The Med proteins of yeast and their function through the RNA polymerase II carboxy-terminal domain (original) (raw)
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Evidence for a Mediator of RNA Polymerase II Transcriptional Regulation Conserved from Yeast to Man
Cell, 2002
S. cerevisiae showing interference between different ac-France tivator proteins (Gill and Ptashne, 1988). Purification of an activity that could relieve this interference led to the identification of a 20 subunit entity termed Mediator Mediator complexes (MED) link transcriptional regula-(Flanagan et al., 1991; Kelleher et al., 1990; Kim et al., tors to RNA polymerase II. Here, we summarize the 1994). A concomitant genetic analysis revealed that the latest advances on the functional organization of yeast Mediator complex is functionally linked to Pol II, notably Mediator. We argue for the existence of a "universal" through a direct physical contact with the C-terminal Mediator structurally conserved from yeast to man, domain (CTD) of its largest subunit (Rpb1) (Thompson et based on an extensive analysis of sequence dataal., 1993). Subsequently, Mediator was shown to directly bases. Finally, we examine the implications of these interact with DNA-tethered transcriptional activators, ilobservations for the physiological roles of metazoan luminating how the latter might trigger gene activation MED subunits. (Barberis et al., 1995; Hengartner et al., 1995). In a welldefined reconstituted system, Mediator proved to be Introduction required for both basal and regulated transcription, as well as to stimulate the phosphorylation of Rpb1 CTD In eukaryotes, regulation of protein-coding gene tranby the TFIIH-associated kinase (Kim et al., 1994; Myers scription occurs through control of both chromatin et al., 1998). Genetic analysis with yeast strains lacking structure and initiation by RNA polymerase II (also individual subunits revealed defects in both transcripknown as Pol II or Pol B) (reviewed by Kornberg, 1999; tional activation and repression, thus establishing a piv-Lee and Young, 2000; Naar et al., 2001). According to the otal role for Mediator in controlling Pol II initiation (Li et cell type and/or developmental stage, the combinatorial al., 1995). Mediator is now viewed as a modular and binding of sequence-specific activators and/or represdynamic interface that connects diverse gene-specific sors to cognate DNA enhancer and promoter elements regulatory proteins to the basal Pol II transcriptional finely regulates transcriptional initiation of class II genes initiation apparatus by acting as signal sensor, inte-(reviewed by Davidson, 2001; Lemon and Tjian, 2000). grator, and processor (Kang et al., 2001). The discovery a decade ago in the yeast S. cerevisiae Mediator 3D Structure and Subunit Composition of a transcriptional mediator multiprotein complex (Me-Mediator can be isolated in a free state or associated diator or MED) and the more recent findings of related with core Pol II in a large complex 2ف( MDa) referred to entities in metazoans have radically changed our view as holoenzyme (Kim et al., 1994; Koleske and Young, of how diverse gene-specific transcriptional activators 1994). As shown in Figure 1, electron microscopy of the and repressors could transmit their regulatory informapurified MED/Pol II holoenzyme complex revealed three tion to the basal Pol II initiation apparatus (reviewed major Mediator domains that wrap around the globular by Bjorklund et al., 2001; Gustafsson and Samuelsson, polymerase, termed head (h), middle (m), and tail (t) 2001; Malik and Roeder, 2000; Myers and Kornberg, (Asturias et al., 1999; Dotson et al., 2000). In contrast, 2000). The purposes of the present review are to summaisolated Mediator appears as a compact structure, indirize the recent advances in our understanding of the cating that it must undergo an extensive conformational structural and functional organization of yeast Mediator, transition to envelope Pol II in the holoenzyme complex to discuss the existence of a "universal" Mediator inter-(Asturias et al., 1999). In agreement with this idea, the face conserved between yeast and humans, based on conformation of an isolated human MED-related coma comprehensive analysis of sequence databases, and plex (i.e., CRSP; see below) dramatically changes deto emphasize insights from recent in vivo studies of pending on the particular activator to which it is bound the physiological roles of MED subunits in multicellular (Taatjes et al., 2002). Further, activator bound CRSP has organisms. recently been shown to adopt a specific CTD bound conformation (Naar et al., 2002). Structural and Functional Organization of the Yeast The S. cerevisiae core MED complex contains five Transcriptional Mediator Complex subunits originally identified in genetic screens for sup-Discovery of Mediator pressors of Rpb1 CTD truncation mutations, i.e., Srb2 The basal Pol II initiation machinery is highly conserved and Srb4-7 (suppressor of RNA polymerase B) (Thompamong eukaryotes and comprises at least 65 proteins, son et al., 1993). Eight other subunits emerged from among them the 12 subunit core polymerase and its disparate genetic screens for positive as well as negacognate general transcription factors (GTFs) TFIIA, -B, tive transcriptional regulators, i.e.,
Functional Interactions within Yeast Mediator and Evidence of Differential Subunit Modifications
Journal of Biological Chemistry, 2002
