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Molecular and Cellular Biology, October 2009, p. 5604-5610, Vol. 29, No. 20
0270-7306/09/$08.00+0     doi:10.1128/MCB.00632-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Gene Activation by Dissociation of an Inhibitor from a Transcriptional Activation Domain{triangledown}

Fenglei Jiang,1 Benjamin R. Frey,2 Margery L. Evans,3 Jordan C. Friel,4 and James E. Hopper1*

Department of Biochemistry, the Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210,1 Molecular Pathology Laboratory, the Ohio State University, 680 Ackerman Road, Columbus, Ohio 43202,2 Department of Molecular, Cellular, and Developmental Biology, University of Michigan, 830 North University Avenue, Ann Arbor, Michigan 48109,3 Department of Molecular Genetics, the Ohio State University, 484 West 12th Avenue, Columbus, Ohio 432104

Received 15 May 2009/ Returned for modification 10 June 2009/ Accepted 31 July 2009

Gal4 is a prototypical eukaryotic transcriptional activator whose recruitment function is inhibited in the absence of galactose by the Gal80 protein through masking of its transcriptional activation domain (AD). A long-standing nondissociation model posits that galactose-activated Gal3 interacts with Gal4-bound Gal80 at the promoter, yielding a tripartite Gal3-Gal80-Gal4 complex with altered Gal80-Gal4 conformation to enable Gal4 AD activity. Some recent data challenge this model, whereas other recent data support the model. To address this controversy, we imaged fluorescent-protein-tagged Gal80, Gal4, and Gal3 in live cells containing a novel GAL gene array. We find that Gal80 rapidly dissociates from Gal4 in response to galactose. Importantly, this dissociation is Gal3 dependent and concurrent with Gal4-activated GAL gene expression. When galactose-triggered dissociation is followed by galactose depletion, preexisting Gal80 reassociates with Gal4, indicating that sequestration of Gal80 by Gal3 contributes to the observed Gal80-Gal4 dissociation. Moreover, the ratio of nuclear Gal80 to cytoplasmic Gal80 decreases in response to Gal80-Gal3 interaction. Taken together, these and other results provide strong support for a GAL gene switch model wherein Gal80 rapidly dissociates from Gal4 through a mechanism that involves sequestration of Gal80 by galactose-activated Gal3.

* Corresponding author. Mailing address: Department of Biochemistry, the Ohio State University, 484 West 12th Avenue, Columbus, OH 43210. Phone: (614) 247-2552. Fax: (614) 292-6773. E-mail: hopper.65{at}osu.edu

{triangledown} Published ahead of print on 3 August 2009.

Molecular and Cellular Biology, October 2009, p. 5604-5610, Vol. 29, No. 20
0270-7306/09/$08.00+0     doi:10.1128/MCB.00632-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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