Amanitin binding to RNA polymerase II in α-amanitin-resistant rat myoblast mutants

Abstract

A series of independent α-amanitin-resistant (Ama R ) mutants have been isolated from diploid and tetraploid strains of L6 rat myoblast cells. With one exception, these mutants contain both α-amanitin-sensitive wild-type and α-amanitin-resistant mutant RNA polymerase II activities. The relative resistance of the mutant enzymes in extracts of the different Ama R isolates, determined by measuring the inhibition constant, K 1 , for α-amanitin, parallels their relative resistance to growth inhibition by the toxin. The binding affinities of the different mutant forms of RNA polymerase II for γ-amanitin, determined by measuring the equilibrium dissociation constant, K d , for γ-[ 3 H]amanitin§ bound to the enzyme, are also reduced relative to the wild-type enzyme, and the K d values are proportional to the K 1 values. Thus the spectrum of Ama R phenotypes in these mutant strains likely reflects the presence of mutations at different sites in the structural gene coding for the α-amanitin binding subunit of RNA polymerase II. No difference has been found between mutant and wild-type forms of the enzyme with respect to their turnover numbers, K m values for UTP, or thermal stabilities. Diploid mutants contain equal amounts of wild-type and mutant forms of RNA polymerase II. This indicates that they possess one wild-type and one mutant allele for the α-amanitin binding subunit of the enzyme, and that both alleles are expressed in a codominant fashion. Similarly, the tetraploid mutants possess three copies of the wild-type and one of the mutant allele. One tetraploid mutant has been isolated which is only slightly resistant to growth inhibition by α-amanitin and does not contain a detectably altered form of RNA polymerase II. This strain may be defective in α-amanitin transport.