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Sample records for catalytic rna structure

  1. 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.

  2. 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.

  3. Structural Basis for Telomerase Catalytic Subunit TERT Binding to RNA Template and Telomeric DNA

    Energy Technology Data Exchange (ETDEWEB)

    Mitchell, M.; Gillis, A; Futahashi, M; Fujiwara, H; Skordalakes, E

    2010-01-01

    Telomerase is a specialized DNA polymerase that extends the 3{prime} ends of eukaryotic linear chromosomes, a process required for genomic stability and cell viability. Here we present the crystal structure of the active Tribolium castaneum telomerase catalytic subunit, TERT, bound to an RNA-DNA hairpin designed to resemble the putative RNA-templating region and telomeric DNA. The RNA-DNA hybrid adopts a helical structure, docked in the interior cavity of the TERT ring. Contacts between the RNA template and motifs 2 and B{prime} position the solvent-accessible RNA bases close to the enzyme active site for nucleotide binding and selectivity. Nucleic acid binding induces rigid TERT conformational changes to form a tight catalytic complex. Overall, TERT-RNA template and TERT-telomeric DNA associations are remarkably similar to those observed for retroviral reverse transcriptases, suggesting common mechanistic aspects of DNA replication between the two families of enzymes.

  4. Crystal Structure of the Catalytic Core of an RNA-Polymerase Ribozyme

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    Shechner, David M.; Grant, Robert A.; Bagby, Sarah C.; Koldobskaya, Yelena; Piccirilli, Joseph A.; Bartel, David P.; (MIT); (HHMI); (UC)

    2010-09-02

    Primordial organisms of the putative RNA world would have required polymerase ribozymes able to replicate RNA. Known ribozymes with polymerase activity best approximating that needed for RNA replication contain at their catalytic core the class I RNA ligase, an artificial ribozyme with a catalytic rate among the fastest of known ribozymes. Here we present the 3.0 angstrom crystal structure of this ligase. The architecture resembles a tripod, its three legs converging near the ligation junction. Interacting with this tripod scaffold through a series of 10 minor-groove interactions (including two A-minor triads) is the unpaired segment that contributes to and organizes the active site. A cytosine nucleobase and two backbone phosphates abut the ligation junction; their location suggests a model for catalysis resembling that of proteinaceous polymerases.

  5. Catalytic distillation structure

    Science.gov (United States)

    Smith, L.A. Jr.

    1984-04-17

    Catalytic distillation structure is described for use in reaction distillation columns, and provides reaction sites and distillation structure consisting of a catalyst component and a resilient component intimately associated therewith. The resilient component has at least about 70 volume % open space and is present with the catalyst component in an amount such that the catalytic distillation structure consists of at least 10 volume % open space. 10 figs.

  6. Domain organization and crystal structure of the catalytic domain of E.coli RluF, a pseudouridine synthase that acts on 23S rRNA.

    Science.gov (United States)

    Sunita, S; Zhenxing, H; Swaathi, J; Cygler, Miroslaw; Matte, Allan; Sivaraman, J

    2006-06-16

    Pseudouridine synthases catalyze the isomerization of uridine to pseudouridine (Psi) in rRNA and tRNA. The pseudouridine synthase RluF from Escherichia coli (E.C. 4.2.1.70) modifies U2604 in 23S rRNA, and belongs to a large family of pseudouridine synthases present in all kingdoms of life. Here we report the domain architecture and crystal structure of the catalytic domain of E.coli RluF at 2.6A resolution. Limited proteolysis, mass spectrometry and N-terminal sequencing indicate that RluF has a distinct domain architecture, with the catalytic domain flanked at the N and C termini by additional domains connected to it by flexible linkers. The structure of the catalytic domain of RluF is similar to those of RsuA and TruB. RluF is a member of the RsuA sequence family of Psi-synthases, along with RluB and RluE. Structural comparison of RluF with its closest structural homologues, RsuA and TruB, suggests possible functional roles for the N-terminal and C-terminal domains of RluF.

  7. Domain organization and crystal structure of the catalytic domain of E.coli RluF, a pseudouridine synthase that acts on 23S rRNA

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    Sunita,S.; Zhenxing, H.; Swaathi, J.; Cygler, M.; Matte, A.; Sivaraman, J.

    2006-01-01

    Pseudouridine synthases catalyze the isomerization of uridine to pseudouridine ({psi}) in rRNA and tRNA. The pseudouridine synthase RluF from Escherichia coli (E.C. 4.2.1.70) modifies U2604 in 23S rRNA, and belongs to a large family of pseudouridine synthases present in all kingdoms of life. Here we report the domain architecture and crystal structure of the catalytic domain of E. coli RluF at 2.6 Angstroms resolution. Limited proteolysis, mass spectrometry and N-terminal sequencing indicate that RluF has a distinct domain architecture, with the catalytic domain flanked at the N and C termini by additional domains connected to it by flexible linkers. The structure of the catalytic domain of RluF is similar to those of RsuA and TruB. RluF is a member of the RsuA sequence family of {psi}-synthases, along with RluB and RluE. Structural comparison of RluF with its closest structural homologues, RsuA and TruB, suggests possible functional roles for the N-terminal and C-terminal domains of RluF.

  8. Crystal structure of the catalytic domain of RluD, the only rRNA pseudouridine synthase required for normal growth of Escherichia coli.

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    Del Campo, Mark; Ofengand, James; Malhotra, Arun

    2004-02-01

    Escherichia coli pseudouridine synthase RluD makes pseudouridines 1911, 1915, and 1917 in the loop of helix 69 in 23S RNA. These are the most highly conserved ribosomal pseudouridines known. Of 11 pseudouridine synthases in E. coli, only cells lacking RluD have severe growth defects and abnormal ribosomes. We have determined the 2.0 A structure of the catalytic domain of RluD (residues 77-326), the first structure of an RluA family member. The catalytic domain folds into a mainly antiparallel beta-sheet flanked by several loops and helices. A positively charged cleft that presumably binds RNA leads to the conserved Asp 139. The RluD N-terminal S4 domain, connected by a flexible linker, is disordered in our structure. RluD is very similar in both catalytic domain structure and active site arrangement to the pseudouridine synthases RsuA, TruB, and TruA. We identify five sequence motifs, two of which are novel, in the RluA, RsuA, TruB, and TruA families, uniting them as one superfamily. These results strongly suggest that four of the five families of pseudouridine synthases arose by divergent evolution. The RluD structure also provides insight into its multisite specificity.

  9. Insights into the catalytic mechanism of 16S rRNA methyltransferase RsmE (m³U1498) from crystal and solution structures.

    Science.gov (United States)

    Zhang, Heng; Wan, Hua; Gao, Zeng-Qiang; Wei, Yong; Wang, Wen-Jia; Liu, Guang-Feng; Shtykova, Eleonora V; Xu, Jian-Hua; Dong, Yu-Hui

    2012-11-01

    RsmE is the founding member of a new RNA methyltransferase (MTase) family responsible for methylation of U1498 in 16S ribosomal RNA in Escherichia coli. It is well conserved across bacteria and plants and may play an important role in ribosomal intersubunit communication. The crystal structure in monomer showed that it consists of two distinct but structurally related domains: the PUA (pseudouridine synthases and archaeosine-specific transglycosylases)-like RNA recognition and binding domain and the conserved MTase domain with a deep trefoil knot. Analysis of small-angle X-ray scattering data revealed that RsmE forms a flexible dimeric conformation that may be essential for substrate binding. The S-adenosyl-l-methionine (AdoMet)-binding characteristic determined by isothermal titration calorimetry suggested that there is only one AdoMet molecule bound in the subunit of the homodimer. In vitro methylation assay of the mutants based on the RsmE-AdoMet-uridylic acid complex model showed key residues involved in substrate binding and catalysis. Comprehensive comparisons of RsmE with closely related MTases, combined with the biochemical experiments, indicated that the MTase domain of one subunit in dimeric RsmE is responsible for binding of one AdoMet molecule and catalytic process while the PUA-like domain in the other subunit is mainly responsible for recognition of one substrate molecule (the ribosomal RNA fragment and ribosomal protein complex). The methylation process is required by collaboration of both subunits, and dimerization is functionally critical for catalysis. In general, our study provides new information on the structure-function relationship of RsmE and thereby suggests a novel catalytic mechanism.

  10. Crystal structure of the RluD pseudouridine synthase catalytic module, an enzyme that modifies 23S rRNA and is essential for normal cell growth of Escherichia coli.

    Science.gov (United States)

    Sivaraman, J; Iannuzzi, Pietro; Cygler, Miroslaw; Matte, Allan

    2004-01-01

    Pseudouridine (5-beta-D-ribofuranosyluracil, Psi) is the most commonly found modified base in RNA. Conversion of uridine to Psi is performed enzymatically in both prokaryotes and eukaryotes by pseudouridine synthases (EC 4.2.1.70). The Escherichia coli Psi-synthase RluD modifies uridine to Psi at positions 1911, 1915 and 1917 within 23S rRNA. RluD also possesses a second function related to proper assembly of the 50S ribosomal subunit that is independent of Psi-synthesis. Here, we report the crystal structure of the catalytic module of RluD (residues 68-326; DeltaRluD) refined at 1.8A to a final R-factor of 21.8% (R(free)=24.3%). DeltaRluD is a monomeric enzyme having an overall mixed alpha/beta fold. The DeltaRluD molecule consists of two subdomains, a catalytic subdomain and C-terminal subdomain with the RNA-binding cleft formed by loops extending from the catalytic sub-domain. The catalytic sub-domain of DeltaRluD has a similar fold as in TruA, TruB and RsuA, with the location of the RNA-binding cleft, active-site and conserved, catalytic Asp residue superposing in all four structures. Superposition of the crystal structure of TruB bound to a T-stem loop with RluD reveals that similar RNA-protein interactions for the flipped-out uridine base would exist in both structures, implying that base-flipping is necessary for catalysis. This observation also implies that the specificity determinants for site-specific RNA-binding and recognition likely reside in parts of RluD beyond the active site.

  11. Engineering Structurally Interacting RNA (sxRNA)

    Science.gov (United States)

    Doyle, Francis; Lapsia, Sameer; Spadaro, Salvatore; Wurz, Zachary E.; Bhaduri-McIntosh, Sumita; Tenenbaum, Scott A.

    2017-01-01

    RNA-based three-way junctions (3WJs) are naturally occurring structures found in many functional RNA molecules including rRNA, tRNA, snRNA and ribozymes. 3WJs are typically characterized as resulting from an RNA molecule folding back on itself in cis but could also form in trans when one RNA, for instance a microRNA binds to a second structured RNA, such as a mRNA. Trans-3WJs can influence the final shape of one or both of the RNA molecules and can thus provide a means for modulating the availability of regulatory motifs including potential protein or microRNA binding sites. Regulatory 3WJs generated in trans represent a newly identified regulatory category that we call structurally interacting RNA or sxRNA for convenience. Here we show that they can be rationally designed using familiar cis-3WJ examples as a guide. We demonstrate that an sxRNA “bait” sequence can be designed to interact with a specific microRNA “trigger” sequence, creating a regulatable RNA-binding protein motif that retains its functional activity. Further, we show that when placed downstream of a coding sequence, sxRNA can be used to switch “ON” translation of that sequence in the presence of the trigger microRNA and the amount of translation corresponded with the amount of microRNA present. PMID:28350000

  12. Method of fabricating a catalytic structure

    Science.gov (United States)

    Rollins, Harry W.; Petkovic, Lucia M.; Ginosar, Daniel M.

    2009-09-22

    A precursor to a catalytic structure comprising zinc oxide and copper oxide. The zinc oxide has a sheet-like morphology or a spherical morphology and the copper oxide comprises particles of copper oxide. The copper oxide is reduced to copper, producing the catalytic structure. The catalytic structure is fabricated by a hydrothermal process. A reaction mixture comprising a zinc salt, a copper salt, a hydroxyl ion source, and a structure-directing agent is formed. The reaction mixture is heated under confined volume conditions to produce the precursor. The copper oxide in the precursor is reduced to copper. A method of hydrogenating a carbon oxide using the catalytic structure is also disclosed, as is a system that includes the catalytic structure.

  13. Functional identification of catalytic metal ion binding sites within RNA.

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    James L Hougland

    2005-09-01

    Full Text Available The viability of living systems depends inextricably on enzymes that catalyze phosphoryl transfer reactions. For many enzymes in this class, including several ribozymes, divalent metal ions serve as obligate cofactors. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s. In the Tetrahymena group I intron, previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified three metal ions (MA, MB, and MC that make five interactions with the ribozyme substrates in the reaction's transition state. Here, we combine substrate atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to develop a powerful, general strategy for defining the ligands of catalytic metal ions within RNA. In applying this strategy to the Tetrahymena group I intron, we have identified the pro-SP phosphoryl oxygen at nucleotide C262 as a ribozyme ligand for MC. Our findings establish a direct connection between the ribozyme core and the functionally defined model of the chemical transition state, thereby extending the known set of transition-state interactions and providing information critical for the application of the recent group I intron crystallographic structures to the understanding of catalysis.

  14. RNA Thermodynamic Structural Entropy.

    Science.gov (United States)

    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

  15. RNA Thermodynamic Structural Entropy.

    Directory of Open Access Journals (Sweden)

    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

  16. Topology of RNA-RNA interaction structures

    DEFF Research Database (Denmark)

    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...

  17. Domain motions of Argonaute, the catalytic engine of RNA interference

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    Wall Michael E

    2007-11-01

    Full Text Available Abstract Background The Argonaute protein is the core component of the RNA-induced silencing complex, playing the central role of cleaving the mRNA target. Visual inspection of static crystal structures already has enabled researchers to suggest conformational changes of Argonaute that might occur during RNA interference. We have taken the next step by performing an all-atom normal mode analysis of the Pyrococcus furiosus and Aquifex aeolicus Argonaute crystal structures, allowing us to quantitatively assess the feasibility of these conformational changes. To perform the analysis, we begin with the energy-minimized X-ray structures. Normal modes are then calculated using an all-atom molecular mechanics force field. Results The analysis reveals low-frequency vibrations that facilitate the accommodation of RNA duplexes – an essential step in target recognition. The Pyrococcus furiosus and Aquifex aeolicus Argonaute proteins both exhibit low-frequency torsion and hinge motions; however, differences in the overall architecture of the proteins cause the detailed dynamics to be significantly different. Conclusion Overall, low-frequency vibrations of Argonaute are consistent with mechanisms within the current reaction cycle model for RNA interference.

  18. Repeated stress increases catalytic TrkB mRNA in rat hippocampus.

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    Nibuya, M; Takahashi, M; Russell, D S; Duman, R S

    1999-05-28

    Northern blot analysis was utilized to distinguish between catalytic and truncated TrkB mRNA on the basis of transcript size. Repeated (10 days), but not acute, immobilization stress significantly increased levels of catalytic TrkB mRNA, but did not influence expression of truncated TrkB transcripts in rat hippocampus. Exposure to another paradigm, a combination of different, unpredictable stressors, also increased levels of catalytic, but not truncated, TrkB mRNA. In situ hybridization analysis demonstrated that chronic stress up-regulated TrkB mRNA in CA1 and CA3 pyramidal and dentate gyrus granule cells layers of hippocampus. As previously reported, both acute and chronic immobilization stress decreased expression of BDNF mRNA, suggesting that up-regulation of catalytic TrkB mRNA may be a compensatory adaptation to repeated stress.

  19. Crystal structure of the RNA component of bacterial ribonuclease P

    Energy Technology Data Exchange (ETDEWEB)

    Torres-Larios, Alfredo; Swinger, Kerren K.; Krasilnikov, Andrey S.; Pan, Tao; Mondragon, Alfonso (NWU); (UC)

    2010-03-08

    Transfer RNA (tRNA) is produced as a precursor molecule that needs to be processed at its 3' and 5' ends. Ribonuclease P is the sole endonuclease responsible for processing the 5' end of tRNA by cleaving the precursor and leading to tRNA maturation. It was one of the first catalytic RNA molecules identified and consists of a single RNA component in all organisms and only one protein component in bacteria. It is a true multi-turnover ribozyme and one of only two ribozymes (the other being the ribosome) that are conserved in all kingdoms of life. Here we show the crystal structure at 3.85 {angstrom} resolution of the RNA component of Thermotoga maritima ribonuclease P. The entire RNA catalytic component is revealed, as well as the arrangement of the two structural domains. The structure shows the general architecture of the RNA molecule, the inter- and intra-domain interactions, the location of the universally conserved regions, the regions involved in pre-tRNA recognition and the location of the active site. A model with bound tRNA is in agreement with all existing data and suggests the general basis for RNA-RNA recognition by this ribozyme.

  20. Crystal structure of the RNA component of bacterial ribonuclease P.

    Science.gov (United States)

    Torres-Larios, Alfredo; Swinger, Kerren K; Krasilnikov, Andrey S; Pan, Tao; Mondragón, Alfonso

    2005-09-22

    Transfer RNA (tRNA) is produced as a precursor molecule that needs to be processed at its 3' and 5' ends. Ribonuclease P is the sole endonuclease responsible for processing the 5' end of tRNA by cleaving the precursor and leading to tRNA maturation. It was one of the first catalytic RNA molecules identified and consists of a single RNA component in all organisms and only one protein component in bacteria. It is a true multi-turnover ribozyme and one of only two ribozymes (the other being the ribosome) that are conserved in all kingdoms of life. Here we show the crystal structure at 3.85 A resolution of the RNA component of Thermotoga maritima ribonuclease P. The entire RNA catalytic component is revealed, as well as the arrangement of the two structural domains. The structure shows the general architecture of the RNA molecule, the inter- and intra-domain interactions, the location of the universally conserved regions, the regions involved in pre-tRNA recognition and the location of the active site. A model with bound tRNA is in agreement with all existing data and suggests the general basis for RNA-RNA recognition by this ribozyme.

  1. Structure of an Rrp6-RNA exosome complex bound to poly(A) RNA

    Energy Technology Data Exchange (ETDEWEB)

    Wasmuth, Elizabeth V.; Januszyk, Kurt; Lima, Christopher D. [MSKCC

    2014-08-20

    The eukaryotic RNA exosome processes and degrades RNA by directing substrates to the distributive or processive 3' to 5' exoribonuclease activities of Rrp6 or Rrp44, respectively. The non-catalytic nine-subunit exosome core (Exo9) features a prominent central channel. Although RNA can pass through the channel to engage Rrp44, it is not clear how RNA is directed to Rrp6 or whether Rrp6 uses the central channel. Here we report a 3.3 Å crystal structure of a ten-subunit RNA exosome complex from Saccharomyces cerevisiae composed of the Exo9 core and Rrp6 bound to single-stranded poly(A) RNA. The Rrp6 catalytic domain rests on top of the Exo9 S1/KH ring above the central channel, the RNA 3' end is anchored in the Rrp6 active site, and the remaining RNA traverses the S1/KH ring in an opposite orientation to that observed in a structure of a Rrp44-containing exosome complex. Solution studies with human and yeast RNA exosome complexes suggest that the RNA path to Rrp6 is conserved and dependent on the integrity of the S1/KH ring. Although path selection to Rrp6 or Rrp44 is stochastic in vitro, the fate of a particular RNA may be determined in vivo by the manner in which cofactors present RNA to the RNA exosome.

  2. Topology of RNA-RNA interaction structures

    CERN Document Server

    Andersen, Jørgen E; Penner, Robert C; Reidys, Christian M

    2011-01-01

    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 for fixed genus only finitely many irreducible shadows. This implies that for fixed genus there are only finitely many classes of interaction structures. In particular the simplest case of genus zero already provides the formalism for certain types of structures that occur in nature and are not covered by other filtrations. This case of genus zero interaction structures is already of practical interest, is studied here in detail and found to be expressed by a multiple context-free grammar extending the usual one for RNA secondary structures. We show that in $O(n^6)$ time and $O(n^4)$ space complexity, this grammar for genus zero interaction structures provides not only minimum free energy solutions but also the complete partit...

  3. Cations and hydration in catalytic RNA: molecular dynamics of the hepatitis delta virus ribozyme.

    Science.gov (United States)

    Krasovska, Maryna V; Sefcikova, Jana; Réblová, Kamila; Schneider, Bohdan; Walter, Nils G; Sponer, Jirí

    2006-07-15

    The hepatitis delta virus (HDV) ribozyme is an RNA enzyme from the human pathogenic HDV. Cations play a crucial role in self-cleavage of the HDV ribozyme, by promoting both folding and chemistry. Experimental studies have revealed limited but intriguing details on the location and structural and catalytic functions of metal ions. Here, we analyze a total of approximately 200 ns of explicit-solvent molecular dynamics simulations to provide a complementary atomistic view of the binding of monovalent and divalent cations as well as water molecules to reaction precursor and product forms of the HDV ribozyme. Our simulations find that an Mg2+ cation binds stably, by both inner- and outer-sphere contacts, to the electronegative catalytic pocket of the reaction precursor, in a position to potentially support chemistry. In contrast, protonation of the catalytically involved C75 in the precursor or artificial placement of this Mg2+ into the product structure result in its swift expulsion from the active site. These findings are consistent with a concerted reaction mechanism in which C75 and hydrated Mg2+ act as general base and acid, respectively. Monovalent cations bind to the active site and elsewhere assisted by structurally bridging long-residency water molecules, but are generally delocalized.

  4. How do ADARs bind RNA? New protein-RNA structures illuminate substrate recognition by the RNA editing ADARs.

    Science.gov (United States)

    Thomas, Justin M; Beal, Peter A

    2017-02-20

    Deamination of adenosine in RNA to form inosine has wide ranging consequences on RNA function including amino acid substitution to give proteins not encoded in the genome. What determines which adenosines in an mRNA are subject to this modification reaction? The answer lies in an understanding of the mechanism and substrate recognition properties of adenosine deaminases that act on RNA (ADARs). Our recent publication of X-ray crystal structures of the human ADAR2 deaminase domain bound to RNA editing substrates shed considerable light on how the catalytic domains of these enzymes bind RNA and promote adenosine deamination. Here we review in detail the deaminase domain-RNA contact surfaces and present models of how full length ADARs, bearing double stranded RNA-binding domains (dsRBDs) and deaminase domains, could process naturally occurring substrate RNAs.

  5. Quasispecies-like behavior observed in catalytic RNA populations evolving in a test tube

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    Lehman Niles

    2010-03-01

    Full Text Available Abstract Background During the RNA World, molecular populations were probably very small and highly susceptible to the force of strong random drift. In conjunction with Muller's Ratchet, this would have imposed difficulties for the preservation of the genetic information and the survival of the populations. Mechanisms that allowed these nascent populations to overcome this problem must have been advantageous. Results Using continuous in vitro evolution experimentation with an increased mutation rate imposed by MnCl2, it was found that clonal 100-molecule populations of ribozymes clearly exhibit certain characteristics of a quasispecies. This is the first time this has been seen with a catalytic RNA. Extensive genotypic sampling from two replicate lineages was gathered and phylogenetic networks were constructed to elucidate the structure of the evolving RNA populations. A common distribution was found in which a mutant sequence was present at high frequency, surrounded by a cloud of mutant with lower frequencies. This is a typical distribution of quasispecies. Most of the mutants in these clouds were connected by short Hamming distance values, indicating their close relatedness. Conclusions The quasispecies nature of mutant RNA clouds facilitates the recovery of genotypes under pressure of being removed from the population by random drift. The empirical populations therefore evolved a genotypic resiliency despite a high mutation rate by adopting the characteristics of quasispecies, implying that primordial RNA pools could have used this strategy to avoid extinction.

  6. Molecular dynamics and mutational analysis of the catalytic and translocation cycle of RNA polymerase

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    Kireeva Maria L

    2012-06-01

    Full Text Available Abstract Background During elongation, multi-subunit RNA polymerases (RNAPs cycle between phosphodiester bond formation and nucleic acid translocation. In the conformation associated with catalysis, the mobile “trigger loop” of the catalytic subunit closes on the nucleoside triphosphate (NTP substrate. Closing of the trigger loop is expected to exclude water from the active site, and dehydration may contribute to catalysis and fidelity. In the absence of a NTP substrate in the active site, the trigger loop opens, which may enable translocation. Another notable structural element of the RNAP catalytic center is the “bridge helix” that separates the active site from downstream DNA. The bridge helix may participate in translocation by bending against the RNA/DNA hybrid to induce RNAP forward movement and to vacate the active site for the next NTP loading. The transition between catalytic and translocation conformations of RNAP is not evident from static crystallographic snapshots in which macromolecular motions may be restrained by crystal packing. Results All atom molecular dynamics simulations of Thermus thermophilus (Tt RNAP reveal flexible hinges, located within the two helices at the base of the trigger loop, and two glycine hinges clustered near the N-terminal end of the bridge helix. As simulation progresses, these hinges adopt distinct conformations in the closed and open trigger loop structures. A number of residues (described as “switch” residues trade atomic contacts (ion pairs or hydrogen bonds in response to changes in hinge orientation. In vivo phenotypes and in vitro activities rendered by mutations in the hinge and switch residues in Saccharomyces cerevisiae (Sc RNAP II support the importance of conformational changes predicted from simulations in catalysis and translocation. During simulation, the elongation complex with an open trigger loop spontaneously translocates forward relative to the elongation complex with a

  7. Shapes of RNA pseudoknot structures

    CERN Document Server

    Reidys, Christian M

    2009-01-01

    In this paper we study $\\mathcal{I}_k$- and $\\mathcal{J}_k$-shapes of $k$-noncrossing, $\\sigma$-canonical RNA structures. These shapes, if induced by RNA secondary structures coincide with the $\\pi$- and $\\pi'$-shapes introduced by \\cite{Giegerich:04ashape}. Using a novel approach we compute the generating functions of $\\mathcal{I}_k$- and $\\mathcal{J}_k$-shapes as well as the generating functions of all $\\mathcal{I}_k$- and $\\mathcal{J}_k$-shapes induced by $k$-noncrossing, $\\sigma$-canonical RNA structures for fixed $n$. By means of singularity analysis of the generating functions, we derive explicit asymptotic expressions and can prove that $\\mathcal{I}_k$- and $\\mathcal{J}_k$-shapes lead to a meaningful categorization of RNA pseudoknot structures.

  8. Structured materials for catalytic and sensing applications

    Science.gov (United States)

    Hokenek, Selma

    been synthesized and characterized to establish the effects of nanoparticle size on catalytic activity in methanol decomposition. The physicochemical properties of the synthesized palladium-nickel nanoparticles will be discussed, as a function of the synthesis parameters. The optical characteristics of the Ag and Pd nanoparticles will be determined, with a view toward tuning the response of the nanoparticles for incorporation in sensors. Analysis of the monometallic palladium particles revealed a dependence of syngas production on nanoparticle size. The peak and steady state TOFs increased roughly linearly with the average nanoparticle diameter. The amount of coke deposited on the particle surfaces was found to be independent on the size of the nanoparticles. Shape control of the nickel-palladium nanoparticles with a high selectivity for (100) and (110) facets (≤ 80%) has been demonstrated. The resulting alloy nanoparticles were found to have homogeneous composition throughout their volume and maintain FCC crystal structure. Substitution of Ni atoms in the Pd lattice at a 1:3 molar ratio was found to induce lattice strains of ~1%. The Ag nanocubes synthesized exhibited behavior very similar to literature values, when taken on their own, exhibiting a pair of distinct absorbance peaks at 350 nm and 455 nm. In physical mixtures with the Pd nanoparticles synthesized, their behavior showed that the peak position of the Ag nanocubes' absorbance in UV-Vis could be tuned based on the relative proportions of the Ag and Pd nanoparticles present in the suspension analysed. The Ag polyhedra synthesized for comparison showed a broad doublet peak throughout the majority of the visible range before testing as a component in a physical mixture with the Pd nanoparticles. The addition of Pd nanoparticles to form a physical mixture resulted in some damping of the doublet peak observed as well as a corresponding shift in the baseline absorbance proportional to the amount of Pd added to

  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. Cleavage mediated by the catalytic domain of bacterial RNase P RNA.

    Science.gov (United States)

    Wu, Shiying; Kikovska, Ema; Lindell, Magnus; Kirsebom, Leif A

    2012-09-14

    Like other RNA molecules, RNase P RNA (RPR) is composed of domains, and these have different functions. Here, we provide data demonstrating that the catalytic (C) domain of Escherichia coli (Eco) RPR when separated from the specificity (S) domain mediates cleavage using various model RNA hairpin loop substrates. Compared to full-length Eco RPR, the rate constant, k(obs), of cleavage for the truncated RPR (CP RPR) was reduced 30- to 13,000-fold depending on substrate. Specifically, the structural architecture of the -1/+73 played a significant role where a C(-1)/G(+73) pair had the most dramatic effect on k(obs). Substitution of A(248) (E. coli numbering), positioned near the cleavage site in the RNase P-substrate complex, with G in the CP RPR resulted in 30-fold improvement in rate. In contrast, strengthening the interaction between the RPR and the 3' end of the substrate only had a modest effect. Interestingly, although deleting the S-domain gave a reduction in the rate, it resulted in a less erroneous RPR with respect to cleavage site selection. These data support and extend our understanding of the coupling between the distal interaction between the S-domain and events at the active site. Our findings will also be discussed with respect to the structure of RPR derived from different organisms.

  11. RNA structure. Structure of the HIV-1 RNA packaging signal.

    Science.gov (United States)

    Keane, Sarah C; Heng, Xiao; Lu, Kun; Kharytonchyk, Siarhei; Ramakrishnan, Venkateswaran; Carter, Gregory; Barton, Shawn; Hosic, Azra; Florwick, Alyssa; Santos, Justin; Bolden, Nicholas C; McCowin, Sayo; Case, David A; Johnson, Bruce A; Salemi, Marco; Telesnitsky, Alice; Summers, Michael F

    2015-05-22

    The 5' leader of the HIV-1 genome contains conserved elements that direct selective packaging of the unspliced, dimeric viral RNA into assembling particles. By using a (2)H-edited nuclear magnetic resonance (NMR) approach, we determined the structure of a 155-nucleotide region of the leader that is independently capable of directing packaging (core encapsidation signal; Ψ(CES)). The RNA adopts an unexpected tandem three-way junction structure, in which residues of the major splice donor and translation initiation sites are sequestered by long-range base pairing and guanosines essential for both packaging and high-affinity binding to the cognate Gag protein are exposed in helical junctions. The structure reveals how translation is attenuated, Gag binding promoted, and unspliced dimeric genomes selected, by the RNA conformer that directs packaging.

  12. Protein Structure Is Related to RNA Structural Reactivity In Vivo.

    Science.gov (United States)

    Tang, Yin; Assmann, Sarah M; Bevilacqua, Philip C

    2016-02-27

    We assessed whether in vivo mRNA structural reactivity and the structure of the encoded protein are related. This is the first investigation of such a relationship that utilizes information on RNA structure obtained in living cells. Based on our recent genome-wide Structure-seq analysis in Arabidopsis thaliana, we report that, as a meta property, regions of individual mRNAs that code for protein domains generally have higher reactivity to DMS (dimethyl sulfate), a chemical that covalently modifies accessible As and Cs, than regions that encode protein domain junctions. This relationship is prominent for proteins annotated for catalytic activity and reversed in proteins annotated for binding and transcription regulatory activity. Upon analyzing intrinsically disordered proteins, we found a similar pattern for disordered regions as compared to ordered regions: regions of individual mRNAs that code for ordered regions have significantly higher DMS reactivity than regions that code for intrinsically disordered regions. Based on these effects, we hypothesize that the decreased DMS reactivity of RNA regions that encode protein domain junctions or intrinsically disordered regions may reflect increased RNA structure that may slow translation, allowing time for the nascent protein domain or ordered region of the protein to fold, thereby reducing protein misfolding. In addition, a drop in DMS reactivity was observed on portions of mRNA sequences that correspond to the C-termini of protein domains, suggesting ribosome protection at these mRNA regions. Structural relationships between mRNAs and their encoded proteins may have evolved to allow efficient and accurate protein folding.

  13. 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 alig...... 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.......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...... two or more sequences. The advantage of this algorithm over those that separate the folding and alignment steps is that it makes better predictions. The disadvantage is that it is slower and requires more computer memory to run. The amount of computational resources needed to run the Sankoff algorithm...

  14. Resolving the Structure of Active Sites on Platinum Catalytic Nanoparticles

    DEFF Research Database (Denmark)

    Chang, Lan Yun; Barnard, Amanda S.; Gontard, Lionel Cervera

    2010-01-01

    Accurate understanding of the structure of active sites is fundamentally important in predicting catalytic properties of heterogeneous nanocatalysts. We present an accurate determination of both experimental and theoretical atomic structures of surface monatomic steps on industrial platinum...

  15. Structure of the Tribolium castaneum Telomerase Catalytic Subunit TERT

    Energy Technology Data Exchange (ETDEWEB)

    Gillis,A.; Schuller, A.; Skordalakes, E.

    2008-01-01

    A common hallmark of human cancers is the overexpression of telomerase, a ribonucleoprotein complex that is responsible for maintaining the length and integrity of chromosome ends. Telomere length deregulation and telomerase activation is an early, and perhaps necessary, step in cancer cell evolution. Here we present the high-resolution structure of the Tribolium castaneum catalytic subunit of telomerase, TERT. The protein consists of three highly conserved domains, organized into a ring-like structure that shares common features with retroviral reverse transcriptases, viral RNA polymerases and B-family DNA polymerases. Domain organization places motifs implicated in substrate binding and catalysis in the interior of the ring, which can accommodate seven to eight bases of double-stranded nucleic acid. Modelling of an RNA-DNA heteroduplex in the interior of this ring demonstrates a perfect fit between the protein and the nucleic acid substrate, and positions the 3'-end of the DNA primer at the active site of the enzyme, providing evidence for the formation of an active telomerase elongation complex.

  16. Combinatorics of RNA structures with pseudoknots.

    Science.gov (United States)

    Jin, Emma Y; Qin, Jing; Reidys, Christian M

    2008-01-01

    In this paper, we derive the generating function of RNA structures with pseudoknots. We enumerate all k-noncrossing RNA pseudoknot structures categorized by their maximal sets of mutually intersecting arcs. In addition, we enumerate pseudoknot structures over circular RNA. For 3-noncrossing RNA structures and RNA secondary structures we present a novel 4-term recursion formula and a 2-term recursion, respectively. Furthermore, we enumerate for arbitrary k all k-noncrossing, restricted RNA structures i.e. k-noncrossing RNA structures without 2-arcs i.e. arcs of the form (i,i+2), for 1< or =i< or =n-2.

  17. Activities of human RRP6 and structure of the human RRP6 catalytic domain

    Energy Technology Data Exchange (ETDEWEB)

    Januszyk, Kurt; Liu, Quansheng; Lima, Christopher D. (SKI)

    2011-08-29

    The eukaryotic RNA exosome is a highly conserved multi-subunit complex that catalyzes degradation and processing of coding and noncoding RNA. A noncatalytic nine-subunit exosome core interacts with Rrp44 and Rrp6, two subunits that possess processive and distributive 3'-to-5' exoribonuclease activity, respectively. While both Rrp6 and Rrp44 are responsible for RNA processing in budding yeast, Rrp6 may play a more prominent role in processing, as it has been demonstrated to be inhibited by stable RNA secondary structure in vitro and because the null allele in budding yeast leads to the buildup of specific structured RNA substrates. Human RRP6, otherwise known as PM/SCL-100 or EXOSC10, shares sequence similarity to budding yeast Rrp6 and is proposed to catalyze 3'-to-5' exoribonuclease activity on a variety of nuclear transcripts including ribosomal RNA subunits, RNA that has been poly-adenylated by TRAMP, as well as other nuclear RNA transcripts destined for processing and/or destruction. To characterize human RRP6, we expressed the full-length enzyme as well as truncation mutants that retain catalytic activity, compared their activities to analogous constructs for Saccharomyces cerevisiae Rrp6, and determined the X-ray structure of a human construct containing the exoribonuclease and HRDC domains that retains catalytic activity. Structural data show that the human active site is more exposed when compared to the yeast structure, and biochemical data suggest that this feature may play a role in the ability of human RRP6 to productively engage and degrade structured RNA substrates more effectively than the analogous budding yeast enzyme.

  18. Crystal structures of trypanosomal histidyl-tRNA synthetase illuminate differences between eukaryotic and prokaryotic homologs

    OpenAIRE

    Merritt, Ethan A.; Arakaki, Tracy L; Gillespie, J Robert; Larson, Eric T.; Kelley, Angela; Mueller, Natascha; Napuli, Alberto J.; Kim, Jessica; Li ZHANG; Verlinde, Christophe L M J; Fan, Erkang; Zucker, Frank; Buckner, Frederick S.; Van Voorhis, Wesley C.; Hol, Wim G. J.

    2010-01-01

    Crystal structures of histidyl-tRNA synthetase from the eukaryotic parasites Trypanosoma brucei and Trypanosoma cruzi provide a first structural view of a eukaryotic form of this enzyme, and reveal differences from bacterial homologs. Histidyl-tRNA synthetases in general contain an extra domain inserted between conserved motifs 2 and 3 of the Class II aminoacyl-tRNA synthetase catalytic core. The current structures show that the three dimensional topology of this domain is very different in b...

  19. Inverse Folding of RNA Pseudoknot Structures

    CERN Document Server

    Gao, James Z M; Reidys, Christian M

    2010-01-01

    Background: RNA exhibits a variety of structural configurations. Here we consider a structure to be tantamount to the noncrossing Watson-Crick and \\pairGU-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, {\\tt RNAinverse}, {\\tt RNA-SSD} as well as {\\tt INFO-RNA} are limited to RNA secondary structures, we present in this paper the inverse folding algorithm {\\tt Inv} which can deal with 3-noncrossing, canonical pseudoknot structures. Results: In this paper we present the inverse folding algorithm {\\tt Inv}. We give a detailed analysis of {\\tt Inv}, including pseudocodes. We show that {\\tt Inv} allows to...

  20. Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA.

    Science.gov (United States)

    Reiter, Nicholas J; Osterman, Amy; Torres-Larios, Alfredo; Swinger, Kerren K; Pan, Tao; Mondragón, Alfonso

    2010-12-09

    Ribonuclease (RNase) P is the universal ribozyme responsible for 5'-end tRNA processing. We report the crystal structure of the Thermotoga maritima RNase P holoenzyme in complex with tRNA(Phe). The 154 kDa complex consists of a large catalytic RNA (P RNA), a small protein cofactor and a mature tRNA. The structure shows that RNA-RNA recognition occurs through shape complementarity, specific intermolecular contacts and base-pairing interactions. Soaks with a pre-tRNA 5' leader sequence with and without metal help to identify the 5' substrate path and potential catalytic metal ions. The protein binds on top of a universally conserved structural module in P RNA and interacts with the leader, but not with the mature tRNA. The active site is composed of phosphate backbone moieties, a universally conserved uridine nucleobase, and at least two catalytically important metal ions. The active site structure and conserved RNase P-tRNA contacts suggest a universal mechanism of catalysis by RNase P.

  1. Asymmetric catalytic synthesis of the proposed structure of trocheliophorolide B.

    Science.gov (United States)

    Trost, Barry M; Quintard, Adrien

    2012-09-01

    A concise catalytic asymmetric synthesis of the proposed structure of trocheliophorolide B is reported. The synthetic sequence notably features an asymmetric acetaldehyde alkynylation, a Ru-catalyzed alder-ene reaction, and a Zn-ProPhenol ynone aldol condensation. Comparison with the reported data suggests a misassignment of the natural product structure.

  2. Predicting RNA structure: advances and limitations.

    Science.gov (United States)

    Hofacker, Ivo L; Lorenz, Ronny

    2014-01-01

    RNA secondary structures can be predicted using efficient algorithms. A widely used software package implementing a large number of computational methods is the ViennaRNA Package. This chapter describes how to use programs from the ViennaRNA Package to perform common tasks such as prediction of minimum free-energy structures, suboptimal structures, or base pairing probabilities, and generating secondary structure plots with reliability annotation. Moreover, we present recent methods to assess the folding kinetics of an RNA via 2D projections of the energy landscape, identification of local minima and energy barriers, or simulation of RNA folding as a Markov process.

  3. RNA structure prediction: progress and perspective

    CERN Document Server

    Shi, Ya-Zhou; Wang, Feng-Hua; Tan, Zhi-Jie

    2014-01-01

    Many recent exciting discoveries have revealed the versatility of RNAs and their importance in a variety of cellular functions which are strongly coupled to RNA structures. To understand the functions of RNAs, some structure prediction models have been developed in recent years. In this review, the progress in computational models for RNA structure prediction is introduced and the distinguishing features of many outstanding algorithms are discussed, emphasizing three dimensional (3D) structure prediction. A promising coarse-grained model for predicting RNA 3D structure, stability and salt effect is also introduced briefly. Finally, we discuss the major challenges in the RNA 3D structure modeling.

  4. Mechanism and substrate specificity of tRNA-guanine transglycosylases (TGTs): tRNA-modifying enzymes from the three different kingdoms of life share a common catalytic mechanism.

    Science.gov (United States)

    Stengl, Bernhard; Reuter, Klaus; Klebe, Gerhard

    2005-11-01

    Transfer RNA-guanine transglycosylases (TGTs) are evolutionarily ancient enzymes, present in all kingdoms of life, catalyzing guanine exchange within their cognate tRNAs by modified 7-deazaguanine bases. Although distinct bases are incorporated into tRNA at different positions in a kingdom-specific manner, the catalytic subunits of TGTs are structurally well conserved. This review provides insight into the sequential steps along the reaction pathway, substrate specificity, and conformational adaptions of the binding pockets by comparison of TGT crystal structures in complex with RNA substrates of a eubacterial and an archaebacterial species. Substrate-binding modes indicate an evolutionarily conserved base-exchange mechanism with a conserved aspartate serving as a nucleophile through covalent binding to C1' of the guanosine ribose moiety in an intermediate state. A second conserved aspartate seems to control the spatial rearrangement of the ribose ring along the reaction pathway and supposedly operates as a general acid/base. Water molecules inside the binding pocket accommodating interaction sites subsequently occupied by polar atoms of substrates help to elucidate substrate-recognition and substrate-specificity features. This emphasizes the role of water molecules as general probes to map binding-site properties for structure-based drug design. Additionally, substrate-bound crystal structures allow the extraction of valuable information about the classification of the TGT superfamily into a subdivision of presumably homologous superfamilies adopting the triose-phosphate isomerase type barrel fold with a standard phosphate-binding motif.

  5. RNA structural analysis by evolving SHAPE chemistry.

    Science.gov (United States)

    Spitale, Robert C; Flynn, Ryan A; Torre, Eduardo A; Kool, Eric T; Chang, Howard Y

    2014-01-01

    RNA is central to the flow of biological information. From transcription to splicing, RNA localization, translation, and decay, RNA is intimately involved in regulating every step of the gene expression program, and is thus essential for health and understanding disease. RNA has the unique ability to base-pair with itself and other nucleic acids to form complex structures. Hence the information content in RNA is not simply its linear sequence of bases, but is also encoded in complex folding of RNA molecules. A general chemical functionality that all RNAs have is a 2'-hydroxyl group in the ribose ring, and the reactivity of the 2'-hydroxyl in RNA is gated by local nucleotide flexibility. In other words, the 2'-hydroxyl is reactive at single-stranded and conformationally flexible positions but is unreactive at nucleotides constrained by base-pairing. Recent efforts have been focused on developing reagents that modify RNA as a function of RNA 2' hydroxyl group reactivity. Such RNA structure probing techniques can be read out by primer extension in experiments termed RNA SHAPE (selective 2'- hydroxyl acylation and primer extension). Herein, we describe the efforts devoted to the design and utilization of SHAPE probes for characterizing RNA structure. We also describe current technological advances that are being applied to utilize SHAPE chemistry with deep sequencing to probe many RNAs in parallel. The merging of chemistry with genomics is sure to open the door to genome-wide exploration of RNA structure and function.

  6. Probing complex RNA structures by mechanical force

    CERN Document Server

    Harlepp, S; Robert, J; Leger, J F; Xayaphoummine, A; Isambert, H; Chatenay, D

    2003-01-01

    RNA secondary structures of increasing complexity are probed combining single molecule stretching experiments and stochastic unfolding/refolding simulations. We find that force-induced unfolding pathways cannot usually be interpretated by solely invoking successive openings of native helices. Indeed, typical force-extension responses of complex RNA molecules are largely shaped by stretching-induced, long-lived intermediates including non-native helices. This is first shown for a set of generic structural motifs found in larger RNA structures, and then for Escherichia coli's 1540-base long 16S ribosomal RNA, which exhibits a surprisingly well-structured and reproducible unfolding pathway under mechanical stretching. Using out-of-equilibrium stochastic simulations, we demonstrate that these experimental results reflect the slow relaxation of RNA structural rearrangements. Hence, micromanipulations of single RNA molecules probe both their native structures and long-lived intermediates, so-called "kinetic traps",...

  7. Structure of RNA 3'-phosphate cyclase bound to substrate RNA.

    Science.gov (United States)

    Desai, Kevin K; Bingman, Craig A; Cheng, Chin L; Phillips, George N; Raines, Ronald T

    2014-10-01

    RNA 3'-phosphate cyclase (RtcA) catalyzes the ATP-dependent cyclization of a 3'-phosphate to form a 2',3'-cyclic phosphate at RNA termini. Cyclization proceeds through RtcA-AMP and RNA(3')pp(5')A covalent intermediates, which are analogous to intermediates formed during catalysis by the tRNA ligase RtcB. Here we present a crystal structure of Pyrococcus horikoshii RtcA in complex with a 3'-phosphate terminated RNA and adenosine in the AMP-binding pocket. Our data reveal that RtcA recognizes substrate RNA by ensuring that the terminal 3'-phosphate makes a large contribution to RNA binding. Furthermore, the RNA 3'-phosphate is poised for in-line attack on the P-N bond that links the phosphorous atom of AMP to N(ε) of His307. Thus, we provide the first insights into RNA 3'-phosphate termini recognition and the mechanism of 3'-phosphate activation by an Rtc enzyme.

  8. Crystal structure of Caulobacter crescentus polynucleotide phosphorylase reveals a mechanism of RNA substrate channelling and RNA degradosome assembly.

    Science.gov (United States)

    Hardwick, Steven W; Gubbey, Tobias; Hug, Isabelle; Jenal, Urs; Luisi, Ben F

    2012-04-01

    Polynucleotide phosphorylase (PNPase) is an exoribonuclease that cleaves single-stranded RNA substrates with 3'-5' directionality and processive behaviour. Its ring-like, trimeric architecture creates a central channel where phosphorolytic active sites reside. One face of the ring is decorated with RNA-binding K-homology (KH) and S1 domains, but exactly how these domains help to direct the 3' end of single-stranded RNA substrates towards the active sites is an unsolved puzzle. Insight into this process is provided by our crystal structures of RNA-bound and apo Caulobacter crescentus PNPase. In the RNA-free form, the S1 domains adopt a 'splayed' conformation that may facilitate capture of RNA substrates. In the RNA-bound structure, the three KH domains collectively close upon the RNA and direct the 3' end towards a constricted aperture at the entrance of the central channel. The KH domains make non-equivalent interactions with the RNA, and there is a marked asymmetry within the catalytic core of the enzyme. On the basis of these data, we propose that structural non-equivalence, induced upon RNA binding, helps to channel substrate to the active sites through mechanical ratcheting. Structural and biochemical analyses also reveal the basis for PNPase association with RNase E in the multi-enzyme RNA degradosome assembly of the α-proteobacteria.

  9. Functional Diversification of Maize RNA Polymerase IV and V subtypes via Alternative Catalytic Subunits

    Energy Technology Data Exchange (ETDEWEB)

    Haag, Jeremy R.; Brower-Toland, Brent; Krieger, Elysia K.; Sidorenko, Lyudmila; Nicora, Carrie D.; Norbeck, Angela D.; Irsigler, Andre; LaRue, Huachun; Brzeski, Jan; Mcginnis, Karen A.; Ivashuta, Sergey; Pasa-Tolic, Ljiljana; Chandler, Vicki L.; Pikaard, Craig S.

    2014-10-01

    Unlike nuclear multisubunit RNA polymerases I, II, and III, whose subunit compositions are conserved throughout eukaryotes, plant RNA polymerases IV and V are nonessential, Pol II-related enzymes whose subunit compositions are still evolving. Whereas Arabidopsis Pols IV and V differ from Pol II in four or five of their 12 subunits, respectively, and differ from one another in three subunits, proteomic ana- lyses show that maize Pols IV and V differ from Pol II in six subunits but differ from each other only in their largest subunits. Use of alternative catalytic second subunits, which are nonredundant for development and paramutation, yields at least two sub- types of Pol IV and three subtypes of Pol V in maize. Pol IV/Pol V associations with MOP1, RMR1, AGO121, Zm_DRD1/CHR127, SHH2a, and SHH2b extend parallels between paramutation in maize and the RNA-directed DNA methylation pathway in Arabidopsis.

  10. SCOR: a Structural Classification of RNA database.

    Science.gov (United States)

    Klosterman, Peter S; Tamura, Makio; Holbrook, Stephen R; Brenner, Steven E

    2002-01-01

    The Structural Classification of RNA (SCOR) database provides a survey of the three-dimensional motifs contained in 259 NMR and X-ray RNA structures. In one classification, the structures are grouped according to function. The RNA motifs, including internal and external loops, are also organized in a hierarchical classification. The 259 database entries contain 223 internal and 203 external loops; 52 entries consist of fully complementary duplexes. A classification of the well-characterized tertiary interactions found in the larger RNA structures is also included along with examples. The SCOR database is accessible at http://scor.lbl.gov.

  11. SCOR: a structural classification of RNA database.

    Energy Technology Data Exchange (ETDEWEB)

    Klosterman, Peter S.; Tamura, Makio; Holbrook, Stephen R.; Brenner, Steven E.

    2001-10-10

    The Structural Classification of RNA (SCOR) database provides a survey of the three-dimensional motifs contained in 259 NMR and X-ray RNA structures. In one classification, the structures are grouped according to function. The RNA motifs, including internal and external loops, are also organized in a hierarchical classification. The 259 database entries contain 223 internal and 203 external loops; 52 entries consist of fully complementary duplexes. A classification of the well-characterized tertiary interactions found in the larger RNA structures is also included along with examples. The SCOR database is accessible at http://scor.lbl.gov.

  12. Predicting RNA Structure Using Mutual Information

    DEFF Research Database (Denmark)

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

    2005-01-01

    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......, 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...... performance of MIfold improves with the number of aligned sequences for certain types of RNA sequences. In addition, we show that, for these sequences, MIfold is more sensitive but less selective than the related RNAalifold structure prediction program and is comparable with the COVE structure prediction...

  13. Structure and catalytic reactivity of Rh oxides

    DEFF Research Database (Denmark)

    Gustafson, J.; Westerström, R.; Resta, A.;

    2009-01-01

    Using a combination of experimental and theoretical techniques, we show that a thin RhO2 surface oxide film forms prior to the bulk Rh2O3 corundum oxide on all close-packed single crystal Rh surfaces. Based on previous reports, we argue that the RhO2 surface oxide also forms on vicinal Rh surfaces...... as well as on Rh nanoparticles. The detailed structure of this film was previously determined using UHV based techniques and density functional theory. In the present paper, we also examine the structure of the bulk Rh2O3 corundum oxide using surface X-ray diffraction. Being armed with this structural...... surface oxide film. In the case of Pt25Rh75(1 0 0), our measurements demonstrate that the formation of bulk Rh2O3 corundum oxide poisons the reaction, and argue that this is also valid for all other Rh surfaces. Our study implies that the CO oxidation reaction over Rh surfaces at realistic conditions...

  14. RNA tertiary structure prediction with ModeRNA.

    Science.gov (United States)

    Rother, Magdalena; Rother, Kristian; Puton, Tomasz; Bujnicki, Janusz M

    2011-11-01

    Noncoding RNAs perform important roles in the cell. As their function is tightly connected with structure, and as experimental methods are time-consuming and expensive, the field of RNA structure prediction is developing rapidly. Here, we present a detailed study on using the ModeRNA software. The tool uses the comparative modeling approach and can be applied when a structural template is available and an alignment of reasonable quality can be performed. We guide the reader through the entire process of modeling Escherichia coli tRNA(Thr) in a conformation corresponding to the complex with an aminoacyl-tRNA synthetase (aaRS). We describe the choice of a template structure, preparation of input files, and explore three possible modeling strategies. In the end, we evaluate the resulting models using six alternative benchmarks. The ModeRNA software can be freely downloaded from http://iimcb.genesilico.pl/moderna/ under the conditions of the General Public License. It runs under LINUX, Windows and Mac OS. It is also available as a server at http://iimcb.genesilico.pl/modernaserver/. The models and the script to reproduce the study from this article are available at http://www.genesilico.pl/moderna/examples/.

  15. 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...... in three dimensions (3D) and used as building blocks assembled manually during a bioinformatic interactive process. Comparing the models to the corresponding crystal structures has validated the method as being powerful to predict the RNA topology and architecture while being less accurate regarding...

  16. Tetrahedral DNA nanostructure-based microRNA biosensor coupled with catalytic recycling of the analyte.

    Science.gov (United States)

    Miao, Peng; Wang, Bidou; Chen, Xifeng; Li, Xiaoxi; Tang, Yuguo

    2015-03-25

    MicroRNAs are not only important regulators of a wide range of cellular processes but are also identified as promising disease biomarkers. Due to the low contents in serum, microRNAs are always difficult to detect accurately . In this study, an electrochemical biosensor for ultrasensitive detection of microRNA based on tetrahedral DNA nanostructure is developed. Four DNA single strands are engineered to form a tetrahedral nanostructure with a pendant stem-loop and modified on a gold electrode surface, which largely enhances the molecular recognition efficiency. Moreover, taking advantage of strand displacement polymerization, catalytic recycling of microRNA, and silver nanoparticle-based solid-state Ag/AgCl reaction, the proposed biosensor exhibits high sensitivity with the limit of detection down to 0.4 fM. This biosensor shows great clinical value and may have practical utility in early diagnosis and prognosis of certain diseases.

  17. Methods of using structures including catalytic materials disposed within porous zeolite materials to synthesize hydrocarbons

    Science.gov (United States)

    Rollins, Harry W.; Petkovic, Lucia M.; Ginosar, Daniel M.

    2011-02-01

    Catalytic structures include a catalytic material disposed within a zeolite material. The catalytic material may be capable of catalyzing a formation of methanol from carbon monoxide and/or carbon dioxide, and the zeolite material may be capable of catalyzing a formation of hydrocarbon molecules from methanol. The catalytic material may include copper and zinc oxide. The zeolite material may include a first plurality of pores substantially defined by a crystal structure of the zeolite material and a second plurality of pores dispersed throughout the zeolite material. Systems for synthesizing hydrocarbon molecules also include catalytic structures. Methods for synthesizing hydrocarbon molecules include contacting hydrogen and at least one of carbon monoxide and carbon dioxide with such catalytic structures. Catalytic structures are fabricated by forming a zeolite material at least partially around a template structure, removing the template structure, and introducing a catalytic material into the zeolite material.

  18. Metal dependence and branched RNA cocrystal structures of the RNA lariat debranching enzyme Dbr1

    Energy Technology Data Exchange (ETDEWEB)

    Clark, Nathaniel E.; Katolik, Adam; Roberts, Kenneth M.; Taylor, Alexander B.; Holloway, Stephen P.; Schuermann, Jonathan P.; Montemayor, Eric J.; Stevens, Scott W.; Fitzpatrick, Paul F.; Damha, Masad J.; Hart, P. John (UW); (Texas); (McGill); (UTSMC); (Cornell); (SC)

    2016-12-06

    Intron lariats are circular, branched RNAs (bRNAs) produced during pre-mRNA splicing. Their unusual chemical and topological properties arise from branch-point nucleotides harboring vicinal 2',5'- and 3',5'-phosphodiester linkages. The 2',5'-bonds must be hydrolyzed by the RNA debranching enzyme Dbr1 before spliced introns can be degraded or processed into small nucleolar RNA and microRNA derived from intronic RNA. Here, we measure the activity of Dbr1 from Entamoeba histolytica by using a synthetic, dark-quenched bRNA substrate that fluoresces upon hydrolysis. Purified enzyme contains nearly stoichiometric equivalents of Fe and Zn per polypeptide and demonstrates turnover rates of ~3 s-1. Similar rates are observed when apo-Dbr1 is reconstituted with Fe(II)+Zn(II) under aerobic conditions. Under anaerobic conditions, a rate of ~4.0 s-1 is observed when apoenzyme is reconstituted with Fe(II). In contrast, apo-Dbr1 reconstituted with Mn(II) or Fe(II) under aerobic conditions is inactive. Diffraction data from crystals of purified enzyme using X-rays tuned to the Fe absorption edge show Fe partitions primarily to the β-pocket and Zn to the α-pocket. Structures of the catalytic mutant H91A in complex with 7-mer and 16-mer synthetic bRNAs reveal bona fide RNA branchpoints in the Dbr1 active site. A bridging hydroxide is in optimal position for nucleophilic attack of the scissile phosphate. The results clarify uncertainties regarding structure/function relationships in Dbr1 enzymes, and the fluorogenic probe permits high-throughput screening for inhibitors that may hold promise as treatments for retroviral infections and neurodegenerative disease.

  19. RNA Secondary Structure Analysis Using RNAstructure.

    Science.gov (United States)

    Mathews, David H

    2014-06-17

    RNAstructure is a user-friendly program for the prediction and analysis of RNA secondary structure. It is available as a Web server, as a program with a graphical user interface, or as a set of command-line tools. The programs are available for Microsoft Windows, Macintosh OS X, or Linux. This unit provides protocols for RNA secondary structure prediction (using the Web server or the graphical user interface) and prediction of high-affinity oligonucleotide biding sites to a structured RNA target (using the graphical user interface).

  20. Three-Dimensional Structure and Catalytic Mechanism of Cytosine Deaminase

    Energy Technology Data Exchange (ETDEWEB)

    R Hall; A Fedorov; C Xu; E Fedorov; S Almo; F Raushel

    2011-12-31

    Cytosine deaminase (CDA) from E. coli is a member of the amidohydrolase superfamily. The structure of the zinc-activated enzyme was determined in the presence of phosphonocytosine, a mimic of the tetrahedral reaction intermediate. This compound inhibits the deamination of cytosine with a K{sub i} of 52 nM. The zinc- and iron-containing enzymes were characterized to determine the effect of the divalent cations on activation of the hydrolytic water. Fe-CDA loses activity at low pH with a kinetic pKa of 6.0, and Zn-CDA has a kinetic pKa of 7.3. Mutation of Gln-156 decreased the catalytic activity by more than 5 orders of magnitude, supporting its role in substrate binding. Mutation of Glu-217, Asp-313, and His-246 significantly decreased catalytic activity supporting the role of these three residues in activation of the hydrolytic water molecule and facilitation of proton transfer reactions. A library of potential substrates was used to probe the structural determinants responsible for catalytic activity. CDA was able to catalyze the deamination of isocytosine and the hydrolysis of 3-oxauracil. Large inverse solvent isotope effects were obtained on k{sub cat} and k{sub cat}/K{sub m}, consistent with the formation of a low-barrier hydrogen bond during the conversion of cytosine to uracil. A chemical mechanism for substrate deamination by CDA was proposed.

  1. Functional Diversification of Maize RNA Polymerase IV and V Subtypes via Alternative Catalytic Subunits

    Directory of Open Access Journals (Sweden)

    Jeremy R. Haag

    2014-10-01

    Full Text Available Unlike nuclear multisubunit RNA polymerases I, II, and III, whose subunit compositions are conserved throughout eukaryotes, plant RNA polymerases IV and V are nonessential, Pol II-related enzymes whose subunit compositions are still evolving. Whereas Arabidopsis Pols IV and V differ from Pol II in four or five of their 12 subunits, respectively, and differ from one another in three subunits, proteomic analyses show that maize Pols IV and V differ from Pol II in six subunits but differ from each other only in their largest subunits. Use of alternative catalytic second subunits, which are nonredundant for development and paramutation, yields at least two subtypes of Pol IV and three subtypes of Pol V in maize. Pol IV/Pol V associations with MOP1, RMR1, AGO121, Zm_DRD1/CHR127, SHH2a, and SHH2b extend parallels between paramutation in maize and the RNA-directed DNA methylation pathway in Arabidopsis.

