An Activator Target in the RNA Polymerase II Holoenzyme

Abstract

Expression of protein-coding genes in eukaryotes involves the recruitment, by transcriptional activator proteins, of a transcription initiation apparatus consisting of greater than 50 polypeptides. Recent genetic and biochemical evidence in yeast suggests that a subset of these proteins, called SRB proteins, are likely targets for transcriptional activators. We demonstrate here, through affinity chromatography, photo-cross-linking, and surface plasmon resonance experiments, that the GAL4 activator interacts directly with the SRB4 subunit of the RNA polymerase II holoenzyme. The GAL4 activation domain binds to two essential segments of SRB4. The physiological relevance of this interaction is confirmed by mutations in SRB4, which occur within its GAL4-binding domain and which restore activation in vivo by a GAL4 derivative bearing a mutant activation domain.

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

“…To determine whether Mediator has a role in recruitment of SAGA or Gal4, we performed ChIP assays in a strain harboring a temperature-sensitive mutation in the Mediator subunit Srb4, which is required for complex integrity (Koh et al 1998;Kang et al 2001). Figure 6C shows, as expected, that inactivation of Srb4 substantially decreased recruitment of Mediator (Srb2) to the UAS.…”

“…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.…”

On the Mechanism of Constitutive Pdr1 Activator-mediated PDR5 Transcription in Saccharomyces cerevisiae

“…To address the role of Mediator in regulating PDR5 transcription, we analyzed the effect of inactivating Srb4 on PDR5 transcription. The Mediator subunit Srb4 is required for the integrity of the complex (45,46). Thermal inactivation of temperature-sensitive Srb4-2 caused a dramatic reduction of PDR5 transcription and, in fact, the effect of the srb4-2 mutation on PDR5 was noticeable even at 25°C (Fig.…”

Genetic Interactions Between Mediator and the Late G1-Specific Transcription Factor Swi6 in Saccharomyces cerevisiae

“…A fourth, less tightly associated module composed of Srb8, -9, -10, and -11 has also been isolated as a separate complex with kinase activity Borggrefe et al 2002). The head module includes eight tightly associated proteins (Koh et al 1998) and is thought to interact with the C-terminal domain (CTD) of RNA polymerase II (Lee and Kim 1998) and with two of the general transcription factors (TBP, TFIIB). The middle module with its eight subunits interacts with the CTD and TFIIE and the Srb8-11 module of the mediator (Kang et al 2001).…”