title: Prolonged α-amanitin treatment of cells for studying mutated polymerases causes degradation of DSIF160 and other proteins.
abstract: A useful method for studying the function of the mammalian RNA polymerase II takes advantage of the extreme sensitivity of its largest subunit, Rpb1, to α-amanitin. Mutations of interest are introduced into an α-amanitin-resistant version of Rpb1, which is then expressed ectopically in cells. The phenotypes of these cells are then examined after inhibiting the endogenous wild-type polymerase with α-amanitin. Here, we show that cells that are enabled to grow in α-amanitin by expression of an α-amanitin-resistant Rpb1 exhibit changes in cell physiology that can lead to misleading experimental outcomes. The changes we have characterized include the accelerated degradation of some proteins, such as DSIF160, and the reduced rate of synthesis of others. In one series of experiments, we examined an α-amanitin-resistant construct, with a mutant C-terminal domain (CTD), that was unable to direct poly(A)-dependent transcription termination in cells growing in α-amanitin. The potential interpretation that the termination defect in this construct is due to the mutation in the CTD was rejected when the construct was found to be termination-competent in cells grown in the absence of α-amanitin. Instead, it appears that certain termination factors become limiting when the cells are grown in α-amanitin, presumably due to the α-amanitin-induced degradation we have characterized and/or to the inadequate transcription of certain genes by the α-amanitin-resistant Rpb1-containing polymerase.
authors.affiliation: Department of Chemistry and Biochemistry, and the Molecular Biology Institute, University of California at Los Angeles, Los Angeles, CA 90095-1569, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Tsao
authors.forename: Noh Jin
authors.initials: NJ
authors.lastname: Park
authors.forename: Anita
authors.initials: A
authors.lastname: Nag
authors.forename: Harold G
authors.initials: HG
authors.lastname: Martinson
mesh_terms: D053959:Alpha-Amanitin / Q000494:pharmacology*; D000818:Animals; D019556:COS Cells; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D009154:Mutation; D010641:Phenotype; D059748:Proteolysis / Q000187:drug effects; D012319:RNA Polymerase II / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D053959:Alpha-Amanitin; D012319:RNA Polymerase II