  2. Modeling of the catalytic core of Arabidopsis thaliana Dicer-like 4 protein and its complex with double-stranded RNA.

    Science.gov (United States)

    Mickiewicz, Agnieszka; Sarzyńska, Joanna; Miłostan, Maciej; Kurzyńska-Kokorniak, Anna; Rybarczyk, Agnieszka; Łukasiak, Piotr; Kuliński, Tadeusz; Figlerowicz, Marek; Błażewicz, Jacek

    2017-02-01

    Plant Dicer-like proteins (DCLs) belong to the Ribonuclease III (RNase III) enzyme family. They are involved in the regulation of gene expression and antiviral defense through RNA interference pathways. A model plant, Arabidopsis thaliana encodes four DCL proteins (AtDCL1-4) that produce different classes of small regulatory RNAs. Our studies focus on AtDCL4 that processes double-stranded RNAs (dsRNAs) into 21 nucleotide trans-acting small interfering RNAs. So far, little is known about the structures of plant DCLs and the complexes they form with dsRNA. In this work, we present models of the catalytic core of AtDCL4 and AtDCL4-dsRNA complex constructed by computational methods. We built a homology model of the catalytic core of AtDCL4 comprising Platform, PAZ, Connector helix and two RNase III domains. To assemble the AtDCL4-dsRNA complex two modeling approaches were used. In the first method, to establish conformations that allow building a consistent model of the complex, we used Normal Mode Analysis for both dsRNA and AtDCL4. The second strategy involved template-based approach for positioning of the PAZ domain and manual arrangement of the Connector helix. Our results suggest that the spatial orientation of the Connector helix, Platform and PAZ relative to the RNase III domains is crucial for measuring dsRNA of defined length. The modeled complexes provide information about interactions that may contribute to the relative orientations of these domains and to dsRNA binding. All these information can be helpful for understanding the mechanism of AtDCL4-mediated dsRNA recognition and binding, to produce small RNA of specific size.

  3. X-ray structure of tRNA pseudouridine synthase TruD reveals an inserted domain with a novel fold.

    Science.gov (United States)

    Ericsson, Ulrika B; Nordlund, Pär; Hallberg, B Martin

    2004-05-01

    Pseudouridine synthases catalyse the isomerisation of uridine to pseudouridine in structural RNA. The pseudouridine synthase TruD, that modifies U13 in tRNA, belongs to a recently identified and large family of pseudouridine synthases present in all kingdoms of life. We report here the crystal structure of Escherichia coli TruD at 2.0 A resolution. The structure reveals an overall V-shaped molecule with an RNA-binding cleft formed between two domains: a catalytic domain and an insertion domain. The catalytic domain has a fold similar to that of the catalytic domains of previously characterised pseudouridine synthases, whereas the insertion domain displays a novel fold.

  4. Designing functional metalloproteins: from structural to catalytic metal sites.

    Science.gov (United States)

    Zastrow, Melissa L; Pecoraro, Vincent L

    2013-09-01

    Metalloenzymes efficiently catalyze some of the most important and difficult reactions in nature. For many years, coordination chemists have effectively used small molecule models to understand these systems. More recently, protein design has been shown to be an effective approach for mimicking metal coordination environments. Since the first designed proteins were reported, much success has been seen for incorporating metal sites into proteins and attaining the desired coordination environment but until recently, this has been with a lack of significant catalytic activity. Now there are examples of designed metalloproteins that, although not yet reaching the activity of native enzymes, are considerably closer. In this review, we highlight work leading up to the design of a small metalloprotein containing two metal sites, one for structural stability (HgS3) and the other a separate catalytic zinc site to mimic carbonic anhydrase activity (ZnN3O). The first section will describe previous studies that allowed for a high affinity thiolate site that binds heavy metals in a way that stabilizes three-stranded coiled coils. The second section will examine ways of preparing histidine rich environments that lead to metal based hydrolytic catalysts. We will also discuss other recent examples of the design of structural metal sites and functional metalloenzymes. Our work demonstrates that attaining the proper first coordination geometry of a metal site can lead to a significant fraction of catalytic activity, apparently independent of the type of secondary structure of the surrounding protein environment. We are now in a position to begin to meet the challenge of building a metalloenzyme systematically from the bottom-up by engineering and analyzing interactions directly around the metal site and beyond.

  5. Structure and folding of the Tetrahymena telomerase RNA pseudoknot

    Science.gov (United States)

    Cash, Darian D.; Feigon, Juli

    2017-01-01

    Telomerase maintains telomere length at the ends of linear chromosomes using an integral telomerase RNA (TER) and telomerase reverse transcriptase (TERT). An essential part of TER is the template/pseudoknot domain (t/PK) which includes the template, for adding telomeric repeats, template boundary element (TBE), and pseudoknot, enclosed in a circle by stem 1. The Tetrahymena telomerase holoenzyme catalytic core (p65-TER-TERT) was recently modeled in our 9 Å resolution cryo-electron microscopy map by fitting protein and TER domains, including a solution NMR structure of the Tetrahymena pseudoknot. Here, we describe in detail the structure and folding of the isolated pseudoknot, which forms a compact structure with major groove U•A-U and novel C•G-A+ base triples. Base substitutions that disrupt the base triples reduce telomerase activity in vitro. NMR studies also reveal that the pseudoknot does not form in the context of full-length TER in the absence of TERT, due to formation of a competing structure that sequesters pseudoknot residues. The residues around the TBE remain unpaired, potentially providing access by TERT to this high affinity binding site during an early step in TERT-TER assembly. A model for the assembly pathway of the catalytic core is proposed. PMID:27899638

  6. Structural Investigation of the GlmS Ribozyme Bound to Its Catalytic Cofactor

    Energy Technology Data Exchange (ETDEWEB)

    Cochrane,J.; Lipchock, S.; Strobel, S.

    2007-01-01

    The GlmS riboswitch is located in the 5'-untranslated region of the gene encoding glucosamine-6-phosphate (GlcN6P) synthetase. The GlmS riboswitch is a ribozyme with activity triggered by binding of the metabolite GlcN6P. Presented here is the structure of the GlmS ribozyme (2.5 {angstrom} resolution) with GlcN6P bound in the active site. The GlmS ribozyme adopts a compact double pseudoknot tertiary structure, with two closely packed helical stacks. Recognition of GlcN6P is achieved through coordination of the phosphate moiety by two hydrated magnesium ions as well as specific nucleobase contacts to the GlcN6P sugar ring. Comparison of this activator bound and the previously published apoenzyme complex supports a model in which GlcN6P does not induce a conformational change in the RNA, as is typical of other riboswitches, but instead functions as a catalytic cofactor for the reaction. This demonstrates that RNA, like protein enzymes, can employ the chemical diversity of small molecules to promote catalytic activity.

  7. Structural alignment of RNA with complex pseudoknot structure.

    Science.gov (United States)

    Wong, Thomas K F; Lam, T W; Sung, Wing-Kin; Cheung, Brenda W Y; Yiu, S M

    2011-01-01

    The secondary structure of an ncRNA molecule is known to play an important role in its biological functions. Aligning a known ncRNA to a target candidate to determine the sequence and structural similarity helps in identifying de novo ncRNA molecules that are in the same family of the known ncRNA. However, existing algorithms cannot handle complex pseudoknot structures which are found in nature. In this article, we propose algorithms to handle two types of complex pseudoknots: simple non-standard pseudoknots and recursive pseudoknots. Although our methods are not designed for general pseudoknots, it already covers all known ncRNAs in both Rfam and PseudoBase databases. An evaluation of our algorithms shows that it is useful to identify ncRNA molecules in other species which are in the same family of a known ncRNA.

  8. Structural biology of bacterial RNA polymerase.

    Science.gov (United States)

    Murakami, Katsuhiko S

    2015-05-11

    Since its discovery and characterization in the early 1960s (Hurwitz, J. The discovery of RNA polymerase. J. Biol. Chem. 2005, 280, 42477-42485), an enormous amount of biochemical, biophysical and genetic data has been collected on bacterial RNA polymerase (RNAP). In the late 1990s, structural information pertaining to bacterial RNAP has emerged that provided unprecedented insights into the function and mechanism of RNA transcription. In this review, I list all structures related to bacterial RNAP (as determined by X-ray crystallography and NMR methods available from the Protein Data Bank), describe their contributions to bacterial transcription research and discuss the role that small molecules play in inhibiting bacterial RNA transcription.

  9. Crystal structure of the bacteriophage P2 integrase catalytic domain.

    Science.gov (United States)

    Skaar, Karin; Claesson, Magnus; Odegrip, Richard; Högbom, Martin; Haggård-Ljungquist, Elisabeth; Stenmark, Pål

    2015-11-30

    Bacteriophage P2 is a temperate phage capable of integrating its DNA into the host genome by site-specific recombination upon lysogenization. Integration and excision of the phage genome requires P2 integrase, which performs recognition, cleavage and joining of DNA during these processes. This work presents the high-resolution crystal structure of the catalytic domain of P2 integrase, and analysis of the structure-function relationship of several previously identified non-functional P2 integrase mutants. The DNA binding area is characterized by a large positively charged patch, harboring key residues. The structure reveals potential for large dimer flexibility, likely essential for rearrangement of DNA strands upon integration and excision of the phage DNA.

  10. 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...

  11. Loops in canonical RNA pseudoknot structures

    CERN Document Server

    Nebel, Markus E; Wang, Rita R

    2009-01-01

    In this paper we compute the limit distributions of the numbers of hairpin-loops, interior-loops and bulges in k-noncrossing RNA structures. The latter are coarse grained RNA structures allowing for cross-serial interactions, subject to the constraint that there are at most k-1 mutually crossing arcs in the diagram representation of the molecule. We prove central limit theorems by means of studying the corresponding bivariate generating functions. These generating functions are obtained by symbolic inflation of Ik5-shapes.

  12. Combinatorics of locally optimal RNA secondary structures.

    Science.gov (United States)

    Fusy, Eric; Clote, Peter

    2014-01-01

    It is a classical result of Stein and Waterman that the asymptotic number of RNA secondary structures is 1.104366∙n-3/2∙2.618034n. Motivated by the kinetics of RNA secondary structure formation, we are interested in determining the asymptotic number of secondary structures that are locally optimal, with respect to a particular energy model. In the Nussinov energy model, where each base pair contributes -1 towards the energy of the structure, locally optimal structures are exactly the saturated structures, for which we have previously shown that asymptotically, there are 1.07427∙n-3/2∙2.35467n many saturated structures for a sequence of length n. In this paper, we consider the base stacking energy model, a mild variant of the Nussinov model, where each stacked base pair contributes -1 toward the energy of the structure. Locally optimal structures with respect to the base stacking energy model are exactly those secondary structures, whose stems cannot be extended. Such structures were first considered by Evers and Giegerich, who described a dynamic programming algorithm to enumerate all locally optimal structures. In this paper, we apply methods from enumerative combinatorics to compute the asymptotic number of such structures. Additionally, we consider analogous combinatorial problems for secondary structures with annotated single-stranded, stacking nucleotides (dangles).

  13. RNA-Puzzles Round II: assessment of RNA structure prediction programs applied to three large RNA structures.

    Science.gov (United States)

    Miao, Zhichao; Adamiak, Ryszard W; Blanchet, Marc-Frédérick; Boniecki, Michal; Bujnicki, Janusz M; Chen, Shi-Jie; Cheng, Clarence; Chojnowski, Grzegorz; Chou, Fang-Chieh; Cordero, Pablo; Cruz, José Almeida; Ferré-D'Amaré, Adrian R; Das, Rhiju; Ding, Feng; Dokholyan, Nikolay V; Dunin-Horkawicz, Stanislaw; Kladwang, Wipapat; Krokhotin, Andrey; Lach, Grzegorz; Magnus, Marcin; Major, François; Mann, Thomas H; Masquida, Benoît; Matelska, Dorota; Meyer, Mélanie; Peselis, Alla; Popenda, Mariusz; Purzycka, Katarzyna J; Serganov, Alexander; Stasiewicz, Juliusz; Szachniuk, Marta; Tandon, Arpit; Tian, Siqi; Wang, Jian; Xiao, Yi; Xu, Xiaojun; Zhang, Jinwei; Zhao, Peinan; Zok, Tomasz; Westhof, Eric

    2015-06-01

    This paper is a report of a second round of RNA-Puzzles, a collective and blind experiment in three-dimensional (3D) RNA structure prediction. Three puzzles, Puzzles 5, 6, and 10, represented sequences of three large RNA structures with limited or no homology with previously solved RNA molecules. A lariat-capping ribozyme, as well as riboswitches complexed to adenosylcobalamin and tRNA, were predicted by seven groups using RNAComposer, ModeRNA/SimRNA, Vfold, Rosetta, DMD, MC-Fold, 3dRNA, and AMBER refinement. Some groups derived models using data from state-of-the-art chemical-mapping methods (SHAPE, DMS, CMCT, and mutate-and-map). The comparisons between the predictions and the three subsequently released crystallographic structures, solved at diffraction resolutions of 2.5-3.2 Å, were carried out automatically using various sets of quality indicators. The comparisons clearly demonstrate the state of present-day de novo prediction abilities as well as the limitations of these state-of-the-art methods. All of the best prediction models have similar topologies to the native structures, which suggests that computational methods for RNA structure prediction can already provide useful structural information for biological problems. However, the prediction accuracy for non-Watson-Crick interactions, key to proper folding of RNAs, is low and some predicted models had high Clash Scores. These two difficulties point to some of the continuing bottlenecks in RNA structure prediction. All submitted models are available for download at http://ahsoka.u-strasbg.fr/rnapuzzles/.

  14. RNA-Puzzles Round II: assessment of RNA structure prediction programs applied to three large RNA structures

    Science.gov (United States)

    Miao, Zhichao; Adamiak, Ryszard W.; Blanchet, Marc-Frédérick; Boniecki, Michal; Bujnicki, Janusz M.; Chen, Shi-Jie; Cheng, Clarence; Chojnowski, Grzegorz; Chou, Fang-Chieh; Cordero, Pablo; Cruz, José Almeida; Ferré-D'Amaré, Adrian R.; Das, Rhiju; Ding, Feng; Dokholyan, Nikolay V.; Dunin-Horkawicz, Stanislaw; Kladwang, Wipapat; Krokhotin, Andrey; Lach, Grzegorz; Magnus, Marcin; Major, François; Mann, Thomas H.; Masquida, Benoît; Matelska, Dorota; Meyer, Mélanie; Peselis, Alla; Popenda, Mariusz; Purzycka, Katarzyna J.; Serganov, Alexander; Stasiewicz, Juliusz; Szachniuk, Marta; Tandon, Arpit; Tian, Siqi; Wang, Jian; Xiao, Yi; Xu, Xiaojun; Zhang, Jinwei; Zhao, Peinan; Zok, Tomasz; Westhof, Eric

    2015-01-01

    This paper is a report of a second round of RNA-Puzzles, a collective and blind experiment in three-dimensional (3D) RNA structure prediction. Three puzzles, Puzzles 5, 6, and 10, represented sequences of three large RNA structures with limited or no homology with previously solved RNA molecules. A lariat-capping ribozyme, as well as riboswitches complexed to adenosylcobalamin and tRNA, were predicted by seven groups using RNAComposer, ModeRNA/SimRNA, Vfold, Rosetta, DMD, MC-Fold, 3dRNA, and AMBER refinement. Some groups derived models using data from state-of-the-art chemical-mapping methods (SHAPE, DMS, CMCT, and mutate-and-map). The comparisons between the predictions and the three subsequently released crystallographic structures, solved at diffraction resolutions of 2.5–3.2 Å, were carried out automatically using various sets of quality indicators. The comparisons clearly demonstrate the state of present-day de novo prediction abilities as well as the limitations of these state-of-the-art methods. All of the best prediction models have similar topologies to the native structures, which suggests that computational methods for RNA structure prediction can already provide useful structural information for biological problems. However, the prediction accuracy for non-Watson–Crick interactions, key to proper folding of RNAs, is low and some predicted models had high Clash Scores. These two difficulties point to some of the continuing bottlenecks in RNA structure prediction. All submitted models are available for download at http://ahsoka.u-strasbg.fr/rnapuzzles/. PMID:25883046

  15. General combinatorics of RNA secondary structure.

    Science.gov (United States)

    Liao, Bo; Wang, Tian-ming

    2004-09-01

    The total number of RNA secondary structures of a given length with minimal hairpin loop length m(m>0) and with minimal stack length l(l>0) is computed, under the assumption that all base pairs can occur. Asymptotics are derived from the determination of recurrence relations of decomposition properties.

  16. Phylogeny and evolution of RNA structure.

    Science.gov (United States)

    Gesell, Tanja; Schuster, Peter

    2014-01-01

    Darwin's conviction that all living beings on Earth are related and the graph of relatedness is tree-shaped has been essentially confirmed by phylogenetic reconstruction first from morphology and later from data obtained by molecular sequencing. Limitations of the phylogenetic tree concept were recognized as more and more sequence information became available. The other path-breaking idea of Darwin, natural selection of fitter variants in populations, is cast into simple mathematical form and extended to mutation-selection dynamics. In this form the theory is directly applicable to RNA evolution in vitro and to virus evolution. Phylogeny and population dynamics of RNA provide complementary insights into evolution and the interplay between the two concepts will be pursued throughout this chapter. The two strategies for understanding evolution are ultimately related through the central paradigm of structural biology: sequence ⇒ structure ⇒ function. We elaborate on the state of the art in modeling both phylogeny and evolution of RNA driven by reproduction and mutation. Thereby the focus will be laid on models for phylogenetic sequence evolution as well as evolution and design of RNA structures with selected examples and notes on simulation methods. In the perspectives an attempt is made to combine molecular structure, population dynamics, and phylogeny in modeling evolution.

  17. Asymptotic Enumeration of RNA Structures with Pseudoknots

    CERN Document Server

    Jin, Emma Y

    2007-01-01

    In this paper we present the asymptotic enumeration of RNA structures with pseudoknots. We develop a general framework for the computation of exponential growth rate and the sub exponential factors for $k$-noncrossing RNA structures. Our results are based on the generating function for the number of $k$-noncrossing RNA pseudoknot structures, ${\\sf S}_k(n)$, derived in \\cite{Reidys:07pseu}, where $k-1$ denotes the maximal size of sets of mutually intersecting bonds. We prove a functional equation for the generating function $\\sum_{n\\ge 0}{\\sf S}_k(n)z^n$ and obtain for $k=2$ and $k=3$ the analytic continuation and singular expansions, respectively. It is implicit in our results that for arbitrary $k$ singular expansions exist and via transfer theorems of analytic combinatorics we obtain asymptotic expression for the coefficients. We explicitly derive the asymptotic expressions for 2- and 3-noncrossing RNA structures. Our main result is the derivation of the formula ${\\sf S}_3(n) \\sim \\frac{10.4724\\cdot 4!}{n(n...

  18. Synthesis, structure characterization and catalytic activity of nickel tungstate nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Pourmortazavi, Seied Mahdi, E-mail: pourmortazavi@yahoo.com [Faculty of Material and Manufacturing Technologies, Malek Ashtar University of Technology, Tehran (Iran, Islamic Republic of); Rahimi-Nasrabadi, Mehdi, E-mail: rahiminasrabadi@gmail.com [Department of Chemistry, Imam Hossein University, Tehran (Iran, Islamic Republic of); Khalilian-Shalamzari, Morteza [Department of Chemistry, Imam Hossein University, Tehran (Iran, Islamic Republic of); Zahedi, Mir Mahdi; Hajimirsadeghi, Seiedeh Somayyeh [Islamic Azad University, Varamin Pishva Branch, Varamin (Iran, Islamic Republic of); Omrani, Ismail [Department of Chemistry, Imam Hossein University, Tehran (Iran, Islamic Republic of)

    2012-12-15

    Graphical abstract: NiWO{sub 4} nanoparticles were prepared via precipitation technique. Experimental parameters of procedure were optimized statistically. Highlights: Black-Right-Pointing-Pointer NiWO{sub 4} spherical nanoparticles were synthesized via direct precipitation method. Black-Right-Pointing-Pointer Taguchi robust design was used for optimization of synthesis reaction parameters. Black-Right-Pointing-Pointer Composition and structural properties of NiWO{sub 4} nanoparticles were characterized. Black-Right-Pointing-Pointer EDAX, XRD, SEM, FT-IR, UV-vis and photoluminescence techniques were employed. Black-Right-Pointing-Pointer Catalytic activity of the product in a cyclo-addition reaction was investigated. - Abstract: Taguchi robust design was applied to optimize experimental parameters for controllable, simple and fast synthesis of nickel tungstate nanoparticles. NiWO{sub 4} nanoparticles were synthesized by precipitation reaction involving addition of nickel ion solution to the tungstate aqueous reagent and then formation of nickel tungstate nucleolus which are insoluble in aqueous media. Effects of various parameters such as nickel and tungstate concentrations, flow rate of reagent addition and reactor temperature on diameter of synthesized nickel tungstate nanoparticles were investigated experimentally by the aid of orthogonal array design. The results for analysis of variance (ANOVA) showed that particle size of nickel tungstate can be effectively tuned by controlling significant variables involving nickel and tungstate concentrations and flow rate; while, temperature of the reactor has a no considerable effect on the size of NiWO{sub 4} particles. The ANOVA results proposed the optimum conditions for synthesis of nickel tungstate nanoparticles via this technique. Also, under optimum condition nanoparticles of NiWO{sub 4} were prepared and their structure and chemical composition were characterized by means of EDAX, XRD, SEM, FT-IR spectroscopy, UV

  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. Combinatorics of locally optimal RNA secondary structures

    CERN Document Server

    Clote, Peter

    2011-01-01

    It is a classical result of Stein and Waterman that the asymptotic number of RNA secondary structures is $1.104366 \\cdot n^{-3/2} \\cdot 2.618034^n$. To provide a better understanding of the kinetics of RNA secondary structure formation, we are interested in determining the asymptotic number of secondary structures that are {\\em locally optimal}, with respect to a particular energy model. In the Nussinov energy model, where each base pair contributes -1 towards the energy of the structure, locally optimal structures are exactly the {\\em saturated} structures, for which we have previously shown that asymptotically, there are $1.07427\\cdot n^{-3/2} \\cdot 2.35467^n$ many saturated structures for a sequence of length $n$. In this paper, we consider the {\\em base stacking energy model}, a mild variant of the Nussinov model, where each stacked base pair contributes -1 toward the energy of the structure. Locally optimal structures with respect to the base stacking energy model are exactly those secondary structures, ...

  1. Structural basis for duplex RNA recognition and cleavage by Archaeoglobus fulgidus C3PO

    Science.gov (United States)

    Parizotto, Eneida A; Lowe, Edward D; Parker, James S

    2013-01-01

    Oligomeric complexes of Trax and Translin proteins, known as C3POs, participate in a variety of eukaryotic nucleic acid metabolism pathways including RNAi and tRNA processing. In RNAi in humans and Drosophila, C3PO activates pre-RISC by removing the passenger strand of the siRNA precursor duplex using nuclease activity present in Trax. It is not known how C3POs engage with nucleic acid substrates. Here we identify a single protein from Archaeoglobus fulgidus that assembles into an octamer with striking similarity to human C3PO. The structure in complex with duplex RNA reveals that the octamer entirely encapsulates a single thirteen base-pair RNA duplex inside a large inner cavity. Trax-like subunit catalytic sites target opposite strands of the duplex for cleavage, separated by seven base pairs. The structure provides insight into the mechanism of RNA recognition and cleavage by an archaeal C3PO-like complex. PMID:23353787

  2. The catalytic and the RNA subunits of human telomerase are required to immortalize equid primary fibroblasts.

    Science.gov (United States)

    Vidale, Pamela; Magnani, Elisa; Nergadze, Solomon G; Santagostino, Marco; Cristofari, Gael; Smirnova, Alexandra; Mondello, Chiara; Giulotto, Elena

    2012-10-01

    Many human primary somatic cells can be immortalized by inducing telomerase activity through the exogenous expression of the human telomerase catalytic subunit (hTERT). This approach has been extended to the immortalization of cell lines from several mammals. Here, we show that hTERT expression is not sufficient to immortalize primary fibroblasts from three equid species, namely donkey, Burchelli's zebra and Grevy's zebra. In vitro analysis of a reconstituted telomerase composed by hTERT and an equid RNA component of telomerase (TERC) revealed a low activity of this enzyme compared to human telomerase, suggesting a low compatibility of equid and human telomerase subunits. This conclusion was also strengthened by comparison of human and equid TERC sequences, which revealed nucleotide differences in key regions for TERC and TERT interaction. We then succeeded in immortalizing equid fibroblasts by expressing hTERT and hTERC concomitantly. Expression of both human telomerase subunits led to telomerase activity and telomere elongation, indicating that human telomerase is compatible with the other equid telomerase subunits and proteins involved in telomere metabolism. The immortalization procedure described herein could be extended to primary cells from other mammals. The availability of immortal cells from endangered species could be particularly useful for obtaining new information on the organization and function of their genomes, which is relevant for their preservation.

  3. Structure of the catalytic domain of the hepatitis C virus NS2-3 protease

    Energy Technology Data Exchange (ETDEWEB)

    Lorenz,I.; Marcotrigiano, J.; Dentzer, T.; Rice, C.

    2006-01-01

    Hepatitis C virus is a major global health problem affecting an estimated 170 million people worldwide. Chronic infection is common and can lead to cirrhosis and liver cancer. There is no vaccine available and current therapies have met with limited success. The viral RNA genome encodes a polyprotein that includes two proteases essential for virus replication. The NS2-3 protease mediates a single cleavage at the NS2/NS3 junction, whereas the NS3-4A protease cleaves at four downstream sites in the polyprotein. NS3-4A is characterized as a serine protease with a chymotrypsin-like fold, but the enzymatic mechanism of the NS2-3 protease remains unresolved. Here we report the crystal structure of the catalytic domain of the NS2-3 protease at 2.3 Angstroms resolution. The structure reveals a dimeric cysteine protease with two composite active sites. For each active site, the catalytic histidine and glutamate residues are contributed by one monomer, and the nucleophilic cysteine by the other. The carboxy-terminal residues remain coordinated in the two active sites, predicting an inactive post-cleavage form. Proteolysis through formation of a composite active site occurs in the context of the viral polyprotein expressed in mammalian cells. These features offer unexpected insights into polyprotein processing by hepatitis C virus and new opportunities for antiviral drug design.

  4. Combinatorics of saturated secondary structures of RNA.

    Science.gov (United States)

    Clote, P

    2006-11-01

    Following Zuker (1986), a saturated secondary structure for a given RNA sequence is a secondary structure such that no base pair can be added without violating the definition of secondary structure, e.g., without introducing a pseudoknot. In the Nussinov-Jacobson energy model (Nussinov and Jacobson, 1980), where the energy of a secondary structure is -1 times the number of base pairs, saturated secondary structures are local minima in the energy landscape, hence form kinetic traps during the folding process. Here we present recurrence relations and closed form asymptotic limits for combinatorial problems related to the number of saturated secondary structures. In addition, Python source code to compute the number of saturated secondary structures having k base pairs can be found at the web servers link of bioinformatics.bc.edu/clotelab/.

  5. RNA structural motif recognition based on least-squares distance.

    Science.gov (United States)

    Shen, Ying; Wong, Hau-San; Zhang, Shaohong; Zhang, Lin

    2013-09-01

    RNA structural motifs are recurrent structural elements occurring in RNA molecules. RNA structural motif recognition aims to find RNA substructures that are similar to a query motif, and it is important for RNA structure analysis and RNA function prediction. In view of this, we propose a new method known as RNA Structural Motif Recognition based on Least-Squares distance (LS-RSMR) to effectively recognize RNA structural motifs. A test set consisting of five types of RNA structural motifs occurring in Escherichia coli ribosomal RNA is compiled by us. Experiments are conducted for recognizing these five types of motifs. The experimental results fully reveal the superiority of the proposed LS-RSMR compared with four other state-of-the-art methods.

  6. Ru Nanoframes with an fcc Structure and Enhanced Catalytic Properties.

    Science.gov (United States)

    Ye, Haihang; Wang, Qingxiao; Catalano, Massimo; Lu, Ning; Vermeylen, Joseph; Kim, Moon J; Liu, Yuzi; Sun, Yugang; Xia, Xiaohu

    2016-04-13

    Noble-metal nanoframes are of great interest to many applications due to their unique open structures. Among various noble metals, Ru has never been made into nanoframes. In this study, we report for the first time an effective method based on seeded growth and chemical etching for the facile synthesis of Ru nanoframes with high purity. The essence of this approach is to induce the preferential growth of Ru on the corners and edges of Pd truncated octahedra as the seeds by kinetic control. The resultant Pd-Ru core-frame octahedra could be easily converted to Ru octahedral nanoframes of ∼2 nm in thickness by selectively removing the Pd cores through chemical etching. Most importantly, in this approach the face-centered cubic (fcc) crystal structure of Pd seeds was faithfully replicated by Ru that usually takes an hcp structure. The fcc Ru nanoframes showed higher catalytic activities toward the reduction of p-nitrophenol by NaBH4 and the dehydrogenation of ammonia borane compared with hcp Ru nanowires with roughly the same thickness.

  7. Structure and Catalytic Behavior of CuO-ZrO-CeO2 Mixed Oxides

    Institute of Scientific and Technical Information of China (English)

    王恩过; 陈诵英

    2002-01-01

    The effect of doping CuO on the structure and properties of zirconia-ceria mixed oxide was studied. The results show that addition of CuO decreases the reduction temperature of ceria, and stabilizes the cubic structure of mixed oxides, and enhances catalytic activity of CuO-ZrO-CeO2 mixed oxides for CO oxidation. Increasing ceria content in the mixed oxides can enhance the catalytic activity, but some impurities such as sulfate make catalytic activity falling. There is little effect of calcination temperature on catalytic activities, implying that these catalysts are effective with good thermal stability.

  8. Crystal structure of yeast DNA polymerase ε catalytic domain.

    Directory of Open Access Journals (Sweden)

    Rinku Jain

    Full Text Available DNA polymerase ε (Polε is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Here we report the ternary structure of the Polε catalytic subunit (Pol2 bound to a nascent G:C base pair (Pol2G:C. Pol2G:C has a typical B-family polymerase fold and embraces the template-primer duplex with the palm, fingers, thumb and exonuclease domains. The overall arrangement of domains is similar to the structure of Pol2T:A reported recently, but there are notable differences in their polymerase and exonuclease active sites. In particular, we observe Ca2+ ions at both positions A and B in the polymerase active site and also observe a Ca2+ at position B of the exonuclease site. We find that the contacts to the nascent G:C base pair in the Pol2G:C structure are maintained in the Pol2T:A structure and reflect the comparable fidelity of Pol2 for nascent purine-pyrimidine and pyrimidine-purine base pairs. We note that unlike that of Pol3, the shape of the nascent base pair binding pocket in Pol2 is modulated from the major grove side by the presence of Tyr431. Together with Pol2T:A, our results provide a framework for understanding the structural basis of high fidelity DNA synthesis by Pol2.

  9. Protein Structure and the Sequential Structure of mRNA

    DEFF Research Database (Denmark)

    Brunak, Søren; Engelbrecht, Jacob

    1996-01-01

    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...... 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...

  10. Random $k$-noncrossing RNA Structures

    CERN Document Server

    Chen, William Y C; Reidys, Christian M

    2009-01-01

    In this paper we derive polynomial time algorithms that generate random $k$-noncrossing matchings and $k$-noncrossing RNA structures with uniform probability. Our approach employs the bijection between $k$-noncrossing matchings and oscillating tableaux and the $P$-recursiveness of the cardinalities of $k$-noncrossing matchings. The main idea is to consider the tableaux sequences as paths of stochastic processes over shapes and to derive their transition probabilities.

  11. The X-ray Structures of Six Octameric RNA Duplexes in the Presence of Different Di- and Trivalent Cations

    Directory of Open Access Journals (Sweden)

    Michelle F. Schaffer

    2016-06-01

    Full Text Available Due to the polyanionic nature of RNA, the principles of charge neutralization and electrostatic condensation require that cations help to overcome the repulsive forces in order for RNA to adopt a three-dimensional structure. A precise structural knowledge of RNA-metal ion interactions is crucial to understand the mechanism of metal ions in the catalytic or regulatory activity of RNA. We solved the crystal structure of an octameric RNA duplex in the presence of the di- and trivalent metal ions Ca2+, Mn2+, Co2+, Cu2+, Sr2+, and Tb3+. The detailed investigation reveals a unique innersphere interaction to uracil and extends the knowledge of the influence of metal ions for conformational changes in RNA structure. Furthermore, we could demonstrate that an accurate localization of the metal ions in the X-ray structures require the consideration of several crystallographic and geometrical parameters as well as the anomalous difference map.

  12. The Influence of RNA Secondary Structure on the Efficiency of siRNA Silencing

    Institute of Scientific and Technical Information of China (English)

    SUN Ying; GUI Jian-bin; CHEN Zhao-xue

    2015-01-01

    In the application of RNAi technology, it is an essential step to design siRNA applicable to target gene. At present, there are many researches and conclusions on siRNA design. This paper aims to the influences of mRNA secondary structure or siRNA antisense-strand secondary structure on siRNA silence efficiency. The paper also discusses the problems and sets out further insights in the research.

  13. Synthesis, structure characterization and catalytic activity of nickel tungstate nanoparticles

    Science.gov (United States)

    Pourmortazavi, Seied Mahdi; Rahimi-Nasrabadi, Mehdi; Khalilian-Shalamzari, Morteza; Zahedi, Mir Mahdi; Hajimirsadeghi, Seiedeh Somayyeh; Omrani, Ismail

    2012-12-01

    Taguchi robust design was applied to optimize experimental parameters for controllable, simple and fast synthesis of nickel tungstate nanoparticles. NiWO4 nanoparticles were synthesized by precipitation reaction involving addition of nickel ion solution to the tungstate aqueous reagent and then formation of nickel tungstate nucleolus which are insoluble in aqueous media. Effects of various parameters such as nickel and tungstate concentrations, flow rate of reagent addition and reactor temperature on diameter of synthesized nickel tungstate nanoparticles were investigated experimentally by the aid of orthogonal array design. The results for analysis of variance (ANOVA) showed that particle size of nickel tungstate can be effectively tuned by controlling significant variables involving nickel and tungstate concentrations and flow rate; while, temperature of the reactor has a no considerable effect on the size of NiWO4 particles. The ANOVA results proposed the optimum conditions for synthesis of nickel tungstate nanoparticles via this technique. Also, under optimum condition nanoparticles of NiWO4 were prepared and their structure and chemical composition were characterized by means of EDAX, XRD, SEM, FT-IR spectroscopy, UV-vis spectroscopy, and photoluminescence. Finally, catalytic activity of the nanoparticles in a cycloaddition reaction was examined.

  14. Polymer supported nickel complex: Synthesis, structure and catalytic application

    Indian Academy of Sciences (India)

    Alekha Kumar Sutar; Tungabidya Maharana; Yasobanta Das; Prasanta Rath

    2014-11-01

    In the present investigation, a new synthetic route for a novel recyclable free [3-MOBdMBn-Ni] and polystyrene-anchored [P-3-MOBdMBn-Ni] nickel complexes is presented. The free and polymer-anchored metal complexes were synthesized by the reaction of nickel (II) with one molar equivalent of unsupported N N′-bis (2-Hydroxy-3-methoxybenzaldehyde) 4-Methylbenzene-1,2-diamine (3-MOBdMBn) or polymersupported (P-3-MOBdMBn) Schiff-base ligand in methanol under nitrogen atmosphere. The advantages of these polymer-supported catalysts are the low cost of catalyst and recyclability up to six times, due to easy availability of materials and simple synthetic route. The higher efficiency of complexation of nickel on the polymer-anchored 3-MOBdMBn Schiff base than the unsupported analogue is another advantage of this catalyst system. The structural study reveals that nickel(II) complex of 3-MOBdMBn is square planar in geometry. The catalytic activity of nickel complex towards the oxidation of phenol was investigated in the presence of hydrogen peroxide. Experimental results indicate that the reactivity of P-3-MOBdMBn-Ni was dramatically affected by the polymer support compared to free 3-MOBdMBn-Ni. The rates of oxidation (R) for unsupported and supported catalysts are 1.37 × 10-6 mole dm-3 s-1 and 2.33 × 10-6 mole dm-3 s-1 respectively.

  15. 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.

    2015-03-01

    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 N6-methyladenosine (m6A) modification genome wide. These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression.

  16. Structures of coxsackievirus, rhinovirus, and poliovirus polymerase elongation complexes solved by engineering RNA mediated crystal contacts.

    Science.gov (United States)

    Gong, Peng; Kortus, Matthew G; Nix, Jay C; Davis, Ralph E; Peersen, Olve B

    2013-01-01

    RNA-dependent RNA polymerases play a vital role in the growth of RNA viruses where they are responsible for genome replication, but do so with rather low fidelity that allows for the rapid adaptation to different host cell environments. These polymerases are also a target for antiviral drug development. However, both drug discovery efforts and our understanding of fidelity determinants have been hampered by a lack of detailed structural information about functional polymerase-RNA complexes and the structural changes that take place during the elongation cycle. Many of the molecular details associated with nucleotide selection and catalysis were revealed in our recent structure of the poliovirus polymerase-RNA complex solved by first purifying and then crystallizing stalled elongation complexes. In the work presented here we extend that basic methodology to determine nine new structures of poliovirus, coxsackievirus, and rhinovirus elongation complexes at 2.2-2.9 Å resolution. The structures highlight conserved features of picornaviral polymerases and the interactions they make with the template and product RNA strands, including a tight grip on eight basepairs of the nascent duplex, a fully pre-positioned templating nucleotide, and a conserved binding pocket for the +2 position template strand base. At the active site we see a pre-bound magnesium ion and there is conservation of a non-standard backbone conformation of the template strand in an interaction that may aid in triggering RNA translocation via contact with the conserved polymerase motif B. Moreover, by engineering plasticity into RNA-RNA contacts, we obtain crystal forms that are capable of multiple rounds of in-crystal catalysis and RNA translocation. Together, the data demonstrate that engineering flexible RNA contacts to promote crystal lattice formation is a versatile platform that can be used to solve the structures of viral RdRP elongation complexes and their catalytic cycle intermediates.

  17. Structures of coxsackievirus, rhinovirus, and poliovirus polymerase elongation complexes solved by engineering RNA mediated crystal contacts.

    Directory of Open Access Journals (Sweden)

    Peng Gong

    Full Text Available RNA-dependent RNA polymerases play a vital role in the growth of RNA viruses where they are responsible for genome replication, but do so with rather low fidelity that allows for the rapid adaptation to different host cell environments. These polymerases are also a target for antiviral drug development. However, both drug discovery efforts and our understanding of fidelity determinants have been hampered by a lack of detailed structural information about functional polymerase-RNA complexes and the structural changes that take place during the elongation cycle. Many of the molecular details associated with nucleotide selection and catalysis were revealed in our recent structure of the poliovirus polymerase-RNA complex solved by first purifying and then crystallizing stalled elongation complexes. In the work presented here we extend that basic methodology to determine nine new structures of poliovirus, coxsackievirus, and rhinovirus elongation complexes at 2.2-2.9 Å resolution. The structures highlight conserved features of picornaviral polymerases and the interactions they make with the template and product RNA strands, including a tight grip on eight basepairs of the nascent duplex, a fully pre-positioned templating nucleotide, and a conserved binding pocket for the +2 position template strand base. At the active site we see a pre-bound magnesium ion and there is conservation of a non-standard backbone conformation of the template strand in an interaction that may aid in triggering RNA translocation via contact with the conserved polymerase motif B. Moreover, by engineering plasticity into RNA-RNA contacts, we obtain crystal forms that are capable of multiple rounds of in-crystal catalysis and RNA translocation. Together, the data demonstrate that engineering flexible RNA contacts to promote crystal lattice formation is a versatile platform that can be used to solve the structures of viral RdRP elongation complexes and their catalytic cycle

  18. Networks of High Mutual Information Define the Structural Proximity of Catalytic Sites: Implications for Catalytic Residue Identification

    DEFF Research Database (Denmark)

    Buslje, Cristina Marino; Teppa, Elin; Di Doménico, Tomas;

    2010-01-01

    . A structural proximity conservation average score (termed pC) was also calculated and demonstrated to carry distinct information from pMI. A catalytic likeliness score (Cls), combining the KL, pC and pMI measures, was shown to lead to significantly improved prediction accuracy. At a specificity of 0...... throughout a given protein family making identification of CR a challenging task. Here, we put forward the hypothesis that CR carry a particular signature defined by networks of close proximity residues with high mutual information (MI), and that this signature can be applied to distinguish functional from...... to significantly outperform both the Shannon entropy and maximal frequency measurements. Residues in the proximity of catalytic sites were shown to be rich in shared MI. A structural proximity MI average score (termed pMI) was demonstrated to be a strong predictor for CR, thus confirming the proposed hypothesis...

  19. Transition State Structure of RNA Depurination by Saporin L3.

    Science.gov (United States)

    Yuan, Hongling; Stratton, Christopher F; Schramm, Vern L

    2016-05-20

    Saporin L3 from the leaves of the common soapwort is a catalyst for hydrolytic depurination of adenine from RNA. Saporin L3 is a type 1 ribosome inactivating protein (RIP) composed only of a catalytic domain. Other RIPs have been used in immunotoxin cancer therapy, but off-target effects have limited their development. In the current study, we use transition state theory to understand the chemical mechanism and transition state structure of saporin L3. In favorable cases, transition state structures guide the design of transition state analogues as inhibitors. Kinetic isotope effects (KIEs) were determined for an A14C mutant of saporin L3. To permit KIE measurements, small stem-loop RNAs that contain an AGGG tetraloop structure were enzymatically synthesized with the single adenylate bearing specific isotopic substitutions. KIEs were measured and corrected for forward commitment to obtain intrinsic values. A model of the transition state structure for depurination of stem-loop RNA (5'-GGGAGGGCCC-3') by saporin L3 was determined by matching KIE values predicted via quantum chemical calculations to a family of intrinsic KIEs. This model indicates saporin L3 displays a late transition state with the N-ribosidic bond to the adenine nearly cleaved, and the attacking water nucleophile weakly bonded to the ribosyl anomeric carbon. The transition state retains partial ribocation character, a feature common to most N-ribosyl transferases. However, the transition state geometry for saporin L3 is distinct from ricin A-chain, the only other RIP whose transition state is known.

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

    OpenAIRE

    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 perfor...

  1. Metal ion binding and function in natural and artificial small RNA enzymes from a structural perspective.

    Science.gov (United States)

    Wedekind, Joseph E

    2011-01-01

    Ribozymes are often perceived as part of an antiquated catalytic arsenal hearkening back to a pre-biotic RNA World that was eventually supplanted by proteins. However, recent genome-wide searches have revealed a plethora of new catalytic RNA motifs that appear to be variations on well-known themes. This suggests that ribozymes have continued to evolve in order to fulfill specific, RNA-essential biological niches. Although such ribozymes are small and catalyze one-step phosphodiester-bond scission reactions, ongoing structure and function analyses at the lab bench have demonstrated that RNA has the capacity for a diverse number of reactions such as carbon-carbon bond formation, and tRNA aminoacylation. Here we describe the fundamental structure and metal binding properties of four naturally occurring RNA enzymes: the hammerhead, hairpin, hepatitis delta virus, and glmS metabolite sensing ribozyme. In addition, we discuss the fold and ion coordination of three artificial ribozymes developed to probe the boundaries of RNA catalysis; these include the leadzyme, the flexizyme, and the Diels-Alder ribozyme. Our approach is to relate structure to function with the knowledge of ideal metal-ion coordination geometry that we have derived herein from surveys of high-resolution small molecule structures. An emergent theme is that natural and artificial ribozymes that catalyze single-step reactions often possess a pre-formed active site. Multivalent ions facilitate RNA active site formation, but can also provide Lewis acid functionality that is necessary for catalysis. When metal ion binding isn't possible, ribozymes make due by ionizing their bases, or by recruiting cofactors that augment their chemical functionality.

  2. Multiple 3D RNA Structure Superposition Using Neighbor Joining.

    Science.gov (United States)

    Hoksza, David; Svozil, Daniel

    2015-01-01

    Recent advances in RNA research and the steady growth of available RNA structures call for bioinformatics methods for handling and analyzing RNA structural data. Recently, we introduced SETTER-a fast and accurate method for RNA pairwise structure alignment. In this paper, we describe MultiSETTER, SETTER extension for multiple RNA structure alignment. MultiSETTER combines SETTER's decomposition of RNA structures into non-overlapping structural subunits with the multiple sequence alignment algorithm ClustalW adapted for the structure alignment. The accuracy of MultiSETTER was assessed by the automatic classification of RNA structures and its comparison to SCOR annotations. In addition, MultiSETTER classification was also compared to multiple sequence alignment-based and secondary structure alignment-based classifications provided by LocARNA and RNADistance tools, respectively. MultiSETTER precompiled Windows libraries, as well as the C++ source code, are freely available from http://siret.cz/multisetter.

  3. Crystal structure of the S. solfataricus archaeal exosome reveals conformational flexibility in the RNA-binding ring.

    Directory of Open Access Journals (Sweden)

    Changrui Lu

    Full Text Available BACKGROUND: The exosome complex is an essential RNA 3'-end processing and degradation machinery. In archaeal organisms, the exosome consists of a catalytic ring and an RNA-binding ring, both of which were previously reported to assume three-fold symmetry. METHODOLOGY/PRINCIPAL FINDINGS: Here we report an asymmetric 2.9 A Sulfolobus solfataricus archaeal exosome structure in which the three-fold symmetry is broken due to combined rigid body and thermal motions mainly within the RNA-binding ring. Since increased conformational flexibility was also observed in the RNA-binding ring of the related bacterial PNPase, we speculate that this may reflect an evolutionarily conserved mechanism to accommodate diverse RNA substrates for degradation. CONCLUSION/SIGNIFICANCE: This study clearly shows the dynamic structures within the RNA-binding domains, which provides additional insights on mechanism of asymmetric RNA binding and processing.

  4. The role of RNA structure at 5' untranslated region in microRNA-mediated gene regulation.

    Science.gov (United States)

    Gu, Wanjun; Xu, Yuming; Xie, Xueying; Wang, Ting; Ko, Jae-Hong; Zhou, Tong

    2014-09-01

    Recent studies have suggested that the secondary structure of the 5' untranslated region (5' UTR) of messenger RNA (mRNA) is important for microRNA (miRNA)-mediated gene regulation in humans. mRNAs that are targeted by miRNA tend to have a higher degree of local secondary structure in their 5' UTR; however, the general role of the 5' UTR in miRNA-mediated gene regulation remains unknown. We systematically surveyed the secondary structure of 5' UTRs in both plant and animal species and found a universal trend of increased mRNA stability near the 5' cap in mRNAs that are regulated by miRNA in animals, but not in plants. Intra-genome comparison showed that gene expression level, GC content of the 5' UTR, number of miRNA target sites, and 5' UTR length may influence mRNA structure near the 5' cap. Our results suggest that the 5' UTR secondary structure performs multiple functions in regulating post-transcriptional processes. Although the local structure immediately upstream of the start codon is involved in translation initiation, RNA structure near the 5' cap site, rather than the structure of the full-length 5' UTR sequences, plays an important role in miRNA-mediated gene regulation.

  5. 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.

  6. Structural properties of cyanase. Denaturation, renaturation, and role of sulfhydryls and oligomeric structure in catalytic activity.

    Science.gov (United States)

    Little, R M; Anderson, P M

    1987-07-25

    Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to give ammonia and bicarbonate. The enzyme is composed of 8-10 identical subunits (Mr = 17,008). The objective of this study was to clarify some of the structural properties of cyanase for the purpose of understanding the relationship between oligomeric structure and catalytic activity. Circular dichroism studies showed that cyanase has a significant amount of alpha-helix and beta-sheet structure. The one sulfhydryl group per subunit does not react with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) unless cyanase is denatured. Denaturation is apparently complete in 10 M urea or 6 M guanidine hydrochloride, but is significantly reduced in 10 M urea by the presence of azide (analog of cyanate) and is incomplete in 8 M urea. Denatured cyanase could be renatured and reactivated (greater than 85%) by removal of denaturants. Reactivation was greatly facilitated by the presence of certain anions, particularly bicarbonate, and by high ionic strength and protein concentration. The catalytic activity of renatured cyanase was associated only with oligomer. Cyanase that had been denatured in the presence of DTNB to give a cyanase-DTNB derivative could also be renatured at 26 degrees C to give active cyanase-DTNB oligomer. The active oligomeric form of the cyanase-DTNB derivative could be converted reversibly to inactive dimer by lowering the temperature to 4 degrees C or by reduction of the ionic strength and removal of monoanions. These results provide evidence that free sulfhydryl groups are not required for catalytic activity and that catalytic activity may be dependent upon oligomeric structure.

  7. Structural alignment of RNA with triple helix structure.

    Science.gov (United States)

    Wong, Thomas K F; Yiu, S M

    2012-04-01

    Structural alignment is useful in identifying members of ncRNAs. Existing tools are all based on the secondary structures of the molecules. There is evidence showing that tertiary interactions (the interaction between a single-stranded nucleotide and a base-pair) in triple helix structures are critical in some functions of ncRNAs. In this article, we address the problem of structural alignment of RNAs with the triple helix. We provide a formal definition to capture a simplified model of a triple helix structure, then develop an algorithm of O(mn(3)) time to align a query sequence (of length m) with known triple helix structure with a target sequence (of length n) with an unknown structure. The resulting algorithm is shown to be useful in identifying ncRNA members in a simulated genome.

  8. The NMR structure of the inhibited catalytic domain of human stromelysin-1.

    Science.gov (United States)

    Gooley, P R; O'Connell, J F; Marcy, A I; Cuca, G C; Salowe, S P; Bush, B L; Hermes, J D; Esser, C K; Hagmann, W K; Springer, J P

    1994-02-01

    The three-dimensional structure of the catalytic domain of stromelysin-1 complexed with an N-carboxyl alkyl inhibitor has been determined by NMR methods. The global fold consists of three helices, a five stranded beta-sheet and a methionine located in a turn near the catalytic histidines, classifying stromelysin-1 as a metzincin. Stromelysin-1 is unique in having two independent zinc binding sites: a catalytic site and a structural site. The inhibitor binds in an extended conformation. The S1' subsite is a deep hydrophobic pocket, whereas S2' appears shallow and S3' open.

  9. Structural basis for catalytically restrictive dynamics of a high-energy enzyme state

    Science.gov (United States)

    Kovermann, Michael; Ådén, Jörgen; Grundström, Christin; Elisabeth Sauer-Eriksson, A.; Sauer, Uwe H.; Wolf-Watz, Magnus

    2015-07-01

    An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or `invisible' states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the molecular topology and conformational dynamics of a catalytically indispensable high-energy state of an adenylate kinase variant. On the basis of matching energy barriers for conformational dynamics and catalytic turnover, it was found that the enzyme's catalytic activity is governed by its dynamic interconversion between the high-energy state and a ground state structure that was determined by X-ray crystallography. Our results show that it is possible to rationally tune enzymes' conformational dynamics and hence their catalytic power--a key aspect in rational design of enzymes catalysing novel reactions.

  10. RNA secondary structures, polygon dissections and clusters

    CERN Document Server

    Marsh, Robert J

    2010-01-01

    We show that the notion of induction introduced by Cassaigne, Ferenczi and Zamboni for trees of relations arising in the context of interval exchange relations can be generalised to the case of an arbitrary number of possible edge labels. We prove that the equivalence classes of its transitive closure can still be characterised via a circular order on the trees of relations in this case. We compute the cardinalities of these equivalence classes and show that the sequence of cardinalities, for a fixed number of possible edge labels, is a convolution of a Fuss-Catalan sequence. As in the original case, the equivalence classes are in bijection with a set of pseudoknot-free secondary structures arising from the study of RNA; we show that a natural subset of this set is in bijection with a set of m-clusters (in the cluster algebra sense).

  11. Structural features of the tmRNA-ribosome interaction.

    Science.gov (United States)

    Bugaeva, Elizaveta Y; Surkov, Serhiy; Golovin, Andrey V; Ofverstedt, Lars-Göran; Skoglund, Ulf; Isaksson, Leif A; Bogdanov, Alexey A; Shpanchenko, Olga V; Dontsova, Olga A

    2009-12-01

    Trans-translation is a process which switches the synthesis of a polypeptide chain encoded by a nonstop messenger RNA to the mRNA-like domain of a transfer-messenger RNA (tmRNA). It is used in bacterial cells for rescuing the ribosomes arrested during translation of damaged mRNA and directing this mRNA and the product polypeptide for degradation. The molecular basis of this process is not well understood. Earlier, we developed an approach that allowed isolation of tmRNA-ribosomal complexes arrested at a desired step of tmRNA passage through the ribosome. We have here exploited it to examine the tmRNA structure using chemical probing and cryo-electron microscopy tomography. Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation.

  12. PRI-Modeler: extracting RNA structural elements from PDB files of protein-RNA complexes.

    Science.gov (United States)

    Han, Kyungsook; Nepal, Chirag

    2007-05-01

    A complete understanding of protein and RNA structures and their interactions is important for determining the binding sites in protein-RNA complexes. Computational approaches exist for identifying secondary structural elements in proteins from atomic coordinates. However, similar methods have not been developed for RNA, due in part to the very limited structural data so far available. We have developed a set of algorithms for extracting and visualizing secondary and tertiary structures of RNA and for analyzing protein-RNA complexes. These algorithms have been implemented in a web-based program called PRI-Modeler (protein-RNA interaction modeler). Given one or more protein data bank files of protein-RNA complexes, PRI-Modeler analyzes the conformation of the RNA, calculates the hydrogen bond (H bond) and van der Waals interactions between amino acids and nucleotides, extracts secondary and tertiary RNA structure elements, and identifies the patterns of interactions between the proteins and RNAs. This paper presents PRI-Modeler and its application to the hydrogen bond and van der Waals interactions in the most representative set of protein-RNA complexes. The analysis reveals several interesting interaction patterns at various levels. The information provided by PRI-Modeler should prove useful for determining the binding sites in protein-RNA complexes. PRI-Modeler is accessible at http://wilab.inha.ac.kr/primodeler/, and supplementary materials are available in the analysis results section at http://wilab.inha.ac.kr/primodeler/.

  13. The mRNA expression of apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G in peripheral blood mononuclear cells in patients with chronic hepatitis C and its regulation by interferon-α

    Institute of Scientific and Technical Information of China (English)

    蔡卫平

    2013-01-01

    Objective To study the mRNA expression of apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G(APOBEC3G) in the peripheral blood mononuclear cells(PBMC) in patients with chronic hepatitis C(CHC) and its regulation by exogenous interferon-α

  14. Classification of non-coding RNA using graph representations ofsecondary structure

    Energy Technology Data Exchange (ETDEWEB)

    Karklin, Yan; Meraz, Richard F.; Holbrook, Stephen R.

    2004-06-07

    Some genes produce transcripts that function directly in regulatory, catalytic, or structural roles in the cell. These non-coding RNAs are prevalent in all living organisms, and methods that aid the understanding of their functional roles are essential. RNA secondary structure, the pattern of base-pairing, contains the critical information for determining the three dimensional structure and function of the molecule. In this work we examine whether the basic geometric and topological properties of secondary structure are sufficient to distinguish between RNA families in a learning framework. First, we develop a labeled dual graph representation of RNA secondary structure by adding biologically meaningful labels to the dual graphs proposed by Gan et al [1]. Next, we define a similarity measure directly on the labeled dual graphs using the recently developed marginalized kernels [2]. Using this similarity measure, we were able to train Support Vector Machine classifiers to distinguish RNAs of known families from random RNAs with similar statistics. For 22 of the 25 families tested, the classifier achieved better than 70% accuracy, with much higher accuracy rates for some families. Training a set of classifiers to automatically assign family labels to RNAs using a one vs. all multi-class scheme also yielded encouraging results. From these initial learning experiments, we suggest that the labeled dual graph representation, together with kernel machine methods, has potential for use in automated analysis and classification of uncharacterized RNA molecules or efficient genome-wide screens for RNA molecules from existing families.

  15. Unified approach to partition functions of RNA secondary structures.

    Science.gov (United States)

    Bundschuh, Ralf

    2014-11-01

    RNA secondary structure formation is a field of considerable biological interest as well as a model system for understanding generic properties of heteropolymer folding. This system is particularly attractive because the partition function and thus all thermodynamic properties of RNA secondary structure ensembles can be calculated numerically in polynomial time for arbitrary sequences and homopolymer models admit analytical solutions. Such solutions for many different aspects of the combinatorics of RNA secondary structure formation share the property that the final solution depends on differences of statistical weights rather than on the weights alone. Here, we present a unified approach to a large class of problems in the field of RNA secondary structure formation. We prove a generic theorem for the calculation of RNA folding partition functions. Then, we show that this approach can be applied to the study of the molten-native transition, denaturation of RNA molecules, as well as to studies of the glass phase of random RNA sequences.

  16. Crystal Structure and Activity of the Endoribonuclease Domain of the piRNA Pathway Factor Maelstrom

    Directory of Open Access Journals (Sweden)

    Naoki Matsumoto

    2015-04-01

    Full Text Available PIWI-interacting RNAs (piRNAs protect the genome from transposons in animal gonads. Maelstrom (Mael is an evolutionarily conserved protein, composed of a high-mobility group (HMG domain and a MAEL domain, and is essential for piRNA-mediated transcriptional transposon silencing in various species, such as Drosophila and mice. However, its structure and biochemical function have remained elusive. Here, we report the crystal structure of the MAEL domain from Drosophila melanogaster Mael, at 1.6 Å resolution. The structure reveals that the MAEL domain has an RNase H-like fold but lacks canonical catalytic residues conserved among RNase H-like superfamily nucleases. Our biochemical analyses reveal that the MAEL domain exhibits single-stranded RNA (ssRNA-specific endonuclease activity. Our cell-based analyses further indicate that ssRNA cleavage activity appears dispensable for piRNA-mediated transcriptional transposon silencing in Drosophila. Our findings provide clues toward understanding the multiple roles of Mael in the piRNA pathway.

  17. 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

  18. Redox status affects the catalytic activity of glutamyl-tRNA synthetase

    DEFF Research Database (Denmark)

    Katz, Assaf; Banerjee, Rajat; de Armas, Merly;

    2010-01-01

    Glutamyl-tRNA synthetases (GluRS) provide Glu-tRNA for different processes including protein synthesis, glutamine transamidation and tetrapyrrole biosynthesis. Many organisms contain multiple GluRSs, but whether these duplications solely broaden tRNA specificity or also play additional roles......, in vitro, GluRS1 activity is reversibly inactivated upon oxidation by hemin and hydrogen peroxide. The targets for oxidation-based inhibition were found to be cysteines from a SWIM zinc-binding motif located in the tRNA acceptor helix-binding domain. tRNA(Glu) was able to protect GluRS1 against oxidative...... inactivation by hemin plus hydrogen peroxide. The sensitivity to oxidation of A. ferrooxidans GluRS1 might provide a means to regulate tetrapyrrole and protein biosynthesis in response to extreme changes in both the redox and heme status of the cell via a single enzyme....

  19. RNA targeting by small molecules: Binding of protoberberine, benzophenanthridine and aristolochia alkaloids to various RNA structures

    Indian Academy of Sciences (India)

    Gopinatha Suresh Kumar

    2012-07-01

    Studies on RNA targeting by small molecules to specifically control certain cellular functions is an area of remarkable current interest. For this purpose, a basic understanding of the molecular aspects of the interaction of small molecules with various RNA structures is essential. Alkaloids are a group of natural products with potential therapeutic utility, and very recently, their interaction with many RNA structures have been reported. Especially noteworthy are the protoberberines and aristolochia alkaloids distributed widely in many botanical families. Many of the alkaloids of these group exhibit excellent binding affinity to many RNA structures that may be exploited to develop RNA targeted therapeutics. This review attempts to present the current status on the understanding of the interaction of these alkaloids with various RNA structures, mainly highlighting the biophysical aspects.

