INTRODUCTION

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This review presents an overview of the RNA polymerase II (RNA pol II) core transcriptional machinery. I discuss promoter elements and then review recent advances pertaining to three classes for transcription factors: general transcription factors (GTFs), transcriptional coactivators, and general transcriptional repressors. I focus on the transcriptional machinery from the yeast Saccharomyces cerevisiae, emphasizing the combined roles of yeast genetics and biochemistry in defining factors and their associated functions. However, this subject cannot be considered separately from the RNA pol II core transcriptional machinery from higher eukaryotic organisms, where results obtained with human, rat and Drosophila systems have often led the way. Although I emphasize the yeast system, I have attempted to integrate information from both yeast and metazoan systems whenever appropriate.

A breakthrough in understanding the mechanism of transcription initiation followed the discovery in the laboratory of Roeder that purified RNA pol II would selectively and accurately initiate transcription from template DNA when supplemented with a crude cell extract (529). This activity provided an assay for the fractionation and subsequent identification of the GTFs, defined as factors required for accurate, basal-level transcription initiation in vitro (311). Similar work defined analogous factors in rats, Drosophila, and yeast, suggesting that the GTFs are indeed "general" factors, required for expression of most, perhaps all, class II genes. Thus, the process of transcription initiation by RNA pol II is highly conserved among eukaryotic organisms, allowing for the experimental advantages offered by different organisms to be exploited to identify and define these factors.

S. cerevisiae has proven to be extraordinarily valuable in these studies. In 1987, Lue and Kornberg established an in vitro transcription system derived from yeast nuclei that would accurately initiate transcription from exogenous template DNA (295). A second in vitro transcription system was derived from yeast whole-cell extracts (541, 542). These systems have been instrumental not only for identifying the GTFs and their functions, but also for defining other transcription factors that influence the rate of transcription initiation.

A second advantage of yeast is the potential to exploit the power of classical and molecular genetic methods to investigate fundamental biological problems. An array of genetic selections has been developed to identify factors affecting RNA pol II transcription. In many cases, these studies have identified novel transcription factors or unexpected activities associated with these factors that had gone undetected by biochemical means. As a notable example, genetic selections for mutants unable to ferment sucrose (snf), for mutants that suppress promoter defects caused by insertion mutations (spt), and for mutants defective in mating-type switching (swi) converged, leading to the discovery of the SWI/SNF chromatin-remodeling complex that facilitates transcriptional activation (reviewed in references 51 and 537). Another important example is the genetic selection for suppressors of the conditional growth defect associated with truncation of the RNA pol II carboxy-terminal repeat domain (srb), which led to the discovery of the RNA pol II holoenzyme (reviewed in reference 261a).

The information available from the extensive collection of well-characterized yeast mutants and the complete sequence of the yeast genome are additional advantages to the yeast system. Accordingly, sequence information for proteins identified biochemically, either from yeast or from other organisms, can be compared to the yeast database. In many cases, a biochemically identified protein corresponds to the product of a gene identified in a genetic selection or screen. This combination of biochemistry and genetics often provides novel insight into protein function. Two remarkable examples are a histone acetyltransferase from Tetrahymena and a histone deacetylase from humans. Sequence analysis of these two proteins revealed similarity to the products of the genetically defined yeast GCN5 and RPD3 genes (38, 481). Although the biochemical function of neither gene had been defined, GCN5 was identified in a genetic selection for transcriptional coactivators whereas RPD3 was identified in a selection for transcriptional repressors. This combination of biochemistry and genetics led to the identification of Gcn5 and Rpd3 as histone acetyltransferase and histone deacetylase, respectively. Moreover, it provided a direct link between histone acetylation/deacetylation and transcriptional activation/repression (reviewed in reference 181).

Eukaryotic promoters can be divided into core elements and regulatory elements (reviewed in reference 456). Core promoter elements define the site for assembly of the transcription preinitiation complex (PIC) and include a TATA sequence, located upstream of the transcription start site, and an initiator sequence (Inr), encompassing the start site. Promoters can include a TATA box, an Inr sequence, or both of these control elements. A third core element, the downstream promoter element (DPE), was initially described in Drosophila and is located about 30 bp downstream of the start site (48). The DPE appears to function, in conjunction with the Inr element, as a TFIID binding site at TATA-less promoters.

Regulatory elements are gene-specific sequences that are located upstream of the core promoter and control the rate of transcription initiation; they include both upstream activation sequences (UAS) and upstream repression sequences (URS), which serve as binding sites for enhancers and repressors of transcription, respectively. In addition, poly(dA-dT) sequences are bidirectional upstream promoter elements that facilitate constitutive gene expression, not as UAS-like elements but apparently by forming a structure that is less stable to repressing nucleosomes. These elements are reviewed below.

