Role of Linkers between Zinc Fingers in Spacing Recognition by Plant TFIIIA-Type Zinc-Finger Proteins

The EPF family of plant TFIIIA-type zinc-finger (ZF) proteins (ZPTs) is characterized by long linkers separating ZF motifs. We previously reported that two-fingered ZPTs bind to two tandem core sites that are separated by several base pairs, each ZF making contact with one core site. Here we report further characterization of DNA-binding activities of ZPTs using four family members, ZPT2-14, ZPT2-7, ZPT2-8, and ZPT2-2, having inter-ZF linkers of different lengths and sequences, to investigate the correlation of the length and/or sequence of the linker with preference for the spacing between core sites in target DNAs. Selected and amplified binding site (SAAB)-imprinting assays and gel mobility shift assays prompted three conclusions. (1) The four ZPTs have common specificity for core binding sites-two AGT(G)/(C)ACTs separated by several nucleotides. (2) The four ZPTs prefer a spacing of 10 bases between the core sites, but each ZPT has its own preference for suboptimal spacing. (3) At a particular spacing, two zinc fingers may bind to the core sites on both strands. The results provide new information about how the diversity in linker length/sequence affects DNA-sequence recognition in this protein family.