Properties of an altered RNA polymerase II activity from an α-amanitin-resistant mouse cell line

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Abstract
.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 differed from the parental cell line in that approximately half the activity was resistant to 0.5 .mu.g/ml .alpha.-amanitin, while the parental enzyme was 50% inhibited at 0.005 .mu.g/mL. Enzymes from A169b and the parental line were purified free of polymerase III and their properties compared. The 2 preparations were identical in their apparent affinities for the 4 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 KI for the parental enzyme was 3.5 .times. 10-9 M; plots of 1/V [velocity] vs. .alpha.-amanitin concentration gave a biphasic curve with A169b enzyme. The 2 apparent KI values were 4.1 .times. 10-9 and 2.1 .times. 10-6 M. The enzyme from A169b showed a 2-fold higher activity on poly[d(AT)] as template, compared to native DNA, than that of the parental enzyme. Other template preferences might be affected, but differences were marginal. These results indicated 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.