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Mapping ATP-dependent activation at a sigma54 promoter
Robert N Leach1, Christopher Gell, Sivaramesh Wigneshweraraj
1Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
The Journal of Biological Chemistry
|August 24, 2006
Summary
Bacterial sigma(54) factors form silent complexes, activating transcription via nucleotide-dependent isomerization. This study reveals sigma(54) domain rearrangements and functional states during activation, crucial for bacterial gene regulation.
Area of Science:
- * Molecular Biology
- * Biochemistry
- * Genetics
Background:
- * Sigma(54) (sigma54) is a bacterial RNA polymerase (RNAP) specificity factor distinct from other sigma factors.
- * Sigma(54) forms a transcriptionally silent closed complex upon promoter binding, requiring an activator protein and ATP hydrolysis for transcriptional activation.
- * Activator proteins bind enhancers and use ATP hydrolysis energy to induce structural changes in sigma(54) and core RNAP, facilitating promoter isomerization.
Purpose of the Study:
- * To investigate the spatial organization of sigma(54)-promoter and sigma(54)-RNAP-promoter complexes.
- * To understand the structural rearrangements of sigma(54) during closed complex formation.
- * To identify functional states of the activator-sigma(54)-RNAP-promoter complex throughout transcriptional activation.
Main Methods:
- * Fluorescence resonance energy transfer (FRET) assays were employed.
- * Sigma(54) single cysteine-mutants labeled with acceptor fluorophores were used.
- * Donor fluorophore-labeled DNA sequences mimicking nifH promoters with mismatches were utilized.
Main Results:
- * Sigma(54) undergoes spatial rearrangements of functionally important domains upon closed complex formation.
- * Nucleotide-dependent alterations in FRET efficiencies identified distinct functional states of the activator-sigma(54)-RNAP-promoter complex.
- * Open complex formation was found to be efficient only upon replacement of a repressive fork junction with downstream melted DNA.
Conclusions:
- * Sigma(54) undergoes significant structural changes during closed complex formation.
- * The study elucidates the dynamic functional states of the sigma(54)-RNAP-promoter complex during the mechano-chemical transduction of transcriptional activation.
- * Efficient open complex formation is dependent on specific DNA structures at the promoter site.
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