Low specificity of metal ion binding in the metal ion core of a folded RNA
Abstract
The structure and activity of nucleic acids depend on their interactions with metal ions. Fundamental to these interactions is the degree of specificity observed between the metal ions and nucleic acids, and a complete description of nucleic acid folding requires that we understand the nature of the interactions with metal ions, including specificity. The prior demonstration that high concentrations of monovalent cations prevent nonspecific association of divalent ions with nucleic acids provides a novel and powerful means to examine site-specific metal ion binding isolated from complicating effects of the ion atmosphere. Using these high monovalent cation solution conditions we have monitored the affinity of a series of divalent metal ions for two site-specific metal ion binding sites in the P4-P6 domain of the Tetrahymena group I intron ribozyme. The metal ion core of this highly structured RNA binds two divalent metal ions under these conditions. Despite multiple metal ion–RNA interactions observed in the X-ray crystallographic structure of P4-P6 RNA at the metal ion binding sites, these sites exhibit low specificity among Mn2+, Mg2+, Ca2+, Ni2+, and Zn2+. Nevertheless, the largest divalent metal ions tested, Sr2+ and Ba2+, were excluded from binding, exhibiting affinities at least two orders of magnitude weaker than observed for the other metal ions. Thus, a picture emerges of two metal ion binding sites, each with a high tolerance for metal ions with different properties but also with limits to accommodation. These limits presumably arise from steric or electrostatic features of the metal ion binding sites.
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Footnotes
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↵1 Present address: Kevin Travers, Affymetrix, Inc., Santa Clara, CA 95051, USA.
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Reprint requests to: Daniel Herschlag; Department of Biochemistry, Stanford University, Stanford, California 94305-5307, USA; e-mail: herschla{at}stanford.edu; fax: (650) 723-6783.
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↵2 To obtain the relative affinities at the two sites, it is assumed that one site has a preference of three-, 10-, or 30-fold, and the affinity for the test ion at the second site is adjusted by the same factor in the opposite direction to reproduce the observed folding midpoint for the titration with that metal ion alone. For example, if one site has a threefold preference for Ca2+, the other site would have a threefold decrease in affinity for Ca2+. For ions with similar affinity in single ion titrations (for example, Mg2+, Ca2+, and Ni2+), this procedure results in one site having a preference for one ion and the other site having a preference for the other ion. When both ions are present, each binds to its preferred site, resulting in folding at a total ion concentration lower than would be seen for either ion on its own. The equation used to generate the expected plots for threefold, 10-fold, and 30-fold preferences for one ion (Equation [8]) is analogous to Equation (7), except that the affinity at each specific metal ion site was treated independently. In this equation, M1 and M2 are the two ions being mixed in the titration, A and B refer to the two specific metal ion binding sites, and KM1 A, KM1 B, KM2 A, and KM2 B refer to the affinities of each ion for each of the two binding sites.
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Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.566007.
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- Received March 17, 2007.
- Accepted May 17, 2007.
- Copyright © 2007 RNA Society
