Yeast global transcriptional regulators Sin4 and Rgr1 are components of mediator complex/RNA polymerase II holoenzyme (original) (raw)
Related papers
Journal of Biological Chemistry, 1998
Mediator was isolated from yeast on the basis of its requirement for transcriptional activation in a fully defined system. We have now identified three new members of mediator in the low molecular mass range by peptide sequence determination. These are the products of the NUT2, CSE2, and MED11 genes. The product of the NUT1 gene is evidently a component of mediator as well. NUT1 and NUT2 were earlier identified as negative regulators of the HO promoter, whereas mutations in CSE2 affect chromosome segregation. MED11 is a previously uncharacterized gene. The existence of these proteins in the mediator complex was verified by copurification and co-immunoprecipitation with RNA polymerase II holoenzyme.
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.,
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...
Mediator Influences Schizosaccharomyces pombe RNA Polymerase II-dependent Transcription in Vitro
Journal of Biological Chemistry, 2003
The fission yeast Schizosaccharomyces pombe has proved an important model system for cross-species comparative studies of many fundamental processes in the eukaryotic cell, such as cell cycle control and DNA replication. The RNA polymerase II transcription machinery is, however, still relatively poorly understood in S. pombe, partially due to the absence of a reconstituted in vitro transcription system. We have now purified S. pombe RNA polymerase II and its general initiation factors TFIIB, TFIIF, TFIIE, and TFIIH to near homogeneity. These factors enable RNA polymerase II to initiate transcription from the S. pombe alcohol dehydrogenase promoter (adh1p) when combined with Saccharomyces cerevisiae TATA-binding protein. We use our reconstituted system to examine effects of Mediator on basal transcription in vitro. S. pombe Mediator exists in two distinct forms, a free form, which contains the spSrb8, spTrap240, spSrb10, and spSrb11 subunits, and a smaller form, which lacks these four subunits and associates with RNA polymerase II to form a holoenzyme. We find that spSrb8/spTrap240/spSrb10/spSrb11 containing Mediator repress basal transcription, whereas Mediator lacking these subunits has a stimulatory effect on transcription. Our findings thus demonstrate that the spSrb8/spTrap240/spSrb10/spSrb11 subcomplex governs the ability of Mediator to stimulate or repress basal transcription in vitro.
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
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.
Architectural Mediator subunits are differentially essential for global transcription in yeast
SummaryThe modular Mediator complex is a coactivator of RNA polymerase II transcription. We show that depletion of the main complex scaffold Med14 or the head module scaffold Med17 is lethal and results in global transcriptional downregulation in yeast, though Med17 removal has a markedly greater negative effect. Depletion of Med14 or Med17 impairs pre-initiation complex (PIC) assembly similarly, suggesting that the differential transcriptional effects observed are not due to differing extents of defective PIC formation. Co-depletion of Med14 and Med17 reduced transcription and TFIIB promoter occupancy similarly to Med17 ablation alone, suggesting that the independent head module can weakly stimulate transcription in vivo, though not to a level that maintains viability. We suggest that, while the structural integrity of complete Mediator and the head module are both important for PIC assembly, the head module additionally promotes optimal PIC function and is thus the key functional ...
The Med proteins of yeast and their function through the RNA polymerase II carboxy-terminal domain
Genes & Development, 1998
Mediator was resolved from yeast as a multiprotein complex on the basis of its requirement for transcriptional activation in a fully defined system. Three groups of mediator polypeptides could be distinguished: the products of five SRB genes, identified as suppressors of carboxy-terminal domain (CTD)-truncation mutants; products of four genes identified as global repressors; and six members of a new protein family, termed Med, thought to be primarily responsible for transcriptional activation. Notably absent from the purified mediator were Srbs 8, 9, 10, and 11, as well as members of the SWI/SNF complex. The CTD was required for function of mediator in vitro, in keeping with previous indications of involvement of the CTD in transcriptional activation in vivo. Evidence for human homologs of several mediator proteins, including Med7, points to similar mechanisms in higher cells.