TATA elements in S. cerevisiae are typically located 40 to 120 bp upstream of the transcription initiation site. This is in contrast to other eukaryotes, including Schizosaccharomyces pombe, where the TATA element is almost always located at a fixed distance of 25 to 30 bp from the start site (reviewed in reference 456). The TATA sequence is the binding site for the TATA binding protein (TBP). TBP-TATA association nucleates the assembly of an approximately 4-MDa transcription preinitiation complex, a step that can be rate limiting for transcription initiation in vivo (257).

Mutational analysis and random selection for functional TATA elements defined TATAAA as the consensus TATA sequence in yeast (74, 441, 539). Many derivatives of this sequence also confer TATA function, albeit with diminished activity. One derivative, TGTAAA, eliminated TATA function and was used to select for TBP derivatives with altered binding specificity (454). TBP^m3, described below, allowed transcription from TGTAAA promoters but not from certain other single-nucleotide derivatives of TATAAA (454). This mutant demonstrated the importance of specific interactions between the TATA element and TBP for efficient initiation. Functional analysis of mutated TATA elements revealed that yeast and human TBP have nearly identical TATA sequence requirements, underscoring the evolutionary conservation of the TBP-TATA interaction (539).

Some yeast promoters contain multiple TATA elements. For example, transcriptional analysis of site-directed mutations defined two functional TATA-like sequences within the CYC1 promoter (281). These two elements differed in sequence; one is denoted -type, and the other is denoted -type. Interestingly, when both elements were present, both were used equally to direct initiation within distinct but overlapping windows. However, if the same type (either  or ) was present at both sites, only the upstream element was used, directing initiation within the upstream window. These results were interpreted to mean that - and -type TATA elements are recognized by different factors of the transcriptional apparatus. However, TBP binds both consensus and nonconsensus TATA elements (178), suggesting that regulatory factors other than TBP might confer differential recognition of closely related TATA elements (281).

Yeast promoters lacking canonical TATA elements (TATA-less promoters) have also been identified. For example, the HIS3 promoter contains two TATA elements, one of which (T_R) conforms to the canonical TATAAA sequence and is responsible for initiation at position +13 in response to activation by Gcn4. The other element (T_C) does not resemble a consensus TBP binding site, directs initiation from position +1, and supports initiation in the absence of activators. Nonetheless, T_C-directed transcription is TBP dependent in vivo (99). Interestingly, the relative utilization of T_C and T_R depends upon the overall level of transcription (223). T_C is preferentially utilized at low levels of transcription, T_C and T_R are utilized equally well at moderate levels of transcription, and T_R is preferentially utilized at high levels of transcription. These results suggest that transcription initiation from weak TATA elements is not mechanistically distinct from that mediated by canonical TATA elements but is determined instead by the overall level of transcription (223).

Transcription from TATA-less promoters remains TBP dependent. Accordingly, the term "TATA-less promoter" denotes relatively weak TBP-DNA affinity rather than a fundamentally distinct promoter element. Nonetheless, the rate-limiting step in PIC assembly at TATA-less promoters is unlikely to be TBP recruitment. Presumably, another component(s) of the core machinery recognizes a promoter structure other than TATA to nucleate PIC assembly. Indeed, TAF_II60 and TAF_II40 from human and Drosophila cells play a direct role in basal transcription by specifically binding the DPE of TATA-less promoters (47, 48).

Inr elements are DNA sequences encompassing transcription start sites. The fixed distance between TATA and the Inr element in eukaryotic organisms other than S. cerevisiae suggests that the Inr is determined simply by spacing from TATA. In contrast, the variable distance between TATA and the Inr in S. cerevisiae implies the existence of specific sequences that permit transcription initiation. Experiments to determine the relationship between TATA and the Inr established that the TATA element defines the window within which initiation can occur but that specific sequences within the window define the Inr element (179, 191, 281, 331, 403). Mutational analyses and surveys of start sites have defined preferred Inr sequences (146), yet there is no clearly defined Inr consensus sequence.

Yeast mutants that affect start site selection have been identified (11, 27, 147, 193, 216, 367). Specific mutations in TFIIB and the largest subunit of RNA pol II shift initiation downstream of normal (27, 367). However, in no case is the downstream site a "new" initiation site. Rather, these sites are normal, albeit minor initiation sites that generally conform to preferred Inr sequences. Thus, defects in TFIIB and RNA pol II do not alter the specificity of Inr element recognition but instead shift the window within which initiation can occur further downstream. Although the mechanism of Inr recognition is unclear, RNA pol II and TFIIB are key players in this process (284, 366).

