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Volume 271, Number 38, Issue of September 20, 1996 pp. 23368-23373
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Target-Sequence Recognition by Separate-type Cys₂/His₂ Zinc Finger Proteins in Plants

(Received for publication, April 9, 1996, and in revised form, June 28, 1996)

Hiroshi Takatsuji and Takashi Matsumoto ^¶

From the Laboratories of  Developmental Biology and ^¶ Protein Engineering, National Institute of Agrobiological Resources, 2-1-2 Kannondai, Tsukuba, Ibaraki 305, Japan

The EPF family is a group of transcription factors containing canonical Cys₂/His₂-type zinc finger motifs that were first discovered in plants. These zinc finger proteins are characterized by two zinc fingers that are separated by spacers of various lengths, which are much longer than typical spacers (HC-link) in cluster-type zinc finger proteins. We describe here direct evidence that the two zinc fingers make contact with two tandemly repeated AGT core sequences that are separated by about 13 base pairs, by contrast to the cluster-type zinc finger proteins that bind to contiguous triplet sequences. DNA binding affinities were sensitive to the spaces between the core sequences, and the sensitivity to the spacing was greatly affected by the DNA sequence between the core sequences, with GC-rich sequences endowing much higher specificity than AT-rich sequences. Among the members of the EPF family, EPF1 was less sensitive to the spacing than EPF2-5. These results suggest that EPFs recognize their cognate target DNAs not only by the sequence of the core sites but also by the spacing between the core sites and, moreover, that different members in the EPF family distinguish their specific target genes by reference to these two parameters. This represents a unique type of target-sequence recognition among Cys₂/His₂-type zinc finger transcription factors. In addition, site-directed mutagenesis studies demonstrated that the two zinc fingers contribute synergistically to the binding to DNA, indicating that both fingers are necessary for the high affinity DNA binding.

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