D domain swapping to build large scale oligomers like the putative hexamer in the packaging RNA of bacteriophage Phi29.

  16. Glassy transition in a disordered model for the RNA secondary structure

    International Nuclear Information System (INIS)

    Pagnani, A.; Parisi, G.; Ricci-Tersenghi, F.

    2000-04-01

    We numerically study a disordered model for the RNA secondary structure and we find that it undergoes a phase transition, with a breaking of the replica symmetry in the low temperature region (like in spin glasses). Our results are based on the exact evaluation of the partition function. (author)

  17. Structural and biochemical studies on ATP binding and hydrolysis by the Escherichia coli RNA chaperone Hfq.

    Directory of Open Access Journals (Sweden)

    Hermann Hämmerle

    Full Text Available In Escherichia coli the RNA chaperone Hfq is involved in riboregulation by assisting base-pairing between small regulatory RNAs (sRNAs and mRNA targets. Several structural and biochemical studies revealed RNA binding sites on either surface of the donut shaped Hfq-hexamer. Whereas sRNAs are believed to contact preferentially the YKH motifs present on the proximal site, poly(A(15 and ADP were shown to bind to tripartite binding motifs (ARE circularly positioned on the distal site. Hfq has been reported to bind and to hydrolyze ATP. Here, we present the crystal structure of a C-terminally truncated variant of E. coli Hfq (Hfq(65 in complex with ATP, showing that it binds to the distal R-sites. In addition, we revisited the reported ATPase activity of full length Hfq purified to homogeneity. At variance with previous reports, no ATPase activity was observed for Hfq. In addition, FRET assays neither indicated an impact of ATP on annealing of two model oligoribonucleotides nor did the presence of ATP induce strand displacement. Moreover, ATP did not lead to destabilization of binary and ternary Hfq-RNA complexes, unless a vast stoichiometric excess of ATP was used. Taken together, these studies strongly suggest that ATP is dispensable for and does not interfere with Hfq-mediated RNA transactions.

  18. Structure-function relationship of substituted bromomethylcoumarins in nucleoside specificity of RNA alkylation.

    Science.gov (United States)

    Kellner, Stefanie; Kollar, Laura Bettina; Ochel, Antonia; Ghate, Manjunath; Helm, Mark

    2013-01-01

    Selective alkylation of RNA nucleotides is an important field of RNA biochemistry, e.g. in applications of fluorescent labeling or in structural probing experiments, yet detailed structure-function studies of labeling agents are rare. Here, bromomethylcoumarins as reactive compounds for fluorescent labeling of RNA are developed as an attractive scaffold on which electronic properties can be modulated by varying the substituents. Six different 4-bromomethyl-coumarins of various substitution patterns were tested for nucleotide specificity of RNA alkylation using tRNA from Escherichia coli as substrate. Using semi-quantitative LC-MS/MS analysis, reactions at mildly acidic and slightly alkaline pH were compared. For all tested compounds, coumarin conjugates with 4-thiouridine, pseudouridine, guanosine, and uridine were identified, with the latter largely dominating. This data set shows that selectivity of ribonucleotide alkylation depends on the substitution pattern of the reactive dye, and even more strongly on the modulation of the reaction conditions. The latter should be therefore carefully optimized when striving to achieve selectivity. Interestingly, the highest selectivity for labeling of a modified nucleoside, namely of 4-thiouridine, was achieved with a compound whose selectivity was somewhat less dependent on reaction conditions than the other compounds. In summary, bromomethylcoumarin derivatives are a highly interesting class of compounds, since their selectivity for 4-thiouridine can be efficiently tuned by variation of substitution pattern and reaction conditions.

  19. The subgenomic promoter of brome mosaic virus folds into a stem-loop structure capped by a pseudo-triloop that is structurally similar to the triloop of the genomic promoter

    DEFF Research Database (Denmark)

    Skov, J.; Gaudin, M.; Podbevsek, P.

    2012-01-01

    In brome mosaic virus, both the replication of the genomic (+)-RNA strands and the transcription of the subgenomic RNA are carried out by the viral replicase. The production of (-)-RNA strands is dependent on the formation of an AUA triloop in the stem-loop C (SLC) hairpin in the 3'-untranslated...... region of the (+)-RNA strands. Two alternate hypotheses have been put forward for the mechanism of subgenomic RNA transcription. One posits that transcription commences by recognition of at least four key nucleotides in the subgenomic promoter by the replicase. The other posits that subgenomic...... transcription starts by binding of the replicase to a hairpin formed by the subgenomic promoter that resembles the minus strand promoter hairpin SLC. In this study, we have determined the three-dimensional structure of the subgenomic promoter hairpin using NMR spectroscopy. The data show that the hairpin...

  20. Structure of the Hantavirus Nucleoprotein Provides Insights into the Mechanism of RNA Encapsidation

    Directory of Open Access Journals (Sweden)

    Daniel Olal

    2016-03-01

    Full Text Available Hantaviruses are etiological agents of life-threatening hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. The nucleoprotein (N of hantavirus is essential for viral transcription and replication, thus representing an attractive target for therapeutic intervention. We have determined the crystal structure of hantavirus N to 3.2 Å resolution. The structure reveals a two-lobed, mostly α-helical structure that is distantly related to that of orthobunyavirus Ns. A basic RNA binding pocket is located at the intersection between the two lobes. We provide evidence that oligomerization is mediated by amino- and C-terminal arms that bind to the adjacent monomers. Based on these findings, we suggest a model for the oligomeric ribonucleoprotein (RNP complex. Our structure provides mechanistic insights into RNA encapsidation in the genus Hantavirus and constitutes a template for drug discovery efforts aimed at combating hantavirus infections.

  1. Molecular structure and thermodynamic predictions to create highly sensitive microRNA biosensors

    International Nuclear Information System (INIS)

    Larkey, Nicholas E.; Brucks, Corinne N.; Lansing, Shan S.; Le, Sophia D.; Smith, Natasha M.; Tran, Victoria; Zhang, Lulu; Burrows, Sean M.

    2016-01-01

    Many studies have established microRNAs (miRNAs) as post-transcriptional regulators in a variety of intracellular molecular processes. Abnormal changes in miRNA have been associated with several diseases. However, these changes are sometimes subtle and occur at nanomolar levels or lower. Several biosensing hurdles for in situ cellular/tissue analysis of miRNA limit detection of small amounts of miRNA. Of these limitations the most challenging are selectivity and sensor degradation creating high background signals and false signals. Recently we developed a reporter+probe biosensor for let-7a that showed potential to mitigate false signal from sensor degradation. Here we designed reporter+probe biosensors for miR-26a-2-3p and miR-27a-5p to better understand the effect of thermodynamics and molecular structures of the biosensor constituents on the analytical performance. Signal changes from interactions between Cy3 and Cy5 on the reporters were used to understand structural aspects of the reporter designs. Theoretical thermodynamic values, single stranded conformations, hetero- and homodimerization structures, and equilibrium concentrations of the reporters and probes were used to interpret the experimental observations. Studies of the sensitivity and selectivity revealed 5–9 nM detection limits in the presence and absence of interfering off-analyte miRNAs. These studies will aid in determining how to rationally design reporter+probe biosensors to overcome hurdles associated with highly sensitive miRNA biosensing. - Highlights: • Challenges facing highly sensitive miRNA biosensor designs are addressed. • Thermodynamic and molecular structure design metrics for reporter+probe biosensors are proposed. • The influence of ideal and non-ideal reporter hairpin structures on reporter+probe formation and signal change are discussed. • 5–9 nM limits of detection were observed with no interference from off-analytes.