The Inr element, as defined in higher eukaryotes, is not simply the DNA sequence encompassing the transcription start site. Rather, an Inr element was initially defined at the TATA-less promoter of the terminal deoxynucleotidyltransferase gene as a core promoter element, distinct from TATA, that can nucleate PIC assembly (442). Inr elements were subsequently identified at many promoters, both TATA-containing and TATA-less, and have been implicated in transcriptional control by directing accurate initiation in a TATA-independent manner (reviewed in reference 530). Proteins that bind Inr elements include CIF (241), YY1 (497), E2F (318), TFII-I, and USF (398, 399), as well as RNA pol II itself (12, 61). The CIF complex includes a homolog of Drosophila TAF_II150 (241), which binds promoter DNA overlapping the Inr region (509) and has been implicated in differential recognition of two tandem Adh promoter elements (187). Furthermore, a recombinant TBP-TAF_II150-TAF_II250 subcomplex is minimally required for efficient utilization of Inr and downstream promoter elements in a reconstituted transcription system (507). Thus, it appears that TFIID can be recruited to a promoter by either of two distinct pathways, one involving TBP-TATA interaction and the other involving TAF-Inr interaction.

It is not clear whether Inr elements that function as distinct core promoter elements to nucleate PIC assembly exist in yeast. There are some intriguing prospects, though. For example, the GAL80 promoter has been reported to include both an Inr element and a TATA element, with these two elements directing initiation at distinct sites. The GAL80 Inr element is functionally portable, and a GAL80 Inr-binding protein has been detected (412). These results suggest that GAL80 transcription is driven by two independent pathways, one Inr dependent and the other TATA-dependent (412). This scenario is reminiscent of transcription at the HIS3 promoter (456). Thus, Inr elements that facilitate transcriptional control might be a universal feature of eukaryotic of RNA pol II transcription.

UAS elements are DNA sequences that function as binding sites for specific transcriptional activators. As such, UAS elements are analogous to metazoan enhancers, functioning in either orientation and at variable distances from the core promoter. An important functional distinction between enhancers and UAS elements is that UAS elements do not function when positioned downstream of the TATA box (168, 455). However, this has been reported for only a few genes and needs to be tested more thoroughly. Once associated with their cognate UAS elements, transcriptional activators facilitate assembly of the PIC, either by direct contact with GTFs or indirectly through coactivators, which in some cases mediate activator-GTF interactions. Consistent with its role in activation, deletion of a UAS element diminishes mRNA synthesis under activating conditions.

URS elements are binding sites for gene-specific transcriptional repressors. URS-repressor complexes can impair transcription by several different mechanisms, including interference with activator-UAS binding; interference with the activation domain of an activator-UAS complex; or by contact with the core transcriptional machinery, a process analogous to activation, albeit with opposite effects (reviewed in reference 233). URS-repressor complexes can also mediate repression indirectly by recruiting another complex that targets either the core transcriptional machinery or histones. For example, several URS-repressor complexes recruit the Ssn6-Tup1 complex, which appears to mediate repression by affecting histone function (214). Transcriptional repression associated with histone deacetylation is another example of this type of repression. In the best-characterized example in yeast, the GC-rich URS1 element binds the Ume6 repressor, which in turn recruits the Sin3-Rpd3 histone deacetylase complex (234). This process is analogous to transcriptional repression mediated by histone deacetylases in metazoan systems (reviewed in reference 359).

Homopolymeric dA-dT sequences are a common feature of yeast promoters and in several cases have been shown to be required for normal levels of transcription in vivo (reviewed in reference 224). Poly(dA-dT) sequences have distinct structural characteristics that impair nucleosome assembly or stability, which led to the proposal that poly(dA-dT) sequences function as promoter elements based on their intrinsic structure, rather than as conventional UAS elements to which sequence-specific transcription factors bind (75). However, a naturally occurring poly(dA-dT) sequence activated transcription in vitro, an effect that could be squelched by addition of a related oligonucleotide (294). This result argued for involvement of a poly(dA-dT)-specific transcription factor. Indeed, a poly(dA-dT)-binding protein, datin, has been identified (538).

The mechanism of poly(dA-dT)-mediated transcriptional activation has been investigated by using a combination of functional assays and probes of chromatin structure. A poly(dA-dT) sequence located upstream of the Gcn4 binding site in the HIS3 promoter stimulated Gcn4p-activated transcription in a length-dependent manner (224). Moreover, datin repressed, rather than stimulated, gene expression, and poly(dG-dC), which also affects nucleosome structure, functioned similarly to poly(dA-dT) (224). These results imply that poly(dA-dT) stimulates transcription as a consequence of its intrinsic structure, rather than as a conventional UAS element. This conclusion is supported and extended by the demonstration that a poly(dA-dT) element located adjacent to the Candida glabrata metal-dependent transcriptional activator gene, AMT1, plays a critical role in transcriptional autoactivation by causing a localized distortion of the nucleosomal DNA, allowing Amt1 to gain access to its cognate promoter element (565). Recently, a whole-genome analysis revealed that poly(dA-dT) tracts are abundant in S. cerevisiae and occur predominantly at unit nucleosomal length both upstream and downstream of open reading frames, leading to the proposal that such tracts modulate nucleosome positioning (382).

