DNA-dependent RNA polymerases from Acanthamoeba castellanii: properties and levels of activity during encystment

Three DNA-dependent RNA polymerases have been isolated and partially purified from trophozoites of Acanthamoeba castellanii. Separated by DEAE-Sephadex chromatography, they have been designated polymerases, I, IIa and IIB according to their alpha-amanitin sensitivity and kinetic properties. I is completely insensitive to alpha-amanitin. IIa and IIb are sensitive to low concentrations (0.1 mug/ml) of alpha-amanitin; however, in order to achieve 100% inhibition much higher concentrations (130 mug/ml) are needed. Both I and II (a or b) have rather broad ionic strength optima (0.06--0.10 M (NH4)2SO4). All three prefer denatured over native DNA (I, 4:1; II, 2:1). Polymerase I utilizes magnesium better than manganese as divalent cation whereas II prefers manganese. When Acanthamoeba is transferred to a medium lacking nutrients, the cells undergo a synchronous differentiation resulting in cyst formation. In general agreement with the decrease in the rate of synthesis of its product (rRNA), the amount of polymerase I decreases relative to the amanitin sensitive polymerase(s). However, the absolute amount of polymerase I does not change. Rather, the levels of the amanitin sensitive enzymes increase during the first 10 h of encystment. Since the overall RNA synthesis rate decreases, these results suggest that the transcription rate is not controlled by specific enzyme levels alone.