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Andrey Kulbachinskiy - One of the best experts on this subject based on the ideXlab platform.
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Factor-specific effects of mutations in the active site of RNA polymerase on RNA Cleavage.
Biochemical and Biophysical Research Communications, 2019Co-Authors: Nataliya Miropolskaya, Andrey Kulbachinskiy, Daria EsyuninaAbstract:Abstract Bacterial RNA polymerase (RNAP) relies on the same active site for RNA synthesis and co-transcriptional RNA proofreading. The intrinsic RNA proofreading activity of RNAP can be greatly stimulated by Gre factors, which bind within the secondary channel and directly participate in the RNA Cleavage reaction in the active site of RNAP. Here, we characterize mutations in Escherichia coli RNAP that differentially affect intrinsic and Gre-stimulated RNA Cleavage. Substitution of a highly conserved arginine residue that contacts nascent RNA upstream of the active site strongly impairs intrinsic and GreA-dependent Cleavage, without reducing GreA affinity or catalytic Mg2+ binding. In contrast, substitutions of several nonconserved residues at the Gre-interacting interface in the secondary channel primarily affect GreB-dependent Cleavage, by decreasing both the catalytic rate and GreB affinity. The results suggest that RNAP residues not directly involved in contacts with the reacting RNA groups or catalytic ions play essential roles in RNA Cleavage and can modulate its regulation by transcription factors.
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Interactions in the active site of Deinococcus radiodurans RNA polymerase during RNA proofreading
Biochemical and Biophysical Research Communications, 2018Co-Authors: Daria Esyunina, Andrey KulbachinskiyAbstract:Abstract Co-transcriptional RNA proofreading by RNA polymerase (RNAP) is essential for accurate mRNA synthesis and reactivation of stalled transcription complexes, which can otherwise compromise genome integrity. RNAP from the stress-resistant bacterium Deinococcus radiodurans exhibits high levels of RNA Cleavage in comparison with RNAP from Escherichia coli, which allows it to remove misincorporated nucleotides with high efficiency. Here, we show that the rate of RNA Cleavage by D. radiodurans RNAP depends on the structure of the (mis)matched RNA 3′-nucleotide and its contacts with the active site. These interactions likely position the reactive phosphodiester bond in the Cleavage-competent conformation, thus facilitating its hydrolysis catalyzed by metal ions in the active center. The universal RNA Cleavage factor GreA largely alleviates defects in RNA Cleavage caused by modifications in the RNA 3′-nucleotide or in its binding pocket in RNAP, suggesting that GreA functionally substitutes for these contacts. The results demonstrate that various RNAPs rely on a conserved mechanism for RNA proofreading, which can be modulated by changes in accessory parts of the active center.
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conserved functions of the trigger loop and gre factors in RNA Cleavage by bacterial RNA polymerases
Journal of Biological Chemistry, 2017Co-Authors: Nataliya Miropolskaya, Daria Esyunina, Andrey KulbachinskiyAbstract:Abstract RNA Cleavage by RNA polymerase (RNAP) is the central step in co-transcriptional RNA proofreading. Bacterial RNAPs were proposed to rely on the same mobile element of the active site, the trigger loop (TL), for both nucleotide addition and RNA Cleavage. RNA Cleavage can also be stimulated by universal Gre factors, which should replace the TL to get access to the RNAP active site. The contributions of the TL and Gre factors to RNA Cleavage reportedly vary between RNAPs from different bacterial species and, probably, different types of transcription complexes. Here, by comparing RNAPs from Escherichia coli (Eco), Deinococcus radiodurans (Dra) and Thermus aquaticus (Taq) we show that the functions of the TL and Gre factors in RNA Cleavage are conserved in various species, with important variations which may be related to extremophilic adaptation. Deletions of the TL strongly impair intrinsic RNA Cleavage by all three RNAPs and eliminate the inter-species differences in the reaction rates. GreA factors activate RNA Cleavage by wild-type RNAPs to similar levels. The rates of GreA-dependent Cleavage are lower for ΔTL RNAP variants, suggesting that the TL contributes to the Gre function. Finally, neither the TL nor GreA can efficiently activate RNA Cleavage in certain types of backtracked transcription complexes suggesting that these complexes adopt a catalytically inactive conformation probably important for transcription regulation.
