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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 (15-17). Mg²⁺ was originally demonstrated to be especially important in folding of tRNA (18-20) 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 (21). Mg²⁺ is required by many ribozymes for organizing RNA or water molecules within active sites, and for stabilizing reactants or transition states (22,23).

Using a standard peroxidase assay (24), we investigated the catalytic abilities of a variety of native nucleic acids to determine if they can catalyze single electron transfer in association with Fe²⁺ instead of Mg²⁺ . In this assay, an organic dye (a reducing agent) is oxidized by hydrogen peroxide (an oxidizing agent) via electron transfer. Specifically, one electron is transferred from 3,3′,5,5′-tetramethylbenzidine (TMB) to hydrogen peroxide, forming a radical cation (TMB• +) according to the equation:

½ (HOOH) + TMB + H+ → H2O + TMB• +.

This reaction is a broadly used assay for detection of electron transfer. Absorbance at 370 nm and 652 nm is used to monitor the course of the reaction.

Catalysis of single electron transfer is observed for a subset of RNAs tested here (Figure 1). Fe²⁺ , RNA, and the exclusion of O₂ are required. In solutions containing Mg²⁺ instead of Fe²⁺ , solutions lacking RNA, or solutions with heat-degraded RNA, catalysis of electron transfer is not detected. Some RNAs catalyze electron transfer much more efficiently than other RNAs, at lower concentration of RNA (0.03-1.0 μM strand, 75-100 μM in nucleotide). For the inefficient RNAs, electron transfer is observable only at high concentration (>500 μM in nucleotide). Relative rates of electron transfer are inferred from the initial slopes of graphs such as those represented in Figure 1. Additional information on materials, methods, reaction rates, RNA stability, and control reactions is found in the online-only methods.

The 23S rRNA gives efficient catalysis of electron transfer at a low concentration of RNA strands (Figure 1), with a greater k_cat (Figure 2) than other RNAs. On a per nucleotide bases, the P4-P6 domain RNA and a mixture of yeast tRNAs are most efficient, catalyzing electron transfer with roughly similar efficiencies. The appropriate choice of concentration units, strand or nucleotide, is dependent on whether the Fe²⁺ binds at a few highly specific sites, or less specifically to many sites on the RNAs.

We examined a diverse set of nucleic acids in an attempt to isolate properties responsible for catalysis of electron transfer in the presence of Fe²⁺ (Figure 1). Closely spaced phosphate groups, as in ATP, are not sufficient to induce catalysis. The regular array of phosphate groups and other structural features of B-form DNA are insufficient for efficient catalysis. We investigated a double-stranded 32 base pair DNA duplex composed of a mixture of C-G and A-T base pairs. The combination of B-DNA and Fe²⁺ does not catalyze electron transfer. To investigate whether a short unstructured RNA catalyzes electron transfer in the presence of Fe²⁺ , we used a small, single-stranded RNA oligomer (5′-GCACU-3′). This short RNA oligomer does not catalyze electron transfer. We investigated the Satellite Tobacco Mosaic Virus (STMV) genomic RNA (25) to determine if a large, partially unstructured RNA is competent to catalyze electron transfer in the presence of Fe²⁺ . This 1058 nucleotide RNA genome does not catalyze electron transfer at 0.5 μM strand, which, in terms of nucleotide concentration, greatly exceeds the concentration where P4-P6 Domain RNA, 23S rRNA and tRNAs are catalytic.

The differential catalysis of single-electron transfer by various RNAs supports a model in which the potential to form well-defined three-dimensional RNA structure is important to catalysis. The ability of RNA to coordinate divalent cations may be significant. In the large ribosomal subunit a subset of associated divalent cations are chelated by up to three phosphate groups of the 23S rRNA (26,27). The P4-P6 domain RNA chelates fewer divalent cations but with analogous coordination geometry (28). tRNA also interacts strongly with divalent cations (18-20). In contrast, RNAs such as nucleotides, small single-stranded RNA, B-DNA, and the STMV RNA genome, which lack the potential to form well-defined structures and do not chelate divalent cations, are much less efficient electron transfer catalysts.
Fe²⁺ can substitute for Mg²⁺ in RNA compaction, as previously demonstrated for the P4-P6 domain RNA, which folds to the same state with either Mg²⁺ or Fe²⁺ ions (13), with common binding sites for either metal (29). However, the low salt conditions that yield the best catalysis here suggest that the RNAs are not fully folded to their native states in our experiments and therefore, the structures of RNA-Fe²⁺ complexes that yield catalysis remain to be fully characterized.

