title: Thio effects and an unconventional metal ion rescue in the genomic hepatitis delta virus ribozyme.
abstract: Metal ion and nucleobase catalysis are important for ribozyme mechanism, but the extent to which they cooperate is unclear. A crystal structure of the hepatitis delta virus (HDV) ribozyme suggested that the pro-RP oxygen at the scissile phosphate directly coordinates a catalytic Mg(2+) ion and is within hydrogen bonding distance of the amine of the general acid C75. Prior studies of the genomic HDV ribozyme, however, showed neither a thio effect nor metal ion rescue using Mn(2+). Here, we combine experiment and theory to explore phosphorothioate substitutions at the scissile phosphate. We report significant thio effects at the scissile phosphate and metal ion rescue with Cd(2+). Reaction profiles with an SP-phosphorothioate substitution are indistinguishable from those of the unmodified substrate in the presence of Mg(2+) or Cd(2+), supporting the idea that the pro-SP oxygen does not coordinate metal ions. The RP-phosphorothioate substitution, however, exhibits biphasic kinetics, with the fast-reacting phase displaying a thio effect of up to 5-fold and the slow-reacting phase displaying a thio effect of ~1000-fold. Moreover, the fast- and slow-reacting phases give metal ion rescues in Cd(2+) of up to 10- and 330-fold, respectively. The metal ion rescues are unconventional in that they arise from Cd(2+) inhibiting the oxo substrate but not the RP substrate. This metal ion rescue suggests a direct interaction of the catalytic metal ion with the pro-RP oxygen, in line with experiments with the antigenomic HDV ribozyme. Experiments without divalent ions, with a double mutant that interferes with Mg(2+) binding, or with C75 deleted suggest that the pro-RP oxygen plays at most a redundant role in positioning C75. Quantum mechanical/molecular mechanical (QM/MM) studies indicate that the metal ion contributes to catalysis by interacting with both the pro-RP oxygen and the nucleophilic 2'-hydroxyl, supporting the experimental findings.
authors.affiliation: Department of Chemistry and Center for RNA Molecular Biology, The Pennsylvania State University , University Park, Pennsylvania 16802, United States.
authors.forename: Pallavi
authors.initials: P
authors.lastname: Thaplyal
authors.forename: Abir
authors.initials: A
authors.lastname: Ganguly
authors.forename: Barbara L
authors.initials: BL
authors.lastname: Golden
authors.forename: Sharon
authors.initials: S
authors.lastname: Hammes-Schiffer
authors.forename: Philip C
authors.initials: PC
authors.lastname: Bevilacqua
mesh_terms: D002104:Cadmium / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain; D003698:Hepatitis Delta Virus / Q000235:genetics*; D008274:Magnesium / Q000737:chemistry*; D056004:Molecular Dynamics Simulation; D009690:Nucleic Acid Conformation; D063086:Organothiophosphates / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry*; D011789:Quantum Theory; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013455:Sulfur / Q000737:chemistry*
chemical_list: D063086:Organothiophosphates; D016337:RNA, Catalytic; D002104:Cadmium; D013455:Sulfur; D008274:Magnesium; D010100:Oxygen