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Lineage-specific variations in the trigger loop modulate RNA proofreading by bacterial RNA polymerases
Nucleic Acids Research, 2016Co-Authors: Daria Esyunina, Danil Pupov, Matti Turtola, I. A. Bass, Saulius Klimašauskas, Georgiy A. Belogurov, Andrey KulbachinskiyAbstract:RNA Cleavage by bacterial RNA polymerase (RNAP) has been implicated in transcriptional proofreading and reactivation of arrested transcription elongation complexes but its molecular mechanism is less understood than the mechanism of nucleotide addition, despite both reactions taking place in the same active site. RNAP from the radioresistant bacterium Deinococcus radiodurans is characterized by highly efficient intrinsic RNA Cleavage in comparison with Escherichia coli RNAP. We find that the enhanced RNA Cleavage activity largely derives from amino acid substitutions in the trigger loop (TL), a mobile element of the active site involved in various RNAP activities. The differences in RNA Cleavage between these RNAPs disappear when the TL is deleted, or in the presence of GreA Cleavage factors, which replace the TL in the active site. We propose that the TL substitutions modulate the RNA Cleavage activity by altering the TL folding and its contacts with substrate RNA and that the resulting differences in transcriptional proofreading may play a role in bacterial stress adaptation.
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Analysis of RNA Cleavage by RNA polymerases from Escherichia coli and Deinococcus radiodurans.
Biochemistry, 2008Co-Authors: Danil Pupov, Nataliya Barinova, Andrey KulbachinskiyAbstract:RNA polymerase can both synthesize and cleave RNA. Both reactions occur at the same catalytic center containing two magnesium ions bound to three aspartic acid residues of the absolutely conserved NADFDGD motif of the RNA polymerase β′ subunit. We have demonstrated that RNA polymerase from Deinococcus radiodurans possesses much higher rate of intrinsic RNA Cleavage than RNA polymerase from Escherichia coli (the difference in the rates is about 15-fold at 20°C). However, these RNA polymerases do not differ in the rates of RNA synthesis. Comparison of the RNA polymerase sequences adjacent to the NADFDGD motif reveals the only amino acid substitution in this region (Glu751 in D. radiodurans vs. Ala455 in E. coli), which is localized in the secondary enzyme channel and can potentially affect the rate of RNA Cleavage. Introduction of the corresponding substitution in the E. coli RNA polymerase leads to a slight (about 2–3-fold) increase in the Cleavage rate, but does not affect RNA synthesis. Thus, the difference in the RNA Cleavage rates between E. coli and D. radiodurans RNA polymerases is likely determined by multiple amino acid substitutions, which do not affect the rate of RNA synthesis and are localized in several regions of the active center.
Daria Esyunina - One of the best experts on this subject based on the ideXlab platform.
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Factor-specific effects of mutations in the active site of RNA polymerase on RNA Cleavage.
Biochemical and Biophysical Research Communications, 2019Co-Authors: Nataliya Miropolskaya, Andrey Kulbachinskiy, Daria EsyuninaAbstract:Abstract Bacterial RNA polymerase (RNAP) relies on the same active site for RNA synthesis and co-transcriptional RNA proofreading. The intrinsic RNA proofreading activity of RNAP can be greatly stimulated by Gre factors, which bind within the secondary channel and directly participate in the RNA Cleavage reaction in the active site of RNAP. Here, we characterize mutations in Escherichia coli RNAP that differentially affect intrinsic and Gre-stimulated RNA Cleavage. Substitution of a highly conserved arginine residue that contacts nascent RNA upstream of the active site strongly impairs intrinsic and GreA-dependent Cleavage, without reducing GreA affinity or catalytic Mg2+ binding. In contrast, substitutions of several nonconserved residues at the Gre-interacting interface in the secondary channel primarily affect GreB-dependent Cleavage, by decreasing both the catalytic rate and GreB affinity. The results suggest that RNAP residues not directly involved in contacts with the reacting RNA groups or catalytic ions play essential roles in RNA Cleavage and can modulate its regulation by transcription factors.