  20. 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.

  1. On the importance of cotranscriptional RNA structure formation.

    Science.gov (United States)

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

    2013-11-01

    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.

  2. 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

    BACKGROUND: Within the last decade a large number of noncoding RNA genes have been identified, but this may only be the tip of the iceberg. Using comparative genomics a large number of sequences that have signals concordant with conserved RNA secondary structures have been discovered in the human...... genome. Moreover, genome wide transcription profiling with tiling arrays indicate that the majority of the genome is transcribed. RESULTS: We have combined tiling array data with genome wide structural RNA predictions to search for novel noncoding and structural RNA genes that are expressed in the human...... of 3 of the hairpin structures and 3 out of 9 high covariance structures in SK-N-AS cells. CONCLUSION: Our results demonstrate that many human noncoding, structured and conserved RNA genes remain to be discovered and that tissue specific tiling array data can be used in combination with computational...

  3. RNA Structural Homology Search with a Succinct Stochastic Grammar Model

    Institute of Scientific and Technical Information of China (English)

    Ying-Lei Song; Ji-Zhen Zhao; Chun-Mei Liu; Kan Liu; Russell Malmberg; Li-Ming Cai

    2005-01-01

    An increasing number of structural homology search tools, mostly based on profile stochastic context-free grammars (SCFGs) have been recently developed for the non-coding RNA gene identification. SCFGs can include statistical biases that often occur in RNA sequences, necessary to profile specific RNA structures for structural homology search. In this paper, a succinct stochastic grammar model is introduced for RNA that has competitive search effectiveness. More importantly, the profiling model can be easily extended to include pseudoknots, structures that are beyond the capability of profile SCFGs. In addition, the model allows heuristics to be exploited, resulting in a significant speed-up for the CYK algorithm-based search.

  4. Structural Basis for the Catalytic Activity of Human Serine/Threonine Protein Phosphatase-5

    Science.gov (United States)

    Swingle, M. R.; Honkanen, R.; Ciszak, E. M.

    2004-01-01

    Serinehhreonine protein phosphatase-5 (PP5) affects many signaling networks that regulate cell growth and cellular responses to stress. Here we report the crystal structure of the PP5 catalytic domain (PP5c) at a resolution of 1.6 A. From this structure we resolved the mechanism for PP5-mediated hydrolysis of phosphoprotein substrates, which requires the precise positioning of two metal ions within a con served Aspn-271-M(sub 1):M(sub 2)-W(sup 1)-His-427-His-304-Asp-274 catalytic motif. The structure of PPSc provides a structural basis for explaining the exceptional catalytic proficiency of protein phosphatases, which are among the most powerful known catalysts. Resolution of the entire C-terminus revealed a novel subdomain, and the structure of the PP5c should also aid development of type-specific inhibitors.

  5. 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.

  6. 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.

  7. Non-Watson Crick base pairs might stabilize RNA structural motifs in ribozymes – A comparative study of group-I intron structures

    Indian Academy of Sciences (India)

    K Chandrasekhar; R Malathi

    2003-09-01

    In recent decades studies on RNA structure and function have gained significance due to discoveries on diversified functions of RNA. A common element for RNA secondary structure formed by series of non-Watson/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 motifs in ribozymes, playing a crucial role in their enzymatic activity. To understand the functional significance of these non-canonical base pairs in catalytic RNA, we analysed the sequences of group-I introns from nuclear genes. The results suggest that they might form the building blocks of folded RNA motifs which are crucial to the catalytic activity of the ribozyme. The conservation of these, as observed from divergent organisms, argues for the presence of non-canonical base pairs as an important requisite for the structure and enzymatic property of ribozymes by enabling them to carry out functions such as replication, polymerase activity etc. in primordial conditions in the absence of proteins.

  8. 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.

  9. Chemical and structural effects of base modifications in messenger RNA

    Science.gov (United States)

    Harcourt, Emily M.; Kietrys, Anna M.; Kool, Eric T.

    2017-01-01

    A growing number of nucleobase modifications in messenger RNA have been revealed through advances in detection and RNA sequencing. Although some of the biochemical pathways that involve modified bases have been identified, research into the world of RNA modification -- the epitranscriptome -- is still in an early phase. A variety of chemical tools are being used to characterize base modifications, and the structural effects of known base modifications on RNA pairing, thermodynamics and folding are being determined in relation to their putative biological roles.

  10. 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

    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....... 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...

  11. Structural Basis for the Catalytic Activity of Human SER/THR Protein Phosphatase-5

    Science.gov (United States)

    Swingle, M. R.; Honkanen, R.; Ciszak, E.

    2004-01-01

    Serinekhreonine protein phosphatase-5 (PP5) affects many signaling networks that regulate cell growth. Here we report the 1.6 Angstrom resolution crystal structure of PP5 catalytic domain with metal and phosphate ions in the active site. The structure reveals a mechanism for PPS-mediated catalysis that requires the precise positioning of two metal ions within a conserved Asp(sup 271)-M(sub 1),-M(sub 2)-His(sup 427)-W(sup 2)-His(sup 304)-Asp(sup 274) catalytic motif, and provides a structural basis for the exceptional catalytic proficiency of protein phosphatases placing them among the most powerful catalysts. Resolution of the entire C-terminus revealed a novel subdomain, and the structure of PP5 should aid development of specific inhibitors.

  12. Structural models of vanadate-dependent haloperoxidases, their reactivity, immobilization on polymer support and catalytic activities

    Indian Academy of Sciences (India)

    Mannar R Maurya

    2011-03-01

    The design of structural and functional models of enzymes vanadate-dependent haloperoxidases (VHPO) and the isolation and/or generation of species having {VO(H2O)}, {VO2}, {VO(OH)} and {VO(O2)} cores, proposed as intermediate(s) during catalytic action, in solution have been studied. Catalytic potential of these complexes have been tested for oxo-transfer as well as oxidative bromination and sulfide oxidation reactions. Some of the oxidovanadium(IV) and dioxidovanadium(V) complexes have been immobilized on polymer support in order to improve their recycle ability during catalytic activities and turn over number. The formulations of the polymer-anchored complexes are based on the respective neat complexes and conclusions drawn from the various characterization studies. These catalysts have successfully been used for all catalytic reactions mentioned above. These catalysts are stable and recyclable.

  13. The 5'-3' Distance of RNA Secondary Structures

    DEFF Research Database (Denmark)

    Han, Hillary S W; Reidys, Christian

    2012-01-01

    Abstract Recently, Yoffe and colleagues observed that the average distances between 5'-3' ends of RNA molecules are very small and largely independent of sequence length. This observation is based on numerical computations as well as theoretical arguments maximizing certain entropy functionals....... In this article, we compute the exact distribution of 5'-3' distances of RNA secondary structures for any finite n. Furthermore, we compute the limit distribution and show that for n = 30 the exact distribution and the limit distribution are very close. Our results show that the distances of random RNA secondary...... structures are distinctively lower than those of minimum free energy structures of random RNA sequences....

  14. Thermodynamics of RNA structures by Wang–Landau sampling

    OpenAIRE

    Lou, Feng; Clote, Peter

    2010-01-01

    Motivation: Thermodynamics-based dynamic programming RNA secondary structure algorithms have been of immense importance in molecular biology, where applications range from the detection of novel selenoproteins using expressed sequence tag (EST) data, to the determination of microRNA genes and their targets. Dynamic programming algorithms have been developed to compute the minimum free energy secondary structure and partition function of a given RNA sequence, the minimum free-energy and partit...

  15. Accurate Classification of RNA Structures Using Topological Fingerprints

    Science.gov (United States)

    Li, Kejie; Gribskov, Michael

    2016-01-01

    While RNAs are well known to possess complex structures, functionally similar RNAs often have little sequence similarity. While the exact size and spacing of base-paired regions vary, functionally similar RNAs have pronounced similarity in the arrangement, or topology, of base-paired stems. Furthermore, predicted RNA structures often lack pseudoknots (a crucial aspect of biological activity), and are only partially correct, or incomplete. A topological approach addresses all of these difficulties. In this work we describe each RNA structure as a graph that can be converted to a topological spectrum (RNA fingerprint). The set of subgraphs in an RNA structure, its RNA fingerprint, can be compared with the fingerprints of other RNA structures to identify and correctly classify functionally related RNAs. Topologically similar RNAs can be identified even when a large fraction, up to 30%, of the stems are omitted, indicating that highly accurate structures are not necessary. We investigate the performance of the RNA fingerprint approach on a set of eight highly curated RNA families, with diverse sizes and functions, containing pseudoknots, and with little sequence similarity–an especially difficult test set. In spite of the difficult test set, the RNA fingerprint approach is very successful (ROC AUC > 0.95). Due to the inclusion of pseudoknots, the RNA fingerprint approach both covers a wider range of possible structures than methods based only on secondary structure, and its tolerance for incomplete structures suggests that it can be applied even to predicted structures. Source code is freely available at https://github.rcac.purdue.edu/mgribsko/XIOS_RNA_fingerprint. PMID:27755571

  16. 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.

  17. Energy profile and secondary structure impact shRNA efficacy

    Directory of Open Access Journals (Sweden)

    Zeng Xiao

    2009-07-01

    Full Text Available Abstract Background RNA interference (RNAi is a cellular mechanism in which a short/small double stranded RNA induces the degradation of its sequence specific target mRNA, leading to specific gene silencing. Since its discovery, RNAi has become a powerful biological technique for gene function studies and drug discovery. The very first requirement of applying RNAi is to design functional small interfering RNA (siRNA that can uniquely induce the degradation of the targeted mRNA. It has been shown that many functional synthetic siRNAs share some common characteristics, such as GC content limitation and free energy preferences at both terminals, etc. Results Our three-phase algorithm was developed to design siRNA on a whole-genome scale based on those identified characteristics of functional siRNA. When this algorithm was applied to design short hairpin RNA (shRNA, the validated success rate of shRNAs was over 70%, which was almost double the rate reported for TRC library. This indicates that the designs of siRNA and shRNA may share the same concerns. Further analysis of the shRNA dataset of 444 designs reveals that the high free energy states of the two terminals have the largest positive impact on the shRNA efficacy. Enforcing these energy characteristics of both terminals can further improve the shRNA design success rate to 83.1%. We also found that functional shRNAs have less probability for their 3' terminals to be involved in mRNA secondary structure formation. Conclusion Functional shRNAs prefer high free energy states at both terminals. High free energy states of the two terminals were found to be the largest positive impact factor on shRNA efficacy. In addition, the accessibility of the 3' terminal is another key factor to shRNA efficacy.

  18. Structural Insights into the Catalytic Mechanism of Escherichia coli Selenophosphate Synthetase

    Energy Technology Data Exchange (ETDEWEB)

    Noinaj, Nicholas; Wattanasak, Rut; Lee, Duck-Yeon; Wally, Jeremy L.; Piszczek, Grzegorz; Chock, P. Boon; Stadtman, Thressa C.; Buchanan, Susan K. (NIH)

    2012-03-26

    Selenophosphate synthetase (SPS) catalyzes the synthesis of selenophosphate, the selenium donor for the biosynthesis of selenocysteine and 2-selenouridine residues in seleno-tRNA. Selenocysteine, known as the 21st amino acid, is then incorporated into proteins during translation to form selenoproteins which serve a variety of cellular processes. SPS activity is dependent on both Mg{sup 2+} and K{sup +} and uses ATP, selenide, and water to catalyze the formation of AMP, orthophosphate, and selenophosphate. In this reaction, the gamma phosphate of ATP is transferred to the selenide to form selenophosphate, while ADP is hydrolyzed to form orthophosphate and AMP. Most of what is known about the function of SPS has derived from studies investigating Escherichia coli SPS (EcSPS) as a model system. Here we report the crystal structure of the C17S mutant of SPS from E. coli (EcSPS{sup C17S}) in apo form (without ATP bound). EcSPS{sup C17S} crystallizes as a homodimer, which was further characterized by analytical ultracentrifugation experiments. The glycine-rich N-terminal region (residues 1 through 47) was found in the open conformation and was mostly ordered in both structures, with a magnesium cofactor bound at the active site of each monomer involving conserved aspartate residues. Mutating these conserved residues (D51, D68, D91, and D227) along with N87, also found at the active site, to alanine completely abolished AMP production in our activity assays, highlighting their essential role for catalysis in EcSPS. Based on the structural and biochemical analysis of EcSPS reported here and using information obtained from similar studies done with SPS orthologs from Aquifex aeolicus and humans, we propose a catalytic mechanism for EcSPS-mediated selenophosphate synthesis.

  19. Crystal Structure of the Catalytic Domain of a Serine Threonine Protein Phosphatase

    Science.gov (United States)

    Swinglel, Mark; Honkanel, Richard; Ciszak, Ewa

    2003-01-01

    Reversible phosphorylation of serine and threonine residues is a well-recognized mechanism in eukaryotic cells for the regulation of cell-cycle progression, cell growth and metabolism. Human serine/threonine phosphatases can be placed into two major families, PPP and PPM. To date the structure on one PPP family member (PPl) has been determined. Here we present the structure of a 323-residue catalytic domain of a second phosphatase belonging to the PPP family of enzyme. catalytic domain of the enzyme has been determined to 1.60Angstrom resolution and refined to R=17.5 and Rfree = 20.8%. The catalytic domain possesses a unique fold consisting of a largely monolithic structure, divisible into closely-associated helical and sheet regions. The catalytic site contains two manganese ions that are involved in substrate binding and catalysis. The enzyme crystallizes as a dimer that completely buries catalytic surfaces of both monomers, Also, the structure shows evidence of some flexibility around the active site cleft that may be related to substrate specificity of this enzyme.

  20. Fabrication of fuel cell electrodes and other catalytic structures

    Science.gov (United States)

    Smith, J.L.

    1987-02-11

    A porous layer of catalyst material suitable for use as an electrode in a molten carbonate fuel cell includes elongated pores substantially extending across the layer thickness. The catalyst layer is prepared by depositing particulate catalyst material into polymeric flocking on a substrate surface by a procedure such as tape casting. The loaded substrate is heated in a series of steps with rising temperatures to set the tape, thermally decompose the substrate with flocking and sinter bond the catalyst particles into a porous catalytic layer with elongated pores across its thickness. Employed as an electrode, the elongated pores provide distribution of reactant gas into contact with catalyst particles wetted by molten electrolyte. 1 fig.

  1. Structure-function analysis of Methanobacterium thermoautotrophicum RNA ligase – engineering a thermostable ATP independent enzyme

    Directory of Open Access Journals (Sweden)

    Zhelkovsky Alexander M

    2012-07-01

    Full Text Available Abstract Background RNA ligases are essential reagents for many methods in molecular biology including NextGen RNA sequencing. To prevent ligation of RNA to itself, ATP independent mutant ligases, defective in self-adenylation, are often used in combination with activated pre-adenylated linkers. It is important that these ligases not have de-adenylation activity, which can result in activation of RNA and formation of background ligation products. An additional useful feature is for the ligase to be active at elevated temperatures. This has the advantage or reducing preferences caused by structures of single-stranded substrates and linkers. Results To create an RNA ligase with these desirable properties we performed mutational analysis of the archaeal thermophilic RNA ligase from Methanobacterium thermoautotrophicum. We identified amino acids essential for ATP binding and reactivity but dispensable for phosphodiester bond formation with 5’ pre-adenylated donor substrate. The motif V lysine mutant (K246A showed reduced activity in the first two steps of ligation reaction. The mutant has full ligation activity with pre-adenylated substrates but retained the undesirable activity of deadenylation, which is the reverse of step 2 adenylation. A second mutant, an alanine substitution for the catalytic lysine in motif I (K97A abolished activity in the first two steps of the ligation reaction, but preserved wild type ligation activity in step 3. The activity of the K97A mutant is similar with either pre-adenylated RNA or single-stranded DNA (ssDNA as donor substrates but we observed two-fold preference for RNA as an acceptor substrate compared to ssDNA with an identical sequence. In contrast, truncated T4 RNA ligase 2, the commercial enzyme used in these applications, is significantly more active using pre-adenylated RNA as a donor compared to pre-adenylated ssDNA. However, the T4 RNA ligases are ineffective in ligating ssDNA acceptors. Conclusions

  2. Crystal structure of complete rhinovirus RNA polymerase suggests front loading of protein primer.

    Science.gov (United States)

    Appleby, Todd C; Luecke, Hartmut; Shim, Jae Hoon; Wu, Jim Z; Cheney, I Wayne; Zhong, Weidong; Vogeley, Lutz; Hong, Zhi; Yao, Nanhua

    2005-01-01

    Picornaviruses utilize virally encoded RNA polymerase and a uridylylated protein primer to ensure replication of the entire viral genome. The molecular details of this mechanism are not well understood due to the lack of structural information. We report the crystal structure of human rhinovirus 16 3D RNA-dependent RNA polymerase (HRV16 3Dpol) at a 2.4-A resolution, representing the first complete polymerase structure from the Picornaviridae family. HRV16 3Dpol shares the canonical features of other known polymerase structures and contains an N-terminal region that tethers the fingers and thumb subdomains, forming a completely encircled active site cavity which is accessible through a small tunnel on the backside of the molecule. The small thumb subdomain contributes to the formation of a large cleft on the front face of the polymerase which also leads to the active site. The cleft appears large enough to accommodate a template:primer duplex during RNA elongation or a protein primer during the uridylylation stage of replication initiation. Based on the structural features of HRV16 3Dpo1 and the catalytic mechanism known for all polymerases, a front-loading model for uridylylation is proposed.

  3. Crystal structures of the catalytic domains of pseudouridine synthases RluC and RluD from Escherichia coli.

    Science.gov (United States)

    Mizutani, Kenji; Machida, Yoshitaka; Unzai, Satoru; Park, Sam-Yong; Tame, Jeremy R H

    2004-04-20

    The most frequent modification of RNA, the conversion of uridine bases to pseudouridines, is found in all living organisms and often in highly conserved locations in ribosomal and transfer RNA. RluC and RluD are homologous enzymes which each convert three specific uridine bases in Escherichia coli ribosomal 23S RNA to pseudouridine: bases 955, 2504, and 2580 in the case of RluC and 1911, 1915, and 1917 in the case of RluD. Both have an N-terminal S4 RNA binding domain. While the loss of RluC has little phenotypic effect, loss of RluD results in a much reduced growth rate. We have determined the crystal structures of the catalytic domain of RluC, and full-length RluD. The S4 domain of RluD appears to be highly flexible or unfolded and is completely invisible in the electron density map. Despite the conserved topology shared by the two proteins, the surface shape and charge distribution are very different. The models suggest significant differences in substrate binding by different pseudouridine synthases.

  4. Principles for Predicting RNA Secondary Structure Design Difficulty.

    Science.gov (United States)

    Anderson-Lee, Jeff; Fisker, Eli; Kosaraju, Vineet; Wu, Michelle; Kong, Justin; Lee, Jeehyung; Lee, Minjae; Zada, Mathew; Treuille, Adrien; Das, Rhiju

    2016-02-27

    Designing RNAs that form specific secondary structures is enabling better understanding and control of living systems through RNA-guided silencing, genome editing and protein organization. Little is known, however, about which RNA secondary structures might be tractable for downstream sequence design, increasing the time and expense of design efforts due to inefficient secondary structure choices. Here, we present insights into specific structural features that increase the difficulty of finding sequences that fold into a target RNA secondary structure, summarizing the design efforts of tens of thousands of human participants and three automated algorithms (RNAInverse, INFO-RNA and RNA-SSD) in the Eterna massive open laboratory. Subsequent tests through three independent RNA design algorithms (NUPACK, DSS-Opt and MODENA) confirmed the hypothesized importance of several features in determining design difficulty, including sequence length, mean stem length, symmetry and specific difficult-to-design motifs such as zigzags. Based on these results, we have compiled an Eterna100 benchmark of 100 secondary structure design challenges that span a large range in design difficulty to help test future efforts. Our in silico results suggest new routes for improving computational RNA design methods and for extending these insights to assess "designability" of single RNA structures, as well as of switches for in vitro and in vivo applications.

  5. SARA: a server for function annotation of RNA structures.

    Science.gov (United States)

    Capriotti, Emidio; Marti-Renom, Marc A

    2009-07-01

    Recent interest in non-coding RNA transcripts has resulted in a rapid increase of deposited RNA structures in the Protein Data Bank. However, a characterization and functional classification of the RNA structure and function space have only been partially addressed. Here, we introduce the SARA program for pair-wise alignment of RNA structures as a web server for structure-based RNA function assignment. The SARA server relies on the SARA program, which aligns two RNA structures based on a unit-vector root-mean-square approach. The likely accuracy of the SARA alignments is assessed by three different P-values estimating the statistical significance of the sequence, secondary structure and tertiary structure identity scores, respectively. Our benchmarks, which relied on a set of 419 RNA structures with known SCOR structural class, indicate that at a negative logarithm of mean P-value higher or equal than 2.5, SARA can assign the correct or a similar SCOR class to 81.4% and 95.3% of the benchmark set, respectively. The SARA server is freely accessible via the World Wide Web at http://sgu.bioinfo.cipf.es/services/SARA/.

  6. RNA-Puzzles Round III: 3D RNA structure prediction of five riboswitches and one ribozyme.

    Science.gov (United States)

    Miao, Zhichao; Adamiak, Ryszard W; Antczak, Maciej; Batey, Robert T; Becka, Alexander J; Biesiada, Marcin; Boniecki, Michał J; Bujnicki, Janusz; Chen, Shi-Jie; Cheng, Clarence Yu; Chou, Fang-Chieh; Ferré-D'Amaré, Adrian R; Das, Rhiju; Dawson, Wayne K; Feng, Ding; Dokholyan, Nikolay V; Dunin-Horkawicz, Stanisław; Geniesse, Caleb; Kappel, Kalli; Kladwang, Wipapat; Krokhotin, Andrey; Łach, Grzegorz E; Major, François; Mann, Thomas H; Magnus, Marcin; Pachulska-Wieczorek, Katarzyna; Patel, Dinshaw J; Piccirilli, Joseph A; Popenda, Mariusz; Purzycka, Katarzyna J; Ren, Aiming; Rice, Greggory M; Santalucia, John; Sarzynska, Joanna; Szachniuk, Marta; Tandon, Arpit; Trausch, Jeremiah J; Tian, Siqi; Wang, Jian; Weeks, Kevin M; Williams, Benfeard; Xiao, Yi; Xu, Xiaojun; Zhang, Dong; Zok, Tomasz; Westhof, Eric

    2017-01-30

    RNA-Puzzles is a collective experiment in blind 3D RNA structure prediction. We report here a third round of RNA-Puzzles. Five puzzles, 4, 8, 12, 13, 14, all structures of riboswitch aptamers and puzzle 7, a ribozyme structure, are included in this round of the experiment. The riboswitch structures include biological binding sites for small molecules (S-adenosyl methionine, cyclic diadenosine monophosphate, 5-amino 4-imidazole carboxamide riboside 5'-triphosphate, glutamine) and proteins (YbxF) and one set describes large conformational changes between ligand-free and ligand-bound states; the Varkud satellite ribozyme is the most recently solved structure of a known large ribozyme. All the puzzles have established biological functions and require structural understanding to appreciate their molecular mechanisms. Through the use of fast-track experimental data, including multidimensional chemical mapping, and accurate prediction of RNA secondary structure, a large portion of the contacts in 3D have been predicted correctly leading to similar topologies for the top ranking predictions. Template-based and homology-derived predictions could predict structures to particularly high accuracies. However, achieving biological insights from de novo prediction of RNA 3D structures still depends on the size and complexity of the RNA. Blind computational predictions of RNA structures already appear to provide useful structural information in many cases. Similar to the previous RNA-Puzzles Round II experiment, the prediction of non-Watson-Crick interactions and the observed high atomic clash scores reveal notable need for algorithm of improvement. All prediction models and assessment results are available at http://ahsoka.u-strasbg.fr/rnapuzzles/.

  7. 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.

  8. Evolutionary rate variation and RNA secondary structure prediction

    DEFF Research Database (Denmark)

    Knudsen, B; Andersen, E S; Damgaard, Christian Kroun

    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...

  9. Predicting RNA secondary structures from sequence and probing data.

    Science.gov (United States)

    Lorenz, Ronny; Wolfinger, Michael T; Tanzer, Andrea; Hofacker, Ivo L

    2016-07-01

    RNA secondary structures have proven essential for understanding the regulatory functions performed by RNA such as microRNAs, bacterial small RNAs, or riboswitches. This success is in part due to the availability of efficient computational methods for predicting RNA secondary structures. Recent advances focus on dealing with the inherent uncertainty of prediction by considering the ensemble of possible structures rather than the single most stable one. Moreover, the advent of high-throughput structural probing has spurred the development of computational methods that incorporate such experimental data as auxiliary information.

  10. siRNA release from pri-miRNA scaffolds is controlled by the sequence and structure of RNA.

    Science.gov (United States)

    Galka-Marciniak, Paulina; Olejniczak, Marta; Starega-Roslan, Julia; Szczesniak, Michal W; Makalowska, Izabela; Krzyzosiak, Wlodzimierz J

    2016-04-01

    shmiRs are pri-miRNA-based RNA interference triggers from which exogenous siRNAs are expressed in cells to silence target genes. These reagents are very promising tools in RNAi in vivo applications due to their good activity profile and lower toxicity than observed for other vector-based reagents such as shRNAs. In this study, using high-resolution northern blotting and small RNA sequencing, we investigated the precision with which RNases Drosha and Dicer process shmiRs. The fidelity of siRNA release from the commonly used pri-miRNA shuttles was found to depend on both the siRNA insert and the pri-miR scaffold. Then, we searched for specific factors that may affect the precision of siRNA release and found that both the structural features of shmiR hairpins and the nucleotide sequence at Drosha and Dicer processing sites contribute to cleavage site selection and cleavage precision. An analysis of multiple shRNA intermediates generated from several reagents revealed the complexity of shmiR processing by Drosha and demonstrated that Dicer selects substrates for further processing. Aside from providing new basic knowledge regarding the specificity of nucleases involved in miRNA biogenesis, our results facilitate the rational design of more efficient genetic reagents for RNAi technology.

  11. Control of chromatin structure by long noncoding RNA

    Science.gov (United States)

    Böhmdorfer, Gudrun; Wierzbicki, Andrzej T.

    2015-01-01

    Long noncoding RNA (lncRNA) is a pivotal factor regulating various aspects of genome activity. Genome regulation via DNA methylation and posttranslational histone modifications is a well-documented function of lncRNA in plants, fungi, and animals. Here, we summarize evidence showing that lncRNA also controls chromatin structure including nucleosome positioning and chromosome looping. We focus on data from plant experimental systems, discussed in the context of other eukaryotes. We explain the mechanisms of lncRNA-controlled chromatin remodeling and the implications of the functional interplay between noncoding transcription and several different chromatin remodelers. We propose that the unique properties of RNA make it suitable for controlling chromatin modifications and structure. PMID:26410408

  12. 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.

  13. The crystal structure of E. coli rRNA pseudouridine synthase RluE.

    Science.gov (United States)

    Pan, Hu; Ho, Joseph D; Stroud, Robert M; Finer-Moore, Janet

    2007-04-13

    Pseudouridine synthase RluE modifies U2457 in a stem of 23 S RNA in Escherichia coli. This modification is located in the peptidyl transferase center of the ribosome. We determined the crystal structures of the C-terminal, catalytic domain of E. coli RluE at 1.2 A resolution and of full-length RluE at 1.6 A resolution. The crystals of the full-length enzyme contain two molecules in the asymmetric unit and in both molecules the N-terminal domain is disordered. The protein has an active site cleft, conserved in all other pseudouridine synthases, that contains invariant Asp and Tyr residues implicated in catalysis. An electropositive surface patch that covers the active site cleft is just wide enough to accommodate an RNA stem. The RNA substrate stem can be docked to this surface such that the catalytic Asp is adjacent to the target base, and a conserved Arg is positioned to help flip the target base out of the stem into the enzyme active site. A flexible RluE specific loop lies close to the conserved region of the stem in the model, and may contribute to substrate specificity. The stem alone is not a good RluE substrate, suggesting RluE makes additional interactions with other regions in the ribosome.

  14. Combinatorics of RNA Secondary Structures with Base Triples.

    Science.gov (United States)

    Müller, Robert; Nebel, Markus E

    2015-07-01

    The structure of RNA has been the subject of intense research over the last decades due to its importance for the correct functioning of RNA molecules in biological processes. Hence, a large number of models for RNA folding and corresponding algorithms for structure prediction have been developed. However, previous models often only consider base pairs, although every base is capable of up to three edge-to-edge interactions with other bases. Recently, Höner zu Siederdissen et al. presented an extended model of RNA secondary structure, including base triples together with a folding algorithm-the first thermodynamics-based algorithm that allows the prediction of secondary structures with base triples. In this article, we investigate the search space processed by this new algorithm, that is, the combinatorics of extended RNA secondary structures with base triples. We present generalized definitions for structural motifs like hairpins, stems, bulges, or interior loops occurring in structures with base triples. Furthermore, we prove precise asymptotic results for the number of different structures (size of search space) and expectations for various parameters associated with structural motifs (typical shape of folding). Our analysis shows that the asymptotic number of secondary structures of size n increases exponentially to [Formula: see text] compared to the classic model by Stein and Waterman for which [Formula: see text] structures exist. A comparison with the classic model reveals large deviations in the expected structural appearance, too. The inclusion of base triples constitutes a significant refinement of the combinatorial model of RNA secondary structure, which, by our findings, is quantitatively characterized. Our results are of special theoretical interest, because a closer look at the numbers involved suggests that extended RNA secondary structures constitute a new combinatorial class not bijective with any other combinatorial objects studied so far.

  15. Alterations in Lipoxygenase and Cyclooxygenase-2 Catalytic Activity and mRNA Expression in Prostate Carcinoma

    Directory of Open Access Journals (Sweden)

    Scott B. Shappell

    2001-01-01

    Full Text Available Recent studies in prostate tissues and especially cell lines have suggested roles for arachidonic acid (AA metabolizing enzymes in prostate adenocarcinoma (Pca development or progression. The goal of this study was to more fully characterize lipoxygenase (LOX and cyclooxygenase-2 (COX-2 gene expression and AA metabolism in benign and malignant prostate using snap-frozen tissues obtained intraoperatively and mRNA analyses and enzyme assays. Formation of 15-hydroxyeicosatetraenoic acid (15-HETE was detected in 23/29 benign samples and 15-LOX-2 mRNA was detected in 21/25 benign samples. In pairs of pure benign and Pca from the same patients, 15-HETE production and 15-LOX-2 mRNA were reduced in Pca versus benign in 9/14 (P=.04 and 14/17 (P=.002, respectively. Under the same conditions, neither 5HETE nor 12-HETE formation was detectable in 29 benign and 24 tumor samples; with a more sensitive assay, traces were detected in some samples, but there was no clear association with tumor tissue. COX-2 mRNA was detected by nuclease protection assay in 7/16 benign samples and 5/16 tumors. In benign and tumor pairs from 10 patients, COX-2 was higher in tumor versus benign in only 2, with similar results by in situ hybridization. Paraffin immunoperoxidase for COX2 was performed in whole mount sections from 87 additional radical prostatectomy specimens, with strong expression in ejaculatory duct as a positive control and corroboration with in situ hybridization. No immunostaining was detected in benign prostate or tumor in 45% of cases. Greater immunostaining in tumor versus benign was present in only 17% of cases, and correlated with high tumor grade (Gleason score 8 and 9 vs. 5 to 7. In conclusion, reduced 15-LOX-2 expression and 15-HETE formation is the most characteristic alteration of AA metabolism in Pca. Increased 12-HETE and 5-HETE formation in Pca were not discernible. Increased COX-2 expression is not a typical abnormality in Pca in general, but

  16. Alterations in lipoxygenase and cyclooxygenase-2 catalytic activity and mRNA expression in prostate carcinoma.

    Science.gov (United States)

    Shappell, S B; Manning, S; Boeglin, W E; Guan, Y F; Roberts, R L; Davis, L; Olson, S J; Jack, G S; Coffey, C S; Wheeler, T M; Breyer, M D; Brash, A R

    2001-01-01

    Recent studies in prostate tissues and especially cell lines have suggested roles for arachidonic acid (AA) metabolizing enzymes in prostate adenocarcinoma (Pca) development or progression. The goal of this study was to more fully characterize lipoxygenase (LOX) and cyclooxygenase-2 (COX-2) gene expression and AA metabolism in benign and malignant prostate using snap-frozen tissues obtained intraoperatively and mRNA analyses and enzyme assays. Formation of 15-hydroxyeicosatetraenoic acid (15-HETE) was detected in 23/29 benign samples and 15-LOX-2 mRNA was detected in 21/25 benign samples. In pairs of pure benign and Pca from the same patients, 15-HETE production and 15-LOX-2 mRNA were reduced in Pca versus benign in 9/14 (P=.04) and 14/17 (P=.002), respectively. Under the same conditions, neither 5-HETE nor 12-HETE formation was detectable in 29 benign and 24 tumor samples; with a more sensitive assay, traces were detected in some samples, but there was no clear association with tumor tissue. COX-2 mRNA was detected by nuclease protection assay in 7/16 benign samples and 5/16 tumors. In benign and tumor pairs from 10 patients, COX-2 was higher in tumor versus benign in only 2, with similar results by in situ hybridization. Paraffin immunoperoxidase for COX-2 was performed in whole mount sections from 87 additional radical prostatectomy specimens, with strong expression in ejaculatory duct as a positive control and corroboration with in situ hybridization. No immunostaining was detected in benign prostate or tumor in 45% of cases. Greater immunostaining in tumor versus benign was present in only 17% of cases, and correlated with high tumor grade (Gleason score 8 and 9 vs. 5 to 7). In conclusion, reduced 15-LOX-2 expression and 15-HETE formation is the most characteristic alteration of AA metabolism in Pca. Increased 12-HETE and 5-HETE formation in Pca were not discernible. Increased COX-2 expression is not a typical abnormality in Pca in general, but occurs in high

  17. Aged nano-structured platinum based catalyst: effect of chemical treatment on adsorption and catalytic activity.

    Science.gov (United States)

    Shim, Wang Geun; Nahm, Seung Won; Park, Hyuk Ryeol; Yun, Hyung Sun; Seo, Seong Gyu; Kim, Sang Chai

    2011-02-01

    To examine the effect of chemical treatment on the adsorption and catalytic activity of nanostructured platinum based catalyst, the aged commercial Pt/AC catalyst was pretreated with sulfuric acid (H2SO4) and a cleaning agent (Hexane). Several reliable methods such as nitrogen adsorption, X-ray powder diffraction (XRD), scanning electron microscopy (SEM) and inductively coupled plasma (ICP) were employed to characterize the aged Pt/AC catalyst and its chemically pretreated Pt/AC catalysts. The catalytic and adsorption activities of nano-structured heterogeneous Pt/AC catalyst were investigated on the basis of toluene oxidation and adsorption isotherm data. In addition, the adsorption isotherms of toluene were used to calculate the adsorption energy distribution functions for the parent catalyst and its pre-treated nano-structured Pt/AC catalysts. It was found that sulfuric acid aqueous treatment can enhance the catalytic performance of aged Pt/AC catalyst toward catalytic oxidation of toluene. It was also shown that a comparative analysis of the energy distribution functions for nano-structured Pt/AC catalysts as well as the pore size distribution provides valuable information about their structural and energetic heterogeneity.

  18. Effect of Alkali Treatment on the Structure and Catalytic Properties of ZSM-5 Zeolite

    Institute of Scientific and Technical Information of China (English)

    Zi Yun LI; Xin SUN; Qiang XIAO; Shou He XIANG

    2005-01-01

    Catalytic properties of ZSM-5 zeolite samples pretreated with NaOH solution have been investigated. The samples are characterized by XRD, SEM, chemical analysis, and N2 adsorption.The results indicate that mesopores are created in ZSM-5 crystals under alkali treatment without change the microporous structure and acidic strength of the zeolite, but the crystallinity is greatly decreased under severe treatment. IR indicates that the concentration of silanol is greatly enriched by alkali treatment. The etherification activities of ZSM-5 zeolites are greatly increased by alkali-treatment. The noticeably improved catalytic activity of treated samples is ascribed to the formation of mesopores and greatly enriched silanol group.

  19. A catalytic intermediate and several flavin redox states stabilized by folate-dependent tRNA methyltransferase from Bacillus subtilis.

    Science.gov (United States)

    Hamdane, Djemel; Guerineau, Vincent; Un, Sun; Golinelli-Pimpaneau, Beatrice

    2011-06-14

    The flavoprotein TrmFO catalyzes the C5 methylation of uridine 54 in the TΨC loop of tRNAs using 5,10-methylenetetrahydrofolate (CH(2)THF) as a methylene donor and FAD as a reducing agent. Here, we report biochemical and spectroscopic studies that unravel the remarkable capability of Bacillus subtilis TrmFO to stabilize, in the presence of oxygen, several flavin-reduced forms, including an FADH(•) radical, and a catalytic intermediate endowed with methylating activity. The FADH(•) radical was characterized by high-field electron paramagnetic resonance and electron nuclear double-resonance spectroscopies. Interestingly, the enzyme exhibited tRNA methylation activity in the absence of both an added carbon donor and an external reducing agent, indicating that a reaction intermediate, containing presumably CH(2)THF and FAD hydroquinone, is present in the freshly purified enzyme. Isolation by acid treatment, under anaerobic conditions, of noncovalently bound molecules, followed by mass spectrometry analysis, confirmed the presence in TrmFO of nonmodified FAD. Addition of formaldehyde to the purified enzyme protects the reduced flavins from decay by probably preventing degradation of CH(2)THF. The absence of air-stable reduced FAD species during anaerobic titration of oxidized TrmFO, performed in the absence or presence of added CH(2)THF, argues against their thermodynamic stabilization but rather implicates their kinetic trapping by the enzyme. Altogether, the unexpected isolation of a stable catalytic intermediate suggests that the flavin-binding pocket of TrmFO is a highly insulated environment, diverting the reduced FAD present in this intermediate from uncoupled reactions.

  20. 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

  1. Crystal structures of trypanosomal histidyl-tRNA synthetase illuminate differences between eukaryotic and prokaryotic homologs.

    Science.gov (United States)

    Merritt, Ethan A; Arakaki, Tracy L; Gillespie, J Robert; Larson, Eric T; Kelley, Angela; Mueller, Natascha; Napuli, Alberto J; Kim, Jessica; Zhang, Li; Verlinde, Christophe L M J; Fan, Erkang; Zucker, Frank; Buckner, Frederick S; van Voorhis, Wesley C; Hol, Wim G J

    2010-03-26

    Crystal structures of histidyl-tRNA synthetase (HisRS) from the eukaryotic parasites Trypanosoma brucei and Trypanosoma cruzi provide a first structural view of a eukaryotic form of this enzyme and reveal differences from bacterial homologs. HisRSs in general contain an extra domain inserted between conserved motifs 2 and 3 of the Class II aminoacyl-tRNA synthetase catalytic core. The current structures show that the three-dimensional topology of this domain is very different in bacterial and archaeal/eukaryotic forms of the enzyme. Comparison of apo and histidine-bound trypanosomal structures indicates substantial active-site rearrangement upon histidine binding but relatively little subsequent rearrangement after reaction of histidine with ATP to form the enzyme's first reaction product, histidyladenylate. The specific residues involved in forming the binding pocket for the adenine moiety differ substantially both from the previously characterized binding site in bacterial structures and from the homologous residues in human HisRSs. The essentiality of the single HisRS gene in T. brucei is shown by a severe depression of parasite growth rate that results from even partial suppression of expression by RNA interference.

  2. Structural basis for full-spectrum inhibition of translational functions on a tRNA synthetase

    Science.gov (United States)

    Fang, Pengfei; Yu, Xue; Jeong, Seung Jae; Mirando, Adam; Chen, Kaige; Chen, Xin; Kim, Sunghoon; Francklyn, Christopher S.; Guo, Min

    2015-01-01

    The polyketide natural product borrelidin displays antibacterial, antifungal, antimalarial, anticancer, insecticidal and herbicidal activities through the selective inhibition of threonyl-tRNA synthetase (ThrRS). How borrelidin simultaneously attenuates bacterial growth and suppresses a variety of infections in plants and animals is not known. Here we show, using X-ray crystal structures and functional analyses, that a single molecule of borrelidin simultaneously occupies four distinct subsites within the catalytic domain of bacterial and human ThrRSs. These include the three substrate-binding sites for amino acid, ATP and tRNA associated with aminoacylation, and a fourth ‘orthogonal’ subsite created as a consequence of binding. Thus, borrelidin competes with all three aminoacylation substrates, providing a potent and redundant mechanism to inhibit ThrRS during protein synthesis. These results highlight a surprising natural design to achieve the quadrivalent inhibition of translation through a highly conserved family of enzymes. PMID:25824639

  3. Revealing structural dynamics in catalytic reactions using ultrafast transient x-ray absorption spectroscopy.

    Energy Technology Data Exchange (ETDEWEB)

    Chen, L. X.; Liu, D.; Chemical Sciences and Engineering Division; Northwestern Univ.

    2009-03-02

    Progression of a typical heterogeneous catalytic process as a function of a reaction parameter such as temperature can often be segmented, according to Fig. 1. Reactants first bind to active sites to form reaction intermediates with increases in temperature and kinetic energy (Region I). Further temperature increase leads to a full 'light-off' of the catalytic conversion and the reaction is dominated by the intra-particulate diffusion (Region II). The characteristic 'light-off' temperature at the boundary of region I and II often defines the activity of the catalyst. At even higher temperature enters the bulk diffusion region (III), where the catalytic reaction is limited mainly by mass transport between different phases. Significant efforts in practical catalyst design involve improving catalytic activities in the kinetic region (I) and reducing the 'light-off' temperature. Reaction rates at the kinetic region are defined by potential saddle points on top of which a series of 'transitional state' complexes are formed between the active sites and the adsorbed reactants (Fig. 2). Capturing structures of the 'transition state complexes' from the active center's perspective will provide ultimate understanding of catalytic mechanisms and insight into new catalyst design. Experimentally, however, it is a very challenging proposition.

  4. Pore Structure and Catalytic Performance of Steam-Dealuminated ZSM-5/Y Composite Zeolites

    Institute of Scientific and Technical Information of China (English)

    GuoJintao; ShenBaojian; ChenHonglin

    2005-01-01

    For investigating the effect of dealumination on the pore structure and catalytic performance, ZSM-5/Y composite zeolites synthesized in situ from NaY gel were dealuminated by steaming at different temperatures. XRD (X-ray diffraction) characterization indicates that the relative crystallinity of the composite zeolites decreases with the increase in Si/Al ratio after steaming. N2 adsorption-desorption suggests that more mesopores are formed while the BET(Brunauer, Emmett and Teller) specific surface area and the micropore specific surface area decrease as the temperature of steaming rises. Daqing heavy oil was used as feedstock to test the catalytic cracking activity of ZSM-5/Y composite zeolites. The experimental results of the catalytic cracking performance reveal that the distribution of products differs due to the different conditions of hydrothermal treatment. Further hydrothermal treatment leads to an increase in the yield of light oil, and a decrease in the yield of gas products and coke.

  5. The tRNA Elbow in Structure, Recognition and Evolution

    Directory of Open Access Journals (Sweden)

    Jinwei Zhang

    2016-01-01

    Full Text Available Prominent in the L-shaped three-dimensional structure of tRNAs is the “elbow” where their two orthogonal helical stacks meet. It has a conserved structure arising from the interaction of the terminal loops of the D- and T-stem-loops, and presents to solution a flat face of a tertiary base pair between the D- and T-loops. In addition to the ribosome, which interacts with the elbow in all three of its tRNA binding sites, several cellular RNAs and many proteins are known to recognize the elbow. At least three classes of non-coding RNAs, namely 23S rRNA, ribonuclease P, and the T-box riboswitches, recognize the tRNA elbow employing an identical structural motif consisting of two interdigitated T-loops. In contrast, structural solutions to tRNA-elbow recognition by proteins are varied. Some enzymes responsible for post-transcriptional tRNA modification even disrupt the elbow structure in order to access their substrate nucleotides. The evolutionary origin of the elbow is mysterious, but, because it does not explicitly participate in the flow of genetic information, it has been proposed to be a late innovation. Regardless, it is biologically essential. Even some viruses that hijack the cellular machinery using tRNA decoys have convergently evolved near-perfect mimics of the tRNA elbow.

  6. Dissecting structural basis of the unique substrate selectivity of human enteropeptidase catalytic subunit.

    Science.gov (United States)

    Ostapchenko, Valeriy G; Gasparian, Marine E; Kosinsky, Yurij A; Efremov, Roman G; Dolgikh, Dmitry A; Kirpichnikov, Mikhail P

    2012-01-01

    Enteropeptidase is a key enzyme in the digestion system of higher animals. It initiates enzymatic cascade cleaving trypsinogen activation peptide after a unique sequence DDDDK. Recently, we have found specific activity of human enteropeptidase catalytic subunit (L-HEP) being significantly higher than that of its bovine ortholog (L-BEP). Moreover, we have discovered that L-HEP hydrolyzed several nonspecific peptidic substrates. In this work, we aimed to further characterize species-specific enteropeptidase activities and to reveal their structural basis. First, we compared hydrolysis of peptides and proteins lacking DDDDK sequence by L-HEP and L-BEP. In each case human enzyme was more efficient, with the highest hydrolysis rate observed for substrates with a large hydrophobic residue in P2-position. Computer modeling suggested enzyme exosite residues 96 (Arg in L-HEP, Lys in L-BEP) and 219 (Lys in L-HEP, Gln in L-BEP) to be responsible for these differences in enteropeptidase catalytic activity. Indeed, human-to-bovine mutations Arg96Lys, Lys219Gln shifted catalytic properties of L-HEP toward those of L-BEP. This effect was amplified in case of the double mutation Arg96Lys/Lys219Gln, but still did not cover the full difference in catalytic activities of human and bovine enzymes. To find a missing link, we studied monopeptide benzyl-arginine-β-naphthylamide hydrolysis. L-HEP catalyzed it with an order lower K (m) than L-BEP, suggesting the monopeptide-binding S1 site input into catalytic distinction between two enteropeptidase species. Together, our findings suggest structural basis of the unique catalytic properties of human enteropeptidase and instigate further studies of its tentative physiological and pathological roles.

  7. Crystal structure of Zika virus NS5 RNA-dependent RNA polymerase

    Science.gov (United States)

    Godoy, Andre S.; Lima, Gustavo M. A.; Oliveira, Ketllyn I. Z.; Torres, Naiara U.; Maluf, Fernando V.; Guido, Rafael V. C.; Oliva, Glaucius

    2017-01-01

    The current Zika virus (ZIKV) outbreak became a global health threat of complex epidemiology and devastating neurological impacts, therefore requiring urgent efforts towards the development of novel efficacious and safe antiviral drugs. Due to its central role in RNA viral replication, the non-structural protein 5 (NS5) RNA-dependent RNA-polymerase (RdRp) is a prime target for drug discovery. Here we describe the crystal structure of the recombinant ZIKV NS5 RdRp domain at 1.9 Å resolution as a platform for structure-based drug design strategy. The overall structure is similar to other flaviviral homologues. However, the priming loop target site, which is suitable for non-nucleoside polymerase inhibitor design, shows significant differences in comparison with the dengue virus structures, including a tighter pocket and a modified local charge distribution. PMID:28345596

  8. Structure of the ATP Synthase Catalytic Complex (F1) from Escherichia coli in an Autoinhibited conformation

    Energy Technology Data Exchange (ETDEWEB)

    G Cingolani; T Duncan

    2011-12-31

    ATP synthase is a membrane-bound rotary motor enzyme that is critical for cellular energy metabolism in all kingdoms of life. Despite conservation of its basic structure and function, autoinhibition by one of its rotary stalk subunits occurs in bacteria and chloroplasts but not in mitochondria. The crystal structure of the ATP synthase catalytic complex (F{sub 1}) from Escherichia coli described here reveals the structural basis for this inhibition. The C-terminal domain of subunit {var_epsilon} adopts a heretofore unknown, highly extended conformation that inserts deeply into the central cavity of the enzyme and engages both rotor and stator subunits in extensive contacts that are incompatible with functional rotation. As a result, the three catalytic subunits are stabilized in a set of conformations and rotational positions distinct from previous F{sub 1} structures.

  9. Structural organizations of yeast RNase P and RNase MRP holoenzymes as revealed by UV-crosslinking studies of RNA-protein interactions.

    Science.gov (United States)

    Khanova, Elena; Esakova, Olga; Perederina, Anna; Berezin, Igor; Krasilnikov, Andrey S

    2012-04-01

    Eukaryotic ribonuclease (RNase) P and RNase MRP are closely related ribonucleoprotein complexes involved in the metabolism of various RNA molecules including tRNA, rRNA, and some mRNAs. While evolutionarily related to bacterial RNase P, eukaryotic enzymes of the RNase P/MRP family are much more complex. Saccharomyces cerevisiae RNase P consists of a catalytic RNA component and nine essential proteins; yeast RNase MRP has an RNA component resembling that in RNase P and 10 essential proteins, most of which are shared with RNase P. The structural organizations of eukaryotic RNases P/MRP are not clear. Here we present the results of RNA-protein UV crosslinking studies performed on RNase P and RNase MRP holoenzymes isolated from yeast. The results indicate locations of specific protein-binding sites in the RNA components of RNase P and RNase MRP and shed light on the structural organizations of these large ribonucleoprotein complexes.

  10. 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.

  11. Coarse-grained modeling of RNA 3D structure.

    Science.gov (United States)

    Dawson, Wayne K; Maciejczyk, Maciej; Jankowska, Elzbieta J; Bujnicki, Janusz M

    2016-07-01

    Functional RNA molecules depend on three-dimensional (3D) structures to carry out their tasks within the cell. Understanding how these molecules interact to carry out their biological roles requires a detailed knowledge of RNA 3D structure and dynamics as well as thermodynamics, which strongly governs the folding of RNA and RNA-RNA interactions as well as a host of other interactions within the cellular environment. Experimental determination of these properties is difficult, and various computational methods have been developed to model the folding of RNA 3D structures and their interactions with other molecules. However, computational methods also have their limitations, especially when the biological effects demand computation of the dynamics beyond a few hundred nanoseconds. For the researcher confronted with such challenges, a more amenable approach is to resort to coarse-grained modeling to reduce the number of data points and computational demand to a more tractable size, while sacrificing as little critical information as possible. This review presents an introduction to the topic of coarse-grained modeling of RNA 3D structures and dynamics, covering both high- and low-resolution strategies. We discuss how physics-based approaches compare with knowledge based methods that rely on databases of information. In the course of this review, we discuss important aspects in the reasoning process behind building different models and the goals and pitfalls that can result.

  12. FRASS: the web-server for RNA structural comparison

    Directory of Open Access Journals (Sweden)

    Carugo Oliviero

    2010-06-01

    Full Text Available Abstract Background The impressive increase of novel RNA structures, during the past few years, demands automated methods for structure comparison. While many algorithms handle only small motifs, few techniques, developed in recent years, (ARTS, DIAL, SARA, SARSA, and LaJolla are available for the structural comparison of large and intact RNA molecules. Results The FRASS web-server represents a RNA chain with its Gauss integrals and allows one to compare structures of RNA chains and to find similar entries in a database derived from the Protein Data Bank. We observed that FRASS scores correlate well with the ARTS and LaJolla similarity scores. Moreover, the-web server can also reproduce satisfactorily the DARTS classification of RNA 3D structures and the classification of the SCOR functions that was obtained by the SARA method. Conclusions The FRASS web-server can be easily used to detect relationships among RNA molecules and to scan efficiently the rapidly enlarging structural databases.

  13. Structure of a functional ribonucleoprotein pseudouridine synthase bound to a substrate RNA

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Bo; Zhou, Jing; Kahen, Elliot; Terns, Rebecca M.; Terns, Michael P.; Li, Hong; (Inst. Mol. BioScience); (FSU); (Georgia)

    2009-09-29

    Box H/ACA small nucleolar and Cajal body ribonucleoprotein particles comprise the most complex pseudouridine synthases and are essential for ribosome and spliceosome maturation. The multistep and multicomponent-mediated enzyme mechanism remains only partially understood. Here we report a crystal structure at 2.35 {angstrom} of a substrate-bound functional archaeal enzyme containing three of the four proteins, Cbf5, Nop10 and L7Ae, and a box H/ACA RNA that reveals detailed information about the protein-only active site. The substrate RNA, containing 5-fluorouridine at the modification position, is fully docked and catalytically rearranged by the enzyme in a manner similar to that seen in two stand-alone pseudouridine synthases. Structural analysis provides a mechanism for plasticity in the diversity of guide RNA sequences used and identifies a substrate-anchoring loop of Cbf5 that also interacts with Gar1 in unliganded structures. Activity analyses of mutated proteins and RNAs support the structural findings and further suggest a role of the Cbf5 loop in regulation of enzyme activity.

  14. Structure of a functional ribonucleoprotein pseudouridine synthase bound to a substrate RNA.

    Science.gov (United States)

    Liang, Bo; Zhou, Jing; Kahen, Elliot; Terns, Rebecca M; Terns, Michael P; Li, Hong

    2009-07-01

    Box H/ACA small nucleolar and Cajal body ribonucleoprotein particles comprise the most complex pseudouridine synthases and are essential for ribosome and spliceosome maturation. The multistep and multicomponent-mediated enzyme mechanism remains only partially understood. Here we report a crystal structure at 2.35 A of a substrate-bound functional archaeal enzyme containing three of the four proteins, Cbf5, Nop10 and L7Ae, and a box H/ACA RNA that reveals detailed information about the protein-only active site. The substrate RNA, containing 5-fluorouridine at the modification position, is fully docked and catalytically rearranged by the enzyme in a manner similar to that seen in two stand-alone pseudouridine synthases. Structural analysis provides a mechanism for plasticity in the diversity of guide RNA sequences used and identifies a substrate-anchoring loop of Cbf5 that also interacts with Gar1 in unliganded structures. Activity analyses of mutated proteins and RNAs support the structural findings and further suggest a role of the Cbf5 loop in regulation of enzyme activity.

  15. 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.

  16. The structure of the RNA m5C methyltransferase YebU from Escherichia coli reveals a C-terminal RNA-recruiting PUA domain.

    Science.gov (United States)

    Hallberg, B Martin; Ericsson, Ulrika B; Johnson, Kenneth A; Andersen, Niels Møller; Douthwaite, Stephen; Nordlund, Pär; Beuscher, Albert E; Erlandsen, Heidi

    2006-07-21

    Nucleotide methylations are the most common type of rRNA modification in bacteria, and are introduced post-transcriptionally by a wide variety of site-specific enzymes. Three 5-methylcytidine (m(5)C) bases are found in the rRNAs of Escherichia coli and one of these, at nucleotide 1407 in 16 S rRNA, is the modification product of the methyltransferase (MTase) YebU (also called RsmF). YebU requires S-adenosyl-l-methionine (SAM) and methylates C1407 within assembled 30 S subunits, but not in naked 16 S rRNA or within tight-couple 70 S ribosomes. Here, we describe the three-dimensional structure of YebU determined 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 RsmB and PH1374. The C-terminal one-third of YebU contains a domain similar to that in pseudouridine synthases and archaeosine-specific transglycosylases (PUA-domain), which was not predicted by sequence alignments. Furthermore, YebU is predicted to contain extended regions of positive electrostatic 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.

  17. Insights into tRNA-Dependent Amidotransferase Evolution and Catalysis from the Structure of the Aquifex aeolicus Enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Jing; Bu, Weishu; Sheppard, Kelly; Kitabatake, Makoto; Kwon, Suk-Tae; Söll, Dieter; Smith, Janet L.; (Yale); (Michigan); (Kyoto)

    2010-08-17

    Many bacteria form Gln-tRNA{sup Gln} and Asn-tRNA{sup ASN} by conversion of the misacylated Glu-tRNA{sup Gln} and Asp-tRNA{sup ASN} species catalyzed by the GatCAB amidotransferase in the presence of ATP and an amide donor (glutamine or asparagine). Here, we report the crystal structures of GatCAB from the hyperthermophilic bacterium Aquifex aeolicus, complexed with glutamine, asparagine, aspartate, ADP, or ATP. In contrast to the Staphylococcus aureus GatCAB, the A. aeolicus enzyme formed acyl-enzyme intermediates with either glutamine or asparagine, in line with the equally facile use by the amidotransferase of these amino acids as amide donors in the transamidation reaction. A water-filled ammonia channel is open throughout the length of the A. aeolicus GatCAB from the GatA active site to the synthetase catalytic pocket in the B-subunit. A non-catalytic Zn{sup 2+} site in the A. aeolicus GatB stabilizes subunit contacts and the ammonia channel. Judged from sequence conservation in the known GatCAB sequences, the Zn{sup 2+} binding motif was likely present in the primordial GatB/E, but became lost in certain lineages (e.g., S. aureus GatB). Two divalent metal binding sites, one permanent and the other transient, are present in the catalytic pocket of the A. aeolicus GatB. The two sites enable GatCAB to first phosphorylate the misacylated tRNA substrate and then amidate the activated intermediate to form the cognate products, Gln-tRNA{sup Gln} or Asn-tRNA{sup ASN}.

  18. Nanostructured, mesoporous Au/TiO2 model catalysts – structure, stability and catalytic properties

    Directory of Open Access Journals (Sweden)

    Matthias Roos

    2011-09-01

    Full Text Available Aiming at model systems with close-to-realistic transport properties, we have prepared and studied planar Au/TiO2 thin-film model catalysts consisting of a thin mesoporous TiO2 film of 200–400 nm thickness with Au nanoparticles, with a mean particle size of ~2 nm diameter, homogeneously distributed therein. The systems were prepared by spin-coating of a mesoporous TiO2 film from solutions of ethanolic titanium tetraisopropoxide and Pluronic P123 on planar Si(100 substrates, calcination at 350 °C and subsequent Au loading by a deposition–precipitation procedure, followed by a final calcination step for catalyst activation. The structural and chemical properties of these model systems were characterized by X-ray diffraction (XRD, transmission electron microscopy (TEM, N2 adsorption, inductively coupled plasma ionization spectroscopy (ICP–OES and X-ray photoelectron spectroscopy (XPS. The catalytic properties were evaluated through the oxidation of CO as a test reaction, and reactivities were measured directly above the film with a scanning mass spectrometer. We can demonstrate that the thin-film model catalysts closely resemble dispersed Au/TiO2 supported catalysts in their characteristic structural and catalytic properties, and hence can be considered as suitable for catalytic model studies. The linear increase of the catalytic activity with film thickness indicates that transport limitations inside the Au/TiO2 film catalyst are negligible, i.e., below the detection limit.

  19. The 5'-3' distance of RNA secondary structures

    CERN Document Server

    Han, Hillary S W

    2011-01-01

    Recently Yoffe {\\it et al.} \\citep{Yoffe} observed that the average distances between 5'-3' ends of RNA molecules are very small and largely independent of sequence length. This observation is based on numerical computations as well as theoretical arguments maximizing certain entropy functionals. In this paper we compute the exact distribution of 5'-3' distances of RNA secondary structures for any finite $n$. We furthermore compute the limit distribution and show that already for $n=30$ the exact distribution and the limit distribution are very close. Our results show that the distances of random RNA secondary structures are distinctively lower than those of minimum free energy structures of random RNA sequences.

