Metal-ion rescue revisited: Biochemical detection of site-bound metal ions important for RNA folding

Abstract

Within the three-dimensional architectures of RNA molecules, divalent metal ions populate specific locations, shedding their water molecules to form chelates. These interactions help the RNA adopt and maintain specific conformations and frequently make essential contributions to function. Defining the locations of these site-bound metal ions remains challenging despite the growing database of RNA structures. Metal-ion rescue experiments have provided a powerful approach to identify and distinguish catalytic metal ions within RNA active sites, but the ability of such experiments to identify metal ions that contribute to tertiary structure acquisition and structural stability is less developed and has been challenged. Herein, we use the well-defined P4-P6 RNA domain of the Tetrahymena group I intron to reevaluate prior evidence against the discriminatory power of metal-ion rescue experiments and to advance thermodynamic descriptions necessary for interpreting these experiments. The approach successfully identifies ligands within the RNA that occupy the inner coordination sphere of divalent metal ions and distinguishes them from ligands that occupy the outer coordination sphere. Our results underscore the importance of obtaining complete folding isotherms and establishing and evaluating thermodynamic models in order to draw conclusions from metal-ion rescue experiments. These results establish metal-ion rescue as a rigorous tool for identifying and dissecting energetically important metal-ion interactions in RNAs that are noncatalytic but critical for RNA tertiary structure.

Protein-free ribosomal RNA folds to a near-native state in the presence of Mg2+

“…4 Mg 2+ has been implicated previously in facilitating these interdomain contacts, 6 whereas moderate monovalent cation concentrations induce 2 structure only. 25 SHAPE results here support formation of 12 out of 14 native domain pair interactions ( Fig. 2E and A role for rProteins is suggested by RNA-RNA interactions that appear to be absent from the rRNA-Mg 2+ state.…”

Kinetic and thermodynamic framework for P4-P6 RNA reveals tertiary motif modularity and modulation of the folding preferred pathway

“…ions bound by the MC are shown as gold spheres (46,50,82). (B) P4-P6 secondary structure and variants used in this study to isolate specific folding intermediates (see also Fig.…”

“…This ability, along with homogenous molecular behavior (ref. 90 and Materials and Methods), allows construction of a folding framework under uniform conditions, avoiding uncertainties and complications from extrapolation of RNA behavior across conditions (62,82,91). Example traces and data quality analyses for all constructs can be found in Dataset S1.…”

“…3A). The local equilibrium between the native and alternative P5c secondary structure of WT P4-P6 and constructs with the native P5c secondary structure enforced by two (Nat+2) or three (Nat+3) base pairs (46,50,77,82). (B) Scatter plot of folding versus unfolding rate constants for WT and Nat+3 P4-P6.…”

The spliceosome catalyzes debranching in competition with reverse of the first chemical reaction

“…In many cases, other metal ions can replace the requirement of Mg 2+ (Chen et al 1997;Sontheimer et al 1997;Shan et al 1999;Frederiksen et al 2012). Figure 2B shows that 4 mM Ca 2+ or Mn 2+ stimulated R1 in the presence of KCl (lanes 10,11) or NaCl (lanes 15,16).…”