Secondary structure and DNA binding by the C-terminal domain of the transcriptional activator

The NifA protein of Klebsiella pneumoniae is required for transcriptional activation of all nitrogen ®xation (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, s54. The isolated NifA CTD is monomeric and binds speci®cally to DNA in vitro as shown by DNase I footprinting. Heteronuclear 3D NMR experi-ments have been used to assign the signals from the protein backbone. Three a-helices have been identi®ed, based on secondary chemical shifts and medium range Hai-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 identi®cation of residues that are most likely to interact with DNA. These occur in the ®nal two helices of the protein, directly con®rming that DNA binding is mediated by a helix±turn±helix motif