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Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction.

ABSTRACT: The hammerhead ribozyme is a self-cleaving RNA broadly dispersed across all kingdoms of life. Although it was the first of the small, nucleolytic ribozymes discovered, the mechanism by which it catalyzes its reaction remains elusive. The nucleobase of G12 is well positioned to be a general base, but it is unclear if or how this guanine base becomes activated for proton transfer. Metal ions have been implicated in the chemical mechanism, but no interactions between divalent metal ions and the cleavage site have been observed crystallographically. To better understand how this ribozyme functions, we have solved crystal structures of wild-type and G12A mutant ribozymes. We observe a pH-dependent conformational change centered around G12, consistent with this nucleotide becoming deprotonated. Crystallographic and kinetic analysis of the G12A mutant reveals a Zn(2+) specificity switch suggesting a direct interaction between a divalent metal ion and the purine at position 12. The metal ion specificity switch and the pH-rate profile of the G12A mutant suggest that the minor imino tautomer of A12 serves as the general base in the mutant ribozyme. We propose a model in which the hammerhead ribozyme rearranges prior to the cleavage reaction, positioning two divalent metal ions in the process. The first metal ion, positioned near G12, becomes directly coordinated to the O6 keto oxygen, to lower the pKa of the general base and organize the active site. The second metal ion, positioned near G10.1, bridges the N7 of G10.1 and the scissile phosphate and may participate directly in the cleavage reaction.

SUBMITTER: Mir A

PROVIDER: S-EPMC4710350 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction.

Mir Aamir A Chen Ji J Robinson Kyle K Lendy Emma E Goodman Jaclyn J Neau David D Golden Barbara L BL

Biochemistry 20151002 41

The hammerhead ribozyme is a self-cleaving RNA broadly dispersed across all kingdoms of life. Although it was the first of the small, nucleolytic ribozymes discovered, the mechanism by which it catalyzes its reaction remains elusive. The nucleobase of G12 is well positioned to be a general base, but it is unclear if or how this guanine base becomes activated for proton transfer. Metal ions have been implicated in the chemical mechanism, but no interactions between divalent metal ions and the cle ...[more]

PMID: 26398724

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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

Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction.

Publications

Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction.

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