A: Protein Composition

Human TFIIA is composed of a, p, and y subunits with molecular weights of 35 kDa, 19 kDa, and 12 kDa respectively. These three subunits are encoded by two genes: TFIIAaP and TFIIAy in higher eukaryotes, and TOA1 and TOA2 in yeast. Human TFIIAaP encodes a 55-kDa precursor protein that is highly conserved with TOA1 in yeast within the N-terminal 54 amino acids and the C-terminal 76 amino acids, but is less conserved in the central part of TFIIA which has been shown to be dispensable for function (Ranish et al, 1992; Kang et al, 1995). The smallest subunit of TFIIA, TFIIAy, is homologous to yeast TOA2 (Ozer et al, 1994; Sun et al, 1994). In higher eukaryotes, proteolytic cleavage of TFIIAaP into TFIIAa and TFIIAP subunits occurs (DeJong and Roeder, 1993; Ma et al, 1993; Yokomori et al, 1993). Originally it was thought that the three independent subunits (a, P, and y) constitute TFIIA activity, since only the cleavage products were detected in cell extracts in association with one another. Recently, intact TFIIAaP protein was detected in association with TFIIAy along with TBP in embryonal carcinoma cells (Mitsiou and Stunnenberg, 2000, 2003). The site for proteolytic cleavage within TFIIAaP was determined by Edman degradation; TFIIAP was found to start at Asp 278 (H0iby et al., 2004). Furthermore, cleaved TFIIAa and TFIIAP were more efficiently degraded than the unprocessed precursor via the ubiquitin-proteasome pathway, suggesting that cleavage and degradation of TFIIA controls the level of TFIIA within the cell to adapt rapidly to the transcriptional needs in responding to environmental changes (Iteiby et al, 2004). Similar to TAF4b, a cell-specific TFIIAa/ß-like factor (ALF) has been identified in human testis (Upadhyaya et al., 1999; Ozer et al., 2000). ALF works in conjunction with TFIIAy to stabilize TBP binding to the promoter (Upadhyaya et al., 2002). Further experiments indicate that ALF is also found in immature oocytes of the frog Xenopus laevis, in which ALF replaces TFIIA during oogenesis (Han et al., 2003).

B: Is TFIIA a Genuine GTF?

The role of TFIIA as a GTF has been controversial. As with TFIID, TFIIA was initially identified as a phosphocellulose column fraction necessary for pol Il-mediated transcription in vitro (Matsui et al., 1980). Early in vitro experiments showed that TFIIA was essential for transcription (Reinberg et al., 1987), while in other in vitro studies TFIIA was shown to be largely dispensable for basal level transcription (Van Dyke et al., 1988; Wu et al., 1998; Pugh, 2000). It has also been suggested that TFIIA stimulates both basal and activated transcription in vitro 2- to 10-fold, but generally only when TFIID, instead of TBP, is used as the promoter-binding factor (Orphanides et al., 1996; Hampsey, 1998; Warfield et al., 2004). In our laboratory, we found TFIIA was not required for either basal or activator-dependent transcription in a highly purified transcription system reconstituted with recombinant GTFs (TFIIB, TFIIE, and TFIIF), recombinant PC4 coactivator, and epitope-tagged multiprotein complexes (pol II, TFIID, and TFIIH), irrespective of whether TFIID or recombinant TBP was used in the assay (Wu et al., 1998). Nevertheless, TFIIA indeed became essential for transcription in a reconstituted system containing partially purified fractions obtained according to the purification scheme outlined in Fig. 2.1. Collectively, these studies suggest that TFIIA mainly functions as an antirepressor to overcome inhibitors present in crude fractions likely by increasing the affinity of TBP or TFIID for DNA (Buratowski et al., 1989; Lee et al., 1992; Imbalzano et al., 1994; Kang et al., 1995), thereby enhancing PIC assembly. TFIIA stabilizes TBP-TATA box interactions through direct contacts with both TBP and DNA (Geiger et al., 1996; Tan et al., 1996). Binding of TFIIA to TBP dimers has also been shown to induce TBP monomer formation and accelerate the kinetics of TBP binding to DNA (Coleman et al., 1999). TFIIA is able to counteract the repressive effects of negative factors such as Drl/NC2ß,

PC3/Dr2, Motl, HMG1, and also the inhibitory activity of TAF1 on TBP binding to DNA (Meisterernst et al., 1991; Inostroza et al., 1992; Merino et al., 1993; Ge and Roeder, 1994; Ozer et al, 1994, 1998b; Orphanides et al., 1996; Auble et al., 1997; Hampsey, 1998; Kokubo et al., 1998; see also the Chapter by Thomas and Chiang, 2005). Interestingly, TAF1 interaction with TFIIA may modulate TFIIA activity, since it has been shown that TAF1 phosphorylates human TFIIA(3 on serine residues important for TBP binding and transcription activity (Solow et al., 2001). Additionally, experiments have shown that the TFIIA-TBP-DNA complex may also be regulated by the transcriptional coactivator p300 through acetylation of TFIIA (Mitsiou and Stunnenberg, 2003).

C: TFIIA as a Coactivator

Many studies have demonstrated that TFIIA interacts with activators, such as Gal4-VP16, Zta, and HTLV-1 Tax (Ozer et al., 1994; Kobayashi et al., 1995; Clemens et al., 1996; Ranish et al., 1999; Dion and Coulombe, 2003), and transcriptional coactivators PC4 and HMG2 (Ge and Roeder, 1994; Sun et al., 1994). TFIIA has also been shown to interact with specific TAFs in TFIID (Yokomori et al., 1993; Kraemer et al., 2001; Robinson et al., 2005). In one proposed model of TFIIA function, interaction of TFIIA with both activators and TFIID may stimulate and stabilize binding of TFIID to DNA as part of an activator-TFIID-TFIIA-DNA complex, thereby enhancing a rate-limiting step for TFIID binding to DNA in the transcriptional process (Wang et al., 1992; Lieberman and Berk, 1994, Chi et al., 1995; Ranish et al., 1999; Dion and Coulombe, 2003). Moreover, TFIIA may upregulate PIC formation by stimulating the functions of both TFIIE and TFIIF (Langelier et al., 2001). In TAF-independent transcriptional activation experiments using a highly purified transcription system, TFIIA could potentiate TBP-mediated activation, suggesting that TFIIA may function as a coactivator especially in the absence of TAFs (Wu et al., 1998). Several studies using immunodepletion of TFIIA subunits in vitro and mutational studies abolishing TFIIA-TBP interactions in yeast have indicated that, similar to TAFs, TFIIA is important for transcription only from a subset of genes and does not seem to be universally required for all gene transcription (Kang et al., 1995; Ozer et al., 1998a; Liu et al., 1999; Stargell et al., 2000).

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