  2. Molecular structure and thermodynamic predictions to create highly sensitive microRNA biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Larkey, Nicholas E.; Brucks, Corinne N.; Lansing, Shan S.; Le, Sophia D.; Smith, Natasha M.; Tran, Victoria; Zhang, Lulu; Burrows, Sean M., E-mail: sean.burrows@oregonstate.edu

    2016-02-25

    Many studies have established microRNAs (miRNAs) as post-transcriptional regulators in a variety of intracellular molecular processes. Abnormal changes in miRNA have been associated with several diseases. However, these changes are sometimes subtle and occur at nanomolar levels or lower. Several biosensing hurdles for in situ cellular/tissue analysis of miRNA limit detection of small amounts of miRNA. Of these limitations the most challenging are selectivity and sensor degradation creating high background signals and false signals. Recently we developed a reporter+probe biosensor for let-7a that showed potential to mitigate false signal from sensor degradation. Here we designed reporter+probe biosensors for miR-26a-2-3p and miR-27a-5p to better understand the effect of thermodynamics and molecular structures of the biosensor constituents on the analytical performance. Signal changes from interactions between Cy3 and Cy5 on the reporters were used to understand structural aspects of the reporter designs. Theoretical thermodynamic values, single stranded conformations, hetero- and homodimerization structures, and equilibrium concentrations of the reporters and probes were used to interpret the experimental observations. Studies of the sensitivity and selectivity revealed 5–9 nM detection limits in the presence and absence of interfering off-analyte miRNAs. These studies will aid in determining how to rationally design reporter+probe biosensors to overcome hurdles associated with highly sensitive miRNA biosensing. - Highlights: • Challenges facing highly sensitive miRNA biosensor designs are addressed. • Thermodynamic and molecular structure design metrics for reporter+probe biosensors are proposed. • The influence of ideal and non-ideal reporter hairpin structures on reporter+probe formation and signal change are discussed. • 5–9 nM limits of detection were observed with no interference from off-analytes.

  3. Considerations in the identification of functional RNA structural elements in genomic alignments

    Directory of Open Access Journals (Sweden)

    Blencowe Benjamin J

    2007-01-01

    Full Text Available Abstract Background Accurate identification of novel, functional noncoding (nc RNA features in genome sequence has proven more difficult than for exons. Current algorithms identify and score potential RNA secondary structures on the basis of thermodynamic stability, conservation, and/or covariance in sequence alignments. Neither the algorithms nor the information gained from the individual inputs have been independently assessed. Furthermore, due to issues in modelling background signal, it has been difficult to gauge the precision of these algorithms on a genomic scale, in which even a seemingly small false-positive rate can result in a vast excess of false discoveries. Results We developed a shuffling algorithm, shuffle-pair.pl, that simultaneously preserves dinucleotide frequency, gaps, and local conservation in pairwise sequence alignments. We used shuffle-pair.pl to assess precision and recall of six ncRNA search tools (MSARI, QRNA, ddbRNA, RNAz, Evofold, and several variants of simple thermodynamic stability on a test set of 3046 alignments of known ncRNAs. Relative to mononucleotide shuffling, preservation of dinucleotide content in shuffling the alignments resulted in a drastic increase in estimated false-positive detection rates for ncRNA elements, precluding evaluation of higher order alignments, which cannot not be adequately shuffled maintaining both dinucleotides and alignment structure. On pairwise alignments, none of the covariance-based tools performed markedly better than thermodynamic scoring alone. Although the high false-positive rates call into question the veracity of any individual predicted secondary structural element in our analysis, we nevertheless identified intriguing global trends in human genome alignments. The distribution of ncRNA prediction scores in 75-base windows overlapping UTRs, introns, and intergenic regions analyzed using both thermodynamic stability and EvoFold (which has no thermodynamic component was

  4. Cyanobacteria contain a structural homologue of the Hfq protein with altered RNA binding properties

    DEFF Research Database (Denmark)

    Bøggild, Andreas; Overgaard, Martin; Valentin-Hansen, Poul

    2009-01-01

    Hfq proteins are common in many species of enterobacteria, where they participate in RNA folding and translational regulation through pairing of small RNAs and messenger RNAs. Hfq proteins share the distinctive Sm fold, and form ring-shaped structures similar to those of the Sm/Lsm proteins...... proteins from the cyanobacteria Synechocystis sp. PCC 6803 and Anabaena PCC 7120 at 1.3 and 2.3 A resolution, respectively, and show that they retain the classic Sm fold despite low sequence conservation. In addition, the intersubunit contacts and RNA-binding site are divergent, and we show biochemically...

  5. Cyanobacteria contain a structural homologue of the Hfq protein with altered RNA-binding properties

    DEFF Research Database (Denmark)

    Bøggild, Andreas; Overgaard, Martin; Valentin-Hansen, Poul

    2009-01-01

    Hfq proteins are common in many species of enterobacteria, where they participate in RNA folding and translational regulation through pairing of small RNAs and messenger RNAs. Hfq proteins share the distinctive Sm fold, and form ring-shaped structures similar to those of the Sm/Lsm proteins...... proteins from the cyanobacteria Synechocystis sp. PCC 6803 and Anabaena PCC 7120 at 1.3 and 2.3 A resolution, respectively, and show that they retain the classic Sm fold despite low sequence conservation. In addition, the intersubunit contacts and RNA-binding site are divergent, and we show biochemically...

  6. Locked nucleoside analogues expand the potential of DNAzymes to cleave structured RNA targets

    Directory of Open Access Journals (Sweden)

    Wengel Jesper

    2006-06-01

    Full Text Available Abstract Background DNAzymes cleave at predetermined sequences within RNA. A prerequisite for cleavage is that the DNAzyme can gain access to its target, and thus the DNAzyme must be capable of unfolding higher-order structures that are present in the RNA substrate. However, in many cases the RNA target sequence is hidden in a region that is too tightly structured to be accessed under physiological conditions by DNAzymes. Results We investigated how incorporation of LNA (locked nucleic acid monomers into DNAzymes improves their ability to gain access and cleave at highly-structured RNA targets. The binding arms of DNAzymes were varied in length and were substituted with up to three LNA and α-L-LNA monomers (forming LNAzymes. For one DNAzyme, the overall cleavage reaction proceeded fifty times faster after incorporation of two α-L-LNA monomers per binding arm (kobs increased from 0.014 min-1 to 0.78 min-1. Conclusion The data demonstrate how hydrolytic performance can be enhanced by design of LNAzymes, and indicate that there are optimal lengths for the binding arms and for the number of modified LNA monomers.

  7. Eddy intrusion of hot plasma into the polar cap and formation of polar-cap arcs

    International Nuclear Information System (INIS)

    Chiu, Y.T.; Gorney, D.J.

    1983-01-01

    We present plasma and electric field data obtained by the S3-3 satellite over the polar caps. We demonstrate that: (1) plasma signatures in the polar cap arc formation region near 5000 km altitude show clear intrusions of plasma sheet (approx.keV) and magneto sheath (approx.100 eV) plasma into a background of low-energy polar cap plasma; (2) the combined plasma and electric field signatures (electron inverted-V, ion beam and delxE<0) are exactly the same as in the evening discrete arc. We interpret this equivalence of polar cap and evening discrete arc signatures as indication that their formation processes are identical. The spatial structures of polar cap electric fields and the associated plasma signatures are consistent with the hypothesis that plasma intrusion into the polar cap takes the form of multiple cellular eddies. This hypothesis provides a unifying view of arc formation and arc configurations

  8. Structure of Hepatitis E Virion-Sized Particle Reveals an RNA-Dependent Viral Assembly Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Xing, L.; Wall, J.; Li, T.-C.; Mayazaki, N.; Simon, M. N.; Moore, M.; Wang, C.-Y.; Takeda, N.; Wakita, T.; Miyamura, T.; Cheng, R. H.

