RNA Folding and Catalysis Mediated by Iron (II)

<div><p>Mg²⁺ shares a distinctive relationship with RNA, playing important and specific roles in the folding and function of essentially all large RNAs. Here we use theory and experiment to evaluate Fe²⁺ in the absence of free oxygen as a replacement for Mg²⁺ in RNA folding and catalysis. We describe both quantum mechanical calculations and experiments that suggest that the roles of Mg²⁺ in RNA folding and function can indeed be served by Fe²⁺. The results of quantum mechanical calculations show that the geometry of coordination of Fe²⁺ by RNA phosphates is similar to that of Mg²⁺. Chemical footprinting experiments suggest that the conformation of the <em>Tetrahymena thermophila</em> Group I intron P4–P6 domain RNA is conserved between complexes with Fe²⁺ or Mg²⁺. The catalytic activities of both the L1 ribozyme ligase, obtained previously by <em>in vitro</em> selection in the presence of Mg²⁺, and the hammerhead ribozyme are enhanced in the presence of Fe²⁺ compared to Mg²⁺. All chemical footprinting and ribozyme assays in the presence of Fe²⁺ were performed under anaerobic conditions. The primary motivation of this work is to understand RNA in plausible early earth conditions. Life originated during the early Archean Eon, characterized by a non-oxidative atmosphere and abundant soluble Fe²⁺. The combined biochemical and paleogeological data are consistent with a role for Fe²⁺ in an RNA World. RNA and Fe²⁺ could, in principle, support an array of RNA structures and catalytic functions more diverse than RNA with Mg²⁺ alone.</p> </div

Addition of Mg²⁺ or Fe²⁺ causes the same changes in the SHAPE reactivity of the P4–P6 domain of the <i>T. thermophila</i> Group 1 intron.

<p>A) Shape profile in presence of 250 mM NaCl and no divalent cations. B) The addition of Mg²⁺ increases the reactivity at the sites indicated with the asterisks and decreases reactivity at other sites. This reaction contains 2.5 mM Mg²⁺ and 250 mM NaCl. C) The addition of Fe²⁺ causes the same changes in SHAPE reactivity as Mg²⁺. This reaction contains 2.5 mM Fe²⁺ and 250 mM NaCl.</p

Conformations of RNA-Mg²⁺ and RNA-Fe²⁺ clamps are nearly identical.

<p>A) RNA-Mg²⁺ clamp from the L1 ribozyme ligase (PDB 2OIU). B) RNA-Mg²⁺ clamp optimized by high level QM calculations. C) An optimized RNA-Fe²⁺ clamp. Each cation (Mg²⁺ or Fe²⁺) is hexacoordinate. Mg²⁺ is shown as a yellow sphere and Fe²⁺ is shown as a green sphere. Water molecules are omitted from the images for clarity. Distances are in Å.</p

Ribozyme activity is enhanced by Fe²⁺ compared to Mg²⁺.

<p>A) L1 ribozyme ligase activity is enhanced in Fe²⁺ compared to Mg²⁺. Ligase reactions were performed under anaerobic conditions at room temperature and 250 mM Na⁺ in 100 µM [Fe²⁺] or 100 µM [Mg²⁺]. The reaction components were first annealed in 50 mM HEPES, pH 8.0, 200 mM sodium acetate by incubating at 90°C for 3 min and cooling to room temperature over 30 min. The ligation reaction was initiated by adding the appropriate cation salt. The Na⁺ only reaction gave no product. Reaction progress was monitored by gel electrophoresis. B) Hammerhead ribozyme activity is enhanced in Fe²⁺ compared to Mg²⁺. Hammerhead ribozyme cleavage reactions were performed under anaerobic conditions at room temperature in 50 mM HEPES, pH 7.5 and 25 µM [Fe²⁺] or 25 µM [Mg²⁺]. Substrate and ribozyme RNA strands were first annealed in 50 mM HEPES buffer by incubating at 90°C for 2 min and cooling to room temperature over 30 min. Cleavage reactions were initiated by addition of FeCl₂ or MgCl₂ from stock solutions. Reactions were monitored by both gel electrophoresis and capillary electrophoresis, which gave similar results.</p