Inner-Sphere Coordination of Divalent Metal Ion with Nucleobase in Catalytic RNA

Abstract

Identification of the function of metal ions and the RNA moieties, particularly nucleobases, that bind metal ions are important questions in RNA catalysis. Here we combine single-atom and abasic substitutions to probe functions of conserved nucleobases in ribonuclease P (RNase P). Structural and biophysical studies of bacterial RNase P propose direct coordination of metal ions by the nucleobases of conserved uridine and guanosine in helix P4 of the RNA subunit (P RNA). To biochemically probe the function of metal ion interactions, we substituted the universally conserved bulged uridine (U51) in the P4 helix of circularly permuted Bacillus subtilis P RNA with 4-thiouridine, 4-deoxyuridine and abasic modifications and G378/379 with 2-aminopurine, N7-deazaguanosine, and 6-thioguanosine. The functional group modifications of U51 decrease RNase P-catalyzed phosphodiester bond cleavage 16- to 23-fold, as measured by the single-turnover cleavage rate constant. The activity of the 4-thiouridine RNase P is partially rescued by addition of Cd(II) or Mn(II) ions. This is the first time a metal-rescue experiment provides evidence for inner-sphere divalent metal ion coordination with a nucleobase. Modifications of G379 modestly decrease the cleavage activity of RNase P, suggesting outer-sphere coordination of O6 on G379 to a metal ion. These data provide biochemical evidence for catalytically important interactions of the P4 helix of P RNA with metal ions, demonstrating that the bulged uridine coordinates at least one catalytic metal ion through an inner-sphere interaction. The combination of single-atom and abasic nucleotide substitutions provides a powerful strategy to probe functions of conserved nucleobases in large RNAs.

Nucleobase carbonyl groups are poor Mg2+inner-sphere binders but excellent monovalent ion binders—a critical PDB survey

“…In the current PDB data set, through replication of errors, the recurrence of a given binding site may unfortunately not warrant its reliability and, therefore, it is important to keep redundancy issues in mind. More specifically, present data imply that the assertion that "the coordination of Mg 2+ by nucleobases should be considered as a significant factor in the stabilization of RNA structure" (Zheng et al 2015) must be approached with caution and should not be used to support claims regarding the implication of nucleobase carbonyl groups in catalytic mechanisms (Liu et al 2017).…”

Structural insight into precursor tRNA processing by yeast ribonuclease P

“…However, pre-tRNA recognition by the much more complex and indispensable protein components of eukaryotic RNase Ps is still poorly understood. Kinetic evidences suggested that a conformational change occurs after the formation of the initial ES complex and before 5′-leader cleavage, transforming the ES complex into an active ES* state (7,9,18,24,25). The ES* complex is stabilized by at least two Mg 2+ ions during the transition from ES to ES*, and the functional groups and metal ions in the active site are believed to be repositioned to coordinate the substrate for catalysis (19,26).…”

“…The phosphodiester bond cleavage is an S N 2-type transesterification reaction, which uses a two-Mg 2+ -ion mechanism that is also used by the group I and group II introns as well as the spliceosome (27)(28)(29)(30). Despite extensive studies, the positions of the catalytic Mg 2+ ions and the composition of the catalytic center in RNase P are still enigmatic because of the lack of structural information of a pre-tRNA substrate-bound RNase P (24,(31)(32)(33)(34)(35)(36)(37)(38)(39). Consequently, the chemical mechanism of this two-Mg 2+ -ion-mediated reaction still awaits to be revealed.…”

Phospholipase D encapsulated into metal-surfactant nanocapsules for enhancing biocatalysis in a two-phase system

“…Metal ions have important effects on enzyme activity. 24,25 In this study, Co 2+ , Ca 2+ , Mg 2+ , Ni 2+ , and Zn 2+ were tested for their ability to form nanocomposites with NaDC and to encapsulate PLD in aqueous solution ( Fig. 1).…”