RNA POLYMERASE II

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Overview

Yeast RNA pol II is composed of 12 subunits encoded by the RPB1 to RPB12 genes (543). There is extensive structural conservation among the subunits of eukaryotic RNA pol II. Indeed, six subunits of human RNA pol II can functionally replace their homologs in yeast (317). The two largest RNA pol II subunits, Rpb1 (~200 kDa) and Rpb2 (~150 kDa), are the most highly conserved subunits. Moreover, Rpb1 and Rpb2 are homologous to the ' and  subunits, respectively, of bacterial RNA polymerase. Rpb3 is related to the  subunit of bacterial RNA polymerase based on partial amino acid sequence similarity, size similarity, identical subunit stoichiometry (two per molecule), and assembly defects associated with mutations in either subunit (543). None of the RNA pol II subunits appears to be closely related to the bacterial -subunit family, although structural and functional similarities between  and certain GTFs have been identified (see below).

The Rpb1, Rpb2, Rpb3, and Rpb11 subunits of RNA pol II are homologous to subunits of RNA polymerases I and III. Moreover, five subunits, Rpb5, Rpb6, Rpb8, Rpb10, and Rpb12, are common to all three RNA polymerases. Only Rpb4, Rpb7, and Rpb9 are unique to RNA pol II. Thus, RNA polymerases are assembled from common as well as class-specific subunits. Ten of the yeast genes encoding RNA pol II subunits are essential for cell viability. Only the RPB4 and RPB9 genes are dispensable, although deletion of either gene confers conditional growth phenotypes.

The sequence similarity between Rpb1 and Rpb2 and the bacterial ' and  subunits occurs in highly conserved domains, designated A to H in Rpb1, and A to I in Rpb2 (555). This structural similarity extends among the two largest subunits for all eukaryotic RNA pol II investigated. Not surprisingly, the structural similarity between Rpb1/' and Rpb2/ extends to functional similarity. Both Rpb1 and ' are involved in DNA binding, whereas Rpb2 and  bind nucleotide substrates.

Many mutations in RPB1 and RPB2 have been isolated and characterized (reviewed in reference 6). Most amino acid replacements are located within the highly conserved domains. Specific mutations in RPB1 and RPB2 affect the accuracy of transcription initiation, demonstrating a role for these subunits in defining start site selection (11, 27, 193). Other mutations in RPB1 and RPB2 confer sensitivity to 6-azauracil (6-AU), a phenotype associated with transcription elongation defects, suggesting that both subunits are also involved in overcoming transcriptional arrest (7, 374). 6-AU-sensitive rpb1 mutants can be suppressed by overexpression of PPR2, the gene encoding the elongation factor SII (7). In the case of RPB2, 6-AU-sensitive alleles encode elongation-defective forms of RNA pol II (374).

The Rpb4 and Rpb7 subunits are functionally related. These two subunits can be dissociated from RNA pol II, and RNA pol II purified from a rpb4 null mutant lacks Rpb7 (122). This form of RNA pol II is indistinguishable from wild-type RNA pol II in an in vitro elongation assay but is inactive in promoter-directed transcription initiation. Furthermore, this form of RNA pol II could be complemented in vitro by an inactive RNA pol II with a defective form of Rpb1. These results demonstrate that Rpb4 and Rpb7 function in transcription initiation and suggest that they can shuttle between RNA pol II molecules (122). Interestingly, rpb4 mutants exhibit substantially impaired growth rates at elevated temperature or under conditions of nutritional deprivation, implicating Rpb4 in tolerance of RNA pol II to stress (80).

Similar mutational analyses are needed to define functions for the other RNA pol II subunits. Limited mutational analysis revealed that Rpb3 is involved in RNA pol II assembly (262). Mutations in RPB9 affect start site selection (147, 148, 216, 460), and in one case an rpb9 allele suppresses a TFIIB defect that affects start site selection (460). Thus, Rpb9, like Rpb1 and Rpb2, affects the accuracy of initiation, perhaps through interaction with TFIIB.