    2010-10-22

    Hepatitis E virus (HEV) induces acute hepatitis in humans with a high fatality rate in pregnant women. There is a need for anti-HEV research to understand the assembly process of HEV native capsid. Here, we produced a large virion-sized and a small T=1 capsid by expressing the HEV capsid protein in insect cells with and without the N-terminal 111 residues, respectively, for comparative structural analysis. The virion-sized capsid demonstrates a T=3 icosahedral lattice and contains RNA fragment in contrast to the RNA-free T=1 capsid. However, both capsids shared common decameric organization. The in vitro assembly further demonstrated that HEV capsid protein had the intrinsic ability to form decameric intermediate. Our data suggest that RNA binding is the extrinsic factor essential for the assembly of HEV native capsids.

  9. Natural and artificial binders of polyriboadenylic acid and their effect on RNA structure

    Directory of Open Access Journals (Sweden)

    Giovanni N. Roviello

    2015-06-01

    Full Text Available The employment of molecular tools with nucleic acid binding ability to specifically control crucial cellular functions represents an important scientific area at the border between biochemistry and pharmaceutical chemistry. In this review we describe several molecular systems of natural or artificial origin, which are able to bind polyriboadenylic acid (poly(rA both in its single-stranded or structured forms. Due to the fundamental role played by the poly(rA tail in the maturation and stability of mRNA, as well as in the initiation of the translation process, compounds able to bind this RNA tract, influencing the mRNA fate, are of special interest for developing innovative biomedical strategies mainly in the field of anticancer therapy.

  10. Assembly of proteins and 5 S rRNA to transcripts of the major structural domains of 23 S rRNA

    DEFF Research Database (Denmark)

    Ostergaard, P; Phan, H; Johansen, L B

    1998-01-01

    The six major structural domains of 23 S rRNA from Escherichia coli, and all combinations thereof, were synthesized as separate T7 transcripts and reconstituted with total 50 S subunit proteins. Analysis by one and two-dimensional gel electrophoresis demonstrated the presence of at least one prim...... approach was used to map the putative binding regions on domain V of protein L9 and the 5 S RNA-L5-L18 complex....

  11. Topological structure of the space of phenotypes: the case of RNA neutral networks.

    Directory of Open Access Journals (Sweden)

    Jacobo Aguirre

    Full Text Available The evolution and adaptation of molecular populations is constrained by the diversity accessible through mutational processes. RNA is a paradigmatic example of biopolymer where genotype (sequence and phenotype (approximated by the secondary structure fold are identified in a single molecule. The extreme redundancy of the genotype-phenotype map leads to large ensembles of RNA sequences that fold into the same secondary structure and can be connected through single-point mutations. These ensembles define neutral networks of phenotypes in sequence space. Here we analyze the topological properties of neutral networks formed by 12-nucleotides RNA sequences, obtained through the exhaustive folding of sequence space. A total of 4(12 sequences fragments into 645 subnetworks that correspond to 57 different secondary structures. The topological analysis reveals that each subnetwork is far from being random: it has a degree distribution with a well-defined average and a small dispersion, a high clustering coefficient, and an average shortest path between nodes close to its minimum possible value, i.e. the Hamming distance between sequences. RNA neutral networks are assortative due to the correlation in the composition of neighboring sequences, a feature that together with the symmetries inherent to the folding process explains the existence of communities. Several topological relationships can be analytically derived attending to structural restrictions and generic properties of the folding process. The average degree of these phenotypic networks grows logarithmically with their size, such that abundant phenotypes have the additional advantage of being more robust to mutations. This property prevents fragmentation of neutral networks and thus enhances the navigability of sequence space. In summary, RNA neutral networks show unique topological properties, unknown to other networks previously described.

  12. RNA Crystallization

    Science.gov (United States)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  13. Microtubule's conformational cap

    DEFF Research Database (Denmark)

    Flyvbjerg, H.

    1999-01-01

    The molecular mechanisms that allow elongation of the unstable microtubule lattice remain unclear. It is usually thought that the GDP-liganded tubulin lattice is capped by a small layer of GTP- or GDP-P(i)-liganded molecules, the so called "GTP-cap". Here, we point-out that the elastic properties...

  14. Fast pairwise structural RNA alignments by pruning of the dynamical programming matrix

    DEFF Research Database (Denmark)

    Havgaard, Jakob Hull; Torarinsson, Elfar; Gorodkin, Jan

    2007-01-01

    and backtracked in a normal fashion. Finally, the FOLDALIGN algorithm has also been updated with a better memory implementation and an improved energy model. With these improvements in the algorithm, the FOLDALIGN software package provides the molecular biologist with an efficient and user-friendly tool...... the advantage of providing the constraints dynamically. This has been included in a new implementation of the FOLDALIGN algorithm for pairwise local or global structural alignment of RNA sequences. It is shown that time and memory requirements are dramatically lowered while overall performance is maintained....... Furthermore, a new divide and conquer method is introduced to limit the memory requirement during global alignment and backtrack of local alignment. All branch points in the computed RNA structure are found and used to divide the structure into smaller unbranched segments. Each segment is then realigned...

  15. Structural Basis for Specific Inhibition of tRNA Synthetase by an ATP Competitive Inhibitor.

    Science.gov (United States)

    Fang, Pengfei; Han, Hongyan; Wang, Jing; Chen, Kaige; Chen, Xin; Guo, Min

    2015-06-18

    Pharmaceutical inhibitors of aminoacyl-tRNA synthetases demand high species and family specificity. The antimalarial ATP-mimetic cladosporin selectively inhibits Plasmodium falciparum LysRS (PfLysRS). How the binding to a universal ATP site achieves the specificity is unknown. Here we report three crystal structures of cladosporin with human LysRS, PfLysRS, and a Pf-like human LysRS mutant. In all three structures, cladosporin occupies the class defining ATP-binding pocket, replacing the adenosine portion of ATP. Three residues holding the methyltetrahydropyran moiety of cladosporin are critical for the specificity of cladosporin against LysRS over other class II tRNA synthetase families. The species-exclusive inhibition of PfLysRS is linked to a structural divergence beyond the active site that mounts a lysine-specific stabilizing response to binding cladosporin. These analyses reveal that inherent divergence of tRNA synthetase structural assembly may allow for highly specific inhibition even through the otherwise universal substrate binding pocket and highlight the potential for structure-driven drug development. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Know Your Enemy: Successful Bioinformatic Approaches to Predict Functional RNA Structures in Viral RNAs

    Science.gov (United States)

    Lim, Chun Shen; Brown, Chris M.

    2018-01-01

    Structured RNA elements may control virus replication, transcription and translation, and their distinct features are being exploited by novel antiviral strategies. Viral RNA elements continue to be discovered using combinations of experimental and computational analyses. However, the wealth of sequence data, notably from deep viral RNA sequencing, viromes, and metagenomes, necessitates computational approaches being used as an essential discovery tool. In this review, we describe practical approaches being used to discover functional RNA elements in viral genomes. In addition to success stories in new and emerging viruses, these approaches have revealed some surprising new features of well-studied viruses e.g., human immunodeficiency virus, hepatitis C virus, influenza, and dengue viruses. Some notable discoveries were facilitated by new comparative analyses of diverse viral genome alignments. Importantly, comparative approaches for finding RNA elements embedded in coding and non-coding regions differ. With the exponential growth of computer power we have progressed from stem-loop prediction on single sequences to cutting edge 3D prediction, and from command line to user friendly web interfaces. Despite these advances, many powerful, user friendly prediction tools and resources are underutilized by the virology community. PMID:29354101

  17. Three RNA recognition motifs participate in RNA recognition and structural organization by the pro-apoptotic factor TIA-1

    Science.gov (United States)

    Bauer, William J.; Heath, Jason; Jenkins, Jermaine L.; Kielkopf, Clara L.

