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J Biol Chem, Vol. 273, Issue 43, 27757-27760, October 23, 1998
From the Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142 and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
The RNA polymerase II holoenzyme is the form of
eukaryotic RNA polymerase II that is recruited to the promoters of
protein-coding genes in living cells. The exact composition of the
holoenzyme is not entirely established, due in part to technical
difficulties associated with purifying intact megadalton size
multiprotein complexes. Nonetheless, yeast and human holoenzyme
preparations have been described that consist of near stoichiometric
levels of most components known to be generally involved in initiation other than TATA-binding protein
(TBP)1 and its associated
factors. We review here the functions of five major components of yeast
RNA polymerase II holoenzymes: core RNA polymerase II, the general
transcription factors (GTFs), the core Srb-mediator complex, the Srb10
cyclin-dependent kinase (CDK) complex, and the Swi-Snf
complex (Table I).
INTRODUCTION
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Introduction
References
RNA polymerase II holoenzyme components in S. cerevisiae
The holoenzyme concept stems from the discovery that Srb proteins are critical for regulated transcription of protein coding genes and the observation that these proteins are tightly associated with a portion of core RNA polymerase II in yeast cells (1). The genes encoding the yeast Srb proteins were discovered through a genetic screen designed to identify components of the transcription apparatus that are involved in the response to transcriptional regulators (2, 3). Attempts to purify these proteins led to the isolation of a large complex containing core RNA polymerase II, a subset of the general transcription factors, and a variety of regulatory proteins (1). This holoenzyme complex had the capacity to initiate transcription and respond to activators when supplemented with additional purified general transcription factors in vitro. A subcomplex dissociated from the holoenzyme, which contains the Srb and additional proteins, reconstituted the response to activators in a defined in vitro transcription system (4). The response to activators is especially significant as in vitro systems reconstituted with yeast GTFs and polymerase alone are not activator-responsive (5, 6). Two of the yeast Srb proteins were found to be required for transcription of most protein-coding genes, and because they are found tightly associated with the holoenzyme, it seems likely that the Srb-containing holoenzyme is the form of RNA polymerase II that is recruited to most promoters in vivo (7).
RNA polymerase II holoenzymes have been purified from many eukaryotic organisms (1, 4, 8-16). The subunit composition of these different preparations differs somewhat, and these differences involve the presence or absence of two types of components: GTFs and regulatory factors. Some protocols lead to the purification of RNA polymerase II holoenzymes containing all of the GTFs (8, 9, 14), whereas other protocols generate holoenzymes in which only a single GTF (TFIIF) remains associated (4). Some yeast holoenzyme preparations contain stoichiometric levels of Swi-Snf (17), whereas others lack any detectable Swi-Snf protein (18). In the present discussion, we will make the simplifying assumption that holoenzymes in living cells resemble the more complex preparations. Thus, the holoenzyme we discuss here is composed of core RNA polymerase II, all the GTFs other than TBP (and its associated proteins), the core Srb-mediator complex, the Srb10 cyclin-dependent kinase complex, and the Swi-Snf complex (Table I).
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Core RNA Polymerase II |
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Eukaryotic core RNA polymerase II (Pol II) was first purified by using transcription assays with promoterless templates (19, 20). The purified enzyme typically has 10-12 subunits and is incapable of specific promoter recognition. Yeast RNA polymerase II consists of 12 subunits, RPB1-RPB12, which range in size from approximately 6 to 200 kDa. A very similar 12-subunit enzyme can be purified from human cells, and numerous subunit-subunit interactions within the polymerase have been delineated (21). An interesting feature of the enzyme is the highly conserved domain at the C terminus of the largest subunit (CTD). This domain contains multiple repeats of the consensus sequence YSPTSPS and, as discussed below, is a substrate for several kinases that have roles in regulation of gene expression.
There is considerable evidence that the eukaryotic enzymes are highly conserved. Core Pol II molecules purified from a wide range of eukaryotes have conserved subunit structure and sequence. Moreover, many yeast Pol II subunit genes can be replaced with their mammalian counterparts in living cells without deleterious effects on cell function (22-26). This level of functional conservation is remarkable considering the thousands of protein coding genes that must be expressed appropriately for normal cellular function and viability.
