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J. Biol. Chem., Vol. 279, Issue 50, 52447-52455, December 10, 2004

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Intact RNA-binding Domains Are Necessary for Structure-specific DNA Binding and Transcription Control by CBTF¹²² during Xenopus Development^*

Garry P. Scarlett, Stuart J. Elgar, Peter D. Cary, Anna M. Noble, Robert L. Orford¶, G. Geoffrey Kneale, and Matthew J. Guille||

From the Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth, PO1 2DT, the Cardiff School of Biosciences, Cardiff University, Cardiff, CF10 3TL, and the ^¶National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, United Kingdom

CBTF¹²² is a subunit of the Xenopus CCAAT box transcription factor complex and a member of a family of double-stranded RNA-binding proteins that function in both transcriptional and post-transcriptional control. Here we identify a region of CBTF¹²² containing the double-stranded RNA-binding domains that is capable of binding either RNA or DNA. We show that these domains bind A-form DNA in preference to B-form DNA and that the -59 to -31 region of the GATA-2 promoter (an in vivo target of CCAAT box transcription factor) adopts a partial A-form structure. Mutations in the RNA-binding domains that inhibit RNA binding also affect DNA binding in vitro. In addition, these mutations alter the ability of CBTF¹²² fusions with engrailed transcription repressor and VP16 transcription activator domains to regulate transcription of the GATA-2 gene in vivo. These data support the hypothesis that the double-stranded RNA-binding domains of this family of proteins are important for their DNA binding both in vitro and in vivo.

Received for publication, June 2, 2004 , and in revised form, September 21, 2004.

^* This work was supported by a Biotechnology and Biological Sciences Research Council studentship (to S. J. E.) and a Wellcome Trust project grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed. E-mail: matthew.guille{at}port.ac.uk.

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