title: Millisecond phase kinetic analysis of elongation catalyzed by human, yeast, and Escherichia coli RNA polymerase.
abstract: Strategies for assembly and analysis of human, yeast, and bacterial RNA polymerase elongation complexes are described, and methods are shown for millisecond phase kinetic analyses of elongation using rapid chemical quench flow. Human, yeast, and bacterial RNA polymerases function very similarly in NTP-Mg2+ commitment and phosphodiester bond formation. A "running start, two-bond, double-quench" protocol is described and its advantages discussed. These studies provide information about stable NTP-Mg2+ loading, phosphodiester bond synthesis, the processive transition between bonds, and sequence-specific effects on transcription elongation dynamics.
authors.affiliation: Gene Regulation and Chromosome Biology Laboratory, National Cancer Institute-Frederick, Bldg. 539, Room 222, Frederick, MD 21702-1201, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Kireeva
authors.forename: Yuri A
authors.initials: YA
authors.lastname: Nedialkov
authors.forename: Xue Qian
authors.initials: XQ
authors.lastname: Gong
authors.forename: Chunfen
authors.initials: C
authors.lastname: Zhang
authors.forename: Yalin
authors.initials: Y
authors.lastname: Xiong
authors.forename: Woo
authors.initials: W
authors.lastname: Moon
authors.forename: Zachary F
authors.initials: ZF
authors.lastname: Burton
authors.forename: Mikhail
authors.initials: M
authors.lastname: Kashlev
mesh_terms: D001483:Base Sequence; D002384:Catalysis; D012321:DNA-Directed RNA Polymerases / Q000502:physiology*; D004586:Electrophoresis / Q000379:methods; D004926:Escherichia coli; D005786:Gene Expression Regulation; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000502:physiology; D012441:Saccharomyces cerevisiae
chemical_list: D012321:DNA-Directed RNA Polymerases