PMID:11842099
| Citation |
Cannon, W, Wigneshweraraj, SR and Buck, M (2002) Interactions of regulated and deregulated forms of the sigma54 holoenzyme with heteroduplex promoter DNA. Nucleic Acids Res. 30:886-93 |
|---|---|
| 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. |
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| Keywords |
Bacterial Proteins/physiology; Base Sequence; Binding Sites; DNA/metabolism; DNA Probes/metabolism; DNA-Binding Proteins; DNA-Directed RNA Polymerases/chemistry; DNA-Directed RNA Polymerases/genetics; DNA-Directed RNA Polymerases/metabolism; Electrophoretic Mobility Shift Assay; Escherichia coli Proteins; Holoenzymes/chemistry; Holoenzymes/genetics; Holoenzymes/metabolism; Kinetics; Mutation; Nucleic Acid Heteroduplexes/metabolism; Promoter Regions, Genetic; Protein Binding; Protein Structure, Tertiary; RNA Polymerase Sigma 54; Sigma Factor/chemistry; Sigma Factor/genetics; Sigma Factor/metabolism; Templates, Genetic; Trans-Activators/physiology; Transcription, Genetic |
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