Highly active template-specific RNA-dependent RNA polymerase from barley leaves infected with brome mosaic virus

Abstract

The extraction of a template-dependent and template-specific RNA-dependent RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from a eukaryotic source is described. The enzyme, extracted from barley leaves infected with brome mosaic virus (BMV), is capable of incorporating high levels of radioactivity into trichloroacetic acid-insoluble products. The purification procedure included solubilization with nonionic detergent and precipitation with polyethylene glycol. The enzyme was more than 50 times more active than was a comparable preparation from mock-inoculated leaves and was stimulated more than 15-fold by the addition of BMV RNA to the reaction. Other viral RNA templates were less than 25% as efficient as was BMV RNA in stimulating UMP incorporation; poly(A), tRNA, and mRNA gave little stimulation and rRNA was inactive. Autoradiographic analysis after electrophoretic separation of the radioactive products from reaction mixtures containing BMV RNA template revealed prominent bands that coelectrophoresed with replicative forms of BMV RNAs. When BMV RNA template was enriched in RNA3 or RNA4, larger proportions of the products were replicative forms of RNA3 or RNA4, respectively.