Ironing Out the RNA World

For at least the last half-billion years, the earth’s biosphere has been rich in free oxygen: a product of photosynthesis. With the rise in free oxygen much of the biologically available iron (Fe2+) was oxidized and deposited in banded iron formations (BIFs) (1). With photosynthesis, the early earth’s abundant Fe2+ was oxidized and sequestered to the extent that current biomass and species diversity in many ecosystems is limited by Fe2+ availability (2).

Living systems are dependent on and must acquire and utilize iron. Yet, iron combined with oxygen is toxic and expensive to manage (3, 4). The concentration of iron in cells is on the order of 100 mM, with iron largely constrained to heme, iron-sulfur clusters, and di-iron or mono-iron centers, or to transporters, carriers, exporters, and concentrators such as ferritins (5). Because the solubility of ferric iron in water or plasma is very low (10-18 M), cells must combat a massive internal to external concentration gradient.

It was not always thus. Life originated and thrived for billions of years on earth in an anoxic environment in which iron was soluble, abundant, and non-toxic. BIF iron was once soluble and is seen by isotopic variations to have participated in ancient biological processes (6). The transition from benign and abundant iron to scarce and toxic iron caused a slow but dramatic shift in biology that required radical transformations in biochemical mechanisms and metabolic pathways.

It is believed that life originated with RNA-based genetic and metabolic systems, i.e. the RNA world (7). Questions naturally arise as to whether Fe2+ interacted extensively with RNA during the long anoxic period before photosynthesis. Could Fe2+ have facilitated ancient RNA folding and catalysis? Was Fe2+ replaced by Mg2+ with the rise of oxygen and an increase in Mg2+ availability? Are the RNA-Fe2+ complexes observed in extant biology (8) a type of molecular fossil from the RNA world? Here we address the structural and catalytic relationship between RNA and Fe2+ in the absence of oxygen. We use quantum mechanical calculations, chemical footprinting and ribozyme assays to demonstrate that Fe2+ can readily substitute for Mg2+ in RNA folding and catalysis. The relationship of Fe2+ with RNA could provide a missing link between the geological record and the RNA world.

Theory shows that RNA conformation is conserved in the presence of Fe2+ or Mg2+. The results of our high-level quantum mechanical (QM) calculations show that the geometry of coordination of Fe2+ by RNA phosphates is similar to that of Mg2+. The RNA-Mg2+ complex characterized here by computation represents the most frequent mode of multidentate coordination of Mg2+ in large RNAs, where a Mg2+ ion is coordinated by oxyanions of adjacent nucleotides (9, 10) (Figure 1A). One observes twenty-five of these RNA-Mg2+ clamps in the Haloarcula marisortui large ribosomal subunit (11), two in the P4-P6 domain of the Tetrahymena thermophila Group 1 intron (12), one in a self-splicing group II intron from Oceanobacillus iheyensis (13), and one in the L1 ribozyme ligase (14). The folding and function of each of these RNAs is Mg2+ dependent.

The conformations of RNA clamps with Mg2+ or Fe2+ determined by high-level theory are virtually indistinguishable. The metal-oxygen distances and angles are nearly identical (Figures 1B &1C). The QM calculations do indicate some subtle differences, however. The energies of complex formation favor Fe2+ over Mg2+ by 1.4 kcal/mol, as revealed by Natural Bond Order analysis (15). More charge is transferred from phosphate to Fe2+ (0.43 e-) than from phosphate to Mg2+ (0.29 e-), suggesting that compared to Mg2+, Fe2+ better activates the phosphorous of RNA to nucleophilic attack. These differences are attributable to the greater accessibility of Fe2+ d-orbitals. The similarities of the interactions of Fe2+ or Mg2+ in complexes with RNA are consistent with the interchangeability of Fe2+ and Mg2+ in phosphate minerals such as lazulite [(Mg2+,Fe2+)(Al3+)2(PO43-)2(OH-)2].

Chemical probing confirms that RNA conformation is conserved in the presence of Fe2+ or Mg2+. The conservation of RNA conformation in association with Fe2+ or Mg2+ predicted in silico is observed in vitro. In solution, RNA conformation can be assayed by chemical footprinting using SHAPE (16) (Selective 2’-Hydroxyl Acylation analyzed by Primer Extension). These chemical footprints are known to be dependent on Mg2+ concentration (16, 17). We used SHAPE to compare RNA conformation with Fe2+ to that with Mg2+. We observe that in the absence of oxygen, the addition of Fe2+ or Mg2+ causes pronounced and nearly identical changes in the SHAPE reactivity of the Tetrahymena Group I intron P4-P6 domain (Figure 2). These results suggest that the ‘magnesium ion core’ of the P4-P6 domain can be recapitulated by Fe2+ in the absence of oxygen.