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The General Transcription Factors |
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General transcription factors were purified for their ability to facilitate specific promoter recognition by core RNA polymerase II. Five such factors were found to be essential for this activity on most promoters used in vitro: TFIID, TFIIB, TFIIF, TFIIH, and TFIIE. As with core RNA polymerase, the GTFs are highly conserved among eukaryotes. The precise roles of the GTFs have been reviewed in detail elsewhere (27) and will not be discussed here.
Based on the genetic and biochemical evidence to date, we favor the model that transcription activation at many promoters involves recruitment of the transcription initiation apparatus in two steps. In this model, recruitment of two complexes is required for activation: a TBP-containing complex (reviewed in Ref. 28) and a holoenzyme containing the remaining GTFs. Although recruitment of either complex could be sufficient to assemble the other, activators may target members from both complexes and/or multiple components within a single complex. This combination of multiple activator-target interactions would result in transcription. This model does not exclude the possibility that transcription initiation at some promoters involves recruitment of components in many steps, and initiation at other promoters can involve recruitment of the entire apparatus in a single step. Indeed, given the large number of promoters present in living cells and the diverse mechanisms known to regulate gene expression, it seems likely that the entire spectrum of possibilities is realized.
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The Core Srb-Mediator Complex |
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Transcription reactions reconstituted in vitro with highly purified RNA polymerase II and GTFs are not responsive to activators. The yeast mediator is a multiprotein complex that was purified for its ability to mediate activation (4). A less complex "core" Srb-mediator complex has recently been purified (29). This core complex consists of 16 polypeptides: Srb2, -4, -5, -6, and -7, Med1, -2, -4, -6, -7, and -8, Gal11, Sin4, Rgr1, Rox3, and Pgd1. These polypeptides can be separated into several functional groups and are described in more detail below.
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Srb2, -4, -5, and -6 Subcomplex |
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The SRB2, -4, -5, and -6 genes were identified genetically as dominant, gain of function, suppressors of yeast RNA polymerase II large subunit CTD truncation mutants (2, 8, 30). CTD truncation does not appear to reduce the stability of core RNA polymerase II (31) but does reduce the ability of the holoenzyme to respond to activators (32-34). It appears that the SRB gain-of-function mutations compensate for CTD truncations by affecting the ability of activators to interact with the holoenzyme (35) and possibly by increasing its stability.
Recombinant Srb2, Srb4, Srb5, and Srb6 can form a stable complex in vitro (35). Within this complex, Srb2 is associated with Srb5, and Srb4 is associated with Srb6. An interaction between Srb2 and Srb4 is responsible for bringing the four proteins together in a single complex. Srb4 and Srb6 are essential for the transcription of most, if not all, protein-coding genes (7). Srb2 and Srb5 increase stable preinitiation complex formation in nuclear extracts but are not essential for all yeast gene expression (8).
Recent genetic and biochemical studies show that Srb4 is a target of the yeast activator Gal4 (35). Affinity chromatography, photocross-linking, and surface plasmon resonance experiments all demonstrated that the Gal4 activator can interact directly with Srb4. The Gal4 activation domain was found to bind to two essential segments of Srb4. The physiological relevance of this interaction was confirmed by isolating gain-of-function mutations in the Gal4-binding domain of Srb4, which restore activation in vivo by a Gal4 derivative bearing a mutant activation domain.
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Srb7 |
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SRB7, an essential gene, was identified through a recessive mutation that restores the viability of yeast CTD truncation mutants (36). The human SRB7 gene has been cloned and is functional in yeast when present as a human-yeast chimera (10). Antibodies against human Srb7 have been used to purify and characterize mammalian holoenzymes (10, 13, 15, 16). The lack of a conditional mutation in Srb7 or an in vitro system in which Srb-mediator activity can be reconstituted with purified recombinant proteins has prevented more detailed analysis of its function.
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Med Proteins |
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The Med proteins are the set of polypeptides that are found in the yeast core mediator complex but which were not previously identified through genetic analysis (Table I) (29, 37). Recent evidence reveals that the Med proteins are associated with the Srb2, -4, -5, and -6 complex via an interaction between Med6 and Srb4 (38).
