Use of Chemical Modification To Elucidate RNA Folding Pathways

David H. Mathews1, Douglas H. Turner1

1 University of Rochester, Rochester, New York
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 11.9
DOI:  10.1002/0471142700.nc1109s09
Online Posting Date:  August, 2002
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Abstract

As discussed in this overview, chemical modification is sensitive to the accessibility of a nucleotide to the solvent, and many nucleotides become less accessible as an RNA folds into its structured form. Chemical modification reagents are therefore suitable for following RNA folding, and can be used to study the kinetics of structure formation on time scales ranging from minutes to hours.

     
 
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Table of Contents

  • Choosing a Transition
  • Characterizing the Transition
  • Determining Folding Kinetics
  • Conclusion
  • Figures
     
 
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Materials

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Figures

Videos

Literature Cited

Literature Cited
   Banerjee, A.R. and Turner, D.H. 1995. The time dependence of chemical modification reveals slow steps in the folding of a group I ribozyme. Biochemistry 34:6504‐6512.
   Banerjee, A.R., Jaeger, J.A., and Turner, D.H. 1993. Thermal unfolding of a group I ribozyme: The low temperature transition is primarily disruption of tertiary structure. Biochemistry 32:153‐163.
   Bevilacqua, P.C., Kierzek, R., Johnson, K.A., and Turner, D.H. 1992. Dynamics of ribozyme binding of substrate revealed by fluorescence‐detected stopped‐flow methods. Science 258:1355‐1358.
   Chaulk, S.G. and MacMillan, A.M. 2000. Characterization of the Tetrahymena ribozyme folding pathway using the kinetic footprinting reagent peroxynitrous acid. Biochemistry 39:2‐8.
   Crothers, D.M., Cole, P.E., Hilbers, C.W., and Schulman, R.G. 1995. The molecular mechanism of thermal unfolding of Escherichia coli formylmethionine transfer RNA. J. Mol. Biol. 87:63‐88.
   Ehresmann, C., Baudin, F., Mougel, M., Romby, P., Ebel, J., and Ehresmann, B. 1987. Probing the structure of RNAs in solution. Nucl. Acids Res. 15:9109‐9128.
   Hilbers, C.W., Robillard, G.T., Shulman, R.G., Blake, R.D., Webb, P.K., Fresco, R., and Riesner, D. 1976. Thermal unfolding of yeast glycine transfer RNA. Biochemistry 15:1874‐1882.
   Inuoe, T. and Cech, T.R. 1985. Secondary structure of the circular form of the Tetrahymena rRNA intervening sequence: A technique for RNA structure analysis using chemical probes and reverse transcriptase. Proc. Natl. Acad. Sci. U.S.A. 82:331‐346.
   Jaeger, L., Westhof, E., and Michel, F. 1993. Monitoring of cooperative unfolding of the sunY group I intron of bacteriophage T4. J. Mol. Biol. 234:648‐652.
   Kent, O., Chaulk, S.G., and MacMillan, A.M. 2000. Kinetic analysis of the M1 RNA folding pathway. J. Mol. Biol. 304:699‐705.
   Mathews, D.H., Banerjee, A.R., Luan, D.D., Eickbush, T.H., and Turner, D.H. 1997. Secondary structure model of the RNA recognized by the reverse transcriptase from the R2 retrotransposable element. RNA 3:1‐16.
   Moazed, D., Stern, S., and Noller, H.F. 1986. Rapid chemical probing of conformation in 16S ribosomal RNA and 30S ribosomal subunits using primer extension. J. Mol. Biol. 187:399‐416.
   Pan, T. and Sosnick, T.R. 1997. Intermediates and kinetic traps in the folding of a large ribozyme revealed by circular dichroism and UV absorbance spectroscopies and catalytic activity. Nat. Struct. Biol. 4:931‐938.
   Sclavi, B., Sullivan, M., Chance, M.R., Brenowitz, M., and Woodson, S.A. 1998. RNA folding at millisecond intervals by synchrotron hydroxyl radical footprinting. Science 279:1940‐1943.
   Tinoco, I. Jr. 1959. Hypochromism in polynucleotides. J. Am. Chem. Soc. 82:4785‐4790.
   Zarrinkar, P.P. and Williamson, J.R. 1994. Kinetic intermediates in RNA folding. Science 265:918‐924.
Key References
   Banerjee and Turner 1995. See above.
  Presents the kinetics of folding of the T. thermophila group I ribozyme as determined by chemical modification with kethoxal.
   Ehresmann, et al; 1987. See above.
  Reviews the activity and use of many popular chemical modification reagents.
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