Probing RNA Structure by Lead Cleavage

Tao Pan1

1 University of Chicago, Chicago, Illinois
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 6.3
DOI:  10.1002/0471142700.nc0603s00
Online Posting Date:  May, 2001
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Abstract

Lead cleavage causes a transesterification reaction that breaks the 5′,3′‐phosphodiester backbone of RNA, leaving a 2′,3′‐cyclic phosphate and a 5′‐hydroxyl. Since the efficiency of the reaction at the 2′‐hydroxyl is related to steric and chemical constraints on particular 2′‐hydroxyls embedded in the RNA, this reaction can be used to examine the structure of individual nucleotides within an RNA molecule. It is a sensitive probe of tertiary RNA structure, provided that Pb2+‐binding sites are created in the tertiary structure.

     
 
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Table of Contents

  • Basic Protocol 1: Probing the RNA Structure by Pb2+ Cleavage
  • Support Protocol 1: 5′ 32P‐Labeling of RNA Including Dephosphorylation
  • Support Protocol 2: 3′ 32P Labeling of RNA Using T4 RNA Ligase and [32P]pCp
  • Support Protocol 3: Optimization of RNA Renaturation
  • Support Protocol 4: Partial Alkaline Hydrolysis and Nuclease T1 Digestion
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Probing the RNA Structure by Pb2+ Cleavage

  Materials
  • 5′ or 3′ 32P‐labeled RNA in water (see Support Protocols protocol 21 and protocol 32)
  • 0.3 M buffer (see Critical Parameters)
  • Urea loading buffer ( appendix 2A)
  • Pb(OAc) 2 stock solution prepared at 10× desired final concentration in reaction (see recipe for 10 mM solution)
  • Heating block
  • 3‐mm filter paper (Whatman)
  • Phosphorimager with appropriate software and phosphor screens
  • Additional reagents and equipment for RNA renaturation (see protocol 4), partial alkaline hydrolysis and T1 nuclease digestion (see protocol 5), and denaturing polyacrylamide gel electrophoresis (see appendix 3B)

Support Protocol 1: 5′ 32P‐Labeling of RNA Including Dephosphorylation

  Materials
  • RNA sample in water
  • 1 M Tris⋅Cl, pH 8.0 ( appendix 2A)
  • 1 U/µL calf‐intestine alkaline phosphatase
  • Soaking buffer: 50 mM potassium acetate ( appendix 2A)/200 mM KCl, pH ∼7
  • 1:1 (v/v) phenol/chloroform ( appendix 2A)
  • Ethanol
  • 10× T4 polynucleotide kinase buffer (see recipe)
  • [γ‐32P]ATP (use highest activity available)
  • T4 polynucleotide kinase
  • Urea loading buffer (see appendix 2A)
  • Polyacrylamide gel containing 8 M urea
  • Sodium acetate, pH 5.2 ( appendix 2A)
  • Micropipettor
  • Speedvac evaporator (e.g., Savant)
  • X‐ray film
  • RNase‐free surgical blade
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (see appendix 3B)
NOTE: If phosphatase treatment is not needed, proceed directly to step .NOTE: All microcentrifugations are performed at full speed.

Support Protocol 2: 3′ 32P Labeling of RNA Using T4 RNA Ligase and [32P]pCp

  Materials
  • 10× T4 polynucleotide kinase buffer (see recipe)
  • [γ‐32P]ATP
  • 3′ cytosine monophosphate (3′ Cp)
  • T4 polynucleotide kinase
  • Dimethyl sulfoxide (DMSO)
  • 100 µM nonradioactive ATP
  • T4 RNA ligase
  • RNA sample in water
  • Soaking buffer: 50 mM potassium acetate ( appendix 2A)/200 mM KCl, pH ∼7
  • Urea loading buffer ( appendix 2A)
  • Additional reagents and equipment for purification of ligated RNA (see protocol 2)
NOTE: If commercial 5′ [32P]pCp (e.g., New England Nuclear) is used, proceed directly to step . Depending on the concentration supplied, use a quantity sufficient to provide the same molar ratio as achieved with [γ‐32P]ATP.

Support Protocol 3: Optimization of RNA Renaturation

  Materials
  • RNA sample in water
  • MES, MOPS, or HEPES buffer
  • MgCl 2 stock solution , prepared at 10× desired final concentration in reaction ( appendix 2A for 1 M stock solution)
  • Heating block
  • Additional materials for nondenaturing polyacrylamide gel electrophoresis (see e.g.,CPMB UNIT )

Support Protocol 4: Partial Alkaline Hydrolysis and Nuclease T1 Digestion

  Materials
  • 5′‐ or 3′‐end‐labeled RNA (≥200,000 cpm; see Support Protocols protocol 21 and protocol 32)
  • 1 mg/mL E. coli tRNA mixture (Sigma), dissolved in water
  • 5× alkaline hydrolysis (AH) buffer: 5 mM glycine/2 mM MgSO 4, pH 9.5
  • 1 U/µL ribonuclease T1, diluted in water from a 100 U/µl stock (store up to ∼1 month at −20°C)
  • Urea loading buffer ( appendix 2A)
  • 100°C water bath
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Literature Cited

Literature Cited
   Behlen, L.S., Sampson, J.R., DiRenzo, A.B., and Uhlenbeck, C. 1990. Lead‐catalyzed cleavage of yeast tRNAPhe mutant. Biochemistry 29:2515‐2523.
   Brown, R., Dewan, J., and Klug, A. 1985. Crystallographic and biochemical investigation of the lead(II)‐catalyzed hydrolysis of yeast phenylalanine tRNA. Biochemistry 24:4785‐4801.
   Hertel, K.J., Herschlag, D., and Uhlenbeck, O.C. 1994. A kinetic and thermodynamic framework for the hammerhead ribozyme reaction. Biochemistry 33:3374‐3385.
   Kragten, J. 1978. Atlas of Metal‐Ligand Equilibria in Aqueous Solution. Halsted Press, Chichester, UK.
   Pan, T. and Uhlenbeck, O.C. 1992. A small metalloribozyme with a two‐step mechanism. Nature 358:560‐563.
   Pan, T., Dichtl, B., and Uhlenbeck, O.C. 1994. Properties of an in vitro selected Pb2+ cleavage motif. Biochemistry 33:9561‐9565.
   Uhlenbeck, O.C. 1995. Keeping RNA happy. RNA 1:4‐6.
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