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Interactions in the active site of Deinococcus radiodurans RNA polymerase during RNA proofreading
Biochemical and Biophysical Research Communications, 2018Co-Authors: Daria Esyunina, Andrey KulbachinskiyAbstract:Abstract Co-transcriptional RNA proofreading by RNA polymerase (RNAP) is essential for accurate mRNA synthesis and reactivation of stalled transcription complexes, which can otherwise compromise genome integrity. RNAP from the stress-resistant bacterium Deinococcus radiodurans exhibits high levels of RNA Cleavage in comparison with RNAP from Escherichia coli, which allows it to remove misincorporated nucleotides with high efficiency. Here, we show that the rate of RNA Cleavage by D. radiodurans RNAP depends on the structure of the (mis)matched RNA 3′-nucleotide and its contacts with the active site. These interactions likely position the reactive phosphodiester bond in the Cleavage-competent conformation, thus facilitating its hydrolysis catalyzed by metal ions in the active center. The universal RNA Cleavage factor GreA largely alleviates defects in RNA Cleavage caused by modifications in the RNA 3′-nucleotide or in its binding pocket in RNAP, suggesting that GreA functionally substitutes for these contacts. The results demonstrate that various RNAPs rely on a conserved mechanism for RNA proofreading, which can be modulated by changes in accessory parts of the active center.
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conserved functions of the trigger loop and gre factors in RNA Cleavage by bacterial RNA polymerases
Journal of Biological Chemistry, 2017Co-Authors: Nataliya Miropolskaya, Daria Esyunina, Andrey KulbachinskiyAbstract:Abstract RNA Cleavage by RNA polymerase (RNAP) is the central step in co-transcriptional RNA proofreading. Bacterial RNAPs were proposed to rely on the same mobile element of the active site, the trigger loop (TL), for both nucleotide addition and RNA Cleavage. RNA Cleavage can also be stimulated by universal Gre factors, which should replace the TL to get access to the RNAP active site. The contributions of the TL and Gre factors to RNA Cleavage reportedly vary between RNAPs from different bacterial species and, probably, different types of transcription complexes. Here, by comparing RNAPs from Escherichia coli (Eco), Deinococcus radiodurans (Dra) and Thermus aquaticus (Taq) we show that the functions of the TL and Gre factors in RNA Cleavage are conserved in various species, with important variations which may be related to extremophilic adaptation. Deletions of the TL strongly impair intrinsic RNA Cleavage by all three RNAPs and eliminate the inter-species differences in the reaction rates. GreA factors activate RNA Cleavage by wild-type RNAPs to similar levels. The rates of GreA-dependent Cleavage are lower for ΔTL RNAP variants, suggesting that the TL contributes to the Gre function. Finally, neither the TL nor GreA can efficiently activate RNA Cleavage in certain types of backtracked transcription complexes suggesting that these complexes adopt a catalytically inactive conformation probably important for transcription regulation.
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Lineage-specific variations in the trigger loop modulate RNA proofreading by bacterial RNA polymerases
Nucleic Acids Research, 2016Co-Authors: Daria Esyunina, Danil Pupov, Matti Turtola, I. A. Bass, Saulius Klimašauskas, Georgiy A. Belogurov, Andrey KulbachinskiyAbstract:RNA Cleavage by bacterial RNA polymerase (RNAP) has been implicated in transcriptional proofreading and reactivation of arrested transcription elongation complexes but its molecular mechanism is less understood than the mechanism of nucleotide addition, despite both reactions taking place in the same active site. RNAP from the radioresistant bacterium Deinococcus radiodurans is characterized by highly efficient intrinsic RNA Cleavage in comparison with Escherichia coli RNAP. We find that the enhanced RNA Cleavage activity largely derives from amino acid substitutions in the trigger loop (TL), a mobile element of the active site involved in various RNAP activities. The differences in RNA Cleavage between these RNAPs disappear when the TL is deleted, or in the presence of GreA Cleavage factors, which replace the TL in the active site. We propose that the TL substitutions modulate the RNA Cleavage activity by altering the TL folding and its contacts with substrate RNA and that the resulting differences in transcriptional proofreading may play a role in bacterial stress adaptation.
