In Vitro Translation of Plant Viral RNA

Karen S. Browning1, Laura Mayberry1

1 University of Texas at Austin, Austin, Texas
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 16K.1
DOI:  10.1002/9780471729259.mc16k01s01
Online Posting Date:  June, 2006
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Abstract

This unit describes the preparation of a wheat germ extract that provides all the soluble components of the plant translational machinery. Many RNA plant viruses have positive‐strand genomes and the viral RNA serves as messenger RNA (mRNA). The preparation of mRNA by in vitro transcription is also described. The translation assay requires optimization of the amount of wheat germ extract, level of mRNA, and the concentration of Mg2+ and K+ for each mRNA. The translational efficiency of RNAs or mutants may be compared (e.g., capped versus uncapped RNAs to measure cap‐independent translation) or the amount/size of the protein product may be determined.

Keywords: translation; protein synthesis; wheat germ; plant virus; mRNA

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Wheat Germ Extract (S30)
  • Support Protocol 1: Preparation of A 2‐Liter G‐25 Column
  • Basic Protocol 2: In Vitro Transcription of Plant Viral RNAs
  • Basic Protocol 3: Protein Synthesis Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Wheat Germ Extract (S30)

  Materials
  • Wheat germ column elution buffer (see recipe)
  • Wheat germ extraction buffer (see recipe)
  • 14.3 M 2‐mercaptoethanol (2‐ME; Sigma)
  • 2‐ liter Sephadex G‐25 column (see protocol 2)
  • Wheat germ (e.g., Randolph & James Flax Mills), chilled: store up to 6 months at −20°C
  • Soybean trypsin inhibitor (STI; Sigma)
  • 50 mM PMSF (see recipe)
  • Powdered dry ice or dry ice/isopropanol bath or liquid nitrogen
  • Blender, chilled
  • Slender kitchen spatula (optional)
  • 50‐ml centrifuge tubes, chilled
  • High‐speed centrifuge (capacity up to ∼250 ml)
  • Cheese cloth

Support Protocol 1: Preparation of A 2‐Liter G‐25 Column

  Materials
  • Sephadex G‐25 slurry (Sigma, cat. no. G‐25‐150)
  • Wheat germ column wash buffer (see recipe)
  • 2.35‐liter (50 × 1200–mm) ACE glass column (e.g., Ace Glass, cat. no. 5820‐58; http://www.aceglass.com)
  • Top and bottom Luer adaptors (Ace Glass, cat. no. 5837‐21) with appropriate tubing and stopcock
  • Buffer reservoir (e.g., 6‐liter flat‐style carboy) with appropriate tubing

Basic Protocol 2: In Vitro Transcription of Plant Viral RNAs

  Materials
  • Plasmid DNA
  • Restriction enzyme and buffer appropriate for linearizing plasmid DNA
  • Sterile water
  • Ambion MegaScript (uncapped RNA) or MessageMachine (capped RNA) T7 or SP6 transcription kit or equivalent
  • Sephadex G100 slurry (see recipe)
  • RNA column elution buffer (see recipe)
  • ACE‐saturated phenol (see recipe)
  • Chloroform
  • 80% and 100% ethanol, −20°C
  • 20‐ml plastic columns and frits (BioRad)
  • Stopcocks (Baxter), sterile
  • 1.5‐ml microcentrifuge tubes, sterile
  • Tabletop centrifuge
  • 13‐ml sterile centrifuge tubes
  • Additional reagents and equipment for purifying DNA (Moore and Dowhan, ) and separating RNA on a urea gel (Ellington and Pollard, )
NOTE: All reagents must be prepared with ultrapure, sterile water to prevent nuclease contamination. Autoclave all buffers, resin, columns, tubes, and other reagents and equipment to prevent nuclease contamination. Gloves should be worn and all efforts made to prevent nuclease contamination from personnel or air sources.

Basic Protocol 3: Protein Synthesis Assay

  Materials
  • Supermix (–Leu or –Met; see recipe)
  • 1 M potassium acetate (see recipe)
  • 0.1 M magnesium acetate (see recipe)
  • 0.6 mM [14C]leucine (∼170 µCi/µmol, ∼200 cpm/pmol) or 0.6 mM [35S]methionine (∼1300 µCi/µmol, ∼ 2000 cpm/pmol)
  • mRNA
  • Wheat germ column elution buffer (see recipe)
  • S30 ( protocol 1)
  • 5% and 1% TCA (see recipe; for filter binding assays) in acid‐resistant squeeze bottles
  • Scintillation fluid (e.g., Econo‐Safe, Research Products International; for filter binding analysis)
  • SDS sample buffer ( appendix 2A; for SDS‐PAGE analysis)
  • 12 × 75–mm glass test tubes (for filter binding assay) or 1.5‐ml microcentrifuge tubes (for SDS‐PAGE analysis)
  • 27°C water bath
  • 90°C heating block (for filter binding analysis)
  • Glass fiber filters (Schleicher and Schuell; for filter binding analysis): wet with 5% TCA prior to use
  • Vacuum filtration manifold (for filter binding analysis)
  • 100°C drying oven (for filter binding analysis)
  • Scintillation vials (for filter binding analysis)
  • Additional reagents and equipment for SDS‐PAGE (Gallagher, )
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Literature Cited

Literature Cited
   Ellington, A. and Pollard, J.D. Jr. 1998. Purification of oligonucleotides using denaturing polyacrylamide 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. 2.12.1‐2.12.7. John Wiley & Sons, Hoboken, N.J.
   Gallagher, S.R. 1999. One dimensional SDS gel electrophoresis of proteins. 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. 10.2A.1‐10.2A.34. John Wiley & Sons, Hoboken, N.J.
   Lax, S.R., Lauer, S.J., Browning, K.S., and Ravel, J.M. 1986. Purification and properties of protein synthesis initiation and elongation factors from wheat germ. Methods Enzymol. 118:109‐128.
   Madin, K., Sawasaki, T., Ogasawara, T., and Endo, Y. 2000. A highly efficient and robust cell‐free protein synthesis system prepared from wheat embryos: Plants apparently contain a suicide system directed at ribosomes. Proc. Natl. Acad. Sci. U.S.A. 97:559‐564.
   Marcu, K. and Dudock, B. 1974. Characterization of a highly efficient protein synthesizing system derived from commercial wheat germ. Nucl. Acids Res. 1:1385‐1390.
   Marcus, A., Efron, D., and Weeks, D.P. 1974. The wheat embryo cell‐free system. Methods Enzymol. 30:749‐754.
   Moore, D. and Dowhan, D. 2002. Purification and concentration of DNA from aqueous solution. 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. 2.1.1‐2.1.10. John Wiley & Sons, Hoboken, N.J.
   Pokrovskaya, I.D. and Gurevich, V.V. 1994. In vitro transcription: Preparative RNA yields in analytical scale reactions. Anal. Biochem. 220:420‐423.
   Roberts, B.E. and Paterson, B.M. 1973. Efficient translation of tobacco mosaic virus RNA and rabbit globin 9S RNA in a cell‐free system from commercial wheat germ. Proc. Natl. Acad. Sci. U.S.A. 70:2330‐2334.
   Struhl, K. 2003. Synthesizing proteins in vitro by transcription and translation of cloned genes. 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. 10.17.1‐10.17.5. John Wiley & Sons, Hoboken, N.J.
   Walthall, B.J., Spremulli, L.L., Lax, S.R., and Ravel, J.M. 1979. Isolation and purification of protein synthesis initiation factors from wheat germ. Methods Enzymol. 60:193‐204.
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