kDaShared with RNAPI, RNAPIII


14 kDa

Forms heterodimer with Rpb3

Rpbl 2

8 kDa

Shared with RNAPI, RNAPIII

B: RNAPII Carboxy-Terminal Domain

The largest subunit of RNAPII, Rpbl, contains a unique C-terminal domain (CTD) consisting of tandem repeats of the highly conserved amino acid sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser (YSPTSPS) which is not found in the RNAPI or RNAPIII enzymes. This repeat sequence varies in length from 26 repeats in yeast RNAPII (Allison et al., 1985) to 52 repeats in mammalian RNAPII (Corden et al., 1985). The CTD is essential for cell viability, as partial or complete deletion of the heptapeptide repeat sequence results in lethality (Gerber et al., 1995; Nonet et al., 1987). The CTD is variably and reversibly phosphorylated at different phases throughout the transcription cycle (Laybourn and Dahmus, 1990; Dahmus, 1994), a process necessary for the recruitment of a variety of factors, including components of the Mediator complex, histone methyltransferases, as well as capping and polyadenylation enzymes, to RNAPII (reviewed in Hampsey and Reinberg, 2003; Maniatis and Reed, 2002).

C: The General Transcription Factors

Enroute to the characterization of the RNAPII holoenzyme, identification and extensive characterization of the general transcription factors (GTFs) necessary for transcription initiation has taken place. The GTFs of RNAPII, including TFIIA, TFIIB, TFIID (composed of TBP and TAFs), TFIIE, TFIIF, and TFIIH, were identified biochemically as factors required for accurate transcription initiation by RNAPII from double stranded DNA templates in vitro (reviewed in Conaway and Conaway, 1993; Zawel and Reinberg, 1993; Orphanides et al., 1996; Roeder, 1996). R. Kornberg and colleagues were able to reconstitute RNAPII transcription initiation from a variety of yeast and mammalian promoters in a system using only purified TBP, TFIIB, -E, -F, -H, and RNAPII (Myers et al., 1997). The addition of mediator complex components (see below) to the preparation, comprising a 44 polypeptide system, resulted in transcription from not only a minimal promoter but also that which could respond to activator proteins bound upstream of the promoter (Kim et al., 1994).

D: Coactivators

Coactivators are distinct from the general transcription factors in that they are dispensable for basal-level transcription in vitro but are required for regulated transcriptional activation. They differ from activators in that they generally do not bind DNA directly in a sequence-specific manner, but act as bridges between DNA binding activator proteins and the GTFs or facilitate chromatin remodeling. Several examples of coactivators that serve as bridges or mediators of transcription by acting in concert with the general transcription machinery will be discussed below.

TFIID, along with RNAPII and other GTFs is sufficient to direct promoter-specific transcription initiation. Cloning and expression of yeast TBP however, revealed that recombinant TBP could replace TFIID in RNAPII directed transcription from TATA-containing promoters in vitro, but these reactions were not responsive to sequence-specific transcriptional activators. In addition they could not direct transcription from TATA-less promoters (Zhou et al., 1992). These results led to a model in which subunits of TFIID other than TBP are required for regulated transcriptional activation. These subunits are referred to as TBP-associated proteins or TAFs (Dynacht et al., 1991, Poon and Weil, 1993; reviewed in Goodrich and Tjian 1994a; Verrijzer and Tjian, 1996). Multiple protein interactions occur between TAFs and the GTFs, as well as TAFs and the activator proteins. In addition, multiple forms of TFIID, which vary in TAF composition and function have been described, including a form of TFIID that plays a role in the repression of transcription (Wade and Jaehning, 1996). Thus, TFIID and its associated TAFs play a critical role in the regulation of transcription by relaying information from activators and repressors to the core transcriptional machinery.

E: The Mediator-Containing Holoenzyme

Earlier studies, as discussed above, centered around the identification and characterization of TAFs in association with TBP. More recent work, however, has focused on characterizing the role(s) of a coactivator component of the RNAPII holoenzyme known as the mediator complex. The first direct evidence for a yeast mediator comes from the results of transcriptional reconstitution experiments in which a mediator was required for in vitro transcription by the activator GAL4-VP16, but had no effect on transcription in the absence of the activator (Flanagan et al., 1991). In addition, Nonet and Young discovered that a series of yeast mutants with partial deletions in the heptapeptide repeat sequence (YSPTSPS) of the largest subunit of RNAPII-the CTD- were temperature-sensitive, cold-sensitive and inositol auxotrophs. A genetic screen for suppressors of the cold-sensitive phenotype of these CTD mutants led to the identification of the Srb (Suppressor of RNA polymerase B) proteins (Nonet and Young, 1989) whose products were shown to reside in a multiprotein complex (Thompson et al., 1993). A

