TFIIA interacts with TFIID via association with TATA-binding protein and TAF40.

Transcription by eukaryotic RNA polymerase II (Pol II) involves the assembly of a preinitiation complex consisting of Pol II and the general transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (for review, see reference 66). An important step of transcription initiation is the binding of TFIID to the core promoter. TFIID, a multisubunit protein complex that is highly conserved among eukaryotes, is composed of the TATA-binding protein (TBP) and over a dozen TBP-associated factors (TAFs) (reviewed in references 26 and 27). TBP mediates promoter recognition through the sequence-specific binding of the TATA element found at many promoters. The importance of the TBP-TATA interaction is illustrated by many studies which demonstrate that recruitment of TBP is a rate-limiting step at a majority of promoters (12, 17, 37, 39, 46, 50). In yeast, 13 TAFs are required for viability, indicating essential roles for individual TAFs (75, 77; reviewed in reference 26). However, the precise functional requirements for the TAFs remain unresolved. In vitro biochemical experiments suggest that TAFs function in higher eukaryotic systems as obligatory coactivators essential for activator response (reviewed in references 9 and 84). In contrast, functional inactivation and depletion studies with certain TAFs in yeast cells demonstrate that the expression of many genes are unaffected by TAF loss, although TAF inactivation results in distinct cell cycle phenotypes (2, 55, 59, 60, 62, 86). In addition, disruption of the TFIID complex with a temperature-sensitive mutation in TBP results in gene-specific transcriptional defects (71), and promoter occupancy studies indicate that TAFs are not present on certain transcriptionally active promoters in vivo (45, 49). Promoter-specific requirements for particular TAFs are further illustrated by whole-genome transcriptional profiles. For example, inactivation of TAF145/130 has no effect on the expression of a majority of genes, while transcription of a subset of genes is affected (31). This TAF dependence was mapped to the core promoter (78), indicating important TAF functions in promoter activity in vivo. In contrast to these gene-specific effects, inactivation of several other TAFs, namely TAF17 (2, 59, 61), TAF40 (44), TAF60 and TAF61/68 (59, 62), and TAF23/25 (76), results in dramatic effects on a large fraction of genes transcribed by Pol II. The requirement for these particular TAFs is not yet understood, but it is clear that individual TAFs may be generally required for transcription while others function at a subset of promoters. To complicate the issue further, it is apparent that certain TAFs in both human and yeast systems can be found in large protein complexes distinct from TFIID, such as the SAGA complex (25) and the SWI/SNF complex (10). Taken together, these studies indicate that different TAFs may have distinct functional roles in transcription, yet the nature of the specific functions for a majority of the TAFs remains to be elucidated. In addition to TAFs, other transcription factors associate with TBP at the core promoter to mediate transcription. One such factor, TFIIA, has been shown to stabilize the interaction between TBP and DNA at the TATA element (reviewed in reference 27). Mutational studies of both TBP and TFIIA demonstrate the importance of the TFIIA-TBP interaction for transcription in vivo (35, 53, 68, 80). TFIIA has been described as a coactivator, since in vitro functions of certain activators are TFIIA dependent (51, 67), and as an antirepressor, because TFIIA can mediate displacement of certain transcriptional inhibitors that act on TBP (4, 5, 22, 32, 42, 52, 56, 58, 69). A growing body of evidence suggests that the functions of TFIIA and TAFs are connected. DNase I footprinting experiments show that the addition of TFIIA alters the DNA protection pattern of TFIID (14, 15, 51). Consistent with these findings, UV-cross-linking experiments indicate that TFIIA induces a conformational change in TFIID that alters specific TAF interactions with the core promoter (64). Moreover, a set of TFIIA mutations that can form a TBP-TFIIA-DNA complex are defective for forming a complex with TFIID (69). Finally, the three-dimensional structure of the human TFIID-TFIIA-TFIIB complex clearly suggests TFIIA-TAF interactions, since TFIIA maps to a large noncentral lobe of TFIID, with TBP being located more centrally in the structure (1). It is not yet understood how this TFIIA-TAF communication is established or which particular TAFs are involved. In this report, we investigate the importance of the interactions between TFIIA and yeast TAFs. We demonstrate a direct interaction between TFIIA and TAF40, as well as a direct interaction between TAF40 and TBP. We also find that mutations in TFIIA that impair the TFIIA-TAF40 interaction result in conditional growth phenotypes and defects in transcription in vivo. These results suggest that TAF40 serves as a link between TFIIA and TFIID functions, and they reveal a new role for TAF40 in RNA Pol II transcriptional regulation.