Mutual Targeting of Mediator and the TFIIH Kinase Kin28

Guidi, Benjamin W.; Bjornsdottir, Gudrun; Hopkins, Daniel; Lacomis, Lynne; Erdjument‐Bromage, Hediye; Tempst, Paul; Myers, Lawrence C.

doi:10.1074/jbc.m404426200

Cited by 43 publications

(41 citation statements)

References 54 publications

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“…The general transcription factors used for all of the reactions in this study came from identical aliquots of a single preparation. The kinase assays with purified Mediator complexes, TFIIK, and GST-CTD were performed as previously described (30).…”

Section: Methodsmentioning

confidence: 99%

“…We have previously shown that the enhancement of CTD phosphorylation by TFIIH can be completely recapitulated in a reaction containing purified Mediator, TFIIK (the kinase module of TFIIH) and a GST-CTD fusion protein (30). We repeated these assays for the purified wild type, ⌬med19(rox3)-HT, and ⌬med19(rox3)-HMT Mediators.…”

Section: The Middle Module Of Mediator Is Required For Activated and mentioning

confidence: 99%

“…Immunoblots show that purified RNA Pol II is retained by the immobilized wild type Mediator but that under identical conditions the ⌬med19(rox3)-HMT Mediator does not retain a detectable amount of RNA Pol II (Fig. 5B) (6, 7) and is also a target of this kinase activity on at least two subunits (30,36). To determine whether the decreased affinity of the ⌬med19(rox3) Mediator for RNA Pol II and the Fig.…”

Section: The Middle Module Of Mediator Is Required For Activated and mentioning

confidence: 99%

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Med19(Rox3) Regulates Intermodule Interactions in the Saccharomyces cerevisiae Mediator Complex

Baidoobonso

Guidi

Myers

2007

Journal of Biological Chemistry

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The Saccharomyces cerevisiae Mediator is a 25-subunit complex that facilitates both transcriptional activation and repression. Structural and functional studies have divided Mediator subunits into four distinct modules. The Head, Middle, and Tail modules form the core functional Mediator complex, whereas a fourth, the Cyc-C module, is variably associated with the core. By purifying Mediator from a strain lacking the Med19(Rox3) subunit, we have found that a complex missing only the Med19(Rox3) subunit can be isolated under mild conditions. Additionally, we have established that the entire Middle module is released when the ⌬med19(rox3) Mediator is purified under more stringent conditions. In contrast to most models of the modular structure of Mediator, we show that release of the Middle module in the ⌬med19(rox3) Mediator leaves a stable complex made up solely of Head and Tail subunits. Both the intact and Head-Tail ⌬med19(rox3) Mediator complexes have defects in enhanced basal transcription, enhanced TFIIH phosphorylation of the CTD, as well as binding of RNA Pol II and the CTD. The largely intact ⌬med19(rox3) complex facilitates activated transcription at levels similar to the wild type Mediator. In the absence of the Middle module, however, the ⌬med19(rox3) Mediator is unable to facilitate activated transcription. Although the Middle module is unnecessary for holding the Head and Tail modules together, it is required for the complex to function as a conduit between activators and the core transcription machinery.

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Section: Methodsmentioning

confidence: 99%

Section: The Middle Module Of Mediator Is Required For Activated and mentioning

confidence: 99%

Section: The Middle Module Of Mediator Is Required For Activated and mentioning

confidence: 99%

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Med19(Rox3) Regulates Intermodule Interactions in the Saccharomyces cerevisiae Mediator Complex

Baidoobonso

Guidi

Myers

2007

Journal of Biological Chemistry

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“…The kinase module has also been shown to negatively regulate transcription, either via Cdk8/Srb10 phosphorylating the pol II CTD prior to formation of the preinitiation complex (PIC) or by its directly phosphorylating DNA-bound activators, thereby leading to their ubiquitin-mediated proteolysis or export from the nucleus (Chi et al 2001). Subsequent to PIC formation, Mediator enhances Kin28 phosphorylation of the pol II CTD at Ser5 (Guidi et al 2004), suggesting a way in which Mediator may positively regulate gene transcription. These and other observations point to a complex role for Mediator as an integrator of intracellular signals.…”

mentioning

confidence: 99%

A Functional Module of Yeast Mediator That Governs the Dynamic Range of Heat-Shock Gene Expression