Although the CTD is essential for cell viability, its function is not entirely clear. There are two forms of RNA pol II in vivo, designated IIO, which is extensively phosphorylated at the CTD, and IIA, which is not phosphorylated. The IIA form preferentially enters the PIC, whereas IIO is found in the elongating complex (reviewed in reference 103). Conversion of IIA to IIO occurs concomitant with or shortly after the transition from initiation to elongation and is accompanied by extensive CTD phosphorylation (292, 346). These results implicate the CTD in conversion of RNA pol II from a form involved in promoter recognition to an elongation-competent form. Nonetheless, a form of RNA pol II lacking the CTD (IIB) is able to initiate transcription from TATA-containing promoters in vitro, although not from TATA-less promoters (2, 40).

The kinase activity of TFIIH can mediate CTD phosphorylation (132, 293, 429), although other kinases, including Cdc2 (83), Ctk1 (274), the Srb10-Srb11 kinase-cyclin pair (285), and P-TEFb (310), have also been implicated in CTD phosphorylation. Drosophila P-TEFb (positive transcription elongation factor b) affects the transition from abortive to productive transcription elongation (310). The catalytic subunit of P-TEFb is homologous to PITALRE, a Cdc2-related protein kinase (564). Human P-TEFb associates with the Tat protein of human immunodeficiency virus type 1 to potentiate transcriptional elongation (307, 564). Thus, multiple kinases appear to mediate phosphorylation of the RNA pol II CTD. Whether these CTD kinases are gene specific or affect different steps in the transition from initiation to elongation remains to be determined.

A phosphatase responsible for dephosphorylation of the CTD has also been identified (66). CTD phosphatase activity is regulated by TFIIB and TFIIF (67). The RAP74 subunit of TFIIF stimulates CTD phosphatase activity, whereas TFIIB inhibits the stimulatory activity of TFIIF. Since the dephosphorylated form of RNA pol II (IIA) preferentially enters the PIC (103), these results suggest that the CTD phosphatase, TFIIF, and TFIIB interact to regulate RNA pol II recycling.

Although the CTD is essential for cell growth, all but 8 to 10 repeats can be deleted from yeast Rpb1 without loss of viability (345, 532). However, strains with a minimum number of CTD repeats exhibit a cold-sensitive growth defect, a phenotype that was exploited to isolate extragenic suppressors of CTD truncations (345). This selection identified SRB genes, which encode components of the SRB-mediator complex required for transcriptional activation (28). Thus, the CTD functions in transcriptional activation.

The CTD has also been implicated in pre-mRNA processing. A speculative model proposed that the negatively charged, hyperphosphorylated CTD of the IIO form of RNA pol II facilitates electrostatic interactions with positively charged regions of certain splicing factors (167). This model has received considerable experimental support. Splicing is inhibited in vitro (557) and in vivo (118) by CTD repeat polypeptides and in vitro by an anti-CTD antibody (557). Furthermore, proteins that might connect the spliceosome to RNA pol II via the CTD have been identified (450, 557). The CTD has also been implicated in 5' capping of mRNA and 3'-end formation. The 5'-capping enzyme specifically binds the phosphorylated IIO form of RNA pol II, suggesting a mechanism for coupling of cap addition to RNA pol II transcription (79, 313, 556). A role for the CTD in 3'-end formation was discovered by the effects of CTD truncations on 3' processing and poly(A) addition and substantiated by the association of cleavage and polyadenylation factors with the CTD (314). These results suggest that the CTD functions as a platform for the recruitment and assembly of factors involved in pre-mRNA processing (reviewed in reference 449).

RNA pol II and the GTFs assemble in a defined order on promoter DNA in vitro (42, 301, 503). These results suggested stepwise assembly of the PIC. However, several GTFs were known to associate with RNA polymerase in the absence of DNA, hinting at the existence of an RNA pol II holoenzyme complex (96). Antibodies directed against SRB proteins provided direct evidence for a holoenzyme complex that includes a subset of the GTFs, as well as SRB proteins (261). Unlike core RNA pol II, holoenzyme responds to transcriptional activators in vitro. Holoenzyme was independently discovered based on the association of RNA pol II with mediator, the protein complex required for transcriptional activation (252). As described in the coactivator section below, mediator includes SRB proteins, MED proteins, and a subcomplex composed of Gal11, Sin4, Rgr1, and Med3. These two holoenzymes are comparable, although one complex is reported to include TFIIB, TFIIF, and TFIIH (261) whereas the other includes TFIIF as the only GTF (252). One holoenzyme preparation is also reported to include Srb8 to Srb11 (194, 285) and the SWI/SNF chromatin remodeling complex (535), whereas the other includes neither of these sets of proteins (57, 252, 330). Regardless of the distinction between these two holoenzyme preparations, a key point is that holoenzyme supports activated transcription with only TBP and other GTFs in reconstituted transcription systems. Transcriptional activation is mediator dependent but TAF independent. This is in contrast to metazoan transcription systems, where activation is TAF dependent (reviewed in reference 183).

