Purification of a heteromeric CCAAT binding protein from Neurospora crassa

Expression of the Neurospora crassa am (NADP-specific glutamate dehydrogenase) gene is controlled by two upstream enhancer-like elements designated URSam alpha and URSam beta. URSam alpha is localized between - 1.3 and - 1.4 kb with respect to the major transcriptional start site. Deletion of a 90 bp sequence containing this element resulted in the loss of approximately 50% of normal glutamate dehydrogenase expression. Gel mobility shift analysis indicated that a nuclear protein from Neurospora binds in a specific manner to sequences within the 90 bp fragment. We have now used a combination of ion-exchange and affinity chromatography to purify this nuclear protein, which we call Am Alpha Binding protein (AAB). The activity was monitored by gel shift analysis. The protein was purified more than 14,000-fold with a yield of approximately 7%. The purified protein appears as a heteromer on denaturing polyacrylamide gel electrophoresis, with only two strong bands visible in silver-stained preparations. One band has an apparent molecular mass of 40 kDa, the other appears as a doublet with an apparent molecular mass of 30 kDa. DNAse I protection analysis indicated a protected region consisting of 30 bp, which contains a CCAAT pentanucleotide motif. Mutagenesis of the CCAAT motif abolished the binding of AAB to the DNA fragment.