title: Alpha-amanitin blocks translocation by human RNA polymerase II.
abstract: Our laboratory has developed methods for transient state kinetic analysis of human RNA polymerase II elongation. In these studies, multiple conformations of the RNA polymerase II elongation complex were revealed by their distinct elongation potential and differing dependence on nucleoside triphosphate substrate. Among these are conformations that appear to correspond to different translocation states of the DNA template and RNA-DNA hybrid. Using alpha-amanitin as a dynamic probe of the RNA polymerase II mechanism, we show that the most highly poised conformation of the elongation complex, which we interpreted previously as the posttranslocated state, is selectively resistant to inhibition with alpha-amanitin. Because initially resistant elongation complexes form only a single phosphodiester bond before being rendered inactive in the following bond addition cycle, alpha-amanitin inhibits elongation at each translocation step.
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319, USA.
authors.forename: Xue Q
authors.initials: XQ
authors.lastname: Gong
authors.forename: Yuri A
authors.initials: YA
authors.lastname: Nedialkov
authors.forename: Zachary F
authors.initials: ZF
authors.lastname: Burton
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000546:Amanitins / Q000494:pharmacology*; D001483:Base Sequence; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D021381:Protein Transport; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013698:Templates, Genetic
chemical_list: D000546:Amanitins; D004791:Enzyme Inhibitors; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D012319:RNA Polymerase II