title: Interactions of regulated and deregulated forms of the sigma54 holoenzyme with heteroduplex promoter DNA.
abstract: The bacterial sigma54 RNA polymerase holoenzyme binds to promoters as a stable closed complex that is silent for transcription unless acted upon by an enhancer-bound activator protein. Using DNA binding and transcription assays the ability of the enhancer-dependent sigma54 holoenzyme to interact with promoter DNA containing various regions of heteroduplex from -12 to -1 was assessed. Different DNA regions important for stabilising sigma54 holoenzyme-promoter interactions, destabilizing binding, limiting template utilisation in activator-dependent transcription and for stable binding of a deregulated form of the holoenzyme lacking sigma54 Region I were identified. It appears that homoduplex structures are required for early events in sigma54 holoenzyme promoter binding and that disruption of a repressive fork junction structure only modestly deregulates transcription. DNA opening from -5 to -1 appears important for stable engagement of the holoenzyme following activation. The regulatory Region I of sigma54 was shown to be involved in interactions with the sequences in the -5 to -1 area.
authors.affiliation: Department of Biological Sciences, Imperial College of Science, Technology and Medicine, Sir Alexander Fleming Building, Imperial College Road, London SW7 2AZ, UK.
authors.forename: Wendy
authors.initials: W
authors.lastname: Cannon
authors.forename: Siva R
authors.initials: SR
authors.lastname: Wigneshweraraj
authors.forename: Martin
authors.initials: M
authors.lastname: Buck
mesh_terms: D001426:Bacterial Proteins / Q000502:physiology; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D015342:DNA Probes / Q000378:metabolism; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D029968:Escherichia coli Proteins; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051877:RNA Polymerase Sigma 54; D012808:Sigma Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013698:Templates, Genetic; D015534:Trans-Activators / Q000502:physiology; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D015342:DNA Probes; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D020035:Holoenzymes; D009692:Nucleic Acid Heteroduplexes; C098954:PspF protein, E coli; D012808:Sigma Factor; D015534:Trans-Activators; C497751:rpoN protein, E coli; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D051877:RNA Polymerase Sigma 54