The mediator complex was purified for its ability to reconstitute activated transcription in an in vitro system with highly purified factors (4); confirmation that the Med proteins contribute to activated transcription in vivo has come from experiments employing a conditional mutation in MED6 (39). Strains harboring a Med6 temperature-sensitive allele are defective for activation of certain genes in vivo. Holoenzymes prepared from this strain show mild defects in basal transcription and more striking defects in activated transcription. Interestingly, these defects can be rescued by addition of recombinant Med6 protein, and the activation defect manifests itself only prior to initiation. That is, after preinitiation complex (PIC) formation, Med6 has no apparent function.
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Gal11, Sin4, Rgr1, Rox3, and Pgd1/Hrs1/Med3 |
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The members of this group of proteins were identified in a variety of yeast genetic screens as a result of their influence on repression of certain genes (40). Mutations in these proteins can cause elevated expression of genes whose promoters lack upstream-activating sequence elements (41-46). In this context, they appear to act as repressors, and they may act to prevent initiation at promoters unless the proper activation signals are present. Further supporting this idea, loss of function mutations in Gal11 and Sin4 relieve repression of GAL genes in the absence of the inducer (42). Sin4 and Rgr1 mutations allow transcription of the HO gene in the absence of the activator Swi5 (47-49).
Mutations in these proteins can also cause a reduction in activation for certain genes in vivo (41, 44, 50-52). For example, GAL gene expression in Gal11 mutants can be activated to just 30% of wild type levels (53). Most of our knowledge of the functions of Gal11, Sin4, Rgr1, Rox3, and Pgd1 comes from genetic studies, so it is not clear whether the primary roles of these proteins involve both positive and negative functions or if their roles in activation are indirect. It seems likely, however, that the primary roles of these proteins are in negative regulation. RNA polymerase II holoenzymes lacking these components do not exhibit activation defects in vitro (54). In cells with deficiencies in these proteins, the extent of derepression of some genes can be 10-fold or greater whereas the degree to which activation is reduced is not as substantial. The increased expression of certain genes because of loss of repression could cause a reduction in normal activated levels of expression at highly inducible genes simply because there is insufficient transcription initiation apparatus to accommodate the demands of the latter set of genes in the cell.
Analysis of deletion and truncation mutants suggests that Gal11, Sin4, Rgr1, and Pgd1 form a subcomplex whose interaction with other components of the mediator is anchored by Rgr1 (54). This biochemical analysis nicely explains the similarity of the mutant phenotypes.
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Srb10 CDK and Associated Polypeptides |
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Srb8, -9, -10, and -11 were identified as recessive suppressors of CTD truncation mutations (36, 55). These loss of function mutations act in a manner opposite to the core mediator Srbs (Srb2, -4, -5, -6, and -7), indicating that they play a negative role in transcription. The genes encoding Srb8, -9, -10, and -11 were also identified as Ssn5, -2, -3, and -8 in a screen developed to identify suppressors of a loss of function in the SNF1 gene, and these genes are essential for complete repression of the glucose-repressed, galactose metabolism genes (GAL ) (56, 57). Loss of function mutations in this subcomplex cause derepression of a wide variety of genes (58). Additionally, Srb10 and -11 function is required for full Tup1-Ssn6-mediated repression (56, 59).
Srb10 and -11 form a cyclin-dependent kinase-cyclin pair that phosphorylates serine 5 of the consensus heptapeptide repeat of the large subunit of RNA polymerase II C-terminal domain (60). Interestingly, this kinase-cyclin pair has the same substrate specificity as the Kin28-Ccl1 kinase-cyclin pair found in the GTF TFIIH. Kin28 has a positive role in transcription, that of producing a phosphorylated form of the enzyme which is associated with active elongation. In contrast, the Srb10 kinase has a negative role in transcription. Srb10 is uniquely capable of phosphorylating the CTD in purified holoenzymes prior to template binding, and this phosphorylation inhibits subsequent transcription by the holoenzyme. Srb10 does not appear to inhibit transcription after formation of a stable PIC. Thus, the transcription initiation apparatus can be regulated positively or negatively via modification of the CTD, depending on the timing of the phosphorylation event. Srb10 and -11 fires before competent PIC formation, thereby repressing transcription, and Kin28-Ccl1 fires after PIC formation, creating an elongation-competent form of polymerase (60).
