Mapping ATP-dependent activation at a σ54 promoter

Abstract

The σ54 promoter specificity factor is distinct from other bacterial RNA polymerase (RNAP) σ factors in that it forms a transcriptionally silent closed complex upon promoter binding. Transcriptional activation occurs through a nucleotidedependent isomerization of σ54, mediated via its interactions with an enhancer-binding activator protein that utilizes the energy released in ATP hydrolysis to effect structural changes in σ54 and core RNA polymerase. The organization of σ54 -promoter and σ54-RNAP- promoter complexes was investigated by fluorescence resonance energy transfer assays using σ54 single cysteine-mutants labeled with an acceptor fluorophore and donor fluorophore-labeled DNA sequences containing mismatches that mimic nifH early- and late-melted promoters. The results show that σ54 undergoes spatial rearrangements of functionally important domains upon closed complex formation. σ54 and σ54-RNAP promoter complexes reconstituted with the different mismatched DNA constructs were assayed by the addition of the activator phage shock protein F in the presence or absence of ATP and of non-hydrolysable analogues. Nucleotide-dependent alterations in fluorescence resonance energy transfer efficiencies identify different functional states of the activator-σ54-RNAP-promoter complex that exist throughout the mechano-chemical transduction pathway of transcriptional activation, i.e. from closed to open promoter complexes. The results suggest that open complex formation only occurs efficiently on replacement of a repressive fork junction with downstream melted DNA. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.