| Abstract: | The NIT2 nitrogen regulatory protein of Neurospora is a DNA binding protein which contains a single Cys2/Cys2 type finger motif followed immediately by a highly basic region. Several different approaches were employed to identify nucleotides which appear to be in contact with NIT2 in the DNA-protein complex. Methylation interference and missing contact analyses with the promoter DNA fragment of the L-amino acid oxidase gene showed that all three purines in both of two GATA core sequences and the single adenine residue in each of the complementary TATC sequences were in intimate contact with NIT2. Modification or loss of the three purine residues located between the two GATA core sequences also significantly reduced NIT2 binding, whereas alteration of purines which flank the binding element showed only minor effects. Chemical modification of all six thymine bases in the two GATA and TATC complement core sequences also strongly affected NIT2 binding. High affinity NIT2 binding sites appear to contain at least two GATA core sequences, with single GATA sequences acting only as weak binding sites. Mobility shift experiments with the DNA fragment upstream of nit-3, the structural gene for nitrate reductase, revealed two DNA-NIT2 protein complexes. In complex I, which is formed first, NIT2 was bound to a pair of GATA sites located at -180. In complex II, the paired GATA sites at -180 plus a single GATA site at -290 were all occupied by NIT2. A DNA fragment containing only the single -290 GATA element bound NIT2 very weakly. The affinity of this single GATA for NIT2 was ten to twenty times greater when it was situated on the same DNA fragment with the distant paired GATA elements than when alone. |