Naoki Sugimoto - One of the best experts on this subject based on the ideXlab platform.
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Novel biomaterials derived from deoxyribozyme and NAPzyme
Macromolecular Symposia, 2003Co-Authors: Yasuhide Okumoto, Tatsuo Ohmichi, Hiroyoshi Fujiki, Junji Kawakami, Shoji Nakashima, Shu-ichi Nakano, Daisuke Miyoshi, Naoki SugimotoAbstract:We report the potential of a small Ca 2+ -dependent deoxyribozyme as a novel biomaterial to distinguish RNA foldings. It is found that an immobilized deoxyribozyme using avidin-biotin interaction cleaves the target site within only single-stranded RNAs. The RNA Cleavage reaction is also detected using the deoxyribozyme SPR sensor chip. Furthermore, we develop a novel NAPzyme (nucleic acid peptide deoxyribozyrne) with its RNA Cleavage function in the absence of divalent metal ions.
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Factors that Contribute to Efficient Catalytic Activity of a Small Ca2+-Dependent Deoxyribozyme in Relation to Its RNA Cleavage Function†
Biochemistry, 2003Co-Authors: Yasuhide Okumoto, Yoshiatsu Tanabe, Naoki SugimotoAbstract:Recently, we found a small Ca2+-dependent deoxyribozyme (unmodified), d(GCCTGGCAG1G2C3T4A5C6A7A8C9G10A11GTCCCT), with Cleavage activity for its RNA substrate, r(AGGGACA↓UGCCAGGC) (↓ denotes the RNA Cleavage site), in the presence of Ca2+ and developed a functional SPR sensor chip with this deoxyribozyme [Okumoto, Y., Ohmichi, T., and Sugimoto, N. (2002) Biochemistry 41, 2769−2773]. In the study presented here, to clarify the factors contributing to the efficient catalytic activity of the unmodified deoxyribozyme, RNA Cleavage reactions were carried out using 24 mutant deoxyribozymes containing one unnatural DNA nucleotide, such as dI (2‘-deoxyinosine), 7-deaza-dG, 2-aminopurine, 7-deaza-dA, 2-amino-dA, dm5C (5-methyl-2‘-deoxycytosine), or dPC (5-propynyl-2‘-deoxycytosine). The Km values (Michaelis constants) with the mutants that lacked N7 and O6 of G1 and O6 of G2 were 4.5 and 6.6 times that of the unmodified one, respectively. The kcat value (Cleavage rate constant) with the mutants that lacked O6 of G1...
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Development of a short Ca2+-dependent deoxyribozyme with RNA Cleavage activity
Nucleic Acids Symposium Series, 1999Co-Authors: Naoki Sugimoto, Yasuhide OkumotoAbstract:We developed a short Ca^+.dependent deoxyribozyme with 11 mer catalytic loop domain (dGGCTACAACGA) that catalyzed site-specific RNA Cleavage reaction between rA and rU. The second-order rate constant of this short deoxyribozyme is 1.7 x 10^ M~l min~l at 37 °C, and this value is very similar to that of the deoxyribozyme (dGGCTAGCTACAACGA) in the presence of Ca 2 + . INTRODUCTION Metal ions play the crucial roles in the catalytic activity of all ribozymes (1), because these ribozymes are considered as metalloenzymes (1,2). The ribozymes have a catalytic loop with a metal binding site. The deletion of nucleotides from the catalytic loop would show higher activity or unique property in the case of the small ribozyme (3). An deoxyribozyme that was derived from a random library by in vitro selection is one of the DNA enzymes that requires Mg^+ and catalyzed site-specific RNA Cleavage reaction (4). The original deoxyribozyme, dGCCTGGCAGiG2C3T4A5T6G7C8 A9C1 oA 11A12C13G14A15GTCCCT, binds to the RNA substrate, rAGGGACA^lUGCCAGGC, and acts as a enzyme in the presence of Ca2+ or Mg2+ so that it cleaves the RNA substrate at one site of rApiU in the asymmetric inteRNAl loop (4). The catalytic efficiency of the original deoxyribozyme in the presence of Mg2+ is very similar to that of Ca2+. In this study, we developed a short metal iondependent deoxyribozyme with site-specific RNA Cleavage activity. MATERIALS AND METHODS Materials The RNA and DNA