Probing RNA Structure and Metal‐Binding Sites Using Terbium(III) Footprinting

Dinari A. Harris1, Nils G. Walter1

1 University of Michigan, Ann Arbor, Michigan
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 6.8
DOI:  10.1002/0471142700.nc0608s13
Online Posting Date:  August, 2003
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The function of an RNA molecule is determined by its overall secondary and tertiary structure. The tertiary structure is facilitated and stabilized by the interaction with metal ions. The current chapter offers a detailed protocol on the use of the lanthanide metal ion terbium(III) as a powerful probe of RNA structure and metal‐binding properties. When incubating RNA with low (micromolar) concentrations of terbium(III), specific backbone scission by partially deprotonated aqueous terbium(III) complexes can be used to detect high‐affinity metal‐binding sites, while incubation with high (millimolar) terbium(III) concentrations cleaves the RNA backbone preferentially at structurally accessible regions, providing a footprint of the RNA secondary and tertiary structure.

Keywords: catalytic RNA; lanthanide ion; metal probing; RNA structure probing; sequencing

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • RNA, gel‐purified (unit 6.1), labeled at 5′ or 3′ end with 32P (conc. ≥50,000 cpm/µL; sp. act. ≥150,000 cpm/pmol)
  • Appropriate buffers to fold RNA (usually Tris, MES, and/or HEPES buffer of desired pH; see Critical Parameters for discussion of optimization)
  • 1 M MgCl 2 ( appendix 2A)
  • 100 mM TbCl 3 (see recipe)
  • 0.5 M EDTA, pH 8.0 ( appendix 2A)
  • 3 M sodium acetate ( appendix 2A)
  • 80% and 100% (v/v) ethanol
  • Urea loading buffer ( appendix 2A)
  • Heating block
  • Water bath at optimized incubation temperature (25° to 45°C)
  • Phosphor screens and phosphor imager with appropriate software (e.g., PhosphorImager Storm 840 with ImageQuant software; Molecular Dynamics)
  • Additional reagents and equipment for partial alkaline hydrolysis and RNase T1 digestion of RNA (unit 6.1, Support Protocol 3), and denaturing polyacrylamide gel electrophoresis ( appendix 3B)
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Literature Cited

Literature Cited
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   Ciesiolka, J., Marciniec, T., and Krzyzosiak, W. 1989. Probing the environment of lanthanide binding sites in yeast tRNA(Phe) by specific metal‐ion‐promoted cleavages. Eur. J. Biochem. 182:445‐450.
   Feig, A.L., Panek, M., Horrocks, W.D. Jr., and Uhlenbeck, O.C. 1999. Probing the binding of Tb(III) and Eu(III) to the hammerhead ribozyme using luminescence spectroscopy. Chem. Biol. 6:801‐810.
   Flynn‐Charlebois, A., Lee, N., and Suga, H. 2001. A single metal ion plays structural and chemical roles in an aminoacyl‐transferase ribozyme. Biochemistry 40:13623‐13632.
   Hargittai, M.R. and Musier‐Forsyth, K. 2000. Use of terbium as a probe of tRNA tertiary structure and folding. RNA 6:1672‐1680.
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   Kaye, N.M., Zahler, N.H., Christian, E.L., and Harris, M.E. 2002. Conservation of helical structure contributes to functional metal ion interactions in the catalytic domain of ribonuclease P RNA. J. Mol. Biol. 324:429‐442.
   Matsumura, K. and Komiyama, M. 1997. Enormously fast RNA hydrolysis by lanthanide(III) ions under physiological conditions: Eminent candidates for novel tools of biotechnology. J. Biochem. 122:387‐394.
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   Sigel, R.K., Vaidya, A., and Pyle, A.M. 2000. Metal ion binding sites in a group II intron core. Nat. Struct. Biol. 7:1111‐1116.
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   Walter, N.G., Yang, N., and Burke, J.M. 2000. Probing non‐selective cation binding in the hairpin ribozyme with Tb(III). J. Mol. Biol. 298:539‐555.
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