There are at least two distinct forms of yeast RNA pol II holoenzyme (reviewed in reference 68). A second form was discovered by a purification strategy based on an RNA pol II affinity column immobilized through the CTD (435). Isolated proteins include TFIIB, TFIIF, TFIIS, and Gal11 but not SRB/mediator components (515). Novel components of this complex include Paf1 and Cdc73 (435, 515), as well as Ccr4 and Hpr1 (68). This form of the holoenzyme affects the expression of a different spectrum of genes and is therefore functionally distinct from the SRB/mediator-containing holoenzyme (436). The percentage of total RNA pol II contained in the Paf1-Cdc73-Ccr4-Hpr1 holoenzyme has not been reported, although this holoenzyme is less abundant than the SRB/mediator holoenzyme (227).

RNA pol II holoenzyme complexes have also been identified in mammalian cells (69, 302, 352). Like the yeast holoenzymes but distinct from core RNA pol II, these complexes are able to respond to transcriptional activators in vitro. In addition to the GTFs, a provocative array of proteins has been found in these enzyme preparations, including DNA repair proteins (302), splicing and polyadenylation factors (314), and even the breast cancer tumor suppressor BRCA1 (427).

The GTFs include TBP, TFIIB, TFIIE, TFIIF, and TFIIH and were identified biochemically as factors required for accurate transcription initiation by RNA pol II from double-stranded DNA templates in vitro (reviewed in references 395 and 558). Fractionation of whole-cell extracts from yeast also identified five factors, designated a, b, d, e, and g, that are required for promoter-specific transcription by RNA pol II (420). The yeast factors are comparable in structure and function to the mammalian GTFs and are now designated by the mammalian nomenclature. These factors are reviewed below and summarized in Table 1.

Order-of-addition experiments demonstrated that PIC assembly is nucleated in vitro by TBP binding to the TATA element followed by binding of TFIIB, RNA pol II-TFIIF, TFIIE, and TFIIH (42; reviewed in references 351 and 395). This scenario was challenged by the discovery of RNA pol II holoenzyme complexes, first in yeast and later in mammalian systems (see the RNA polymerase II section, above). These findings suggest that at least some GTFs associate with RNA pol II prior to promoter binding and have implications for the mechanisms of transcriptional regulation. Whether PIC assembly occurs in a stepwise manner or by holoenzyme recruitment, the order-of-addition experiments provided valuable information about GTF interactions within the PIC. A schematic representation of the PIC is presented in Fig. 1; a crystallographic representation of a DNA-TBP-TFIIA-TFIIB complex is presented in Fig. 2.

View larger version (26K): [in this window] [in a new window]   FIG. 1.   Schematic depiction of the transcription PIC. PIC assembly is nucleated by TBP binding to the TATA box, inducing a sharp bend in the DNA template, followed by association of TFIIB, RNA pol II/TFIIF, TFIIE, and TFIIH. Each pattern denotes a distinct general transcription factor. Subunit composition is indicated, except for TFIIH (9 subunits) and RNA pol II (12 subunits). Although PIC assembly can occur by stepwise addition of the general transcription factors (GTFs) in vitro, the discovery of RNA pol II holoenzyme complexes that include GTFs suggests that stepwise assembly might not occur in vivo.

View larger version (44K): [in this window] [in a new window]   FIG. 2.   Tertiary structure of a TATA-TBP-TFIIA-TFIIB complex. The amino- and carboxy-terminal direct repeats of TBP are light and dark blue, respectively. The amino- and carboxy-terminal repeats of core-TFIIB are red and orange, respectively. The Toa1 and Toa2 subunits of TFIIA are green and yellow, respectively. Reprinted from reference 351 with permission of the publisher.

Overview. TBP is a universal transcription factor, required for initiation by all three eukaryotic RNA polymerases (reviewed in reference 198). TBP was identified as a subunit of TFIID, the large (~750-kDa) multisubunit complex composed of TBP and TBP-associated factors (TAFs). Whereas TBP functions in basal level transcription, TFIID is required for response to transcriptional activators in metazoan in vitro transcription systems (reviewed in references 50, 378, and 508). Direct evidence that TBP plays a general role in eukaryotic transcription came from Comai et al., who found TBP to be an essential subunit of the RNA pol I transcription factor SL1 (93). TBP was also identified as a subunit of the RNA pol III general factor TFIIIB (215, 239, 290, 440, 473, 533). Furthermore, mutations in yeast TBP diminished expression by all three RNA polymerases in vivo (99, 426). Thus, TBP plays a requisite role in transcription initiation by all three RNA polymerases, functioning as a common subunit of SL1, TFIID, and TFIIIB.

