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.