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Molecular and Cellular Biology, November 1998, p. 6447-6456, Vol. 18, No. 11
Howard Hughes Medical Institute, Department
of Molecular Biology and Genetics and Department of Neuroscience, Johns
Hopkins University School of Medicine, Baltimore, Maryland 21205
Received 4 March 1998/Returned for modification 27 May
1998/Accepted 5 August 1998
Roaz, a rat C2H2 zinc finger protein, plays
a role in the regulation of olfactory neuronal differentiation through
its interaction with the Olf-1/EBF transcription factor family. An
additional role for the Roaz/Olf-1/EBF heterodimeric protein is
suggested by its ability to regulate gene activation at a distinct
promoter lacking Olf-1/EBF-binding sites. Using an in vitro
binding-site selection assay (Selex), we demonstrate that Roaz protein
binds to novel inverted perfect or imperfect repeats of GCACCC
separated by 2 bp. We show that Roaz is capable of binding to a
canonical consensus recognition sequence with high affinity
(Kd = 3 nM). Analysis of the structural
requirement for protein dimerization and DNA binding by Roaz reveals
the role of specific zinc finger motifs in the Roaz protein for
homodimerization and heterodimerization with the Olf-1/EBF
transcription factor. The DNA-binding domain of Roaz is mapped to the
N-terminal 277 amino acids, containing the first seven zinc finger
motifs, which confers weak monomeric binding to a single half site and
a stronger dimeric binding to the inverted repeat in a
binding-site-dependent manner. Full-length protein can form dimers on
both the inverted repeat and direct repeat but not on a single half
site. These findings support the role of the TFIIIA-type Zn fingers in
both protein-protein interaction and protein-DNA interaction and
suggest distinct functions for specific motifs in proteins with a large
number of zinc finger structures.
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification of DNA Recognition Sequences and Protein
Interaction Domains of the Multiple-Zn-Finger Protein Roaz
and
*
Corresponding author. Mailing address: Department of
Molecular Biology and Genetics, School of Medicine, Johns Hopkins
University, 801 P.C.T.B., 725 N. Wolfe St., Baltimore, MD 21205-2185.
Present address: National Institute of Neurological Disorders and
Stroke, National Institutes of Health, Bethesda, MD 20892.
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