Incorporation of [³H]UMP into yeast spheroplasts in a hypotonic solution

Incorporation of radioactive UMP ([³H]-uridine-5′ monophosphate) into RNA was examined in spheroplasts of Saccharomyces cerevisiae in a hypotonic solution. The reaction, which also takes place in cells lacking mitochondrial DNA, is inhibited by ethidium bromide (EB) (250 μg/ml), requires divalent ions, and is only slightly affected by 50 μg/ml of α-amanitin, an inhibitor of RNA polymerase II. This latter result can be explained by the following hypotheses. (1) RNA polymerase II has low activity under those conditions. (2) α-Amanitin inhibits RNA synthesis only at the level of initiation of transcription, and insensitivity of [³H]UMP incorporation to the drug results from the lack of initiation in our experimental system. (3) In contrast to its in vitro state, RNA polymerase II in vivo may be resistant to α-amanitin, perhaps due to factors bound to the enzyme.