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Nucleic Acids Research, 2002, Vol. 30, No. 4 1016-1028
© 2002 Oxford University Press

Correlating protein footprinting with mutational analysis in the bacterial transcription factor {sigma}54 ({sigma}N)

Siva R. Wigneshweraraj, Paul Casaz1 and Martin Buck*

Department of Biological Sciences, Imperial College of Science, Technology and Medicine, Sir Alexander Fleming Building, Imperial College Road, London SW7 2AZ, UK and 1Paratek Pharmaceuticals, 75 Kneeland Street, Boston, MA 02111, USA

Protein footprints of the enhancer-dependent {sigma}54 protein, upon binding the Escherichia coli RNA polymerase core enzyme or upon forming closed promoter complexes, identified surface-exposed residues in {sigma}54 of potential functional importance at the interface between {sigma}54 and core RNA polymerases (RNAP) or DNA. We have now characterised alanine and glycine substitution mutants at several of these positions. Properties of the mutant {sigma}54s correlate protein footprints to activity. Some mutants show elevated DNA binding suggesting that promoter binding by holoenzyme may be limited to enable normal functioning. One such mutant (F318A) within the DNA binding domain of {sigma}54 shows a changed interaction with the promoter regulatory region implicated in transcription silencing and fails to silence transcription in vitro. It appears specifically defective in preferentially binding to a repressive DNA structure believed to restrict RNA polymerase isomerisation and is largely intact for activator responsiveness. Two mutants, one in the regulatory region I and the other within core interacting sequences of {sigma}54, failed to stably bind the activator in the presence of ADP-aluminium fluoride, an analogue of ATP in the transition state for hydrolysis. Overall, the data presented describe a collection {sigma}54 mutants that have escaped previous analysis and display an array of properties which allows the role of surface-exposed residues in the regulation of open complex formation and promoter DNA binding to be better understood. Their properties support the view that the interface between {sigma}54 and core RNAP is functionally specialised.

* To whom correspondence should be addressed. Tel: +44 20 7594 5442; Fax: +44 20 7594 5419; Email: m.buck{at}ic.ac.uk


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