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Project Progress

In RNA World models of evolution, RNA was once the primary biopolymer of genetics and catalysis (4). Ancient RNA-based life would have inhabited an earth with abundant soluble iron and no free oxygen (5,6). Anoxic life persisted for around 1.0-1.5 billion years before photosynthesis began producing substantial free oxygen. The ‘great oxidation’ led to Fe²⁺/O₂ mediated cellular damage (7) and depletion of soluble iron from the biosphere (8). We have proposed hypothesize that Fe2+ was an RNA cofactor on the ancient earth when iron was benign and abundant, and that Fe²⁺ was replaced by Mg²⁺ during the great oxidation. Our RNA-Fe²⁺ to RNA-Mg²⁺ hypothesis is in close analogy with known metal substitutions in some metalloproteins (9-14). An ancestral ribonucleotide reductase (RNR), for example, spawned di-iron, di-manganese, and iron-manganese RNRs (15).

Without cations, RNA is essentially incapable of molecular recognition or catalysis. RNA requires cations in the form of Na⁺, K⁺ and Mg²⁺ for folding and function (16-18). Mg²⁺ was originally demonstrated to be especially important in folding of tRNA (19-21) and is now known to be critical for folding of compact RNAs. Mg²⁺ ions neutralize the negative charge of the RNA backbone and bind specifically to complex structural features of RNA (22). Mg²⁺ is required by many ribozymes for organizing RNA or water molecules within active sites, and for stabilizing reactants or transition states (23,24).

We reported that replacement of Mg²⁺ by Fe²⁺ alters and expands the functional capabilities of some biological RNAs to include redox-activity (2). Previously Suga used in vitro selection to obtain redox-activity of RNA. Suga’s activity requires covalent attachment of substrate to RNA (25). Sen showed that RNA can enhance the redox-activity of iron-protoporphyrin IX (26). By contrast, we observe that some of the most abundant and evolutionarily conserved RNAs have intrinsic redox functionality that is activated simply by interaction with Fe²⁺. Our results here, combined with previous observations of binding of Fe2⁺ to RNA in vivo (27,28), suggest a biological collaboration of iron and RNA. We propose that RNA function, in analogy with protein function, can be fully understood only in the context of association with a range of possible metals. Our results add a new dimension to the RNA World hypothesis. RNA sequence space is clearly more densely occupied, with a broader array of function, than previously expected. This expansion of the apparent catalytic power of RNA suggests that sophisticated biochemical transformations, such as reduction of ribonucleotides to deoxyribonucleotides, were possible in an RNA world.

The Center is currently testing the hypothesis that replacement of Fe²⁺ by Mg²⁺ in RNA assemblies has not been universal. We have established a program to broadly characterize the biology and biochemistry of RNA in high Fe²⁺/low O₂ environments simulating the ancient earth. We gave set up an anaerobic chamber that will allow use to systematic study the properties of RNA in ancient earth conditions. We predict that Fe²⁺ remains an important mediator of RNA structure and function, conferring a rich variety of structures and enzymatic activities to RNA. We will isolate ribosomes and other RNAs from primitive microbes isolated from anoxic Fe²⁺-rich ecosystems and characterize their metal cofactors by highly sensitive analytical techniques. We will search for extant Fe²⁺-dependent ribozymes in diverse microbes of ancient lineage isolated from low oxygen, high iron niches. We will extend our investigations of Fe²⁺ as an RNA cofactor to models of ever more primitive ancestral ribosomes.

References
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