    2012-01-01

    T-cell intracellular antigen-1 (TIA-1) regulates developmental and stress-responsive pathways through distinct activities at the levels of alternative pre-mRNA splicing and mRNA translation. The TIA-1 polypeptide contains three RNA recognition motifs (RRMs). The central RRM2 and C-terminal RRM3 associate with cellular mRNAs. The N-terminal RRM1 enhances interactions of a C-terminal Q-rich domain of TIA-1 with the U1-C splicing factor, despite linear separation of the domains in the TIA-1 sequence. Given the expanded functional repertoire of the RRM family, it was unknown whether TIA-1 RRM1 contributes to RNA binding as well as documented protein interactions. To address this question, we used isothermal titration calorimetry and small-angle X-ray scattering (SAXS) to dissect the roles of the TIA-1 RRMs in RNA recognition. Notably, the fas RNA exhibited two binding sites with indistinguishable affinities for TIA-1. Analyses of TIA-1 variants established that RRM1 was dispensable for binding AU-rich fas sites, yet all three RRMs were required to bind a polyU RNA with high affinity. SAXS analyses demonstrated a `V' shape for a TIA-1 construct comprising the three RRMs, and revealed that its dimensions became more compact in the RNA-bound state. The sequence-selective involvement of TIA-1 RRM1 in RNA recognition suggests a possible role for RNA sequences in regulating the distinct functions of TIA-1. Further implications for U1-C recruitment by the adjacent TIA-1 binding sites of the fas pre-mRNA and the bent TIA-1 shape, which organizes the N- and C-termini on the same side of the protein, are discussed. PMID:22154808

  18. Population genetic structure and its implications for adaptive variation in memory and the hippocampus on a continental scale in food-caching black-capped chickadees.

    Science.gov (United States)

    Pravosudov, V V; Roth, T C; Forister, M L; Ladage, L D; Burg, T M; Braun, M J; Davidson, B S

    2012-09-01

    Food-caching birds rely on stored food to survive the winter, and spatial memory has been shown to be critical in successful cache recovery. Both spatial memory and the hippocampus, an area of the brain involved in spatial memory, exhibit significant geographic variation linked to climate-based environmental harshness and the potential reliance on food caches for survival. Such geographic variation has been suggested to have a heritable basis associated with differential selection. Here, we ask whether population genetic differentiation and potential isolation among multiple populations of food-caching black-capped chickadees is associated with differences in memory and hippocampal morphology by exploring population genetic structure within and among groups of populations that are divergent to different degrees in hippocampal morphology. Using mitochondrial DNA and 583 AFLP loci, we found that population divergence in hippocampal morphology is not significantly associated with neutral genetic divergence or geographic distance, but instead is significantly associated with differences in winter climate. These results are consistent with variation in a history of natural selection on memory and hippocampal morphology that creates and maintains differences in these traits regardless of population genetic structure and likely associated gene flow. Published 2012. This article is a US Government work and is in the public domain in the USA.

  19. Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography

    Science.gov (United States)

    Stagno, J. R.; Liu, Y.; Bhandari, Y. R.; Conrad, C. E.; Panja, S.; Swain, M.; Fan, L.; Nelson, G.; Li, C.; Wendel, D. R.; White, T. A.; Coe, J. D.; Wiedorn, M. O.; Knoska, J.; Oberthuer, D.; Tuckey, R. A.; Yu, P.; Dyba, M.; Tarasov, S. G.; Weierstall, U.; Grant, T. D.; Schwieters, C. D.; Zhang, J.; Ferré-D'Amaré, A. R.; Fromme, P.; Draper, D. E.; Liang, M.; Hunter, M. S.; Boutet, S.; Tan, K.; Zuo, X.; Ji, X.; Barty, A.; Zatsepin, N. A.; Chapman, H. N.; Spence, J. C. H.; Woodson, S. A.; Wang, Y.-X.

    2017-01-01

    Riboswitches are structural RNA elements that are generally located in the 5‧ untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time. Here we use femtosecond X-ray free electron laser (XFEL) pulses to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of ‘mix-and-inject’ time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes.

  20. Structural basis of the non-coding RNA RsmZ acting as a protein sponge.

    Science.gov (United States)

    Duss, Olivier; Michel, Erich; Yulikov, Maxim; Schubert, Mario; Jeschke, Gunnar; Allain, Frédéric H-T

    2014-05-29

    MicroRNA and protein sequestration by non-coding RNAs (ncRNAs) has recently generated much interest. In the bacterial Csr/Rsm system, which is considered to be the most general global post-transcriptional regulatory system responsible for bacterial virulence, ncRNAs such as CsrB or RsmZ activate translation initiation by sequestering homodimeric CsrA-type proteins from the ribosome-binding site of a subset of messenger RNAs. However, the mechanism of ncRNA-mediated protein sequestration is not understood at the molecular level. Here we show for Pseudomonas fluorescens that RsmE protein dimers assemble sequentially, specifically and cooperatively onto the ncRNA RsmZ within a narrow affinity range. This assembly yields two different native ribonucleoprotein structures. Using a powerful combination of nuclear magnetic resonance and electron paramagnetic resonance spectroscopy we elucidate these 70-kilodalton solution structures, thereby revealing the molecular mechanism of the sequestration process and how RsmE binding protects the ncRNA from RNase E degradation. Overall, our findings suggest that RsmZ is well-tuned to sequester, store and release RsmE and therefore can be viewed as an ideal protein 'sponge'.

  1. Structure of a class II TrmH tRNA-modifying enzyme from Aquifex aeolicus

    International Nuclear Information System (INIS)

    Pleshe, Elizabeth; Truesdell, John; Batey, Robert T.

    2005-01-01

    The crystal structure of Aquifex aeolicus TrmH, a member of the a/b-knot superfamily responsible for O methylation of G18 of tRNAs, was determined to 1.85 Å resolution using the molecular-replacement method. Biological RNAs contain a variety of post-transcriptional modifications that facilitate their efficient function in the cellular environment. One of the two most common forms of modification is methylation of the 2′-hydroxyl group of the ribose sugar, which is performed by a number of S-adenosylmethionine (SAM) dependent methyltransferases. In bacteria, many of these modifications in tRNA and rRNA are carried out by the α/β-knot superfamily of enzymes, whose SAM-binding pocket is created by a characteristic deep trefoil knot. TrmH, an enzyme found throughout all three kingdoms of life, modifies the universally conserved guanosine 18 position of tRNA. The crystal structure of TrmH from the thermophilic bacterium Aquifex aeolicus has been determined at 1.85 Å resolution using data collected from a synchrotron-radiation source. The protein reveals a fold typical of members of the SpoU clan of proteins, a subfamily of the α/β-knot superfamily, with α-helical extensions at the N- and C-termini that are likely to be involved in tRNA binding

  2. Structure across the northeastern margin of Flemish Cap, offshore Newfoundland from Erable multichannel seismic reflection profiles: evidence for a transtensional rifting environment

    Science.gov (United States)

    Welford, J. Kim; Hall, Jeremy; Sibuet, Jean-Claude; Srivastava, Shiri P.

    2010-11-01

    We present the results from processing and interpreting nine multichannel seismic reflection lines collected during the 1992 Erable experiment over the northeastern margin of Flemish Cap offshore Newfoundland. These lines, combined into five cross-sections, provide increased seismic coverage over this lightly probed section of the margin and reveal tectonically significant along-strike variations in the degree and compartmentalization of crustal thinning. Similar to the southeastern margins of Flemish Cap and the Grand Banks, a transitional zone of exhumed serpentinized mantle is interpreted between thinned continental and oceanic crust. The 25 km wide transitional zone bears similarities to the 120 km wide transitional zone interpreted as exhumed serpentinized mantle on the conjugate Irish Atlantic margin but the significant width difference is suggestive of an asymmetric conjugate pair. A 40-50 km wide zone of inferred strike-slip shearing is interpreted and observed to extend along most of the northeastern margin of Flemish Cap. Individual shear zones (SZs) may represent extensions of SZs and normal faults within the Orphan Basin providing further evidence for the rotation and displacement of Flemish Cap out of Orphan Basin. The asymmetry between the Flemish Cap and Irish conjugate pairs is likely due in large part to the rotation and displacement of Flemish Cap which resulted in the Flemish Cap margin displaying features of both a strike-slip margin and an extensional margin.

