Torsional Constraints on the Formation of Open Promoter Complexes on DNA Minicircles Carrying σ54-Dependent Promoters

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Abstract
A topoisomer gel retardation assay has been used to examine the topological requirements for the formation of open promoter complexes on DNA minicircles carrying σ54-dependent promoters. In the absence of intercalators, individual topoisomers carrying both the nifL and nifF promoters could be resolved as discrete species by electrophoresis, but exhibited anomalous electrophoretic behavior at relatively high negative superhelical density, indicative of a structural transition. In the presence of phosphorylated activator protein NTRC, ATP, and σ54 RNA polymerase holoenzyme, discrete topoisomer shifts were detected associated with the formation of open promoter complexes. At the nifL promoter open complexes could be formed on all negatively supercoiled topoisomers examined as well as on nicked circular DNA, but not on the ΔLk = 0 topoisomer or positively supercoiled DNA. Minicircles carrying the σ54-dependent glnAp2 promoter could not be resolved in the electrophoresis system, but using a combination of potassium permanganate footprinting and topoisomerase I relaxation assays, we found in contrast to the nifL promoter, that open complexes were formed not only on negatively supercoiled topoisomers but also on relaxed minicircles and the ΔLk = +1 topoisomer. These results indicate there is a thermodynamic barrier to the formation of open complexes on DNA minicircles carrying the nifL promoter which is not evident at glnAp2.

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