Deletion of the N-terminal region of the AREA protein is correlated with a derepressed phenotype with respect to nitrogen metabolite repression
1997
Lamb, H.K. | Dodds, A.L. | Swatman, D.R. | Cairns, E. | Hawkins, A.R.
The entire areA gene and a truncated version lacking the sequence encoding the N-terminal 389 amino acids were expressed from the qutE promoter and terminator in an Aspergillus nidulans strain with the endogenous areA gene deleted. This expression system was used to decouple the effects of transcription regulation and mRNA stability mediated by the native promoter and terminator from any posttranslational modulation of AREA activity. Both the full-length AREA protein and the truncated form were able to function in the deletion strain, conferring the ability to use alternate nitrogen sources. Transformants containing the entire areA gene had a repressible phenotype with respect to nitrogen metabolite repression, whereas those containing the truncated form of the areA gene had a derepressed phenotype. The truncated areA gene was expressed in an A. nidulans strain containing a normally regulated wild-type areA gene, and transformants displayed a quinate-inducible nitrogen metabolite derepressed phenotype. Northern blot analysis of transformed strains showed that areA-specific mRNAs of the expected sizes were being produced. The truncated AREA protein was overproduced in Escherichia coli as a fusion protein and purified to homogeneity by a single-step immobilized metal affiinity chromatography, and the purified protein was shown to bind specifically to the niaD promoter. Revised sequences of the 5' region of the areA gene and the entire meaB gene are reported.
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