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Nucleic Acids Research, 2002, Vol. 30, No. 18 3972-3980
© 2002 Oxford University Press

Secondary structure and DNA binding by the C-terminal domain of the transcriptional activator NifA from Klebsiella pneumoniae

Pampa Ray1,2, K. John Smith1, Rosemary A. Parslow1, Ray Dixon2 and Eva I. Hyde*,1

1 School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK and 2 Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK

*To whom correspondence should be addressed. Tel: +44 121 414 5393; Fax: +44 121 414 5982; Email: e.i.hyde{at}bham.ac.uk
Present address:
Pampa Ray, Department of Biological Sciences, Imperial College of Science, Technology and Medicine, London SW7 2AZ, UK

The NifA protein of Klebsiella pneumoniae is required for transcriptional activation of all nitrogen fixation (nif) operons except the regulatory nifLA genes. At these operons, NifA binds to an upstream activator sequence (UAS), with the consensus TGT-N10-ACA, via a C-terminal DNA-binding domain (CTD). Binding of the activator to this upstream enhancer-like sequence allows NifA to interact with RNA polymerase containing the alternative sigma factor, {sigma}54. The isolated NifA CTD is monomeric and binds specifically to DNA in vitro as shown by DNase I footprinting. Heteronuclear 3D NMR experiments have been used to assign the signals from the protein backbone. Three {alpha}-helices have been identified, based on secondary chemical shifts and medium range H{alpha}i-NHi + 1, and NHi-NHi + 1 NOEs. On addition of DNA containing a half-site UAS, several changes are observed in the NMR spectra, allowing the identification of residues that are most likely to interact with DNA. These occur in the final two helices of the protein, directly confirming that DNA binding is mediated by a helix–turn–helix motif.


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