A TATA-binding protein mutant defective for TFIID complex formation in vivo.

Transcriptional initiation by eukaryotic RNA polymerase II (Pol II) requires general transcription factors (TFIID, -A, -B, -E, -F, and -H) that nucleate on the TATA promoter element to form the preinitiation complex that is necessary for accurate positioning and initiation by Pol II (for a review, see reference 34). This process begins with the sequence-specific binding of TFIID, a multiprotein complex composed of TATA-binding protein (TBP) and approximately 10 RNA Pol II-specific TBP-associated factors (TAFs) (reviewed in reference 5). TAFs are a set of phylogenetically conserved proteins found in humans, flies, and yeast, ranging in molecular mass from 15 to 250 kDa (for a recent review, see reference 49). Recruitment of TBP, presumably in the TFIID complex, has been proposed to be a rate-limiting step in formation of the preinitiation complex (6, 8, 25, 26, 60). Two distinct mechanisms have been proposed for assembly of the preinitiation complex, depending on the presence or absence of a canonical TATA element (5). Initiation from a TATA-containing promoter is dependent on strong TBP-DNA contacts for recognition and binding of TFIID to the TATA box. However, for TATA-less promoters, sequence-specific recognition of the core promoter by TBP does not occur, suggesting that TAF-DNA interactions play an important role (5). In vitro, TFIID but not TBP is able to recognize and support transcription from TATA-less promoters (62). Moreover, in Drosophila TFIID, specific TAFs have been implicated in making contacts with DNA in sequences overlapping the transcription start site (4, 5, 52). In addition, mutations that hinder the ability of TBP to recognize a TATA box do not affect transcription from TATA-less promoters even though TFIID is required (29). Consistent with this, depletion of certain individual yeast TAFs in vivo causes a reduction in transcription from promoters lacking consensus TATA boxes (31, 32). Thus, it seems that transcription from TATA-less promoters does not depend on strong TBP-DNA interactions but rather depends on TAF-DNA and TAF-TBP interactions. In addition to their role in TATA-less transcription, TAFs have been implicated in the response to activators. Transcriptional activators function by increasing recruitment and stabilizing the Pol II machinery at promoters (reviewed in references 40, 48, and 53). In vitro, activators can interact with many components of the transcription machinery, including TBP, TAFs, TFIIA, TFIIB, TFIIF, TFIIH, Pol II, and the SAGA complex (12, 15, 17, 19, 21, 28, 39, 47, 61). Initial biochemical studies suggested that TAFs function as coactivators required for activator-dependent recruitment of TBP, since TFIID but not TBP could support high levels of transcription in the presence of an activator. Further, it was hypothesized that TAFs can serve as direct targets for activators, because individual TAFs can interact directly with activator proteins (for reviews, see references 5 and 53), and partial TFIID complexes reconstituted from a subset of TAFs can mediate activator-dependent transcription (7). However, TAFs are not absolutely required for the response to activators in vitro, because activation can occur in reactions lacking TAFs (24, 27, 37, 58, 59). In vivo depletion studies indicate that certain individual TAFs are not generally required for response to activators (31, 54). Instead, depletion of certain TAFs results in specific effects on transcription, depending on the particular TAF targeted. In the case of depletion of TAF130, a loss of transcription from genes lacking a canonical TATA element (31), from genes encoding small-subunit ribosomal proteins (RPS genes), and from various cyclin genes (43, 55) was observed. The TAF130 dependence of these genes is mediated by the core promoter, not the initiator or the activator binding sites. In mammalian cells, TAF250, the homolog of yeast TAF130, is important for transcription of selected genes, including those involved in cell cycle control (50, 56). Taken together, these data suggest that TAFs play a critical role in gene-specific as well as TATA-less transcription. Aside from their role in TFIID, certain TAFs are present in the yeast SAGA and mammalian PCAF histone acetyltransferase complexes (16, 38). In vivo depletion of TAF61/68, a component of yeast SAGA, indicates that this TAF is necessary for normal enzymatic activity but is dispensable for interaction with TBP or activation domains. Furthermore, depletion of TAF17, which is also present in SAGA, has broad effects on transcription in yeast cells (1, 30, 32). Thus, it seems that TAFs can serve a variety of roles in the transcription process, depending on the complexes in which they reside. Here we present the characterization of a TBP mutant that is defective for TFIID formation in vivo. Our studies differ from previous in vivo studies in that we analyzed transcription after TFIID disruption which removes multiple TAFs, not after individual TAF depletion. This approach permits the removal of TAFs from TFIID but not from SAGA or other TAF-containing complexes. We show that TFIID disruption does not prevent the response to acidic activators, but it causes transcriptional defects at certain cell cycle-dependent and TATA-less promoters. These results support previous findings that the TBP-TAF interactions may not be essential for activated transcription in vivo and may instead play a role in gene-specific transcription.