  20. Structural characterization of the catalytic site of a Nilaparvata lugens delta-class glutathione transferase.

    Science.gov (United States)

    Yamamoto, Kohji; Higashiura, Akifumi; Hossain, Md Tofazzal; Yamada, Naotaka; Shiotsuki, Takahiro; Nakagawa, Atsushi

    2015-01-15

    Glutathione transferases (GSTs) are a major class of detoxification enzymes that play a central role in the defense against environmental toxicants and oxidative stress. Here, we studied the crystal structure of a delta-class glutathione transferase from Nilaparvata lugens, nlGSTD, to gain insights into its catalytic mechanism. The structure of nlGSTD in complex with glutathione, determined at a resolution of 1.7Å, revealed that it exists as a dimer and its secondary and tertiary structures are similar to those of other delta-class GSTs. Analysis of a complex between nlGSTD and glutathione showed that the bound glutathione was localized to the glutathione-binding site. Site-directed mutagenesis of nlGSTD mutants indicated that amino acid residues Ser11, His52, Glu66, and Phe119 contribute to catalytic activity.

  1. Thermodynamics of RNA structures by Wang–Landau sampling

    Science.gov (United States)

    Lou, Feng; Clote, Peter

    2010-01-01

    Motivation: Thermodynamics-based dynamic programming RNA secondary structure algorithms have been of immense importance in molecular biology, where applications range from the detection of novel selenoproteins using expressed sequence tag (EST) data, to the determination of microRNA genes and their targets. Dynamic programming algorithms have been developed to compute the minimum free energy secondary structure and partition function of a given RNA sequence, the minimum free-energy and partition function for the hybridization of two RNA molecules, etc. However, the applicability of dynamic programming methods depends on disallowing certain types of interactions (pseudoknots, zig-zags, etc.), as their inclusion renders structure prediction an nondeterministic polynomial time (NP)-complete problem. Nevertheless, such interactions have been observed in X-ray structures. Results: A non-Boltzmannian Monte Carlo algorithm was designed by Wang and Landau to estimate the density of states for complex systems, such as the Ising model, that exhibit a phase transition. In this article, we apply the Wang-Landau (WL) method to compute the density of states for secondary structures of a given RNA sequence, and for hybridizations of two RNA sequences. Our method is shown to be much faster than existent software, such as RNAsubopt. From density of states, we compute the partition function over all secondary structures and over all pseudoknot-free hybridizations. The advantage of the WL method is that by adding a function to evaluate the free energy of arbitary pseudoknotted structures and of arbitrary hybridizations, we can estimate thermodynamic parameters for situations known to be NP-complete. This extension to pseudoknots will be made in the sequel to this article; in contrast, the current article describes the WL algorithm applied to pseudoknot-free secondary structures and hybridizations. Availability: The WL RNA hybridization web server is under construction at http

  2. Novel Biochemical Tools for Probing HIV RNA Structure.

    Science.gov (United States)

    Rausch, Jason W; Sztuba-Solinska, Joanna; Lusvarghi, Sabrina; Le Grice, Stuart F J

    2016-01-01

    Functional analysis of viral RNA requires knowledge of secondary structure arrangements and tertiary base interactions. Thus, high-throughput and comprehensive methods for assessing RNA structure are highly desirable. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) has proven highly useful for modeling the secondary structures of HIV and other retroviral RNAs in recent years. This technology is not without its limitations however, as SHAPE data can be severely compromised when the RNA under study is structurally heterogeneous. In addition, the method reveals little information regarding the three-dimensional (3D) organization of an RNA. This chapter outlines four detailed SHAPE-related methodologies that circumvent these limitations. "Ensemble" and "in-gel" variations of SHAPE permit structural analysis of individual conformers within structurally heterogeneous mixtures of RNA, while probing strategies that utilize "through-space" cleavage reagents such as methidiumpropyl-EDTA (MPE) and peptides appended with an ATCUN (amino terminal copper/nickel binding motif) can provide insight into 3D organization. Combinational application of these techniques provides a formidable arsenal for exploring the structures of HIV RNAs and associated nucleoprotein complexes.

  3. Computational analysis of RNA structures with chemical probing data.

    Science.gov (United States)

    Ge, Ping; Zhang, Shaojie

    2015-06-01

    RNAs play various roles, not only as the genetic codes to synthesize proteins, but also as the direct participants of biological functions determined by their underlying high-order structures. Although many computational methods have been proposed for analyzing RNA structures, their accuracy and efficiency are limited, especially when applied to the large RNAs and the genome-wide data sets. Recently, advances in parallel sequencing and high-throughput chemical probing technologies have prompted the development of numerous new algorithms, which can incorporate the auxiliary structural information obtained from those experiments. Their potential has been revealed by the secondary structure prediction of ribosomal RNAs and the genome-wide ncRNA function annotation. In this review, the existing probing-directed computational methods for RNA secondary and tertiary structure analysis are discussed.

  4. Structure of the catalytic domain of Plasmodium falciparum ARF GTPase-activating protein (ARFGAP)

    Energy Technology Data Exchange (ETDEWEB)

    Cook, William J.; Senkovich, Olga; Chattopadhyay, Debasish (UAB)

    2012-03-26

    The crystal structure of the catalytic domain of the ADP ribosylation factor GTPase-activating protein (ARFGAP) from Plasmodium falciparum has been determined and refined to 2.4 {angstrom} resolution. Multiwavelength anomalous diffraction (MAD) data were collected utilizing the Zn{sup 2+} ion bound at the zinc-finger domain and were used to solve the structure. The overall structure of the domain is similar to those of mammalian ARFGAPs. However, several amino-acid residues in the area where GAP interacts with ARF1 differ in P. falciparum ARFGAP. Moreover, a number of residues that form the dimer interface in the crystal structure are unique in P. falciparum ARFGAP.

  5. Structure of the Cmr2 Subunit of the CRISPR-Cas RNA Silencing Complex

    Energy Technology Data Exchange (ETDEWEB)

    Cocozaki, Alexis I.; Ramia, Nancy F.; Shao, Yaming; Hale, Caryn R.; Terns, Rebecca M.; Terns, Michael P.; Li, Hong (FSU); (Georgia)

    2012-08-10

    Cmr2 is the largest and an essential subunit of a CRISPR RNA-Cas protein complex (the Cmr complex) that cleaves foreign RNA to protect prokaryotes from invading genetic elements. Cmr2 is thought to be the catalytic subunit of the effector complex because of its N-terminal HD nuclease domain. Here, however, we report that the HD domain of Cmr2 is not required for cleavage by the complex in vitro. The 2.3 {angstrom} crystal structure of Pyrococcus furiosus Cmr2 (lacking the HD domain) reveals two adenylyl cyclase-like and two {alpha}-helical domains. The adenylyl cyclase-like domains are arranged as in homodimeric adenylyl cyclases and bind ADP and divalent metals. However, mutagenesis studies show that the metal- and ADP-coordinating residues of Cmr2 are also not critical for cleavage by the complex. Our findings suggest that another component provides the catalytic function and that the essential role by Cmr2 does not require the identified ADP- or metal-binding or HD domains in vitro.

  6. 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.

  7. Catalytic Thr or Ser Residue Modulates Structural Switches in 2-Cys Peroxiredoxin by Distinct Mechanisms

    Science.gov (United States)

    Tairum, Carlos A.; Santos, Melina Cardoso; Breyer, Carlos A.; Geyer, R. Ryan; Nieves, Cecilia J.; Portillo-Ledesma, Stephanie; Ferrer-Sueta, Gerardo; Toledo, José Carlos; Toyama, Marcos H.; Augusto, Ohara; Netto, Luis E. S.; de Oliveira, Marcos A.

    2016-01-01

    Typical 2-Cys Peroxiredoxins (2-Cys Prxs) reduce hydroperoxides with extraordinary rates due to an active site composed of a catalytic triad, containing a peroxidatic cysteine (CP), an Arg, and a Thr (or Ser). 2-Cys Prx are involved in processes such as cancer; neurodegeneration and host-pathogen interactions. During catalysis, 2-Cys Prxs switch between decamers and dimers. Analysis of 2-Cys Prx structures in the fully folded (but not locally unfolded) form revealed a highly conserved, non-conventional hydrogen bond (CH-π) between the catalytic triad Thr of a dimer with an aromatic residue of an adjacent dimer. In contrast, structures of 2-Cys Prxs with a Ser in place of the Thr do not display this CH-π bond. Chromatographic and structural data indicate that the Thr (but not Ser) destabilizes the decamer structure in the oxidized state probably through steric hindrance. As a general trend, mutations in a yeast 2-Cys Prx (Tsa1) favoring the dimeric state also displayed a decreased catalytic activity. Remarkably, yeast naturally contains Thr-Ser variants (Tsa1 and Tsa2, respectively) with distinct oligomeric stabilities in their disulfide states. PMID:27629822

  8. Stochastic sampling of the RNA structural alignment space

    OpenAIRE

    2009-01-01

    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 virtu...

  9. The Structural Basis of Ribozyme-Catalyzed RNA Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Robertson, M.P.; Scott, W.G.; /UC, Santa Cruz

    2007-07-12

    Life originated, according to the RNA World hypothesis, from self-replicating ribozymes that catalyzed ligation of RNA fragments. We have solved the 2.6 angstrom crystal structure of a ligase ribozyme that catalyzes regiospecific formation of a 5' to 3' phosphodiester bond between the 5'-triphosphate and the 3'-hydroxyl termini of two RNA fragments. Invariant residues form tertiary contacts that stabilize a flexible stem of the ribozyme at the ligation site, where an essential magnesium ion coordinates three phosphates. The structure of the active site permits us to suggest how transition-state stabilization and a general base may catalyze the ligation reaction required for prebiotic RNA assembly.

  10. In Situ Synthesis of Bimetallic Hybrid Nanocatalysts on a Paper-Structured Matrix for Catalytic Applications

    Directory of Open Access Journals (Sweden)

    Hirotaka Koga

    2011-11-01

    Full Text Available Bimetallic nanoparticles have attracted significant attention as their electrochemical and catalytic properties being superior to those of the individual component nanoparticles. In this study, gold-silver hybrid nanoparticles (AuAgNPs with an Aucore-Agshell nanostructure were successfully synthesized on zinc oxide (ZnO whiskers. The as-prepared nanocatalyst, denoted AuAgNPs@ZnO whisker, exhibits an excellent catalytic efficiency in the aqueous reduction of 4-nitrophenol to 4-aminophenol; the turnover frequency was up to 40 times higher than that of each component nanoparticle. Their unique features were attributed to the electronic ligand effect at the bimetallic interface. In addition, the AuAgNPs were synthesized on a ZnO whisker-containing paper with a fiber-network microstructure, which was prepared via a papermaking technique. The paper-structured AuAgNPs composite possessed both a paper-like practical utility and a good catalytic performance. Furthermore, the on-paper synthesis process for these bimetallic nanocatalysts is facile. These easy-to-handle nanocatalyst hybrid composites are expected to find a wide range of applications in various chemical and catalytic processes.

  11. An alternative RNA polymerase I structure reveals a dimer hinge.

    Science.gov (United States)

    Kostrewa, Dirk; Kuhn, Claus-D; Engel, Christoph; Cramer, Patrick

    2015-09-01

    RNA polymerase I (Pol I) is the central, 14-subunit enzyme that synthesizes the ribosomal RNA (rRNA) precursor in eukaryotic cells. The recent crystal structure of Pol I at 2.8 Å resolution revealed two novel elements: the `expander' in the active-centre cleft and the `connector' that mediates Pol I dimerization [Engel et al. (2013), Nature (London), 502, 650-655]. Here, a Pol I structure in an alternative crystal form that was solved by molecular replacement using the original atomic Pol I structure is reported. The resulting alternative structure lacks the expander but still shows an expanded active-centre cleft. The neighbouring Pol I monomers form a homodimer with a relative orientation distinct from that observed previously, establishing the connector as a hinge between Pol I monomers.

  12. Ion-mediated RNA structural collapse: effect of spatial confinement

    CERN Document Server

    Tan, Zhi-Jie

    2013-01-01

    RNAs are negatively charged molecules residing in macromolecular crowding cellular environments. Macromolecular confinement can influence the ion effects in RNA folding. In this work, using the recently developed tightly bound ion model for ion fluctuation and correlation, we investigate the confinement effect on the ion-mediated RNA structural collapse for a simple model system. We found that, for both Na$^+$ and Mg$^{2+}$, ion efficiencies in mediating structural collapse/folding are significantly enhanced by the structural confinement. Such an enhancement in the ion efficiency is attributed to the decreased electrostatic free energy difference between the compact conformation ensemble and the (restricted) extended conformation ensemble due to the spatial restriction.

  13. Topological classification and enumeration of RNA structures by genus

    DEFF Research Database (Denmark)

    Andersen, Jørgen Ellegaard; Penner, Robert; Reidys, C. M.

    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...... stack size with nucleotides so that no two consecutive nucleotides are basepaired and show that is algebraic. In particular, we prove that , where . Thus, for stack size at least two, the genus only enters through the sub-exponential factor, and the slow growth rate compared to the number of RNA...

  14. Probing Structural and Catalytic Characteristics of Galactose Oxidase Confined in Nanoscale Chemical Environments

    DEFF Research Database (Denmark)

    Ikemoto, Hideki; Mossin, Susanne; Ulstrup, Jens;

    2014-01-01

    Galactose oxidase (GAOX) is a special metalloenzyme in terms of its active site structure and catalytic mechanisms. This work reports a study where the enzyme confined in a nanoscale chemical environment provided by mesoporous silicas (MPS) is probed. Two types of MPS, i.e. SBA-15 and MCF, were...... synthesized and used to accommodate GAOX. SBA-15-ROD is rod-shaped particles with periodically ordered nanopores (9.5 nm), while MCF has a mesocellular foam-like structure with randomly distributed pores (23 nm) interconnected by smaller windows (8.8 nm). GAOX is non-covalently confined in SBA-15- ROD, while...... constant (KM) of the enzyme is largely unchanged upon immobilization, while the turnover number (kcat) is slightly reduced. The overall catalytic efficiency, represented by the ratio of kcat/KM, is retained around 70% and 60% for SBA-15 and MCF immobilization, respectively. The thermal resistance...

  15. Structural and population genetic determinants of RNA secondary structure evolution

    OpenAIRE

    Piskol, Robert

    2011-01-01

    Since their discovery, RNA molecules have been shown to carry functions that extend far beyond their initially ascribed role as intermediates in protein biosynthesis. These noncoding RNAs (ncRNAs) are involved in fundamental cellular processes including the regulation of gene expression and maintenance of genome stability. In most cases the biogenesis or function of the RNA molecule is only possible if the molecule folds into a characteristic two- and three-dimensional shape via formation of ...

  16. 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.

  17. 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...

  18. Widespread purifying selection on RNA structure in mammals.

    Science.gov (United States)

    Smith, Martin A; Gesell, Tanja; Stadler, Peter F; Mattick, John S

    2013-09-01

    Evolutionarily conserved RNA secondary structures are a robust indicator of purifying selection and, consequently, molecular function. Evaluating their genome-wide occurrence through comparative genomics has consistently been plagued by high false-positive rates and divergent predictions. We present a novel benchmarking pipeline aimed at calibrating the precision of genome-wide scans for consensus RNA structure prediction. The benchmarking data obtained from two refined structure prediction algorithms, RNAz and SISSIz, were then analyzed to fine-tune the parameters of an optimized workflow for genomic sliding window screens. When applied to consistency-based multiple genome alignments of 35 mammals, our approach confidently identifies >4 million evolutionarily constrained RNA structures using a conservative sensitivity threshold that entails historically low false discovery rates for such analyses (5-22%). These predictions comprise 13.6% of the human genome, 88% of which fall outside any known sequence-constrained element, suggesting that a large proportion of the mammalian genome is functional. As an example, our findings identify both known and novel conserved RNA structure motifs in the long noncoding RNA MALAT1. This study provides an extensive set of functional transcriptomic annotations that will assist researchers in uncovering the precise mechanisms underlying the developmental ontologies of higher eukaryotes.

  19. Steady-state NTPase activity of Dengue virus NS3: number of catalytic sites, nucleotide specificity and activation by ssRNA.

    Directory of Open Access Journals (Sweden)

    J Jeremías Incicco

    Full Text Available Dengue virus nonstructural protein 3 (NS3 unwinds double stranded RNA driven by the free energy derived from the hydrolysis of nucleoside triphosphates. This paper presents the first systematic and quantitative characterization of the steady-state NTPase activity of DENV NS3 and their interaction with ssRNA. Substrate curves for ATP, GTP, CTP and UTP were obtained, and the specificity order for these nucleotides - evaluated as the ratio (kcat /KM - was GTP[Formula: see text]ATP[Formula: see text]CTP [Formula: see text] UTP, which showed that NS3 have poor ability to discriminate between different NTPs. Competition experiments between the four substrates indicated that all of them are hydrolyzed in one and the same catalytic site of the enzyme. The effect of ssRNA on the ATPase activity of NS3 was studied using poly(A and poly(C. Both RNA molecules produced a 10 fold increase in the turnover rate constant (kcat and a 100 fold decrease in the apparent affinity (KM for ATP. When the ratio [RNA bases]/[NS3] was between 0 and [Formula: see text]20 the ATPase activity was inhibited by increasing both poly(A and poly(C. Using the theory of binding of large ligands (NS3 to a one-dimensional homogeneous lattice of infinite length (RNA we tested the hypothesis that inhibition is the result of crowding of NS3 molecules along the RNA lattices. Finally, we discuss why this hypothesis is consistent with the idea that the ATPase catalytic cycle is tightly coupled to the movement of NS3 helicase along the RNA.

  20. MWW-type titanosilicate synthesis, structural modification and catalytic applications to green oxidations

    CERN Document Server

    Wu, Peng; Xu, Le; Liu, Yueming; He, Mingyuan

    2013-01-01

    This book provides a comprehensive review of a new generation of selective oxidation titanosilicate catalysts with the MWW topology (Ti-MWW) based on the research achievements of the past 12 years. It gives an overview of the synthesis, structure modification and catalytic properties of Ti-MWW. Ti-MWW can readily be prepared by means of direct hydrothermal synthesis with crystallization-supporting agents, using dual-structure-directing agents and a dry-gel conversion technique. It also can be post-synthesized through unique reversible structure transformation and liquid-phase isomorphous subst

  1. Fabrication and catalytic tests of MCM-22/silicon carbide structured catalysts.

    Science.gov (United States)

    Gu, Lijun; Ma, Ding; Hu, Gang; Wu, Jingjing; Wang, Hongxia; Sun, Changyong; Yao, Songdong; Shen, Wenjie; Bao, Xinhe

    2010-10-28

    The structured catalyst of zeolite MCM-22/silicon carbide (SiC) was prepared for the first time through the in situ hydrothermal synthesis approach. The zeolite loading of the structured catalyst could be tuned by changing the synthesis time and applying alkali pre-treatment of SiC substrate. An additional silica layer formed on SiC substrate after the precalcination treatment facilitated the crystallization of MCM-22 zeolite on the SiC substrate. The MCM-22/SiC structured catalyst thus prepared exhibited good catalytic performance in the methane dehydroaromatization reaction.

  2. In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits

    Science.gov (United States)

    Mühleip, Alexander W.; Dewar, Caroline E.; Schnaufer, Achim; Kühlbrandt, Werner; Davies, Karen M.

    2017-01-01

    We used electron cryotomography and subtomogram averaging to determine the in situ structures of mitochondrial ATP synthase dimers from two organisms belonging to the phylum euglenozoa: Trypanosoma brucei, a lethal human parasite, and Euglena gracilis, a photosynthetic protist. At a resolution of 32.5 Å and 27.5 Å, respectively, the two structures clearly exhibit a noncanonical F1 head, in which the catalytic (αβ)3 assembly forms a triangular pyramid rather than the pseudo-sixfold ring arrangement typical of all other ATP synthases investigated so far. Fitting of known X-ray structures reveals that this unusual geometry results from a phylum-specific cleavage of the α subunit, in which the C-terminal αC fragments are displaced by ∼20 Å and rotated by ∼30° from their expected positions. In this location, the αC fragment is unable to form the conserved catalytic interface that was thought to be essential for ATP synthesis, and cannot convert γ-subunit rotation into the conformational changes implicit in rotary catalysis. The new arrangement of catalytic subunits suggests that the mechanism of ATP generation by rotary ATPases is less strictly conserved than has been generally assumed. The ATP synthases of these organisms present a unique model system for discerning the individual contributions of the α and β subunits to the fundamental process of ATP synthesis. PMID:28096380

  3. Template Synthesis of Noble Metal Nanocrystals with Unusual Crystal Structures and Their Catalytic Applications.

    Science.gov (United States)

    Fan, Zhanxi; Zhang, Hua

    2016-12-20

    Noble metal nanocrystals own high chemical stability, unique plasmonic and distinctive catalytic properties, making them outstanding in many applications. However, their practical applications are limited by their high cost and scarcity on the earth. One promising strategy to solve these problems is to boost their catalytic performance in order to reduce their usage amount. To realize this target, great research efforts have been devoted to the size-, composition-, shape- and/or architecture-controlled syntheses of noble metal nanocrystals during the past two decades. Impressively, recent experimental studies have revealed that the crystal structure of noble metal nanocrystals can also significantly affect their physicochemical properties, such as optical, magnetic, catalytic, mechanical, electrical and electronic properties. Therefore, besides the well-established size, composition, shape, and architecture control, the rise of crystal structure-controlled synthesis of noble metal nanocrystals will open up new opportunities to further improve their functional properties, and thus promote their potential applications in energy conversion, catalysis, biosensing, information storage, surface enhanced Raman scattering, waveguide, near-infrared photothermal therapy, controlled release, bioimaging, biomedicine, and so on. In this Account, we review the recent research progress on the crystal structure control of noble metal nanocrystals with a template synthetic approach and their crystal structure-dependent catalytic properties. We first describe the template synthetic methods, such as epitaxial growth and galvanic replacement reaction methods, in which a presynthesized noble metal nanocrystal with either new or common crystal structure is used as the template to direct the growth of unusual crystal structures of other noble metals. Significantly, the template synthetic strategy described here provides an efficient, simple and straightforward way to synthesize unusual

  4. Structure of a dimeric crenarchaeal Cas6 enzyme with an atypical active site for CRISPR RNA processing

    Science.gov (United States)

    Reeks, Judith; Sokolowski, Richard D.; Graham, Shirley; Liu, Huanting; Naismith, James H.; White, Malcolm F.

    2013-01-01

    The competition between viruses and hosts is played out in all branches of life. Many prokaryotes have an adaptive immune system termed ‘CRISPR’ (clustered regularly interspaced short palindromic repeats) which is based on the capture of short pieces of viral DNA. The captured DNA is integrated into the genomic DNA of the organism flanked by direct repeats, transcribed and processed to generate crRNA (CRISPR RNA) that is loaded into a variety of effector complexes. These complexes carry out sequence-specific detection and destruction of invading mobile genetic elements. In the present paper, we report the structure and activity of a Cas6 (CRISPR-associated 6) enzyme (Sso1437) from Sulfolobus solfataricus responsible for the generation of unit-length crRNA species. The crystal structure reveals an unusual dimeric organization that is important for the enzyme's activity. In addition, the active site lacks the canonical catalytic histidine residue that has been viewed as an essential feature of the Cas6 family. Although several residues contribute towards catalysis, none is absolutely essential. Coupled with the very low catalytic rate constants of the Cas6 family and the plasticity of the active site, this suggests that the crRNA recognition and chaperone-like activities of the Cas6 family should be considered as equal to or even more important than their role as traditional enzymes. PMID:23527601

  5. The Expected Order of Saturated RNA Secondary Structures

    CERN Document Server

    Jin, Emma Yu

    2011-01-01

    We show the expected order of RNA saturated secondary structures of size $n$ is $\\log_4n(1+O(\\frac{\\log_2n}{n}))$, if we select the saturated secondary structure uniformly at random. Furthermore, the order of saturated secondary structures is sharply concentrated around its mean. As a consequence saturated structures and structures in the traditional model behave the same with respect to the expected order. Thus we may conclude that the traditional model has already drawn the right picture and conclusions inferred from it with respect to the order (the overall shape) of a structure remain valid even if enforcing saturation (at least in expectation).

  6. 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.

  7. Alphavirus RNA synthesis and non-structural protein functions.

    Science.gov (United States)

    Rupp, Jonathan C; Sokoloski, Kevin J; Gebhart, Natasha N; Hardy, Richard W

    2015-09-01

    The members of the genus Alphavirus are positive-sense RNA viruses, which are predominantly transmitted to vertebrates by a mosquito vector. Alphavirus disease in humans can be severely debilitating, and depending on the particular viral species, infection may result in encephalitis and possibly death. In recent years, alphaviruses have received significant attention from public health authorities as a consequence of the dramatic emergence of chikungunya virus in the Indian Ocean islands and the Caribbean. Currently, no safe, approved or effective vaccine or antiviral intervention exists for human alphavirus infection. The molecular biology of alphavirus RNA synthesis has been well studied in a few species of the genus and represents a general target for antiviral drug development. This review describes what is currently understood about the regulation of alphavirus RNA synthesis, the roles of the viral non-structural proteins in this process and the functions of cis-acting RNA elements in replication, and points to open questions within the field.

  8. Three-dimensional tertiary structure of yeast phenylalanine transfer RNA

    Science.gov (United States)

    Kim, S. H.; Sussman, J. L.; Suddath, F. L.; Quigley, G. J.; Mcpherson, A.; Wang, A. H. J.; Seeman, N. C.; Rich, A.

    1974-01-01

    Results of an analysis and interpretation of a 3-A electron density map of yeast phenylalanine transfer RNA. Some earlier detailed assignments of nucleotide residues to electron density peaks are found to be in error, even though the overall tracing of the backbone conformation of yeast phenylalanine transfer RNA was generally correct. A new, more comprehensive interpretation is made which makes it possible to define the tertiary interactions in the molecule. The new interpretation makes it possible to visualize a number of tertiary interactions which not only explain the structural role of most of the bases which are constant in transfer RNAs, but also makes it possible to understand in a direct and simple fashion the chemical modification data on transfer RNA. In addition, this pattern of tertiary interactions provides a basis for understanding the general three-dimensional folding of all transfer RNA molecules.

  9. Approaches to link RNA secondary structures with splicing regulation

    DEFF Research Database (Denmark)

    Plass, Mireya; Eyras, Eduardo

    2014-01-01

    by facilitating or hindering the interaction with factors and small nuclear ribonucleoproteins (snRNPs) that regulate splicing. Moreover, the secondary structure could play a fundamental role in the splicing of yeast species, which lack many of the regulatory splicing factors present in metazoans. This chapter......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...

  10. The Three-Dimensional Structure and Catalytic Mechanism of Cytosine Deaminase†

    Science.gov (United States)

    Hall, Richard S.; Fedorov, Alexander A.; Xu, Chengfu; Fedorov, Elena V.; Almo, Steven C.; Raushel, Frank M.

    2011-01-01

    Cytosine deaminase (CDA) from E. coli is a member of the amidohydrolase superfamily. The structure of the zinc-activated enzyme was determined in the presence of phosphonocytosine, a mimic of the tetrahedral reaction intermediate. This compound inhibits the deamination of cytosine with a Ki of 52 nM. The zinc and iron containing enzymes were characterized to determine the effect of the divalent cations on activation of the hydrolytic water. Fe-CDA loses activity at low pH with a kinetic pKa of 6.0 and Zn-CDA has a kinetic pKa of 7.3. Mutation of Gln-156 decreased the catalytic activity by more than 5 orders of magnitude, supporting its role in substrate binding. Mutation of Glu-217, Asp-313, and His-246 significantly decreased catalytic activity supporting the role of these three residues in activation of the hydrolytic water molecule and facilitation of proton transfer reactions. A library of potential substrates was used to probe the structural determinants responsible for catalytic activity. CDA was able to catalyze the deamination of isocytosine and the hydrolysis of 3-oxauracil. Large inverse solvent isotope effects were obtained on kcat and kcat/Km, consistent with the formation of a low-barrier hydrogen bond during the conversion of cytosine to uracil. A chemical mechanism for substrate deamination by CDA was proposed. PMID:21545144

  11. Structural Basis for Telomerase RNA Recognition and RNP Assembly by the Holoenzyme La Family Protein p65

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Mahavir; Wang, Zhonghua; Koo, Bon-Kyung; Patel, Anooj; Cascio, Duilio; Collins, Kathleen; Feigon, Juli (UCLA); (UCB)

    2012-07-01

    Telomerase is a ribonucleoprotein complex essential for maintenance of telomere DNA at linear chromosome ends. The catalytic core of Tetrahymena telomerase comprises a ternary complex of telomerase RNA (TER), telomerase reverse transcriptase (TERT), and the essential La family protein p65. NMR and crystal structures of p65 C-terminal domain and its complex with stem IV of TER reveal that RNA recognition is achieved by a combination of single- and double-stranded RNA binding, which induces a 105{sup o} bend in TER. The domain is a cryptic, atypical RNA recognition motif with a disordered C-terminal extension that forms an {alpha} helix in the complex necessary for hierarchical assembly of TERT with p65-TER. This work provides the first structural insight into biogenesis and assembly of TER with a telomerase-specific protein. Additionally, our studies define a structurally homologous domain (xRRM) in genuine La and LARP7 proteins and suggest a general mode of RNA binding for biogenesis of their diverse RNA targets.

  12. Pressure effects on catalytic properties and structural stability of human paraoxonase

    Energy Technology Data Exchange (ETDEWEB)

    Clery-Barraud, C; Leva, J; Bakdouri, N E; Renault, F; Masson, P; Rochu, D [Departement de Toxicologie, Centre de Recherches du Service de Sante des Armees, BP 87, 38702 La Tronche cedex (France)], E-mail: cclerybarraud@crssa.net

    2008-07-15

    Human paraoxonase (HuPON1) is a candidate as catalytic bioscavenger for pre-treatment and therapy of poisoning by organophosphate compounds. HuPON1 is a hydrophobic protein associated with a partner, the human phosphate binding protein (HPBP) in plasma high density lipoproteins. The relationship between the composition and the size of multimeric states of HuPON1 is not well understood. Moreover the effect of HPBP's presence on enzyme catalytic mechanisms and stability is unclear. We investigated the effect of hydrostatic pressure and temperature on structural stability and activity of different PON1 preparations (hybrid recombinant PON1, natural HuPON1 free of its partner or in the presence of 50% w/w HPBP). We showed that PON1 exists under several multimeric forms and that the binding of HPBP amends the size of the hetero-oligomeric states and exerts a stabilizing effect on the activity of PON1.

  13. Pressure effects on catalytic properties and structural stability of human paraoxonase

    Science.gov (United States)

    Cléry-Barraud, C.; Leva, J.; Bakdouri, N. E.; Renault, F.; Masson, P.; Rochu, D.

    2008-07-01

    Human paraoxonase (HuPON1) is a candidate as catalytic bioscavenger for pre-treatment and therapy of poisoning by organophosphate compounds. HuPON1 is a hydrophobic protein associated with a partner, the human phosphate binding protein (HPBP) in plasma high density lipoproteins. The relationship between the composition and the size of multimeric states of HuPON1 is not well understood. Moreover the effect of HPBP's presence on enzyme catalytic mechanisms and stability is unclear. We investigated the effect of hydrostatic pressure and temperature on structural stability and activity of different PON1 preparations (hybrid recombinant PON1, natural HuPON1 free of its partner or in the presence of 50% w/w HPBP). We showed that PON1 exists under several multimeric forms and that the binding of HPBP amends the size of the hetero-oligomeric states and exerts a stabilizing effect on the activity of PON1.

  14. Structures of the human poly (ADP-ribose glycohydrolase catalytic domain confirm catalytic mechanism and explain inhibition by ADP-HPD derivatives.

    Directory of Open Access Journals (Sweden)

    Julie A Tucker

    Full Text Available Poly(ADP-ribose glycohydrolase (PARG is the only enzyme known to catalyse hydrolysis of the O-glycosidic linkages of ADP-ribose polymers, thereby reversing the effects of poly(ADP-ribose polymerases. PARG deficiency leads to cell death whilst PARG depletion causes sensitisation to certain DNA damaging agents, implicating PARG as a potential therapeutic target in several disease areas. Efforts to develop small molecule inhibitors of PARG activity have until recently been hampered by a lack of structural information on PARG. We have used a combination of bio-informatic and experimental approaches to engineer a crystallisable, catalytically active fragment of human PARG (hPARG. Here, we present high-resolution structures of the catalytic domain of hPARG in unliganded form and in complex with three inhibitors: ADP-ribose (ADPR, adenosine 5'-diphosphate (hydroxymethylpyrrolidinediol (ADP-HPD and 8-n-octyl-amino-ADP-HPD. Our structures confirm conservation of overall fold amongst mammalian PARG glycohydrolase domains, whilst revealing additional flexible regions in the catalytic site. These new structures rationalise a body of published mutational data and the reported structure-activity relationship for ADP-HPD based PARG inhibitors. In addition, we have developed and used biochemical, isothermal titration calorimetry and surface plasmon resonance assays to characterise the binding of inhibitors to our PARG protein, thus providing a starting point for the design of new inhibitors.

  15. The crystal structure of the catalytic domain of a eukaryotic guanylate cyclase

    Directory of Open Access Journals (Sweden)

    Marletta Michael A

    2008-10-01

    Full Text Available Abstract Background Soluble guanylate cyclases generate cyclic GMP when bound to nitric oxide, thereby linking nitric oxide levels to the control of processes such as vascular homeostasis and neurotransmission. The guanylate cyclase catalytic module, for which no structure has been determined at present, is a class III nucleotide cyclase domain that is also found in mammalian membrane-bound guanylate and adenylate cyclases. Results We have determined the crystal structure of the catalytic domain of a soluble guanylate cyclase from the green algae Chlamydomonas reinhardtii at 2.55 Å resolution, and show that it is a dimeric molecule. Conclusion Comparison of the structure of the guanylate cyclase domain with the known structures of adenylate cyclases confirms the close similarity in architecture between these two enzymes, as expected from their sequence similarity. The comparison also suggests that the crystallized guanylate cyclase is in an inactive conformation, and the structure provides indications as to how activation might occur. We demonstrate that the two active sites in the dimer exhibit positive cooperativity, with a Hill coefficient of ~1.5. Positive cooperativity has also been observed in the homodimeric mammalian membrane-bound guanylate cyclases. The structure described here provides a reliable model for functional analysis of mammalian guanylate cyclases, which are closely related in sequence.

  16. Crystal Structure of a Novel Viral Protease with a Serine/Lysine Catalytic Dyad Mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Feldman,A.; Lee, J.; Delmas, B.; Paetzel, M.

    2006-01-01

    The blotched snakehead virus (BSNV), an aquatic birnavirus, encodes a polyprotein (NH2-pVP2-X-VP4-VP3-COOH) that is processed through the proteolytic activity of its own protease (VP4) to liberate itself and the viral proteins pVP2, X and VP3. The protein pVP2 is further processed by VP4 to give rise to the capsid protein VP2 and four structural peptides. We report here the crystal structure of a VP4 protease from BSNV, which displays a catalytic serine/lysine dyad in its active site. This is the first crystal structure of a birnavirus protease and the first crystal structure of a viral protease that utilizes a lysine general base in its catalytic mechanism. The topology of the VP4 substrate binding site is consistent with the enzymes substrate specificity and a nucleophilic attack from the si-face of the substrates scissile bond. Despite low levels of sequence identity, VP4 shows similarities in its active site to other characterized Ser/Lys proteases such as signal peptidase, LexA protease and Lon protease. Together, the structure of VP4 provides insights into the mechanism of a recently characterized clan of serine proteases that utilize a lysine general base and reveals the structure of potential targets for antiviral therapy, especially for other related and economically important viruses, such as infectious bursal disease virus in poultry and infectious pancreatic necrosis virus in aquaculture.

  17. Identification of consensus RNA secondary structures using suffix arrays

    Directory of Open Access Journals (Sweden)

    Nguyen Truong

    2006-05-01

    Full Text Available Abstract Background The identification of a consensus RNA motif often consists in finding a conserved secondary structure with minimum free energy in an ensemble of aligned sequences. However, an alignment is often difficult to obtain without prior structural information. Thus the need for tools to automate this process. Results We present an algorithm called Seed to identify all the conserved RNA secondary structure motifs in a set of unaligned sequences. The search space is defined as the set of all the secondary structure motifs inducible from a seed sequence. A general-to-specific search allows finding all the motifs that are conserved. Suffix arrays are used to enumerate efficiently all the biological palindromes as well as for the matching of RNA secondary structure expressions. We assessed the ability of this approach to uncover known structures using four datasets. The enumeration of the motifs relies only on the secondary structure definition and conservation only, therefore allowing for the independent evaluation of scoring schemes. Twelve simple objective functions based on free energy were evaluated for their potential to discriminate native folds from the rest. Conclusion Our evaluation shows that 1 support and exclusion constraints are sufficient to make an exhaustive search of the secondary structure space feasible. 2 The search space induced from a seed sequence contains known motifs. 3 Simple objective functions, consisting of a combination of the free energy of matching sequences, can generally identify motifs with high positive predictive value and sensitivity to known motifs.

  18. Dynamics in Sequence Space for RNA Secondary Structure Design.

    Science.gov (United States)

    Matthies, Marco C; Bienert, Stefan; Torda, Andrew E

    2012-10-01

    We have implemented a method for the design of RNA sequences that should fold to arbitrary secondary structures. A popular energy model allows one to take the derivative with respect to composition, which can then be interpreted as a force and used for Newtonian dynamics in sequence space. Combined with a negative design term, one can rapidly sample sequences which are compatible with a desired secondary structure via simulated annealing. Results for 360 structures were compared with those from another nucleic acid design program using measures such as the probability of the target structure and an ensemble-weighted distance to the target structure.

  19. Genome-wide analyses of Epstein-Barr virus reveal conserved RNA structures and a novel stable intronic sequence RNA

    OpenAIRE

    2013-01-01

    Background Epstein-Barr virus (EBV) is a human herpesvirus implicated in cancer and autoimmune disorders. Little is known concerning the roles of RNA structure in this important human pathogen. This study provides the first comprehensive genome-wide survey of RNA and RNA structure in EBV. Results Novel EBV RNAs and RNA structures were identified by computational modeling and RNA-Seq analyses of EBV. Scans of the genomic sequences of four EBV strains (EBV-1, EBV-2, GD1, and GD2) and of the clo...

  20. RNA Ligase Structures Reveal the Basis for RNA Specificity and Conformational Changes that Drive Ligation Forward

    Energy Technology Data Exchange (ETDEWEB)

    Nandakumar,J.; Shuman, S.; Lima, C.

    2006-01-01

    T4 RNA ligase 2 (Rnl2) and kinetoplastid RNA editing ligases exemplify a family of RNA repair enzymes that seal 3'OH/5'PO{sub 4} nicks in duplex RNAs via ligase adenylylation (step 1), AMP transfer to the nick 5'PO{sub 4} (step 2), and attack by the nick 3'OH on the 5'-adenylylated strand to form a phosphodiester (step 3). Crystal structures are reported for Rnl2 at discrete steps along this pathway: the covalent Rnl2-AMP intermediate; Rnl2 bound to an adenylylated nicked duplex, captured immediately following step 2; and Rnl2 at an adenylylated nick in a state poised for step 3. These structures illuminate the stereochemistry of nucleotidyl transfer and reveal how remodeling of active-site contacts and conformational changes propel the ligation reaction forward. Mutational analysis and comparison of nick-bound structures of Rnl2 and human DNA ligase I highlight common and divergent themes of substrate recognition that can explain their specialization for RNA versus DNA repair.

  1. Knockdown of glutamate cysteine ligase catalytic subunit by siRNA causes the gold nanoparticles-induced cytotoxicity in lung cancer cells.

    Directory of Open Access Journals (Sweden)

    Min Liu

    Full Text Available Gold nanoparticles (GNPs have shown promising medical applications in cancer treatment involved in the regulation of intracellular redox balance. Previously, we have reported that GNPs can trigger apoptosis and necrosis in human lung cancer cells (A549 when L-buthionine-sulfoximine (BSO was used to decrease the expression of intracellular glutathione (GSH. Herein, we investigated the cytotoxicity of GNPs toward lung cancer cells under the glutamate cysteine ligase catalytic subunit (GCLC was silenced by siRNA. Our results showed that GNPs cause apoptosis and necrosis in cells transfected with GCLC siRNA by elevating intracellular reactive oxygen species (ROS. These findings demonstrated that the regulation of glutathione synthesis by GCLC siRNA in A549 cells can initiate the gold nanoparticles-induced cytotoxicity.

  2. The stability and catalytic activity of W13@Pt42 core-shell structure.

    Science.gov (United States)

    Huo, Jin-Rong; Wang, Xiao-Xu; Li, Lu; Cheng, Hai-Xia; Su, Yan-Jing; Qian, Ping

    2016-10-19

    This paper reports a study of the electronic properties, structural stability and catalytic activity of the W13@Pt42 core-shell structure using the First-principles calculations. The degree of corrosion of W13@Pt42 core-shell structure is simulated in acid solutions and through molecular absorption. The absorption energy of OH for this structure is lower than that for Pt55, which inhibits the poison effect of O containing intermediate. Furthermore we present the optimal path of oxygen reduction reaction catalyzed by W13@Pt42. Corresponding to the process of O molecular decomposition, the rate-limiting step of oxygen reduction reaction catalyzed by W13@Pt42 is 0.386 eV, which is lower than that for Pt55 of 0.5 eV. In addition by alloying with W, the core-shell structure reduces the consumption of Pt and enhances the catalytic efficiency, so W13@Pt42 has a promising perspective of industrial application.

  3. Structure-based engineering increased the catalytic turnover rate of a novel phenazine prenyltransferase.

    Directory of Open Access Journals (Sweden)

    Georg Zocher

    Full Text Available Prenyltransferases (PTs catalyze the regioselective transfer of prenyl moieties onto aromatic substrates in biosynthetic pathways of microbial secondary metabolites. Therefore, these enzymes contribute to the chemical diversity of natural products. Prenylation is frequently essential for the pharmacological properties of these metabolites, including their antibiotic and antitumor activities. Recently, the first phenazine PTs, termed EpzP and PpzP, were isolated and biochemically characterized. The two enzymes play a central role in the biosynthesis of endophenazines by catalyzing the regiospecific prenylation of 5,10-dihydrophenazine-1-carboxylic acid (dhPCA in the secondary metabolism of two different Streptomyces strains. Here we report crystal structures of EpzP in its unliganded state as well as bound to S-thiolodiphosphate (SPP, thus defining the first three-dimensional structures for any phenazine PT. A model of a ternary complex resulted from in silico modeling of dhPCA and site-directed mutagenesis. The structural analysis provides detailed insight into the likely mechanism of phenazine prenylation. The catalytic mechanism suggested by the structure identifies amino acids that are required for catalysis. Inspection of the structures and the model of the ternary complex furthermore allowed us to rationally engineer EpzP variants with up to 14-fold higher catalytic reaction rate compared to the wild-type enzyme. This study therefore provides a solid foundation for additional enzyme modifications that should result in efficient, tailor-made biocatalysts for phenazines production.

  4. The stability and catalytic activity of W13@Pt42 core-shell structure

    Science.gov (United States)

    Huo, Jin-Rong; Wang, Xiao-Xu; Li, Lu; Cheng, Hai-Xia; Su, Yan-Jing; Qian, Ping

    2016-01-01

    This paper reports a study of the electronic properties, structural stability and catalytic activity of the W13@Pt42 core-shell structure using the First-principles calculations. The degree of corrosion of W13@Pt42 core-shell structure is simulated in acid solutions and through molecular absorption. The absorption energy of OH for this structure is lower than that for Pt55, which inhibits the poison effect of O containing intermediate. Furthermore we present the optimal path of oxygen reduction reaction catalyzed by W13@Pt42. Corresponding to the process of O molecular decomposition, the rate-limiting step of oxygen reduction reaction catalyzed by W13@Pt42 is 0.386 eV, which is lower than that for Pt55 of 0.5 eV. In addition by alloying with W, the core-shell structure reduces the consumption of Pt and enhances the catalytic efficiency, so W13@Pt42 has a promising perspective of industrial application. PMID:27759038

  5. The stability and catalytic activity of W13@Pt42 core-shell structure

    Science.gov (United States)

    Huo, Jin-Rong; Wang, Xiao-Xu; Li, Lu; Cheng, Hai-Xia; Su, Yan-Jing; Qian, Ping

    2016-10-01

    This paper reports a study of the electronic properties, structural stability and catalytic activity of the W13@Pt42 core-shell structure using the First-principles calculations. The degree of corrosion of W13@Pt42 core-shell structure is simulated in acid solutions and through molecular absorption. The absorption energy of OH for this structure is lower than that for Pt55, which inhibits the poison effect of O containing intermediate. Furthermore we present the optimal path of oxygen reduction reaction catalyzed by W13@Pt42. Corresponding to the process of O molecular decomposition, the rate-limiting step of oxygen reduction reaction catalyzed by W13@Pt42 is 0.386 eV, which is lower than that for Pt55 of 0.5 eV. In addition by alloying with W, the core-shell structure reduces the consumption of Pt and enhances the catalytic efficiency, so W13@Pt42 has a promising perspective of industrial application.

  6. Plant coilin: structural characteristics and RNA-binding properties.

    Directory of Open Access Journals (Sweden)

    Valentine Makarov

    Full Text Available Cajal bodies (CBs are dynamic subnuclear compartments involved in the biogenesis of ribonucleoproteins. Coilin is a major structural scaffolding protein necessary for CB formation, composition and activity. The predicted secondary structure of Arabidopsis thaliana coilin (Atcoilin suggests that the protein is composed of three main domains. Analysis of the physical properties of deletion mutants indicates that Atcoilin might consist of an N-terminal globular domain, a central highly disordered domain and a C-terminal domain containing a presumable Tudor-like structure adjacent to a disordered C terminus. Despite the low homology in amino acid sequences, a similar type of domain organization is likely shared by human and animal coilin proteins and coilin-like proteins of various plant species. Atcoilin is able to bind RNA effectively and in a non-specific manner. This activity is provided by three RNA-binding sites: two sets of basic amino acids in the N-terminal domain and one set in the central domain. Interaction with RNA induces the multimerization of the Atcoilin molecule, a consequence of the structural alterations in the N-terminal domain. The interaction with RNA and subsequent multimerization may facilitate coilin's function as a scaffolding protein. A model of the N-terminal domain is also proposed.

  7. SCOR: Structural Classification of RNA, version 2.0.

    Science.gov (United States)

    Tamura, Makio; Hendrix, Donna K; Klosterman, Peter S; Schimmelman, Nancy R B; Brenner, Steven E; Holbrook, Stephen R

    2004-01-01

    SCOR, the Structural Classification of RNA (http://scor.lbl.gov), is a database designed to provide a comprehensive perspective and understanding of RNA motif three-dimensional structure, function, tertiary interactions and their relationships. SCOR 2.0 represents a major expansion and introduces a new classification organization. The new version represents the classification as a Directed Acyclic Graph (DAG), which allows a classification node to have multiple parents, in contrast to the strictly hierarchical classification used in SCOR 1.2. SCOR 2.0 supports three types of query terms in the updated search engine: PDB or NDB identifier, nucleotide sequence and keyword. We also provide parseable XML files for all information. This new release contains 511 RNA entries from the PDB as of 15 May 2003. A total of 5880 secondary structural elements are classified: 2104 hairpin loops and 3776 internal loops. RNA motifs reported in the literature, such as 'Kink turn' and 'GNRA loops', are now incorporated into the structural classification along with definitions and descriptions.

  8. SCOR: Structural classification of RNA, Version 2.0

    Energy Technology Data Exchange (ETDEWEB)

    Tamura, Makio; Hendrix, Donna K.; Klosterman, Peter

    2003-10-03

    SCOR (http://scor.lbl.gov), the Structural Classification of RNA, is a database designed to provide a comprehensive perspective and understanding of RNA motif three-dimensional structure, function, tertiary interactions, and their relationships. SCOR 2.0 represents a major expansion and introduces a wholly new classification system. The new version represents the classification as a Directed Acyclic Graph (DAG), which allows a classification node to have multiple parents, in contrast to the strictly hierarchical classification used in SCOR 1.2. SCOR 2.0 supports three types of query terms in the updated search engine: PDB or NDB identifier, nucleotide sequence, and keyword. We also provide parseable XML files for all information. This new release contains 511RNA entries from the PDB as of 15 May 2003. A total of 5,880 secondary structural elements are classified; 2,104 hairpin loops and 3,776 internal loops. RNA motifs reported in the literature, such as ''Kinkturn'' and ''GNRA loops,'' are now incorporated into the structural classification along with definitions and descriptions.

  9. Improved prediction of RNA tertiary structure with insights into native state dynamics.

    Science.gov (United States)

    Bida, John Paul; Maher, L James

    2012-03-01

    The importance of RNA tertiary structure is evident from the growing number of published high resolution NMR and X-ray crystallographic structures of RNA molecules. These structures provide insights into function and create a knowledge base that is leveraged by programs such as Assemble, ModeRNA, RNABuilder, NAST, FARNA, Mc-Sym, RNA2D3D, and iFoldRNA for tertiary structure prediction and design. While these methods sample native-like RNA structures during simulations, all struggle to capture the native RNA conformation after scoring. We propose RSIM, an improved RNA fragment assembly method that preserves RNA global secondary structure while sampling conformations. This approach enhances the quality of predicted RNA tertiary structure, provides insights into the native state dynamics, and generates a powerful visualization of the RNA conformational space. RSIM is available for download from http://www.github.com/jpbida/rsim.

  10. Expected distance between terminal nucleotides of RNA secondary structures.

    Science.gov (United States)

    Clote, Peter; Ponty, Yann; Steyaert, Jean-Marc

    2012-09-01

    In "The ends of a large RNA molecule are necessarily close", Yoffe et al. (Nucleic Acids Res 39(1):292-299, 2011) used the programs RNAfold [resp. RNAsubopt] from Vienna RNA Package to calculate the distance between 5' and 3' ends of the minimum free energy secondary structure [resp. thermal equilibrium structures] of viral and random RNA sequences. Here, the 5'-3' distance is defined to be the length of the shortest path from 5' node to 3' node in the undirected graph, whose edge set consists of edges {i, i + 1} corresponding to covalent backbone bonds and of edges {i, j} corresponding to canonical base pairs. From repeated simulations and using a heuristic theoretical argument, Yoffe et al. conclude that the 5'-3' distance is less than a fixed constant, independent of RNA sequence length. In this paper, we provide a rigorous, mathematical framework to study the expected distance from 5' to 3' ends of an RNA sequence. We present recurrence relations that precisely define the expected distance from 5' to 3' ends of an RNA sequence, both for the Turner nearest neighbor energy model, as well as for a simple homopolymer model first defined by Stein and Waterman. We implement dynamic programming algorithms to compute (rather than approximate by repeated application of Vienna RNA Package) the expected distance between 5' and 3' ends of a given RNA sequence, with respect to the Turner energy model. Using methods of analytical combinatorics, that depend on complex analysis, we prove that the asymptotic expected 5'-3' distance of length n homopolymers is approximately equal to the constant 5.47211, while the asymptotic distance is 6.771096 if hairpins have a minimum of 3 unpaired bases and the probability that any two positions can form a base pair is 1/4. Finally, we analyze the 5'-3' distance for secondary structures from the STRAND database, and conclude that the 5'-3' distance is correlated with RNA sequence length.

  11. Predicting loop-helix tertiary structural contacts in RNA pseudoknots.

    Science.gov (United States)

    Cao, Song; Giedroc, David P; Chen, Shi-Jie

    2010-03-01

    Tertiary interactions between loops and helical stems play critical roles in the biological function of many RNA pseudoknots. However, quantitative predictions for RNA tertiary interactions remain elusive. Here we report a statistical mechanical model for the prediction of noncanonical loop-stem base-pairing interactions in RNA pseudoknots. Central to the model is the evaluation of the conformational entropy for the pseudoknotted folds with defined loop-stem tertiary structural contacts. We develop an RNA virtual bond-based conformational model (Vfold model), which permits a rigorous computation of the conformational entropy for a given fold that contains loop-stem tertiary contacts. With the entropy parameters predicted from the Vfold model and the energy parameters for the tertiary contacts as inserted parameters, we can then predict the RNA folding thermodynamics, from which we can extract the tertiary contact thermodynamic parameters from theory-experimental comparisons. These comparisons reveal a contact enthalpy (DeltaH) of -14 kcal/mol and a contact entropy (DeltaS) of -38 cal/mol/K for a protonated C(+)*(G-C) base triple at pH 7.0, and (DeltaH = -7 kcal/mol, DeltaS = -19 cal/mol/K) for an unprotonated base triple. Tests of the model for a series of pseudoknots show good theory-experiment agreement. Based on the extracted energy parameters for the tertiary structural contacts, the model enables predictions for the structure, stability, and folding pathways for RNA pseudoknots with known or postulated loop-stem tertiary contacts from the nucleotide sequence alone.

  12. Canonical RNA Pseudoknot Structures with Arc Length $\\geq 4$

    CERN Document Server

    Reidys, Christian M; Zhao, Albus Y Y

    2010-01-01

    In this paper, we compute the generating function of the arguably most important target class of folding algorithms into RNA pseudoknot structures. This class consists of $k$-noncrossing, canonical RNA structures having minimum arc length four and generalizes directly the canonical secondary structures, studied by Schuster {\\it et al.} \\cite{Schuster:98}. The combinatorics of this class is important since, in analogy to the case of secondary structures, generic properties of genotype phenotype maps into RNA pseudoknot structures, like shape space covering \\cite{Schuster:94} and neutral networks \\cite{Reidys:97a} are a result of the combinatorics and not of the particulars of energy parameters. Let ${\\sf Q}_k(n)$ denote the number of these structures over $n$ vertices. We derive exact enumeration results as well as the asymptotic formula ${\\sf Q}_k(n)\\sim c_k n^{-(k-1)^2-\\frac{k-1}{2}}(\\gamma_{\\theta,k})^{-n}$ for $k=3, ..., 9$ and derive a new proof of Schuster's result, ${\\sf Q}_2(n)\\sim 1.4848\\, n^{-3/2}\\,1...

  13. Visualizing Transient Low-Populated Structures of RNA

    Science.gov (United States)

    Dethoff, Elizabeth A.; Petzold, Katja; Chugh, Jeetender; Casiano-Negroni, Anette; Al-Hashimi, Hashim M.

    2012-01-01

    The visualization of RNA conformational changes has provided fundamental insights into how regulatory RNAs carry out their biological functions. The RNA structural transitions that have been characterized to date involve long-lived species that can be captured by structure characterization techniques. Here, we report the Nuclear Magnetic Resonance visualization of RNA transitions towards invisible ‘excited states’ (ES), which exist in too little abundance (2–13%) and for too short periods of time (45–250 μs) to allow structural characterization by conventional techniques. Transitions towards ESs result in localized rearrangements in base-pairing that alter building block elements of RNA architecture, including helix-junction-helix motifs and apical loops. The ES can inhibit function by sequestering residues involved in recognition and signaling or promote ATP-independent strand exchange. Thus, RNAs do not adopt a single conformation, but rather exist in rapid equilibrium with alternative ESs, which can be stabilized by cellular cues to affect functional outcomes. PMID:23041928

  14. Stable RNA hairpins in 88 coding regions of human mRNA

    Institute of Scientific and Technical Information of China (English)

    PAN Min; WANG Chuanming; LIU Ciquan

    2004-01-01

    RNA hairpins containing UNCG, GNRA, CUUG (N=A, U, C or G, R=G or A) loops are unusually thermodynamic stable and conserved structures. The structural features of these hairpin loops are very special, and they play very important roles in vivo. They are prevalent in rRNA, catalytic RNA and non-coding mRNA. However, the 5′ C(UUCG)G 3′ hairpin is not found in the folding structure of 88 human mRNA coding regions. It is also different from rRNA in that there is no preference for certain sequences among tetraloops in these 88 mRNA folding structures.

  15. Atomic structure of a folate/FAD-dependent tRNA T54 methyltransferase.

    Science.gov (United States)

    Nishimasu, Hiroshi; Ishitani, Ryuichiro; Yamashita, Koki; Iwashita, Chikako; Hirata, Akira; Hori, Hiroyuki; Nureki, Osamu

    2009-05-19

    tRNAs from all 3 phylogenetic domains have a 5-methyluridine at position 54 (T54) in the T-loop. The methyl group is transferred from S-adenosylmethionine by TrmA methyltransferase in most Gram-negative bacteria and some archaea and eukaryotes, whereas it is transferred from 5,10-methylenetetrahydrofolate (MTHF) by TrmFO, a folate/FAD-dependent methyltransferase, in most Gram-positive bacteria and some Gram-negative bacteria. However, the catalytic mechanism remains unclear, because the crystal structure of TrmFO has not been solved. Here, we report the crystal structures of Thermus thermophilus TrmFO in its free form, tetrahydrofolate (THF)-bound form, and glutathione-bound form at 2.1-, 1.6-, and 1.05-A resolutions, respectively. TrmFO consists of an FAD-binding domain and an insertion domain, which both share structural similarity with those of GidA, an enzyme involved in the 5-carboxymethylaminomethylation of U34 of some tRNAs. However, the overall structures of TrmFO and GidA are basically different because of their distinct domain orientations, which are consistent with their respective functional specificities. In the THF complex, the pteridin ring of THF is sandwiched between the flavin ring of FAD and the imidazole ring of a His residue. This structure provides a snapshot of the folate/FAD-dependent methyl transfer, suggesting that the transferring methylene group of MTHF is located close to the redox-active N5 atom of FAD. Furthermore, we established an in vitro system to measure the methylation activity. Our TrmFO-tRNA docking model, in combination with mutational analyses, suggests a catalytic mechanism, in which the methylene of MTHF is directly transferred onto U54, and then the exocyclic methylene of U54 is reduced by FADH(2).

  16. Structural analyses of Legionella LepB reveal a new GAP fold that catalytically mimics eukaryotic RasGAP

    Institute of Scientific and Technical Information of China (English)

    Qin Yu; Liyan Hu; Qing Yao; Yongqun Zhu; Na Dong; Da-Cheng Wang; Feng Shao

    2013-01-01

    Rab GTPases are emerging targets of diverse bacterial pathogens.Here,we perform biochemical and structural analyses of LepB,a Rab GTPase-activating protein (GAP) effector from Legionellapneumophila.We map LepB GAP domain to residues 313-618 and show that the GAP domain is Rab1 specific with a catalytic activity higher than the canonical eukaryotic TBC GAP and the newly identified VirA/EspG family of bacterial RabGAP effectors.Exhaustive mutation analyses identify Arg444 as the arginine finger,but no catalytically essential glutamine residues.Crystal structures of LepB313-618 alone and the GAP domain of Legionella drancourtii LepB in complex with Rab1-GDP-AIF3 support the catalytic role of Arg444,and also further reveal a 3D architecture and a GTPase-binding mode distinct from all known GAPs.Glu449,structurally equivalent to TBC RabGAP glutamine finger in apo-LepB,undergoes a drastic movement upon Rab1 binding,which induces Rab1 Gin70 side-chain flipping towards GDP-AIF3 through a strong ionic interaction.This conformationally rearranged Gln70 acts as the catalytic cis-glutamine,therefore uncovering an unexpected RasGAP-like catalytic mechanism for LepB.Our studies highlight an extraordinary structural and catalytic diversity of RabGAPs,particularly those from bacterial pathogens.

  17. 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

  18. Structural and functional insights into caseinolytic proteases reveal an unprecedented regulation principle of their catalytic triad.

    Science.gov (United States)

    Zeiler, Evelyn; List, Anja; Alte, Ferdinand; Gersch, Malte; Wachtel, Rudolf; Poreba, Marcin; Drag, Marcin; Groll, Michael; Sieber, Stephan A

    2013-07-09

    Caseinolytic proteases (ClpPs) are large oligomeric protein complexes that contribute to cell homeostasis as well as virulence regulation in bacteria. Although most organisms possess a single ClpP protein, some organisms encode two or more ClpP isoforms. Here, we elucidated the crystal structures of ClpP1 and ClpP2 from pathogenic Listeria monocytogenes and observe an unprecedented regulation principle by the catalytic triad. Whereas L. monocytogenes (Lm)ClpP2 is both structurally and functionally similar to previously studied tetradecameric ClpP proteins from Escherichia coli and Staphylococcus aureus, heptameric LmClpP1 features an asparagine in its catalytic triad. Mutation of this asparagine to aspartate increased the reactivity of the active site and led to the assembly of a tetradecameric complex. We analyzed the heterooligomeric complex of LmClpP1 and LmClpP2 via coexpression and subsequent labeling studies with natural product-derived probes. Notably, the LmClpP1 peptidase activity is stimulated 75-fold in the complex providing insights into heterooligomerization as a regulatory mechanism. Collectively, our data point toward different preferences for substrates and inhibitors of the two ClpP enzymes and highlight their structural and functional characteristics.