holoenzyme that supported a response to activator proteins with purified basal GTFs was identified and contained a core mediator complex consisting of Srb2, 4, 5, and 6, as well as Galll, and Sugl (Kim et al., 1994; Koleske and Young, 1994). Other members of this complex have been purified and include Srb7, Medsl, 2, 4, 6, 7, and 8, Sin4, Rgrl, Rox3, and Pgdl (Myers et al., 1998) as well as Nutl, Nut2, Cse2, and Medll (Gustafsson et al., 1998). Genetic and biochemical studies have revealed that many of these proteins affect both positive (the Srb core proteins 2, 4, 5, 6 , and 7) and negative (Srb8, 9, 10, and 11) regulation of transcription (Hengartner, et al., 1995; Holstege et al., 1998; Myers et al., 1999; Han et al., 1999; also see review in Carlson, 1997) and, as is the case with Srb4 and Srb6, are required for transcription of most, if not all, protein-encoding genes (Holstege et al., 1998; Thompson and Young, 1995). Members of this mediator complex can also exist in a large number of subcomplexes in association with the RNAPII holoenzyme, including a Sin4 subcomplex containing Sin4, Galll, Pgdl, and Med2 (Li et al., 1995; Myers et al., 1999) and an Srb subcomplex comprised of Srb2, Srb4, Srb5, Srb6, Rox3, Med6, Med8, and Medll (Lee and Kim, 1998; Kang et al., 2001; Koh et al., 1998) thus adding to the great complexity and broad range of regulatory response activities within transcription by RNA polymerase II (reviewed in Chang and Jaehning, 1997; Myer and Young 1998; Malik and Roeder, 2000).

A current view of the Mediator is one of an evolutionarily conserved and ubiquitously expressed complex of more than twenty subunits that connects the initiating form of RNAPII to DNA-binding regulatory factors in response to environmental signals. Indeed, much progress has been made in the characterization of several forms of a mammalian Mediator complex, and cross-species comparisons have detected metazoan homologs of nearly all yeast Mediator components. Srb/Med-containing complexes, including the thyroid hormone receptor-associated proteins/ SRB-Med containing cofactor (TRAP/SMCC), vitamin D receptor-interacting proteins (DRIP), positive cofactor 2 (PC2), the cofactor required for Spl activation (CRSP), and the activator-recruited factor-large (ARC-L) complexes have been isolated (reviewed in Conaway et al., 2005). The complexes are highly homologous to components of the yeast Mediator. In light of these findings, a common nomenclature has recently been established under the recommendation of a large group of scientists working inside and outside of the transcription field, to clarify, across species, the conserved nature of Mediator proteins (Bourbon et al., 2004). This new nomenclature has replaced the names of many of the previously identified proteins with the acronym MED, which acknowledges the discovery of Med complexes in yeast.

In addition to Mediator, some yeast holoenzyme preparations contain substoichometric levels of Swi-Snf complex components (Wilson, et al., 1996). The Swi-Snf complex has been purified (Cairns et al., 1994; Cote et al., 1995; Treich et al., 1995) and has an ATP-dependent chromatin destabilizing activity allowing for the relief of chromatin-based transcription repression (Winston and Carlson, 1992; Peterson and Tamkun, 1995). In addition, histone acetyltransferase activity, most likely carried out by the Nutl subunit of the Mediator, has been found associated with the holoenzyme (Lorch et al., 2000). Therefore, a form of the holoenzyme can be linked to transcriptional activation through chromatin remodeling.

F: The Pafl Complex

The Pafl complex can be distinguished from the Srb/Med-containing holoenzyme by its composition and the subset of genes it regulates. A novel collection of RNAPII associated proteins (RAPs) was isolated by immobilizing antibodies against an unphosphorylated form of the CTD of RNA polymerase on a Sepharose column. The eluted RAP fraction from a transcriptionally active yeast whole cell extract did not contain the holoenzyme's core mediator components Srb2, 4, 5, and 6, Galll, and Sugl, but did include the known GTFs, TFIIB, and three subunits of TFIIF, as well as the transcription elongation factor TFIIS. Also isolated as RAPs were two novel factors, Pafl and Cdc73 (Wade et al., 1996). Blast searches of peptide sequences revealed that Pafl was an uncharacterized open reading frame, and Cdc73 was the product of a previously identified gene encoding a yeast mating pheromone signaling element (Reed et al., 1988). Mutations in the nonessential PAF1 and CDC73 genes affect cell growth and the abundance of transcripts from a subset of genes (Shi et al., 1996; Chang, et al., 1999; Porter et al., 2002). The isolation of several proteins associated with RNAPII in a whole cell fraction that did not contain components of the Srb-containing mediator complex raised the possibility that Pafl and Cdc73 may be associating as a second, alternative form of the RNAPII holoenzyme with distinct, but overlapping roles in transcription. Thus, in order to determine the relationship between Pafl, Cdc73 and the previously identified Srb-containing holoenzyme, studies were pursued to isolate a Pafl complex. Glutathione S-transferase (GST)-tagged forms of Pafl, Cdc73 were used in glutathione agarose chromatography, and an RNAPII-

associating complex was identified that contained Pafl and Cdc73, as well as several components also found in the Srb-containing form of the holoenzyme, TFIIF, TFIIB, and Galll. Srb proteins, TFIIS, TFIIH, and TBP were not isolated in this complex (Shi et al., 1997). Tfg2, a subunit of TFIIF, was also GST-tagged and used to isolate proteins that may be associated with both the Pafl complex and the Srb- mediator complex. In this case, Srb proteins, as well as Pafl and Cdc73 were isolated, indicating that Pafl-Cdc73 and the Srbs define two separate, but partially redundant complexes (Shi et al., 1997).

