In vitro translation of plant viral RNA.
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 Mg(2+) 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.
Browning
Karen S
KS
University of Texas at Austin, Austin, Texas.
Mayberry
Laura
L
University of Texas at Austin, Austin, Texas.
D010936:Plant Extracts / Q000235:genetics / Q000378:metabolism; D010942:Plant Viruses / Q000235:genetics* / Q000378:metabolism; D014176:Protein Biosynthesis; D012328:RNA Viruses / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D014908:Triticum / Q000235:genetics / Q000378:metabolism
D010936:Plant Extracts; D012367:RNA, Viral
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.