Dataset Information

Separate metal requirements for loop interactions and catalysis in the extended hammerhead ribozyme.

ABSTRACT: An extended hammerhead ribozyme derived from Schistosoma mansoni, including conserved loops in stems I and II, has been examined to directly monitor the relationship between docking of loops and its activity using site-directed spin labeling (SDSL) and EPR spectroscopy. Dynamics with EPR spectroscopy and fast-quench kinetics measurements have shown that the docking of stems I and II occurs at low Mg2+ concentrations ([Mg2+]1/2,dock = 0.7 mM, 0.1 M NaCl), but a much weaker Mg2+ interaction ([Mg2+]1/2,cat approximately 90 mM) increases activity to very high maximum rates of approximately 1 s-1 at 0.1 M Na+ and pH 7.0.

SUBMITTER: Kim NK

PROVIDER: S-EPMC3230267 | biostudies-literature | 2005 Oct

REPOSITORIES: biostudies-literature

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Separate metal requirements for loop interactions and catalysis in the extended hammerhead ribozyme.

Kim Nak-Kyoon NK Murali Ayaluru A DeRose Victoria J VJ

Journal of the American Chemical Society 20051001 41

An extended hammerhead ribozyme derived from Schistosoma mansoni, including conserved loops in stems I and II, has been examined to directly monitor the relationship between docking of loops and its activity using site-directed spin labeling (SDSL) and EPR spectroscopy. Dynamics with EPR spectroscopy and fast-quench kinetics measurements have shown that the docking of stems I and II occurs at low Mg2+ concentrations ([Mg2+]1/2,dock = 0.7 mM, 0.1 M NaCl), but a much weaker Mg2+ interaction ([Mg2+ ...[more]

PMID: 16218578

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Project description:Potential metal interactions with the cleavage site of a minimal hammerhead ribozyme (mHHRz) were probed using (31)P NMR-detected Cd(2+) titration studies of HHRz constructs containing a phosphorothioate (PS) modification at the cleavage site. The mHHRz nucleophile position was replaced by either a 2'-F or a 2'-NH(2) in order to block cleavage activity during the study. The 2'-F/PS cleavage site mHHRz construct, in which the 2'-F should closely imitate the atom size and electronegativity of a 2'-OH, demonstrates low levels of metal ion association (<1 ppm (31)P chemical shift changes). This observation indicates that having an atom size and electrostatic properties that are similar to the 2'-OH are not the governing factors in allowing metal interactions with the scissile phosphate of the mHHRz. With a 2'-NH(2) substitution, a large upfield change in (31)P NMR chemical shift of the phosphorothioate peak (Delta approximately 3 ppm with 6 equiv of added Cd(2+)) indicates observable Cd(2+) interactions with the substituted site. Since a 2'-NH(2), but not a 2'-F, can serve as a metal ligand, these data suggest that a metal ion interaction with the HHRz cleavage site may include both the scissile phosphate and the 2' nucleophile. Control samples in which the 2'-NH(2)/PS unit is placed either next to the mHHRz cleavage site (at U16.1), in a duplex, or in a (am)U(PS)U dinucleotide show much weaker interactions with Cd(2+). Results with these control samples indicate that simply the presence of a 2'-NH(2)/PS unit does not create a strong metal binding site, reinforcing the possibility that the 2'-NH(2)-moderated Cd-PS interaction is specific to the mHHRz cleavage site. Upfield chemical shifts of both (31)P and H-2' (1)H resonances in (am)U(PS)U are observed with addition of Cd(2+), consistent with the predicted metal coordination to both 2'-NH(2) and phosphorothioate ligands. These data suggest that metal ion association with the HHRz cleavage site may include an interaction with the 2'-OH nucleophile.

Dataset Information

Separate metal requirements for loop interactions and catalysis in the extended hammerhead ribozyme.

Publications

Separate metal requirements for loop interactions and catalysis in the extended hammerhead ribozyme.

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