  3. Viroids: from genotype to phenotype just relying on RNA sequence and structural motifs

    Directory of Open Access Journals (Sweden)

    Ricardo eFlores

    2012-06-01

    Full Text Available As a consequence of two unique physical properties, small size and circularity, viroid RNAs do not code for proteins and thus depend on RNA sequence/structural motifs for interacting with host proteins that mediate their invasion, replication, spread, and circumvention of defensive barriers. Viroid genomes fold up on themselves adopting collapsed secondary structures wherein stretches of nucleotides stabilized by Watson-Crick pairs are flanked by apparently unstructured loops. However, compelling data show that they are instead stabilized by alternative non-canonical pairs and that specific loops in the rod-like secondary structure, characteristic of Potato spindle tuber viroid and most other members of the family Pospiviroidae, are critical for replication and systemic trafficking. In contrast, rather than folding into a rod-like secondary structure, most members of the family Avsunvioidae adopt multibranched conformations occasionally stabilized by kissing loop interactions critical for viroid viability in vivo. Besides these most stable secondary structures, viroid RNAs alternatively adopt during replication transient metastable conformations containing elements of local higher-order structure, prominent among which are the hammerhead ribozymes catalyzing a key replicative step in the family Avsunvioidae, and certain conserved hairpins that also mediate replication steps in the family Pospiviroidae. Therefore, different RNA structures ⎯either global or local ⎯ determine different functions, thus highlighting the need for in-depth structural studies on viroid RNAs.

  4. Seismic reflection and structuring characterization of deep aquifer system in the Dakhla syncline (Cap Bon, North-Eastern Tunisia)

    Science.gov (United States)

    Bellali, Abir; Jarraya Horriche, Faten; Gabtni, Hakim; Bédir, Mourad

    2018-04-01

    The Dakhla syncline is located in the North-Eastern Tunisia. It is bounded by Abd El Rahmene anticline to the North-West, El Haouaria Graben to the North-East, Grombalia Graben to the South-West and the Mediterranean Sea to the East. The main aquifer reservoirs of Dakhla syncline are constituted by stacks of fluvial to deltaic Neogene sequences and carbonates. The interpretation of eight seismic reflection profiles, calibrated by wire line logging data of three oil wells, hydraulic wells and geologic field sections highlighted the impact of tectonics on the structuring geometry of aquifers and their distribution in elevated structures and subsurface depressions. Lithostratigraphic correlations and seismic profiles analysis through the syncline show that the principal aquifers are thickest within the central and northern part of the study area and thinnest to the southern part of the syncline. Seismic sections shows that the fracture/fault pattern in this syncline is mainly concentrated along corridors with a major direction of NW-SE and secondary directions of N-S, E-W and NE-SW with different release. This is proved by the complexity structure of Eastern Tunisia, resulted from the interaction between the African and Eurasiatic plates. Isochron maps of aquifers systems exhibited the structuring of this syncline in sub-surface characterized by important lateral and vertical geometric and thickness variations. Seismic sections L1, L2, L3, L4, L5 and petroleum wells showed an heterogeneous multilayer aquifers of Miocene formed by the arrangement of ten sandstone bodies, separated by impermeable clay packages. Oligo-Miocene deposits correspond to the most great potential aquifers, with respectively an average transmissivity estimated: Somaa aquifer 6.5 10-4 m2/s, Sandstone level aquifer 2.6 10-3 m2/s, Beglia aquifer 1.1 10-3 m2/s, Ain Ghrab aquifer 1.3 10-4 m2/s and Oligocene aquifer 2 10-3 m2/s. The interpretation of spatial variations of seismic units and the

  5. Structural biology. Structures of the CRISPR-Cmr complex reveal mode of RNA target positioning

    NARCIS (Netherlands)

    Taylor, D.W.; Zhu, Y.; Staals, R.H.J.; Kornfeld, J.E.; Shinkai, A.; Oost, van der J.; Nogales, E.; Doudna, J.A.

    2015-01-01

    Adaptive immunity in bacteria involves RNA-guided surveillance complexes that use CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target invasive nucleic acids for degradation. Whereas type I and type II CRISPR-Cas

  6. Predicting Consensus Structures for RNA Alignments Via Pseudo-Energy Minimization

    Directory of Open Access Journals (Sweden)

    Junilda Spirollari

    2009-01-01

    Full Text Available Thermodynamic processes with free energy parameters are often used in algorithms that solve the free energy minimization problem to predict secondary structures of single RNA sequences. While results from these algorithms are promising, an observation is that single sequence-based methods have moderate accuracy and more information is needed to improve on RNA secondary structure prediction, such as covariance scores obtained from multiple sequence alignments. We present in this paper a new approach to predicting the consensus secondary structure of a set of aligned RNA sequences via pseudo-energy minimization. Our tool, called RSpredict, takes into account sequence covariation and employs effective heuristics for accuracy improvement. RSpredict accepts, as input data, a multiple sequence alignment in FASTA or ClustalW format and outputs the consensus secondary structure of the input sequences in both the Vienna style Dot Bracket format and the Connectivity Table format. Our method was compared with some widely used tools including KNetFold, Pfold and RNAalifold. A comprehensive test on different datasets including Rfam sequence alignments and a multiple sequence alignment obtained from our study on the Drosophila X chromosome reveals that RSpredict is competitive with the existing tools on the tested datasets. RSpredict is freely available online as a web server and also as a jar file for download at http:// datalab.njit.edu/biology/RSpredict.

  7. Small angle scattering study of the structure and organization of RNA and protein in Brome Mosaic Virus (BMV)

    Science.gov (United States)

    Das, Narayan C.; Warren, Garfield T.; Cheng, Si; Kao, C. Cheng; Ni, Peng; Dragnea, Bogdan; Sokol, Paul E.

    2012-02-01

    Brome mosaic virus (BMV) is a small icosahedral of the alpha virus-like superfamily of RNA with a segmented positive-strand RNA genome and a mean diameter ˜ 268å that offers high levels of RNA synthesis and virus production in plants. BMV also tightly regulates the packaging of its four RNAs (RNA1 through RNA4) into three separate particles; RNA1 and RNA2 are encapsidated separately while one copy each of RNA3 and RNA4 are normally packaged together. Small angle neutron scattering (SANS) and small angle X-ray scattering (SAXS) were applied to study the size, shape and protein-RNA organization of BMV. D2O/H2O mixture was used to enhance contrast in SANS measurement. The radial distribution of BMV from the Fourier transform of scattering spectrum gives a clear indication of RNA packing, and distribution and their structure in the BMV. The result reveals that the virus is about 266 å in diameter and is composed of RNA inside the virion coated with a protein shell.

  8. Production of HIV-1 vif mRNA Is Modulated by Natural Nucleotide Variations and SLSA1 RNA Structure in SA1D2prox Genomic Region

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    Masako Nomaguchi

    2017-12-01

    Full Text Available Genomic RNA of HIV-1 contains localized structures critical for viral replication. Its structural analysis has demonstrated a stem-loop structure, SLSA1, in a nearby region of HIV-1 genomic splicing acceptor 1 (SA1. We have previously shown that the expression level of vif mRNA is considerably altered by some natural single-nucleotide variations (nSNVs clustering in SLSA1 structure. In this study, besides eleven nSNVs previously identified by us, we totally found nine new nSNVs in the SLSA1-containing sequence from SA1, splicing donor 2, and through to the start codon of Vif that significantly affect the vif mRNA level, and designated the sequence SA1D2prox (142 nucleotides for HIV-1 NL4-3. We then examined by extensive variant and mutagenesis analyses how SA1D2prox sequence and SLSA1 secondary structure are related to vif mRNA level. While the secondary structure and stability of SLSA1 was largely changed by nSNVs and artificial mutations introduced to restore the original NL4-3 form from altered ones by nSNVs, no clear association of the two SLSA1 properties with vif mRNA level was observed. In contrast, when naturally occurring SA1D2prox sequences that contain multiple nSNVs were examined, we attained significant inverse correlation between the vif level and SLSA1 stability. These results may suggest that SA1D2prox sequence adapts over time, and also that the altered SA1D2prox sequence, SLSA1 stability, and vif level are mutually related. In total, we show here that the entire SA1D2prox sequence and SLSA1 stability critically contribute to the modulation of vif mRNA level.