oligonucleotides used here were synthesized chemically on a solid support using phosphoramidite procedures and purified by HPLC as described previously (5). The blocked unnatural nucleotide monomers were purchased from Glen Research. The RNA substrate was 5'-end labeled with [y-32p] ATP by the T4 ploynucleotide kinase method. Cleavage Reactions Kinetic measurements were done under multipleturnover conditions in a buffer containing 50 mM Tris-HCl and 25 mM divalent metal ions (pH 8.0) at 37 °C. After Cleavage reactions were stopped, the Cleavage products and the RNA substrates were separated by electrophoresis on 20% polyacrylamide denaturing gels. The RNA Cleavage yields were determined by quantition of radioactivity in the bands of the 5'-end labeled products and the RNA substrate with a Bio-Image Analyzer model BAS 2000 (Fuji Film, Tokyo). Km and &cat values were calculated from EadieHofstee plots. RESULTS AND DISCUSSION The catalytic efficiency (&cat/^m value) of the original deoxyribozyme in the presence 282 Nucleic Acids Symposium Series No. 42
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Development of a short Ca2+-dependent deoxyribozyme with RNA Cleavage activity.
Nucleic acids symposium series, 1999Co-Authors: Naoki Sugimoto, Yasuhide OkumotoAbstract:We developed a short Ca2+-dependent deoxyribozyme with 11 mer catalytic loop domain (dGGCTACAACGA) that catalyzed site-specific RNA Cleavage reaction between rA and rU. The second-order rate constant of this short deoxyribozyme is 1.7 x 10(7) M(-1) min(-1) at 37 degrees C, and this value is very similar to that of the deoxyribozyme (dGGCTAGCTACAACGA) in the presence of Ca2+.
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Effect of metal ions and sequence of deoxyribozymes on their RNA Cleavage activity
Journal of The Chemical Society-perkin Transactions 1, 1999Co-Authors: Naoki Sugimoto, Yasuhide Okumoto, Tatsuo OhmichiAbstract:Catalytic DNA is a promising class of nucleic acid enzyme for possible use as a therapeutic agent and is also interesting in comparison with catalytic RNAs. In this study, we investigated the effect of metal ions and sequence on an original deoxyribozyme, d(GCCTGGCAG1G2C3T4A5G6C7T8A9C10A11A12C13G14A15GTCCCT), which binds to an RNA substrate, r(AGGGACA↓UGCCAGGC), cleaving the RNA substrate at one site indicated by the arrow. The results show that the ability of metal ions to promote the RNA Cleavage reaction by the original deoxyribozyme is Mn2+ > Mg2+ >Ca2+ Ba2+. This result is very similar to the previous tendency observed in the case of a hammerhead ribozyme. Thus, these results suggest that RNA Cleavage by the deoxyribozyme dependence on metal ions for catalysis is similar to that with ribozymes. On the other hand, a nucleotide deletion from the active domain of the original deoxyribozyme results in a novel deoxyribozyme with high Ca2+-dependency, a situation which is not observed with the original deoxyribozyme or hammerhead ribozymes.
Yasuhide Okumoto - One of the best experts on this subject based on the ideXlab platform.
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Novel biomaterials derived from deoxyribozyme and NAPzyme
Macromolecular Symposia, 2003Co-Authors: Yasuhide Okumoto, Tatsuo Ohmichi, Hiroyoshi Fujiki, Junji Kawakami, Shoji Nakashima, Shu-ichi Nakano, Daisuke Miyoshi, Naoki SugimotoAbstract:We report the potential of a small Ca 2+ -dependent deoxyribozyme as a novel biomaterial to distinguish RNA foldings. It is found that an immobilized deoxyribozyme using avidin-biotin interaction cleaves the target site within only single-stranded RNAs. The RNA Cleavage reaction is also detected using the deoxyribozyme SPR sensor chip. Furthermore, we develop a novel NAPzyme (nucleic acid peptide deoxyribozyrne) with its RNA Cleavage function in the absence of divalent metal ions.
