Mutational analysis of the Acropora millepora PaxD paired domain highlights the importance of the linker region for DNA binding.
Pax transcription factors are found in animals, from simple sponges to insects and vertebrates. The defining feature of Pax proteins is the DNA-binding paired domain (PD), which consists of two helix-turn-helix subdomains, joined with a linker region. Despite high specificity in vivo, the paired domains of different Pax proteins bind similar consensus DNA sequences in vitro. Using bandshift techniques, we show here that the paired domain of the Acropora millepora PaxD protein, which unambiguously belongs to the Pax3/7 group, does not bind to three defined paired domain-binding sites. Domain swapping experiments and site-directed mutagenesis identified two amino acid residues in the linker region of the paired domain as critical to DNA binding; G70 and S71 are highly conserved in Pax proteins, but differ in PaxD (L70 and N71). The PaxD data thus highlight the importance of the linker region, and particularly G70 and S71, in DNA binding by Pax proteins.
Nordström
Karin
K
Department of Neuroscience, Pharmacology, Uppsala University, Box 593, Uppsala 751 24, Sweden. Karin.Nordstrom@cob.lu.se
Scholten
Ingo
I
Nordström
Johanna
J
Larhammar
Dan
D
Miller
David
D
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D003063:Cnidaria / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D014644:Genetic Variation; D005998:Glycine / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D000072041:PAX3 Transcription Factor; D051763:PAX7 Transcription Factor; D051761:Paired Box Transcription Factors; D011485:Protein Binding; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D000072041:PAX3 Transcription Factor; D051763:PAX7 Transcription Factor; D051761:Paired Box Transcription Factors; D014157:Transcription Factors; C068686:Pax3 protein, mouse; D012694:Serine; D004247:DNA; D005998:Glycine