NifA protein synthesized in vitro binds to the upstream activator sequence in the promoter of the Klebsiella oxytoca nifB gene.

Abstract

The nitrogen fixation (nif) genes of various bacteria are transcribed with an RNApolymerase modulated by the alternative activated by the NifA protein.1* Several analyses of the Klebsiella pneumoniae nifYL promoter2'3) showed that not only binding of the NifA protein to the upstream activator sequence integration host factor (IHF) to the promoter region was essential for completetranscriptional activation. Here we demonstrate the binding ofKlebsiella oxytoca NifA protein synthesized in vitro to the UASof ni/B promoter and the interaction of IHF with the nifB promoter. The NifA protein was synthesized by a coupled transcription and translation4* of an artificial nifA gene, whose expression wascontrolled by the promoter of the chlor-amphenicol acetyl transferase gene, on pNOA1015)using S30 extract of Escherichia coli MRE600purchased from Amersham. To assay its DNAbinding activity, this trans-lation mixture was directly incubated with a DNAprobe