TFIIA and TATA-binding protein (TBP) associate directly at the TATA element of genes transcribed by RNA polymerase II. In vivo, TBP is complexed with approximately 14 TBP-associated factors (TAFs) to form the general transcription factor TFIID. How TFIIA and TFIID communicate is not well understood. We show that in addition to making direct contacts with TBP, yeast TAF40 interacts directly and specifically with TFIIA. Mutational analyses of the Toa2 subunit of TFIIA indicate that loss of functional interaction between TFIIA and TAF40 results in conditional growth phenotypes and defects in transcription. These results demonstrate that the TFIIA-TAF40 interaction is important in vivo and indicate a functional role for TAF40 as a bridging factor between TFIIA and TFIID.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 TA...

The physiological role of TFIIA was investigated by analyzing transcription in a yeast strain that contains a TATA-binding protein (TBP) mutant (N2-1) defective for interacting with TFIIA. In cells containing N2-1, transcription from a set of artificial his3 promoters dependent on different activators is generally reduced by a similar extent, indicating that TFIIA function is largely nonselective for activators. In addition, TATA element utilization, a core promoter function, is altered at his3 promoters dependent on weak activators. Genomic expression analysis reveals that 3% of the genes are preferentially affected by a factor of 4 or more. Chimeras of affected promoters indicate that the sensitivity to the TFIIA-TBP interaction can map either to the upstream or core promoter region. Unlike wild-type TBP or TFIIA, the N2-1 derivative does not activate transcription when artificially recruited to the promoter via a heterologous DNA binding domain, indicating that TFIIA is important for transcription even in the absence of an activation domain. Taken together, these results suggest that TFIIA plays an important role in both activatordependent and core promoter functions in vivo. Further, they suggest that TFIIA function may not be strictly related to the recruitment of TBP to promoters but may also involve a step after TBP recruitment. Initiation of RNA polymerase (pol)1 II transcription requires the assembly of a large complex of proteins that must interact at the promoter in a productive manner (1, 2). Formation of this complex is accelerated by activators that bind to the promoter and aid in recruitment of the components in the complex. The first step in promoter recognition is binding of TFIID to the TATA element. TFIID is a multiprotein complex containing TATA-binding protein (TBP) and TBP-associated factors (3). TFIIA stabilizes the TBP-TATA interaction (4 -7) by interacting directly with the TBP and DNA flanking the TATA element (8, 9). TFIIA also counteracts several negative regulators of transcription that specifically target TBP. It inhibits the abilities of Mot1 and NC2 to dissociate TBP from the TATA element (10 -13), and it blocks the inhibition of TBP binding to the TATA element by the N-terminal domain of TBP-associated factor 130 (14). Thus, there are several mechanisms by which TFIIA functions at core promoters in vitro.Although TFIIA is not required for in vitro transcription using highly purified components, activated transcription is often stimulated by TFIIA. This is in accord with the observations that TFIIA can interact directly with activation domains in vitro (15-17) and that TFIIA is required for activator-dependent stabilization of the TFIID⅐TATA complex (16, 18 -20). A simple model is that the activator-dependent TFIID⅐TFIIA complex is formed rapidly and stably on the TATA element, thereby serving as an efficient scaffold for the remainder of the initiation complex. Alternatively, TFIIA could act as a coactivator, conveying information between the activator and TBP. In this regard, in...

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