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• W2148420129 abstract "Although the Hammerhead ribozyme (HHRz) has long been used as a model system in the field of ribozyme enzymology, several details of its mechanism are still not well understood. In particular, significant questions remain concerning the disposition and role of catalytic metals in the HHRz. Previous metal-rescue experiments using a “minimal” HHRz resulted in prediction of a catalytic metal that is bound in the A9/G10.1 site in the ground state of the reaction and that bridges to the scissile phosphate further along the reaction pathway. “Native” or extended HHRz constructs contain tertiary contacts that stabilize a more compact structure at moderate ionic strength. We performed Cd 2+ rescue experiments on an extended HHRz from Schistosoma mansoni using stereo-pure scissile phosphorothioate-substituted substrates in order to determine whether a metal ion makes contact with the scissile phosphate in the ground state or further along the reaction coordinate. Inhibition in Ca 2+ /Mg 2+ and rescue by thiophilic Cd 2+ was specific for the R p –S stereoisomer of the scissile phosphate. The affinity of the rescuing Cd 2+ , measured in two different ionic strength backgrounds, increased fourfold to 17-fold when the pro-R p oxygen is replaced by sulfur. These data support a model in which the rescuing metal ion makes a ground-state interaction with the scissile phosphate in the native HHRz. The resulting model for Mg 2+ activation in the HHRz places a metal ion in contact with the scissile phosphate, where it may provide ground-state electrostatic activation of the substrate." @default.
• W2148420129 created "2016-06-24" @default.
• W2148420129 creator A5032772666 @default.
• W2148420129 creator A5090646137 @default.
• W2148420129 date "2011-11-28" @default.
• W2148420129 modified "2023-10-18" @default.
• W2148420129 title "Ground-state coordination of a catalytic metal to the scissile phosphate of a tertiary-stabilized Hammerhead ribozyme" @default.
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• W2148420129 doi "https://doi.org/10.1261/rna.030239.111" @default.
• W2148420129 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3261738" @default.
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