  9. Evaluation of the suitability of free-energy minimization using nearest-neighbor energy parameters for RNA secondary structure prediction

    Directory of Open Access Journals (Sweden)

    Cobaugh Christian W

    2004-08-01

    Full Text Available Abstract Background A detailed understanding of an RNA's correct secondary and tertiary structure is crucial to understanding its function and mechanism in the cell. Free energy minimization with energy parameters based on the nearest-neighbor model and comparative analysis are the primary methods for predicting an RNA's secondary structure from its sequence. Version 3.1 of Mfold has been available since 1999. This version contains an expanded sequence dependence of energy parameters and the ability to incorporate coaxial stacking into free energy calculations. We test Mfold 3.1 by performing the largest and most phylogenetically diverse comparison of rRNA and tRNA structures predicted by comparative analysis and Mfold, and we use the results of our tests on 16S and 23S rRNA sequences to assess the improvement between Mfold 2.3 and Mfold 3.1. Results The average prediction accuracy for a 16S or 23S rRNA sequence with Mfold 3.1 is 41%, while the prediction accuracies for the majority of 16S and 23S rRNA structures tested are between 20% and 60%, with some having less than 20% prediction accuracy. The average prediction accuracy was 71% for 5S rRNA and 69% for tRNA. The majority of the 5S rRNA and tRNA sequences have prediction accuracies greater than 60%. The prediction accuracy of 16S rRNA base-pairs decreases exponentially as the number of nucleotides intervening between the 5' and 3' halves of the base-pair increases. Conclusion Our analysis indicates that the current set of nearest-neighbor energy parameters in conjunction with the Mfold folding algorithm are unable to consistently and reliably predict an RNA's correct secondary structure. For 16S or 23S rRNA structure prediction, Mfold 3.1 offers little improvement over Mfold 2.3. However, the nearest-neighbor energy parameters do work well for shorter RNA sequences such as tRNA or 5S rRNA, or for larger rRNAs when the contact distance between the base-pairs is less than 100 nucleotides.

  10. High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states.

    Directory of Open Access Journals (Sweden)

    Kevin A Wilkinson

    2008-04-01

    Full Text Available Replication and pathogenesis of the human immunodeficiency virus (HIV is tightly linked to the structure of its RNA genome, but genome structure in infectious virions is poorly understood. We invent high-throughput SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension technology, which uses many of the same tools as DNA sequencing, to quantify RNA backbone flexibility at single-nucleotide resolution and from which robust structural information can be immediately derived. We analyze the structure of HIV-1 genomic RNA in four biologically instructive states, including the authentic viral genome inside native particles. Remarkably, given the large number of plausible local structures, the first 10% of the HIV-1 genome exists in a single, predominant conformation in all four states. We also discover that noncoding regions functioning in a regulatory role have significantly lower (p-value < 0.0001 SHAPE reactivities, and hence more structure, than do viral coding regions that function as the template for protein synthesis. By directly monitoring protein binding inside virions, we identify the RNA recognition motif for the viral nucleocapsid protein. Seven structurally homologous binding sites occur in a well-defined domain in the genome, consistent with a role in directing specific packaging of genomic RNA into nascent virions. In addition, we identify two distinct motifs that are targets for the duplex destabilizing activity of this same protein. The nucleocapsid protein destabilizes local HIV-1 RNA structure in ways likely to facilitate initial movement both of the retroviral reverse transcriptase from its tRNA primer and of the ribosome in coding regions. Each of the three nucleocapsid interaction motifs falls in a specific genome domain, indicating that local protein interactions can be organized by the long-range architecture of an RNA. High-throughput SHAPE reveals a comprehensive view of HIV-1 RNA genome structure, and further

  11. TurboFold: Iterative probabilistic estimation of secondary structures for multiple RNA sequences

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    Sharma Gaurav

    2011-04-01

    Full Text Available Abstract Background The prediction of secondary structure, i.e. the set of canonical base pairs between nucleotides, is a first step in developing an understanding of the function of an RNA sequence. The most accurate computational methods predict conserved structures for a set of homologous RNA sequences. These methods usually suffer from high computational complexity. In this paper, TurboFold, a novel and efficient method for secondary structure prediction for multiple RNA sequences, is presented. Results TurboFold takes, as input, a set of homologous RNA sequences and outputs estimates of the base pairing probabilities for each sequence. The base pairing probabilities for a sequence are estimated by combining intrinsic information, derived from the sequence itself via the nearest neighbor thermodynamic model, with extrinsic information, derived from the other sequences in the input set. For a given sequence, the extrinsic information is computed by using pairwise-sequence-alignment-based probabilities for co-incidence with each of the other sequences, along with estimated base pairing probabilities, from the previous iteration, for the other sequences. The extrinsic information is introduced as free energy modifications for base pairing in a partition function computation based on the nearest neighbor thermodynamic model. This process yields updated estimates of base pairing probability. The updated base pairing probabilities in turn are used to recompute extrinsic information, resulting in the overall iterative estimation procedure that defines TurboFold. TurboFold is benchmarked on a number of ncRNA datasets and compared against alternative secondary structure prediction methods. The iterative procedure in TurboFold is shown to improve estimates of base pairing probability with each iteration, though only small gains are obtained beyond three iterations. Secondary structures composed of base pairs with estimated probabilities higher than a

  12. Layered sphere-shaped TiO₂ capped with gold nanoparticles on structural defects and their catalysis of formaldehyde oxidation.

    Science.gov (United States)

    Ma, Chunyan; Pang, Guanglong; He, Guangzhi; Li, Yang; He, Chi; Hao, Zhengping

    2016-01-01

    We describe here a one-step method for the synthesis of Au/TiO2 nanosphere materials, which were formed by layered deposition of multiple anatase TiO2 nanosheets. The Au nanoparticles were stabilized by structural defects in each TiO2 nanosheet, including crystal steps and edges, thereby fixing the Au-TiO2 perimeter interface. Reactant transfer occurred along the gaps between these TiO2 nanosheet layers and in contact with catalytically active sites at the Au-TiO2 interface. The doped Au induced the formation of oxygen vacancies in the Au-TiO2 interface. Such vacancies are essential for generating active oxygen species (*O(-)) on the TiO2 surface and Ti(3+) ions in bulk TiO2. These ions can then form Ti(3+)-O(-)-Ti(4+) species, which are known to enhance the catalytic activity of formaldehyde (HCHO) oxidation. These studies on structural and oxygen vacancy defects in Au/TiO2 samples provide a theoretical foundation for the catalytic mechanism of HCHO oxidation on oxide-supported Au materials. Copyright © 2015. Published by Elsevier B.V.

  13. The interaction between the iron-responsive element binding protein and its cognate RNA is highly dependent upon both RNA sequence and structure.

