title: Interactions of transcription inhibitors with the Escherichia coli RNA polymerase-lacUV5 promoter open complex.
abstract: The interactions of transcription inhibitors with the open complex composed of Escherichia coli RNA polymerase and the lacUV5 promoter have been studied using gel retardation, the chemical nuclease activity of the cuprous complexes of 1,10-phenanthroline (OP) and its derivatives, and steady-state kinetics. Gel retardation shows that two inhibitors, the 2:1 2,9-dimethyl-1,10-phenanthroline-cuprous complex [(2,9-Me2OP)2Cu+] and rifampicin, bind stably to the open-complex. (2,9-Me2OP)2Cu+ blocks scission by the chemical nuclease by interfering with the binding of its redox-active isosteres. Rifampicin does not block scission by the cuprous complexes of 3,4,7,8-tetramethyl-OP, 4-phenyl-OP, and OP but does perturb scission by the cuprous complex of 5-phenyl-OP. Organic ligands including intercalating agents and groove binders (e.g., daunomycin, di(amidinophenyl)indole (DAPI), actinomycin D, distamycin, 9-aminoacridine, mithramycin, and chromomycin A3), which bind to free DNA with high affinity, do not form stable ternary complexes with the open-complex. Gel retardation experiments demonstrate that they promote dissociation of the enzyme from the promoter. The greater sensitivity of enzymatic catalysis to inhibitor concentration relative to polymerase binding suggests that these ligands form metastable, catalytically inactive ternary complexes with RNA polymerase and the promoter.
authors.affiliation: Department of Biological Chemistry, School of Medicine, University of California-Los Angeles 90024-1570.
authors.forename: A
authors.initials: A
authors.lastname: Mazumder
authors.forename: D M
authors.initials: DM
authors.lastname: Perrin
authors.forename: D
authors.initials: D
authors.lastname: McMillin
authors.forename: D S
authors.initials: DS
authors.lastname: Sigman
mesh_terms: D001483:Base Sequence; D003300:Copper / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000187:drug effects* / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D007364:Intercalating Agents / Q000494:pharmacology; D008024:Ligands; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009942:Organometallic Compounds / Q000494:pharmacology*; D010618:Phenanthrolines / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D012293:Rifampin / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects*
chemical_list: D004269:DNA, Bacterial; D007364:Intercalating Agents; D008024:Ligands; D009942:Organometallic Compounds; D010618:Phenanthrolines; C078638:copper-neocuproine complex; D003300:Copper; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin