Optimal conditions for cell-free synthesis of citrus exocortis viroid and the question of specificity of RNA polymerase activity

Abstract

Cell-free synthesis of citrus exocortis viroid (CEV) in nuclei-rich preparations from infected Gynura aurantiaca was optimum at 18-24 degrees C. Incubation of reaction mixtures at higher temperatures (30-36 degrees C) resulted in an increase of CEV linear molecules and the recovery of incomplete or nicked newly synthesized RNA species. Although the Mg(2+) optimum (2.5-5 mM) for CEV synthesis was lower than that for total [(32)P]CMP incorporation (10 mM), the response to Mn(2+) ion was distinctly different. Whereas maximum total activity was observed in 1 mM Mn(2+) with a pronounced reduction (80%) in 5 mM Mn(2+), CEV synthesis was maintained in 1-15 mM Mn(2+). Inhibition of alpha-amanitin-sensitive CEV synthesis in 200 mM (NH(4))(2)SO(4) resembles the reaction of RNA polymerase II on a free nucleic acid template. However, detection of trace levels of alpha-amanitin-resistant CEV synthesis activity inhibited by low (NH(4))(2)SO(4) concentrations (25 mM) suggests the possible involvement of RNA polymerase I- and/or III-like activity.