The Athb-1 and −2 HD-Zip domains homodimerize forming complexes of different DNA binding specificities.

Abstract

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.