Abstract
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.
Highlights
The Cys2/His2 zinc finger proteins, first discovered in the transcription factor IIIA of Xenopus [1], represent an important class of eucaryotic regulatory proteins
More than one zinc finger forms a cluster within the DNA binding domain of transcription factors, and the fingers are separated by short spacers of seven amino acids, known as HC-links [4]
One of the features of the structures of these proteins is that the two fingers are separated by spacers of different lengths, with 44 and 61 amino acids in EPF2–5 and EPF1, respectively, between the second His in the first finger and the first Cys in the second finger
Summary
Construction of Plasmids—A truncated form of the gene for EPF2–5 (residues 66 –210) was created for expression in a rabbit reticulocyte. For production of the truncated form of EPF2–5 in Escherichia coli, the same region of the EPF2–5 gene as described above was amplified by polymerase chain reactions to create additional restriction sites, using an upstream primer that contained a SmaI site and a downstream primer that contained an XhoI site. The products of polymerase chain reaction were digested with SmaI and XhoI and inserted between the XmnI and SalI sites in an expression vector pMAL-c2 (New England Biolabs, Beverly, MA), in-frame with the maltose-binding protein (MBP) gene upstream of the XmnI site to yield pMAL-EPF2–5. The eluted proteins were concentrated and subjected to proteolytic digestion with factor Xa (1%, w/w; New England Biolabs) at 30 °C for 16 h, which removed the amino-terminal MBP domain from the fusion protein to leave a truncated form of EPF 2–5 with no junction sequence (EPF2–5ZF-E). Autoradiography was carried out with an image scanner (BAS 2000; Fuji Photo Film, Kanagawa)
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