It is possible to recruit RNA polymerase II to a target promoter and, thus, activate transcription by fusing Mediator subunits to a DNA binding domain. To investigate functional interactions within Mediator, we have tested such fusions of the lexA DNA binding domain to Med1, Med2, Gal11, Srb7, and Srb10 in wild type, med1, med2, gal11, sin4, srb8, srb10, and srb11 strains. We found that lexA-Med2 and lexA-Gal11 are strong activators that are independent of all Mediator subunits tested. lexA-Srb10 is a weak activator that depends on Srb8 and Srb11. lexA-Med1 and lexA-Srb7 are both cryptic activators that become active in the absence of Srb8, Srb10, Srb11, or Sin4. An unexpected finding was that lexA-VP16 differs from Gal4-VP16 in that it is independent of the activator binding Mediator module. Both lexA-Med1 and lexA-Srb7 are stably associated with Med4 and Med8, which suggests that they are incorporated into Mediator. Med4 and Med8 exist in two mobility forms that differ in their association with lexA-Med1 and lexA-Srb7. Within purified Mediator, Med4 is present as a phosphorylated lower mobility form. Taken together, these results suggest that assembly of Mediator is a multistep process that involves conversion of both Med4 and Med8 to their low mobility forms. RNA polymerase II (pol II) 1 transcribes all protein-encoding genes and some small nuclear RNA genes in eukaryotes. The yeast pol II holoenzyme (1) consists of a catalytic core enzyme of 12 subunits (2, 3) whose crystal structure has been solved (4, 5) and a regulatory Mediator complex comprising 20 subunits. All subunits of the Mediator have now been identified, and 13 of them are encoded by previously known genes. The remaining seven subunits are novel proteins named Med1, Med2, Med4, Med6, Med7, Med8, and Med11 (6-12). Transcription can be reconstituted in vitro from highly purified pol II core enzyme and five general transcription factors, which are the TATA
Journal of Biological Chemistry, 2003
The multiprotein Mediator complex is a coactivator required for activation of RNA polymerase II transcription by DNA bound transcription factors. We previously identified and partially purified a mammalian Mediator complex from rat liver nuclei (Brower, C.Analysis by tandem mass spectrometry of proteins present in the most highly purified rat Mediator fractions led to the identification of a collection of new mammalian Mediator subunits, as well as several potential Mediator subunits including a previously uncharacterized protein encoded by the FLJ10193 open reading frame. In this study, we present direct biochemical evidence that the FLJ10193 protein, which we designate Med25, is a bona fide subunit of the mammalian Mediator complex. In addition, we present evidence that Med25 shares structural and functional properties with Saccharomyces cerevisiae Mediator subunit Cse2 and may be a mammalian Cse2 ortholog. Taken together, our findings identify a novel mammalian Mediator subunit and shed new light on the architecture of the mammalian Mediator complex.
Mechanisms of Mediator complex action in transcriptional activation
Cellular and Molecular Life Sciences, 2013
The transcriptional control of genes transcribed by RNA polymerase II (Pol II) depends on diverse transcriptional regulators that include transcriptional activators, a set of general transcription factors (GTFs), and transcription cofactors [1]. The initiation of transcription generally begins when genespecific activators bind to their cognate sites, or when prebound activators are modified, leading to the recruitment of the general transcription machinery comprising a host of GTFs and RNA Pol II to form the pre-initiation complex (PIC). To accomplish this task, activators interact with a number of cofactors to communicate the activation signal to RNA Pol II and the GTFs. GTFs, comprising TFIIA, B, D, E, F, and H, are defined by their requirement for accurate and efficient transcription from all (or nearly all) Pol II transcribed genes [2, 3]. The transcription cofactors (coactivators and corepressors), which by definition do not directly bind to specific DNA sequences, act at specific subsets of genes and can be broadly categorized into two classes. The first class consists of complexes or proteins that modify the histones on promoter DNA or remodel the chromatin in an ATP dependent manner-for example, the Swi/Snf complex in yeast and CBP in mammalian cells [4, 5]. The other class includes general cofactors such as the Mediator complex that function principally to recruit or activate GTFs and other key components needed for transcriptional activation or repression. Some cofactors belong to both categories: the SAGA complex, for example, activates genes in yeast both by recruiting TBP via the Spt3/Spt8 sub-module and by acetylating histones, and the requirement for these two functions varies at different genes [6, 7]. Mediator plays a central role in the eukaryotic transcription program and the mechanisms of its action known so far seem to be conserved. Mediator is a large multiprotein Abstract Mediator is a large multisubunit complex that plays a central role in the regulation of RNA Pol II transcribed genes. Conserved in overall structure and function among eukaryotes, Mediator comprises 25-30 protein subunits that reside in four distinct modules, termed head, middle, tail, and CDK8/kinase. Different subunits of Mediator contact other transcriptional regulators including activators, co-activators, general transcription factors, subunits of RNA Pol II, and specifically modified histones, leading to the regulated expression of target genes. This review is focused on the interactions of specific Mediator subunits with diverse transcription regulators and how those interactions contribute to Mediator function in transcriptional activation.
