2013 Annual Science Report
Georgia Institute of Technology Reporting | SEP 2012 – AUG 2013
Ironing Out the RNA World
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 hypothesis is supported by our observations (1,2) that (i) RNA folding is conserved between complexes with Fe2+ and Mg2+ and (ii) at least some phosphoryl transfer ribozymes are more active in the presence of Fe2+ than Mg2+. We have shown that reversing the putative metal substitution in an anoxic environment, by removing Mg2+ and adding Fe2+, expands the catalytic repertoire of some RNAs. Fe2+ can confer on RNA a previously uncharacterized ability to catalyze single electron transfer. Catalysis is specific, in that it is dependent on the type of RNA. The 23S rRNA and tRNA, some of the most abundant and ancient RNAs (3), are found to be efficient electron transfer ribozymes in the presence of Fe2+. Therefore, the catalytic competence of ancient RNAs may have been greater in early earth conditions than in extant conditions, and the experiments described here may be reviving latent function. The Center is currently testing the hypothesis that replacement of Fe2+ by Mg2+ in RNA assemblies has not been universal.
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.
1. Athavale, S.S., Petrov, A.S., Hsiao, C., Watkins, D., Prickett, C.D., Gossett, J.J., Lie, L., Bowman, J.C., O’Neill, E., Bernier, C.R. et al. (2012) RNA Folding and Catalysis Mediated by Iron (II). PLoS ONE, 7, e38024.
2. Hsiao, C., Chou, I.-C., Okafor, C.D., Bowman, J.C., O’Neill, E.B., Athavale, S.S., Petrov, A.S., Hud, N.V., Wartell, R.M., Harvey, S.C. et al. (2013) Iron(II) Plus RNA Can Catalyze Electron Transfer. Nature Chemistry, 5, 525-528.
3. Fox, G.E. (2010) Origin and Evolution of the Ribosome. Cold Spring Harb. Perspect. Biol., 2, a003483.
4. In Atkins, J. F., Gesteland, R. F. and Cech, T. R. (eds.). Cold Spring Harbor Laboratory Press.
5. Anbar, A.D. (2008) Oceans. Elements and Evolution. Science, 322, 1481-1483.
6. Hazen, R.M. and Ferry, J.M. (2010) Mineral Evolution: Mineralogy in the Fourth Dimension. Elements, 6, 9-12.
7. Prousek, J. (2007) Fenton Chemistry in Biology and Medicine. Pure Appl. Chem., 79, 2325-2338.
8. Klein, C. (2005) Some Precambrian Banded Iron-Formations (BIFs) from around the World: Their Age, Geologic Setting, Mineralogy, Metamorphism, Geochemistry, and Origin. Am. Mineral., 90, 1473-1499.
9. Aguirre, J.D. and Culotta, V.C. (2012) Battles with Iron: Manganese in Oxidative Stress Protection. J. Biol. Chem., 287, 13541-13548.
10. Ushizaka, S., Kuma, K. and Suzuki, K. (2011) Effects of Mn and Fe on Growth of a Coastal Marine Diatom Talassiosira Weissflogii in the Presence of Precipitated Fe(III) Hydroxide and EDTA-Fe(III) Complex. Fish. Sci., 77, 411-424.
11. Martin, J.E. and Imlay, J.A. (2011) The Alternative Aerobic Ribonucleotide Reductase of Escherichia Coli, Nrdef, Is a Manganese-Dependent Enzyme That Enables Cell Replication During Periods of Iron Starvation. Mol. Microbiol., 80, 319-334.
12. Cotruvo, J.A. and Stubbe, J. (2011) Class I Ribonucleotide Reductases: Metallocofactor Assembly and Repair in Vitro and in Vivo. Annu. Rev. Biochem, 80, 733-767.
13. Anjem, A., Varghese, S. and Imlay, J.A. (2009) Manganese Import Is a Key Element of the Oxyr Response to Hydrogen Peroxide in Escherichia Coli. Mol. Microbiol., 72, 844-858.