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Factors that Contribute to Efficient Catalytic Activity of a Small Ca2+-Dependent Deoxyribozyme in Relation to Its RNA Cleavage Function†
Biochemistry, 2003Co-Authors: Yasuhide Okumoto, Yoshiatsu Tanabe, Naoki SugimotoAbstract:Recently, we found a small Ca2+-dependent deoxyribozyme (unmodified), d(GCCTGGCAG1G2C3T4A5C6A7A8C9G10A11GTCCCT), with Cleavage activity for its RNA substrate, r(AGGGACA↓UGCCAGGC) (↓ denotes the RNA Cleavage site), in the presence of Ca2+ and developed a functional SPR sensor chip with this deoxyribozyme [Okumoto, Y., Ohmichi, T., and Sugimoto, N. (2002) Biochemistry 41, 2769−2773]. In the study presented here, to clarify the factors contributing to the efficient catalytic activity of the unmodified deoxyribozyme, RNA Cleavage reactions were carried out using 24 mutant deoxyribozymes containing one unnatural DNA nucleotide, such as dI (2‘-deoxyinosine), 7-deaza-dG, 2-aminopurine, 7-deaza-dA, 2-amino-dA, dm5C (5-methyl-2‘-deoxycytosine), or dPC (5-propynyl-2‘-deoxycytosine). The Km values (Michaelis constants) with the mutants that lacked N7 and O6 of G1 and O6 of G2 were 4.5 and 6.6 times that of the unmodified one, respectively. The kcat value (Cleavage rate constant) with the mutants that lacked O6 of G1...
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Development of a short Ca2+-dependent deoxyribozyme with RNA Cleavage activity
Nucleic Acids Symposium Series, 1999Co-Authors: Naoki Sugimoto, Yasuhide OkumotoAbstract:We developed a short Ca^+.dependent deoxyribozyme with 11 mer catalytic loop domain (dGGCTACAACGA) that catalyzed site-specific RNA Cleavage reaction between rA and rU. The second-order rate constant of this short deoxyribozyme is 1.7 x 10^ M~l min~l at 37 °C, and this value is very similar to that of the deoxyribozyme (dGGCTAGCTACAACGA) in the presence of Ca 2 + . INTRODUCTION Metal ions play the crucial roles in the catalytic activity of all ribozymes (1), because these ribozymes are considered as metalloenzymes (1,2). The ribozymes have a catalytic loop with a metal binding site. The deletion of nucleotides from the catalytic loop would show higher activity or unique property in the case of the small ribozyme (3). An deoxyribozyme that was derived from a random library by in vitro selection is one of the DNA enzymes that requires Mg^+ and catalyzed site-specific RNA Cleavage reaction (4). The original deoxyribozyme, dGCCTGGCAGiG2C3T4A5T6G7C8 A9C1 oA 11A12C13G14A15GTCCCT, binds to the RNA substrate, rAGGGACA^lUGCCAGGC, and acts as a enzyme in the presence of Ca2+ or Mg2+ so that it cleaves the RNA substrate at one site of rApiU in the asymmetric inteRNAl loop (4). The catalytic efficiency of the original deoxyribozyme in the presence of Mg2+ is very similar to that of Ca2+. In this study, we developed a short metal iondependent deoxyribozyme with site-specific RNA Cleavage activity. MATERIALS AND METHODS Materials The RNA and DNA oligonucleotides used here were synthesized chemically on a solid support using phosphoramidite procedures and purified by HPLC as described previously (5). The blocked unnatural nucleotide monomers were purchased from Glen Research. The RNA substrate was 5'-end labeled with [y-32p] ATP by the T4 ploynucleotide kinase method. Cleavage Reactions Kinetic measurements were done under multipleturnover conditions in a buffer containing 50 mM Tris-HCl and 25 mM divalent metal ions (pH 8.0) at 37 °C. After Cleavage reactions were stopped, the Cleavage products and the RNA substrates were separated by electrophoresis on 20% polyacrylamide denaturing gels. The RNA Cleavage yields were determined by quantition of radioactivity in the bands of the 5'-end labeled products and the RNA substrate with a Bio-Image Analyzer model BAS 2000 (Fuji Film, Tokyo). Km and &cat values were calculated from EadieHofstee plots. RESULTS AND DISCUSSION The catalytic efficiency (&cat/^m value) of the original deoxyribozyme in the presence 282 Nucleic Acids Symposium Series No. 42
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Development of a short Ca2+-dependent deoxyribozyme with RNA Cleavage activity.
