The Negative-Acting NMR Regulatory Protein of Neurospora crassa Binds to and Inhibits the DNA-Binding Activity of the Positive-Acting Nitrogen Regulatory Protein NIT2

Abstract

Structural genes of the nitrogen regulatory circuit of the filamentous fungus Neurospora crassa are under the control of both positive and negative regulatory proteins. NIT2, the major positive-acting nitrogen regulatory protein, activates the expression of structural genes within the nitrogen circuit. NIT2 binds to upstream activation sites which contain at least two GATA core elements in the promoter regions of the nitrogen-controlled structural genes, and activates their transcription, possibly by way of acidic activation domains. The mechanism by which a putative negative-acting regulator, NMR, mediates nitrogen repression of the various structural genes has remained unclear. In the studies reported here, a direct interaction between the NIT2 and NMR proteins has been demonstrated by the use of two different experimental approaches. The yeast two-hybrid system was used to show NIT2-NMR-specific binding in vivo; an independent in vitro assay for protein-protein binding also demonstrated a specific interaction between NIT2 and NMR. Two separate regions of the NIT2 protein, both of which appear to exist as alpha-helices, make direct contact with the NMR protein. One of these alpha-helical regions occurs within the zinc finger DNA-binding domain of NIT2. Mutant NIT2 proteins with amino acid substitutions in the zinc finger motif do not bind to NMR. Mobility shift experiments revealed that the NMR protein inhibits NIT2 DNA binding in vitro. We suggest that NMR carries out its negative regulatory role by directly binding to NIT2, and thereby blocking the function of NIT2 by inhibiting its DNA-binding activity.