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    • Albert Einstein College of Medicine (AECOM)
    • Albert Einstein College of Medicine: Doctoral Dissertations
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    Isolation and characterization of two enzymatic activities involved in RNA metabolism

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    Date
    1988
    Author
    Arenas, Jaime
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    Abstract
    The work presented in this thesis describes the isolation and characterization of an RNA cyclase and a RNA debranching enzyme from Hela cell extracts.;A. RNA cyclase catalyzes the conversion of 3{dollar}\sp\prime{dollar}-phosphate ends of RNA chains to the 2{dollar}\sp\prime{dollar},3{dollar}\sp\prime{dollar}-cyclic phosphate derivative. We have purified this activity from the cytosolic fraction from HeLa cells approximately 6000 fold. The formation of 1 mol of 2{dollar}\sp\prime{dollar},3{dollar}\sp\prime{dollar}-cyclic ends is associated with the hydrolysis of 1 mol of ATP to AMP and PPi.;This enzyme is likely to be involved in tRNA splicing in animal cells providing the 2{dollar}\sp\prime{dollar},3{dollar}\sp\prime{dollar}-cyclic phosphate end required by the HeLa RNA ligase.;B. The branched RNA molecules, RNA lariats, produced during the splicing of nuclear mRNA precursors are generated by the formation of a 2{dollar}\sp\prime{dollar}-5{dollar}\sp\prime{dollar} phosphodiester bond between the 5{dollar}\sp\prime{dollar}-phosphate end of the intron and the 2{dollar}\sp\prime{dollar}-hydroxyl moiety of a specific A residue near its 3{dollar}\sp\prime{dollar} end. The 2{dollar}\sp\prime{dollar}-5{dollar}\sp\prime{dollar} and 3{dollar}\sp\prime{dollar}-5{dollar}\sp\prime{dollar} phosphodiester bonds of the branch are resistant to the action of various known RNases. An RNA debranching activity have been purified 700-fold from the cytosolic fraction of HeLa cells. This activity catalyzes the specific hydrolysis of the 2{dollar}\sp\prime{dollar}-5{dollar}\sp\prime{dollar} phosphodiester bond of branched RNA yielding a 2{dollar}\sp\prime{dollar}-hydroxyl group and a 5{dollar}\sp\prime{dollar}-phosphate end. The reaction catalyzed by the purified fraction requires a divalent cation and is optimal at pH 7.0. This fraction is a valuable reagent for the identification of lariat molecules among the splicing products.
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    https://yulib002.mc.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:8823302
    https://hdl.handle.net/20.500.12202/3230
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    • Albert Einstein College of Medicine: Doctoral Dissertations [1674]

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