A novel heptameric sequence (TTAGTAA) is the binding site for a protein required for high level expression of pcbAB, the first gene of the penicillinbiosynthesis in Penicillium chrysogenum

The first two genes pcbAB and pcbC of the penicillin biosynthesis pathway a re expressed from a 1.01-kilobase bidirectional promoter region. A series o f sequential deletions were made in the pcbAB promoter region, and the cons tructions with the modified promoters coupled to the lacZ reporter gene wer e introduced as single copies at the pyrG locus in Penicillium chrysogenum npe10, Three regions, boxes A, B, and C, produced a significant decrease in expression of the reporter gene when deleted. Protein-DNA complexes were o bserved by using the electrophoretic mobility shift assay with boxes A and B (complexes AGI, BG1, BG2, and BL1) but not with box C, Uracil interferenc e assay showed that a protein in P. chrysogenum cell extracts interacts wit h the thymines in a palindromic heptanucleotide TTAGTAA. Point mutations an d deletion of the entire TTAGTAA sequence supported the involvement of this sequence in the binding of a transcriptional activator named penicillin tr anscriptional activator 1 (PTA1). In vivo studies using constructions carry ing point mutations in the TTAGTAA sequence (or a deletion of the complete heptanucleotide) confirmed that this intact sequence is required for high l evel expression of the pcbAB gene. The TTAGTAA sequence resembles the targe t sequence of BASS (PHO2), a factor required for expression of several gene s in yeasts.