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

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 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.

Localization of an alpha-amanitin resistance mutation in the gene encoding the largest subunit of mouse RNA polymerase II.

“…Surviving colonies were ex-VOL. 7,1987 588 BARTOLOMEI AND CORDEN panded, and their RNA polymerase activities were assayed (51) to determine the level of resistance. Cell lysates were assayed in the presence of increasing concentrations of a-amanitin, and the activity was expressed as a fraction of the counts incorporated when no amanitin was present.…”

“…7,1987 on April 4, 2019 by guest http://mcb.asm.org/ 592 BARTOLOMEI AND CORDEN structure and function. First, the study of this mutation and other a-amanitin resistance mutations will elucidate the mechanism by which a-amanitin binds and inhibits RNA polymerase.…”

“…a-Amanitin binds to RNA polymerase II with an equilibrium association constant of approximately 1010 M-1 and inhibits transcription by blocking elongation of the enzyme after the formation of a phosphodiester bond (13,54). The binding of a single toxin molecule to RNA polymerase II is sufficient to inhibit transcription (13).Mutations that give rise to an a-amanitin-resistant phenotype have been described for cell lines from several species (7,11,29,50,51). In these cell lines, the mutation conferring a-amanitin resistance renders the enzyme 5 to 1,000 times more resistant to a-amanitin.…”

“…Mutations that give rise to an a-amanitin-resistant phenotype have been described for cell lines from several species (7,11,29,50,51). In these cell lines, the mutation conferring a-amanitin resistance renders the enzyme 5 to 1,000 times more resistant to a-amanitin.…”

Transcription of chick genes by mammalian RNA polymerase II in chick erythrocyte‐mammalian cell heterokaryons

“…Alpha amanitin resistant variants have been isolated from Chinese hamster ovary cells (Chan et al, 1972;Ingles et al, 1976;Ingles, 1978;Lobban et al, 1976), murine lymphoblastoid cells (Bryant et al, 1977), baby hamster kidney (BHK) cells (Amati et al, 1975), and from rat L6 myoblasts (Somers et al, 1975;Crerar et al, 1977a,b). The basis for resistance has been related to an altered RNA polymcrasc I1 molecule.…”