Mapping ATP-dependent activation at a sigma54 promoter.
The sigma(54) promoter specificity factor is distinct from other bacterial RNA polymerase (RNAP) sigma factors in that it forms a transcriptionally silent closed complex upon promoter binding. Transcriptional activation occurs through a nucleotide-dependent isomerization of sigma(54), mediated via its interactions with an enhancer-binding activator protein that utilizes the energy released in ATP hydrolysis to effect structural changes in sigma(54) and core RNA polymerase. The organization of sigma(54)-promoter and sigma(54)-RNAP-promoter complexes was investigated by fluorescence resonance energy transfer assays using sigma(54) 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 sigma(54) undergoes spatial rearrangements of functionally important domains upon closed complex formation. sigma(54) and sigma(54)-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-sigma(54)-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 down-stream melted DNA.
Leach
Robert N
RN
Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Gell
Christopher
C
Wigneshweraraj
Sivaramesh
S
Buck
Martin
M
Smith
Alastair
A
Stockley
Peter George
PG
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D051877:RNA Polymerase Sigma 54 / Q000235:genetics* / Q000378:metabolism*
D004269:DNA, Bacterial; D020033:Protein Isoforms; D000255:Adenosine Triphosphate; D051877:RNA Polymerase Sigma 54