Thermal Methods for the Analysis of RNA Folding Pathways

David E. Draper1, Yury V. Bukhman1, Thomas C. Gluick1

1 Johns Hopkins University, Baltimore, Maryland
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 11.3
DOI:  10.1002/0471142700.nc1103s02
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Once a model of the secondary structure of an RNA has been deduced, thermal melting analysis can be used to determine whether the model accounts for all intramolecular interactions of the RNA, or whether noncanonical and tertiary interactions make the structure more stable than predicted, or link parts of the structure in unexpected ways. It is also useful to determine the pH, salt, and temperature ranges under which the RNA adopts a stably folded structure, or to analyze unfolding pathways. This unit discusses sample preparation, instrumentation, and theoretical background. It also provide a sample analysis of tRNA unfolding.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Experimental Considerations
  • Theoretical Background
  • Example Analysis: tRNA Unfolding
  • Experimental Tests of an Unfolding Pathway
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Albergo, D.D., Marky, L.A., Breslauer, K.J., and Turner, D.H. 1981. Thermodynamics of (dG‐dC)3 double‐helix formation in water and deuterium oxide. Biochemistry 20:1409‐1413.
   Chory, J. and Pollard, J.D. Jr. 2000. Separation of small DNA fragments by conventional gel electrophoresis. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 2.7.1‐2.7.8. John Wiley & Sons, New York.
   Cole, P.E., Yang, S.K., and Crothers, D.M. 1972. Conformational changes of transfer ribonucleic acid: Equilibrium phase diagrams. Biochemistry 11:4358‐4368.
   Conn, G.L., Gutell, R.R., and Draper, D.E. 1998. A functional ribosomal RNA tertiary structure involves a base triple interaction. Biochemistry 37:11980‐11988.
   Correll, C.C., Freeborn, B., Moore, P.B., and Steitz, T.A. 1997. Metals, motifs, and recognition in the crystal structure of a 5S rRNA domain. Cell 91:705‐711.
   Crothers, D.M., Cole, P.E., Hilbers, C.W., and Shulman, R.G. 1974. The molecular mechanism of thermal unfolding of Escherichia coli formylmethionine transfer RNA. J. Mol. Biol. 87:63‐88.
   Draper, D.E. and Gluick, T.C. 1995. Melting studies of RNA unfolding and RNA‐ligand interactions. Methods Enzymol. 250:281‐305.
   Draper, D.E., White, S.A., and Kean, J.M. 1988. Preparation of specific ribosomal RNA fragments. Methods Enzymol. 164:221‐237.
   Ehresmann, C., Baudin, F., Mougel, M., Romby, P., Ebel, J.‐P., and Ehresmann, B. 1987. Probing the structure of RNAs in solution. Nucl. Acids Res. 15:9109‐9128.
   Freier, S.M., Burger, B.J., Alkema, D., Neilson, T., and Turner, D.H. 1983. Effects of 3′ dangling end stacking on the stability of GGCC and CCGG double helices. Biochemistry 22:6198‐6202.
   Gluick, T.C. and Draper, D.E. 1997. Folding of an mRNA pseudoknot required for stop codon readthrough: Effects of mono‐ and divalent ions on stability. Biochemistry 36:16173‐16186.
   Gluick, T.C., Gerstner, R.G., and Draper, D.E. 1997. Effects of Mg2+, K+, and H+ on an equilibrium between alternative conformations of an RNA pseudoknot. J. Mol. Biol. 270:451‐463.
   Good, N.E. and Izawa, S. 1972. Hydrogen ion buffers. Methods Enzymol. 24:53‐68.
   Goodwin, J.T., Osborne, S.E., Scholle, E.J., and Glick, G.D. 1996. Design, synthesis, and analysis of yeast tRNAPhe analogs possessing intra‐ and inter‐helical disulfide cross‐links. J. Am. Chem. Soc. 118:5207‐5215.
   Gurevich, V.V. 1996. Use of bacteriophage RNA polymerase in RNA synthesis. Methods Enzymol. 275:382‐397.
   Gutell, R.R., Power, A., Hertz, G.Z., Putz, E.J., and Stormo, G.D. 1992. Identifying constraints on the higher‐order structure of RNA: Continued development and application of comparative sequence analysis methods. Nucl. Acids Res. 20:5785‐5795.
   Heus, H.A. and Pardi, R. 1991. Structural features that give rise to the unusual stability of RNA hairpins containing GNRA loops. Science 252:191‐194.
   Hou, Y.M., Sterner, T., and Jansen, M. 1995. Permutation of a pair of tertiary nucleotides in a transfer RNA. Biochemistry 34:2978‐2984.
   Laing, L.G. and Draper, D.E. 1994. Thermodynamics of RNA folding in a highly conserved ribosomal RNA domain. J. Mol. Biol. 237:560‐576.
   Petersheim, M. and Turner, D.H. 1983. Base‐stacking and base‐pairing contributions to helix stability: Thermodynamics of double‐helix formation with CCGG, CCGGp,CCGGAp, CCGGUp, and ACCGGUp. Biochemistry 22:265‐263.
   Press, W.H., Teukolsky, S.A., Vetterling, W.T., and Flannery, B.P. 1992. Numerical Recipes in C. Cambridge University Press, Cambridge.
   Puglisi, J.D. and Tinoco, I. Jr. 1989. Absorbance melting curves of RNA. Methods Enzymol. 180:304.
   Römer, R. and Hach, R. 1975. tRNA conformation and magnesium binding: A study of yeast phenylalanine‐specific tRNA by a fluorescent indicator and differential melting curves. Eur. J. Biochem. 55:271‐284.
   Scaringe, S.A., Francklyn, C., and Usman, N. 1990. Chemical synthesis of biologically active oligoribonucleotides using beta‐cyanoethyl protected ribonucleoside phosphoramidites. Nucl. Acids Res. 18:5433‐441.
   Serra, M.J. and Turner, D.H. 1995. Predicting thermodynamic properties of RNA. Methods Enzymol. 259:242‐261.
   Stein, A. and Crothers, D.M. 1976. Conformational changes of transfer RNA: The role of magnesium(II). Biochemistry 15:160‐167.
   Tang, C.K. and Draper, D.E. 1989. An unusual mRNA pseudoknot structure is recognized by a protein translational repressor. Cell 57:531‐536.
   Williams, A.P., Longfellow, C.E., Freier, S.M., Kierzek, R., and Turner, D.H. 1985. Laser temperature‐jump, spectroscopic, and thermodynamic study of salt effects on duplex formation by dGCATGC. Biochemistry 28:4283‐4291.
   Wyman, J. and Gill, S. 1990. Binding and Linkage. Functional Chemistry of Biological Macromolecules. University Science Books, Mill Valley, Calif.
   Zuker, M. 1989. On finding all suboptimal foldings of an RNA molecule. Science 244:48‐52.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library