Rapid Magnesium Chelation as a Method to Study Real‐Time Tertiary Unfolding of RNA

Emily J. Maglott1, Gary D. Glick1

1 University of Michigan, Ann Arbor, Michigan
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 11.7
DOI:  10.1002/0471142700.nc1107s06
Online Posting Date:  November, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit describes a method to measure the unfolding of RNA tertiary structure on a millisecond time scale. A stopped‐flow spectrophotometer is used to measure the rate of unfolding induced by the addition of EDTA to an RNA whose tertiary structure has been stabilized in the presence of magnesium ions. Using this methodology, rate constants for unfolding of tertiary or secondary structure can be obtained over a range of temperatures, and these values can be used to construct Arrhenius and Eyring plots, from which activation energy, Arrhenius pre‐exponential factor, and enthalpy and entropy of activation can be obtained. These data provide information about the energy of the transition state and the energy barriers between secondary and tertiary structure, which is necessary for predicting RNA tertiary structure from secondary structure.

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

Table of Contents

  • Basic Protocol 1: Measurement of Unfolding Rates of RNA Tertiary Structure
  • Support Protocol 1: Folding and Equilibration of RNA Samples
  • Support Protocol 2: Flow Dialysis of RNASamples
  • Support Protocol 3: Determination of Tertiary Unfolding Rates and Activation Parameters
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Measurement of Unfolding Rates of RNA Tertiary Structure

  Materials
  • EDTA buffer (see recipe) at EDTA concentration needed to chelate all Mg2+ in RNA sample
  • Mg2+ buffer (see recipe)
  • Folded RNA sample (see protocol 2)
  • Syringe filters, 0.22‐µm
  • 10‐mL and 3‐mL luer‐tip syringes (e.g., Fisher)
  • Parafilm
  • Stopped‐flow spectrophotometer (e.g., Applied Photophysics SX18.MV or equivalent instruments from Olis Instruments or Hi‐Tech Scientific)
  • Side‐arm Erlenmeyer flask
  • Additional reagents and equipment for folding RNA (see protocol 2) and analyzing kinetic traces to determine unfolding rates and activation parameters (see protocol 4)

Support Protocol 1: Folding and Equilibration of RNA Samples

  Materials
  • Dialyzed RNA sample (see protocol 3)
  • Mg2+ buffer (see recipe)
  • 75°C water bath
  • Spectrophotometer

Support Protocol 2: Flow Dialysis of RNASamples

  Materials
  • Mg2+ buffer (see recipe)
  • Argon source
  • Dry RNA sample of interest
  • Bottle‐top filtration apparatus containing a 0.22‐µm filter (e.g., Corning)
  • 10‐well microdialyzer (Spectrum)
  • Peristaltic pump
  • Cellulose ester membrane for use with 10‐well microdialyzer (Spectrum), MWCO 5000

Support Protocol 3: Determination of Tertiary Unfolding Rates and Activation Parameters