Singh

Erkine²,

Kremer³

et al. 2006

Genetics

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We report the results of a genetic screen designed to identify transcriptional coregulators of yeast heatshock factor (HSF). This sequence-specific activator is required to stimulate both basal and induced transcription; however, the identity of factors that collaborate with HSF in governing noninduced heatshock gene expression is unknown. In an effort to identify these factors, we isolated spontaneous extragenic suppressors of hsp82-DHSE1, an allele of HSP82 that bears a 32-bp deletion of its high-affinity HSF-binding site, yet retains its two low-affinity HSF sites. Nearly 200 suppressors of the null phenotype of hsp82-DHSE1 were isolated and characterized, and they sorted into six expression without heat-shock element (EWE) complementation groups. Strikingly, all six groups contain alleles of genes that encode subunits of Mediator. Three of the six subunits, Med7, Med10/Nut2, and Med21/Srb7, map to Mediator's middle domain; two subunits, Med14/Rgr1 and Med16/Sin4, to its tail domain; and one subunit, Med19/Rox3, to its head domain. Mutations in genes encoding these factors enhance not only the basal transcription of hsp82-DHSE1, but also that of wild-type heat-shock genes. In contrast to their effect on basal transcription, the more severe ewe mutations strongly reduce activated transcription, drastically diminishing the dynamic range of heat-shock gene expression. Notably, targeted deletion of other Mediator subunits, including the negative regulators Cdk8/Srb10, Med5/Nut1, and Med15/Gal11 fail to derepress hsp82-DHSE1. Taken together, our data suggest that the Ewe subunits constitute a distinct functional module within Mediator that modulates both basal and induced heat-shock gene transcription. W HEN exposed to thermal or chemical stress, organisms respond by vigorously transcribing genes encoding heat-shock proteins (HSPs). HSPs function as molecular chaperones and protect the cellalong with ubiquitin, proteases, metallothioneins, and antioxidant enzymes-from damage caused by the expression of misfolded proteins. In the yeast Saccharomyces cerevisiae, the expression of heat-responsive genes is stimulated by the sequence-specific transcriptional activator heat-shock factor (HSF) Hsf1 (ScHSF) (Sorger and Pelham 1988;Nieto-Sotelo et al. 1990;Sorger 1990). In response to metabolic, oxidative, or osmotic stress, the transcription of a number of HSP genes is additionally enhanced by the gene-specific activators Msn2/Msn4 and Skn7 (Boy-Marcotte et al. 1998;Treger et al. 1998;Gasch et al. 2000;Raitt et al. 2000;Amoros and Estruch 2001;Kandror et al. 2004). Nonetheless, the only activator known to promote basal heat-shock gene transcription is HSF (McDaniel et al. 1989;Park and Craig 1989;Erkine et al. 1996). Whether this basal expression is an indirect consequence of HSF's role in establishing and maintaining a nucleosomeremodeled (''nucleosome-free'') structure over the transcription start site Erkine et al. 1996), or whether HSF plays a more direct role in recruiting transcriptional coactivators under n...

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“…Additionally, yeast Kin28 phosphorylates two subunits of Mediator (i.e., Med4 and Rgr1/Med14), and although the functionally of these modifications is unknown, it has been demonstrated that Mediator significantly enhances the phosphorylation of RNAPII CTD by Kin28 [94,96]. In fact, in vitro assembly of TFIIH into a pre-initiation complex requires Mediator [139], and following transcription initiation, phosphorylation of Ser5 by Kin28 parallels with the release of Mediator from the CTD of RNAPII as promoter clearance occurs [80].…”

Section: Cdk7/cych and Kin28/ccl1-tfb3mentioning

confidence: 99%