Abstract
The folding thermodynamics and kinetics for the ribozyme from Bacillus subtilis RNase P are analyzed using circular dichroism and UV absorbance spectroscopies and catalytic activity. At 37 °C, the addition of Mg2+ (Kd ∼ 50 μM) to the unfolded state produces an intermediate state within 1 ms which contains a comparable amount of secondary structure as the native ribozyme. The subsequent transition to the native state (Kd[Mg] ∼ 0.8 mM, Hill coefficient ∼ 3.5) has a half-life of hundreds of seconds as measured by circular dichroism at 278 nm and by a ribozyme activity assay. Surprisingly, the formation of the native structure is accelerated strongly by the addition of a denaturant; ∼30-fold at 4.5 M urea. Thus, the rate-limiting step entails the disruption of a considerable number of interactions. The folding of this, and presumably other large RNAs, is slow due to the structural rearrangement of kinetically trapped species. Taken together with previous submillisecond relaxation kinetics of tRNA tertiary structure, we suggest that error-free RNA folding can be on the order of milliseconds.
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References
Crothers, D.M., Cole, P.E., Hilbers, C.W. & Shulman, R.G. The molecular mechanism of thermal unfolding E. coli tRNAfmet . J. Mol. Biol. 87, 63–88 (1974).
Celander, D.W. & Cech, T.R. Visualizing the higher order folding of a catalytic RNA molecule. Science 251, 401–407 (1991).
Banerjee, A.R., Jaeger, J.A. & Turner, D.H. Thermal unfolding of a group I ribozyme: the low-temperature transition is primarily disruption of tertiary structure. Biochemistry 32, 153–163 (1993).
Laing, L.G. & Draper, D.E. Thermodynamics of RNA folding in a conserved ribosomal RNA domain. J. Mol. Biol. 237, 560–576 (1994).
Murphy, F.L. & Cech, T.R. An independently folding domain of RNA tertiary structure within the Tetrahymena ribozyme . Biochemistry 32, 5291–5300 (1993).
Cate, J.H. et al. Crystal structure of a group I ribozyme domain: principles of RNA packing. Science 273, 1678–1685 (1996).
Pan, T. Higher order folding and domain analysis of the ribozyme from Bacillus subtilis ribonuclease P, Biochemistry 34, 902–909 (1995).
Loria, A. & Pan, T. Domain structure of the ribozyme from eubacterial ribonuclease P. RNA 2, 551–563 (1996).
Doherty, E.A. & Doudna, J.A. The P4-P6 domain directs higher order folding of the Tetrahymena ribozyme core. Biochemistry 36, 3159–3169 (1997).
Draper, D.E. Parallel worlds. Nature Struct Biol. 3, 397–400 (1996).
Thirumalai, D. & Woodson, S.A. Kinetics of folding of proteins and RNA. Acc. Chem. Res. 29, 433–439 (1996).
Cole, P.E. & Crothers, D.M. Conformational changes of transfer ribonucleic acid. Relaxation kinetics of the early melting transition of tRNAMet(E. coli) . Biochemistry 11, 4368–4374 (1972).
Urbanke, C., Romer, R. & Maass, G. Tertiary structure of tRNAPhe (Yeast): kinetics and electrostatic repulsion. Eur. J. Biochem. 55, 439–444 (1975).
Cole, P.E., Yang, S.K. & Crothers, D.M. Conformational changes of transfer ribonucleic acid. Equilibrium phase diagrams. Biochemistry 11, 4358–4368 (1972).
Lynch, D.C. & Schimmel, P.R. Cooperative binding of magnesium to transfer RNA studied by a fluorescent probe. Biochemistry 13, 1841–1852 (1974).
Zarrinkar, P.P. & Williamson, J.R. Kinetic intermediates in RNA folding. Science 265, 918–924 (1994).
Banerjeee, A.R. & Turner, D.H. The time dependence of chemical modification reveals slow steps in the folding of a group I ribozyme. Biochemistry 34, 6504–6512 (1995).
Zarrinkar, P.P. & Williamson, J.R. The kinetic folding pathway of the Tetrahymena ribozyme reveals possible similarities between RNA and protein folding. Nature Struct. Biol. 3, 432–438 (1996).
Zarrinkar, P.P., Wang, J. & Williamson, J.R. Slow folding kinetics of RNase P RNA. RNA 2, 564–573 (1996).
Downs, W.D. & Cech, T.R. Kinetic pathway for folding of the Tetrahymena ribozyme revealed by three UV-inducible crosslinks. RNA 2, 718–732 (1996).
Emerick, V.L., Pan, J. & Woodson, S.A. Analysis of rate-determining Conformational changes during self-splicing of the Tetrahymena intron. Biochemistry 35, 13469–13477 (1996).
Sclavi, B., Woodson, S., Sullivan, M., Chance, M.R. & Brenowitz, M. Time- resolved synchrotron X-ray “footprinting”, a new approach to the study of nucleic acid structure and function: application to protein-DNA interactions and RNA folding. J. Mol. Biol. 266, 144–159 (1997).