14. Wolfe-Simon, F., Starovoytov, V., Reinfelder, J.R., Schofield, O. and Falkowski, P.G. (2006) Localization and Role of Manganese Superoxide Dismutase in a Marine Diatom. Plant Physiol., 142, 1701-1709.
15. Torrents, E., Aloy, P., Gibert, I. and Rodriguez-Trelles, F. (2002) Ribonucleotide Reductases: Divergent Evolution of an Ancient Enzyme. J. Mol. Evol., 55, 138-152.
16. Bowman, J.C., Lenz, T.K., Hud, N.V. and Williams, L.D. (2012) Cations in Charge: Magnesium Ions in RNA Folding and Catalysis Curr. Opin. Struct. Biol., 22, 262-272.
17. Auffinger, P., Grover, N. and Westhof, E. (2011) Metal Ion Binding to RNA. Met Ions Life Sci, 9, 1-35.
18. Brion, P. and Westhof, E. (1997) Hierarchy and Dynamics of RNA Folding. Annu. Rev. Biophys. Biomol. Struct., 26, 113-137.
19. Stein, A. and Crothers, D.M. (1976) Conformational Changes of Transfer RNA. The Role of Magnesium(II). Biochemistry, 15, 160-168.
20. Lynch, D.C. and Schimmel, P.R. (1974) Cooperative Binding of Magnesium to Transfer Ribonucleic Acid Studied by a Fluorescent Probe. Biochemistry, 13, 1841-1852.
21. Lindahl, T., Adams, A. and Fresco, J.R. (1966) Renaturation of Transfer Ribonucleic Acids through Site Binding of Magnesium. Proc. Natl. Acad. Sci. U.S.A., 55, 941-948.
22. Petrov, A.S., Bernier, C.R., Hsiao, C.L., Okafor, C.D., Tannenbaum, E., Stern, J., Gaucher, E., Schneider, D., Hud, N.V., Harvey, S.C. et al. (2012) RNA-Magnesium-Protein Interactions in Large Ribosomal Subunit. J. Phys. Chem. B, 116, 8113-8120.
23. Butcher, S.E. (2011) The Spliceosome and Its Metal Ions. Met Ions Life Sci, 9, 235-251.
24. Johnson-Buck, A.E., McDowell, S.E. and Walter, N.G. (2011) Metal Ions: Supporting Actors in the Playbook of Small Ribozymes. Met Ions Life Sci, 9, 175-196.
25. Tsukiji, S., Pattnaik, S.B. and Suga, H. (2004) Reduction of an Aldehyde by a Nadh/Zn2+ -Dependent Redox Active Ribozyme. J. Am. Chem. Soc., 126, 5044-5045.
26. Sen, D. and Poon, L.C. (2011) RNA and DNA Complexes with Hemin [Fe(III) Heme] Are Efficient Peroxidases and Peroxygenases: How Do They Do It and What Does It Mean? Crit. Rev. Biochem. Mol. Biol., 46, 478-492.
27. Ma, J., Haldar, S., Khan, M.A., Sharma, S.D., Merrick, W.C., Theil, E.C. and Goss, D.J. (2012) Fe2+ Binds Iron Responsive Element-RNA, Selectively Changing Protein-Binding Affinities and Regulating mRNA Repression and Activation. Proc. Natl. Acad. Sci. U.S.A., 109, 8417-8422.
28. Serra, M.J., Baird, J.D., Dale, T., Fey, B.L., Retatagos, K. and Westhof, E. (2002) Effects of Magnesium Ions on the Stabilization of RNA Oligomers of Defined Structures. RNA, 8, 307-323.
PROJECT INVESTIGATORS:Stephen Harvey
PROJECT MEMBERS:Shreyas Athavale
RELATED OBJECTIVES:Objective 4.1
Earth's early biosphere.
Production of complex life.