Specific DNA recognition by the Aspergillus nidulans three zinc finger transcription factor PacC.
The three zinc fingers of PacC, the transcription factor mediating pH regulation in Aspergillus nidulans, are necessary and sufficient to recognise specifically the target ipnA2 site. Missing nucleoside footprints confirmed the core target (double-stranded) hexanucleotide 5'-GCCAAG-3'. Any base substitution resulted in substantial or complete loss of binding, excepting A5 (partially replaceable by G). A T preceding the hexanucleotide enhanced binding. Interference footprinting indicates that the four Gs and A4 participate in specific contacts and that five pyrimidines are essential for binding. The size of the target sequence and the amino acid sequence of finger 1 suggested that its probe helix would not participate in base-specific contacts. Using site-directed mutagenesis and analogy to GLI, we propose that finger 1 crucially interacts with finger 2, a pair of conserved Trp residues in the Cys knuckles contacting hydrophobically. Finger 2 would also participate in extensive base contacts with the 5' moiety of the hexanucleotide. The specificity mutation Lys159Gln shows that finger 3 binds the 3' moiety of the hexanucleotide. Replacement of residues in positions +3 (His128Asn) and +2 (Gln155Lys) of the reading helices of fingers 2 and 3, respectively, prevented binding. Our biochemical and molecular data plus modelling using previously determined zinc finger-DNA complexes, predict specific contacts of fingers 2 and 3 to ipnA2. Our data indicate compact organisation of the PacC-ipnA2 complex (with nearly every base involved in specific contacts), illustrate the binding versatility of zinc finger domains and should facilitate analysis of other PacC family members, including Saccharomyces cerevisiae RIM1.
Espeso
E A
EA
Centro de Investigaciones Biológicas del CSIC, Madrid, Spain.
Tilburn
J
J
Sánchez-Pulido
L
L
Brown
C V
CV
Valencia
A
A
Arst
H N
HN
Peñalva
M A
MA
D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000737:chemistry*; D001482:Base Composition; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D005656:Fungal Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides; D011487:Protein Conformation; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers
D005656:Fungal Proteins; D009841:Oligonucleotides; C092394:PacC protein, Aspergillus; D014157:Transcription Factors; D004247:DNA