    Science.gov (United States)

    Jaffrey, S R; Haile, D J; Klausner, R D; Harford, J B

    1993-09-25

    To assess the influence of RNA sequence/structure on the interaction RNAs with the iron-responsive element binding protein (IRE-BP), twenty eight altered RNAs were tested as competitors for an RNA corresponding to the ferritin H chain IRE. All changes in the loop of the predicted IRE hairpin and in the unpaired cytosine residue characteristically found in IRE stems significantly decreased the apparent affinity of the RNA for the IRE-BP. Similarly, alteration in the spacing and/or orientation of the loop and the unpaired cytosine of the stem by either increasing or decreasing the number of base pairs separating them significantly reduced efficacy as a competitor. It is inferred that the IRE-BP forms multiple contacts with its cognate RNA, and that these contacts, acting in concert, provide the basis for the high affinity of this interaction.

  14. Characterization and visualization of RNA secondary structure Boltzmann ensemble via information theory.

    Science.gov (United States)

    Lin, Luan; McKerrow, Wilson H; Richards, Bryce; Phonsom, Chukiat; Lawrence, Charles E

    2018-03-05

    The nearest neighbor model and associated dynamic programming algorithms allow for the efficient estimation of the RNA secondary structure Boltzmann ensemble. However because a given RNA secondary structure only contains a fraction of the possible helices that could form from a given sequence, the Boltzmann ensemble is multimodal. Several methods exist for clustering structures and finding those modes. However less focus is given to exploring the underlying reasons for this multimodality: the presence of conflicting basepairs. Information theory, or more specifically mutual information, provides a method to identify those basepairs that are key to the secondary structure. To this end we find most informative basepairs and visualize the effect of these basepairs on the secondary structure. Knowing whether a most informative basepair is present tells us not only the status of the particular pair but also provides a large amount of information about which other pairs are present or not present. We find that a few basepairs account for a large amount of the structural uncertainty. The identification of these pairs indicates small changes to sequence or stability that will have a large effect on structure. We provide a novel algorithm that uses mutual information to identify the key basepairs that lead to a multimodal Boltzmann distribution. We then visualize the effect of these pairs on the overall Boltzmann ensemble.

  15. An Algorithm for Template-Based Prediction of Secondary Structures of Individual RNA Sequences

    Czech Academy of Sciences Publication Activity Database

    Pánek, Josef; Modrák, Martin; Schwarz, Marek

    2017-01-01

    Roč. 8, OCT 10 (2017), s. 1-11, č. článku 147. ISSN 1664-8021 R&D Projects: GA ČR GA15-00885S; GA MŠk(CZ) LM2015047 Institutional support: RVO:61388971 Keywords : RNA * secondary structure * homology Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 3.789, year: 2016

  16. A fast and robust iterative algorithm for prediction of RNA pseudoknotted secondary structures

    Science.gov (United States)

    2014-01-01

    Background Improving accuracy and efficiency of computational methods that predict pseudoknotted RNA secondary structures is an ongoing challenge. Existing methods based on free energy minimization tend to be very slow and are limited in the types of pseudoknots that they can predict. Incorporating known structural information can improve prediction accuracy; however, there are not many methods for prediction of pseudoknotted structures that can incorporate structural information as input. There is even less understanding of the relative robustness of these methods with respect to partial information. Results We present a new method, Iterative HFold, for pseudoknotted RNA secondary structure prediction. Iterative HFold takes as input a pseudoknot-free structure, and produces a possibly pseudoknotted structure whose energy is at least as low as that of any (density-2) pseudoknotted structure containing the input structure. Iterative HFold leverages strengths of earlier methods, namely the fast running time of HFold, a method that is based on the hierarchical folding hypothesis, and the energy parameters of HotKnots V2.0. Our experimental evaluation on a large data set shows that Iterative HFold is robust with respect to partial information, with average accuracy on pseudoknotted structures steadily increasing from roughly 54% to 79% as the user provides up to 40% of the input structure. Iterative HFold is much faster than HotKnots V2.0, while having comparable accuracy. Iterative HFold also has significantly better accuracy than IPknot on our HK-PK and IP-pk168 data sets. Conclusions Iterative HFold is a robust method for prediction of pseudoknotted RNA secondary structures, whose accuracy with more than 5% information about true pseudoknot-free structures is better than that of IPknot, and with about 35% information about true pseudoknot-free structures compares well with that of HotKnots V2.0 while being significantly faster. Iterative HFold and all data used in

  17. Insights into the effect of N-acetyl-L-cysteine-capped CdTe quantum dots on the structure and activity of human serum albumin by spectroscopic techniques

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Haoyu; Yang, Xudan; Li, Meng; Han, Songlin; Liu, Yingxue [School of Environmental Science and Engineering, Shandong University, China-America CRC for Environment & Health, Shandong Province, 27# Shanda South Road, Jinan 250100 (China); Tan, Xuejie [School of Chemistry and Pharmaceutical Engineering, Qilu University of Technology, Jinan, Shandong Province 250353 (China); Liu, Chunguang, E-mail: chunguangliu2013@sdu.edu.cn [School of Environmental Science and Engineering, Shandong University, China-America CRC for Environment & Health, Shandong Province, 27# Shanda South Road, Jinan 250100 (China); Liu, Rutao [School of Environmental Science and Engineering, Shandong University, China-America CRC for Environment & Health, Shandong Province, 27# Shanda South Road, Jinan 250100 (China)

    2015-11-15

    Quantum dots (QDs) are a kind of nanostructured semiconductor crystals with the size range of 1–10 nm. Their unique photophysical properties and potential toxicity to human health have aroused wide concern of scientists and general public. However, the interaction mechanism of QDs on human serum albumin (HSA, the vital protein in human blood) from both structural and functional perspectives is rarely reported. In the present work, effects of N-acetyl-L-cysteine-capped CdTe quantum dots with fluorescence emission peak at 612 nm (QDs-612) on the conformation and function of HSA were investigated by spectroscopic methods, molecular docking study and esterase activity assay. The hydrophobic interaction between HSA and QDs-612 was spontaneous with the binding constants calculated to be 6.85×10{sup 5} L mol{sup −1} (298 K) and 8.89×10{sup 5} L mol{sup −1} (308 K). The binding of QDs-612 to HSA induced the static quenching of fluorescence and the changes of secondary structure and microenvironment of Tyr-411 residue, which resulted in serious decrease on the hydrolysis of substrate p-nitrophenylacetate in esterase activity assay of HSA. This work confirms the possibility on direct interaction of QDs-612 with HSA and obtains a possible mechanism of relationship between conformation and function of HSA. - Highlights: • The interaction between CdTe QDs (QDs-612) and HSA is spontaneous. • The predominant force of the binding is hydrophobic interaction. • The interaction changes the secondary structure of HSA. • Tyr-411 residue of HSA expose to a hydrophilic environment. • The esterase activity of HSA decreases by adding QDs-612.

  18. Fine-grained parallelism accelerating for RNA secondary structure prediction with pseudoknots based on FPGA.

    Science.gov (United States)

    Xia, Fei; Jin, Guoqing

    2014-06-01

    PKNOTS is a most famous benchmark program and has been widely used to predict RNA secondary structure including pseudoknots. It adopts the standard four-dimensional (4D) dynamic programming (DP) method and is the basis of many variants and improved algorithms. Unfortunately, the O(N(6)) computing requirements and complicated data dependency greatly limits the usefulness of PKNOTS package with the explosion in gene database size. In this paper, we present a fine-grained parallel PKNOTS package and prototype system for accelerating RNA folding application based on FPGA chip. We adopted a series of storage optimization strategies to resolve the "Memory Wall" problem. We aggressively exploit parallel computing strategies to improve computational efficiency. We also propose several methods that collectively reduce the storage requirements for FPGA on-chip memory. To the best of our knowledge, our design is the first FPGA implementation for accelerating 4D DP problem for RNA folding application including pseudoknots. The experimental results show a factor of more than 50x average speedup over the PKNOTS-1.08 software running on a PC platform with Intel Core2 Q9400 Quad CPU for input RNA sequences. However, the power consumption of our FPGA accelerator is only about 50% of the general-purpose micro-processors.