The mammalian Mediator complex and its role in transcriptional regulation
Trends in Biochemical Sciences, 2005
Cover: Synthesis of mRNA in eukaryotes is carried out by RNA polymerase II (pol II), the enzyme that provides the catalytic activity for RNA polymerization. To recognise promoter DNA and initiate transcription, pol II must associate with at least five general transcription factors that, together, assemble on a promoter to form the transcription preinitiation complex. The multiprotein Mediator complex functions as an essential regulatory component of this transcription machinery and has a crucial role in the activation and repression of eukaryotic mRNA synthesis. This special issue focuses on Mediator in a series of reviews commissioned by Roger Kornberg of the TiBS Editorial Board. (The cover image is the structural interaction of Mediator and pol II, termed the 'holoenzyme complex'.)
The yeast Mediator complex and its regulation
Trends in Biochemical Sciences, 2005
The Mediator complex acts as a bridge, conveying regulatory information from enhancers and other control elements to the basal RNA polymerase II transcription machinery. Mediator is required for the regulated transcription of nearly all RNA polymerase II-dependent genes in Saccharomyces cerevisiae, and post-translational modifications of specific Mediator subunits can affect global patterns of gene transcription. Mediator is a conserved co-regulator of gene transcription The Saccharomyces cerevisiae Mediator complex was originally identified as a requirement for activatordependent stimulation of RNA polymerase II (pol II) transcription [1,2]. The S. cerevisiae Mediator complex comprises 21 subunits and it is found both in free form and as a holoenzyme in a complex with pol II [3,4]. Mediator structure and function seems to be conserved in mammalian cells and a unifying nomenclature was recently proposed [5]. Here, we describe recent advances in our understanding of yeast Mediator and its regulation by post-translational modification.
Genome-wide association of mediator and RNA polymerase II in wild-type and mediator mutant yeast
Molecular and cellular biology, 2015
Mediator is a large, multisubunit complex that is required for essentially all mRNA transcription in eukaryotes. In spite of the importance of Mediator, the range of its targets and how it is recruited to these is not well understood. Previous work showed that in Saccharomyces cerevisiae, Mediator contributes to transcriptional activation by two distinct mechanisms, one depending on the tail module triad and favoring SAGA-regulated genes, and the second occurring independently of the tail module and favoring TFIID-regulated genes. Here, we use chromatin immunoprecipitation sequencing (ChIP-seq) to show that dependence on tail module subunits for Mediator recruitment and polymerase II (Pol II) association occurs preferentially at SAGA-regulated over TFIID-regulated genes on a genome-wide scale. We also show that recruitment of tail module subunits to active gene promoters continues genome-wide when Mediator integrity is compromised in med17 temperature-sensitive (ts) yeast, demonstra...
The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation
Nature Reviews Genetics, 2010
The Mediator is an evolutionarily conserved, multiprotein complex that is a key regulator of protein-coding genes. In metazoan cells, multiple pathways that are responsible for homeostasis, cell growth and differentiation converge on the Mediator through transcriptional activators and repressors that target one or more of the almost 30 subunits of this complex. Besides interacting directly with RNA polymerase II, Mediator has multiple functions and can interact with and coordinate the action of numerous other co-activators and co-repressors, including those acting at the level of chromatin. These interactions ultimately allow the Mediator to deliver outputs that range from maximal activation of genes to modulation of basal transcription to long-term epigenetic silencing. The unique transcription programmes of protein-coding genes are dependent on regulatory sequences that are bound by a wide range of gene-and tissue-specific transcriptional factors, which respond to developmental and environmental signals. However, the programme is interpreted largely through the precisely orchestrated recruitment of co-activators by these specific transcription factors 1. To take effect, the regulatory signals must be properly relayed to the general RNA polymerase II (Pol II) machinery that is responsible for transcribing protein-coding genes. This machinery is involved in the formation and function of a preinitiation complex (PIC), which-in addition to Pol II-consists of the general transcription factors (GTFs) TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH. Consistent with a broad two-step mechanism for transcription (FIG. 1), studies over the past two decades have identified numerous co-activators that facilitate the initial penetration of the chromatin barrier to allow the transcriptional machinery access to the regulatory sequences or directly target PIC components. Of the limited number of co-activators that are known to directly target Pol II, the 30-subunit Mediator complex has emerged as potentially the most crucial by virtue of its widespread and multifaceted role across the transcriptome (BOX 1). Box 1 Generality of Mediator action A long-standing issue concerns whether Mediator is required for expression of all genes in a genome. The earliest data from genome-wide analyses in yeast indicated that inactivation of the core MED17 subunit affects transcription of the majority of genes in yeast cells; indeed, the phenotype of these mutants was found to be essentially