Ribozyme activity is enhanced by Fe2+ compared to Mg2+. We investigated the catalytic activity of the L1 ribozyme ligase in the presence of Mg2+ and Fe2+ (in the absence of oxygen). This ligase catalyzes formation of a phosphodiester linkage during which the 3′-hydroxyl group of an RNA substrate attacks the a-phosphorus of the ribozyme 5′-triphosphate (14). This ribozyme was obtained by Robertson and Ellington by in vitro selection in the presence of high [Mg2+] (60 mM), and has been described as Mg2+-dependent (14). The initial rate of ligation is over 10-fold higher in 100 mM Fe2+ than in 100 mM Mg2+ (Figure 3). The Fe2+ advantage holds for essentially any equimolar concentration of the two divalent cations. Achieving an equivalent rate of reaction requires around a 10-fold greater [Mg2+] than [Fe2+]. As expected, this ligase is inactive in Na+ alone. To explore whether enhancement of ribozyme activity by Fe2+ is a general effect, we also examined the hammerhead ribozyme.  Fe2+, compared to Mg2+, causes a significant enhancement of activity of that natural ribozyme.

Some of the first studies of RNA folding showed that magnesium is required for formation of tertiary structure of tRNA (18). It is now known that magnesium plays specific roles in folding of essentially all large RNAs (19, 20). Magnesium shares a distinctive geometric and electronic relationship with phosphate groups. The ionic radius of Mg2+ is small, the charge density is high, the coordination geometry is strictly octahedral, the preferred ligands are charged or neutral oxygens, and the hydration enthalpy is large (21-23). In comparison with group I ions, calcium, or polyamines (24), magnesium has a greater affinity for phosphate oxygens, and can bind to them with well-defined geometry.

Here we provide support for a model of the RNA world, extending into the early protein/DNA/RNA world, in which Fe2+, not Mg2+, was the predominant divalent partner for RNA. The conformations of RNA clamps with Mg2+ or Fe2+ determined by high-level quantum mechanical calculations are virtually indistinguishable. The metal-oxygen distances and angles are nearly identical (Figures 1B &1C). Facile substitution of Mg2+ by Fe2+ is confirmed by chemical probing experiments that show that changes in SHAPE reactivity of RNA induced by association with Mg2+ or Fe2+ are very similar (Figures 2B and 2C). And finally, at equimolar concentrations of Mg2+ or Fe2+, the initial rate of ligation observed for the L1 ribozyme ligase is over 10-fold higher with Fe2+ than Mg2+ (Figure 3).

The RNA world is hypothesized to have occurred during the early Archean eon. During this time it is thought that Mg2+ marine concentration was attenuated by submarine hydrothermal systems associated with higher heat flow (25, 26) and more vigorous seafloor spreading (27, 28), and by reduced Mg2+ delivery to the oceans by smaller continental landmasses. During the early Archean eon, Fe2+ would have been soluble and non-toxic because the environment was anoxic. The [Fe2+]marine in the early Archean is largely circumscribed in current models by PCO2 in the atmosphere (Fe2+ precipitates as siderite: Fe2+CO3-). Early earth PCO2 is inferred using estimates of greenhouse effects, sun luminosity, earth albedo and temperatures required to maintain liquid oceans. Recent indications of a low albedo early earth dispense with requirements for high greenhouse PCO2 (29). If so, [Fe2+]marine could have been as high as 100-1000 mM (30, 31).

The combined biochemical and geological data are consistent with an RNA-Fe2+ world. It is nearly inconceivable that RNA in the early Archean eon escaped extensive interaction with Fe2+. Our observations of Fe2+-mediated RNA folding and catalysis, in combination with the paleogeological models, suggest that RNA functioned and evolved in association with Fe2+.  This RNA-Fe2+ world could have supported an array of RNA structures and catalytic functions far more diverse than that of a RNA-Mg2+ world. Complexes of RNA with Fe2+ offer the prospect of a rich redox chemistry for ancient ribozymes.





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