Properties of an altered RNA polymerase II activity from an alpha-amanitin-resistant mouse cell line.
alpha-Amanitin-resistant clones were selected in the mouse lymphoblastoid cell line L5178Y. One resistant clone, named A169b, was recloned and the properties of its DNA-dependent RNA polymerases were examined. The RNA polymerase II activity from A169b differs from the parental cell line in that approximately half the activity is resistant to 0.5 microgram/mL alpha-amanitin, while the parental enzyme is 50% inhibited at 0.005 microgram/mL. The enzymes from A169b and the parental line were purified free of polymerase III and their properties compared. The two preparations were identical in their apparent affinities for the four nucleoside triphosphates, in their salt and divalent cation preferences, and in their preference for denatured over native DNA. They differed in their response to alpha-amanitin. The apparent K1 for the parental enzyme was 3.5 X 10(-9) M; plots of 1/V vs. alpha-amanitin concentration gave a biphasic curve with A169b enzyme. The two apparent K1 values were 4.1 X 10(-9) and 2.1 X 10(-6) M. In addition, the enzyme from A169b showed a twofold higher activity on poly [d(AT)] as template, compared to native DNA, than that of the parental enzyme. Other template preferences may be affected, but differences were marginal. These results indicate that mutation to alpha-amanitin resistance may alter other enzymatic parameters; such mutations may be helpful in elucidating structure-function relationships in these complex enzymes.
D000546:Amanitins / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002999:Clone Cells; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004351:Drug Resistance; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009154:Mutation; D012319:RNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism*; D012320:RNA Polymerase III / Q000302:isolation & purification / Q000378:metabolism; D013698:Templates, Genetic
D000546:Amanitins; D008345:Manganese; D012319:RNA Polymerase II; D012321:DNA-Directed RNA Polymerases; D012320:RNA Polymerase III; D008274:Magnesium