Nucleic acids symposium series, 1999Co-Authors: Naoki Sugimoto, Yasuhide OkumotoAbstract:We developed a short Ca2+-dependent deoxyribozyme with 11 mer catalytic loop domain (dGGCTACAACGA) that catalyzed site-specific RNA Cleavage reaction between rA and rU. The second-order rate constant of this short deoxyribozyme is 1.7 x 10(7) M(-1) min(-1) at 37 degrees C, and this value is very similar to that of the deoxyribozyme (dGGCTAGCTACAACGA) in the presence of Ca2+.
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Effect of metal ions and sequence of deoxyribozymes on their RNA Cleavage activity
Journal of The Chemical Society-perkin Transactions 1, 1999Co-Authors: Naoki Sugimoto, Yasuhide Okumoto, Tatsuo OhmichiAbstract:Catalytic DNA is a promising class of nucleic acid enzyme for possible use as a therapeutic agent and is also interesting in comparison with catalytic RNAs. In this study, we investigated the effect of metal ions and sequence on an original deoxyribozyme, d(GCCTGGCAG1G2C3T4A5G6C7T8A9C10A11A12C13G14A15GTCCCT), which binds to an RNA substrate, r(AGGGACA↓UGCCAGGC), cleaving the RNA substrate at one site indicated by the arrow. The results show that the ability of metal ions to promote the RNA Cleavage reaction by the original deoxyribozyme is Mn2+ > Mg2+ >Ca2+ Ba2+. This result is very similar to the previous tendency observed in the case of a hammerhead ribozyme. Thus, these results suggest that RNA Cleavage by the deoxyribozyme dependence on metal ions for catalysis is similar to that with ribozymes. On the other hand, a nucleotide deletion from the active domain of the original deoxyribozyme results in a novel deoxyribozyme with high Ca2+-dependency, a situation which is not observed with the original deoxyribozyme or hammerhead ribozymes.
Bertrand Seraphin - One of the best experts on this subject based on the ideXlab platform.
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endonucleolytic RNA Cleavage by a eukaryotic exosome
Nature, 2008Co-Authors: Alice Lebreton, Rafal Tomecki, Andrzej Dziembowski, Bertrand SeraphinAbstract:The exosome is a major eukaryotic nuclease located in both the nucleus and the cytoplasm that contributes to the processing, quality control and/or turnover of a large number of cellular RNAs. This large macromolecular assembly has been described as a 3'-->5' exonuclease and shown to contain a nine-subunit ring structure evolutionarily related to archaeal exosome-like complexes and bacterial polynucleotide phosphorylases. Recent results have shown that, unlike its prokaryotic counterparts, the yeast and human ring structures are catalytically inactive. In contrast, the exonucleolytic activity of the yeast exosome core was shown to be mediated by the RNB domain of the eukaryote-specific Dis3 subunit. Here we show, using in vitro assays, that yeast Dis3 has an additional endoribonuclease activity mediated by the PIN domain located at the amino terminus of this multidomain protein. Simultaneous inactivation of the endonucleolytic and exonucleolytic activities of the exosome core generates a synthetic growth phenotype in vivo, supporting a physiological function for the PIN domain. This activity is responsible for the Cleavage of some natural exosome substrates, independently of exonucleolytic degradation. In contrast with current models, our results show that eukaryotic exosome cores have both endonucleolytic and exonucleolytic activities, mediated by two distinct domains of the Dis3 subunit. The mode of action of eukaryotic exosome cores in RNA processing and degradation should be reconsidered, taking into account the cooperation between its multiple ribonucleolytic activities.