Further analysis also identified Hprl and Ccr4 as associated with the Pafl complex but not the Srb-containing Mediator complex (Chang et. al., 1999). Both Ccr4 and Hprl have been found in other transcription-related complexes, have been demonstrated to have genetic interactions with the transcription machinery (Fan et al., 1996; Liu et al., 1998), and have been implicated in the transcription of subsets of genes (Denis and Malvar., 1990; Draper et al, 1994; Zhu et al, 1995). In addition, more recent studies using immunoaffinity chromatography and mass spectrometry have identified Ctr9, Rtfl, and Leol as components of the Pafl complex (Koch et al, 1999; Krogan, et al, 2002; Mueller and Jaehning, 2002; Squazzo et al, 2002). CTR9 (CDP1) was isolated in a screen for mutants that failed to activate transcription of the cell cycle regulated Gi cyclins (Koch et al, 1999), and a role for Ctr9, as well as Pafl, has been established in the regulation of Gi cyclin expression (Koch, et al, 1999; Porter, et al, 2002). RTF I was originally identified in a screen for extragenic suppressors of a TBP mutant with defects in DNA binding specificity (Stolinski et al, 1997).

The continuing characterization of members of the Pafl complex has linked the complex to a role in transcriptional regulation. While PAF1 and CDC73 are not essential genes in yeast, paflA mutants are temperature-sensitive, slow-growing, and have an enlarged cell morphology. cdc73A . mutants are also temperature-sensitive and grow slightly slower than wild type cells. The initial use of differential display, followed by more recent microarray analyses of a pafl A mutant demonstrated that the Pafl complex is important in the regulation of expression of a subset of genes. Many genetic interactions between the members of the Pafl complex and with members of the Srb-containing holoenzyme also exist: several combinations of deletions within complex members result in lethality, and pafl A and ccr4A are lethal in combination with srb5A. This indicates that factors within and between the two complexes may have overlapping essential functions.

More recent work has indicated that the Pafl complex consists of the core components Pafl, Ctr9, Cdc73, Rtfl, and Leol (Mueller et al., 2002; Squazzo et al., 2002). It appears to be associated with RNAPII throughout the transcription cycle (Pokholok et al., 2002; Simic et al., 2002; Mueller et al., 2004) (Fig. 3.1) and localizes to promoter regions of active genes (Wade et al., 1996; Shi et al., 1997; Pokholok et al., 2002; Mueller et al., 2004). The Pafl complex is also found at the 3' and 5' ends of genes with a distribution and abundance very similar to that of the elongating RNAPII (Pokholok et al., 2002; Simic et al., 2002; Mueller et al., 2004). The Pafl complex has been found to associate with initiation factors (TFIIB, TFIIF (Wade et al., 1996, Shi et al., 1997)), elongation factors (TFIIF, Spt5, Dstl (Mueller et al., 2002; Squazzo et al., 2002; Krogan et al., 2002b)), chromatin remodeling and modifying factors (Chdl, Setl (Simic et al., 2003; Krogan et al., 2003a)), and factors involved in mRNA processing and export (Hprl, Sub2 (Chang et al., 1999; Mueller et al., 2004)). The fact that the Pafl, RNAPII complex is clearly biochemically distinct from the Srb/mediator form of RNAPII establishes that it probably joins the transcription complex after the initial recruitment event (Shi et al., 1997) (see Fig. 3.1). From that point on, until RNAPII reaches the poly(A) site for 3' end processing (Kim et al., 2004), it accompanies the RNA polymerase, possibly serving as a "platform" for the dynamic association of additional factors during transcription elongation (Gerber and Shilatifard, 2003). In addition, recent evidence suggests that it may also function in the coordination of transcription and downstream events leading to proper mRNA biogenesis and maturation (Mueller et al., 2004).

RNA Polymerase II and Transcription Elongation

The transition between transcription initiation and elongation, although separated by a significant phase in transcription regulation (promoter clearance), is not well defined, and it may be difficult to distinguish between the end of one event and the beginning of the other. During promoter clearance, the appearance of a stalled RNAPII-DNA complex and the production of short RNA products, abortive initiation, occurs. At this point in the transcription cycle, a stable transcription elongation complex has not yet formed (Kireeva, et al., 2000), and RNAPII often slips (Pal and Luse, 2002). Luse and colleagues have also found that once an RNA transcript of 23 nucleotides has formed, slippage no longer occurs and a stable, elongating form of RNAPII

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