  19. Structure of the catalytic domain of the Clostridium thermocellum cellulase CelT.

    Science.gov (United States)

    Kesavulu, Muppuru M; Tsai, Jia Yin; Lee, Hsiao Lin; Liang, Po Huang; Hsiao, Chwan Deng

    2012-03-01

    Cellulases hydrolyze cellulose, a major component of plant cell walls, to oligosaccharides and monosaccharides. Several Clostridium species secrete multi-enzyme complexes (cellulosomes) containing cellulases. C. thermocellum CelT, a family 9 cellulase, lacks the accessory module(s) necessary for activity, unlike most other family 9 cellulases. Therefore, characterization of the CelT structure is essential in order to understand its catalytic mechanism. Here, the crystal structure of free CelTΔdoc, the catalytic domain of CelT, is reported at 2.1 Å resolution. Its structure differs in several aspects from those of other family 9 cellulases. CelTΔdoc contains an additional α-helix, α-helices of increased length and two additional surface-exposed β-strands. It also contains three calcium ions instead of one as found in C. cellulolyticum Cel9M. CelTΔdoc also has two flexible loops at the open end of its active-site cleft. Movement of these loops probably allows the substrate to access the active site. CelT is stable over a wide range of pH and temperature conditions, suggesting that CelT could be used to convert cellulose biomass into biofuel.

  20. Catalytic and structural diversity of the fluazifop-inducible glutathione transferases from Phaseolus vulgaris.

    Science.gov (United States)

    Chronopoulou, Evangelia; Madesis, Panagiotis; Asimakopoulou, Basiliki; Platis, Dimitrios; Tsaftaris, Athanasios; Labrou, Nikolaos E

    2012-06-01

    Plant glutathione transferases (GSTs) comprise a large family of inducible enzymes that play important roles in stress tolerance and herbicide detoxification. Treatment of Phaseolus vulgaris leaves with the aryloxyphenoxypropionic herbicide fluazifop-p-butyl resulted in induction of GST activities. Three inducible GST isoenzymes were identified and separated by affinity chromatography. Their full-length cDNAs with complete open reading frame were isolated using RACE-RT and information from N-terminal amino acid sequences. Analysis of the cDNA clones showed that the deduced amino acid sequences share high homology with GSTs that belong to phi and tau classes. The three isoenzymes were expressed in E. coli and their substrate specificity was determined towards 20 different substrates. The results showed that the fluazifop-inducible glutathione transferases from P. vulgaris (PvGSTs) catalyze a broad range of reactions and exhibit quite varied substrate specificity. Molecular modeling and structural analysis was used to identify key structural characteristics and to provide insights into the substrate specificity and the catalytic mechanism of these enzymes. These results provide new insights into catalytic and structural diversity of GSTs and the detoxifying mechanism used by P. vulgaris.

  1. Relation between the structure and catalytic activity for automotive emissions. Use of x-ray anomalous dispersion effect

    CERN Document Server

    Mizuki, J; Tanaka, H

    2003-01-01

    The employment of the X-ray anomalous dispersion effect allows us to detect the change in structure of catalytic converters with the environment exposed. Here we show that palladium atoms in a perovskite crystal move into and out of the crystal by anomalous X-ray diffraction and absorption techniques. This movement of the precious metal plays an important role to keep the catalytic activity long-lived. (author)

  2. Investigating the Synthesis, Structure, and Catalytic Properties of Versatile Gold-Based Nanocatalvsts

    Science.gov (United States)

    Pretzer, Lori A.

    Transition metal nanomaterials are used to catalyze many chemical reactions, including those key to environmental, medicinal, and petrochemical fields. Improving their catalytic properties and lifetime would have significant economic and environmental rewards. Potentially expedient options to make such advancements are to alter the shape, size, or composition of transition metal nanocatalysts. This work investigates the relationships between structure and catalytic properties of synthesized Au, Pd-on-Au, and Au-enzyme model transition metal nanocatalysts. Au and Pd-on-Au nanomaterials were studied due to their wide-spread application and structure-dependent electronic and geometric properties. The goal of this thesis is to contribute design procedures and synthesis methods that enable the preparation of more efficient transition metal nanocatalysts. The influence of the size and composition of Pd-on-Au nanoparticles (NPs) was systematically investigated and each was found to affect the catalyst's surface structure and catalytic properties. The catalytic hydrodechlorination of trichloroethene and reduction of 4-nitrophenol by Pd-on-Au nanoparticles were investigated as these reactions are useful for environmental and pharmaceutical synthesis applications, respectively. Structural characterization revealed that the dispersion and oxidation state of surface Pd atoms are controlled by the Au particle size and concentration of Pd. These structural changes are correlated with observed Pd-on-Au NP activities for both probe reactions, providing new insight into the structure-activity relationships of bimetallic nanocatalysts. Using the structure-dependent electronic properties of Au NPs, a new type of light-triggered biocatalyst was prepared and used to remotely control a model biochemical reaction. This biocatalyst consists of a model thermophilic glucokinase enzyme covalently attached to the surface of Au nanorods. The rod-like shape of the Au nanoparticles made the

  3. RNA structures, genomic organization and selection of recombinant HIV.

    Science.gov (United States)

    Simon-Loriere, Etienne; Rossolillo, Paola; Negroni, Matteo

    2011-01-01

    Recombination is an evolutionary mechanism intrinsic to the evolution of many RNA viruses. In retroviruses and notably in the case of HIV, recombination is so frequent that it can be considered as part of its mode of replication. This process not only plays a central role in shaping HIV genetic diversity worldwide, but has also been involved in immune escape and development of resistance to antiviral treatments. Recombination does not create new mutations in the existing genetic repertoire of the virus, but creates new combinations of pre-existing polymorphisms. The simultaneous insertion of multiple substitutions in a single replication cycle leaves little room for the progressive coevolution of regions of proteins, RNA or, more in general, genomes, to accommodate these drastic sequence changes. Therefore, recombination, while allowing the virus to rapidly explore larger sequence space than the slow accumulation of point mutations, also runs the risk of generating non functional viruses. Recombination is the consequence of a switch in the template used during reverse transcription and is promoted by the presence of structured regions in the genomic RNA template. In this review, we discuss new observations suggesting that the distribution of RNA structures along the HIV genome may enhance recombination rates in regions where the resultant progeny is less likely to be impaired, and could therefore maximize the evolutionary value of this source of genetic diversity.

  4. Interaction of Bacillus subtilis Polynucleotide Phosphorylase and RNase Y: STRUCTURAL MAPPING AND EFFECT ON mRNA TURNOVER.

    Science.gov (United States)

    Salvo, Elizabeth; Alabi, Shanique; Liu, Bo; Schlessinger, Avner; Bechhofer, David H

    2016-03-25

    Polynucleotide phosphorylase (PNPase), a 3'-to-5' phosphorolytic exoribonuclease, is thought to be the primary enzyme responsible for turnover ofBacillus subtilismRNA. The role of PNPase inB. subtilismRNA decay has been analyzed previously by comparison of mRNA profiles in a wild-type strainversusa strain that is deleted forpnpA, the gene encoding PNPase. Recent studies have provided evidence for a degradosome-like complex inB. subtilisthat is built around the major decay-initiating endonuclease, RNase Y, and there is ample evidence for a strong interaction between PNPase and RNase Y. The role of the PNPase-RNase Y interaction in the exonucleolytic function of PNPase needs to be clarified. We sought to construct aB. subtilisstrain containing a catalytically active PNPase that could not interact with RNase Y. Mapping studies of the PNPase-RNase Y interaction were guided by a homology model ofB. subtilisPNPase based on the known structure of theEscherichia coliPNPase in complex with an RNase E peptide. Mutations inB. subtilisresidues predicted to be involved in RNase Y binding showed a loss of PNPase-RNase Y interaction. Two mRNAs whose decay is dependent on RNase Y and PNPase were examined in strains containing full-length PNPase that was either catalytically active but unable to interact with RNase Y, or catalytically inactive but able to interact with RNase Y. At least for these two mRNAs, disruption of the PNPase-RNase Y interaction did not appear to affect mRNA turnover.

  5. Dissecting the chemical interactions and substrate structural signatures governing RNA polymerase II trigger loop closure by synthetic nucleic acid analogues

    DEFF Research Database (Denmark)

    Xu, Liang; Butler, Kyle Vincent; Chong, Jenny;

    2014-01-01

    The trigger loop (TL) of RNA polymerase II (Pol II) is a conserved structural motif that is crucial for Pol II catalytic activity and transcriptional fidelity. The TL remains in an inactive open conformation when the mismatched substrate is bound. In contrast, TL switches from an inactive open...... state to a closed active state to facilitate nucleotide addition upon the binding of the cognate substrate to the Pol II active site. However, a comprehensive understanding of the specific chemical interactions and substrate structural signatures that are essential to this TL conformational change...... II. This study reveals novel insights into understanding the molecular basis of TL conformational transition upon substrate binding during Pol II transcription. This synthetic chemical biology approach may be extended to understand the mechanisms of other RNA polymerases as well as other nucleic acid...

  6. Prediction of RNA Secondary Structure Based on Particle Swarm Optimization

    Institute of Scientific and Technical Information of China (English)

    LIU Yuan-ning; DONG Hao; ZHANG Hao; WANG Gang; LI Zhi; CHEN Hui-ling

    2011-01-01

    A novel method for the prediction of RNA secondary structure was proposed based on the particle swarm optimization(PSO). PSO is known to be effective in solving many different types of optimization problems and known for being able to approximate the global optimal results in the solution space. We designed an efficient objective function according to the minimum free energy, the number of selected stems and the average length of selected stems. We calculated how many legal stems there were in the sequence, and selected some of them to obtain an optimal result using PSO in the right of the objective function. A method based on the improved particle swarm optimization(IPSO) was proposed to predict RNA secondary structure, which consisted of three stages. The first stage was applied to e ncoding the source sequences, and to exploring all the legal stems. Then, a set of encoded stems were created in order to prepare input data for the second stage. In the second stage, IPSO was responsible for structure selection. At last, the optimal result was obtained from the secondary structures selected via IPSO. Nine sequences from the comparative RNA website were selected for the evaluation of the proposed method. Compared with other six methods, the proposed method decreased the complexity and enhanced the sensitivity and specificity on the basis of the experiment results.

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

    CERN Document Server

    Han, Hillary S W

    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 structures derived via the Turner energy parameters contain only finitely many complex irreducible substructures and just minor parameter changes produce a class of mfe-structures that contain a large number of small irreducibles. We localize the exact point where the distribution of irreducibles experiences 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 and space reduction and that the sparsifcation of the folding of structures with modified parameters leads to a lin...

  8. Exoelectrogenic biofilm as a template for sustainable formation of a catalytic mesoporous structure

    KAUST Repository

    Yates, Matthew D.

    2014-06-04

    © 2014 Wiley Periodicals, Inc. Actively respiring biofilms of Geobacter sulfurreducens were used as a biotemplate to form a palladium mesoporous layer directly on an electrode surface. Cells and proteins within the biofilm acted as the reductant and stabilizer to facilitate the reduction, dispersion, and attachment of palladium nanoparticles to the electrode surface without using synthetic chemicals. © 2014 Wiley Periodicals, Inc. Mesoporous structures can increase catalytic activity by maximizing the ratio of surface area to volume, but current synthesis techniques utilize expensive polymers and toxic chemicals. A Geobacter sulfurreducens biofilm was used as a sustainable template to form mesoporous Pd structures while eliminating the need for synthetic chemicals. The bulk of the biofilm material was removed by thermal treatments after nanoparticle formation, producing a catalytic Pd mesoporous (pore size 9.7±0.1nm) structure attached to the graphite electrode with a 1.5-2μm thick backbone composed of nanoparticles (~200nm). A control electrode electrochemically plated with Pd in the absence of a biofilm exhibited a variable planar Pd base (~0.5-3μm thick) with sporadic Pd extrusions (~2μm across, 1-5μm tall) from the surface. The biotemplated mesoporous structure produced 15-20% higher stable current densities during H2 oxidation tests than the electrochemically plated control electrode, even though 30% less Pd was present in the biotemplated catalyst. These results indicate that electroactive biofilms can be used as a sustainable base material to produce nanoporous structures without the need for synthetic polymers. Biotechnol. Bioeng. 2014;111: 2349-2354.

  9. The interaction between the yeast telomerase RNA and the Est1 protein requires three structural elements.

    Science.gov (United States)

    Lubin, Johnathan W; Tucey, Timothy M; Lundblad, Victoria

    2012-09-01

    In the budding yeast Saccharomyces cerevisiae, the telomerase enzyme is composed of a 1.3-kb TLC1 RNA that forms a complex with Est2 (the catalytic subunit) and two regulatory proteins, Est1 and Est3. Previous work has identified a conserved 5-nt bulge, present in a long helical arm of TLC1, which mediates binding of Est1 to TLC1. However, increased expression of Est1 can bypass the consequences of removal of this RNA bulge, indicating that there are additional binding site(s) for Est1 on TLC1. We report here that a conserved single-stranded internal loop immediately adjacent to the bulge is also required for the Est1-RNA interaction; furthermore, a TLC1 variant that lacks this internal loop but retains the bulge cannot be suppressed by Est1 overexpression, arguing that the internal loop may be a more critical element for Est1 binding. An additional structural feature consisting of a single-stranded region at the base of the helix containing the bulge and internal loop also contributes to recognition of TLC1 by Est1, potentially by providing flexibility to this helical arm. Association of Est1 with each of these TLC1 motifs was assessed using a highly sensitive biochemical assay that simultaneously monitors the relative levels of the Est1 and Est2 proteins in the telomerase complex. The identification of three elements of TLC1 that are required for Est1 association provides a detailed view of this particular protein-RNA interaction.

  10. 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...... structures derived via the Turner energy parameters contain only finitely many complex irreducible substructures and just minor parameter changes produce a class of mfe-structures that contain a large number of small irreducibles. We localize the exact point where the distribution of irreducibles experiences...... 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...

  11. Base pairing in RNA structures: A computational analysis of structural aspects and interaction energies

    Indian Academy of Sciences (India)

    Purshotam Sharma; Abhijit Mitra; Sitansh Sharma; Harjinder Singh

    2007-09-01

    The base pairing patterns in RNA structures are more versatile and completely different as compared to DNA. We present here results of ab-initio studies of structures and interaction energies of eight selected RNA base pairs reported in literature. Interaction energies, including BSSE correction, of hydrogen added crystal geometries of base pairs have been calculated at the HF/6-31G∗∗ level. The structures and interaction energies of the base pairs in the crystal geometry are compared with those obtained after optimization of the base pairs. We find that the base pairs become more planar on full optimization. No change in the hydrogen bonding pattern is seen. It is expected that the inclusion of appropriate considerations of many of these aspects of RNA base pairing would significantly improve the accuracy of RNA secondary structure prediction.

  12. Two Classes of Bacterial IMPDHs according to Their Quaternary Structures and Catalytic Properties

    Science.gov (United States)

    Alexandre, Thomas; Rayna, Bertrand; Munier-Lehmann, Hélène

    2015-01-01

    Inosine-5′-monophosphate dehydrogenase (IMPDH) occupies a key position in purine nucleotide metabolism. In this study, we have performed the biochemical and physico-chemical characterization of eight bacterial IMPDHs, among which six were totally unexplored. This study led to a classification of bacterial IMPDHs according to the regulation of their catalytic properties and their quaternary structures. Class I IMPDHs are cooperative enzymes for IMP, which are activated by MgATP and are octameric in all tested conditions. On the other hand, class II IMPDHs behave as Michaelis-Menten enzymes for both substrates and are tetramers in their apo state or in the presence of IMP, which are shifted to octamers in the presence of NAD or MgATP. Our work provides new insights into the IMPDH functional regulation and a model for the quaternary structure modulation is proposed. PMID:25706619

  13. 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.

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

    Science.gov (United States)

    Okada, Yohei; Saito, Yutaka; Sato, Kengo; Sakakibara, Yasubumi

    2011-01-01

    Identification of non-protein-coding RNAs (ncRNAs) in genomes is a crucial task for not only molecular cell biology but also bioinformatics. Secondary structures of ncRNAs are employed as a key feature of ncRNA analysis since biological functions of ncRNAs are deeply related to their secondary structures. Although the minimum free energy (MFE) structure of an RNA sequence is regarded as the most stable structure, MFE alone could not be an appropriate measure for identifying ncRNAs since the free energy is heavily biased by the nucleotide composition. Therefore, instead of MFE itself, several alternative measures for identifying ncRNAs have been proposed such as the structure conservation index (SCI) and the base pair distance (BPD), both of which employ MFE structures. However, these measurements are unfortunately not suitable for identifying ncRNAs in some cases including the genome-wide search and incur high false discovery rate. In this study, we propose improved measurements based on SCI and BPD, applying generalized centroid estimators to incorporate the robustness against low quality multiple alignments. Our experiments show that our proposed methods achieve higher accuracy than the original SCI and BPD for not only human-curated structural alignments but also low quality alignments produced by CLUSTAL W. Furthermore, the centroid-based SCI on CLUSTAL W alignments is more accurate than or comparable with that of the original SCI on structural alignments generated with RAF, a high quality structural aligner, for which twofold expensive computational time is required on average. We conclude that our methods are more suitable for genome-wide alignments which are of low quality from the point of view on secondary structures than the original SCI and BPD.

  15. 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.

  16. Preparation, structure and catalytic performances of rare earth catalytic materials%稀土催化材料的制备、结构及催化性能

    Institute of Scientific and Technical Information of China (English)

    詹望成; 郭耘; 郭杨龙; 龚学庆; 王艳芹; 卢冠忠

    2012-01-01

    The research and development of rare earth catalytic materials will greatly promote the high-efficiency utilization of abundant rare earth elements, such as La and Ce. Rare earth elements have under-filled 4f orbits and lanthanide contraction, resulting in their unique catalytic performances when used as an active component or the carrier of catalysts. This paper reviews the progress in the preparation, structural and catalytic properties of rare earth catalytic materials, containing rare earth oxides, rare earth composite oxides, rare earth-noble metal catalysts and rare earth modified porous materials. The effects of rare earth elements on the structures, activities and stabilities of the concerned catalysts are mainly described. As well as, the interactions between rare earth metals and transition metals or noble metals are also elaborated, which are critical for the performances of the catalysts. Finally, the development thinking for rare earth catalytic materials is put forwarded and outlooked.%稀土催化材料的研究和发展为La和Ce等高丰度轻稀土元素的高质、高效利用提供了有效的途径.稀土元素具有未充满电子的4f轨道和镧系收缩等特征,作为催化剂的活性组分或载体使用时表现出独特的催化性能.本文从稀土氧化物、稀土复合氧化物、稀土-贵金属催化剂、稀土改性多孔催化材料等稀土催化材料出发,重点介绍和讨论了稀土的添加对催化剂的结构、活性和稳定性等的影响,阐述了稀土与过渡金属及氧化物、稀土与贵金属之间的相互作用,及对催化剂催化性能的影响.并对稀土催化材料的研究和发展提出了思考和展望.

  17. Statistical mechanics of secondary structures formed by random RNA sequences.

    Science.gov (United States)

    Bundschuh, R; Hwa, T

    2002-03-01

    The formation of secondary structures by a random RNA sequence is studied as a model system for the sequence-structure problem omnipresent in biopolymers. Several toy energy models are introduced to allow detailed analytical and numerical studies. First, a two-replica calculation is performed. By mapping the two-replica problem to the denaturation of a single homogeneous RNA molecule in six-dimensional embedding space, we show that sequence disorder is perturbatively irrelevant, i.e., an RNA molecule with weak sequence disorder is in a molten phase where many secondary structures with comparable total energy coexist. A numerical study of various models at high temperature reproduces behaviors characteristic of the molten phase. On the other hand, a scaling argument based on the external statistics of rare regions can be constructed to show that the low-temperature phase is unstable to sequence disorder. We performed a detailed numerical study of the low-temperature phase using the droplet theory as a guide, and characterized the statistics of large-scale, low-energy excitations of the secondary structures from the ground state structure. We find the excitation energy to grow very slowly (i.e., logarithmically) with the length scale of the excitation, suggesting the existence of a marginal glass phase. The transition between the low-temperature glass phase and the high-temperature molten phase is also characterized numerically. It is revealed by a change in the coefficient of the logarithmic excitation energy, from being disorder dominated to being entropy dominated.

  18. MTO Schiff-base complexes: synthesis, structures and catalytic applications in olefin epoxidation.

    Science.gov (United States)

    Zhou, Ming-Dong; Zhao, Jin; Li, Jun; Yue, Shuang; Bao, Chang-Nian; Mink, Janos; Zang, Shu-Liang; Kühn, Fritz E

    2007-01-01

    Several Schiff-base ligands readily form complexes with methyltrioxorhenium(VII) (MTO) by undergoing a hydrogen transfer from a ligand-bound OH group to a ligand N atom. The resulting complexes are stable at room temperature and can be handled and stored in air without problems. Due to the steric demands of the ligands they display distorted trigonal-bipyramidal structures in the solid state, as shown by X-ray crystallography, with the O(-) moiety binding to the Lewis acidic Re atom and the Re-bound methyl group being located either in cis or trans position to the Schiff base. In solution, however, the steric differences seem not to be maintained, as can be deduced from (17)O NMR spectroscopy. Furthermore, the Schiff-base ligands exchange with donor ligands. Nevertheless, the catalytic behaviour is influenced significantly by the Schiff bases coordinated to the MTO moiety, which lead either to high selectivities and good activities or to catalyst decomposition. A large excess of ligand, in contrast to the observations with aromatic N-donor ligands, is detrimental to the catalytic performance as it leads to catalyst decomposition.

  19. Analysis of the Staphylococcus aureus DgkB structure reveals a common catalytic mechanism for the soluble diacylglycerol kinases.

    Science.gov (United States)

    Miller, Darcie J; Jerga, Agoston; Rock, Charles O; White, Stephen W

    2008-07-01

    Soluble diacylglycerol (DAG) kinases function as regulators of diacylglycerol metabolism in cell signaling and intermediary metabolism. We report the structure of a DAG kinase, DgkB from Staphylococcus aureus, both as the free enzyme and in complex with ADP. The molecule is a tight homodimer, and each monomer comprises two domains with the catalytic center located within the interdomain cleft. Two distinctive features of DkgB are a structural Mg2+ site and an associated Asp*water*Mg2+ network that extends toward the active site locale. Site-directed mutagenesis revealed that these features play important roles in the catalytic mechanism. The key active site residues and the components of the Asp*water*Mg2+ network are conserved in the catalytic cores of the mammalian signaling DAG kinases, indicating that these enzymes use the same mechanism and have similar structures as DgkB.

  20. Analysis of the Staphylococcus aureus DgkB Structure Reveals a Common Catalytic Mechanism for the Soluble Diacylglycerol Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Miller, Darcie J.; Jerga, Agoston; Rock, Charles O.; White, Stephen W. (SJCH)

    2008-08-11

    Soluble diacylglycerol (DAG) kinases function as regulators of diacylglycerol metabolism in cell signaling and intermediary metabolism. We report the structure of a DAG kinase, DgkB from Staphylococcus aureus, both as the free enzyme and in complex with ADP. The molecule is a tight homodimer, and each monomer comprises two domains with the catalytic center located within the interdomain cleft. Two distinctive features of DkgB are a structural Mg{sup 2+} site and an associated Asp{center_dot}water{center_dot}Mg{sup 2+} network that extends toward the active site locale. Site-directed mutagenesis revealed that these features play important roles in the catalytic mechanism. The key active site residues and the components of the Asp{center_dot}water{center_dot}Mg{sup 2+} network are conserved in the catalytic cores of the mammalian signaling DAG kinases, indicating that these enzymes use the same mechanism and have similar structures as DgkB.

  1. GTfold: Enabling parallel RNA secondary structure prediction on multi-core desktops

    DEFF Research Database (Denmark)

    Swenson, M Shel; Anderson, Joshua; Ash, Andrew

    2012-01-01

    Accurate and efficient RNA secondary structure prediction remains an important open problem in computational molecular biology. Historically, advances in computing technology have enabled faster and more accurate RNA secondary structure predictions. Previous parallelized prediction programs achie...

  2. RNA Secondary Structure Modulates FMRP’s Bi-Functional Role in the MicroRNA Pathway

    Directory of Open Access Journals (Sweden)

    Phillip Kenny

    2016-06-01

    Full Text Available MicroRNAs act by post-transcriptionally regulating the gene expression of 30%–60% of mammalian genomes. MicroRNAs are key regulators in all cellular processes, though the mechanism by which the cell activates or represses microRNA-mediated translational regulation is poorly understood. In this review, we discuss the RNA binding protein Fragile X Mental Retardation Protein (FMRP and its role in microRNA-mediated translational regulation. Historically, FMRP is known to function as a translational suppressor. However, emerging data suggests that FMRP has both an agonistic and antagonistic role in regulating microRNA-mediated translational suppression. This bi-functional role is dependent on FMRP’s interaction with the RNA helicase Moloney leukemia virus 10 (MOV10, which modifies the structural landscape of bound mRNA, therefore facilitating or inhibiting its association with the RNA-Induced Silencing Complex.

  3. 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.

  4. RNA nanoparticles come of age

    Institute of Scientific and Technical Information of China (English)

    John J.Rossi

    2011-01-01

    @@ RNA has multiple functions in nature, including informa- tional transfer (mRNA) Ill, adaptor function (tRNAs) [2], guide functions (snRNAs, snoRNAs) [3,4]catalytic func- tion (ribozymes and the large ribosomal RNA) [5-7], and environmental sensing (riboswitehes) [8].In contrast, DNA only serves as an information storage molecule, and proteins serve as structural and enzymatic molecules.

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

    Science.gov (United States)

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

    2016-01-01

    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 mutations in vivo.

  6. 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

  7. 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.

  8. 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…

  9. Modeling RNA topological structures using small angle X-ray scattering.

    Science.gov (United States)

    Bhandari, Yuba R; Jiang, Wei; Stahlberg, Eric A; Stagno, Jason R; Wang, Yun-Xing

    2016-07-01

    Detailed understanding of the structure and function relationship of RNA requires knowledge about RNA three-dimensional (3D) topological folding. However, there are very few unique RNA entries in structure databases. This is due to challenges in determining 3D structures of RNA using conventional methods, such as X-ray crystallography and NMR spectroscopy, despite significant advances in both of these technologies. Computational methods have come a long way in accurately predicting the 3D structures of small (topological structures, including a new method that combines secondary structural information and SAXS data to sample conformations generated through hierarchical moves of commonly observed RNA motifs.

  10. The La protein functions redundantly with tRNA modification enzymes to ensure tRNA structural stability.

    Science.gov (United States)

    Copela, Laura A; Chakshusmathi, Ghadiyaram; Sherrer, R Lynn; Wolin, Sandra L

    2006-04-01

    Although the La protein stabilizes nascent pre-tRNAs from nucleases, influences the pathway of pre-tRNA maturation, and assists correct folding of certain pre-tRNAs, it is dispensable for growth in both budding and fission yeast. Here we show that the Saccharomyces cerevisiae La shares functional redundancy with both tRNA modification enzymes and other proteins that contact tRNAs during their biogenesis. La is important for growth in the presence of mutations in either the arginyl tRNA synthetase or the tRNA modification enzyme Trm1p. In addition, two pseudouridine synthases, PUS3 and PUS4, are important for growth in strains carrying a mutation in tRNA(Arg)(CCG) and are essential when La is deleted in these strains. Depletion of Pus3p results in accumulation of the aminoacylated mutant tRNA(Arg)(CCG) in nuclei, while depletion of Pus4p results in decreased stability of the mutant tRNA. Interestingly, the degradation of mutant unstable forms of tRNA(Arg)(CCG) does not require the Trf4p poly(A) polymerase, suggesting that yeast cells possess multiple pathways for tRNA decay. These data demonstrate that La functions redundantly with both tRNA modifications and proteins that associate with tRNAs to achieve tRNA structural stability and efficient biogenesis.

  11. RNA 3D Structure Modeling by Combination of Template-Based Method ModeRNA, Template-Free Folding with SimRNA, and Refinement with QRNAS.

    Science.gov (United States)

    Piatkowski, Pawel; Kasprzak, Joanna M; Kumar, Deepak; Magnus, Marcin; Chojnowski, Grzegorz; Bujnicki, Janusz M

    2016-01-01

    RNA encompasses an essential part of all known forms of life. The functions of many RNA molecules are dependent on their 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. To address this problem, computational structure prediction methods were developed that either utilize information derived from known structures of other RNA molecules (by way of template-based modeling) or attempt to simulate the physical process of RNA structure formation (by way of template-free modeling). All computational methods suffer from various limitations that make theoretical models less reliable than high-resolution experimentally determined structures. This chapter provides a protocol for computational modeling of RNA 3D structure that overcomes major limitations by combining two complementary approaches: template-based modeling that is capable of predicting global architectures based on similarity to other molecules but often fails to predict local unique features, and template-free modeling that can predict the local folding, but is limited to modeling the structure of relatively small molecules. Here, we combine the use of a template-based method ModeRNA with a template-free method SimRNA. ModeRNA requires a sequence alignment of the target RNA sequence to be modeled with a template of the known structure; it generates a model that predicts the structure of a conserved core and provides a starting point for modeling of variable regions. SimRNA can be used to fold small RNAs (models for larger RNAs that have a correctly modeled core. ModeRNA can be either downloaded, compiled and run locally or run through a web interface at http://genesilico.pl/modernaserver/ . SimRNA is currently available to download for local use as a precompiled software package at http://genesilico.pl/software/stand-alone/simrna and as a

  12. Probing RNA Native Conformational Ensembles with Structural Constraints.

    Science.gov (United States)

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

    2016-05-01

    Noncoding ribonucleic acids (RNA) play a critical role in a wide variety of cellular processes, ranging from regulating gene expression to post-translational modification and protein synthesis. Their activity is modulated by highly dynamic exchanges between three-dimensional conformational 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 by distance constraints in the tertiary structure. The dimensionality reduction enables efficient exploration of conformational space. We show that the conformational distributions obtained with our method broadly sample the conformational landscape observed in NMR experiments. Compared to normal mode analysis-based exploration, our procedure diffuses faster through the experimental ensemble while also accessing conformational substates to greater precision. Our results suggest that conformational sampling with a highly reduced but fully atomistic representation of noncoding RNA expresses key features of their dynamic nature.

  13. Local Exact Pattern Matching for Non-Fixed RNA Structures.

    Science.gov (United States)

    Amit, Mika; Backofen, Rolf; Heyne, Steffen; Landau, Gad M; Möhl, Mathias; Otto, Christina; Will, Sebastian

    2014-01-01

    Detecting local common sequence-structure regions of RNAs is a biologically important problem. Detecting such regions allows biologists to identify functionally relevant similarities between the inspected molecules. We developed dynamic programming algorithms for finding common structure-sequence patterns between two RNAs. The RNAs are given by their sequence and a set of potential base pairs with associated probabilities. In contrast to prior work on local pattern matching of RNAs, we support the breaking of arcs. This allows us to add flexibility over matching only fixed structures; potentially matching only a similar subset of specified base pairs. We present an O(n(3)) algorithm for local exact pattern matching between two nested RNAs, and an O(n(3) log n) algorithm for one nested RNA and one bounded-unlimited RNA. In addition, an algorithm for approximate pattern matching is introduced that for two given nested RNAs and a number k, finds the maximal local pattern matching score between the two RNAs with at most k mismatches in O(n(3)k(2)) time. Finally, we present an O(n(3)) algorithm for finding the most similar subforest between two nested RNAs.

  14. Structural changes in nanostructured catalytic oxides monitored by Raman spectroscopy: Effect of the laser heating

    Science.gov (United States)

    Oliveira, Alcemira C.; da Silva, Antonio N.; Junior, Jose Alves L.; Freire, Paulo T. C.; Oliveira, Alcineia C.; Filho, Josué M.

    2017-03-01

    The laser power effects on the structural properties of nanostructured oxides were studied by Raman spectroscopy. The nanostructured CeO2, ZrO2, SnO2, TiO2 and MnOx oxides were prepared by a nanocasting route and characterized through various physicochemical techniques. The structural features of the solids were accompanied by varying the incident laser power from 2.0 to 9.1 mW. The laser caused local heating on the surface of the nanostructured solids and influenced on their particle sizes. The CeO2, TiO2 and MnOx spectra exhibited particle size changes due to thermal effects. Elevated laser power up to 9.1 mW accelerated the sintering of CeO2, TiO2 and MnOx particles in contrast to SnO2 counterparts. Simultaneously, the creation of defects in the aforesaid oxide structures was suggested upon increasing the laser power from 2.0 to 9.1 mW. The phase transformation from MnOx-related phases to α-Mn2O3 and the oxidation of these phases were observed. Tetragonal ZrO2 showed a very stable structure under laser heating, envisaging further catalytic applications upon using mild laser power.

  15. Crystal structures of catalytic and regulatory subunits of rat protein kinase CK2

    Institute of Scientific and Technical Information of China (English)

    ZHOU WeiHong; SHEN YueQuan; QIN XiaoHong; YAN XiaoJie; XIE XingQiao; LI Liang; FANG ShaSha; LONG JiaFu; ADELMAN John; TANG Wei-Jen

    2009-01-01

    Protein kinase CK2 consists of two catalytic subunits (CK2α) and two regulatory subunits (CK2β). Here,we report the crystal structures of rat CK2α mutant (rCK2α-△C, 1-335) and CK2β(rCK2β). The overall topology of rCK2α-△C and rCK2βare very similar to the human enzyme, although large structural dif-ferences could be observed in the N-terminal domain of rCK2α-△C. Our reported structure of rCK2α-△C is in the close conformation state while the counterpart hCK2α is in the open conformation state, indi-cating the conformation of CK2α molecule has high plasticity. The structure of rCK2β represents the conformation of free CK2β. Upon CK2α binding, the C-terminal region undergoes a drastic conforma-tional change. The major region of interaction within the interface of CK2αCK2β may serve as a bridge to transmit the conformational change and thus regulate the activity of CK2α.

  16. Catalytic activity of bimetallic catalysts highly sensitive to the atomic composition and phase structure at the nanoscale.

    Science.gov (United States)

    Shan, Shiyao; Petkov, Valeri; Prasai, Binay; Wu, Jinfang; Joseph, Pharrah; Skeete, Zakiya; Kim, Eunjoo; Mott, Derrick; Malis, Oana; Luo, Jin; Zhong, Chuan-Jian

    2015-12-07

    The ability to determine the atomic arrangement in nanoalloy catalysts and reveal the detailed structural features responsible for the catalytically active sites is essential for understanding the correlation between the atomic structure and catalytic properties, enabling the preparation of efficient nanoalloy catalysts by design. Herein we describe a study of CO oxidation over PdCu nanoalloy catalysts focusing on gaining insights into the correlation between the atomic structures and catalytic activity of nanoalloys. PdCu nanoalloys of different bimetallic compositions are synthesized as a model system and are activated by a controlled thermochemical treatment for assessing their catalytic activity. The results show that the catalytic synergy of Pd and Cu species evolves with both the bimetallic nanoalloy composition and temperature of the thermochemical treatment reaching a maximum at a Pd : Cu ratio close to 50 : 50. The nanoalloys are characterized structurally by ex situ and in situ synchrotron X-ray diffraction, including atomic pair distribution function analysis. The structural data show that, depending on the bimetallic composition and treatment temperature, PdCu nanoalloys adopt two different structure types. One features a chemically ordered, body centered cubic (B2) type alloy consisting of two interpenetrating simple cubic lattices, each occupied with Pd or Cu species alone, and the other structure type features a chemically disordered, face-centered cubic (fcc) type of alloy wherein Pd and Cu species are intermixed at random. The catalytic activity for CO oxidation is strongly influenced by the structural features. In particular, it is revealed that the prevalence of chemical disorder in nanoalloys with a Pd : Cu ratio close to 50 : 50 makes them superior catalysts for CO oxidation in comparison with the same nanoalloys of other bimetallic compositions. However, the catalytic synergy can be diminished if the Pd50Cu50 nanoalloys undergo

  17. Sequence-structure-function relationships of a tRNA (m7G46) methyltransferase studied by homology modeling and site-directed mutagenesis.

    Science.gov (United States)

    Purta, Elzbieta; van Vliet, Françoise; Tricot, Catherine; De Bie, Lara G; Feder, Marcin; Skowronek, Krzysztof; Droogmans, Louis; Bujnicki, Janusz M

    2005-05-15

    The Escherichia coli TrmB protein and its Saccharomyces cerevisiae ortholog Trm8p catalyze the S-adenosyl-L-methionine-dependent formation of 7-methylguanosine at position 46 (m7G46) in tRNA. To learn more about the sequence-structure-function relationships of these enzymes we carried out a thorough bioinformatics analysis of the tRNA:m7G methyltransferase (MTase) family to predict sequence regions and individual amino acid residues that may be important for the interactions between the MTase and the tRNA substrate, in particular the target guanosine 46. We used site-directed mutagenesis to construct a series of alanine substitutions and tested the activity of the mutants to elucidate the catalytic and tRNA-recognition mechanism of TrmB. The functional analysis of the mutants, together with the homology model of the TrmB structure and the results of the phylogenetic analysis, revealed the crucial residues for the formation of the substrate-binding site and the catalytic center in tRNA:m7G MTases.

  18. 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.

  19. Avian reovirus L2 genome segment sequences and predicted structure/function of the encoded RNA-dependent RNA polymerase protein

    Directory of Open Access Journals (Sweden)

    Xu Wanhong

    2008-12-01

    Full Text Available Abstract Background The orthoreoviruses are infectious agents that possess a genome comprised of 10 double-stranded RNA segments encased in two concentric protein capsids. Like virtually all RNA viruses, an RNA-dependent RNA polymerase (RdRp enzyme is required for viral propagation. RdRp sequences have been determined for the prototype mammalian orthoreoviruses and for several other closely-related reoviruses, including aquareoviruses, but have not yet been reported for any avian orthoreoviruses. Results We determined the L2 genome segment nucleotide sequences, which encode the RdRp proteins, of two different avian reoviruses, strains ARV138 and ARV176 in order to define conserved and variable regions within reovirus RdRp proteins and to better delineate structure/function of this important enzyme. The ARV138 L2 genome segment was 3829 base pairs long, whereas the ARV176 L2 segment was 3830 nucleotides long. Both segments were predicted to encode λB RdRp proteins 1259 amino acids in length. Alignments of these newly-determined ARV genome segments, and their corresponding proteins, were performed with all currently available homologous mammalian reovirus (MRV and aquareovirus (AqRV genome segment and protein sequences. There was ~55% amino acid identity between ARV λB and MRV λ3 proteins, making the RdRp protein the most highly conserved of currently known orthoreovirus proteins, and there was ~28% identity between ARV λB and homologous MRV and AqRV RdRp proteins. Predictive structure/function mapping of identical and conserved residues within the known MRV λ3 atomic structure indicated most identical amino acids and conservative substitutions were located near and within predicted catalytic domains and lining RdRp channels, whereas non-identical amino acids were generally located on the molecule's surfaces. Conclusion The ARV λB and MRV λ3 proteins showed the highest ARV:MRV identity values (~55% amongst all currently known ARV and MRV

  20. Structural analyses of Avocado sunblotch viroid reveal differences in the folding of plus and minus RNA strands.

    Science.gov (United States)

    Delan-Forino, Clémentine; Deforges, Jules; Benard, Lionel; Sargueil, Bruno; Maurel, Marie-Christine; Torchet, Claire

    2014-01-29

    Viroids are small pathogenic circular single-stranded RNAs, present in two complementary sequences, named plus and minus, in infected plant cells. A high degree of complementarities between different regions of the RNAs allows them to adopt complex structures. Since viroids are naked non-coding RNAs, interactions with host factors appear to be closely related to their structural and catalytic characteristics. Avocado sunblotch viroid (ASBVd), a member of the family Avsunviroidae, replicates via a symmetric RNA-dependant rolling-circle process, involving self-cleavage via hammerhead ribozymes. Consequently, it is assumed that ASBVd plus and minus strands adopt similar structures. Moreover, by computer analyses, a quasi-rod-like secondary structure has been predicted. Nevertheless, secondary and tertiary structures of both polarities of ASBVd remain unsolved. In this study, we analyzed the characteristic of each strand of ASBVd through biophysical analyses. We report that ASBVd transcripts of plus and minus polarities exhibit differences in electrophoretic mobility under native conditions and in thermal denaturation profiles. Subsequently, the secondary structures of plus and minus polarities of ASBVd were probed using the RNA-selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) method. The models obtained show that both polarities fold into different structures. Moreover, our results suggest the existence of a kissing-loop interaction within the minus strand that may play a role in in vivo viroid life cycle.

  1. Structural Analyses of Avocado sunblotch viroid Reveal Differences in the Folding of Plus and Minus RNA Strands

    Science.gov (United States)

    Delan-Forino, Clémentine; Deforges, Jules; Benard, Lionel; Sargueil, Bruno; Maurel, Marie-Christine; Torchet, Claire

    2014-01-01

    Viroids are small pathogenic circular single-stranded RNAs, present in two complementary sequences, named plus and minus, in infected plant cells. A high degree of complementarities between different regions of the RNAs allows them to adopt complex structures. Since viroids are naked non-coding RNAs, interactions with host factors appear to be closely related to their structural and catalytic characteristics. Avocado sunblotch viroid (ASBVd), a member of the family Avsunviroidae, replicates via a symmetric RNA-dependant rolling-circle process, involving self-cleavage via hammerhead ribozymes. Consequently, it is assumed that ASBVd plus and minus strands adopt similar structures. Moreover, by computer analyses, a quasi-rod-like secondary structure has been predicted. Nevertheless, secondary and tertiary structures of both polarities of ASBVd remain unsolved. In this study, we analyzed the characteristic of each strand of ASBVd through biophysical analyses. We report that ASBVd transcripts of plus and minus polarities exhibit differences in electrophoretic mobility under native conditions and in thermal denaturation profiles. Subsequently, the secondary structures of plus and minus polarities of ASBVd were probed using the RNA-selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) method. The models obtained show that both polarities fold into different structures. Moreover, our results suggest the existence of a kissing-loop interaction within the minus strand that may play a role in in vivo viroid life cycle. PMID:24481250

  2. Structural Analyses of Avocado sunblotch viroid Reveal Differences in the Folding of Plus and Minus RNA Strands

    Directory of Open Access Journals (Sweden)

    Clémentine Delan-Forino

    2014-01-01

    Full Text Available Viroids are small pathogenic circular single-stranded RNAs, present in two complementary sequences, named plus and minus, in infected plant cells. A high degree of complementarities between different regions of the RNAs allows them to adopt complex structures. Since viroids are naked non-coding RNAs, interactions with host factors appear to be closely related to their structural and catalytic characteristics. Avocado sunblotch viroid (ASBVd, a member of the family Avsunviroidae, replicates via a symmetric RNA-dependant rolling-circle process, involving self-cleavage via hammerhead ribozymes. Consequently, it is assumed that ASBVd plus and minus strands adopt similar structures. Moreover, by computer analyses, a quasi-rod-like secondary structure has been predicted. Nevertheless, secondary and tertiary structures of both polarities of ASBVd remain unsolved. In this study, we analyzed the characteristic of each strand of ASBVd through biophysical analyses. We report that ASBVd transcripts of plus and minus polarities exhibit differences in electrophoretic mobility under native conditions and in thermal denaturation profiles. Subsequently, the secondary structures of plus and minus polarities of ASBVd were probed using the RNA-selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE method. The models obtained show that both polarities fold into different structures. Moreover, our results suggest the existence of a kissing-loop interaction within the minus strand that may play a role in in vivo viroid life cycle.

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

    Science.gov (United States)

    Han, Hillary S W; Reidys, Christian M

    2012-10-01

    In this article 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 is not sequence-specific, we prove the following dichotomy result. Mfe structures derived via the Turner energy parameters contain only finitely many complex irreducible substructures, and just minor parameter changes produce a class of mfe structures that contain a large number of small irreducibles. We localize the exact point at which the distribution of irreducibles experiences 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 and space reduction, and that the sparsification of the folding of structures with modified parameters leads to a linear time and space reduction. We, furthermore, identify the limit distribution at the phase transition as a Rayleigh distribution.

  4. Targeting Non-Catalytic Cysteine Residues Through Structure-Guided Drug Discovery.

    Science.gov (United States)

    Hallenbeck, Kenneth K; Turner, David M; Renslo, Adam R; Arkin, Michelle R

    2017-01-01

    The targeting of non-catalytic cysteine residues with small molecules is drawing increased attention from drug discovery scientists and chemical biologists. From a biological perspective, genomic and proteomic studies have revealed the presence of cysteine mutations in several oncogenic proteins, suggesting both a functional role for these residues and also a strategy for targeting them in an 'allele specific' manner. For the medicinal chemist, the structure-guided design of cysteine- reactive molecules is an appealing strategy to realize improved selectivity and pharmacodynamic properties in drug leads. Finally, for chemical biologists, the modification of cysteine residues provides a unique means to probe protein structure and allosteric regulation. Here, we review three applications of cysteinemodifying small molecules: 1) the optimization of existing drug leads, 2) the discovery of new lead compounds, and 3) the use of cysteine-reactive molecules as probes of protein dynamics. In each case, structure-guided design plays a key role in determining which cysteine residue(s) to target and in designing compounds with the proper geometry to enable both covalent interaction with the targeted cysteine and productive non-covalent interactions with nearby protein residues.

  5. Catalytic conversion of aliphatic alcohols on carbon nanomaterials: The roles of structure and surface functional groups

    Science.gov (United States)

    Tveritinova, E. A.; Zhitnev, Yu. N.; Chernyak, S. A.; Arkhipova, E. A.; Savilov, S. V.; Lunin, V. V.

    2017-03-01

    Carbon nanomaterials with the structure of graphene and different compositions of the surface groups are used as catalysts for the conversion of C2-C4 aliphatic alcohols. The conversions of ethanol, propanol- 1, propanol-2, butanol-1, butanol-2, and tert-butanol on carbon nanotubes, nanoflakes, and nanoflakes doped with nitrogen are investigated. Oxidized and nonoxidized multiwalled carbon nanotubes, nanoflakes, and nanoflakes doped with nitrogen are synthesized. X-ray diffraction analysis, X-ray photoelectron spectroscopy, scanning and transmission electronic microscopies, Brunauer-Emmett-Teller method, derivatographic analyses, and the pulsed microcatalytic method are used to characterize comprehensively the prepared catalysts. It was established that all of the investigated carbon nanomaterials (with the exception of nondoped carbon nanoflakes) are bifunctional catalysts for the conversion of aliphatic alcohols, and promote dehydration reactions with the formation of olefins and dehydrogenation reactions with the formation of aldehydes or ketones. Nanoflakes doped with nitrogen are inert with respect to secondary alcohols and tert-butanol. The role of oxygen-containing and nitrogen-containing surface groups, and of the geometrical structure of the carbon matrix of graphene nanocarbon materials in the catalytic conversion of aliphatic alcohols, is revealed. Characteristics of the conversion of aliphatic alcohols that are associated with their structure are identified.

  6. Structure of the Photo-catalytically Active Surface of SrTiO 3

    Energy Technology Data Exchange (ETDEWEB)

    Plaza, Manuel; Huang, Xin; Ko, J. Y. Peter; Shen, Mei; Simpson, Burton H.; Rodríguez-López, Joaquín; Ritzert, Nicole L.; Letchworth-Weaver, Kendra; Gunceler, Deniz; Schlom, Darrell G.; Arias, Tomás A.; Brock, Joel D.; Abruña, Héctor D.

    2016-06-29

    A major goal of energy research is to use visible light to cleave water directly, without an applied voltage, into hydrogen and oxygen. Although SrTiO3 requires ultraviolet light, after four decades, it is still the "gold standard" for the photo-catalytic splitting of water. It is chemically robust and can carry out both hydrogen and oxygen evolution reactions without an applied bias. While ultrahigh vacuum surface science techniques have provided useful insights, we still know relatively little about the structure of these electrodes in contact with electrolytes under operating conditions. Here, we report the surface structure evolution of a n-SrTiO3 electrode during water splitting, before and after "training" with an applied positive bias. Operando high-energy X-ray reflectivity measurements demonstrate that training the electrode irreversibly reorders the surface. Scanning electrochemical microscopy at open circuit correlates this training with a 3-fold increase of the activity toward the photo-induced water splitting. A novel first-principles joint density functional theory simulation, constrained to the X-ray data via a generalized penalty function, identifies an anatase-like structure as the more active, trained surface.

  7. [Chemical structure of bioethanol lignin by low-temperature alkaline catalytic hydrothermal treatment].

    Science.gov (United States)

    Liu, Xiao-Huan; Zhang, Ming-Ming; Wang, Ji-Fu; Xu, Yu-Zhi; Wang, Chun-Peng; Chu, Fu-Xiang

    2013-11-01

    In order to improve the reaction activity of bioethanol lignin, we investigated the activation of bioethanol lignin by a hydrothermal treatment method. Catalytic hydrothermal treatment of bioethanol lignin was performed at 180 degrees C for 3 h in the presence of alkaline solutions (NaOH, Na2 CO3, KOH and K2 CO3), the change in bioethanol lignin structures was studied comparatively by FTIR, 1H NMR,GPC and elemental analysis. FTIR spectra showed that after alkali hydrothermal treatment, the band at 1 375 cm(-1) attributed to the phenolic hydroxyl groups increased, and the band intensity at 1 116 cm(-1) attributed to the ether bond decreased. On the other hand, the band at 1 597 and 1 511 cm(-1) attributed to aromatic skeletal vibration remained almost unchanged. 1H NMR spectra showed that after alkali hydrothermal treatment, the number of aromatic methoxyl is increased, and based on the increment of the content of phenolic hydroxyl, the catalytic activity can be ranked as follows: KOH > NaOH > K2 CO3 > Na2 CO3. Especially for KOH, the increment of the content of phenolic hydroxyl was 170%, because the ion radius of potassium cation is bigger than sodium cation, so the potassium cations more easily formed cation adducts with lignin. GPC results showed that the molecular weight of alkali hydrothermal treatment lignin decreased and the molecular distribution got wider. Elemental analysis showed that hydrothermal treatment could break the interlinkage between lignin and protein, which can reduce the protein content and increase the purity of lignin, meanwhile, the content of O and H both decreased,while C fell, indicating that the bioethanol lignin had suffered a decarbonylation reaction. This is the most benefit of the lignin as a substitute for phenol.

  8. Structural functionality, catalytic mechanism modeling and molecular allergenicity of phenylcoumaran benzylic ether reductase, an olive pollen (Ole e 12) allergen

    Science.gov (United States)

    Jimenez-Lopez, Jose C.; Kotchoni, Simeon O.; Hernandez-Soriano, Maria C.; Gachomo, Emma W.; Alché, Juan D.

    2013-10-01

    Isoflavone reductase-like proteins (IRLs) are enzymes with key roles in the metabolism of diverse flavonoids. Last identified olive pollen allergen (Ole e 12) is an IRL relevant for allergy amelioration, since it exhibits high prevalence among atopic patients. The goals of this study are the characterization of (A) the structural-functionality of Ole e 12 with a focus in its catalytic mechanism, and (B) its molecular allergenicity by extensive analysis using different molecular computer-aided approaches covering (1) physicochemical properties and functional-regulatory motifs, (2) sequence analysis, 2-D and 3D structural homology modeling comparative study and molecular docking, (3) conservational and evolutionary analysis, (4) catalytic mechanism modeling, and (5) sequence, structure-docking based B-cell epitopes prediction, while T-cell epitopes were predicted by inhibitory concentration and binding score methods. Structural-based detailed features, phylogenetic and sequences analysis have identified Ole e 12 as phenylcoumaran benzylic ether reductase. A catalytic mechanism has been proposed for Ole e 12 which display Lys133 as one of the conserved residues of the IRLs catalytic tetrad (Asn-Ser-Tyr-Lys). Structure characterization revealed a conserved protein folding among plants IRLs. However, sequence polymorphism significantly affected residues involved in the catalytic pocket structure and environment (cofactor and substrate interaction-recognition). It might also be responsible for IRLs isoforms functionality and regulation, since micro-heterogeneities affected physicochemical and posttranslational motifs. This polymorphism might have large implications for molecular differences in B- and T-cells epitopes of Ole e 12, and its identification may help designing strategies to improve the component-resolving diagnosis and immunotherapy of pollen and food allergy through development of molecular tools.

  9. Crystal structure of the anti-viral APOBEC3G catalytic domain and functional implications

    Energy Technology Data Exchange (ETDEWEB)

    Holden, Lauren G.; Prochnow, Courtney; Chang, Y. Paul; Bransteitter, Ronda; Chelico, Linda; Sen, Udayaditya; Stevens, Raymond C.; Goodman, Myron F.; Chen, Xiaojiang S. (USC); (Scripps)

    2009-04-07

    The APOBEC family members are involved in diverse biological functions. APOBEC3G restricts the replication of human immunodeficiency virus (HIV), hepatitis B virus and retroelements by cytidine deamination on single-stranded DNA or by RNA binding. Here we report the high-resolution crystal structure of the carboxy-terminal deaminase domain of APOBEC3G (APOBEC3G-CD2) purified from Escherichia coli. The APOBEC3G-CD2 structure has a five-stranded {beta}-sheet core that is common to all known deaminase structures and closely resembles the structure of another APOBEC protein, APOBEC2. A comparison of APOBEC3G-CD2 with other deaminase structures shows a structural conservation of the active-site loops that are directly involved in substrate binding. In the X-ray structure, these APOBEC3G active-site loops form a continuous 'substrate groove' around the active centre. The orientation of this putative substrate groove differs markedly (by 90 degrees) from the groove predicted by the NMR structure. We have introduced mutations around the groove, and have identified residues involved in substrate specificity, single-stranded DNA binding and deaminase activity. These results provide a basis for understanding the underlying mechanisms of substrate specificity for the APOBEC family.

  10. Crystal Structure of the Human Pol α B Subunit in Complex with the C-terminal Domain of the Catalytic Subunit.

    Science.gov (United States)

    Suwa, Yoshiaki; Gu, Jianyou; Baranovskiy, Andrey G; Babayeva, Nigar D; Pavlov, Youri I; Tahirov, Tahir H

    2015-06-05

    In eukaryotic DNA replication, short RNA-DNA hybrid primers synthesized by primase-DNA polymerase α (Prim-Pol α) are needed to start DNA replication by the replicative DNA polymerases, Pol δ and Pol ϵ. The C terminus of the Pol α catalytic subunit (p180C) in complex with the B subunit (p70) regulates the RNA priming and DNA polymerizing activities of Prim-Pol α. It tethers Pol α and primase, facilitating RNA primer handover from primase to Pol α. To understand these regulatory mechanisms and to reveal the details of human Pol α organization, we determined the crystal structure of p70 in complex with p180C. The structured portion of p70 includes a phosphodiesterase (PDE) domain and an oligonucleotide/oligosaccharide binding (OB) domain. The N-terminal domain and the linker connecting it to the PDE domain are disordered in the reported crystal structure. The p180C adopts an elongated asymmetric saddle shape, with a three-helix bundle in the middle and zinc-binding modules (Zn1 and Zn2) on each side. The extensive p180C-p70 interactions involve 20 hydrogen bonds and a number of hydrophobic interactions resulting in an extended buried surface of 4080 Å(2). Importantly, in the structure of the p180C-p70 complex with full-length p70, the residues from the N-terminal to the OB domain contribute to interactions with p180C. The comparative structural analysis revealed both the conserved features and the differences between the human and yeast Pol α complexes.

  11. Structure and dynamics of the HIV-1 frameshift element RNA.

    Science.gov (United States)

    Low, Justin T; Garcia-Miranda, Pablo; Mouzakis, Kathryn D; Gorelick, Robert J; Butcher, Samuel E; Weeks, Kevin M

    2014-07-08

    The HIV-1 ribosomal frameshift element is highly structured, regulates translation of all virally encoded enzymes, and is a promising therapeutic target. The prior model for this motif contains two helices separated by a three-nucleotide bulge. Modifications to this model were suggested by SHAPE chemical probing of an entire HIV-1 RNA genome. Novel features of the SHAPE-directed model include alternate helical conformations and a larger, more complex structure. These structural elements also support the presence of a secondary frameshift site within the frameshift domain. Here, we use oligonucleotide-directed structure perturbation, probing in the presence of formamide, and in-virion experiments to examine these models. Our data support a model in which the frameshift domain is anchored by a stable helix outside the conventional domain. Less stable helices within the domain can switch from the SHAPE-predicted to the two-helix conformation. Translational frameshifting assays with frameshift domain mutants support a functional role for the interactions predicted by and specific to the SHAPE-directed model. These results reveal that the HIV-1 frameshift domain is a complex, dynamic structure and underscore the importance of analyzing folding in the context of full-length RNAs.

  12. 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

    The function of non-coding RNA genes largely depends on their secondary structure and the interaction with other molecules. Thus, an accurate prediction of secondary structure and RNA-RNA interaction is essential for the understanding of biological roles and pathways associated with a specific RN...

  13. An RNA secondary structure prediction method based on minimum and suboptimal free energy structures.

    Science.gov (United States)

    Fu, Haoyue; Yang, Lianping; Zhang, Xiangde

    2015-09-07

    The function of an RNA-molecule is mainly determined by its tertiary structures. And its secondary structure is an important determinant of its tertiary structure. The comparative methods usually give better results than the single-sequence methods. Based on minimum and suboptimal free energy structures, the paper presents a novel method for predicting conserved secondary structure of a group of related RNAs. In the method, the information from the known RNA structures is used as training data in a SVM (Support Vector Machine) classifier. Our method has been tested on the benchmark dataset given by Puton et al. The results show that the average sensitivity of our method is higher than that of other comparative methods such as CentroidAlifold, MXScrana, RNAalifold, and TurboFold.

  14. Correlation between the Structure and Catalytic Activity of [Cp*Rh(Substituted Bipyridine)] Complexes for NADH Regeneration.

    Science.gov (United States)

    Ganesan, Vinothkumar; Sivanesan, Dharmalingam; Yoon, Sungho

    2017-02-06

    A series of water-soluble half-sandwich [Cp*Rh(III)(N^N)Cl](+) (Cp* = pentamethylcyclopentadiene, N^N-substituted 2,2'-bipyridine) complexes containing electron-donating substituents around the 2,2'-bipyridyl ligand were synthesized and fully characterized for the regioselective reduction of nicotinamide coenzyme (NAD(+)). The influence of the positional effect of the substituents on the structural, electrochemical, and catalytic properties of the catalyst was systematically studied in detail. The catalytic efficiency of the substituted bipyridine Cp*Rh(III) complexes are inversely correlated with their redox potentials. The 5,5'-substituted bipyridine Cp*Rh(III) complex, which had the lowest reduction potential, most effectively regenerated NADH with a turnover frequency of 1100 h(-1). Detailed kinetic studies on the generation of intermediate(s) provide valuable mechanistic insight into this catalytic cycle and help to direct the future design strategy of corresponding catalysts.

  15. Oscillatory Behavior during the Catalytic Partial Oxidation of Methane: Following Dynamic Structural Changes of Palladium Using the QEXAFS Technique

    DEFF Research Database (Denmark)

    Stoetzel, Jan; Frahm, Ronald; Kimmerle, Bertram

    2012-01-01

    Pd/Al2O3 catalysts oscillate between ignition and extinction of the catalytic partial oxidation of methane when they are exposed to a 2:1 reaction mixture of methane and oxygen. The oscillations of the catalytic performance and the structure of Pd/Al2O3 catalysts in a fixed-bed reactor were...... of the Pd particles at increasing age of the catalyst was observed, which leads to a lower oscillation frequency. Effects of particle size, oven temperature, and oxygen/methane ratio on the oscillation behavior were studied in detail. The deactivation period (reoxidation of Pd) was much less influenced...... by the oven temperature than the ignition behavior of the catalytic partial oxidation of methane. This indicates that deactivation is caused by an autoreduction of the palladium at the beginning of the catalyst bed due to the high temperature achieved by total oxidation of methane....

