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Mol Cell Biol, May 1998, p. 2629-2639, Vol. 18, No. 5
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Cooperative Pho2-Pho4 Interactions at the PHO5 Promoter Are Critical for Binding of Pho4 to UASp1 and for Efficient Transactivation by Pho4 at UASp2

Slobodan Barbaric,1,dagger Martin Münsterkötter,1 Colin Goding,2 and Wolfram Hörz1,*

Institut für Physiologische Chemie, Universität München, D-80336 Munich, Germany,1 and Eukaryotic Transcription Laboratory, Marie Curie Research Institute, Surrey RH8 0TL, United Kingdom2

Received 24 October 1997/Returned for modification 2 December 1997/Accepted 17 February 1998

The activation of the PHO5 gene in Saccharomyces cerevisiae in response to phosphate starvation critically depends on two transcriptional activators, the basic helix-loop-helix protein Pho4 and the homeodomain protein Pho2. Pho4 acts through two essential binding sites corresponding to the regulatory elements UASp1 and UASp2. Mutation of either of them results in a 10-fold decrease in promoter activity, and mutation of both sites renders the promoter totally uninducible. The role of Pho4 appears relatively straightforward, but the mechanism of action of Pho2 had remained elusive. By in vitro footprinting, we have recently mapped multiple Pho2 binding sites adjacent to the Pho4 sites, and by mutating them individually or in combination, we now show that each of them contributes to PHO5 promoter activity. Their function is not only to recruit Pho2 to the promoter but to allow cooperative binding of Pho4 together with Pho2. Cooperativity requires DNA binding of Pho2 to its target sites and Pho2-Pho4 interactions. A Pho4 derivative lacking the Pho2 interaction domain is unable to activate the promoter, but testing of UASp1 and UASp2 individually in a minimal CYC1 promoter reveals a striking difference between the two UAS elements. UASp1 is fully inactive, presumably because the Pho4 derivative is not recruited to its binding site. In contrast, UASp2 activates strongly in a Pho2-independent manner. From in vivo footprinting experiments and activity measurements with a promoter variant containing two UASp2 elements, we conclude that at UASp2, Pho2 is mainly required for the ability of Pho4 to transactivate.

* Corresponding author. Mailing address: Institut für Physiologische Chemie, Universität München, Schillerstr. 44, D-80336 Munich, Germany. Phone: 89-5996 420. Fax: 89-5996 440. E-mail: Hoerz{at}bio.med.uni-muenchen.de.

dagger Present address: Laboratory of Biochemistry, Faculty of Food Technology and Biotechnology, University of Zagreb, 10000 Zagreb, Croatia.

Mol Cell Biol, May 1998, p. 2629-2639, Vol. 18, No. 5
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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