THE ISOLATED CATALYTIC DOMAIN OF NIFA, A BACTERIAL ENHANCER-BINDING PROTEIN, ACTIVATES TRANSCRIPTION IN-VITRO - ACTIVATION IS INHIBITED BY NIFL

The NIFA protein of Klebsiella pneumoniae is required for transcriptio n of all nif (nitrogen fixation) operons except the regulatory nifLA o peron itself. NIFA activates transcription of nif operons by the alter native holoenzyme form of RNA polymerase, sigma54-holoenzyme, in a nuc leoside triphosphate (NTP)-dependent manner. NIFL antagonizes the acti on of NIFA in the presence of molecular oxygen or combined nitrogen. T he NIFA protein of K. pneumoniae is composed of three domains: an N-te rminal domain with unclear function, a central catalytic domain, and a C-terminal DNA-binding domain. We report that the isolated central do main of NIFA activates transcription in vitro and that this activation requires NTP with a hydrolyzable beta-gamma bond, as does activation by intact NIFA. Transcriptional activation by the isolated central dom ain has the heat lability characteristic of intact NIFA and is inhibit ed by NIFL. The central domain has an NTPase activity that is also hea t-labile but is not inhibited by NIFL. Taken together, these results i mply that NIFL interferes with contact between NIFA and sigma54-holoen zyme.