A novel heptameric sequence (TTAGTAA) is the binding site for a protein required for high level expression of pcbAB, the first gene of the penicillin biosynthesis in Penicillium chrysogenum.
The first two genes pcbAB and pcbC of the penicillin biosynthesis pathway are expressed from a 1.01-kilobase bidirectional promoter region. A series of sequential deletions were made in the pcbAB promoter region, and the constructions with the modified promoters coupled to the lacZ reporter gene were 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 observed by using the electrophoretic mobility shift assay with boxes A and B (complexes AG1, BG1, BG2, and BL1) but not with box C. Uracil interference assay showed that a protein in P. chrysogenum cell extracts interacts with the thymines in a palindromic heptanucleotide TTAGTAA. Point mutations and deletion of the entire TTAGTAA sequence supported the involvement of this sequence in the binding of a transcriptional activator named penicillin transcriptional activator 1 (PTA1). In vivo studies using constructions carrying point mutations in the TTAGTAA sequence (or a deletion of the complete heptanucleotide) confirmed that this intact sequence is required for high level expression of the pcbAB gene. The TTAGTAA sequence resembles the target sequence of BAS2 (PHO2), a factor required for expression of several genes in yeasts.
Kosalková
K
K
University of León, Faculty of Biology, Area of Microbiology, 24071 León, Spain.
Marcos
A T
AT
Fierro
F
F
Hernando-Rico
V
V
Gutiérrez
S
S
Martín
J F
JF
D019677:Artificial Gene Fusion; D001483:Base Sequence; D001665:Binding Sites; D004271:DNA, Fungal; D004268:DNA-Binding Proteins / Q000378:metabolism; D016296:Mutagenesis; D010406:Penicillins / Q000096:biosynthesis*; D010408:Penicillium chrysogenum / Q000235:genetics* / Q000378:metabolism; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D010406:Penicillins; D010453:Peptide Synthases; C095368:alpha-aminoadipyl-cysteinyl-valine synthetase