Concerted inhibition of the transcriptional activation functions of the enhancer-binding protein NIFA by the anti-activator NIFL

The Azotobacter vinelandii NIFL regulatory flavoprotein responds to the red ox, energy and nitrogen status of the cell to inhibit transcriptional activ ation by the sigma (N)-dependent enhancer binding protein, NIFA, via the fo rmation of a NIFL-NIFA protein complex. The NIFA protein contains three dom ains: an N-terminal domain of unknown function; a central catalytic domain required to couple nucleotide hydrolysis to activation of the sigma (N)-RNA polymerase holoenzyme; and a C-terminal DNA-binding domain. We report that truncated NIFA proteins that either lack the amino-terminal domain or cont ain only the isolated central domain remain responsive to inhibition by NIF L but, in contrast to native NIFA, continue to hydrolyse nucleotides when N IFL is present. We also report that NIFL is competent to inhibit the DNA-bi nding function of NIFA. Taken together, these results suggest that NIFL inh ibits NIFA via a concerted mechanism in which DNA binding, catalytic activi ty and, potentially, interaction with the polymerase are controlled by NIFL in order to prevent transcriptional activation under detrimental environme ntal conditions.