The catalytic nature of these electron transfer reactions is supported by standard enzyme assays. Since the amount of enzyme in a reaction mixture can be limiting, enzyme-catalyzed reaction rates plateau rather than increase monotonically with increasing substrate concentration. For both 23S rRNA and the P4-P6 Domain RNA, the electron transfer reaction does indeed saturate (Figures 2A & 2C).
Kinetic parameters for single electron transfer by 23S rRNA and the P4-P6 Domain RNA with Fe²⁺ were determined by fitting data to a Michaelis-Menten model (Figure 2). Non-linear regression analysis of the experimental data shows that for P4-P6 domain RNA, k_cat = 2.2 10^-2/s, K_m = 4.15 10^-6 M, k_cat /K_m = 5.3 10³/M⋅s. For the 23S rRNA, k_cat =1.28/s, K_m = 1.75 10^-5 M, k_cat /K_m = 7.3 10⁴/M⋅s. However, if the RNA is not saturated with Fe²⁺ , the data underestimate the true k_cat.

Previously Breaker isolated a series of hammerhead ribozymes that cleave RNA upon binding to divalent metal cations (30). Those phosphoryl-transfer ribozymes show linear relationship of log rate versus log [cation] for RNA cleavage. Slopes of either one or two in the log-log graphs were interpreted as indicating either one or two cations being required for catalysis. Using Breaker’s approach here, for the P4-P6 Domain RNA, the log of the rate varies linearly with the log [Fe²⁺ ] (Figure 2E). The slope is approximately one. Although more complex models cannot be excluded, these observations are consistent with a mechanism in which occupancy of a single Fe²⁺ site confers catalytic activity to the RNA.

We observe that replacement of Mg²⁺ by Fe²⁺ alters and expands the functional capabilities of some biological RNAs to include redox-activity. Previously Suga used in vitro selection to obtain redox-activity of RNA. Suga’s activity requires covalent attachment of substrate to RNA (31). Sen showed that RNA can enhance the redox-activity of iron-protoporphyrin IX (32). 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 (33,34), 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.

References 1. In Atkins, J. F., Gesteland, R. F. and Cech, T. R. (eds.). Cold Spring Harbor Laboratory Press.

2. Anbar, A.D. (2008) Oceans. Elements and Evolution. Science (New York, N.Y.), 322, 1481-1483.

3. Hazen, R.M. and Ferry, J.M. (2010) Mineral Evolution: Mineralogy in the Fourth Dimension. Elements, 6, 9-12.

4. Prousek, J. (2007) Fenton Chemistry in Biology and Medicine. Pure Appl. Chem., 79, 2325-2338.

5. 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.

6. Aguirre, J.D. and Culotta, V.C. (2012) Battles with Iron: Manganese in Oxidative Stress Protection. J. Biol. Chem., 287, 13541-13548.

7. 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.

8. 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.

9. 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.

10. 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.

11. 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.

12. 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.

13. 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.

14. Fox, G.E. (2010) Origin and Evolution of the Ribosome. Cold Spring Harb. Perspect. Biol., 2, a003483.

15. 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.

16. Auffinger, P., Grover, N. and Westhof, E. (2011) Metal Ion Binding to RNA. Met Ions Life Sci, 9, 1-35.

17. Brion, P. and Westhof, E. (1997) Hierarchy and Dynamics of RNA Folding. Annu. Rev. Biophys. Biomol. Struct., 26, 113-137.

18. Stein, A. and Crothers, D.M. (1976) Conformational Changes of Transfer RNA. The Role of Magnesium(II). Biochemistry, 15, 160-168.

19. 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.

20. 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.

21. 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.

22. Butcher, S.E. (2011) The Spliceosome and Its Metal Ions. Met Ions Life Sci, 9, 235-251.

23. 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.

24. Josephy, P.D., Eling, T. and Mason, R.P. (1982) The Horseradish Peroxidase-Catalyzed Oxidation of 3,5,3’,5’-Tetramethylbenzidine. Free Radical and Charge-Transfer Complex Intermediates. J. Biol. Chem., 257, 3669-3675.

25. Larson, S.B., Day, J., Greenwood, A. and McPherson, A. (1998) Refined Structure of Satellite Tobacco Mosaic Virus at 1.8 Å Resolution. J. Mol. Biol., 277, 37-59.

26. Hsiao, C. and Williams, L.D. (2009) A Recurrent Magnesium-Binding Motif Provides a Framework for the Ribosomal Peptidyl Transferase Center. Nucleic Acids Res., 37, 3134-3142.

27. Klein, D.J., Moore, P.B. and Steitz, T.A. (2004) The Contribution of Metal Ions to the Structural Stability of the Large Ribosomal Subunit. RNA, 10, 1366-1379.

28. Cate, J.H., Hanna, R.L. and Doudna, J.A. (1997) A Magnesium Ion Core at the Heart of a Ribozyme Domain. Nat. Struct. Biol., 4, 553-558.

29. Berens, C., Streicher, B., Schroeder, R. and Hillen, W. (1998) Visualizing Metal-Ion-Binding Sites in Group I Introns by Iron(II)-Mediated Fenton Reactions. Chem. Biol., 5, 163-175.

30. Zivarts, M., Liu, Y. and Breaker, R.R. (2005) Engineered Allosteric Ribozymes That Respond to Specific Divalent Metal Ions. Nucleic Acids Res., 33, 622-631.

31. 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.

32. 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.

33. 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.

34. 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-a Publication of the Rna Society, 8, 307-323.