Evidence That Gal11 Protein Is a Target of the Gal4 Activation Domain in the Mediator

Abstract

The mediator is an approximately 20 protein complex that is essential for the transcription of most genes in yeast. It is contacted by a number of gene-specific activators, but the details of these interactions are not well understood in most cases. Here, evidence is presented that the mediator component Gal11 represents at least one target of the Gal4 activation domain (AD). Deletion of Gal11 is shown to decrease the affinity of the Gal4 AD for the mediator, and direct binding of an N-terminal domain of Gal11 with the Gal4 AD is demonstrated. Quantitative studies, however, indicate that the K(D) of the 1:1 Gal4 AD--Gal11 complex is modest. Combined with in vivo data showing that Delta gal11 cells exhibit reduced, but still significant, Gal4-mediated gene expression, these results suggest that the dimeric activator might also contact another protein in the mediator in addition to Gal11.

In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer

“…For example, in some assays direct interactions between Gal4 and Mediator have been detected (Koh et al 1998;Park et al 2000;Jeong et al 2001). Consistent with this possibility, Bryant and Ptashne (2003) reported that Mediator was recruited to the GAL1 promoter in an spt20⌬ mutant, prompting the suggestion that in the initial stages of activation, SAGA and Mediator are independently contacted and recruited by Gal4.…”

“…The well-characterized acidic activator Gal4 is responsible for the transcriptional stimulation of GAL genes, such as GAL1, which contain Gal4-binding sites in their promoters (Johnston 1987;Johnston and Carlson 1992;Dudley et al 1999). A variety of transcriptional components have been proposed to be the target of Gal4 including TBP (Melcher and Johnston 1995;Wu et al 1996), TFIIB (Wu et al 1996), Srb4 (Koh et al 1998;Park et al 2000), Gal11 (Jeong et al 2001), the Swi/Snf complex (Neely et al 2002), and the SAGA (Spt/Ada/ Gcn5/acetyltransferase) complex (Bhaumik and Green 2001;Brown et al 2001;Larschan and Winston 2001). These proposals are based on either in vitro protein interaction studies or inferences from various indirect in vivo experiments, and to date there is no definitive evidence that Gal4 directly interacts with any of these putative targets in vivo.…”

Rapid GAL Gene Switch of Saccharomyces cerevisiae Depends on Nuclear Gal3, Not Nucleocytoplasmic Trafficking of Gal3 and Gal80

“…Gal4 is found associated with the UAS GAL DNA site(s) of the GAL genes in both absence and presence of galactose, but in the absence of galactose it is unable to activate transcription due to masking of its ADII by Gal80 (Torchia et al 1984;Lue et al 1987;Ma and Ptashne 1987;Chasman and Kornberg 1990;Leuther and Johnston 1992;Mizutani and Tanaka 2003). Galactose facilitates association of the Gal3 and the Gal80 proteins, an event that brings about dissociation of Gal80 from Gal4 (Peng and Hopper 2002;Jiang et al 2009) and subsequent Gal4-mediated recruitment of RNA polymerase to UAS GAL -associated promoters (Torchia and Danzinger 1980;Johnston et al 1987;Lue et al 1987;Chasman and Kornberg 1990;Yun et al 1991;Leuther and Johnston 1992;Parthun and Jaehning 1992;Melcher and Johnston 1995;Suzuki-Fujimoto et al 1996;Wu et al 1996;Blank et al 1997;Yano and Fukasawa 1997;Koh et al 1998;Platt and Reece 1998;Vollenbroich et al 1999;Bhaumik and Green 2001;Jeong et al 2001;Larschan and Winston 2001;Melcher and Xu 2001;Carrozza et al 2002;Timson et al 2002;Bryant and Ptashne 2003;Menezes et al 2003).…”

A target essential for the activity of a nonacidic yeast transcriptional activator

“…Of the four promoters we have tested, Gal1, His3, Pho5, and Cyc1, all are activated efficiently by activators bearing the P201 activating region (data not shown). Gal11 also has been proposed to be a target for various acidic activators (37)(38)(39). If so, that contact is not essential, because deletion of Gal11 has only a modest (2-5-fold) effect on activation in those cases.…”