  19. Genomic mid-range inhomogeneity correlates with an abundance of RNA secondary structures

    Directory of Open Access Journals (Sweden)

    Song Jun

    2008-06-01

    Full Text Available Abstract Background Genomes possess different levels of non-randomness, in particular, an inhomogeneity in their nucleotide composition. Inhomogeneity is manifest from the short-range where neighboring nucleotides influence the choice of base at a site, to the long-range, commonly known as isochores, where a particular base composition can span millions of nucleotides. A separate genomic issue that has yet to be thoroughly elucidated is the role that RNA secondary structure (SS plays in gene expression. Results We present novel data and approaches that show that a mid-range inhomogeneity (~30 to 1000 nt not only exists in mammalian genomes but is also significantly associated with strong RNA SS. A whole-genome bioinformatics investigation of local SS in a set of 11,315 non-redundant human pre-mRNA sequences has been carried out. Four distinct components of these molecules (5'-UTRs, exons, introns and 3'-UTRs were considered separately, since they differ in overall nucleotide composition, sequence motifs and periodicities. For each pre-mRNA component, the abundance of strong local SS ( Conclusion We demonstrate that the excess of strong local SS in pre-mRNAs is linked to the little explored phenomenon of genomic mid-range inhomogeneity (MRI. MRI is an interdependence between nucleotide choice and base composition over a distance of 20–1000 nt. Additionally, we have created a public computational resource to support further study of genomic MRI.

  20. UPF201 Archaeal Specific Family Members Reveals Structural Similarity to RNA-Binding Proteins but Low Likelihood for RNA-Binding Function

    Energy Technology Data Exchange (ETDEWEB)

    Rao, K.N.; Swaminathan, S.; Burley, S. K.

    2008-12-11

    We have determined X-ray crystal structures of four members of an archaeal specific family of proteins of unknown function (UPF0201; Pfam classification: DUF54) to advance our understanding of the genetic repertoire of archaea. Despite low pairwise amino acid sequence identities (10-40%) and the absence of conserved sequence motifs, the three-dimensional structures of these proteins are remarkably similar to one another. Their common polypeptide chain fold, encompassing a five-stranded antiparallel {beta}-sheet and five {alpha}-helices, proved to be quite unexpectedly similar to that of the RRM-type RNA-binding domain of the ribosomal L5 protein, which is responsible for binding the 5S- rRNA. Structure-based sequence alignments enabled construction of a phylogenetic tree relating UPF0201 family members to L5 ribosomal proteins and other structurally similar RNA binding proteins, thereby expanding our understanding of the evolutionary purview of the RRM superfamily. Analyses of the surfaces of these newly determined UPF0201 structures suggest that they probably do not function as RNA binding proteins, and that this domain specific family of proteins has acquired a novel function in archaebacteria, which awaits experimental elucidation.

  1. Macrophage Capping Protein CapG Is a Putative Oncogene Involved in Migration and Invasiveness in Ovarian Carcinoma

    Directory of Open Access Journals (Sweden)

    J. Glaser

    2014-01-01

    Full Text Available The actin binding protein CapG modulates cell motility by interacting with the cytoskeleton. CapG is associated with tumor progression in different nongynecologic tumor entities and overexpression in breast cancer cell lines correlates with a more invasive phenotype in vitro. Here, we report a significant CapG overexpression in 18/47 (38% of ovarian carcinomas (OC analyzed by qRealTime-PCR analyses. Functional analyses in OC cell lines through siRNA mediated CapG knockdown and CapG overexpression showed CapG-dependent cell migration and invasiveness. A single nucleotide polymorphism rs6886 inside the CapG gene was identified, affecting a CapG phosphorylation site and thus potentially modifying CapG function. The minor allele frequency (MAF of SNP rs6886 (c.1004A/G was higher and the homozygous (A/A, His335 genotype was significantly more prevalent in patients with fallopian tube carcinomas (50% as in controls (10%. With OC being one of the most lethal cancer diseases, the detection of novel biomarkers such as CapG could reveal new diagnostic and therapeutic targets. Moreover, in-depth analyses of SNP rs6886 related to FTC and OC will contribute to a better understanding of carcinogenesis and progression of OC.

  2. CENTRIFUGE END CAP

    Science.gov (United States)

    Beams, J.W.; Snoddy, L.B.

    1960-08-01

    An end cap for ultra-gas centrifuges is designed to impart or remove angular momentum to or from the gas and to bring the entering gas to the temperature of the gas inside the centrifuge. The end cap is provided with slots or fins for adjusting the temperature and the angular momentum of the entering gas to the temperature and momentum of the gas in the centrifuge and is constructed to introduce both the inner and the peripheral stream into the centrifuge.

  3. Convergence of Domain Architecture, Structure, and Ligand Affinity in Animal and Plant RNA-Binding Proteins.

    Science.gov (United States)

    Dias, Raquel; Manny, Austin; Kolaczkowski, Oralia; Kolaczkowski, Bryan

    2017-06-01

    Reconstruction of ancestral protein sequences using phylogenetic methods is a powerful technique for directly examining the evolution of molecular function. Although ancestral sequence reconstruction (ASR) is itself very efficient, downstream functional, and structural studies necessary to characterize when and how changes in molecular function occurred are often costly and time-consuming, currently limiting ASR studies to examining a relatively small number of discrete functional shifts. As a result, we have very little direct information about how molecular function evolves across large protein families. Here we develop an approach combining ASR with structure and function prediction to efficiently examine the evolution of ligand affinity across a large family of double-stranded RNA binding proteins (DRBs) spanning animals and plants. We find that the characteristic domain architecture of DRBs-consisting of 2-3 tandem double-stranded RNA binding motifs (dsrms)-arose independently in early animal and plant lineages. The affinity with which individual dsrms bind double-stranded RNA appears to have increased and decreased often across both animal and plant phylogenies, primarily through convergent structural mechanisms involving RNA-contact residues within the β1-β2 loop and a small region of α2. These studies provide some of the first direct information about how protein function evolves across large gene families and suggest that changes in molecular function may occur often and unassociated with major phylogenetic events, such as gene or domain duplications. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  4. Essential Structural and Functional Roles of the Cmr4 Subunit in RNA Cleavage by the Cmr CRISPR-Cas Complex

    Directory of Open Access Journals (Sweden)

    Nancy F. Ramia

    2014-12-01

    Full Text Available Summary: The Cmr complex is the multisubunit effector complex of the type III-B clustered regularly interspaced short palindromic repeats (CRISPR-Cas immune system. The Cmr complex recognizes a target RNA through base pairing with the integral CRISPR RNA (crRNA and cleaves the target at multiple regularly spaced locations within the complementary region. To understand the molecular basis of the function of this complex, we have assembled information from electron microscopic and X-ray crystallographic structural studies and mutagenesis of a complete Pyrococcus furiosus Cmr complex. Our findings reveal that four helically packed Cmr4 subunits, which make up the backbone of the Cmr complex, act as a platform to support crRNA binding and target RNA cleavage. Interestingly, we found a hook-like structural feature associated with Cmr4 that is likely the site of target RNA binding and cleavage. Our results also elucidate analogies in the mechanisms of crRNA and target molecule binding by the distinct Cmr type III-A and Cascade type I-E complexes. : Ramia et al. show that the helical core of the type III-B Cmr CRISPR-Cas effector complex, made up of multiple Cmr4 subunits, forms the platform for a corresponding number of cleavages of the target RNA. Comparison with the type I-E Cascade structure reveals strikingly similar mechanisms of crRNA and target binding.

  5. Structure of a Complete Mediator-RNA Polymerase II Pre-Initiation Complex.

    Science.gov (United States)

    Robinson, Philip J; Trnka, Michael J; Bushnell, David A; Davis, Ralph E; Mattei, Pierre-Jean; Burlingame, Alma L; Kornberg, Roger D

    2016-09-08

    A complete, 52-protein, 2.5 million dalton, Mediator-