  Materials
  • Graphical analysis software (e.g., Kaleidagraph from Synergy Software)
  • Statistical analysis software (e.g., SAS from SAS Institute)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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.
   Bassi, G.S., Murchie, A.I.H., and Lilley, D.M.J. 1996. The ion‐induced folding of the hammerhead ribozyme: Core sequence changes that perturb folding into the active conformation. RNA 2:756‐768.
   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.
   Bevilacqua, P.C., Li, Y., and Turner, D.H. 1994. Fluorescence‐detected stopped flow with a pyrene labeled substrate reveals that guanosine facilitates docking of the 5′ cleavage site into a high free energy binding mode in theTetrahymena ribozyme. Biochemistry 33:11340‐11348.
   Butcher, S.E. and Burke, J.M. 1994. A photo‐cross‐linkable tertiary structure motif found in functionally distinct RNA molecules is essential for catalytic function of the hairpin ribozyme. Biochemistry 33:992‐994.
   Celander, D.W. and Cech, T.R. 1991. Visualizing the higher order folding of a catalytic RNA molecule. Science 251:401‐407.
   Chance, M.R., Sclavi, B., Woodson, S.A., and Brenowitz, M. 1997. Examining the conformational dynamics of macromolecules with time‐resolved synchrotron x‐ray footprinting. Structure 5:865‐869.
   Christian, E.L. and Yarus, M. 1993. Metal coordination sites that contribute to structure and catalysis in the group I intron from Tetrahymena. Biochemistry 32:4475‐4480.
   Cole, P.E. and Crothers, D.M. 1972. Conformational changes of transfer ribonucleic acid: Relaxation kinetics of the early melting transition of methionine transfer ribonucleic acid (Escherichia coli). Biochemistry 11:4368‐4374.
   Cole, P.E., Yang, S.K., and Crothers, D.M. 1972. Conformational changes of transfer ribonucleic acid: Equilibrium phase diagrams. Biochemistry 11:4358‐4368.
   Coutts, S.M., Riesner, D., Römer, R., Rabl, C.R., and Maass, G. 1975. Kinetics of conformational changes in tRNAPhe (yeast) as studied by the fluorescence of the Y‐base and of formycin substituted for the 3′‐terminal adenosine. Biophys. Chem. 3:275‐289.
   Crothers, D.M. and Cole, P.E. 1978. Conformational changes of tRNA. In TransferRNA (S. Altman, ed.) pp. 196‐247. MIT Press, Cambridge, Mass.
   Downs, W.D. and Cech, T.R. 1996. Kinetic pathway for folding of the Tetrahymena ribozyme revealed by three UV‐inducible crosslinks. RNA 2:718‐732.
   Draper, D.E., Xing, Y., and Laing, L.G. 1995. Thermodynamics of RNA unfolding: Stabilization of a ribosomal RNA tertiary structure by thiostrepton and ammonium ion. J. Mol. Biol. 249:231‐238.
   Emerick, V.L. and Woodson, S.A. 1994. Fingerprinting the folding of group I precursor RNA. Proc. Natl. Acad. Sci. U.S.A. 91:9675‐9679.
   Emerick, V.L., Pan, J., and Woodson, S.A. 1996. Analysis of rate‐determining conformational changes during self‐splicing of the Tetrahymena intron. Biochemistry 35:13469‐13477.
   Fersht, A. 1985. Enzyme Structure and Mechanism, 2nd Ed., p. 122 W.H. Freeman, New York.
   Jaeger, L., Westhof, E., and Michel, F. 1993. Monitoring of the cooperative unfolding of the sunY group I intron of bacteriophage T4. J. Mol. Biol. 234:331‐346.
   Kazakov, S. and Altman, S. 1991. Site‐specific cleavage by metal ion cofactors and inhibitors of M1 RNA, the catalytic subunit of RNase P fromEscherichia coli. Proc. Natl. Acad. Sci.U.S.A. 88:9193‐9197.
   Laing, L.G., Gluick, T.C., and Draper, D.E. 1994. Stabilization of RNA structure by Mg ions: Specific and non‐specific effects. J.Mol. Biol. 237:577‐587.
   LeCuyer, K.A. and Crothers, D.M. 1993. The Leptomonas collosoma spliced leader RNA can switch between two alternate structural forms. Biochemistry 32:5301‐5311.
   LeCuyer, K.A. and Crothers, D.M. 1994. Kinetics of an RNA conformational switch. Proc. Natl. Acad. Sci. U.S.A. 91:9373‐3377.
   Levy, J., Rialdi, G., and Biltonen, R. 1972. Thermodynamic studies of transfer ribonucleic acids. II. Characterization of the thermal unfolding of yeast phenylalanine‐specific transfer ribonucleic acid. Biochemistry 11:4138‐4144.
   Lu, M. and Draper, D.E. 1994. Bases defining an ammonium and magnesium ion‐dependent tertiary structure within the large subunit ribosomal RNA. J. Mol. Biol. 244:572‐585.
   Maglott, E.J. 1998. Structural and kinetic studies of tertiary folding of an unmodified transfer RNA. Ph.D. Thesis, University of Michigan.
   Maglott, E.J. and Glick, G.D. 1997. A new method to monitor the rate of conformational transitions in RNA. Nucl. Acids Res. 25:3297‐3301.
   Maglott, E.J., Deo, S.S., Pryzkorska, A., and Glick, G.D. 1998. Conformational transitions of an unmodified tRNA: Implications for RNA folding. Biochemistry 46:16349‐16359.
   Maglott, E.J., Goodwin, J.T., and Glick, G.D. 1999. Probing an RNA tertiary structure unfolding transition state. J. Am. Chem.Soc. 121:7461‐7492.
   Narlikar, G.J., and Herschlag, D. 1996. Isolation of a local tertiary folding transition in the context of a globally folded RNA. Nat. Struct. Biol. 3:701‐709.
   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.
   Pyle, A.M. 1993. Ribozymes: A distinct class of metalloenzymes. Science 261:709‐714.
   Qin, P.Z. and Pyle, A.M. 1997. Stopped‐flow fluorescence spectroscopy of a group II intron ribozyme reveals that domain 1 is an independent folding unit with a requirement for specific Mg2+ ions in the tertiary structure. Biochemistry 36:4718‐4730.
   Riesner, D., Römer, R., and Maass, G. 1970. Kinetic study of the three conformational transitions of alanine specific transfer RNA from yeast. Eur. J. Biochem. 15:85‐91.
   Riesner, D., Maass, G., Thiebe, R., Philippsen, P., and Zachau, H.G. 1973. The conformational transitions in yeast tRNAPhe as studied with tRNAPhe fragments. Eur. J. Biochem. 36:76‐88.
   Sclavi, B., Woodson, S., Sullivan, M., Chance, M.R., and Brenowitz, M. 1997. 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.
   Sclavi, B., Sullivan, M., Chance, M.R., Brenowitz, M., and Woodson, S. 1998. RNA folding at millisecond intervals by synchrotron hydroxyl radical footprinting. Science 279:1940‐1943.
   Szewczak, A.A. and Cech, T.R. 1997. An RNA internal loop acts as a hinge to facilitate ribozyme folding and catalysis. RNA 3:838‐849.
   Uhlenbeck, O.C. 1987. A small catalytic oligoribonucleotide. Nature 328:596‐601.
   Urbanke, C., Römer, R., and Maass, , G. 1973. The binding of ethidium bromide to different conformations of tRNA: Unfolding of tertiary structure. Eur. J. Biochem. 33:511‐516.
   Urbanke, C., Römer, R., and Maass, G. 1975. Tertiary structure of tRNAPhe (yeast): Kinetics and electrostatic repulsion. Eur. J. Biochem. 55:439‐444.
   Walpole, R.E. and Myers, R.H. 1978. Probability and Statistics for Engineers and Scientists 2nd ed. Macmillan, New York.
   Yang, S.K. and Crothers, D.M. 1942. Conformational changes of transfer ribonucleic acid: Comparison of the early melting transition of two tyrosine‐specific transfer ribonucleic acids. Biochemistry 11:4375‐4381.
   Zarrinkar, P.P. and Williamson, J.R. 1994. Kinetic intermediates in RNA folding. Science 265:918‐924.
   Zarrinkar, P.P., Wang, J., and Williamson, J.R. 1996. Slow folding kinetics of RNase P RNA. RNA 2:564‐573.
   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