Cantor, C.R. & Schimmel, P.R. In Biophysical chemistry, Part II: techniques for the study of biological structure and function 381–405 (W.H. Freeman & Company, S.F.; 1980).
Gray, D.M., Hung, S.-H. & Johnson, K.H. Absorption and circular dichroism spectroscopy of nucleic acid duplexes and triplexes. Meths. Enz. 246, 19–34 (1995).
Manning, G.S. The molecular theory of polyelectrolyte solutions with applications to the electrostatic properties of polynucleotides. Q. Rev. Biophys. 11, 179–246 (1978).
Matthews, C.R. Effect of point mutations on the folding of globular proteins. Meths. Enz. 154, 498–511 (1987).
Pace, C.N. The stability of globular proteins. CRC Crit. Rev. biochem. 3:1–4–, (1975).
Quigley, G.J. & Rich, A. Structural domains of tranfer RNA molecules. Science 194, 796–806 (1976).
Pan, J., Thirumalai, D. & Woodson, S.A. Folding of RNA involves parallel pathways. J. Mol. Biol. in the press (1997).
Jackson, S.E. & Fersht, A.R. Folding of chymotrypsin inhibitor 2. 1. Evidence for a two-state transition. Biochemistry 30, 10428–10435 (1991).
Sosnick, T.R., Mayne, L. & Englander, S.W. Molecular collapse: The rate- limiting step in 2-state cytochrome c folding. Proteins: Struct. Funct Genet. 24, 413–426 (1996).
Sosnick, T.R., Mayne, L., Hiller, R. & Englander, S.W. The barriers in protein folding. Nature Struct. Biol. 1, 149–156 (1994).
Baldwin, R.L. The nature of protein folding pathways: The classical versus the new view. J. Biomol. NMR 5, 103–109 (1995).
Abkevich, V.I., Gutin, A.M. & Shakhnovich, E.I. Specific nucleus as the transition state for protein folding: Evidence from the lattice model. Biochemistry 33, 10026–10036 (1994).
Itzhaki, L.S., Otzen, D.E. & Fersht, A.R. The structure of the transition state for folding of chymotrypsin inhibitor 2 analysed by protein engineering methods: evidence for a nucleation-condensation mechanism for protein folding. J. Mol. Biol. 254, 260–88 (1995).
Guo, Z.Y. & Thirumalai, D. Kinetics of protein folding: Nucleation mechanism, time scales, and pathways. Biopolymers 36, 83–102 (1995).
Bai, Y., Sosnick, T.R., Mayne, L. & Englander, S.W. Protein folding intermediates: Native-state hydrogen exchange. Science 269, 192–197 (1995).
Hamada, D., Segawa, S., Goto, Y. Non-native α-helical intermediate in the refolding of β-lactoglobin, a predominantly β-sheet protein. Nature Struct. Biol. 3, 868–873 (1996).
Bryngelson, J.D. & Wolynes, P.G. Intermediates and barrier crossing in a random energy model (with applications to protein folding). J. Phys. Chem. 93, 6902–6915 (1989).
Abkevich, V.I., Gutin, A.M. & Shakhnovich, E.I. Free energy landscape for protein folding kinetics: Intermediates, traps, and multiple pathways in theory and lattice moldel simulations. J. Chem. Phys. 101:6052–6062 (1994).
Bryngelson, J.D., Onuchic, J.N., Socci, N.D. & Wolynes, P.G. funnels, pathways and the energy landscape of protein folding: A synthesis. Proteins: Struc. Funct. Genet. 21, 167–195 (1995).
Dill, K.A. & Chan, H.S. From Levinthal to pathways to funnels. Nature Struct. Biol. 4: 10–19 (1997).
Milligan, J.F., Groebe, D.R., Witherell, G.W. & Uhlenbeck, O.C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 15, 8783–8798 (1987).
Pan, T. & Zhong, K. Selection of circularly permuted ribozymes from Bacillus subtilis RNase P by substrate binding. Biochemistry 33, 14207–14212 (1994).
Beebe, J.A. & Fierke, C.A. A kinetic mechanism for cleavage of precursor tRNAAsp catalyzed by the RNA component of Bacillus subtilis ribonuclease P. Biochemistry 33, 10294–10304 (1994).
Loria, A. & Pan, T. Recognition of the T stem-loop of a pre-tRNA substrate by the ribozyme from Bacillus subtilis ribonuclease P. Biochemistry 36, 6317–6235 (1997).
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Pan, T., Sosnick, T. Intermediates and kinetic traps in the folding of a large ribozyme revealed by circular dichroism and UV absorbance spectroscopies and catalytic activity. Nat Struct Mol Biol 4, 931–938 (1997). https://doi.org/10.1038/nsb1197-931
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DOI: https://doi.org/10.1038/nsb1197-931
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