The intergenic region between the divergently transcribed niiA and niaD genes of Aspergillus nidulans contains multiple NirA binding sites which act bidirectionally.

The niaD and niiA genes of Aspergillus nidulans, which code, respectively, for nitrate and nitrite reductases, are divergently transcribed, and their ATGs are separated by 1,200 bp. The genes are under the control of the positively acting NirA transcription factor, which mediates nitrate induction. The DNA binding domain of NirA was expressed as a fusion protein with the glutathione S-transferase of Schistosoma japonicum. Gel shift and footprint experiments have shown that in the intergenic region there are four binding sites for the NirA transcription factor. These sites can be represented by the nonpalindromic consensus 5'CTCCGHGG3'. Making use of a bidirectional expression vector, we have analyzed the role of each of the sites in niaD and niiA expression. The sites were numbered from the niiA side. It appeared that site 1 is necessary for the inducibility of niiA only, while sites 2, 3, and to a lesser extent 4 (which is nearer to and strongly affects niaD) act bidirectionally. The results also suggest that of the 10 binding sites for the AreA protein, which mediates nitrogen metabolite repression, those which are centrally located are physiologically important. The insertion of an unrelated upstream activating sequence into the intergenic region strongly affected the expression of both genes, irrespective of the orientation in which the element was inserted.