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. 1. Introduction The EPF proteins form a subfamily of TFIIIA-type zinc-finger (ZF) proteins (ZPTs) of plants [1, 2]. The TFIIIA-type ZF motif is a sequence of , in which two cysteines and two histidines tetrahedrally coordinate a zinc atom to form a compact structure containing a β-hairpin and an α-helix (ββα motif), and the other conserved residues are packed to form a hydrophobic core [3–10]. Generally, in animals, multiple ZF motifs are present as tandem arrays linked by a conserved short sequence, the HC-link [11–13], and the ZF proteins interact with contiguous sets of triplet sequences, with each ZF making contact with 3–5 base pairs in the major groove of DNA. The ZPTs have 1, 2, 3, or 4？ZF motifs [14]. In most ZF motifs of ZPTs, a highly conserved sequence, QALGGH, is located within DNA-contacting surfaces [2, 14]. Since ZPT2-1 (renamed from EPF1) was first identified in petunia as a DNA-binding protein that interacts with a petal-specific promoter of the enolpyruvylshikimate-3-phosphate synthase gene [15], several EPF1-like ZPTs have been reported in various plant species [16, 17]. In Arabidopsis, a model plant whose genome has been sequenced, an estimated 64？ZF genes containing the QALGGH motif are encoded [16]. Two-fingered ZPTs have been implicated in various important regulatory processes. Some of these proteins are implicated in plant