Srb8 and -9, although not essential for Srb10 and -11 kinase activity in vitro, are required for their stable association with the holoenzyme; holoenzyme preparations from Srb8 deletion strains lack Srb10 and -11.2 It should prove interesting to identify the control mechanisms for the Srb10 CDK.
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Swi-Snf |
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Components of the Swi-Snf complex were first identified in two genetic screens: the loss of ability to switch mating types (Swi) and the loss of ability to utilize sucrose as a carbon source (Snf). The 11-subunit Swi-Snf complex has been purified (61-63) and exhibits an ATP-dependent chromatin-destabilizing activity (62). The Swi-Snf complex is believed to antagonize chromatin-based repression of transcription in vivo (64, 65).
The Swi-Snf complex has an intrinsic, nonspecific DNA binding activity (66), posing the quandary of how the complex can be effectively targeted to specific promoter regions when needed. This problem is underscored by recent enzymological studies that show that the purified Swi-Snf enzyme requires over 4 min to remodel one nucleosome on a nucleosome array (67). At this rate, the complex would require over 35 h to randomly remodel the nucleosomes present in the nucleus (67). Clearly, this must be a targeted event. The finding that Swi-Snf is an accessory subcomplex of the holoenzyme (17) provides an attractive model to explain how Swi-Snf is directed to chromosomal regions that require local remodeling of chromatin structure for appropriate gene expression.
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Holoenzyme Subcomplexes and Gene Regulation |
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The transcription apparatus is recruited to promoters under the control of DNA-binding gene-specific regulators, and these can have positive or negative regulatory functions (68, 69). It seems likely that certain holoenzyme components provide specific interaction surfaces for activators and others for repressors. For example, the transcriptional activator Gal4 contacts Srb4, and we speculate that a promoter-specific factor contacts Srb10 to effect negative regulation at those promoters.
We presume that the complex machinery in the eukaryotic Pol II holoenzyme exists in part to permit a broad range of regulatory capabilities. The ability to recruit this intricate machinery to promoters provides the opportunity to regulate the same apparatus in different ways at different promoters, yet allows for coregulation of sets of genes and the flexibility necessary to respond to changing environments. Genome-wide expression monitoring with mutations in various holoenzyme subunits, together with further study of the interactions between transcriptional regulators and holoenzyme components, could reveal how this is accomplished.
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Holoenzymes and Subcomplexes in Higher Eukaryotes |
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Many of the yeast holoenzyme components discussed here have known homologues in higher eukaryotic cells. As mentioned previously, core RNA polymerase II and the GTFs are highly conserved among eukaryotes. Human Swi-Snf complexes have been purified that share structural and functional attributes with their yeast counterpart (70). Components of the yeast core Srb-mediator complex and the Srb10 cyclin-dependent kinase complex are also found in mammalian cells. These include human Srb7 (hSrb7), human Med6, and human Srb10 and -11 (CDK8-cyclin C)(9-11, 13-16). Thus, the gene regulatory mechanisms at work in the yeast Saccharomyces cerevisiae are likely conserved in all eukaryotes. The isolation and characterization of mammalian Srb-mediator complexes should lead to a more sophisticated understanding of these regulatory mechanisms and might help uncover the mechanisms behind some cell-type specific differences in gene expression.
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FOOTNOTES |
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* This minireview will be reprinted in the 1998 Minireview Compendium, which will be available in December, 1998.
To whom correspondence should be addressed: Whitehead Inst. for
Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142. Tel.:
617-258-5218; Fax: 617-258-0376; E-mail: young{at}wi.mit.edu.
The abbreviations used are: TBP, TATA-binding protein; GTF, general transcription factor; CDK, cyclin-dependent kinase; Pol, polymerase; CTD, C-terminal domain; PIC, preinitiation complex.
2 S.-M. Liao and R. A. Young, unpublished data.
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