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Molecular and Cellular Biology, June 2000, p. 4359-4370, Vol. 20, No. 12
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Mechanism for Specificity by HMG-1 in Enhanceosome Assembly

Katharine B. Ellwood, Yi-Meng Yen, Reid C. Johnson, and Michael Carey*

Department of Biological Chemistry, University of California at Los Angeles School of Medicine, Los Angeles, California 90095-1737

Received 8 March 2000/Accepted 29 March 2000

Assembly of enhanceosomes requires architectural proteins to facilitate the DNA conformational changes accompanying cooperative binding of activators to a regulatory sequence. The architectural protein HMG-1 has been proposed to bind DNA in a sequence-independent manner, yet, paradoxically, it facilitates specific DNA binding reactions in vitro. To investigate the mechanism of specificity we explored the effect of HMG-1 on binding of the Epstein-Barr virus activator ZEBRA to a natural responsive promoter in vitro. DNase I footprinting, mutagenesis, and electrophoretic mobility shift assay reveal that HMG-1 binds cooperatively with ZEBRA to a specific DNA sequence between two adjacent ZEBRA recognition sites. This binding requires a strict alignment between two adjacent ZEBRA sites and both HMG boxes of HMG-1. Our study provides the first demonstration of sequence-dependent binding by a nonspecific HMG-box protein. We hypothesize how a ubiquitous, nonspecific architectural protein can function in a specific context through the use of rudimentary sequence recognition coupled with cooperativity. The observation that an abundant architectural protein can bind DNA cooperatively and specifically has implications towards understanding HMG-1's role in mediating DNA transactions in a variety of enzymological systems.

* Corresponding author. Mailing address: Department of Biological Chemistry, UCLA School of Medicine, Box 1737, Los Angeles, CA 90095-1737. Phone: (310) 206-7859. Fax: (310) 206-9598. E-mail: mcarey{at}ucla.edu.

Molecular and Cellular Biology, June 2000, p. 4359-4370, Vol. 20, No. 12
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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