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Tertiary structure of TBP-2 from Arabidopsis thaliana. The three-dimensional structure is viewed perpendicular to the intramolecular twofold symmetry axis. The -helices (H1, H2 and H1', H2') and -sheets (S1 to S5 and S1' to S5') are labeled and can be correlated with the TBP amino acid replacement data summarized in Table 2. Reprinted from reference 342 with permission of the publisher.

TBP-TATA interactions. Several mutational studies of TBP have addressed the domains and residues that bind DNA. Random mutagenesis of SPT15 identified dominant mutations that diminished DNA binding (387). These included V71E, R105C, T112K, and F116Y single-residue derivatives. Each of these replacements occurs in the N-terminal direct repeat domain of TBP. Site-directed replacements of the analogous residues in the C-terminal repeat, V161E, R196C, V203K, and F207Y, also inhibited DNA binding, suggesting that DNA binding is partitioned between the two direct repeats (387). This prediction was confirmed by the crystal structure of the TATA-TBP complex (246, 251).

TBP-TFIIA interactions. The core domain of TBP includes helices located at the ends of each direct repeat (H2, H2') (Fig. 3). These structures are components of the convex surface of TBP, comprising the seat of the saddle, suggesting that the saddle mediates protein-protein interactions (251). Indeed, mutational analysis of basic residues within the H2 helix demonstrated that residues K133/K138 and K133/K145 are required for interaction with TFIIA, as defined by gel shift analysis (46).

TBP-TFIIB interactions. Site-directed mutations were generated in the C-terminal core domain of yeast TBP in an attempt to define residues critical for GAL4-VP16 activation (248). Three replacements, L114K, L189K, and K211L, selectively blocked activation with no effect on basal transcription in vitro. Each of these TBP derivatives was defective in GAL4-VP16-mediated recruitment of TFIIB to the promoter complex and one, L189K, disrupted TBP-TFIIB interaction. These results were interpreted to mean that GAL4-VP16 activation involves TBP recruitment, as well as stabilization or isomerization of an activation-specific TATA-TBP-TFIIB complex (248). A role for TBP-TFIIB interaction in transcriptional activation is consistent with activator-mediated recruitment of TFIIB to preinitiation complexes in vitro (82) and with a model invoking activator-dependent conformational changes in TBP-TFIIB interaction (discussed in reference 175).

Other TBP derivatives. In the same genetic selection that uncovered the activation-defective K138T/Y139A double replacement that affects TBP-TFIIA interaction (447), four activation-defective single replacements, F148H, T153I, E236P, and F237D, were found (446). These replacements lie on the convex surface of TBP. None affects TATA binding, and the F148H, T153I, and E236P replacements do not affect interaction with TFIIA, TFIIB, or glutathione S-transferase-VP16. These activation defects were at least partially rescued by artificial recruitment of the F148H, T153I, and F273D derivatives to the promoter. These results were interpreted as evidence for a second step in transcriptional activation in vivo, one involving recruitment of TBP to the promoter and the other involving activator interaction with another component of the PIC after TBP recruitment (446). This conclusion is consistent with a mutational analysis of human TBP, where activation-defective mutations were identified on the convex surface of TBP that did not affect interaction with either TFIIA or TFIIB (39).

Suppressors of TBP derivatives. The extensive collection of well-defined TBP derivatives is a lucrative source of primary mutations for isolation of suppressors. In one study, dosage-dependent suppression of the temperature sensitivity associated with the K133L/K138L double mutant identified a TFIIB homolog that is a component of the RNA pol III transcriptional machinery (45). In another case, the same factor was isolated as a suppressor of the P65S replacement in TBP (86). Consistent with its role in RNA polymerase III transcription, the same factor was isolated as a dominant suppressor of a tRNA "A block" promoter mutation (291). The gene encoding this factor is designated BRF1/TDS4/PCF4 and encodes the 67-kDa subunit of TFIIB. The N-terminal half of Brf1/Tds4/Pcf4 is significantly similar to TFIIB, an observation that underscores the similarity of RNA pol II and pol III transcriptional systems and suggests that Brf1/Tds4/Pcf4 is a key factor distinguishing the polymerase specificity of a gene (45, 86).

Overview. Yeast TFIIB is a monomer of 38 kDa encoded by the SUA7 gene. SUA7 was initially identified in a genetic selection for suppressors of a translational defect at the CYC1 locus (366). Recessive sua7 mutations shifted transcription start site selection downstream of normal such that the translational impediment was eliminated from the cyc1 transcript. This result demonstrated that TFIIB functions in transcription start site selection in vivo.

