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.degree.-24.degree. C. Incubation of reaction mixtures at higher temperatures (30.degree.-36.degree. C) resulted in an increase of CEV linear molecules and the recovery of incomplete or nicked newly synthesized RNA species. Although the Mg2+ optimum (2.5-5 mM) for CEV synthesis was lower than that for total [32P]CMP incorporation (10 mM), the response to Mn2+ ion was distinctly different. Whereas maximum total activity was observed in 1 mM Mn2+ with a pronounced reduction (80%) in 5 mM Mn2+, CEV synthesis was maintained in 1-15 mM Mn2+. Inhibition of .alpha.-amanitin-sensitive CEV synthesis in 200 mM (NH4)2SO4 resembles the reaction of RNA polymerase II on a free nucleic acid template. Detection of trace levels of .alpha.-amanitin-resistant CEV synthesis activity inhibited by low (NH4)2SO4 concentrations (25 mM) suggests the possible involvement of RNA polymerase I- and/or III-like activity.