%0 Journal Article
%T Role of Linkers between Zinc Fingers in Spacing Recognition by Plant TFIIIA-Type Zinc-Finger Proteins
%A Setsuko Fukushima
%A Michiteru Yoshida
%A Hiroshi Takatsuji
%J Journal of Amino Acids
%D 2012
%I Hindawi Publishing Corporation
%R 10.1155/2012/848037
%X 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¡ªtwo 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 ¦Â-hairpin and an ¦Á-helix (¦Â¦Â¦Á motif), and the other conserved residues are packed to form a hydrophobic core [3¨C10]. Generally, in animals, multiple ZF motifs are present as tandem arrays linked by a conserved short sequence, the HC-link [11¨C13], and the ZF proteins interact with contiguous sets of triplet sequences, with each ZF making contact with 3¨C5 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
%U http://www.hindawi.com/journals/jaa/2012/848037/