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Molecular and Cellular Biology, November 1998, p. 6447-6456, Vol. 18, No. 11
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

Robert Y. L. Tsaidagger and Randall R. Reed*

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.

* 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.

dagger Present address: National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.

Molecular and Cellular Biology, November 1998, p. 6447-6456, Vol. 18, No. 11
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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