Visualization of intermediary transcription states in the complex between Escherichia coli DNA-dependent RNA polymerases and a promoter-carrying DNA fragment using the gel retardation method
- PMID: 3525164
- DOI: 10.1111/j.1432-1033.1986.tb09793.x
Visualization of intermediary transcription states in the complex between Escherichia coli DNA-dependent RNA polymerases and a promoter-carrying DNA fragment using the gel retardation method
Abstract
DNA-dependent RNA polymerase in complex with a DNA fragment was analyzed by electrophoresis in non-denaturing gels as core enzyme, holoenzyme, during initiation and elongation. The DNA fragment carried the promoter A1 of the phage T7. The stoichiometry between holoenzyme and promoter and between sigma and core enzyme in complex with DNA was determined. Holoenzyme bound as a monomer to the DNA, whereas core enzyme formed aggregates before binding to the DNA. If the molar ratio of holoenzyme to DNA exceeded 0.5:1 a second holoenzyme molecule interacted with the DNA fragment with diminished affinity. A large difference in the frictional coefficient of the holoenzyme-promoter and the core enzyme-DNA complex indicated a drastic conformational difference between the two types of complexes. The stability of the holoenzyme-promoter complex decreased with decreasing temperature, accompanied by at least partial dissociation of holoenzyme into core enzyme and sigma factor. Addition of nucleoside triphosphates did not change the electrophoretic mobility of the complex if abortive transcription only was allowed, but increased it after addition of all four nucleoside triphosphates owing to release of the sigma factor.
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