Start site selection. The effects of sua7 on start site selection are not limited to the CYC1 gene but also affect other genes, including ADH1 (27, 366). Interestingly, the start site shift is always downstream of normal. Furthermore, the downstream start sites are never "new" sites but represent enhanced initiation at minor sites. Therefore, TFIIB defects shift the window within which certain start sites are recognized rather than altering the specificity of start site recognition. Also, the downstream shift is not a consequence of alternative TATA usage. For example, none of the sua7 suppressors compensate for Ty element insertions at the HIS4 or LYS2 loci, which is the basis for isolation of the spt class of suppressors (536).

Suppressors of TFIIB derivatives. In an effort to identify factors that interact with TFIIB, the cold-sensitive phenotype of the sua7-1 mutant was exploited to isolate extragenic suppressors. Cold sensitivity is often associated with defects in assembly of multisubunit complexes (reviewed in reference 180); therefore, suppressors of cold sensitivity seemed likely to identify other components of the PIC or perhaps factors that facilitate PIC assembly.

The subunits of TFIIF were identified as RAP30 and RAP74 based on affinity of these two proteins for RNA pol II (52). RAP30 and RAP74 were required for accurate transcription initiation from several promoters, defining the RAP30-RAP74 complex as a general initiation factor that binds RNA pol II (141). Although TFIIF was not initially identified as one of the four HeLa cell chromatographic fractions (TFIIA, TFIIB, TFIID, and TFIIE) required for accurate initiation by RNA pol II (418), further purification resolved the TFIIE fraction into two factors, TFIIE and TFIIF, both of which are essential for initiation (142). Purification of the TFIIF fraction defined two subunits, identical to RAP30 and RAP74 (139). TFIIF was also identified in human cells as factor FC (253), in rat cells as factor (95), and in Drosophila cells as factor 5 (377). In each case, these factors were required for specific initiation of transcription in vitro.

TFIIF has several characteristics reminiscent of bacterial  factors. These include tight binding of TFIIF to RNA polymerase; suppression of nonspecific binding of RNA pol II to DNA; and stabilization of the PIC (96, 166). Both subunits exhibit limited sequence similarity to bacterial  factors (149, 315, 444, 459, 552). It is not clear whether these structural similarities reflect functional similarities. However, human RAP30-RAP74 binds E. coli RNA polymerase and can be displaced by ⁷⁰ (315). Moreover, polymerase binding is attributed to region 2.1 of ⁷⁰, which is the region of similarity between ⁷⁰ and RAP30 (315). Thus, RAP30 appears to be partially analogous to bacterial ⁷⁰.

TFIIF probably does not play a significant role in promoter selectivity but contributes to PIC stability. Photo-cross-linking studies have defined the topology of a TBP-TFIIB-TFIIF-RNA pol II-TFIIE promoter complex. RAP74 and RAP30 bind promoter DNA between the TATA box and start site, a region where TFIIE and RNA pol II also cross-link (102, 249, 390). RAP74 also binds DNA upstream of TATA, inducing a conformational change that affects the position of RNA pol II relative to the DNA template (144).

In addition to its role in initiation, TFIIF functions in transcriptional elongation by suppressing transient pausing of the polymerase (24, 32, 142, 225, 377). Although RAP30 and RAP74 were initially thought to function exclusively in initiation and in elongation, respectively, both subunits are now known to function in both processes (476).

Yeast TFIIF (factor g) stably associates with RNA polymerase, is required for specific initiation at all promoters tested, and is composed of three subunits with apparent molecular masses of 105, 54, and 30 kDa (197). The genes encoding the two largest subunits of TFIIF were cloned based on the partial sequence of the purified proteins (196). Analysis of the deduced amino acid sequences of TFG1 and TFG2 revealed 50 and 51% similarity to human RAP74 and RAP30, respectively, thereby confirming the relationship between factor g and TFIIF. TFG1 and TFG2 are present in single copy, and both are essential for cell viability (196, 459). TFG3 encodes the 30-kDa subunit and is identical to ANC1, a gene originally identified based on genetic interaction with ACT1 (531). Tfg3 is less tightly associated with the complex than are Tfg1 and Tfg2 and has no counterpart in mammalian TFIIF, although Tfg3 is similar in sequence to the leukemogenic proteins ENL and AF-9 (55). Tfg3 is found in RNA pol II holoenzyme complexes (252, 261, 515), probably as a component of TFIIF. Interestingly, Tfg3 is identical to the TAF_II30 subunit of the yeast TFIID complex (371) and to the Swp29 subunit of the SWI/SNF chromatin-remodeling complex (55). As such, Tfg3 establishes a connection between basal and regulatory components of the transcriptional machinery (55, 196). Tfg3 is the only yeast GTF that is not essential for cell viability (196), perhaps because yeast contains a Tfg3 homolog (YOR213c), which is a subunit of the RSC chromatin-r