title: Ribozyme cleavage of a 2,5-phosphodiester linkage: mechanism and a restricted divalent metal-ion requirement.
abstract: The natural substrate cleaved by the hepatitis delta virus (HDV) ribozyme contains a 3',5'-phosphodiester linkage at the cleavage site; however, a 2',5'-linked ribose-phosphate backbone can also be cleaved by both trans-acting and self-cleaving forms of the HDV ribozyme. With substrates containing either linkage, the HDV ribozyme generated 2',3'-cyclic phosphate and 5'-hydroxyl groups suggesting that the mechanisms of cleavage in both cases were by a nucleophilic attack on the phosphorus center by the adjacent hydroxyl group. Divalent metal ion was required for cleavage of either linkage. However, although the 3',5'-linkage was cleaved slightly faster in Ca2+ than in Mg2+, the 2',5'-linkage was cleaved in Mg2+ (or Mn2+) but not Ca2+. This dramatic difference in metal-ion specificity is strongly suggestive of a crucial metal-ion interaction at the active site. In contrast to the HDV ribozymes, cleavage at a 2',5'-phosphodiester bond was not efficiently catalyzed by the hammerhead ribozyme. The relaxed linkage specificity of the HDV ribozymes may be due in part to lack of a rigid binding site for sequences 5' to the cleavage site.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27705, USA.
authors.forename: I H
authors.initials: IH
authors.lastname: Shih
authors.forename: M D
authors.initials: MD
authors.lastname: Been
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D003698:Hepatitis Delta Virus / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D010755:Organophosphates / Q000378:metabolism; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D017423:Sequence Analysis, RNA; D013997:Time Factors; D015534:Trans-Activators / Q000235:genetics
chemical_list: D009841:Oligonucleotides; D010755:Organophosphates; D016337:RNA, Catalytic; D012367:RNA, Viral; D015534:Trans-Activators; D008274:Magnesium; D002118:Calcium