  16. Structural Basis for the Catalytic Activity of Human Serine/Threonine Protein Phosphatase type 5 (PP5)

    Science.gov (United States)

    Swingle, Mark R.; Ciszak, Ewa M.; Honkanen, Richard E.

    2004-01-01

    Serine/threonine protein phosphatase-5 (PP5) is a member of the PPP-gene family of protein phosphatases that is widely expressed in mammalian tissues and is highly conserved among eukaryotes. PP5 associates with several proteins that affect signal transduction networks, including the glucocorticoid receptor (GR)-heat shock protein-90 (Hsp90)-heterocomplex, the CDC16 and CDC27 subunits of the anaphase-promoting complex, elF2alpha kinase, the A subunit of PP2A, the G12-alpha / G13-alpha subunits of heterotrimeric G proteins and DNA-PK. The catalytic domain of PP5 (PP5c) shares 35-45% sequence identity with the catalytic domains of other PPP-phosphatases, including protein phosphatase-1 (PP1), -2A (PP2A), -2B / calcineurin (PP2B), -4 (PP4), -6 (PP6), and -7 (PP7). Like PP1, PP2A and PP4, PP5 is also sensitive to inhibition by okadaic acid, microcystin, cantharidin, tautomycin, and calyculin A. Here we report the crystal structure of the PP5 catalytic domain (PP5c) at a resolution of 1.6 angstroms. From this structure we propose a mechanism for PP5-mediated hydrolysis of phosphoprotein substrates, which requires the precise positioning of two metal ions within a conserved Asp(sup 271)-M(sub 1):M(sub 2)-W(sup 1)-His(sup 304)-Asp(sup 274) catalytic motif. The structure of PP5c provides a possible structural basis for explaining the exceptional catalytic proficiency of protein phosphatases, which are among the most powerful known catalysts. Resolution of the entire C-terminus revealed a novel subdomain, and the structure of the PP5c should also aid development of type-specific inhibitors.

  17. Displaying the information contents of structural RNA alignments: the structure logos

    DEFF Research Database (Denmark)

    Gorodkin, Jan; Heyer, L.J.; Brunak, Søren

    1997-01-01

    We extend the standard `Sequence Logo' method of Schneider and Stevens to incorporate prior frequencies on the bases, allow for gaps in the alignments, and indicate the mutual information of base-paired regions in RNA. Given an alignment of RNA sequences with the base pairings indicated, the prog......We extend the standard `Sequence Logo' method of Schneider and Stevens to incorporate prior frequencies on the bases, allow for gaps in the alignments, and indicate the mutual information of base-paired regions in RNA. Given an alignment of RNA sequences with the base pairings indicated......, the program will calculate the information at each position, including the mutual information of the base pairs, and display the results in a `Structure Logo'. Alignments without base pairing can also be displayed in a `Sequence Logo', but still allowing gaps and incorporating prior frequencies if desired...

  18. Surface structure and catalytic activity of electrodeposited Ni-Fe-Co-Mo alloy electrode by partially leaching Mo and Fe

    Institute of Scientific and Technical Information of China (English)

    LUO Bei-ping; GONG Zhu-qing; REN Bi-ye; YANG Yu-fang; CHEN Meng-jun

    2006-01-01

    Ni-Fe-Mo-Co alloy electrode was prepared in a citrate solution by electrodeposition, and then Mo and Fe were partially leached out from the electrode in 30% KOH solution. The unique surface micromorphology of a hive-like structure was obtained with an average pore size of about 50 nm. The electrode has a very large real surface area and a stable structure. The effects of sodium molybdate concentration on the composition, surface morphology, and structure of electrodes were analyzed by EDS, SEM and XRD. The polarization curves of the different electrodes show that the catalytic activity of electrodes is strongly correlated with the mole fraction of alloy elements (Ni, Fe, Mo, Co), and the addition of cobalt element to Ni-Fe-Mo alloy improves the catalytic activity. The Ni35.63Fe24.67Mo23.52Co16.18 electrode has the best activity for hydrogen evolution reaction(HER), with an over-potential of 66.2 mV, in 30% KOH at 80 ℃ and 200 mA/cm2. The alloy maintains its good catalytic activity for HER during continuous or intermittent electrolysis. Its electrochemical activity and catalytic stability are much higher than the other iron-group with Mo alloy electrodes.

  19. A ligand-directed divergent catalytic approach to establish structural and functional scaffold diversity

    Science.gov (United States)

    Lee, Yen-Chun; Patil, Sumersing; Golz, Christopher; Strohmann, Carsten; Ziegler, Slava; Kumar, Kamal; Waldmann, Herbert

    2017-02-01

    The selective transformation of different starting materials by different metal catalysts under individually optimized reaction conditions to structurally different intermediates and products is a powerful approach to generate diverse molecular scaffolds. In a more unified albeit synthetically challenging strategy, common starting materials would be exposed to a common metal catalysis, leading to a common intermediate and giving rise to different scaffolds by tuning the reactivity of the metal catalyst through different ligands. Herein we present a ligand-directed synthesis approach for the gold(I)-catalysed cycloisomerization of oxindole-derived 1,6-enynes that affords distinct molecular scaffolds following different catalytic reaction pathways. Varying electronic properties and the steric demand of the gold(I) ligands steers the fate of a common intermediary gold carbene to selectively form spirooxindoles, quinolones or df-oxindoles. Investigation of a synthesized compound collection in cell-based assays delivers structurally novel, selective modulators of the Hedgehog and Wnt signalling pathways, autophagy and of cellular proliferation.

  20. Crystal structure of catalytic domain of the initiation factor 2B epsilon subunit

    DEFF Research Database (Denmark)

    Boesen, Thomas; Mohammad, Sarah S.; Pavitt, Graham D.;

    this motif is involved in binding to the N-terminal part of the eIF2β subunit The aliphatic residues in the AA box motifs are involved in specific contacts in the hydrophobic core of the C-terminal helices important for maintaining the overall structure, whereas acidic residues in the motifs form a clustered......-terminal two helices contain the catalytic part of the domain, whereas the C-terminal six helices harbor the two Aromatic Acidic (AA) box motifs. This motif is also found in initiation factor 5, the GTPase activator protein of eIF2, and furthermore in mammalian initiation factor 4G. In eIF2B and eIF5......, surface exposed acidic patch which might interact with the lysine boxes of eIF2β. Interestingly, Tryptophan 699 was found to be solvent exposed and involved in crystal packing. This residue could possibly be important for the specific interaction with eIF2β. Furthermore, the structure shows the location...

  1. FASTR: A novel data format for concomitant representation of RNA sequence and secondary structure information

    Indian Academy of Sciences (India)

    Tungadri Bose; Anirban Dutta; Mohammed Mh; Hemang Gandhi; Sharmila S Mande

    2015-09-01

    Given the importance of RNA secondary structures in defining their biological role, it would be convenient for researchers seeking RNA data if both sequence and structural information pertaining to RNA molecules are made available together. Current nucleotide data repositories archive only RNA sequence data. Furthermore, storage formats which can frugally represent RNA sequence as well as structure data in a single file, are currently unavailable. This article proposes a novel storage format, `FASTR’, for concomitant representation of RNA sequence and structure. The storage efficiency of the proposed FASTR format has been evaluated using RNA data from various microorganisms. Results indicate that the size of FASTR formatted files (containing both RNA sequence as well as structure information) are equivalent to that of FASTA-format files, which contain only RNA sequence information. RNA secondary structure is typically represented using a combination of a string of nucleotide characters along with the corresponding dot-bracket notation indicating structural attributes. `FASTR’ – the novel storage format proposed in the present study enables a frugal representation of both RNA sequence and structural information in the form of a single string. In spite of having a relatively smaller storage footprint, the resultant `fastr’ string(s) retain all sequence as well as secondary structural information that could be stored using a dot-bracket notation. An implementation of the `FASTR’ methodology is available for download at http://metagenomics.atc.tcs.com/compression/fastr.

  2. 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.

  3. Trade-offs between tRNA abundance and mRNA secondary structure support smoothing of translation elongation rate

    Science.gov (United States)

    Gorochowski, Thomas E.; Ignatova, Zoya; Bovenberg, Roel A.L.; Roubos, Johannes A.

    2015-01-01

    Translation of protein from mRNA is a complex multi-step process that occurs at a non-uniform rate. Variability in ribosome speed along an mRNA enables refinement of the proteome and plays a critical role in protein biogenesis. Detailed single protein studies have found both tRNA abundance and mRNA secondary structure as key modulators of translation elongation rate, but recent genome-wide ribosome profiling experiments have not observed significant influence of either on translation efficiency. Here we provide evidence that this results from an inherent trade-off between these factors. We find codons pairing to high-abundance tRNAs are preferentially used in regions of high secondary structure content, while codons read by significantly less abundant tRNAs are located in lowly structured regions. By considering long stretches of high and low mRNA secondary structure in Saccharomyces cerevisiae and Escherichia coli and comparing them to randomized-gene models and experimental expression data, we were able to distinguish clear selective pressures and increased protein expression for specific codon choices. The trade-off between secondary structure and tRNA-concentration based codon choice allows for compensation of their independent effects on translation, helping to smooth overall translational speed and reducing the chance of potentially detrimental points of excessively slow or fast ribosome movement. PMID:25765653

  4. Regulatory Impact of RNA Secondary Structure across the Arabidopsis Transcriptome[W][OA

    Science.gov (United States)

    Li, Fan; Zheng, Qi; Vandivier, Lee E.; Willmann, Matthew R.; Chen, Ying; Gregory, Brian D.

    2012-01-01

    The secondary structure of an RNA molecule plays an integral role in its maturation, regulation, and function. However, the global influence of this feature on plant gene expression is still largely unclear. Here, we use a high-throughput, sequencing-based, structure-mapping approach in conjunction with transcriptome-wide sequencing of rRNA-depleted (RNA sequencing), small RNA, and ribosome-bound RNA populations to investigate the impact of RNA secondary structure on gene expression regulation in Arabidopsis thaliana. From this analysis, we find that highly unpaired and paired RNAs are strongly correlated with euchromatic and heterochromatic epigenetic histone modifications, respectively, providing evidence that secondary structure is necessary for these RNA-mediated posttranscriptional regulatory pathways. Additionally, we uncover key structural patterns across protein-coding transcripts that indicate RNA folding demarcates regions of protein translation and likely affects microRNA-mediated regulation of mRNAs in this model plant. We further reveal that RNA folding is significantly anticorrelated with overall transcript abundance, which is often due to the increased propensity of highly structured mRNAs to be degraded and/or processed into small RNAs. Finally, we find that secondary structure affects mRNA translation, suggesting that this feature regulates plant gene expression at multiple levels. These findings provide a global assessment of RNA folding and its significant regulatory effects in a plant transcriptome. PMID:23150631

  5. Crystal structures of Trypanosoma brucei oligopeptidase B broaden the paradigm of catalytic regulation in prolyl oligopeptidase family enzymes.

    Science.gov (United States)

    Canning, Peter; Rea, Dean; Morty, Rory E; Fülöp, Vilmos

    2013-01-01

    Oligopeptidase B cleaves after basic amino acids in peptides up to 30 residues. As a virulence factor in bacteria and trypanosomatid pathogens that is absent in higher eukaryotes, this is a promising drug target. Here we present ligand-free open state and inhibitor-bound closed state crystal structures of oligopeptidase B from Trypanosoma brucei, the causative agent of African sleeping sickness. These (and related) structures show the importance of structural dynamics, governed by a fine enthalpic and entropic balance, in substrate size selectivity and catalysis. Peptides over 30 residues cannot fit the enzyme cavity, preventing the complete domain closure required for a key propeller Asp/Glu to fix the catalytic His and Arg in the catalytically competent conformation. This size exclusion mechanism protects larger peptides and proteins from degradation. Similar bacterial prolyl endopeptidase and archael acylaminoacyl peptidase structures demonstrate this mechanism is conserved among oligopeptidase family enzymes across all three domains of life.

  6. Crystal structures of Trypanosoma brucei oligopeptidase B broaden the paradigm of catalytic regulation in prolyl oligopeptidase family enzymes.

    Directory of Open Access Journals (Sweden)

    Peter Canning

    Full Text Available Oligopeptidase B cleaves after basic amino acids in peptides up to 30 residues. As a virulence factor in bacteria and trypanosomatid pathogens that is absent in higher eukaryotes, this is a promising drug target. Here we present ligand-free open state and inhibitor-bound closed state crystal structures of oligopeptidase B from Trypanosoma brucei, the causative agent of African sleeping sickness. These (and related structures show the importance of structural dynamics, governed by a fine enthalpic and entropic balance, in substrate size selectivity and catalysis. Peptides over 30 residues cannot fit the enzyme cavity, preventing the complete domain closure required for a key propeller Asp/Glu to fix the catalytic His and Arg in the catalytically competent conformation. This size exclusion mechanism protects larger peptides and proteins from degradation. Similar bacterial prolyl endopeptidase and archael acylaminoacyl peptidase structures demonstrate this mechanism is conserved among oligopeptidase family enzymes across all three domains of life.

  7. Identification and structural analysis of a novel snoRNA gene cluster from Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A Z2 snoRNA gene cluster,consisting of four antisense snoRNA genes, was identified from Arabidopsis thaliana. The sequence and structural analysis showed that the Z2 snoRNA gene cluster might be transcribed as a polycistronic precursor from an upstream promoter, and the intergenic spacers of the gene cluster encode the 'hairpin' structures similar to the processing recognition signals of yeast Saccharomyces cerevisiae polycistronic snoRNA precursor. The results also revealed that plant snoRNA gene with multiple copies is a characteristic in common, and provides a good system for further revealing the transcription and expression mechanism of plant snoRNA gene cluster.

  8. Role of pri-miRNA tertiary structure in miR-17~92 miRNA biogenesis.

    Science.gov (United States)

    Chaulk, Steven G; Thede, Gina L; Kent, Oliver A; Xu, Zhizhong; Gesner, Emily M; Veldhoen, Richard A; Khanna, Suneil K; Goping, Ing Swie; MacMillan, Andrew M; Mendell, Joshua T; Young, Howard S; Fahlman, Richard P; Glover, J N Mark

    2011-01-01

    MicroRNAs (miRNAs) regulate gene expression in a variety of biological pathways such as development and tumourigenesis. miRNAs are initially expressed as long primary transcripts (pri-miRNAs) that undergo sequential processing by Drosha and then Dicer to yield mature miRNAs. miR-17~92 is a miRNA cluster that encodes 6 miRNAs and while it is essential for development it also has reported oncogenic activity. To date, the role of RNA structure in miRNA biogenesis has only been considered in terms of the secondary structural elements required for processing of pri-miRNAs by Drosha. Here we report that the miR-17~92 cluster has a compact globular tertiary structure where miRNAs internalized within the core of the folded structure are processed less efficiently than miRNAs on the surface of the structure. Increased miR-92 expression resulting from disruption of the compact miR-17~92 structure results in increased repression of integrin α5 mRNA, a known target of miR-92a. In summary, we describe the first example of pri-miRNA structure modulating differential expression of constituent miRNAs.

  9. Strong epistatic selection on the RNA secondary structure of HIV.

    Directory of Open Access Journals (Sweden)

    Raquel Assis

    2014-09-01

    Full Text Available A key question in evolutionary genomics is how populations navigate the adaptive landscape in the presence of epistasis, or interactions among loci. This problem can be directly addressed by studying the evolution of RNA secondary structures, for which there is constraint to maintain pairing between Watson-Crick (WC sites. Replacement of a nucleotide at one site of a WC pair reduces fitness by disrupting binding, which can be restored via a compensatory replacement at the interacting site. Here, I present the first genome-scale analysis of epistasis on the RNA secondary structure of human immunodeficiency virus type 1 (HIV-1. Comparison of polymorphism frequencies at ancestrally conserved sites reveals that selection against replacements is ∼ 2.7 times stronger at WC than at non-WC sites, such that nearly 50% of constraint can be attributed to epistasis. However, almost all epistatic constraint is due to selection against conversions of WC pairs to unpaired (UP nucleotides, whereas conversions to GU wobbles are only slightly deleterious. This disparity is also evident in pairs with second-site compensatory replacements; conversions from UP nucleotides to WC pairs increase median fitness by ∼ 4.2%, whereas conversions from GU wobbles to WC pairs only increase median fitness by ∼ 0.3%. Moreover, second-site replacements that convert UP nucleotides to GU wobbles also increase median fitness by ∼ 4%, indicating that such replacements are nearly as compensatory as those that restore WC pairing. Thus, WC peaks of the HIV-1 epistatic adaptive landscape are connected by high GU ridges, enabling the viral population to rapidly explore distant peaks without traversing deep UP valleys.

  10. Pairwise local structural alignment of RNA sequences with sequence similarity less than 40%

    DEFF Research Database (Denmark)

    Havgaard, Jakob Hull; Lyngsø, Rune B.; Stormo, Gary D.

    2005-01-01

    Motivation: Searching for non-coding RNA (ncRNA) genes and structural RNA elements (eleRNA) are major challenges in gene finding todya as these often are conserved in structure rather than in sequence. Even though the number of available methods is growing, it is still of interest to pairwise....... The structure prediction performance for a family is typically around 0.7 using Matthews correlation coefficient. In case (2), the algorithm is successful at locating RNA families with an average sensitivity of 0.8 and a positive predictive value of 0.9 using a BLAST-like hit selection scheme. Availability...

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

    Science.gov (United States)

    Helbo, Alexandra Søgaard; Lay, Fides D.; Jones, Peter A.; Liang, Gangning; Grønbæk, Kirsten

    2017-02-01

    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-resolution analysis to show substantial differences in chromatin structure of pol II and pol III promoters, and between subtypes of pol III genes. Notably, the nucleosome depleted region at the transcription start site of pol III genes extends past the termination sequences, resulting in nucleosome free gene bodies. The +1 nucleosome is located further downstream than at pol II genes and furthermore displays weak positioning. The variable position of the +1 location is seen not only within individual cell populations and between cell types, but also between different pol III promoter subtypes, suggesting that the +1 nucleosome may be involved in the transcriptional regulation of pol III genes. We find that expression and DNA methylation patterns correlate with distinct accessibility patterns, where DNA methylation associates with the silencing and inaccessibility at promoters. Taken together, this study provides the first high-resolution map of nucleosome positioning and occupancy at human pol III promoters at specific loci and genome wide.

  12. High-throughput SHAPE and hydroxyl radical analysis of RNA structure and ribonucleoprotein assembly.

    Science.gov (United States)

    McGinnis, Jennifer L; Duncan, Caia D S; Weeks, Kevin M

    2009-01-01

    RNA folds to form complex structures vital to many cellular functions. Proteins facilitate RNA folding at both the secondary and tertiary structure levels. An absolute prerequisite for understanding RNA folding and ribonucleoprotein (RNP) assembly reactions is a complete understanding of the RNA structure at each stage of the folding or assembly process. Here we provide a guide for comprehensive and high-throughput analysis of RNA secondary and tertiary structure using SHAPE and hydroxyl radical footprinting. As an example of the strong and sometimes surprising conclusions that can emerge from high-throughput analysis of RNA folding and RNP assembly, we summarize the structure of the bI3 group I intron RNA in four distinct states. Dramatic structural rearrangements occur in both secondary and tertiary structure as the RNA folds from the free state to the active, six-component, RNP complex. As high-throughput and high-resolution approaches are applied broadly to large protein-RNA complexes, other proteins previously viewed as making simple contributions to RNA folding are also likely to be found to exert multifaceted, long-range, cooperative, and nonadditive effects on RNA folding. These protein-induced contributions add another level of control, and potential regulatory function, in RNP complexes.

  13. 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...

  14. Silencing structural and nonstructural genes in baculovirus by RNA interference.

    Science.gov (United States)

    Flores-Jasso, C Fabian; Valdes, Victor Julian; Sampieri, Alicia; Valadez-Graham, Viviana; Recillas-Targa, Felix; Vaca, Luis

    2004-06-01

    We review several aspects of RNAi and gene silencing with baculovirus. We show that the potency of RNAi in Spodoptera frugiperda (Sf21) insect cells correlates well with the efficiency of transfection of the siRNA. Using a fluorescein-labeled siRNA we found that the siRNA localized in areas surrounding the endoplasmic reticulum (ER). Both long (700 nucleotides long) and small ( approximately 25 nucleotides long) interfering RNAs were equally effective in initiating RNA interference (RNAi), and the duration of the interfering effect was indistinguishable. Even though RNAi in Sf21 cells is very effective, in vitro experiments show that these cells fragment the long dsRNA into siRNA poorly, when compared to HEK cells. Finally, we show that in vivo inhibition of baculovirus infection with dsRNA homologous to genes that are essential for baculovirus infectivity depends strongly on the amount of dsRNA used in the assays. Five hundred nanogram of dsRNA directly injected into the haemolymph of insects prevent animal death to over 95%. In control experiments, over 96% of insects not injected with dsRNA or injected with an irrelevant dsRNA died within a week. These results demonstrate the efficiency of dsRNA for in vivo prevention of a viral infection by virus that is very cytotoxic and lytic in animals.

  15. Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Jian-Ching; Rebrin, Igor [Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (United States); Klichko, Vladimir; Orr, William C. [Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275 (United States); Sohal, Rajindar S., E-mail: sohal@usc.edu [Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90033 (United States)

    2010-10-08

    Research highlights: {yields} Cytochrome c oxidase loses catalytic activity during the aging process. {yields} Abundance of seven nuclear-encoded subunits of cytochrome c oxidase decreased with age in Drosophila. {yields} Cytochrome c oxidase is specific intra-mitochondrial site of age-related deterioration. -- Abstract: The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H{sub 2}O{sub 2} generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-, and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc, and VIII, indicated that the abundance these polypeptides decreased, ranging from 11% to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology.

  16. Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function.

    Directory of Open Access Journals (Sweden)

    Cristian Del Campo

    2015-10-01

    Full Text Available Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown. Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, we unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage. This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation.

  17. Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function.

    Science.gov (United States)

    Del Campo, Cristian; Bartholomäus, Alexander; Fedyunin, Ivan; Ignatova, Zoya

    2015-10-01

    Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown. Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, we unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage. This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation.

  18. SimRNAweb: a web server for RNA 3D structure modeling with optional restraints.

    Science.gov (United States)

    Magnus, Marcin; Boniecki, Michał J; Dawson, Wayne; Bujnicki, Janusz M

    2016-07-08

    RNA function in many biological processes depends on the formation of three-dimensional (3D) structures. However, RNA structure is difficult to determine experimentally, which has prompted the development of predictive computational methods. Here, we introduce a user-friendly online interface for modeling RNA 3D structures using SimRNA, a method that uses a coarse-grained representation of RNA molecules, utilizes the Monte Carlo method to sample the conformational space, and relies on a statistical potential to describe the interactions in the folding process. SimRNAweb makes SimRNA accessible to users who do not normally use high performance computational facilities or are unfamiliar with using the command line tools. The simplest input consists of an RNA sequence to fold RNA de novo. Alternatively, a user can provide a 3D structure in the PDB format, for instance a preliminary model built with some other technique, to jump-start the modeling close to the expected final outcome. The user can optionally provide secondary structure and distance restraints, and can freeze a part of the starting 3D structure. SimRNAweb can be used to model single RNA sequences and RNA-RNA complexes (up to 52 chains). The webserver is available at http://genesilico.pl/SimRNAweb.

  19. Different structure and mRNA expression of Entamoeba invadens chitinases in the encystation and excystation.

    Science.gov (United States)

    Makioka, Asao; Kumagai, Masahiro; Hiranuka, Kazushi; Kobayashi, Seiki; Takeuchi, Tsutomu

    2011-08-01

    Entamoeba histolytica forms chitin-walled cysts during encystation process, where formation of the cyst wall needs not only chitin synthase but also chitinase. During excystation, quadruplet amoebae emerge from the chitin-walled cysts by dissolving the wall, so that chitinase may be necessary for excystation process as well. There is, however, no report on chitinase expression during excystation. In this study, we used Entamoeba invadens, a reptilian amoeba, as a model for encystation and excystation of E. histolytica, and studied chitinase mRNA expression in those processes. Although expression of three E. invadens chitinases designated EiChit1, EiChit2, and EiChit3 during encystation has been reported, we identified another enzyme named as EiChit4 in the E. invadens genome database. Therefore, we investigated the primary structure and mRNA expression of these four chitinases of Ei in the excystation as well as the encystation by real-time reverse transcription polymerase chain reaction (RT-PCR). Like EiChit1, EiChit4 had an 8 × Cys chitin-binding domain (CBD) and a hydrophilic spacer between the CBD and catalytic domain, and was also closer to EiChit1 than EiChit2 and EiChit3 in the phylogenetic tree. During encystation, the expression of all four chitinases increased in the early phase; the increase in EiChit1 and EiChit4 was much higher than in EiChit2 and EiChit3. Then, the expression of all four chitinases sharply decreased in the later phase. In cysts, EiChit1 was most abundantly expressed and EiChit4 was at a lower level, while the expressions of EiChit2 and EiChit3 were virtually absent. Following the induction of excystation, mRNA levels of EiChit1 and EiChit4 in cysts 5 h after induction were significantly lower than those in cysts before induction, while those of EiChit2 and EiChit3 were remarkably higher than before induction. The mRNAs of only EiChit2 and EiChit3 remarkably increased when the excystation was induced in the presence of cytochalasin D

  20. Structural basis for recognition of cognate tRNA by tyrosyl-tRNA synthetase from three kingdoms.

    Science.gov (United States)

    Tsunoda, Masaru; Kusakabe, Yoshio; Tanaka, Nobutada; Ohno, Satoshi; Nakamura, Masashi; Senda, Toshiya; Moriguchi, Tomohisa; Asai, Norio; Sekine, Mitsuo; Yokogawa, Takashi; Nishikawa, Kazuya; Nakamura, Kazuo T

    2007-01-01

    The specific aminoacylation of tRNA by tyrosyl-tRNA synthetases (TyrRSs) relies on the identity determinants in the cognate tRNA(Tyr)s. We have determined the crystal structure of Saccharomyces cerevisiae TyrRS (SceTyrRS) complexed with a Tyr-AMP analog and the native tRNA(Tyr)(GPsiA). Structural information for TyrRS-tRNA(Tyr) complexes is now full-line for three kingdoms. Because the archaeal/eukaryotic TyrRSs-tRNA(Tyr)s pairs do not cross-react with their bacterial counterparts, the recognition modes of the identity determinants by the archaeal/eukaryotic TyrRSs were expected to be similar to each other but different from that by the bacterial TyrRSs. Interestingly, however, the tRNA(Tyr) recognition modes of SceTyrRS have both similarities and differences compared with those in the archaeal TyrRS: the recognition of the C1-G72 base pair by SceTyrRS is similar to that by the archaeal TyrRS, whereas the recognition of the A73 by SceTyrRS is different from that by the archaeal TyrRS but similar to that by the bacterial TyrRS. Thus, the lack of cross-reactivity between archaeal/eukaryotic and bacterial TyrRS-tRNA(Tyr) pairs most probably lies in the different sequence of the last base pair of the acceptor stem (C1-G72 vs G1-C72) of tRNA(Tyr). On the other hand, the recognition mode of Tyr-AMP is conserved among the TyrRSs from the three kingdoms.

  1. Structural and catalytic effects of an invariant purine substitution in the hammerhead ribozyme: implications for the mechanism of acid-base catalysis.

    Science.gov (United States)

    Schultz, Eric P; Vasquez, Ernesto E; Scott, William G

    2014-09-01

    The hammerhead ribozyme catalyzes RNA cleavage via acid-base catalysis. Whether it does so by general acid-base catalysis, in which the RNA itself donates and abstracts protons in the transition state, as is typically assumed, or by specific acid-base catalysis, in which the RNA plays a structural role and proton transfer is mediated by active-site water molecules, is unknown. Previous biochemical and crystallographic experiments implicate an invariant purine in the active site, G12, as the general base. However, G12 may play a structural role consistent with specific base catalysis. To better understand the role of G12 in the mechanism of hammerhead catalysis, a 2.2 Å resolution crystal structure of a hammerhead ribozyme from Schistosoma mansoni with a purine substituted for G12 in the active site of the ribozyme was obtained. Comparison of this structure (PDB entry 3zd4), in which A12 is substituted for G, with three previously determined structures that now serve as important experimental controls, allows the identification of structural perturbations that are owing to the purine substitution itself. Kinetic measurements for G12 purine-substituted schistosomal hammerheads confirm a previously observed dependence of rate on the pK(a) of the substituted purine; in both cases inosine, which is similar to G in pK(a) and hydrogen-bonding properties, is unexpectedly inactive. Structural comparisons indicate that this may primarily be owing to the lack of the exocyclic 2-amino group in the G12A and G12I substitutions and its structural effect upon both the nucleotide base and phosphate of A9. The latter involves the perturbation of a previously identified and well characterized metal ion-binding site known to be catalytically important in both minimal and full-length hammerhead ribozyme sequences. The results permit it to be suggested that G12 plays an important role in stabilizing the active-site structure. This result, although not inconsistent with the potential

  2. Structural visualization of the p53/RNA polymerase II assembly.

    Science.gov (United States)

    Singh, Sameer K; Qiao, Zhen; Song, Lihua; Jani, Vijay; Rice, William; Eng, Edward; Coleman, Robert A; Liu, Wei-Li

    2016-11-15

    The master tumor suppressor p53 activates transcription in response to various cellular stresses in part by facilitating recruitment of the transcription machinery to DNA. Recent studies have documented a direct yet poorly characterized interaction between p53 and RNA polymerase II (Pol II). Therefore, we dissected the human p53/Pol II interaction via single-particle cryo-electron microscopy, structural docking, and biochemical analyses. This study reveals that p53 binds Pol II via the Rpb1 and Rpb2 subunits, bridging the DNA-binding cleft of Pol II proximal to the upstream DNA entry site. In addition, the key DNA-binding surface of p53, frequently disrupted in various cancers, remains exposed within the assembly. Furthermore, the p53/Pol II cocomplex displays a closed conformation as defined by the position of the Pol II clamp domain. Notably, the interaction of p53 and Pol II leads to increased Pol II elongation activity. These findings indicate that p53 may structurally regulate DNA-binding functions of Pol II via the clamp domain, thereby providing insights into p53-regulated Pol II transcription.

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

    DEFF Research Database (Denmark)

    He, Yangzi

    -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...... initiation factor (eIF)4A, a DEAD-box RNA helicase, as well as ancillary factors eIF4B and eIF4G. In higher eukaryotes, scanning of mRNAs containing stable secondary structures in the 5’ UTR furthermore requires DHX29, another DEAH/RHA helicase. I participated in characterizing the synergistic activation...

  4. Structural Dynamics as a Contributor to Error-prone Replication by an RNA-dependent RNA Polymerase*

    Science.gov (United States)

    Moustafa, Ibrahim M.; Korboukh, Victoria K.; Arnold, Jamie J.; Smidansky, Eric D.; Marcotte, Laura L.; Gohara, David W.; Yang, Xiaorong; Sánchez-Farrán, María Antonieta; Filman, David; Maranas, Janna K.; Boehr, David D.; Hogle, James M.; Colina, Coray M.; Cameron, Craig E.

    2014-01-01

    RNA viruses encoding high- or low-fidelity RNA-dependent RNA polymerases (RdRp) are attenuated. The ability to predict residues of the RdRp required for faithful incorporation of nucleotides represents an essential step in any pipeline intended to exploit perturbed fidelity as the basis for rational design of vaccine candidates. We used x-ray crystallography, molecular dynamics simulations, NMR spectroscopy, and pre-steady-state kinetics to compare a mutator (H273R) RdRp from poliovirus to the wild-type (WT) enzyme. We show that the nucleotide-binding site toggles between the nucleotide binding-occluded and nucleotide binding-competent states. The conformational dynamics between these states were enhanced by binding to primed template RNA. For the WT, the occluded conformation was favored; for H273R, the competent conformation was favored. The resonance for Met-187 in our NMR spectra reported on the ability of the enzyme to check the correctness of the bound nucleotide. Kinetic experiments were consistent with the conformational dynamics contributing to the established pre-incorporation conformational change and fidelity checkpoint. For H273R, residues comprising the active site spent more time in the catalytically competent conformation and were more positively correlated than the WT. We propose that by linking the equilibrium between the binding-occluded and binding-competent conformations of the nucleotide-binding pocket and other active-site dynamics to the correctness of the bound nucleotide, faithful nucleotide incorporation is achieved. These studies underscore the need to apply multiple biophysical and biochemical approaches to the elucidation of the physical basis for polymerase fidelity. PMID:25378410

  5. Structure-Guided Control of siRNA Off-Target Effects.

    Science.gov (United States)

    Suter, Scott R; Sheu-Gruttadauria, Jessica; Schirle, Nicole T; Valenzuela, Rachel; Ball-Jones, Alexi A; Onizuka, Kazumitsu; MacRae, Ian J; Beal, Peter A

    2016-07-20

    Short interfering RNAs (siRNAs) are promising therapeutics that make use of the RNA interference (RNAi) pathway, but liabilities arising from the native RNA structure necessitate chemical modification for drug development. Advances in the structural characterization of components of the human RNAi pathway have enabled structure-guided optimization of siRNA properties. Here we report the 2.3 Å resolution crystal structure of human Argonaute 2 (hAgo2), a key nuclease in the RNAi pathway, bound to an siRNA guide strand bearing an unnatural triazolyl nucleotide at position 1 (g1). Unlike natural nucleotides, this analogue inserts deeply into hAgo2's central RNA binding cleft and thus is able to modulate pairing between guide and target RNAs. The affinity of the hAgo2-siRNA complex for a seed-only matched target was significantly reduced by the triazolyl modification, while the affinity for a fully matched target was unchanged. In addition, siRNA potency for off-target repression was reduced (4-fold increase in IC50) by the modification, while on-target knockdown was improved (2-fold reduction in IC50). Controlling siRNA on-target versus microRNA (miRNA)-like off-target potency by projection of substituent groups into the hAgo2 central cleft from g1 is a new approach to enhance siRNA selectivity with a strong structural rationale.

  6. In situ structures of the segmented genome and RNA polymerase complex inside a dsRNA virus

    Science.gov (United States)

    Zhang, Xing; Ding, Ke; Yu, Xuekui; Chang, Winston; Sun, Jingchen; Hong Zhou, Z.

    2015-11-01

    Viruses in the Reoviridae, like the triple-shelled human rotavirus and the single-shelled insect cytoplasmic polyhedrosis virus (CPV), all package a genome of segmented double-stranded RNAs (dsRNAs) inside the viral capsid and carry out endogenous messenger RNA synthesis through a transcriptional enzyme complex (TEC). By direct electron-counting cryoelectron microscopy and asymmetric reconstruction, we have determined the organization of the dsRNA genome inside quiescent CPV (q-CPV) and the in situ atomic structures of TEC within CPV in both quiescent and transcribing (t-CPV) states. We show that the ten segmented dsRNAs in CPV are organized with ten TECs in a specific, non-symmetric manner, with each dsRNA segment attached directly to a TEC. The TEC consists of two extensively interacting subunits: an RNA-dependent RNA polymerase (RdRP) and an NTPase VP4. We find that the bracelet domain of RdRP undergoes marked conformational change when q-CPV is converted to t-CPV, leading to formation of the RNA template entry channel and access to the polymerase active site. An amino-terminal helix from each of two subunits of the capsid shell protein (CSP) interacts with VP4 and RdRP. These findings establish the link between sensing of environmental cues by the external proteins and activation of endogenous RNA transcription by the TEC inside the virus.

  7. RNA secondary structures regulate three steps of Rho-dependent transcription termination within a bacterial mRNA leader.

    Science.gov (United States)

    Kriner, Michelle A; Groisman, Eduardo A

    2017-01-25

    Transcription termination events in bacteria often require the RNA helicase Rho. Typically, Rho promotes termination at the end of coding sequences, but it can also terminate transcription within leader regions to implement regulatory decisions. Rho-dependent termination requires initial recognition of a Rho utilization (rut) site on a nascent RNA by Rho's primary binding surface. However, it is presently unclear what factors determine the location of transcription termination, how RNA secondary structures influence this process and whether mechanistic differences distinguish constitutive from regulated Rho-dependent terminators. We previously demonstrated that the 5' leader mRNA of the Salmonella corA gene can adopt two mutually exclusive conformations that dictate accessibility of a rut site to Rho. We now report that the corA leader also controls two subsequent steps of Rho-dependent termination. First, the RNA conformation that presents an accessible rut site promotes pausing of RNA polymerase (RNAP) at a single Rho-dependent termination site over 100 nt downstream. Second, an additional RNA stem-loop promotes Rho activity and controls the location at which Rho-dependent termination occurs, despite having no effect on initial Rho binding to the corA leader. Thus, the multi-step nature of Rho-dependent termination may facilitate regulation of a given coding region by multiple cytoplasmic signals.

  8. Oscillatory behaviour of catalytic properties, structure and temperature during the catalytic partial oxidation of methane on Pd/Al2O3

    DEFF Research Database (Denmark)

    Kimmerle, B.; Baiker, A.; Grunwaldt, Jan-Dierk

    2010-01-01

    Pd/Al2O3 catalysts showed an oscillatory behaviour during the catalytic partial oxidation (CPO) of methane, which was investigated simultaneously by IR-thermography, X-ray absorption spectroscopy, and online mass-spectrometry to correlate the temperature, state of the catalyst and catalytic...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. Exon structure requirements for yeast tRNA ligase

    Institute of Scientific and Technical Information of China (English)

    刘建华; 金由辛; 王德宝

    1997-01-01

    Different nucleotides were introduced into nucleotides 32, 37 and 38 of yeast tRNAphe precursors via oligonucleotide directed mutations. Pre-tRNAs were prepared using T7-transcription in vitro and spliced with the purified yeast tRNA endonuclease and tRNA ligase. It is demonstrated that tRNA ligase activities will be inhibited by the 5’-double-stranded end of 3’-halves.

  11. Synthesis Strategies to Design Structures for Catalytic Applications%硅基催化结构合成的策略

    Institute of Scientific and Technical Information of China (English)

    Harold H. KUNG; Mayfair C. KUNG

    2008-01-01

    Two approaches to synthesize silicon-based catalytic structures that aim at capturing the properties and functionalities of natural enzymes are described in this brief review: unit-by-unit synthesis of macromolecular units and templating/imprinting synthesis of nanocages. The unit-by-unit approach mimics the peptide synthesis method, offers atomic control of the structure, but is inefficient in synthesizing large structures such as nanocages. The templating/imprinting method is more suitable for nanocages at the sacrifice of atomic control, and the nanocages obtained are shown to possess properties exhibited by enzyme cavities.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-07-25

    Prohead RNA (pRNA) is an essential component in the assembly and operation of the powerful bacteriophage {psi}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 {angstrom} 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 {psi}29 DNA.

  13. Structural delineation of stem-loop RNA binding by human TAF15 protein.

    Science.gov (United States)

    Kashyap, Maruthi; Ganguly, Akshay Kumar; Bhavesh, Neel Sarovar

    2015-11-27

    Human TATA binding protein associated factor 2 N (TAF15) and Fused in sarcoma (FUS) are nucleic acid binding proteins belonging to the conserved FET family of proteins. They are involved in diverse processes such as pre-mRNA splicing, mRNA transport, and DNA binding. The absence of information regarding the structural mechanism employed by the FET family in recognizing and discriminating their cognate and non-cognate RNA targets has hampered the attainment of consensus on modes of protein-RNA binding for this family. Our study provides a molecular basis of this RNA recognition using a combination of solution-state NMR spectroscopy, calorimetry, docking and molecular dynamics simulation. Analysis of TAF15-RRM solution structure and its binding with stem-loop RNA has yielded conclusive evidence of a non-canonical mode of RNA recognition. Rather than classical stacking interactions that occur across nitrogen bases and aromatic amino acids on ribonucleoprotein sites, moderate-affinity hydrogen bonding network between the nitrogen bases in the stem-loop RNA and a concave face on the RRM surface primarily mediate TAF15-RRM RNA interaction. We have compared the binding affinities across a set of single-stranded RNA oligonucleotides to conclusively establish that RNA binding is dependent upon structural elements in the RNA rather than sequence.

  14. Mechanistic Insights into the Structure-Dependent Selectivity of Catalytic Furfural Conversion on Platinum Catalysts

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Qiuxia; Wang, Jianguo; Wang, Yang-Gang; Mei, Donghai

    2015-11-01

    The effects of structure and size on the selectivity of catalytic furfural conversion over supported Pt catalysts in the presence of hydrogen have been studied using first principles density functional theory (DFT) calculations and microkinetic modeling. Four Pt model systems, i.e., periodic Pt(111), Pt(211) surfaces, as well as small nanoclusters (Pt13 and Pt55) are chosen to represent the terrace, step, and corner sites of Pt nanoparticles. Our DFT results show that the reaction routes for furfural hydrogenation and decarbonylation are strongly dependent on the type of reactive sites, which lead to the different selectivity. On the basis of the size-dependent site distribution rule, we correlate the site distributions as a function of the Pt particle size. Our microkinetic results indicate the critical particle size that controls the furfural selectivity is about 1.0 nm, which is in good agreement with the reported experimental value under reaction conditions. This work was supported by National Basic Research Program of China (973 Program) (2013CB733501) and the National Natural Science Foundation of China (NSFC-21306169, 21176221, 21136001, 21101137 and 91334103). This work was also partially supported by the US Department of Energy (DOE), the Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences & Biosciences. Pacific Northwest National Laboratory (PNNL) is a multiprogram national laboratory operated for DOE by Battelle. Computing time was granted by the grand challenge of computational catalysis of the William R. Wiley Environmental Molecular Sciences Laboratory (EMSL). EMSL is a national scientific user facility located at Pacific Northwest National Laboratory (PNNL) and sponsored by DOE’s Office of Biological and Environmental Research.

  15. Crystal structure of Bacillus subtilis TrmB, the tRNA (m7G46) methyltransferase.

    Science.gov (United States)

    Zegers, Ingrid; Gigot, Daniel; van Vliet, Françoise; Tricot, Catherine; Aymerich, Stéphane; Bujnicki, Janusz M; Kosinski, Jan; Droogmans, Louis

    2006-01-01

    The structure of Bacillus subtilis TrmB (BsTrmB), the tRNA (m7G46) methyltransferase, was determined at a resolution of 2.1 A. This is the first structure of a member of the TrmB family to be determined by X-ray crystallography. It reveals a unique variant of the Rossmann-fold methyltransferase (RFM) structure, with the N-terminal helix folded on the opposite site of the catalytic domain. The architecture of the active site and a computational docking model of BsTrmB in complex with the methyl group donor S-adenosyl-L-methionine and the tRNA substrate provide an explanation for results from mutagenesis studies of an orthologous enzyme from Escherichia coli (EcTrmB). However, unlike EcTrmB, BsTrmB is shown here to be dimeric both in the crystal and in solution. The dimer interface has a hydrophobic core and buries a potassium ion and five water molecules. The evolutionary analysis of the putative interface residues in the TrmB family suggests that homodimerization may be a specific feature of TrmBs from Bacilli, which may represent an early stage of evolution to an obligatory dimer.

  16. Mutagenesis of the catalytic and cleavage site residues of the hypovirus papain-like proteases p29 and p48 reveals alternative processing and contributions to optimal viral RNA accumulation.

    Science.gov (United States)

    Jensen, Kenneth S; Nuss, Donald L

    2014-10-01

    The positive-stranded RNA genome of the prototypic virulence-attenuating hypovirus CHV-1/EP713 contains two open reading frames (ORF), each encoding an autocatalytic papain-like leader protease. Protease p29, derived from the N-terminal portion of ORF A, functions as a suppressor of RNA silencing, while protease p48, derived from the N-terminal portion of ORF B, is required for viral RNA replication. The catalytic and cleavage site residues required for autoproteolytic processing have been functionally mapped in vitro for both proteases but not confirmed in the infected fungal host. We report here the mutagenesis of the CHV-1/EP713 infectious cDNA clone to define the requirements for p29 and p48 cleavage and the role of autoproteolysis in the context of hypovirus replication. Mutation of the catalytic cysteine and histidine residues for either p29 or p48 was tolerated but reduced viral RNA accumulation to ca. 20 to 50% of the wild-type level. Mutation of the p29 catalytic residues caused an accumulation of unprocessed ORF A product p69. Surprisingly, the release of p48 from the ORF B-encoded polyprotein was not prevented by mutation of the p48 catalytic and cleavage site residues and was independent of p29. The results show that, while dispensable for hypovirus replication, the autocatalytic processing of the leader proteases p29 and p48 contributes to optimal virus RNA accumulation. The role of the predicted catalytic residues in autoproteolytic processing of p29 was confirmed in the infected host, while p48 was found to also undergo alternative processing independent of the encoded papain-like protease activities. Importance: Hypoviruses are positive-strand RNA mycoviruses that attenuate virulence of their pathogenic fungal hosts. The prototypic hypovirus CHV-1/EP713, which infects the chestnut bight fungus Cryphonetria parasitica, encodes two papain-like autocatalytic leader proteases, p29 and p48, that also have important functions in suppressing the RNA

  17. 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.

  18. Theoretical study of fMet-tRNA and fAla-tRNA structures by using quantum calculation

    Directory of Open Access Journals (Sweden)

    M. Noei

    2016-11-01

    Full Text Available In the prokaryotes, protein synthesis always starts with N-formylmethionine amino acid. Comparison of this amino acid with other amino acids is attempted and that is why formylmethionine is always the first amino acid to begin protein synthesis, in this paper we added a formyl group to alanine amino acid and then studied it when attached to the tRNA molecule and compared this structure with formylmethionine-tRNA structure. The quantum chemical calculations have performed using Gaussian 03 suite of programs. The fAla-tRNA and fMet-tRNA structures have fully optimized at the HF and B3LYP levels with 3–21G∗ and 6–31G∗ basis sets as well as MP2/3–21G∗ level and theoretically solvent effects on the structures were investigated. Then we studied electronic structures of the compounds using Natural Bond Orbital (NBO analysis and calculated NMR parameters at the gas-phase. Frequency analysis was also calculated at the HF and B3LYP/3-21G∗ levels in the different solvents in 298.15 K, 310.15 K temperatures and 1.00 atmosphere pressure.

  19. 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

  20. Small-angle X-ray scattering: a bridge between RNA secondary structures and three-dimensional topological structures

    Energy Technology Data Exchange (ETDEWEB)

    Fang, Xianyang [National Inst. of Health (NIH), Bethesda, MD (United States). National Cancer Inst., NCI Small Angle X-ray Scattering Core Facility; Stagno, Jason R. [National Inst. of Health (NIH), Bethesda, MD (United States). National Cancer Inst., Protein-Nucleic Acid Interaction Section, Structural Biophysics Lab.; Bhandari, Yuba R. [National Inst. of Health (NIH), Bethesda, MD (United States). National Cancer Inst., Protein-Nucleic Acid Interaction Section, Structural Biophysics Lab.; Zuo, Xiaobing [Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS); Wang, Yun-Xing [National Inst. of Health (NIH), Bethesda, MD (United States). National Cancer Inst., NCI Small Angle X-ray Scattering Core Facility; National Inst. of Health (NIH), Bethesda, MD (United States). National Cancer Inst., Protein-Nucleic Acid Interaction Section, Structural Biophysics Lab.

    2015-02-01

    Whereas the structures of small to medium-sized well folded RNA molecules often can be determined by either X-ray crystallography or NMR spectroscopy, obtaining structural information for large RNAs using experimental, computational, or combined approaches remains a major interest and challenge. RNA is very sensitive to small-angle X-ray scattering (SAXS) due to high electron density along phosphate-sugar backbones, whose scattering contribution dominates SAXS intensity. For this reason, SAXS is particularly useful in obtaining global RNA structural information that outlines backbone topologies and, therefore, molecular envelopes. Such information is extremely valuable in bridging the gap between the secondary structures and three-dimensional topological structures of RNAmolecules, particularly those that have proven difficult to study using other structuredetermination methods. Here we review published results of RNA topological structures derived from SAXS data or in combination with other experimental data, as well as details on RNA sample preparation for SAXS experiments.

  1. 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/.

  2. mRNA pseudoknot structures can act as ribosomal roadblocks

    DEFF Research Database (Denmark)

    Hansen, Jesper Tholstrup; Oddershede, Lene Broeng; Sørensen, Michael Askvad

    2012-01-01

    Several viruses utilize programmed ribosomal frameshifting mediated by mRNA pseudoknots in combination with a slippery sequence to produce a well defined stochiometric ratio of the upstream encoded to the downstream-encoded protein. A correlation between the mechanical strength of mRNA pseudoknot...

  3. 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.

  4. RNA Structure Refinement using the ERRASER-Phenix pipeline

    Science.gov (United States)

    Chou, Fang-Chieh; Echols, Nathaniel; Terwilliger, Thomas C.; Das, Rhiju

    2015-01-01

    Summary The final step of RNA crystallography involves the fitting of coordinates into electron density maps. The large number of backbone atoms in RNA presents a difficult and tedious challenge, particularly when experimental density is poor. The ERRASER-Phenix pipeline can improve an initial set of RNA coordinates automatically based on a physically realistic model of atomic-level RNA interactions. The pipeline couples diffraction-based refinement in Phenix with the Rosetta-based real-space refinement protocol ERRASER (Enumerative Real-Space Refinement ASsisted by Electron density under Rosetta). The combination of ERRASER and Phenix can improve the geometrical quality of RNA crystallographic models while maintaining or improving the fit to the diffraction data (as measured by Rfree). Here we present a complete tutorial for running ERRASER-Phenix through the Phenix GUI, from the command-line, and via an application in the Rosetta On-line Server that Includes Everyone (ROSIE). PMID:26227049

  5. Dynamics of translation by single ribosomes through mRNA secondary structures

    OpenAIRE

    Chen, Chunlai; Zhang, Haibo; Broitman, Steven L.; Reiche, Michael; Farrell, Ian; Cooperman, Barry S.; Goldman, Yale E.

    2013-01-01

    During protein synthesis, the ribosome translates nucleotide triplets in single-stranded mRNA into polypeptide sequences. Strong downstream mRNA secondary (2°) structures, which must be unfolded for translation, can slow or even halt protein synthesis. Here we employ single molecule fluorescence resonance energy transfer to determine reaction rates for specific steps within the elongation cycle as the Escherichia coli ribosome encounters stem loop or pseudoknot mRNAstructures. Downstream ...

  6. Effect of structure and surface properties on the catalytic activity of nanodiamond in the conversion of 1,2-dichloroethane

    Science.gov (United States)

    Tveritinova, E. A.; Zhitnev, Yu. N.; Kulakova, I. I.; Maslakov, K. I.; Nesterova, E. A.; Kharlanov, A. N.; Ivanov, A. S.; Savilov, S. V.; Lunin, V. V.

    2015-04-01

    The catalytic activity of a detonation nanodiamond and its Ni-containing forms in the conversion of 1,2-dichloroethane is studied and compared with the activity of other carbon and nanocarbon materials: carbon nanotubes, "Dalan" synthetic diamond, and fluorinated graphite. The surface and structure of the carbon materials are characterized using XRD, diffuse reflectance IR spectroscopy, XPS, BET, and TPR. The catalytic properties of the materials are studied using the pulsed microcatalytic method. It is found that the synthetic diamond, the nanodiamond, and its Ni-containing forms are catalysts for dichloroethane conversion in a nitrogen atmosphere, where the main product is ethylene. It is noted that the catalytic activity of deactivated diamond catalysts is restored after hydrogen treatment. It is shown that the carbon structure of the nanodiamond and the "Dalan" synthetic diamond with hydrogen groups located on it plays a key role in the dichloroethane conversion. It is found that the nanodiamond acts simultaneously as a catalyst and an adsorbent of chlorine-containing products of dichloroethane conversion.

  7. Reconstitution and structure of a bacterial Pnkp1RnlHen1 RNA repair complex

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Pei; Selvadurai, Kiruthika; Huang , Raven H. (UIUC)

    2016-01-22

    Ribotoxins cleave essential RNAs for cell killing, and RNA repair neutralizes the damage inflicted by ribotoxins for cell survival. We report a new bacterial RNA repair complex that performs RNA repair linked to immunity. This new RNA repair complex is a 270-kDa heterohexamer composed of three proteins—Pnkp1, Rnl and Hen1—that are required to repair ribotoxin-cleaved RNA in vitro. The crystal structure of the complex reveals the molecular architecture of the heterohexamer as two rhomboid-shaped ring structures of Pnkp1–Rnl–Hen1 heterotrimer fused at the Pnkp1 dimer interface. The four active sites required for RNA repair are located on the inner rim of each ring. Furthermore, the architecture and the locations of the active sites of the Pnkp1–Rnl–Hen1 heterohexamer suggest an ordered series of repair reactions at the broken RNA ends that confer immunity to recurrent damage.

  8. Sulfur-Functionalized N-Heterocyclic Carbene Complexes of Pd(II: Syntheses, Structures and Catalytic Activities

    Directory of Open Access Journals (Sweden)

    Dan Yuan

    2012-03-01

    Full Text Available N-heterocyclic carbenes (NHCs can be easily modified by introducing functional groups at the nitrogen atoms, which leads to versatile coordination chemistry as well as diverse catalytic applications of the resulting complexes. This article summarizes our contributions to the field of NHCs bearing different types of sulfur functions, i.e., thioether, sulfoxide, thiophene, and thiolato. The experimental evidence for the truly hemilabile coordination behavior of a Pd(II thioether-NHC complex has been reported as well. In addition, complexes bearing rigid CSC-pincer ligands have been synthesized and the reasons for pincer versus pseudo-pincer formation investigated. Incorporation of the electron-rich thiolato function resulted in the isolation of structurally diverse complexes. The catalytic activities of selected complexes have been tested in Suzuki-Miyaura, Mizoroki-Heck and hydroamination reactions.

  9. Two Gd(III) coordination polymers based on a flexible tricarboxylate: Syntheses, structures, luminescence and catalytic properties

    Science.gov (United States)

    Zhu, Yu; Zhu, Min; Liu, Pan; Xia, Li; Wu, Yunlong; Xie, Jimin

    2017-02-01

    Two Gadolinium coordination polymers {[Gd·(TTTA)·(H2O)2]·2H2O}n (1) and [Gd·(TTTA)·DMF]n (2) have been synthesized based on Gd(NO3)3·6H2O and the flexible tripodal ligand 2,2‧,2″-[1,3,5-triazine-2,4,6-triyltris(thio)]tris-acetic acid (H3TTTA) under hydrothermal conditions. In the structure of 1, the tridentate TTTA3- ligands connect the dimeric metal centers into an infinite one dimensional chain. While 2 shows a two dimensional network with tridentate TTTA3- ligands. The two complexes all quench the emission spectra after coordinated to the Gd3+ ions. The catalytic results indicate that two complexes show excellent activities for the cyanosilylation of benzaldehyde and its derivatives. Besides, 2 is better than 1 in the catalytic reactions due to more possibility of metal open sites.

  10. Structure effect of molybdenum (5) complexes on its activity in appearance of catalytic polarographic currents of chlorate- and perchlorate ions

    Energy Technology Data Exchange (ETDEWEB)

    Zajtsev, P.M.; Zhdanov, S.I.; Dergacheva, E.N.; Savchenko, E.N.; Nikolaeva, T.D. (Vsesoyuznyj Nauchno-Issledovatel' skij Inst. Khimicheskikh Reaktivov i Osobo Chistykh Veshchestv, Moscow (USSR))

    1982-08-01

    Polarographic behaviour and reactivity of synthesized molybdenum (5) in reactions, conditioning catalytic currents of ClO/sub 3//sup -/ and ClO/sub 4//sup -/ have been studied. Their comparison with similar characteristics for molybdenum (5) appearing in the process of Mo (6) solution polarography is made. For the purpose a salt of molybdenum (5) in H/sub 2/SO/sub 4/, HCl and HClO/sub 4/ solutions have been synthesized by electrochemical and chemical ways. It has been established that in reactions conditioning catalytic currents of chlorate- and perchlorate-ions the preservation of structure of Mo (6) complex in the Mo (5) complex formed, i.e. processes of Mo (5) complex ageing, plays a very significant role.

  11. 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 R...

  12. 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.

  13. Structural basis for the modular recognition of single-stranded RNA by PPR proteins

    Science.gov (United States)

    Yin, Ping; Li, Quanxiu; Yan, Chuangye; Liu, Ying; Liu, Junjie; Yu, Feng; Wang, Zheng; Long, Jiafu; He, Jianhua; Wang, Hong-Wei; Wang, Jiawei; Zhu, Jian-Kang; Shi, Yigong; Yan, Nieng

    2013-12-01

    Pentatricopeptide repeat (PPR) proteins represent a large family of sequence-specific RNA-binding proteins that are involved in multiple aspects of RNA metabolism. PPR proteins, which are found in exceptionally large numbers in the mitochondria and chloroplasts of terrestrial plants, recognize single-stranded RNA (ssRNA) in a modular fashion. The maize chloroplast protein PPR10 binds to two similar RNA sequences from the ATPI-ATPH and PSAJ-RPL33 intergenic regions, referred to as ATPH and PSAJ, respectively. By protecting the target RNA elements from 5' or 3' exonucleases, PPR10 defines the corresponding 5' and 3' messenger RNA termini. Despite rigorous functional characterizations, the structural basis of sequence-specific ssRNA recognition by PPR proteins remains to be elucidated. Here we report the crystal structures of PPR10 in RNA-free and RNA-bound states at resolutions of 2.85 and 2.45Å, respectively. In the absence of RNA binding, the nineteen repeats of PPR10 are assembled into a right-handed superhelical spiral. PPR10 forms an antiparallel, intertwined homodimer and exhibits considerable conformational changes upon binding to its target ssRNA, an 18-nucleotide PSAJ element. Six nucleotides of PSAJ are specifically recognized by six corresponding PPR10 repeats following the predicted code. The molecular basis for the specific and modular recognition of RNA bases A, G and U is revealed. The structural elucidation of RNA recognition by PPR proteins provides an important framework for potential biotechnological applications of PPR proteins in RNA-related research areas.

  14. Bias in ligation-based small RNA sequencing library construction is determined by adaptor and RNA structure.

    Directory of Open Access Journals (Sweden)

    Ryan T Fuchs

    Full Text Available High-throughput sequencing (HTS has become a powerful tool for the detection of and sequence characterization of microRNAs (miRNA and other small RNAs (sRNA. Unfortunately, the use of HTS data to determine the relative quantity of different miRNAs in a sample has been shown to be inconsistent with quantitative PCR and Northern Blot results. Several recent studies have concluded that the major contributor to this inconsistency is bias introduced during the construction of sRNA libraries for HTS and that the bias is primarily derived from the adaptor ligation steps, specifically where single stranded adaptors are sequentially ligated to the 3' and 5'-end of sRNAs using T4 RNA ligases. In this study we investigated the effects of ligation bias by using a pool of randomized ligation substrates, defined mixtures of miRNA sequences and several combinations of adaptors in HTS library construction. We show that like the 3' adaptor ligation step, the 5' adaptor ligation is also biased, not because of primary sequence, but instead due to secondary structures of the two ligation substrates. We find that multiple secondary structural factors influence final representation in HTS results. Our results provide insight about the nature of ligation bias and allowed us to design adaptors that reduce ligation bias and produce HTS results that more accurately reflect the actual concentrations of miRNAs in the defined starting material.

  15. Noble metal-based bimetallic nanoparticles: the effect of the structure on the optical, catalytic and photocatalytic properties.

