The Athb-1 and -2 HD-Zip domains homodimerize forming complexes of different DNA binding specificities.
The Arabidopsis Athb-1 and -2 proteins are characterized by the presence of a homeodomain (HD) with a closely linked leucine zipper motif (Zip). We have suggested that the HD-Zip motif could, via dimerization of the leucine zippers, recognize dyad-symmetric DNA sequences. Here we report an analysis of the DNA binding properties of the Athb-1 homeodomain-leucine zipper (HD-Zip-1) domain in vitro. DNA binding analysis performed using random-sequence DNA templates showed that the HD-Zip-1 domain, but not the Athb-1 HD alone, binds to DNA. The HD-Zip-1 domain recognizes a 9 bp dyad-symmetric sequence [CAAT(A/T)ATTG], as determined by selecting high-affinity binding sites from random-sequence DNA. Gel retardation assays demonstrated that the HD-Zip-1 domain binds to DNA as a dimer. Moreover, the analysis of the DNA binding activity of Athb-1 derivatives indicated that a correct spatial relationship between the HD and the Zip is essential for DNA binding. Finally, we determined that the Athb-2 HD-Zip domain recognizes a distinct 9 bp dyad-symmetric sequence [CAAT(G/C)ATTG]. A model of DNA binding by the HD-Zip proteins is proposed.
Sessa
G
G
Dipartimento di Genetica e Biologia Molecolare, Università di Roma La Sapienza, Italy.
Morelli
G
G
Ruberti
I
I
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D018398:Homeodomain Proteins; D016350:Leucine Zippers; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D010940:Plant Proteins / Q000096:biosynthesis / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D016385:TATA Box; D014158:Transcription, Genetic
D029681:Arabidopsis Proteins; D004268:DNA-Binding Proteins; C069386:HB-2 protein, Arabidopsis; D018398:Homeodomain Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; D005982:Glutathione Transferase