CHARACTERIZATION OF A VIRION-ASSOCIATED RNA POLYMERASE FROM KILLER YEAST

Abstract

Publisher Summary This chapter discusses characterization of a virion-associated RNA polymerase from killer yeast. Killer strains of Saccharomyces cerevisiae contain a 1.7 × 106 dalton double-stranded RNA plasmid; such strains secrete a protein toxin that kills strains lacking the plasmid—sensitives—but to which the killers are immune. The killer plasmid and two larger double-stranded RNA species are present in intracellular virus-like particles. Yeast strains in the stationary phase of growth with ethanol as the carbon source contain a DNA-independent RNA polymerase activity that copurifies with these virions. This activity requires all four ribonucleotide triphosphates and catalyzes the incorporation of all four into single-stranded RNA based on nuclease sensitivity. The reaction requires Mg++, is sensitive to sulfhydryl reagents and high levels of monovalent cation, and is insensitive to DNase (0.5 mg/ml), α-amanitin (100 μg/ml), actinomycin D (100 μg/ml), and thiolutin (50 μg/ml). Pyrophosphate (5 mM) completely blocks incorporation, which is also inhibited by the intercalating agent ethidium bromide.