    Science.gov (United States)

    Zaleska-Medynska, Adriana; Marchelek, Martyna; Diak, Magdalena; Grabowska, Ewelina

    2016-03-01

    Nanoparticles composed of two different metal elements show novel electronic, optical, catalytic or photocatalytic properties from monometallic nanoparticles. Bimetallic nanoparticles could show not only the combination of the properties related to the presence of two individual metals, but also new properties due to a synergy between two metals. The structure of bimetallic nanoparticles can be oriented in random alloy, alloy with an intermetallic compound, cluster-in-cluster or core-shell structures and is strictly dependent on the relative strengths of metal-metal bond, surface energies of bulk elements, relative atomic sizes, preparation method and conditions, etc. In this review, selected properties, such as structure, optical, catalytic and photocatalytic of noble metals-based bimetallic nanoparticles, are discussed together with preparation routes. The effects of preparation method conditions as well as metal properties on the final structure of bimetallic nanoparticles (from alloy to core-shell structure) are followed. The role of bimetallic nanoparticles in heterogeneous catalysis and photocatalysis are discussed. Furthermore, structure and optical characteristics of bimetallic nanoparticles are described in relation to the some features of monometallic NPs. Such a complex approach allows to systematize knowledge and to identify the future direction of research.

  16. The structure of a rigorously conserved RNA element within the SARS virus genome.

    Directory of Open Access Journals (Sweden)

    Michael P Robertson

    2005-01-01

    Full Text Available We have solved the three-dimensional crystal structure of the stem-loop II motif (s2m RNA element of the SARS virus genome to 2.7-A resolution. SARS and related coronaviruses and astroviruses all possess a motif at the 3' end of their RNA genomes, called the s2m, whose pathogenic importance is inferred from its rigorous sequence conservation in an otherwise rapidly mutable RNA genome. We find that this extreme conservation is clearly explained by the requirement to form a highly structured RNA whose unique tertiary structure includes a sharp 90 degrees kink of the helix axis and several novel longer-range tertiary interactions. The tertiary base interactions create a tunnel that runs perpendicular to the main helical axis whose interior is negatively charged and binds two magnesium ions. These unusual features likely form interaction surfaces with conserved host cell components or other reactive sites required for virus function. Based on its conservation in viral pathogen genomes and its absence in the human genome, we suggest that these unusual structural features in the s2m RNA element are attractive targets for the design of anti-viral therapeutic agents. Structural genomics has sought to deduce protein function based on three-dimensional homology. Here we have extended this approach to RNA by proposing potential functions for a rigorously conserved set of RNA tertiary structural interactions that occur within the SARS RNA genome itself. Based on tertiary structural comparisons, we propose the s2m RNA binds one or more proteins possessing an oligomer-binding-like fold, and we suggest a possible mechanism for SARS viral RNA hijacking of host protein synthesis, both based upon observed s2m RNA macromolecular mimicry of a relevant ribosomal RNA fold.

  17. 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.

  18. EFFECT OF MORPHOLOGICAL STRUCTURE OF AMINOMETHYL POLYSTYRENE RESIN ON THE CATALYTIC PROPERTIES OF POLYMER-SUPPORTED RUTHENIUM COMPLEXES

    Institute of Scientific and Technical Information of China (English)

    Ren-ren Wang; Jia-qin Wang; Ce Luo; Zhe Zhang; Bi-tao Su; Zi-qiang Lei

    2009-01-01

    Polymer-supported ruthenium complexes (p)-Phen-Ru-①,(p)-Phen-Ru-②,(P)-Phen-Ru-③,(p)-Phen-Ru-④,morphological structures as supports.A variety of alcohols were oxidized efficiently into the corresponding ketones,carboxylic acids or aldehydes with iodosylbenzene (PhIO) catalyzed by aminomethyl polystyrene-supported ruthenium complexes under mild reaction conditions in acetonitrile.The influences of morphological structure of the polymer supporters on the catalytic properties of these metal complexes were investigated in detail.

  19. Crystal Structure of a Trapped Catalytic Intermediate Suggests that Forced Atomic Proximity Drives the Catalysis of mIPS

    OpenAIRE

    Neelon, Kelly; Roberts, Mary F.; Stec, Boguslaw

    2011-01-01

    1-L-myo-inositol-phosphate synthase (mIPS) catalyzes the first step of the unique, de novo pathway of inositol biosynthesis. However, details about the complex mIPS catalytic mechanism, which requires oxidation, enolization, intramolecular aldol cyclization, and reduction, are not fully known. To gain further insight into this mechanism, we determined the crystal structure of the wild-type mIPS from Archaeoglobus fulgidus at 1.7 Å, as well as the crystal structures of three active-site mutant...

  20. Cold Spring Harbor symposia on quantitative biology: Volume 52, Evolution of catalytic function

    Energy Technology Data Exchange (ETDEWEB)

    1987-01-01

    This document contains 97 papers presented at the symposium. The primary topic was the evolution of the catalytic function. Speakers discussed the evolution of genetic apparatus, the primordial soup, the anatomy of RNA, RNA templates, protein assembly, protein structure, cofactors, ribosomes, exons, and introns. Individual papers were processed separately for the data base. (TEM)

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

    Science.gov (United States)

    Fernández-García, Yaiza; Reguera, Juan; Busch, Carola; Witte, Gregor; Sánchez-Ramos, Oliberto; Betzel, Christian; Cusack, Stephen; Günther, Stephan; Reindl, Sophia

    2016-06-01

    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.

  2. 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.

  3. PSRna: Prediction of small RNA secondary structures based on reverse complementary folding method.

    Science.gov (United States)

    Li, Jin; Xu, Chengzhen; Wang, Lei; Liang, Hong; Feng, Weixing; Cai, Zhongxi; Wang, Ying; Cong, Wang; Liu, Yunlong

    2016-08-01

    Prediction of RNA secondary structures is an important problem in computational biology and bioinformatics, since RNA secondary structures are fundamental for functional analysis of RNA molecules. However, small RNA secondary structures are scarce and few algorithms have been specifically designed for predicting the secondary structures of small RNAs. Here we propose an algorithm named "PSRna" for predicting small-RNA secondary structures using reverse complementary folding and characteristic hairpin loops of small RNAs. Unlike traditional algorithms that usually generate multi-branch loops and 5[Formula: see text] end self-folding, PSRna first estimated the maximum number of base pairs of RNA secondary structures based on the dynamic programming algorithm and a path matrix is constructed at the same time. Second, the backtracking paths are extracted from the path matrix based on backtracking algorithm, and each backtracking path represents a secondary structure. To improve accuracy, the predicted RNA secondary structures are filtered based on their free energy, where only the secondary structure with the minimum free energy was identified as the candidate secondary structure. Our experiments on real data show that the proposed algorithm is superior to two popular methods, RNAfold and RNAstructure, in terms of sensitivity, specificity and Matthews correlation coefficient (MCC).

  4. Immobilization of rhodium complexes at thiolate monolayers on gold surfaces : Catalytic and structural studies

    NARCIS (Netherlands)

    Belser, T; Stöhr, Meike; Pfaltz, A

    2005-01-01

    Chiral rhodium-diphosphine complexes have been incorporated into self-assembled thiolate monolayers (SAMS) on gold colloids. Catalysts of this type are of interest because they combine properties of homogeneous and heterogeneous systems. In addition, it should be possible to influence the catalytic

  5. Surface Structure and Catalytic Performance of Ni-Fe Catalyst for Low-Temperature CO Hydrogenation

    Directory of Open Access Journals (Sweden)

    Fanhui Meng

    2014-01-01

    Full Text Available Catalysts 16NixFe/Al2O3 (x is 0, 1, 2, 4, 6, 8 were prepared by incipient wetness impregnation method and the catalytic performance for the production of synthetic natural gas (SNG from CO hydrogenation in slurry-bed reactor were studied. The catalysts were characterized by BET, XRD, UV-Vis DRS, H2-TPR, CO-TPD, and XPS, and the results showed that the introduction of iron improved the dispersion of Ni species, weakened the interaction between Ni species and support and decreased the reduction temperature and that catalyst formed Ni-Fe alloy when the content of iron exceeded 2%. Experimental results revealed that the addition of iron to the catalyst can effectively improve the catalytic performance of low-temperature CO methanation. Catalyst 16Ni4Fe/Al2O3 with the iron content of 4% exhibited the best catalytic performance, the conversion of CO and the yield of CH4 reached 97.2% and 84.9%, respectively, and the high catalytic performance of Ni-Fe catalyst was related to the property of formed Ni-Fe alloy. Further increase of iron content led to enhancing the water gas shift reaction.

  6. A fast selenium derivatization strategy for crystallization and phasing of RNA structures

    OpenAIRE

    Olieric, Vincent; Rieder, Ulrike; Lang, Kathrin; Serganov, Alexander; Schulze-Briese, Clemens; Micura, Ronald; Dumas, Philippe; Ennifar, Eric

    2009-01-01

    Site-specific 2′-methylseleno RNA labeling is a promising tool for tackling the phase problem in RNA crystallography. We have developed an efficient strategy for crystallization and structure determination of RNA and RNA/protein complexes based on preliminary crystallization screening of 2′-OCH3-modified RNA sequences, prior to the replacement of 2′-OCH3 groups with their 2′-SeCH3 counterparts. The method exploits the similar crystallization properties of 2′-OCH3- and 2′-SeCH3-modified RNAs a...

  7. Structural comparisons of the nucleoprotein from three negative strand RNA virus families

    Directory of Open Access Journals (Sweden)

    Tsao Jun

    2007-07-01

    Full Text Available Abstract Structures of the nucleoprotein of three negative strand RNA virus families, borna disease virus, rhabdovirus and influenza A virus, are now available. Structural comparisons showed that the topology of the RNA binding region from the three proteins is very similar. The RNA was shown to fit into a cavity formed by the two distinct domains of the RNA binding region in the rhabdovirus nucleoprotein. Two helices connecting the two domains characterize the center of the cavity. The nucleoproteins contain at least 5 conserved helices in the N-terminal domain and 3 conserved helices in the C-terminal domain. Since all negative strand RNA viruses are required to have the ribonucleoprotein complex as their active genomic templates, it is perceivable that the (5H+3H structure is a common motif in the nucleoprotein of negative strand RNA viruses.

  8. Structures of the Bacterial Ribosome in Classical and Hybrid States of tRNA Binding

    Energy Technology Data Exchange (ETDEWEB)

    Dunkle, Jack A.; Wang, Leyi; Feldman, Michael B.; Pulk, Arto; Chen, Vincent B.; Kapral, Gary J.; Noeske, Jonas; Richardson, Jane S.; Blanchard, Scott C.; Cate, Jamie H. Doudna (Cornell); (UCB); (Duke)

    2011-09-06

    During protein synthesis, the ribosome controls the movement of tRNA and mRNA by means of large-scale structural rearrangements. We describe structures of the intact bacterial ribosome from Escherichia coli that reveal how the ribosome binds tRNA in two functionally distinct states, determined to a resolution of {approx}3.2 angstroms by means of x-ray crystallography. One state positions tRNA in the peptidyl-tRNA binding site. The second, a fully rotated state, is stabilized by ribosome recycling factor and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit site. The structures help to explain how the ratchet-like motion of the two ribosomal subunits contributes to the mechanisms of translocation, termination, and ribosome recycling.

  9. Electrical, thermal, catalytic and magnetic properties of nano-structured materials and their applications

    Science.gov (United States)

    Liu, Zuwei

    properties are briefly reviewed in Chapter One, including the concepts of ferro-magnetism, plasmonics, photocatalysis, thermal emission, and Raman spectra of carbon nanotubes. In Chapter Two, we focus on the magnetic properties of ferro-magnetic cobalt nanowires with high crystalline quality synthesized via a low voltage electro-deposition method. The crystal structure of these Co nanowires is characterized by high resolution transmission electron microscopy and X-ray diffraction. The magnetic properties of individual nanowires and nanowire arrays are investigated by magnetic force microscope (MFM) and superconducting quantum interference device (SQUID) measurements. A theoretical model is developed to explain these experimental observations. In Chapter Three, we exploit the strong plasmon resonance of gold nanoparticles. We also demonstrate a new method for patterning SERS (surface enhanced Raman spectroscopy) aggregates of gold nanoparticles by using a focused laser beam to optically trap the nanoparticles in a water suspension. Raman spectroscopy is used to estimate the temperature in the laser spot during the in-situ aggregation, by measuring the Raman peak of the hydroxyl bond of water. In Chapter Four, we demonstrate plasmonic enhancement of photocatalytic water splitting under visible illumination by integrating strongly plasmonic Au nanoparticles with strongly catalytic TiO2. Electromagnetic simulations indicate that the near-field optical enhancement increases the electron-hole pair generation rate at the surface of the TiO2, thus increasing the amount of photo-generated charge contributing to catalysis. Our results suggest that enhancement factors many times larger than this are possible if this mechanism can be optimized. In Chapter Five, we study the Raman spectra and thermal emission spectra of individual suspended carbon nanotubes induced by electrical heating. Semiconducting and metallic devices exhibit different spectra, based on their distinctive band

  10. 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

  11. Catalytic Features of the Botulinum Neurotoxin A Light Chain Revealed by High Resolution Structure of an Inhibitory Peptide Complex

    Energy Technology Data Exchange (ETDEWEB)

    Silvaggi,N.; Wilson, D.; Tzipori, S.; Allen, K.

    2008-01-01

    The Clostridium botulinum neurotoxin serotype A light chain (BoNT/A-LC) is a Zn(II)-dependent metalloprotease that blocks the release of acetylcholine at the neuromuscular junction by cleaving SNAP-25, one of the SNARE proteins required for exocytosis. Because of the potential for use of the toxin in bioterrorism and the increasingly widespread application of the toxin in the medical field, there is significant interest in the development of small-molecule inhibitors of the metalloprotease. Efforts to design such inhibitors have not benefited from knowledge of how peptides bind to the active site since the enzyme-peptide structures available previously either were not occupied in the vicinity of the catalytic Zn(II) ion or did not represent the product of SNAP-25 substrate cleavage. Herein we report the 1.4 Angstroms-resolution X-ray crystal structure of a complex between the BoNT/A-LC and the inhibitory peptide N-Ac-CRATKML, the first structure of the light chain with an inhibitory peptide bound at the catalytic Zn(II) ion. The peptide is bound with the Cys S? atom coordinating the metal ion. Surprisingly, the cysteine sulfur is oxidized to the sulfenic acid form. Given the unstable nature of this species in solution, is it likely that oxidation occurs on the enzyme. In addition to the peptide-bound structure, we report two structures of the unliganded light chain with and without the Zn(II) cofactor bound at 1.25 and 1.20 Angstroms resolution, respectively. The two structures are nearly identical, confirming that the Zn(II) ion plays a purely catalytic role. Additionally, the structure of the Zn(II)-bound uncomplexed enzyme allows identification of the catalytic water molecule and a second water molecule that occupies the same position as the peptidic oxygen in the tetrahedral intermediate. This observation suggests that the enzyme active site is prearranged to stabilize the tetrahedral intermediate of the protease reaction.

  12. Crystal structures at 2.5 Angstrom resolution of seryl-tRNA synthetase complexed with two analogs of seryl adenylate

    DEFF Research Database (Denmark)

    Belrhali, H.; Yaremchuk, A.; Tukalo, M.;

    1994-01-01

    Crystal structures of seryl-tRNA synthetase from Thermus thermophilus complexed with two different analogs of seryl adenylate have been determined at 2.5 Angstrom resolution. The first complex is between the enzyme and seryl-hydroxamate-AMP (adenosine monophosphate), produced enzymatically...... in a deep hydrophilic cleft formed by the antiparallel beta sheet and surrounding loops of the synthetase catalytic domain. Four regions in the primary sequence are involved in the interactions, including the motif 2 and 3 regions of class 2 synthetases. Apart from the specific recognition of the serine...

  13. Structure and Activity of an Aminoacyl-tRNA Synthetase that Charges tRNA with Nitro-Tryptophan

    Energy Technology Data Exchange (ETDEWEB)

    Buddha,M.; Crane, B.

    2005-01-01

    The most divergent of two tryptophanyl tRNA synthetases (TrpRS II) found in Deinococcus radiodurans interacts with a nitric oxide synthase protein that produces 4-nitro-tryptophan (4-NRP). TrpRS II efficiently charges transfer RNATrp with 4-NRP and 5-hydroxy-tryptophan (5-HRP). The crystal structures of TrpRS II bound to tryptophan and 5-HRP reveal residue substitutions that accommodate modified indoles. A class of auxiliary bacterial TrpRSs conserve this capacity to charge tRNA with nonstandard amino acids.

  14. Structure of human aspartyl aminopeptidase complexed with substrate analogue: insight into catalytic mechanism, substrate specificity and M18 peptidase family

    Directory of Open Access Journals (Sweden)

    Chaikuad Apirat

    2012-06-01

    Full Text Available Abstract Backround Aspartyl aminopeptidase (DNPEP, with specificity towards an acidic amino acid at the N-terminus, is the only mammalian member among the poorly understood M18 peptidases. DNPEP has implicated roles in protein and peptide metabolism, as well as the renin-angiotensin system in blood pressure regulation. Despite previous enzyme and substrate characterization, structural details of DNPEP regarding ligand recognition and catalytic mechanism remain to be delineated. Results The crystal structure of human DNPEP complexed with zinc and a substrate analogue aspartate-β-hydroxamate reveals a dodecameric machinery built by domain-swapped dimers, in agreement with electron microscopy data. A structural comparison with bacterial homologues identifies unifying catalytic features among the poorly understood M18 enzymes. The bound ligands in the active site also reveal the coordination mode of the binuclear zinc centre and a substrate specificity pocket for acidic amino acids. Conclusions The DNPEP structure provides a molecular framework to understand its catalysis that is mediated by active site loop swapping, a mechanism likely adopted in other M18 and M42 metallopeptidases that form dodecameric complexes as a self-compartmentalization strategy. Small differences in the substrate binding pocket such as shape and positive charges, the latter conferred by a basic lysine residue, further provide the key to distinguishing substrate preference. Together, the structural knowledge will aid in the development of enzyme-/family-specific aminopeptidase inhibitors.

  15. Hierarchical folding of multiple sequence alignments for the prediction of structures and RNA-RNA interactions

    DEFF Research Database (Denmark)

    Seemann, Ernst Stefan; Richter, Andreas S.; Gorodkin, Jan;

    2010-01-01

    Background: Many regulatory non-coding RNAs (ncRNAs) function through complementary binding with mRNAs or other ncRNAs, e.g., microRNAs, snoRNAs and bacterial sRNAs. Predicting these RNA interactions is essential for functional studies of putative ncRNAs or for the design of artificial RNAs. Many...

  16. Dynamics of translation by single ribosomes through mRNA secondary structures.

    Science.gov (United States)

    Chen, Chunlai; Zhang, Haibo; Broitman, Steven L; Reiche, Michael; Farrell, Ian; Cooperman, Barry S; Goldman, Yale E

    2013-05-01

    During protein synthesis, the ribosome translates nucleotide triplets in single-stranded mRNA into polypeptide sequences. Strong downstream mRNA secondary structures, which must be unfolded for translation, can slow or even halt protein synthesis. Here we used single-molecule fluorescence resonance energy transfer to determine reaction rates for specific steps within the elongation cycle as the Escherichia coli ribosome encounters stem-loop or pseudoknot mRNA secondary structures. Downstream stem-loops containing 100% GC base pairs decrease the rates of both tRNA translocation within the ribosome and deacylated tRNA dissociation from the ribosomal exit site (E site). Downstream stem-loops or pseudoknots containing both GC and AU pairs also decrease the rate of tRNA dissociation, but they have little effect on tRNA translocation rate. Thus, somewhat unexpectedly, unfolding of mRNA secondary structures is more closely coupled to E-site tRNA dissociation than to tRNA translocation.

  17. Hierarchical folding of multiple sequence alignments for the prediction of structures and RNA-RNA interactions

    Directory of Open Access Journals (Sweden)

    Gorodkin Jan

    2010-05-01

    Full Text Available Abstract Background Many regulatory non-coding RNAs (ncRNAs function through complementary binding with mRNAs or other ncRNAs, e.g., microRNAs, snoRNAs and bacterial sRNAs. Predicting these RNA interactions is essential for functional studies of putative ncRNAs or for the design of artificial RNAs. Many ncRNAs show clear signs of undergoing compensating base changes over evolutionary time. Here, we postulate that a non-negligible part of the existing RNA-RNA interactions contain preserved but covarying patterns of interactions. Methods We present a novel method that takes compensating base changes across the binding sites into account. The algorithm works in two steps on two pre-generated multiple alignments. In the first step, individual base pairs with high reliability are found using the PETfold algorithm, which includes evolutionary and thermodynamic properties. In step two (where high reliability base pairs from step one are constrained as unpaired, the principle of cofolding is combined with hierarchical folding. The final prediction of intra- and inter-molecular base pairs consists of the reliabilities computed from the constrained expected accuracy scoring, which is an extended version of that used for individual multiple alignments. Results We derived a rather extensive algorithm. One of the advantages of our approach (in contrast to other RNA-RNA interaction prediction methods is the application of covariance detection and prediction of pseudoknots between intra- and inter-molecular base pairs. As a proof of concept, we show an example and discuss the strengths and weaknesses of the approach.

  18. 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 Anders Edvin;

    2011-01-01

    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...... 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...

  19. 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....

  20. 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.

  1. Tertiary structure mapping of the pri-miRNA miR-17~92.

    Science.gov (United States)

    Chaulk, Steven G; Fahlman, Richard P

    2014-01-01

    The understanding of RNA in regulating gene expression has exploded over the past 15 years. MicroRNAs (miRNAs) have vastly expanded the role of RNA in gene regulation beyond spliceosomal, ribosomal, and messenger RNAs. Approximately one half of miRNAs are polycistronic, where two or more miRNAs are encoded on a single pri-miRNA transcript, termed a miRNA cluster. The six miRNAs of the miR-17~92 cluster are contained within a ~800 nucleotide region within intron 3 of the cl13orf25 ~7 kb pri-miRNA transcript. We recently reported on the tertiary structured domain of miR-17~92 and its role in modulating miRNA biogenesis. The key finding was that the cluster structure explained the differential processing of the miRNA hairpins by Drosha. This work demonstrated the need to consider pri-miRNA tertiary structure in miRNA biogenesis. Since biochemical structure probing is typically performed on relatively short RNAs (≤200 nucleotides), we had to adapt these methodologies for application on large RNAs (~800 nucleotide miR-17~92 pri-miRNA). We present here our adaptation of a protection footprinting method using ribonucleases to probe the structure of the ~800 nucleotide miR-17~92 pri-miRNA. We outline the technical difficulties involved in probing large RNAs and data visualization using denaturing polyacrylamide gel electrophoresis and how we adapted the existing approaches to probe large RNAs. The methodology outlined here is generally applicable to large RNAs including long noncoding RNAs (lncRNA).

  2. Crystal structure of the single-stranded RNA binding protein HutP from Geobacillus thermodenitrificans.

    Science.gov (United States)

    Thiruselvam, Viswanathan; Sivaraman, Padavattan; Kumarevel, Thirumananseri; Ponnuswamy, Mondikalipudur Nanjappagounder

    2014-04-18

    RNA binding proteins control gene expression by the attenuation/antitermination mechanism. HutP is an RNA binding antitermination protein. It regulates the expression of hut operon when it binds with RNA by modulating the secondary structure of single-stranded hut mRNA. HutP necessitates the presence of l-histidine and divalent metal ion to bind with RNA. Herein, we report the crystal structures of ternary complex (HutP-l-histidine-Mg(2+)) and EDTA (0.5 M) treated ternary complex (HutP-l-histidine-Mg(2+)), solved at 1.9 Å and 2.5 Å resolutions, respectively, from Geobacillus thermodenitrificans. The addition of 0.5 M EDTA does not affect the overall metal-ion mediated ternary complex structure and however, the metal ions at the non-specific binding sites are chelated, as evidenced from the results of structural features.

  3. A fast selenium derivatization strategy for crystallization and phasing of RNA structures

    Science.gov (United States)

    Olieric, Vincent; Rieder, Ulrike; Lang, Kathrin; Serganov, Alexander; Schulze-Briese, Clemens; Micura, Ronald; Dumas, Philippe; Ennifar, Eric

    2009-01-01

    Site-specific 2′-methylseleno RNA labeling is a promising tool for tackling the phase problem in RNA crystallography. We have developed an efficient strategy for crystallization and structure determination of RNA and RNA/protein complexes based on preliminary crystallization screening of 2′-OCH3-modified RNA sequences, prior to the replacement of 2′-OCH3 groups with their 2′-SeCH3 counterparts. The method exploits the similar crystallization properties of 2′-OCH3- and 2′-SeCH3-modified RNAs and has been successfully validated for two test cases. In addition, our data show that 2′-SeCH3-modified RNA have an increased resistance to X-ray radiolysis in comparison with commonly used 5-halogen-modified RNA, which permits collection of experimental electron density maps of remarkable quality. PMID:19228585

  4. A detailed view of a ribosomal active site: the structure of the L11-RNA complex.

    Science.gov (United States)

    Wimberly, B T; Guymon, R; McCutcheon, J P; White, S W; Ramakrishnan, V

    1999-05-14

    We report the crystal structure of a 58 nucleotide fragment of 23S ribosomal RNA bound to ribosomal protein L11. This highly conserved ribonucleoprotein domain is the target for the thiostrepton family of antibiotics that disrupt elongation factor function. The highly compact RNA has both familiar and novel structural motifs. While the C-terminal domain of L11 binds RNA tightly, the N-terminal domain makes only limited contacts with RNA and is proposed to function as a switch that reversibly associates with an adjacent region of RNA. The sites of mutations conferring resistance to thiostrepton and micrococcin line a narrow cleft between the RNA and the N-terminal domain. These antibiotics are proposed to bind in this cleft, locking the putative switch and interfering with the function of elongation factors.

  5. Identification and structural analysis of a novel snoRNA gene cluster from Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    周惠; 孟清; 屈良鹄

    2000-01-01

    A 22 snoRNA gene cluster, consisting of four antisense snoRNA genes, was identified from Arabidopsis thaliana. The sequence and structural analysis showed that the 22 snoRNA gene cluster might be transcribed as a polycistronic precursor from an upstream promoter, and the in-tergenic spacers of the gene cluster encode the ’hairpin’ structures similar to the processing recognition signals of yeast Saccharomyces cerevisiae polycistronic snoRNA precursor. The results also revealed that plant snoRNA gene with multiple copies is a characteristic in common, and provides a good system for further revealing the transcription and expression mechanism of plant snoRNA gene cluster.

  6. 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

    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......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...... 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...

  7. Novel catalytic applications of carbon nanofibers on sintered metal fibers filters as structured supports

    OpenAIRE

    2009-01-01

    Supported metal catalysts are important from both an industrial and a scientific point of view. They are used, amongst others, in large-scale processes such as catalytic reforming, hydrotreating, polymerization reactions and hydrogenations. Often, these catalysts consist of nanosized metal particles deposited on a suitable support, which acts as an anchor for the active phase and, in several cases, contributes to improve the overall catalyst performances. The growth of carbon nanofibers on si...

  8. Novel catalytic applications of carbon nanofibers on sintered metal fibers filters as structured supports

    OpenAIRE

    2008-01-01

    Supported metal catalysts are important from both an industrial and a scientific point of view. They are used, amongst others, in large-scale processes such as catalytic reforming, hydrotreating, polymerization reactions and hydrogenations. Often, these catalysts consist of nanosized metal particles deposited on a suitable support, which acts as an anchor for the active phase and, in several cases, contributes to improve the overall catalyst performances. The growth of carbon nanofibers on si...

  9. New Structure Sheds Light on Selective HIV-1 Genomic RNA Packaging.

    Science.gov (United States)

    Olson, Erik D; Cantara, William A; Musier-Forsyth, Karin

    2015-08-24

    Two copies of unspliced human immunodeficiency virus (HIV)-1 genomic RNA (gRNA) are preferentially selected for packaging by the group-specific antigen (Gag) polyprotein into progeny virions as a dimer during the late stages of the viral lifecycle. Elucidating the RNA features responsible for selective recognition of the full-length gRNA in the presence of an abundance of other cellular RNAs and spliced viral RNAs remains an area of intense research. The recent nuclear magnetic resonance (NMR) structure by Keane et al. [1] expands upon previous efforts to determine the conformation of the HIV-1 RNA packaging signal. The data support a secondary structure wherein sequences that constitute the major splice donor site are sequestered through base pairing, and a tertiary structure that adopts a tandem 3-way junction motif that exposes the dimerization initiation site and unpaired guanosines for specific recognition by Gag. While it remains to be established whether this structure is conserved in the context of larger RNA constructs or in the dimer, this study serves as the basis for characterizing large RNA structures using novel NMR techniques, and as a major advance toward understanding how the HIV-1 gRNA is selectively packaged.

  10. New Structure Sheds Light on Selective HIV-1 Genomic RNA Packaging

    Directory of Open Access Journals (Sweden)

    Erik D. Olson

    2015-08-01

    Full Text Available Two copies of unspliced human immunodeficiency virus (HIV-1 genomic RNA (gRNA are preferentially selected for packaging by the group-specific antigen (Gag polyprotein into progeny virions as a dimer during the late stages of the viral lifecycle. Elucidating the RNA features responsible for selective recognition of the full-length gRNA in the presence of an abundance of other cellular RNAs and spliced viral RNAs remains an area of intense research. The recent nuclear magnetic resonance (NMR structure by Keane et al. [1] expands upon previous efforts to determine the conformation of the HIV-1 RNA packaging signal. The data support a secondary structure wherein sequences that constitute the major splice donor site are sequestered through base pairing, and a tertiary structure that adopts a tandem 3-way junction motif that exposes the dimerization initiation site and unpaired guanosines for specific recognition by Gag. While it remains to be established whether this structure is conserved in the context of larger RNA constructs or in the dimer, this study serves as the basis for characterizing large RNA structures using novel NMR techniques, and as a major advance toward understanding how the HIV-1 gRNA is selectively packaged.

  11. Sequence, structure, and stacking: specifics of tRNA anchoring to the T box riboswitch.

    Science.gov (United States)

    Grigg, Jason C; Ke, Ailong

    2013-12-01

    The term riboswitch usually refers to small molecule sensing regulatory modules in the 5' untranslated regions of a mRNA. They are typically comprised of separate ligand binding and regulatory domains. The T box riboswitch is unique from other identified riboswitches because its effector is an essential macromolecule, tRNA. It senses the aminoacylation state of tRNA to regulate genes involved in a variety of functions relating to amino acid metabolism and tRNA aminoacylation. T box riboswitches performs an intuitively simple process using a complex structured RNA element and, until recently, the underlying mechanisms were poorly understood. Only two sequence-specific contacts had been previously identified: (1) between the specifier sequence (codon) and the tRNA anticodon and (2) between an anti-terminator stem loop and the tRNA acceptor arm CCA tail. tRNA aminoacylation blocks the latter interaction and therefore serves as the switch between termination and anti-termination. Outside of these two contacts, the structure and functions of T box riboswitches have come to light in some recent studies. We recently described the X-ray crystal structure of the highly conserved T box riboswitch distal Stem I region and demonstrated that this region interacts with the tRNA elbow to anchor it to the riboswitch. Independently, Lehmann et al. used sequence homology search to arrive at a similar model for Stem I-tRNA interactions. The model was further supported by two recent structures of the Stem I-tRNA complex, determined independently by our group and by Zhang and Ferré-D'Amaré. This article highlights some of these contributions to synthesize an updated model for tRNA recognition by the T box riboswitch.

  12. mRNA secondary structures fold sequentially but exchange rapidly in vivo.

    Directory of Open Access Journals (Sweden)

    Elisabeth M Mahen

    2010-02-01

    Full Text Available RNAs adopt defined structures to perform biological activities, and conformational transitions among alternative structures are critical to virtually all RNA-mediated processes ranging from metabolite-activation of bacterial riboswitches to pre-mRNA splicing and viral replication in eukaryotes. Mechanistic analysis of an RNA folding reaction in a biological context is challenging because many steps usually intervene between assembly of a functional RNA structure and execution of a biological function. We developed a system to probe mechanisms of secondary structure folding and exchange directly in vivo using self-cleavage to monitor competition between mutually exclusive structures that promote or inhibit ribozyme assembly. In previous work, upstream structures were more effective than downstream structures in blocking ribozyme assembly during transcription in vitro, consistent with a sequential folding mechanism. However, upstream and downstream structures blocked ribozyme assembly equally well in vivo, suggesting that intracellular folding outcomes reflect thermodynamic equilibration or that annealing of contiguous sequences is favored kinetically. We have extended these studies to learn when, if ever, thermodynamic stability becomes an impediment to rapid equilibration among alternative RNA structures in vivo. We find that a narrow thermodynamic threshold determines whether kinetics or thermodynamics govern RNA folding outcomes in vivo. mRNA secondary structures fold sequentially in vivo, but exchange between adjacent secondary structures is much faster in vivo than it is in vitro. Previous work showed that simple base-paired RNA helices dissociate at similar rates in vivo and in vitro so exchange between adjacent structures must occur through a different mechanism, one that likely involves facilitation of branch migration by proteins associated with nascent transcripts.

  13. Structure of the human protein kinase CK2 catalytic subunit CK2α' and interaction thermodynamics with the regulatory subunit CK2β

    DEFF Research Database (Denmark)

    Bischoff, Nils; Olsen, Birgitte; Raaf, Jennifer;

    2011-01-01

    the limited biochemical knowledge about the second paralog (CK2α'), we developed a well-soluble catalytically active full-length mutant of human CK2α', characterized it by Michaelis-Menten kinetics and isothermal titration calorimetry, and determined its crystal structure to a resolution of 2 Å. The affinity......Protein kinase CK2 (formerly "casein kinase 2") is composed of a central dimer of noncatalytic subunits (CK2β) binding two catalytic subunits. In humans, there are two isoforms of the catalytic subunit (and an additional splicing variant), one of which (CK2α) is well characterized. To supplement...

  14. 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 BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

  15. Evasion of the innate immune response: the Old World alphavirus nsP2 protein induces rapid degradation of Rpb1, a catalytic subunit of RNA polymerase II.

    Science.gov (United States)

    Akhrymuk, Ivan; Kulemzin, Sergey V; Frolova, Elena I

    2012-07-01

    The Old World alphaviruses are emerging human pathogens with an ability to cause widespread epidemics. The latest epidemic of Chikungunya virus, from 2005 to 2007, affected over 40 countries in Africa, Asia, and Europe. The Old World alphaviruses are highly cytopathic and known to evade the cellular antiviral response by inducing global inhibition of transcription in vertebrate cells. This function was shown to be mediated by their nonstructural nsP2 protein; however, the detailed mechanism of this phenomenon has remained unknown. Here, we report that nsP2 proteins of Sindbis, Semliki Forest, and Chikungunya viruses inhibit cellular transcription by inducing rapid degradation of Rpb1, a catalytic subunit of the RNAPII complex. This degradation of Rpb1 is independent of the nsP2-associated protease activity, but, instead, it proceeds through nsP2-mediated Rpb1 ubiquitination. This function of nsP2 depends on the integrity of the helicase and S-adenosylmethionine (SAM)-dependent methyltransferase-like domains, and point mutations in either of these domains abolish Rpb1 degradation. We go on to show that complete degradation of Rpb1 in alphavirus-infected cells occurs within 6 h postinfection, before other previously described virus-induced changes in cell physiology, such as apoptosis, autophagy, and inhibition of STAT1 phosphorylation, are detected. Since Rpb1 is a subunit that catalyzes the polymerase reaction during RNA transcription, degradation of Rpb1 plays an indispensable role in blocking the activation of cellular genes and downregulating cellular antiviral response. This indicates that the nsP2-induced degradation of Rpb1 is a critical mechanism utilized by the Old World alphaviruses to subvert the cellular antiviral response.

  16. Requirement of catalytically active Tyk2 and accessory signals for the induction of TRAIL mRNA by IFN-beta.

    Science.gov (United States)

    Rani, M R Sandhya; Pandalai, Sudha; Shrock, Jennifer; Almasan, Alex; Ransohoff, Richard M

    2007-09-01

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L) mRNA was induced preferentially by interferon (IFN)-beta but not IFN-alpha in human fibrosarcoma and primary fibroblast cells. To characterize the signaling components mediating the IFN subtype-specific induction of this gene, we used mutant cell lines lacking individual components involved in signaling by type I IFNs. TRAIL was not induced by IFN-beta in mutant cell lines U2A, U3A, U4A, U5A, and U6A, which lack, respectively, IFN regulatory factor-9 (IRF-9), Stat1, Jak1, IFNAR-2.2, and Stat2, indicating transcription factor IFN-stimulated gene factor 3 (ISGF3) was essential for the induction of this gene. TRAIL was not induced by IFN-beta in U1A (Tyk2 null) or U1A.R930 cells (that express a kinase-deficient point mutant of Tyk2) but was induced in U1A.wt-5 cells (U1A cells expressing wild-type Tyk2), indicating that Tyk2 protein and kinase activity were both required for induction of the gene. Biochemical and genetic analyses revealed the requirement of transcription factor NF-kappa B and phosphoinositide 3-kinase (PI3K) but not extracellular signal-regulated kinase (ERK) for the induction of TRAIL by IFN-beta. Furthermore, the antiproliferative but not antiviral effects of IFN-beta required catalytically active Tyk2, suggesting that expression of genes, such as TRAIL, may play an important role in mediating the biologic effects of IFNs.

  17. Structure-guided systems-level engineering of oxidation-prone methionine residues in catalytic domain of an alkaline α-amylase from Alkalimonas amylolytica for significant improvement of both oxidative stability and catalytic efficiency.

    Directory of Open Access Journals (Sweden)

    Haiquan Yang

    Full Text Available High oxidative stability and catalytic efficiency are required for the alkaline α-amylases to keep the enzymatic performance under the harsh conditions in detergent industries. In this work, we attempted to significantly improve both the oxidative stability and catalytic efficiency of an alkaline α-amylase from Alkalimonas amylolytica by engineering the five oxidation-prone methionine residues around the catalytic domain via a systematic approach. Specifically, based on the tertiary structure analysis, five methionines (Met 145, Met 214, Met 229, Met 247 and Met 317 were individually substituted with oxidation-resistant threonine, isoleucine and alaline, respectively. Among the created 15 mutants, 7 mutants M145A, M145I, M214A, M229A, M229T, M247T and M317I showed significantly enhanced oxidative stability or catalytic efficiency. In previous work, we found that the replacement of M247 with leucine could significantly improve the oxidative stability. Thus, these 8 positive mutants (M145A, M145I, M214A, M229A, M229T, M247T, M247L and M317I were used to conduct the second round of combinational mutations. Among the constructed 85 mutants (25 two-point mutants, 36 three-point mutants, 16 four-point mutants and 8 five-point mutants, the mutant M145I-214A-229T-247T-317I showed a 5.4-fold increase in oxidative stability and a 3.0-fold increase in catalytic efficiency. Interestingly, the specific activity, alkaline stability and thermal stability of this mutant were also increased. The increase of salt bridge and hydrogen bonds around the catalytic domain contributed to the significantly improved catalytic efficiency and stability, as revealed by the three-dimensional structure model of wild-type alkaline α-amylase and its mutant M145I-214A-229T-247T-317I. With the significantly improved oxidative stability and catalytic efficiency, the mutant M145I-214A-229T-247T-317I has a great potential as a detergent additive, and this structure-guided systems

  18. Structure-guided systems-level engineering of oxidation-prone methionine residues in catalytic domain of an alkaline α-amylase from Alkalimonas amylolytica for significant improvement of both oxidative stability and catalytic efficiency.

    Science.gov (United States)

    Yang, Haiquan; Liu, Long; Shin, Hyun-dong; Li, Jianghua; Du, Guocheng; Chen, Jian

    2013-01-01

    High oxidative stability and catalytic efficiency are required for the alkaline α-amylases to keep the enzymatic performance under the harsh conditions in detergent industries. In this work, we attempted to significantly improve both the oxidative stability and catalytic efficiency of an alkaline α-amylase from Alkalimonas amylolytica by engineering the five oxidation-prone methionine residues around the catalytic domain via a systematic approach. Specifically, based on the tertiary structure analysis, five methionines (Met 145, Met 214, Met 229, Met 247 and Met 317) were individually substituted with oxidation-resistant threonine, isoleucine and alaline, respectively. Among the created 15 mutants, 7 mutants M145A, M145I, M214A, M229A, M229T, M247T and M317I showed significantly enhanced oxidative stability or catalytic efficiency. In previous work, we found that the replacement of M247 with leucine could significantly improve the oxidative stability. Thus, these 8 positive mutants (M145A, M145I, M214A, M229A, M229T, M247T, M247L and M317I) were used to conduct the second round of combinational mutations. Among the constructed 85 mutants (25 two-point mutants, 36 three-point mutants, 16 four-point mutants and 8 five-point mutants), the mutant M145I-214A-229T-247T-317I showed a 5.4-fold increase in oxidative stability and a 3.0-fold increase in catalytic efficiency. Interestingly, the specific activity, alkaline stability and thermal stability of this mutant were also increased. The increase of salt bridge and hydrogen bonds around the catalytic domain contributed to the significantly improved catalytic efficiency and stability, as revealed by the three-dimensional structure model of wild-type alkaline α-amylase and its mutant M145I-214A-229T-247T-317I. With the significantly improved oxidative stability and catalytic efficiency, the mutant M145I-214A-229T-247T-317I has a great potential as a detergent additive, and this structure-guided systems engineering

  19. 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.

  20. Effect of Sequence of Impregnation on the Structure and Catalytic Properties of Cu-Co-Al-Oxide System

    Institute of Scientific and Technical Information of China (English)

    Suzan A.Ali

    2004-01-01

    Twelve samples of Co-Cu/Al2O3 were prepared by impregnating Al2O3 with cobalt salt followed by copper salt or vice versa. The composition of the prepared samples varied in the molar ratios 1:1, 1:2 and 1:3 with respect to CuO:CoO or CoO:CuO, while Al2O3 content was kept at about 13-15 mol. The prepared solids were calcined at diflerent temperatures and the products were characterized by means of XRD-analysis. The catalytic activity of the calcined solids was tested in H2O2 decomposition. The XRD-analysis revealed that the sequence of impregnation affects much the structure of the samples. The loading of alumina with cobalt followed by copper salts produced sample with structure differs from that for sample firstly treated with copper followed by cobalt salts. XRD- analysis showed the formation of crystalline spinel Co1-x Cux Al2O4 with nearly the same crystal structure as CoAl2O4 even with high copper content. The examination of catalytic activity of these samples showed that catalysts with Co-loaded over Cu were more active than catalysts with Cu loaded over Co. In all cases the double oxides loaded over Al2O3 were more active than the single oxide over Al2O3.

  1. Minimum-free-energy distribution of RNA secondary structures: Entropic and thermodynamic properties of rare events

    Science.gov (United States)

    Wolfsheimer, S.; Hartmann, A. K.

    2010-08-01

    We study the distribution of the minimum free energy (MFE) for the Turner model of pseudoknot free RNA secondary structures over ensembles of random RNA sequences. In particular, we are interested in those rare and intermediate events of unexpected low MFEs. Generalized ensemble Markov-chain Monte Carlo methods allow us to explore the rare-event tail of the MFE distribution down to probabilities such as 10-70 and to study the relationship between the sequence entropy and structural properties for sequence ensembles with fixed MFEs. Entropic and structural properties of those ensembles are compared with natural RNA of the same reduced MFE ( z score).

  2. Structural and biochemical insights into 2′-O-methylation at the 3′-terminal nucleotide of RNA by Hen1

    Energy Technology Data Exchange (ETDEWEB)

    Chan, Chio Mui; Zhou, Chun; Brunzelle, Joseph S.; Huang, Raven H.; (UIUC); (NWU)

    2010-01-28

    Small RNAs of {approx}20-30 nt have diverse and important biological roles in eukaryotic organisms. After being generated by Dicer or Piwi proteins, all small RNAs in plants and a subset of small RNAs in animals are further modified at their 3'-terminal nucleotides via 2'-O-methylation, carried out by the S-adenosylmethionine-dependent methyltransferase (MTase) Hen1. Methylation at the 3' terminus is vital for biological functions of these small RNAs. Here, we report four crystal structures of the MTase domain of a bacterial homolog of Hen1 from Clostridium thermocellum and Anabaena variabilis, which are enzymatically indistinguishable from the eukaryotic Hen1 in their ability to methylate small single-stranded RNAs. The structures reveal that, in addition to the core fold of the MTase domain shared by other RNA and DNA MTases, the MTase domain of Hen1 possesses a motif and a domain that are highly conserved and are unique to Hen1. The unique motif and domain are likely to be involved in RNA substrate recognition and catalysis. The structures allowed us to construct a docking model of an RNA substrate bound to the MTase domain of bacterial Hen1, which is likely similar to that of the eukaryotic counterpart. The model, supported by mutational studies, provides insight into RNA substrate specificity and catalytic mechanism of Hen1.

  3. The impact of CRISPR repeat sequence on structures of a Cas6 protein-RNA complex

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ruiying; Zheng, Han; Preamplume, Gan; Shao, Yaming; Li, Hong [FSU

    2012-03-15

    The repeat-associated mysterious proteins (RAMPs) comprise the most abundant family of proteins involved in prokaryotic immunity against invading genetic elements conferred by the clustered regularly interspaced short palindromic repeat (CRISPR) system. Cas6 is one of the first characterized RAMP proteins and is a key enzyme required for CRISPR RNA maturation. Despite a strong structural homology with other RAMP proteins that bind hairpin RNA, Cas6 distinctly recognizes single-stranded RNA. Previous structural and biochemical studies show that Cas6 captures the 5' end while cleaving the 3' end of the CRISPR RNA. Here, we describe three structures and complementary biochemical analysis of a noncatalytic Cas6 homolog from Pyrococcus horikoshii bound to CRISPR repeat RNA of different sequences. Our study confirms the specificity of the Cas6 protein for single-stranded RNA and further reveals the importance of the bases at Positions 5-7 in Cas6-RNA interactions. Substitutions of these bases result in structural changes in the protein-RNA complex including its oligomerization state.

  4. Finding Common Sequence and Structure Motifs in a set of RNA sequences

    DEFF Research Database (Denmark)

    Gorodkin, Jan; Heyer, Laurie J.; Stormo, Gary D.

    1997-01-01

    We present a computational scheme to search for the most common motif, composed of a combination of sequence and structure constraints, among a collection of RNA sequences. The method uses a simplified version of the Sankoff algorithm for simultaneous folding and alignment of RNA sequences...

  5. Computational methods toward accurate RNA structure prediction using coarse-grained and all-atom models.

    Science.gov (United States)

    Krokhotin, Andrey; Dokholyan, Nikolay V

    2015-01-01

    Computational methods can provide significant insights into RNA structure and dynamics, bridging the gap in our understanding of the relationship between structure and biological function. Simulations enrich and enhance our understanding of data derived on the bench, as well as provide feasible alternatives to costly or technically challenging experiments. Coarse-grained computational models of RNA are especially important in this regard, as they allow analysis of events occurring in timescales relevant to RNA biological function, which are inaccessible through experimental methods alone. We have developed a three-bead coarse-grained model of RNA for discrete molecular dynamics simulations. This model is efficient in de novo prediction of short RNA tertiary structure, starting from RNA primary sequences of less than 50 nucleotides. To complement this model, we have incorporated additional base-pairing constraints and have developed a bias potential reliant on data obtained from hydroxyl probing experiments that guide RNA folding to its correct state. By introducing experimentally derived constraints to our computer simulations, we are able to make reliable predictions of RNA tertiary structures up to a few hundred nucleotides. Our refined model exemplifies a valuable benefit achieved through integration of computation and experimental methods.

  6. Crystal structure of the DNA cytosine deaminase APOBEC3F: the catalytically active and HIV-1 Vif-binding domain.

    Science.gov (United States)

    Bohn, Markus-Frederik; Shandilya, Shivender M D; Albin, John S; Kouno, Takahide; Anderson, Brett D; McDougle, Rebecca M; Carpenter, Michael A; Rathore, Anurag; Evans, Leah; Davis, Ahkillah N; Zhang, Jingying; Lu, Yongjian; Somasundaran, Mohan; Matsuo, Hiroshi; Harris, Reuben S; Schiffer, Celia A

    2013-06-04

    Human APOBEC3F is an antiretroviral single-strand DNA cytosine deaminase, susceptible to degradation by the HIV-1 protein Vif. In this study the crystal structure of the HIV Vif binding, catalytically active, C-terminal domain of APOBEC3F (A3F-CTD) was determined. The A3F-CTD shares structural motifs with portions of APOBEC3G-CTD, APOBEC3C, and APOBEC2. Residues identified to be critical for Vif-dependent degradation of APOBEC3F all fit within a predominantly negatively charged contiguous region on the surface of A3F-CTD. Specific sequence motifs, previously shown to play a role in Vif susceptibility and virion encapsidation, are conserved across APOBEC3s and between APOBEC3s and HIV-1 Vif. In this structure these motifs pack against each other at intermolecular interfaces, providing potential insights both into APOBEC3 oligomerization and Vif interactions.

  7. Prediction of RNA secondary structures: from theory to models and real molecules

    Science.gov (United States)

    Schuster, Peter

    2006-05-01

    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. Catalytic Metal Free Production of Large Cage Structure Carbon Particles: A Candidate for Hydrogen Storage

    Science.gov (United States)

    Kimura, Yuki; Nuth, Joseph A., III; Ferguson, Frank T.

    2005-01-01

    We will demonstrate that carbon particles consisting of large cages can be produced without catalytic metal. The carbon particles were produced in CO gas as well as by introduction of 5% methane gas into the CO gas. The gas-produced carbon particles were able to absorb approximately 16.2 wt% of hydrogen. This value is 2.5 times higher than the 6.5 wt% goal for the vehicular hydrogen storage proposed by the Department of Energy in the USA. Therefore, we believe that this carbon particle is an excellent candidate for hydrogen storage for fuel cells.

  9. RNA helicases

    OpenAIRE

    Owttrim, George W.

    2013-01-01

    Similar to proteins, RNA molecules must fold into the correct conformation and associate with protein complexes in order to be functional within a cell. RNA helicases rearrange RNA secondary structure and RNA-protein interactions in an ATP-dependent reaction, performing crucial functions in all aspects of RNA metabolism. In prokaryotes, RNA helicase activity is associated with roles in housekeeping functions including RNA turnover, ribosome biogenesis, translation and small RNA metabolism. In...

  10. Application of RNA secondary structure in siRNA design%RNA二级结构在siRNA设计中的应用

    Institute of Scientific and Technical Information of China (English)

    桂坚斌; 孙迎; 高武

    2012-01-01

    RNA干扰(RNA interference,RNAi)是一种基因沉默现象,通过短双链RNA(small interfering RNA,siRNA)降解靶基因的信使RNA(message RNA,mRNA)以抑制基因的表达.在RNAi的应用中,设计高效的siRNA对RNAi的成功有重要作用.关于siRNA的设计已有很多设计原则可供参考,如MPI准则、Tuschl法则等.在此基础上,亦有研究进一步考察了 RNA二级结构对siRNA沉默效率的影响,并建立相关模型进行分析,如氢键系数、排斥环模型等.本文就RNA二级结构在siRNA设计过程中模型的建立及结论的验证做出论述与分析.%RNA interference (RNAi) is a kind of gene silencing. Small interfering RNA (siRNA) is used to suppress gene expression by degrading the message RNA (mRNA) of the target gene. Effective siRNAs are important in RNAi experiments. There are a lot of principles for siRNA design,such as MPI basic principles and Tuschl principles and so on. On the basis of these studies, some have made further research on the relationship between RNA secondary structure and silencing efficiency of siRNA,and several models have been established,such as H-b index,spelling loops and so on. In this paper,we discuss and analyze the studies of RNA secondary structure in siRNA design.

  11. 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/.

  12. Alphavirus RNA synthesis and non-structural protein functions

    OpenAIRE

    Rupp, Jonathan C.; Sokoloski, Kevin J.; Gebhart, Natasha N.; Hardy, Richard W.

    2015-01-01

    The members of the genus Alphavirus are positive-sense RNA viruses, which are predominantly transmitted to vertebrates by a mosquito vector. Alphavirus disease in humans can be severely debilitating, and depending on the particular viral species, infection may result in encephalitis and possibly death. In recent years, alphaviruses have received significant attention from public health authorities as a consequence of the dramatic emergence of chikungunya virus in the Indian Ocean islands and ...

  13. 4-hydroxyphenylpyruvate dioxygenase catalysis: identification of catalytic residues and production of a hydroxylated intermediate shared with a structurally unrelated enzyme.

    Science.gov (United States)

    Raspail, Corinne; Graindorge, Matthieu; Moreau, Yohann; Crouzy, Serge; Lefèbvre, Bertrand; Robin, Adeline Y; Dumas, Renaud; Matringe, Michel

    2011-07-22

    4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxyphenylpyruvate (HPP) into homogentisate. HPPD is the molecular target of very effective synthetic herbicides. HPPD inhibitors may also be useful in treating life-threatening tyrosinemia type I and are currently in trials for treatment of Parkinson disease. The reaction mechanism of this key enzyme in both plants and animals has not yet been fully elucidated. In this study, using site-directed mutagenesis supported by quantum mechanical/molecular mechanical theoretical calculations, we investigated the role of catalytic residues potentially interacting with the substrate/intermediates. These results highlight the following: (i) the central role of Gln-272, Gln-286, and Gln-358 in HPP binding and the first nucleophilic attack; (ii) the important movement of the aromatic ring of HPP during the reaction, and (iii) the key role played by Asn-261 and Ser-246 in C1 hydroxylation and the final ortho-rearrangement steps (numbering according to the Arabidopsis HPPD crystal structure 1SQD). Furthermore, this study reveals that the last step of the catalytic reaction, the 1,2 shift of the acetate side chain, which was believed to be unique to the HPPD activity, is also catalyzed by a structurally unrelated enzyme.

  14. Regulation of primate lentiviral RNA dimerization by structural entrapment

    Directory of Open Access Journals (Sweden)

    Lodmell J Stephen

    2008-07-01

    Full Text Available Abstract Background Genomic RNA dimerization is an important process in the formation of an infectious lentiviral particle. One of the signals involved is the stem-loop 1 (SL1 element located in the leader region of lentiviral genomic RNAs which also plays a role in encapsidation and reverse transcription. Recent studies revealed that HIV types 1 and 2 leader RNAs adopt different conformations that influence the presentation of RNA signals such as SL1. To determine whether common mechanisms of SL1 regulation exist among divergent lentiviral leader RNAs, here we compare the dimerization properties of SIVmac239, HIV-1, and HIV-2 leader RNA fragments using homologous constructs and experimental conditions. Prior studies from several groups have employed a variety of constructs and experimental conditions. Results Although some idiosyncratic differences in the dimerization details were observed, we find unifying principles in the regulation strategies of the three viral RNAs through long- and short-range base pairing interactions. Presentation and efficacy of dimerization through SL1 depends strongly upon the formation or dissolution of the lower stem of SL1 called stem B. SL1 usage may also be down-regulated by long-range interactions involving sequences between SL1 and the first codons of the gag gene. Conclusion Despite their sequence differences, all three lentiviral RNAs tested in this study showed a local regulation of dimerization through the stabilization of SL1.

  15. RF sputtered CuO thin films: Structural, optical and photo-catalytic behavior

    Science.gov (United States)

    Al-Ghamdi, Attieh A.; Khedr, M. H.; Shahnawaze Ansari, M.; Hasan, P. M. Z.; Abdel-wahab, M. Sh.; Farghali, A. A.

    2016-07-01

    Nanocrystalline CuO thin films were deposited for 600, 1200 and 1800 s on glass substrate using RF magnetron sputtering technique. The films deposited at room temperature were crystalline and showed Tenorite phase of CuO. The increase in average particle size from 6.67 nm to 9.09 nm and the thickness from 160 nm to 490 nm was observed with the increase in deposition time. The optical band gap was decreased from 2.2 eV to 1.73 eV as the film thickness was increased. The intensity of PL peak showed its maximum for the film deposited for 600 s and minimum for 1800 s. Some unusual emission peaks were observed due to the quantization effect and lattice/surface defects. The CuO films with different thicknesses could be used as photo-catalysts for the degradation of Methylene blue (MB) from the wastewater. Under the exposure of 200 W energy of tungsten lamp, CuO thin films showed excellent photo-catalytic activities. CuO thin film of minimum thickness of around 160 nm responded as a best catalyst for MB degradation. The films were very stable and have a speciality to be recycled without much loss of their photo-catalytic activity. These characteristics have proved the high possibility of commercial applications of CuO thin films in environmental remediation.

  16. Synthesis, structural properties and catalytic activity of MgO-SnO2 nanocatalysts

    Science.gov (United States)

    Perveen, Hina; Farrukh, Muhammad Akhyar; Khaleeq-ur-Rahman, Muhammad; Munir, Badar; Tahir, Muhammad Ashraf

    2015-01-01

    Surfactant controlled synthesis of magnesium oxide-tin oxide (MgO-SnO2) nanocatalysts was carried out via the hydrothermal method. Concentration of sodium dodecyl sulfate (SDS) was varied while all other reaction conditions were kept constant same for this purpose. Furthermore, MgO-SnO2 nanocatalysts were also prepared by changing the precursor's concentration. These precursors are magnesium nitrate Mg(NO3)2 · 6H2O and tin chloride (SnCl4 · 5H2O). The influence of these reaction parameters on the sizes and morphology of the nanocatalysts were studied by using Fourier transform infrared (FTIR) spectroscopy, Scanning electron microscopy-Energy dispersive X-ray (SEM-EDX), Powder X-ray diffraction (XRD), Transmission electron microscopy and Thermo gravimetric analysis (TGA). The catalytic efficiency of MgO-SnO2 was checked against 2,4-dinitrophenylhydrazine (DNPH), which is an explosive compound. The nanocatalysts were found as a good catalyst to degrade the DNPH. Catalytic activity of nanocatalysts was observed up to 19.13% for the degradation DNPH by using UV-spectrophotometer.

  17. Structural basis of RNA recognition and activation by innate immune receptor RIG-I

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Fuguo; Ramanathan, Anand; Miller, Matthew T.; Tang, Guo-Qing; Gale, Jr., Michael; Patel, Smita S.; Marcotrigiano, Joseph (Rutgers); (RWJ-Med); (UW-MED)

    2012-05-29

    Retinoic-acid-inducible gene-I (RIG-I; also known as DDX58) is a cytoplasmic pathogen recognition receptor that recognizes pathogen-associated molecular pattern (PAMP) motifs to differentiate between viral and cellular RNAs. RIG-I is activated by blunt-ended double-stranded (ds)RNA with or without a 5'-triphosphate (ppp), by single-stranded RNA marked by a 5'-ppp and by polyuridine sequences. Upon binding to such PAMP motifs, RIG-I initiates a signalling cascade that induces innate immune defences and inflammatory cytokines to establish an antiviral state. The RIG-I pathway is highly regulated and aberrant signalling leads to apoptosis, altered cell differentiation, inflammation, autoimmune diseases and cancer. The helicase and repressor domains (RD) of RIG-I recognize dsRNA and 5'-ppp RNA to activate the two amino-terminal caspase recruitment domains (CARDs) for signalling. Here, to understand the synergy between the helicase and the RD for RNA binding, and the contribution of ATP hydrolysis to RIG-I activation, we determined the structure of human RIG-I helicase-RD in complex with dsRNA and an ATP analogue. The helicase-RD organizes into a ring around dsRNA, capping one end, while contacting both strands using previously uncharacterized motifs to recognize dsRNA. Small-angle X-ray scattering, limited proteolysis and differential scanning fluorimetry indicate that RIG-I is in an extended and flexible conformation that compacts upon binding RNA. These results provide a detailed view of the role of helicase in dsRNA recognition, the synergy between the RD and the helicase for RNA binding and the organization of full-length RIG-I bound to dsRNA, and provide evidence of a conformational change upon RNA binding. The RIG-I helicase-RD structure is consistent with dsRNA translocation without unwinding and cooperative binding to RNA. The structure yields unprecedented insight into innate immunity and has a broader impact on other areas of biology, including

  18. Compositions, structures, and catalytic activities of CeO₂@Cu₂O nanocomposites prepared by the template-assisted method.

