In the filamentous fungus Neurospora crassa, both the global-acting regulatory protein NIT2 and the pathway-specific regulatory protein NIT4 are required to turn on the expression of the nit-3 gene, which encodes nitrate reductase, the first enzyme in the nitrate assimilatory pathway. Three NIT2 binding sites and two NIT4 binding sites have been identified in the 1.3-kb nit-3 promoter region via mobility shift and footprinting experiments with NIT2--galactosidase and NIT4--Gactosidase fusion proteins. Quantitative mobility shift assays were used to examine the affinity of individual NIT2 binding sites for the native NIT2 protein present in N. crassa nuclear extracts. In vivo analysis of nit-3 promoter 5 deletion constructs and individual NIT2 and NIT4 binding-site deletions or mutations revealed that all of the NIT2 and NIT4 binding sites are required for the full level of expression of the nit-3 gene. A cluster of two NIT2 and two NIT4 binding sites located more than 1 kb upstream of the translational start site is required for nit-3 expression, and one NIT2 binding site and one NIT4 site, which are immediately adjacent to each other, are of particular functional importance. A significant NIT2-NIT4 protein-protein interaction might occur upon their binding to nearby sites.The filamentous fungus Neurospora crassa is able to utilize secondary nitrogen sources such as nitrate, purines, and various amino acids when preferred primary nitrogen sources such as ammonia, glutamine, or glutamate are not available (14,15). A series of unlinked structural genes which encode various nitrogen catabolic enzymes must be turned on by global-acting as well as pathway-specific regulatory factors for secondary nitrogen source assimilation. When nitrate is present as the sole nitrogen source, nitrogen catabolic repression is lifted, and nitrate serves as an inducer for expression of nitrate assimilation pathway genes, nit-3 and nit-6, which encode nitrate reductase and nitrite reductase, respectively. Expression of nit-3 and nit-6 is turned on by the global positive-acting nitrogen regulatory factor NIT2 and the pathway-specific regulatory protein NIT4.NIT2 is a 110-kDa regulatory protein with a single Cys-X 2 -Cys-X 17 -Cys-X 2 -Cys-type zinc finger DNA binding motif (5, 6). Three NIT2 binding sites have been identified in the nit-3 promoter; one is located near the start site for the nit-3 gene, whereas the other two binding sites are far upstream, approximately 1 kb away from the nit-3 gene (7). NIT2 DNA binding is dependent on core GATA sequence elements (2). NIT4 is a 120-kDa positive-acting regulatory protein with a single GAL4-like Zn(II)Cys 6 -type zinc finger DNA binding motif (20). Two NIT4 binding sites have also been identified in the nit-3 promoter region (4). In addition to the proximal NIT2 binding site, a cluster of two NIT2 and two NIT4 binding sites is located far upstream of the nit-3 gene, as diagrammed in Fig. 1.In this study, we report the results of quantitative in vitro electrophoretic mobility shift assa...