Applicability of urea in the thermodynamic analysis of secondary and tertiary RNA folding.
The equilibrium folding of a series of self-complementary RNA duplexes and the unmodified yeast tRNA(Phe) is studied as a function of urea and Mg(2+) concentration with optical spectroscopies and chemical modification under isothermal conditions. Via application of standard methodologies from protein folding, the folding free energy and its dependence on urea concentration, the m value, are determined. The free energies of the RNA duplexes obtained from the urea titrations are in good agreement with those calculated from thermal melting studies [Freier, S. I., et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9373]. The m value correlates with the length of the RNA duplex and is not sensitive to ionic conditions and temperature. The folding of the unmodified yeast tRNA(Phe) can be described by two Mg(2+)-dependent transitions, the second of which corresponds to the formation of the native tertiary structure as confirmed by hydroxyl radical protection and partial nuclease digestion. Both transitions are sensitive to urea and have m values of 0.94 and 1.70 kcal mol(-)(1) M(-)(1), respectively. Although the precise chemical basis of urea denaturation of RNA is uncertain, the m values for the duplexes and tRNA(Phe) are proportional to the amount of the surface area buried in the folding transition. This proportionality, 0.099 cal mol(-)(1) M(-)(1) A(-)(2), is very similar to that observed for proteins, 0.11 cal mol(-)(1) M(-)(1) A(-)(2) [Myers, J., Pace, N., and Scholtz, M. (1995) Protein Sci. 4, 2138]. These results indicate that urea titration can be used to measure both the free energy and the magnitude of an RNA folding transition.
Shelton
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Department of Chemistry, Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637, USA.
Sosnick
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TR
Pan
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D001483:Base Sequence; D002942:Circular Dichroism; D004722:Endoribonucleases / Q000737:chemistry; D017665:Hydroxyl Radical / Q000737:chemistry; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000138:chemical synthesis; D012313:RNA / Q000138:chemical synthesis / Q000737:chemistry*; D012331:RNA, Fungal / Q000737:chemistry; D012360:RNA, Transfer, Phe / Q000737:chemistry; D006163:Ribonuclease T1 / Q000737:chemistry; D012441:Saccharomyces cerevisiae; D013816:Thermodynamics; D017186:Titrimetry; D014508:Urea / Q000737:chemistry*
D009692:Nucleic Acid Heteroduplexes; D012331:RNA, Fungal; D012360:RNA, Transfer, Phe; D017665:Hydroxyl Radical; D012313:RNA; D014508:Urea; D004722:Endoribonucleases; C032825:ribonuclease V(1); D006163:Ribonuclease T1