    Science.gov (United States)

    Bao, Huizhi; Zhang, Zhenhua; Hua, Qing; Huang, Weixin

    2014-06-10

    CeO2@Cu2O nanocomposites were prepared from Cu2O cubes and octahedra by the template-assisted method involving the liquid (Ce(IV))-solid (Cu2O) interfacial reaction. Their compositions, structures, and catalytic activities in CO oxidation were studied in detail. Under the same reaction conditions, CeO2@Cu2O nanocomposites prepared from cubic and octahedral Cu2O templates exhibit different compositions and structures. With an increasing amount of Ce(IV) reactant, a smooth CeO2-CuOx shell develops on the surface of Cu2O cubes and eventually void cubic core/multishell Cu2O/CeO2-CuOx nanocomposites form; however, a rough CeO2-CuOx shell develops on the surface of Cu2O octahedra, and eventually hollow octahedral CeO2-CuOx nanocages form. The formation of different compositions and structures of CeO2@Cu2O nanocomposites was correlated with the different exposed crystal planes and surface reactivities of Cu2O cubes and octahedra. The catalytic activity of CeO2@Cu2O nanocomposites in CO oxidation depends on their compositions and structures. The most active CeO2@Cu2O nanocomposites become active at 70 °C and achieve a 100% CO conversion at 170 °C. These results broaden the versatility of Cu2O nanocrystals as the sacrificial template for the fabrication of novel nanocomposites with core/shell and hollow nanostructures and exemplify the morphology effect of Cu2O nanocrystals in liquid-solid interfacial reactions with respect to the composition, structure, and properties of nanocomposites prepared by the template-assisted method.

  19. Structure-infectivity analysis of the human rhinovirus genomic RNA 3' non-coding region.

    OpenAIRE

    1996-01-01

    The specific recognition of genomic positive strand RNAS as templates for the synthesis of intermediate negative strands by the picornavirus replication machinery is presumably mediated by cis-acting sequences within the genomic RNA 3' non-coding region (NCR). A structure-infectivity analysis was conducted on the 44 nt human rhinovirus 14 (HRV14) 3' NCR to identify the primary sequence and/or secondary structure determinants required for viral replication. Using biochemical RNA secondary stru...

  20. Prediction of viral microRNA precursors based on human microRNA precursor sequence and structural features.

    Science.gov (United States)

    Kumar, Shiva; Ansari, Faraz A; Scaria, Vinod

    2009-08-20

    MicroRNAs (small approximately 22 nucleotide long non-coding endogenous RNAs) have recently attracted immense attention as critical regulators of gene expression in multi-cellular eukaryotes, especially in humans. Recent studies have proved that viruses also express microRNAs, which are thought to contribute to the intricate mechanisms of host-pathogen interactions. Computational predictions have greatly accelerated the discovery of microRNAs. However, most of these widely used tools are dependent on structural features and sequence conservation which limits their use in discovering novel virus expressed microRNAs and non-conserved eukaryotic microRNAs. In this work an efficient prediction method is developed based on the hypothesis that sequence and structure features which discriminate between host microRNA precursor hairpins and pseudo microRNAs are shared by viral microRNA as they depend on host machinery for the processing of microRNA precursors. The proposed method has been found to be more efficient than recently reported ab-initio methods for predicting viral microRNAs and microRNAs expressed by mammals.

  1. Conservation of mRNA secondary structures may filter out mutations in Escherichia coli evolution.

    Science.gov (United States)

    Chursov, Andrey; Frishman, Dmitrij; Shneider, Alexander

    2013-09-01

    Recent reports indicate that mutations in viral genomes tend to preserve RNA secondary structure, and those mutations that disrupt secondary structural elements may reduce gene expression levels, thereby serving as a functional knockout. In this article, we explore the conservation of secondary structures of mRNA coding regions, a previously unknown factor in bacterial evolution, by comparing the structural consequences of mutations in essential and nonessential Escherichia coli genes accumulated over 40 000 generations in the course of the 'long-term evolution experiment'. We monitored the extent to which mutations influence minimum free energy (MFE) values, assuming that a substantial change in MFE is indicative of structural perturbation. Our principal finding is that purifying selection tends to eliminate those mutations in essential genes that lead to greater changes of MFE values and, therefore, may be more disruptive for the corresponding mRNA secondary structures. This effect implies that synonymous mutations disrupting mRNA secondary structures may directly affect the fitness of the organism. These results demonstrate that the need to maintain intact mRNA structures imposes additional evolutionary constraints on bacterial genomes, which go beyond preservation of structure and function of the encoded proteins.

  2. Transcripts with in silico predicted RNA structure are enriched everywhere in the mouse brain

    Directory of Open Access Journals (Sweden)

    Seemann Stefan E

    2012-05-01

    Full Text Available Abstract Background Post-transcriptional control of gene expression is mostly conducted by specific elements in untranslated regions (UTRs of mRNAs, in collaboration with specific binding proteins and RNAs. In several well characterized cases, these RNA elements are known to form stable secondary structures. RNA secondary structures also may have major functional implications for long noncoding RNAs (lncRNAs. Recent transcriptional data has indicated the importance of lncRNAs in brain development and function. However, no methodical efforts to investigate this have been undertaken. Here, we aim to systematically analyze the potential for RNA structure in brain-expressed transcripts. Results By comprehensive spatial expression analysis of the adult mouse in situ hybridization data of the Allen Mouse Brain Atlas, we show that transcripts (coding as well as non-coding associated with in silico predicted structured probes are highly and significantly enriched in almost all analyzed brain regions. Functional implications of these RNA structures and their role in the brain are discussed in detail along with specific examples. We observe that mRNAs with a structure prediction in their UTRs are enriched for binding, transport and localization gene ontology categories. In addition, after manual examination we observe agreement between RNA binding protein interaction sites near the 3’ UTR structures and correlated expression patterns. Conclusions Our results show a potential use for RNA structures in expressed coding as well as noncoding transcripts in the adult mouse brain, and describe the role of structured RNAs in the context of intracellular signaling pathways and regulatory networks. Based on this data we hypothesize that RNA structure is widely involved in transcriptional and translational regulatory mechanisms in the brain and ultimately plays a role in brain function.

  3. Predicting RNA Secondary Structure Using Profile Stochastic Context-Free Grammars and Phylogenic Analysis

    Institute of Scientific and Technical Information of China (English)

    Xiao-Yong Fang; Zhi-Gang Luo; Zheng-Hua Wang

    2008-01-01

    Stochastic context-free grammars (SCFGs) have been applied to predicting RNA secondary structure. The prediction of RNA secondary structure can be facilitated by incorporating with comparative sequence analysis. However,most of existing SCFG-based methods lack explicit phylogenic analysis of homologous RNA sequences, which is probably the reason why these methods are not ideal in practical application. Hence, we present a new SCFG-based method by integrating phylogenic analysis with the newly defined profile SCFG. The method can be summarized as: 1) we define a new profile SCFG, M, to depict consensus secondary structure of multiple RNA sequence alignment; 2) we introduce two distinct hidden Markov models, λ and λ', to perform phylogenic analysis of homologous RNA sequences. Here, λ is for non-structural regions of the sequence and λ' is for structural regions of the sequence; 3) we mergeλ and λ' in to M todevise a combined model for prediction of RNA secondary structure. We tested our method on data sets constructed from the Rfam database. The sensitivity and specificity of our method are more accurate than those of the predictions by Pfold.

  4. Reaction of psoralen with RNA: specificity and use as a probe for secondary-structure analysis

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, J.F.

    1982-09-01

    A variety of techniques has been used to study how psoralen and its derivatives react with RNA. This information has then been used to analyze the secondary structure of different ribosomal RNAs. Paper electrophoresis at pH 3.5 and 8.8 and HPLC has been used to get high-resolution separation of RNA-psoralen adducts. The separated adducts have been analyzed and shown to be primarily uridine adducts with the psoralen reacted at the furan end. The stereochemistry of the major adducts was determined by NMR. The effect of structural transitions on the number and type of adducts was found for several polymers. The effect of psoralen structure on cross linking ability was analyzed. Charged derivatives formed monoadducts very efficiently but did not produce the level of crosslinking obtainable with lower levels of reaction with uncharged derivatives. Secondary structure analysis of D. melanogaster 5S RNA yielded two definite and two tentative crosslinks which support the generally accepted models for 5S structure. Analysis of E. coli 16S RNA by gel techniques yielded 13 cross-links. Evidence is also presented for an interaction between eukaryotic mRNA (5' cap structure) and 18S RNA (hypermodified base am psi) which serves a function analogous to the Shine-Dalgarno sequence in pro karyotes.

  5. 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, includi

  6. 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...

  7. A model of 3D-structure of H+,K+-ATPase catalytic subunit derived by homology modeling

    Institute of Scientific and Technical Information of China (English)

    Dong YAN; Yuan-dong HU; Song LI; Mao-sheng CHENG

    2004-01-01

    AIM: To build a model of 3D-structure of H+, K+-ATPase catalytic subunit for theoretical study and anti-ulcer drug design. METHODS: The model was built on the basis of structural data from the Ca2+-ATPase. Structurally conserved regions were defined by amino acid sequence comparisons, optimum interconnecting loops were selected from the protein databank, and amino (N)- and carboxyl (C)-terminal ends were generated as random coil structures. Applying molecular mechanics method then minimized the model energy. Molecular dynamics technique was used to do further structural optimization. RESULTS: The model of 3D-structure of H+, K+-ATPase was derived. The model is reasonable according to several validation criteria. There were ten transmembrane helices (TM1-TM 10) in the model and inhibitor-binding site was identified on the TM5-8 riched negatively charged residues.CONCLUSION: The 3D-structure model from our study is informative to guide future molecular biology study about H+, K+-ATPase and drug design based on database searching.

  8. Crystal structure of a trapped catalytic intermediate suggests that forced atomic proximity drives the catalysis of mIPS.

    Science.gov (United States)

    Neelon, Kelly; Roberts, Mary F; Stec, Boguslaw

    2011-12-07

    1-L-myo-inositol-phosphate synthase (mIPS) catalyzes the first step of the unique, de novo pathway of inositol biosynthesis. However, details about the complex mIPS catalytic mechanism, which requires oxidation, enolization, intramolecular aldol cyclization, and reduction, are not fully known. To gain further insight into this mechanism, we determined the crystal structure of the wild-type mIPS from Archaeoglobus fulgidus at 1.7 Å, as well as the crystal structures of three active-site mutants. Additionally, we obtained the structure of mIPS with a trapped 5-keto-glucose-6-phosphate intermediate at 2 Å resolution by a novel (to our knowledge) process of activating the crystal at high temperature. A comparison of all of the crystal structures of mIPS described in this work suggests a novel type of catalytic mechanism that relies on the forced atomic proximity of functional groups. The lysine cluster is contained in a small volume in the active site, where random motions of these side chains are responsible for the progress of the complex multistep reaction as well as for the low rate of catalysis. The mechanism requires that functional groups of Lys-274, Lys-278, Lys-306, and Lys-367 assume differential roles in the protonation/deprotonation steps that must occur during the mIPS reaction. This mechanism is supported by the complete loss of activity of the enzyme caused by the Leu-257 mutation to Ala that releases the lysine containment.

  9. Oscillatory behaviour of catalytic properties, structure and temperature during the catalytic partial oxidation of methane on Pd/Al(2)O(3).

    Science.gov (United States)

    Kimmerle, Bertram; Baiker, Alfons; Grunwaldt, Jan-Dierk

    2010-03-14

    Pd/Al(2)O(3) catalysts showed an oscillatory behaviour during the catalytic partial oxidation (CPO) of methane, which was investigated simultaneously by IR-thermography, X-ray absorption spectroscopy, and online mass-spectrometry to correlate the temperature, state of the catalyst and catalytic performance. The following stages were observed: (i) build-up of a temperature maximum in the first half of the catalyst bed, (ii) reduction of palladium in the end zone of the catalyst bed with a front moving toward the entrance zone, (iii) strong hot spot formation accompanied by reduction of palladium due to self-reduction leading to extinction of the process. The latter was the key driver for the oscillations and thus gave additional insight into the mechanism of partial methane oxidation.

  10. Mechanical unfolding of RNA: From hairpins to structures with internal multiloops

    CERN Document Server

    Hyeon, Changbong

    2007-01-01

    Mechanical unfolding of RNA structures, ranging from hairpins to ribozymes, using laser optical tweezer (LOT) experiments have begun to reveal the features of the energy landscape that cannot be easily explored using conventional experiments. Upon application of constant force ($f$), RNA hairpins undergo cooperative transitions from folded to unfolded states whereas subdomains of ribozymes unravel one at a time. Here, we use a self-organized polymer (SOP) model and Brownian dynamics simulations to probe mechanical unfolding at constant force and constant-loading rate of four RNA structures of varying complexity. Our work shows (i) the response of RNA to force is largely determined by the native structure; (ii) only by probing mechanical unfolding over a wide range of forces can the underlying energy landscape be fully explored.

  11. Synthesis,structure and catalytic behavior of yttrium complexes bearing a diaminobis(phenolate) ligand

    Institute of Scientific and Technical Information of China (English)

    SONG FengKui; YAN ChunHui; SUN HongMei; YAO YingMing; SHEN Qi; ZHANG Yong

    2009-01-01

    Yttrium complexes stabilized by a diaminobis(phenolate) ligand were synthesized and their catalytic behavior was explored. Reaction of YCI3 with 1 equiv of LNa2 [L=Me2NCH2CH2N{CH2-(2-O-C6H2-tBu2-3,5)}2]gave the yttrium chloride LYCI(THF) (1) in 92% yield. Complex 1 can be used as starting material to prepare the yttrium amido derivative. Complex 1 reacted with 1 equiv of LiNPh2 in THF to afford the expected yttrium amido complex LYNPh2 (2) in high yield. Both of complexes 1 and 2 have been well detected by elemental analysis,NMR spectra and single-crystal X-ray analysis. It was found that complex 2 can efficiently initiate the ring-opening polymerization of L-lactide and ε-caprolactone,and a controlled manner is observed in the former case.

  12. Structural basis for dsRNA recognition and interferon antagonism by Ebola VP35

    Energy Technology Data Exchange (ETDEWEB)

    Leung, Daisy W.; Prins, Kathleen C.; Borek, Dominika M.; Farahbakhsh, Mina; Tufariello, JoAnn M.; Ramanan, Parameshwaran; Nix, Jay C.; Helgeson, Luke A.; Otwinowski, Zbyszek; Honzatko, Richard B.; Basler, Christopher F.; Amarasinghe, Gaya K. (Sinai); (Iowa State); (LBNL); (UTSMC)

    2010-03-12

    Ebola viral protein 35 (VP35), encoded by the highly pathogenic Ebola virus, facilitates host immune evasion by antagonizing antiviral signaling pathways, including those initiated by RIG-I-like receptors. Here we report the crystal structure of the Ebola VP35 interferon inhibitory domain (IID) bound to short double-stranded RNA (dsRNA), which together with in vivo results reveals how VP35-dsRNA interactions contribute to immune evasion. Conserved basic residues in VP35 IID recognize the dsRNA backbone, whereas the dsRNA blunt ends are 'end-capped' by a pocket of hydrophobic residues that mimic RIG-I-like receptor recognition of blunt-end dsRNA. Residues critical for RNA binding are also important for interferon inhibition in vivo but not for viral polymerase cofactor function of VP35. These results suggest that simultaneous recognition of dsRNA backbone and blunt ends provides a mechanism by which Ebola VP35 antagonizes host dsRNA sensors and immune responses.

  13. Intergenic regions of Borrelia plasmids contain phylogenetically conserved RNA secondary structure motifs

    Directory of Open Access Journals (Sweden)

    Delihas Nicholas

    2009-03-01

    Full Text Available Abstract Background Borrelia species are unusual in that they contain a large number of linear and circular plasmids. Many of these plasmids have long intergenic regions. These regions have many fragmented genes, repeated sequences and appear to be in a state of flux, but they may serve as reservoirs for evolutionary change and/or maintain stable motifs such as small RNA genes. Results In an in silico study, intergenic regions of Borrelia plasmids were scanned for phylogenetically conserved stem loop structures that may represent functional units at the RNA level. Five repeat sequences were found that could fold into stable RNA-type stem loop structures, three of which are closely linked to protein genes, one of which is a member of the Borrelia lipoprotein_1 super family genes and another is the complement regulator-acquiring surface protein_1 (CRASP-1 family. Modeled secondary structures of repeat sequences display numerous base-pair compensatory changes in stem regions, including C-G→A-U transversions when orthologous sequences are compared. Base-pair compensatory changes constitute strong evidence for phylogenetic conservation of secondary structure. Conclusion Intergenic regions of Borrelia species carry evolutionarily stable RNA secondary structure motifs. Of major interest is that some motifs are associated with protein genes that show large sequence variability. The cell may conserve these RNA motifs whereas allow a large flux in amino acid sequence, possibly to create new virulence factors but with associated RNA motifs intact.

  14. Tuning the structural and catalytic properties of ceria by doping with Zr4+, La3+ and Eu3+ cations

    Indian Academy of Sciences (India)

    T Vinodkumar; D Naga Durgasri; Swamy Maloth; Benjaram M Reddy

    2015-07-01

    This work attempts to gain information about the role of trivalent and tetravalent dopants on the structural and catalytic properties of ceria (CeO2). In this study, we have prepared Zr4+, La3+, and Eu3+ doped ceria (CZ, CL, and CE) by coprecipitation method and calcined at 773 K. The physicochemical characterization was achieved by using various techniques, namely, X-ray powder diffraction (XRD), Brunauer-Emmett-Teller (BET) surface area, high resolution transmission electron microscopy (HRTEM), Raman spectroscopy, temperature programmed reduction (TPR), X-ray photoelectron spectroscopy (XPS), and electron spin resonance (ESR) spectroscopy. The catalytic efficiency for soot oxidation was evaluated by thermogravimetric (TG) method and compared with undoped CeO2. Doped CeO2 catalysts decreased the soot oxidation temperature by more than 158 K compared to pure ceria. This is ascribed to mutual interaction and synergistic effect between the dopant species and the ceria. Among the synthesized nanocatalysts, the CE sample exhibited better performance. The observed better activity of CE was attributed to the presence of more number of oxygen vacancies, a high specific surface area, and easy reducibility as confirmed from Raman, BET surface area, and TPR measurements, respectively.

  15. A computational analysis of the structural determinants of APOBEC3's catalytic activity and vulnerability to HIV-1 Vif.

    Science.gov (United States)

    Shandilya, Shivender M D; Bohn, Markus-Frederik; Schiffer, Celia A

    2014-12-01

    APOBEC3s (A3) are Zn(2+) dependent cytidine deaminases with diverse biological functions and implications for cancer and immunity. Four of the seven human A3s restrict HIV by 'hypermutating' the reverse-transcribed viral genomic DNA. HIV Virion Infectivity Factor (Vif) counters this restriction by targeting A3s to proteasomal degradation. However, there is no apparent correlation between catalytic activity, Vif binding, and sequence similarity between A3 domains. Our comparative structural analysis reveals features required for binding Vif and features influencing polynucleotide deaminase activity in A3 proteins. All Vif-binding A3s share a negatively charged surface region that includes residues previously implicated in binding the highly-positively charged Vif. Additionally, catalytically active A3s share a positively charged groove near the Zn(2+) coordinating active site, which may accommodate the negatively charged polynucleotide substrate. Our findings suggest surface electrostatics, as well as the spatial extent of substrate accommodating region, are critical determinants of substrate and Vif binding across A3 proteins with implications for anti-retroviral and anti-cancer therapeutic design.

  16. Structured Perovskite-Based Catalysts and Their Application as Three-Way Catalytic Converters—A Review

    Directory of Open Access Journals (Sweden)

    Sylvain Keav

    2014-07-01

    Full Text Available Automotive Three-Way Catalysts (TWC were introduced more than 40 years ago. Despite that, the development of a sustainable TWC still remains a critical research topic owing to the increasingly stringent emission regulations together with the price and scarcity of precious metals. Among other material classes, perovskite-type oxides are known to be valuable alternatives to conventionally used TWC compositions and have demonstrated to be suitable for a wide range of automotive applications, ranging from TWC to Diesel Oxidation Catalysts (DOC, from NOx Storage Reduction catalysts (NSR to soot combustion catalysts. The interest in these catalysts has been revitalized in the past ten years by the introduction of the concept of catalyst regenerability of perovskite-based TWC, which is in principle well applicable to other catalytic processes as well, and by the possibility to reduce the amounts of critical elements, such as precious metals without seriously lowering the catalytic performance. The aim of this review is to show that perovskite-type oxides have the potential to fulfil the requirements (high activity, stability, and possibility to be included into structured catalysts for implementation in TWC.

  17. Catalytic activity for nitrate electroreduction of nano-structured polypyrrole films electrochemically synthesized onto a copper electrode

    Science.gov (United States)

    Phuong Thoa Nguyen, Thi; Thinh Nguyen, Viet; Hai Le, Viet

    2010-03-01

    Polypyrrole film was synthesized electrochemically onto a copper electrode in oxalate, oxalic acid and salicylic acid solutions. The electrochemical oxidation of pyrrole to form polypyrrole film and the electroreduction of nitrate and nitrite ions at synthesized Ppy modified copper electrodes (Ppy/Cu) in potassium chloride aqueous solutions were studied by cyclic voltammetry. Polypyrrole nano-porous film formation and the activity of the modified Ppy/Cu electrode for nitrate reduction were found to be dependent on the synthesis medium and conditions: pH; content and concentrations of the electrolytes; pyrrole concentration; electrode potential; electrolysis duration; drying time and temperature for finishing the Ppy/Cu electrode and immersion time in water for storing the Ppy/Cu electrode before use. High catalytic activity for nitrate reduction was found for composite electrodes with nano-porous structured Ppy films. The Ppy/Cu electrodes prepared in oxalate buffer and salicylic acid solutions perform more stable catalytic activity for nitrate reduction; their service life is about ten times longer than for an electrode prepared in oxalic acid solution.

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

    Science.gov (United States)

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

    2017-02-17

    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 towards small, symmetric purine loops, and thus poorly predict asymmetric, pyrimidine-rich loops with more than 6 nucleotides that occur frequently in viral RNA. This paper presents new thermodynamic data for 40 pyrimidine loops, many of which can form UU or protonated CC base pairs. Protonated cytosine and uracil 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 more than 6 nucleotides 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 2x2 have been measured (Mathews 2004). 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.

  19. 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.

  20. The mechanism of E. coli RNA polymerase regulation by ppGpp is suggested by the structure of their complex.

    Science.gov (United States)

    Zuo, Yuhong; Wang, Yeming; Steitz, Thomas A

    2013-05-01

    Guanosine tetraphosphate (ppGpp) is an alarmone that enables bacteria to adapt to their environment. It has been known for years that ppGpp acts directly on RNA polymerase (RNAP) to alter the rate of transcription, but its exact target site is still under debate. Here we report a crystal structure of Escherichia coli RNAP holoenzyme in complex with ppGpp at 4.5 Å resolution. The structure reveals that ppGpp binds at an interface between the shelf and core modules on the outer surface of RNAP, away from the catalytic center and the nucleic acid binding path. Bound ppGpp connects these two pivotal modules that may restrain the opening of the RNAP cleft. A detailed mechanism of action of ppGpp is proposed in which ppGpp prevents the closure of the active center that is induced by the binding of NTP, which could slow down nucleotide addition cycles and destabilize the initial transcription complexes.

  1. Structural and Biochemical Characterization of a Cyanobacterial PP2C Phosphatase Reveals Insights into Catalytic Mechanism and Substrate Recognition

    Directory of Open Access Journals (Sweden)

    Yunlong Si

    2016-04-01

    Full Text Available PP2C-type phosphatases play roles in signal transduction pathways related to abiotic stress. The cyanobacterial PP2C-type phosphatase tPphA specifically dephosphorylates the PII protein, which is a key regulator in cyanobacteria adapting to nitrogen-deficient environments. Previous studies have shown that residue His39 of tPphA is critical for the enzyme’s recognition of the PII protein; however, the manner in which this residue determines tPphA substrate specificity is unknown. Here, we solved the crystal structure of H39A, a tPphA variant. The structure revealed that the mutation of residue His39 to alanine changes the conformation and the flexibility of the loop in which residue His39 is located, and these changes affect the substrate specificity of tPphA. Moreover, previous studies have assumed that the FLAP subdomain and the third metal (M3 of tPphA could mutually influence each other to regulate PP2C catalytic activity and substrate specificity. However, despite the variable conformations adopted by the FLAP subdomain, the position of M3 was consistent in the tPphA structure. These results indicate that the FLAP subdomain does not influence M3 and vice versa. In addition, a small screen of tPphA inhibitors was performed. Sanguinarine and Ni2+ were found to be the most effective inhibitors among the assayed chemicals. Finally, the dimeric form of tPphA was stabilized by cross-linkers and still exhibited catalytic activity towards p-nitrophenyl phosphate.

  2. Structural Basis on the Catalytic Reaction Mechanism of Novel 1,2-Alpha L-Fucosidase (AFCA) From Bifidobacterium Bifidum

    Energy Technology Data Exchange (ETDEWEB)

    Nagae, M.; Tsuchiya, A.; Katayama, T.; Yamamoto, K.; Wakatsuki, S.; Kato, R.

    2009-06-03

    1,2-alpha-L-fucosidase (AfcA), which hydrolyzes the glycosidic linkage of Fucalpha1-2Gal via an inverting mechanism, was recently isolated from Bifidobacterium bifidum and classified as the first member of the novel glycoside hydrolase family 95. To better understand the molecular mechanism of this enzyme, we determined the x-ray crystal structures of the AfcA catalytic (Fuc) domain in unliganded and complexed forms with deoxyfuconojirimycin (inhibitor), 2'-fucosyllactose (substrate), and L-fucose and lactose (products) at 1.12-2.10 A resolution. The AfcA Fuc domain is composed of four regions, an N-terminal beta region, a helical linker, an (alpha/alpha)6 helical barrel domain, and a C-terminal beta region, and this arrangement is similar to bacterial phosphorylases. In the complex structures, the ligands were buried in the central cavity of the helical barrel domain. Structural analyses in combination with mutational experiments revealed that the highly conserved Glu566 probably acts as a general acid catalyst. However, no carboxylic acid residue is found at the appropriate position for a general base catalyst. Instead, a water molecule stabilized by Asn423 in the substrate-bound complex is suitably located to perform a nucleophilic attack on the C1 atom of L-fucose moiety in 2'-fucosyllactose, and its location is nearly identical near the O1 atom of beta-L-fucose in the products-bound complex. Based on these data, we propose and discuss a novel catalytic reaction mechanism of AfcA.

  3. Structure of the catalytic domain of the Tannerella forsythia matrix metallopeptidase karilysin in complex with a tetrapeptidic inhibitor.

    Science.gov (United States)

    Guevara, Tibisay; Ksiazek, Miroslaw; Skottrup, Peter Durand; Cerdà-Costa, Núria; Trillo-Muyo, Sergio; de Diego, Iñaki; Riise, Erik; Potempa, Jan; Gomis-Rüth, F Xavier

    2013-05-01

    Karilysin is the only metallopeptidase identified as a virulence factor in the odontopathogen Tannerella forsythia owing to its deleterious effect on the host immune response during bacterial infection. The very close structural and sequence-based similarity of its catalytic domain (Kly18) to matrix metalloproteinases suggests that karilysin was acquired by horizontal gene transfer from an animal host. Previous studies by phage display identified peptides with the consensus sequence XWFPXXXGGG (single-letter amino-acid codes; X represents any residue) as karilysin inhibitors with low-micromolar binding affinities. Subsequent refinement revealed that inhibition comparable to that of longer peptides could be achieved using the tetrapeptide SWFP. To analyze its binding, the high-resolution crystal structure of the complex between Kly18 and SWFP was determined and it was found that the peptide binds to the primed side of the active-site cleft in a substrate-like manner. The catalytic zinc ion is clamped by the α-amino group and the carbonyl O atom of the serine, thus distantly mimicking the general manner of binding of hydroxamate inhibitors to metallopeptidases and contributing, together with three zinc-binding histidines from the protein scaffold, to an octahedral-minus-one metal-coordination sphere. The tryptophan side chain penetrates the deep partially water-filled specificity pocket of Kly18. Together with previous serendipitous product complexes of Kly18, the present results provide the structural determinants of inhibition of karilysin and open the field for the design of novel inhibitory strategies aimed at the treatment of human periodontal disease based on a peptidic hit molecule.

  4. Histone gene expression and histone mRNA 3' end structure in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Pettitt Jonathan

    2007-06-01

    Full Text Available Abstract Background Histone protein synthesis is essential for cell proliferation and required for the packaging of DNA into chromatin. In animals, histone proteins are provided by the expression of multicopy replication-dependent histone genes. Histone mRNAs that are processed by a histone-specific mechanism to end after a highly conserved RNA hairpin element, and lack a poly(A tail. In vertebrates and Drosophila, their expression is dependent on HBP/SLBP that binds to the RNA hairpin element. We showed previously that these cis and trans acting regulators of histone gene expression are conserved in C. elegans. Here we report the results of an investigation of the histone mRNA 3' end structure and of histone gene expression during C. elegans development. Results Sequence analysis of replication-dependent histone genes revealed the presence of several highly conserved sequence elements in the 3' untranslated region of histone pre-mRNAs, including an RNA hairpin element and a polyadenylation signal. To determine whether in C. elegans histone mRNA 3' end formation occurs at this polyadenylation signal and results in polyadenylated histone mRNA, we investigated the mRNA 3' end structure of histone mRNA. Using poly(A selection, RNAse protection and sequencing of histone mRNA ends, we determined that a majority of C. elegans histone mRNAs lack a poly(A tail and end three to six nucleotides after the hairpin structure, after an A or a U, and have a 3' OH group. RNAi knock down of CDL-1, the C. elegans HBP/SLBP, does not significantly affect histone mRNA levels but severely depletes histone protein levels. Histone gene expression varies during development and is reduced in L3 animals compared to L1 animals and adults. In adults, histone gene expression is restricted to the germ line, where cell division occurs. Conclusion Our findings indicate that the expression of C. elegans histone genes is subject to control mechanisms similar to the ones in other

  5. Brr2p RNA helicase with a split personality: insights into structure and function.

    Science.gov (United States)

    Hahn, Daniela; Beggs, Jean D

    2010-08-01

    RNA helicases are involved in many cellular processes. Pre-mRNA splicing requires eight different DExD/H-box RNA helicases, which facilitate spliceosome assembly and remodelling of the intricate network of RNA rearrangements that are central to the splicing process. Brr2p, one of the spliceosomal RNA helicases, stands out through its unusual domain architecture. In the present review we highlight the advances made by recent structural and biochemical studies that have important implications for the mechanism and regulation of Brr2p activity. We also discuss the involvement of human Brr2 in retinitis pigmentosa, a degenerative eye disease, and how its functions in splicing might connect to the molecular pathology of the disease.

  6. Dynamics of human telomerase RNA structure revealed by antisense oligonucleotide technique.

    Science.gov (United States)

    Vasilkova, Daria V; Azhibek, Dulat M; Zatsepin, Timofei S; Naraikina, Yulia V; Prassolov, Vladimir S; Prokofjeva, Maria M; Zvereva, Maria I; Rubtsova, Maria P

    2013-12-01

    Telomeres are the nucleoprotein complexes that cap the linear chromosome ends. Telomerase is a ribonucleoprotein that maintains telomere length in stem, embryonic and cancer cells. Somatic cells don't contain active telomerase and telomere function as mitotic clock and telomere length determines the number of cell divisions. Telomerase RNA (TER) contains the template for telomere synthesis and serves as a structural scaffold for holoenzyme assembly. We compared different oligonucleotide based methods for telomerase RNA inhibition, such as antisense oligonucleotides, knockdown by transient siRNA transfection and silencing by miRNA derived from short expressed RNA hairpin in HEK293 cells. All of these methods were applied to different TER regions. Our results revealed that CR2/CR3 domain of TER is accessible in vitro and in vivo and could serve as an optimal site for oligonucleotide-based telomerase silencing.

  7. Comparative analysis of mt LSU rRNA secondary structures of Odonates: structural variability and phylogenetic signal.

    Science.gov (United States)

    Misof, B; Fleck, G

    2003-12-01

    Secondary structures of the most conserved part of the mt 16S rRNA gene, domains IV and V, have been recently analysed in a comparative study. However, full secondary structures of the mt LSU rRNA molecule are published for only a few insect species. The present study presents full secondary structures of domains I, II, IV and V of Odonates and one representative of mayflies, Ephemera sp. The reconstructions are based on a comparative approach and minimal consensus structures derived from sequence alignments. The inferred structures exhibit remarkable similarities to the published Drosophila melanogaster model, which increases confidence in these structures. Structural variance within Odonates is homoplastic, and neighbour-joining trees based on tree edit distances do not correspond to any of the phylogenetically expected patterns. However, despite homoplastic quantitative structural variation, many similarities between Odonates and Ephemera sp. suggest promising character sets for higher order insect systematics that merit further investigations.

  8. Dynamic Contacts of U2, RES, Cwc25, Prp8 and Prp45 Proteins with the Pre-mRNA Branch-Site and 3' Splice Site during Catalytic Activation and Step 1 Catalysis in Yeast Spliceosomes.

    Directory of Open Access Journals (Sweden)

    Cornelius Schneider

    Full Text Available Little is known about contacts in the spliceosome between proteins and intron nucleotides surrounding the pre-mRNA branch-site and their dynamics during splicing. We investigated protein-pre-mRNA interactions by UV-induced crosslinking of purified yeast B(act spliceosomes formed on site-specifically labeled pre-mRNA, and analyzed their changes after conversion to catalytically-activated B* and step 1 C complexes, using a purified splicing system. Contacts between nucleotides upstream and downstream of the branch-site and the U2 SF3a/b proteins Prp9, Prp11, Hsh49, Cus1 and Hsh155 were detected, demonstrating that these interactions are evolutionarily conserved. The RES proteins Pml1 and Bud13 were shown to contact the intron downstream of the branch-site. A comparison of the B(act crosslinking pattern versus that of B* and C complexes revealed that U2 and RES protein interactions with the intron are dynamic. Upon step 1 catalysis, Cwc25 contacts with the branch-site region, and enhanced crosslinks of Prp8 and Prp45 with nucleotides surrounding the branch-site were observed. Cwc25's step 1 promoting activity was not dependent on its interaction with pre-mRNA, indicating it acts via protein-protein interactions. These studies provide important insights into the spliceosome's protein-pre-mRNA network and reveal novel RNP remodeling events during the catalytic activation of the spliceosome and step 1 of splicing.

  9. Principles for understanding the accuracy of SHAPE-directed RNA structure modeling.

    Science.gov (United States)

    Leonard, Christopher W; Hajdin, Christine E; Karabiber, Fethullah; Mathews, David H; Favorov, Oleg V; Dokholyan, Nikolay V; Weeks, Kevin M

    2013-01-29

    Accurate RNA structure modeling is an important, incompletely solved, challenge. Single-nucleotide resolution SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) yields an experimental measurement of local nucleotide flexibility that can be incorporated as pseudo-free energy change constraints to direct secondary structure predictions. Prior work from our laboratory has emphasized both the overall accuracy of this approach and the need for nuanced interpretation of modeled structures. Recent studies by Das and colleagues [Kladwang, W., et al. (2011) Biochemistry 50, 8049; Nat. Chem. 3, 954], focused on analyzing six small RNAs, yielded poorer RNA secondary structure predictions than expected on the basis of prior benchmarking efforts. To understand the features that led to these divergent results, we re-examined four RNAs yielding the poorest results in this recent work: tRNA(Phe), the adenine and cyclic-di-GMP riboswitches, and 5S rRNA. Most of the errors reported by Das and colleagues reflected nonstandard experiment and data processing choices, and selective scoring rules. For two RNAs, tRNA(Phe) and the adenine riboswitch, secondary structure predictions are nearly perfect if no experimental information is included but were rendered inaccurate by the SHAPE data of Das and colleagues. When best practices were used, single-sequence SHAPE-directed secondary structure modeling recovered ~93% of individual base pairs and >90% of helices in the four RNAs, essentially indistinguishable from the results of the mutate-and-map approach with the exception of a single helix in the 5S rRNA. The field of experimentally directed RNA secondary structure prediction is entering a phase focused on the most difficult prediction challenges. We outline five constructive principles for guiding this field forward.

  10. Crystal structures of wild-type Trichoderma reesei Cel7A catalytic domain in open and closed states

    Energy Technology Data Exchange (ETDEWEB)

    Bodenheimer, Annette M. [Molecular and Structural Biochemistry Department, North Carolina State University, Raleigh NC USA; Neutron Sciences Directorate, Oak Ridge National Laboratory, TN USA; Meilleur, Flora [Molecular and Structural Biochemistry Department, North Carolina State University, Raleigh NC USA; Neutron Sciences Directorate, Oak Ridge National Laboratory, TN USA

    2016-11-07

    Trichoderma reesei Cel7A efficiently hydrolyses cellulose. We report here the crystallographic structures of the wild-type TrCel7A catalytic domain (CD) in an open state and, for the first time, in a closed state. Molecular dynamics (MD) simulations indicate that the loops along the CD tunnel move in concerted motions. Together, the crystallographic and MD data suggest that the CD cycles between the tense and relaxed forms that are characteristic of work producing enzymes. Analysis of the interactions formed by R251 provides a structural rationale for the concurrent decrease in product inhibition and catalytic efficiency measured for product-binding site mutants.

  11. 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

    2009-07-01

    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/.

  12. Evaluation of several lightweight stochastic context-free grammars for RNA secondary structure prediction

    Directory of Open Access Journals (Sweden)

    Eddy Sean R

    2004-06-01

    Full Text Available Abstract Background RNA secondary structure prediction methods based on probabilistic modeling can be developed using stochastic context-free grammars (SCFGs. Such methods can readily combine different sources of information that can be expressed probabilistically, such as an evolutionary model of comparative RNA sequence analysis and a biophysical model of structure plausibility. However, the number of free parameters in an integrated model for consensus RNA structure prediction can become untenable if the underlying SCFG design is too complex. Thus a key question is, what small, simple SCFG designs perform best for RNA secondary structure prediction? Results Nine different small SCFGs were implemented to explore the tradeoffs between model complexity and prediction accuracy. Each model was tested for single sequence structure prediction accuracy on a benchmark set of RNA secondary structures. Conclusions Four SCFG designs had prediction accuracies near the performance of current energy minimization programs. One of these designs, introduced by Knudsen and Hein in their PFOLD algorithm, has only 21 free parameters and is significantly simpler than the others.

  13. Ru(0) and Ru(II) nitrosyl pincer complexes: structure, reactivity, and catalytic activity.

    Science.gov (United States)

    Fogler, Eran; Iron, Mark A; Zhang, Jing; Ben-David, Yehoshoa; Diskin-Posner, Yael; Leitus, Gregory; Shimon, Linda J W; Milstein, David

    2013-10-07

    Despite considerable interest in ruthenium carbonyl pincer complexes and their substantial catalytic activity, there has been relatively little study of the isoelectronic ruthenium nitrosyl complexes. Here we describe the synthesis and reactivity of several complexes of this type as well as the catalytic activity of complex 6. Reaction of the PNP ligand (PNP = 2,6-bis((t)Bu2PCH2)pyridine) with RuCl3(NO)(PPh3)2 yielded the Ru(II) complex 3. Chloride displacement by BAr(F-) (BAr(F-) = tetrakis(3,5-bis(trifluoromethyl)phenyl)borate) gave the crystallographicaly characterized, linear NO Ru(II) complex 4, which upon treatment with NaBEt3H yielded the Ru(0) complexes 5. The crystallographically characterized Ru(0) square planar complex 5·BF4 bears a linear NO ligand located trans to the pyridilic nitrogen. Further treatment of 5·BF4 with excess LiOH gave the crystallographicaly characterized Ru(0) square planar, linear NO complex 6. Complex 6 catalyzes the dehydrogenative coupling of alcohols to esters, reaching full conversion under air or under argon. Reaction of the PNN ligand (PNN = 2-((t)Bu2PCH2)-6-(Et2NCH2)pyridine) with RuCl3(NO)(H2O)2 in ethanol gave an equilibrium mixture of isomers 7a and 7b. Further treatment of 7a + 7b with 2 equivalent of sodium isopropoxide gave the crystallographicaly characterized, bent-nitrosyl, square pyramidal Ru(II) complex 8. Complex 8 was also synthesized by reaction of PNN with RuCl3(NO)(H2O)2 and Et3N in ethanol. Reaction of the "long arm" PN(2)N ligand (PN(2)N = 2-((t)Bu2PCH2-)-6-(Et2NCH2CH2)pyridine) with RuCl3(NO)(H2O)2 in ethanol gave complex 9, which upon treatment with 2 equiv of sodium isopropoxide gave complex 10. Complex 10 was also synthesized directly by reaction of PN(2)N with RuCl3(NO)(H2O)2 and a base in ethanol. A noteworthy aspect of these nitrosyl complexes is their preference for the Ru(0) oxidization state over Ru(II). This preference is observed with both aromatized and dearomatized pincer ligands, in

  14. 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.

  15. Structural biology. Crystal structure of a CRISPR RNA-guided surveillance complex bound to a ssDNA target.

    Science.gov (United States)

    Mulepati, Sabin; Héroux, Annie; Bailey, Scott

    2014-09-19

    In prokaryotes, RNA derived from type I and type III CRISPR loci direct large ribonucleoprotein complexes to destroy invading bacteriophage and plasmids. In Escherichia coli, this 405-kilodalton complex is called Cascade. We report the crystal structure of Cascade bound to a single-stranded DNA (ssDNA) target at a resolution of 3.03 angstroms. The structure reveals that the CRISPR RNA and target strands do not form a double helix but instead adopt an underwound ribbon-like structure. This noncanonical structure is facilitated by rotation of every sixth nucleotide out of the RNA-DNA hybrid and is stabilized by the highly interlocked organization of protein subunits. These studies provide insight into both the assembly and the activity of this complex and suggest a mechanism to enforce fidelity of target binding.

  16. Structural characterization, antibacterial and catalytic effect of iron oxide nanoparticles synthesised using the leaf extract of Cynometra ramiflora

    Science.gov (United States)

    Groiss, Silvia; Selvaraj, Raja; Varadavenkatesan, Thivaharan; Vinayagam, Ramesh

    2017-01-01

    In the present investigation, the leaf extract of Cynometra ramiflora was used to synthesize iron oxide nanoparticles. Within minutes of adding iron sulphate to the leaf extract, iron oxide nanoparticles were formed and thus, the method is very simple and fast. UV-VIS spectra showed the strong absorption band in the visible region. SEM images showed discrete spherical shaped particles and EDS spectra confirmed the iron and oxygen presence. The XRD results depicted the crystalline structure of iron oxide nanoparticles. FT-IR spectra portrayed the existence of functional groups of phytochemicals which are probably involved in the formation and stabilization of nanoparticles. The iron oxide nanoparticles exhibited effective inhibition against E. coli and S. epidermidis which may find its applications in the antibacterial drug development. Furthermore, the catalytic activity of the nanoparticles as Fenton-like catalyst was successfully investigated for the degradation of Rhodamine-B dye. This outcome could play a prominent role in the wastewater treatment.

  17. Crystal Structure of 12-Lipoxygenase Catalytic-Domain-Inhibitor Complex Identifies a Substrate-Binding Channel for Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Shu; Mueser, Timothy C.; Marnett, Lawrence J.; Funk, Jr., Max O. (Toledo); (Vanderbilt)

    2014-10-02

    Lipoxygenases are critical enzymes in the biosynthesis of families of bioactive lipids including compounds with important roles in the initiation and resolution of inflammation and in associated diseases such as diabetes, cardiovascular disease, and cancer. Crystals diffracting to high resolution (1.9 {angstrom}) were obtained for a complex between the catalytic domain of leukocyte 12-lipoxygenase and the isoform-specific inhibitor, 4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP). In the three-dimensional structure of the complex, the inhibitor occupied a new U-shaped channel open at one end to the surface of the protein and extending past the redox-active iron site that is essential for catalysis. In models, the channel accommodated arachidonic acid, defining the binding site for the substrate of the catalyzed reaction. There was a void adjacent to the OPP binding site connecting to the surface of the enzyme and providing a plausible access channel for the other substrate, oxygen.

  18. Structure prediction of the EcoRV DNA methyltransferase based on mutant profiling, secondary structure analysis, comparison with known structures of methyltransferases and isolation of catalytically inactive single mutants.

    Science.gov (United States)

    Jeltsch, A; Sobotta, T; Pingoud, A

    1996-05-01

    The EcoRV DNA methyltransferase (M.EcoRV) is an alpha-adenine methyltransferase. We have used two different programs to predict the secondary structure of M.EcoRV. The resulting consensus prediction was tested by a mutant profiling analysis. 29 neutral mutations of M.EcoRV were generated by five cycles of random mutagenesis and selection for active variants to increase the reliability of the prediction and to get a secondary structure prediction for some ambiguously predicted regions. The predicted consensus secondary structure elements could be aligned to the common topology of the structures of the catalytic domains of M.HhaI and M.TaqI. In a complementary approach we have isolated nine catalytically inactive single mutants. Five of these mutants contain an amino acid exchange within the catalytic domain of M.EcoRV (Val2-Ala, Lys81Arg, Cys192Arg, Asp193Gly, Trp231Arg). The Trp231Arg mutant binds DNA similarly to wild-type M.EcoRV, but is catalytically inactive. Hence this mutant behaves like a bona fide active site mutant. According to the structure prediction, Trp231 is located in a loop at the putative active site of M.EcoRV. The other inactive mutants were insoluble. They contain amino acid exchanges within the conserved amino acid motifs X, III or IV in M.EcoRV confirming the importance of these regions.

  19. 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.

  20. Structural insights into the mechanism of the DEAH-box RNA helicase Prp43

    Science.gov (United States)

    Tauchert, Marcel J; Fourmann, Jean-Baptiste; Lührmann, Reinhard; Ficner, Ralf

    2017-01-01

    The DEAH-box helicase Prp43 is a key player in pre-mRNA splicing as well as the maturation of rRNAs. The exact modus operandi of Prp43 and of all other spliceosomal DEAH-box RNA helicases is still elusive. Here, we report crystal structures of Prp43 complexes in different functional states and the analysis of structure-based mutants providing insights into the unwinding and loading mechanism of RNAs. The Prp43•ATP-analog•RNA complex shows the localization of the RNA inside a tunnel formed by the two RecA-like and C-terminal domains. In the ATP-bound state this tunnel can be transformed into a groove prone for RNA binding by large rearrangements of the C-terminal domains. Several conformational changes between the ATP- and ADP-bound states explain the coupling of ATP hydrolysis to RNA translocation, mainly mediated by a β-turn of the RecA1 domain containing the newly identified RF motif. This mechanism is clearly different to those of other RNA helicases. DOI: http://dx.doi.org/10.7554/eLife.21510.001 PMID:28092261

  1. PRince: a web server for structural and physicochemical analysis of Protein-RNA interface

    Science.gov (United States)

    Barik, Amita; Mishra, Abhishek; Bahadur, Ranjit Prasad

    2012-01-01

    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. PMID:22689640

  2. Inhibition of hepatitis C virus replication by single-stranded RNA structural mimics

    Institute of Scientific and Technical Information of China (English)

    Robert; Smolic; Martina; Smolic; John; H; Andorfer; Catherine; H; Wu; Robert; M; Smith; George; Y; Wu

    2010-01-01

    AIM: To examine the effect of hepatitis C virus (HCV) structural mimics of regulatory regions of the genome on HCV replication.METHODS: HCV RNA structural mimics were constructed and tested in a HCV genotype 1b aBB7 replicon,and a Japanese fulminant hepatitis-1 (JFH-1) HCV genotype 2a infection model.All sequences were computer-predicted to adopt stem-loop structures identical to the corresponding elements in full-length viral RNA.Huh7.5 cells bearing the BB7 replicon or infected with JFH-1 virus were trans...

  3. Synthesis, Crystal Structure and Catalytic Behavior of 1-Ethyl-3-benyl-imidazolyl Tetranuclear N-Heterocyclic Carbene Silver Bromide

    Institute of Scientific and Technical Information of China (English)

    WANG Zhi-Guo; SU Zhi-Xian; BIAN Qing-Quan; LIU Si-Man; LIU Ting

    2012-01-01

    The title complex [Ag(carbene)2]2[Ag2Br4] has been synthesized by the reaction of Ag2O with 1-ethyl-3-benyl-imidazolium bromide in DMSO at room temperature, and characterized by elemental analysis, 1H NMR and single-crystal X-ray diffraction analysis. It crystallizes in triclinic, space group P with a = 10.1597(10), b =11.0646(11), c = 13.0245(14) , α = 102.230(2), β = 90.606, γ = 113.9250(10)o, V = 1300.3(2) 3, Mr = 748.06, Z = 2, Dc = 1.911 g/cm3, μ(MoKα) = 4.60 mm-1 and F(000) = 728. The structure was refined to R = 0.0316 and wR = 0.0835 for 3744 observed reflections with I 〉 2σ(I). The title compound crystallizes as a centrosymmetric tetranuclear compound. One half of the molecule comprises the asymmetric unit of the structure. The Ag(1) atom is nearly linear or T-shaped when the Ag(1)-Ag(2) interaction is taken into consideration, which is bi-coordinated by two carbene carbon atoms. The Ag(2) atom adopts tetrahedral geometry. The catalytic behavior of the title complex has been investigated, and the results indicate it has a highly catalytic activation for L-lactide polymerization.

  4. Hierarchical ZSM-11 with intergrowth structures:Synthesis,characterization and catalytic properties

    Institute of Scientific and Technical Information of China (English)

    Qingjun; Yu; Chaoyue; Cui; Qiang; Zhang; Jing; Chen; Yang; Li; Jinpeng; Sun; Chunyi; Li; Qiukai; Cui; Chaohe; Yang; Honghong; Shan

    2013-01-01

    Hierarchical ZSM-11 microspheres with intercrystalline mesoporous properties and rod-like crystals intergrowth morphology have been synthesized using a spot of tetrabutylammonium as a single template.XRD,FTIR,SEM,TEM and N2 adsorption analysis revealed that each individual particle was composed of nanosized rod crystals inserting each other and the intercrystalline voids existing among rods gave a significant mesopore size distribution.Steam treatment result demonstrated the excellent hydrothermal stability of samples.Various crystallization modes including constant temperature crystallization (one-stage crystallization) and two-stage temperature-varying crystallization with different 1st stage durations were investigated.The results suggested that the crystallization modes were mainly responsible for the adjustable particle size and textural properties of samples while the small amount of tetrabutylammonium bromide was mainly used to direct the formation of both ZSM-11 framework and its intergrowth morphology.Furthermore,the performance of optimal ZSM-11 as an active component for the catalytic pyrolysis of heavy oil was also investigated.Compared with the commercial pyrolysis catalyst,the hierarchical ZSM-11 catalyst exhibited a high selectivity to desired products(LPG+gasoline+diesel),as well as a much lower dry gas and coke yield,plus a high selectivity and yield of light olefins(C=3 C=4)and very poor selectivity to benzene.Therefore,fully open micropore-mesopore connectivity would make such hierarchically porous ZSM-11 zeolites very attractive for applications in clean petrochemical catalysis field.

  5. Preparation of bitumen from charkchemical tar by catalytic oxidation and study of its structure and properties

    Directory of Open Access Journals (Sweden)

    Zhaksyntay Kairbekov

    2012-12-01

    Full Text Available Optimal conditions (temperature, time are determined for processes of oxidation of chark-chemical tar in the presence of FеСl3·6H2O catalyst. In the course of oxidation during increase of quantity of catalyst from 0.4 mass. % to 1.0 mass. % in the composition of initial tar the amount of tar decreased from 42,75 mass.% to 28,56 mass.%, the amount of hydrocarbons from 30,18 mass.% to  28,92 mass.%, and the quantity of asphaltene increased from 15,84 mass.% to   38 mass.%. The  values of physical-mechanical indicators of obtained products in the presence of 0,8 mass.% to 1,0 mass.% catalyst corresponds to the requirements of the standard, hence they may be quantitatively attributed to viscous construction oil bitumen of mark BH 70/30, BN 90/10. Chemical transformation in the composition of obtained products during catalytic oxidation is proved by the results of IR-spectroscopic analysis.

  6. R3D Align: global pairwise alignment of RNA 3D structures using local superpositions

    Science.gov (United States)

    Rahrig, Ryan R.; Leontis, Neocles B.; Zirbel, Craig L.

    2010-01-01

    Motivation: Comparing 3D structures of homologous RNA molecules yields information about sequence and structural variability. To compare large RNA 3D structures, accurate automatic comparison tools are needed. In this article, we introduce a new algorithm and web server to align large homologous RNA structures nucleotide by nucleotide using local superpositions that accommodate the flexibility of RNA molecules. Local alignments are merged to form a global alignment by employing a maximum clique algorithm on a specially defined graph that we call the ‘local alignment’ graph. Results: The algorithm is implemented in a program suite and web server called ‘R3D Align’. The R3D Align alignment of homologous 3D structures of 5S, 16S and 23S rRNA was compared to a high-quality hand alignment. A full comparison of the 16S alignment with the other state-of-the-art methods is also provided. The R3D Align program suite includes new diagnostic tools for the structural evaluation of RNA alignments. The R3D Align alignments were compared to those produced by other programs and were found to be the most accurate, in comparison with a high quality hand-crafted alignment and in conjunction with a series of other diagnostics presented. The number of aligned base pairs as well as measures of geometric similarity are used to evaluate the accuracy of the alignments. Availability: R3D Align is freely available through a web server http://rna.bgsu.edu/R3DAlign. The MATLAB source code of the program suite is also freely available for download at that location. Supplementary information: Supplementary data are available at Bioinformatics online. Contact: r-rahrig@onu.edu PMID:20929913

  7. Crystal structure of pseudouridine synthase RluA: indirect sequence readout through protein-induced RNA structure.

    Science.gov (United States)

    Hoang, Charmaine; Chen, Junjun; Vizthum, Caroline A; Kandel, Jason M; Hamilton, Christopher S; Mueller, Eugene G; Ferré-D'Amaré, Adrian R

    2006-11-17

    RluA is a dual-specificity enzyme responsible for pseudouridylating 23S rRNA and several tRNAs. The 2.05 A resolution structure of RluA bound to a substrate RNA comprising the anticodon stem loop of tRNA(Phe) reveals that enzyme binding induces a dramatic reorganization of the RNA. Instead of adopting its canonical U turn conformation, the anticodon loop folds into a new structure with a reverse-Hoogsteen base pair and three flipped-out nucleotides. Sequence conservation, the cocrystal structure, and the results of structure-guided mutagenesis suggest that RluA recognizes its substrates indirectly by probing RNA loops for their ability to adopt the reorganized fold. The planar, cationic side chain of an arginine intercalates between the reverse-Hoogsteen base pair and the bottom pair of the anticodon stem, flipping the nucleotide to be modified into the active site of RluA. Sequence and structural comparisons suggest that pseudouridine synthases of the RluA, RsuA, and TruA families employ an equivalent arginine for base flipping.

  8. Role of RNA secondary structure in emergence of compartment specific hepatitis B virus immune escape variants

    OpenAIRE

    Datta, Sibnarayan; Chakravarty, Runu

    2016-01-01

    AIM To investigate the role of subgenotype specific RNA secondary structure in the compartment specific selection of hepatitis B virus (HBV) immune escape mutations. METHODS This study was based on the analysis of the specific observation of HBV subgenotype A1 in the serum/plasma, while subgenotype A2 with G145R mutation in the peripheral blood leukocytes (PBLs). Genetic variability found among the two subgenotypes was used for prediction and comparison of the full length pregenomic RNA (pgRN...

  9. 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

  10. Homology modeling and analysis of structure predictions of the bovine rhinitis B virus RNA dependent RNA polymerase (RdRp).

    Science.gov (United States)

    Rai, Devendra K; Rieder, Elizabeth

    2012-01-01

    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 (3D(pol)). In the present study we carried out phylogenetic analysis, structure based sequence alignment and prediction of three-dimensional structure of BRBV 3D(pol) using a combination of different computational tools. Model structures of BRBV 3D(pol) were verified for their stereochemical quality and accuracy. The BRBV 3D(pol) 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 3D(pol) (PDB, 1U09 and 2E9Z). The closest proximity of BRBV and FMDV 3D(pol) as compared to human rhinovirus type 16 (HRV-16) and rabbit hemorrhagic disease virus (RHDV) 3D(pols) 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 3D(pol) compared to FMDV 3D(pol), indicate that despite a very high homology to FMDV 3D(pol), BRBV 3D(pol) may adopt a different mechanism for handling its substrates and adapting to physiological requirements. Our findings will be valuable in the design of structure-function interventions and identification of molecular targets for drug design applicable

  11. FoldAtlas: a repository for genome-wide RNA structure probing data

    Science.gov (United States)

    Norris, Matthew; Kwok, Chun Kit; Cheema, Jitender; Hartley, Matthew; Morris, Richard J.; Aviran, Sharon; Ding, Yiliang

    2017-01-01

    Summary: Most RNA molecules form internal base pairs, leading to a folded secondary structure. Some of these structures have been demonstrated to be functionally significant. High-throughput RNA structure chemical probing methods generate millions of sequencing reads to provide structural constraints for RNA secondary structure prediction. At present, processed data from these experiments are difficult to access without computational expertise. Here we present FoldAtlas, a web interface for accessing raw and processed structural data across thousands of transcripts. FoldAtlas allows a researcher to easily locate, view, and retrieve probing data for a given RNA molecule. We also provide in silico and in vivo secondary structure predictions for comparison, visualized in the browser as circle plots and topology diagrams. Data currently integrated into FoldAtlas are from a new high-depth Structure-seq data analysis in Arabidopsis thaliana, released with this work. Availability and Implementation: The FoldAtlas website can be accessed at www.foldatlas.com. Source code is freely available at github.com/mnori/foldatlas under the MIT license. Raw reads data are available under the NCBI SRA accession SRP066985. Contact: yiliang.ding@jic.ac.uk or matthew.norris@jic.ac.uk. Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27663500

  12. Keeping Uracil Out of DNA: Physiological Role, Structure and Catalytic Mechanism of dUTPases

    OpenAIRE

    2009-01-01

    The thymine-uracil exchange constitutes one of the major chemical differences between DNA and RNA. Although these two bases form the same Watson-Crick base pairs with adenine and are equivalent for both information storage and transmission, uracil incorporation in DNA is usually a mistake that needs to be excised. There are two ways for uracil to appear in DNA: thymine replacement and cytosine deamination. Most DNA polymerases readily incorporate dUMP as well as dTMP depending solely on the a...

  13. Elastic network models capture the motions apparent within ensembles of RNA structures.

    Science.gov (United States)

    Zimmermann, Michael T; Jernigan, Robert L

    2014-06-01

    The role of structure and dynamics in mechanisms for RNA becomes increasingly important. Computational approaches using simple dynamics models have been successful at predicting the motions of proteins and are often applied to ribonucleo-protein complexes but have not been thoroughly tested for well-packed nucleic acid structures. In order to characterize a true set of motions, we investigate the apparent motions from 16 ensembles of experimentally determined RNA structures. These indicate a relatively limited set of motions that are captured by a small set of principal components (PCs). These limited motions closely resemble the motions computed from low frequency normal modes from elastic network models (ENMs), either at atomic or coarse-grained resolution. Various ENM model types, parameters, and structure representations are tested here against the experimental RNA structural ensembles, exposing differences between models for proteins and for folded RNAs. Differences in performance are seen, depending on the structure alignment algorithm used to generate PCs, modulating the apparent utility of ENMs but not significantly impacting their ability to generate functional motions. The loss of dynamical information upon coarse-graining is somewhat larger for RNAs than for globular proteins, indicating, perhaps, the lower cooperativity of the less densely packed RNA. However, the RNA structures show less sensitivity to the elastic network model parameters than do proteins. These findings further demonstrate the utility of ENMs and th