Differential requirement of SAGA components for recruitment of TATA-box-binding protein to promoters in vivo.

The expression of most eukaryotic genes is controlled at the level of transcription initiation. Transcription by RNA polymerase II requires binding of the TATA-box binding protein (TBP) and the assembly of multiple basal transcription factors into a preinitiation complex (PIC) at the core promoter. The primary promoter recognition factor is TFIID, a complex that consists of TBP and at least 14 TBP-associated factors (TAFs). Transcription of eukaryotic protein-coding genes also often involves the action of transcriptional activator proteins (activators), which bind to specific cis-acting promoter elements. Activators function by stimulating PIC assembly via a mechanism that is thought to involve a direct interaction with one or more components of the transcription machinery. Recent studies in Saccharomyces cerevisiae have demonstrated the presence of two distinct classes of promoters: those that depend on multiple TAFs for transcription (TAF dependent) and those that have no apparent TAF requirement (TAF independent) (24, 28). At TAF-dependent promoters, TAFs are present at levels comparable to that of TBP and are required for the delivery of TBP to the core promoter. The recruitment of TAFs to these promoters is activator dependent. At TAF-independent promoters, TAFs are not required for transcriptional activity or TBP recruitment (28). The mechanism by which TBP is recruited to TAF-independent promoters is an intriguing problem that researchers are only now beginning to understand. One pathway for activating TAF-independent promoters involves the SAGA (Spt-Ada-Gcn5-acetyltransferase) complex (6, 25). SAGA is a large multiprotein complex that is required for the normal transcription of approximately 10% of yeast genes (26). The role of SAGA in transcription activation has been studied extensively at GAL1, a TAF-independent promoter that requires SAGA (13, 34, 37). At the GAL1 promoter, SAGA is first recruited by the Gal4p activator to the upstream activating sequence (UAS), and the UAS-bound SAGA then facilitates the binding of TBP to the core promoter, thereby stimulating PIC assembly and transcription (6, 25). SAGA is essential for GAL1 transcription: if SAGA is not recruited to the UAS, the PIC is not assembled and transcription does not occur (6, 25). SAGA is thought to function by serving as an adaptor that directly contacts one or more components of the PIC. However, the detailed molecular mechanism by which SAGA interacts with the transcriptional machinery and stimulates PIC assembly remains to be elucidated. In yeast, the nonessential components of SAGA can be classified into three groups on the basis of their distinct mutant phenotypes: (i) Ada1p, Spt7p, and Spt20p; (ii) Spt3p and Spt8p; and (iii) Gcn5p, Ada2p, and Ada3p (8, 18, 20, 22, 34, 37). Gcn5p, the most extensively characterized component of SAGA, possesses a histone acetyltransferase (HAT) activity (36). The SAGA complex also includes a subset of TAFs: TAF5, TAF6, TAF9, TAF10, and TAF12 (formerly known as TAF90, TAF60, TAF17, TAF25, and TAF61/68, respectively [38]). Several of these TAFs have demonstrable roles in SAGA function and hence transcriptional stimulation (17, 32). Finally, SAGA also contains the ATM/PI-3-kinase-related protein Tra1p, which has been reported to be a direct target of certain activators (7). Collectively, these observations suggest that SAGA components make distinct contributions to the function of the complete complex. To understand in greater detail the roles of individual SAGA components in transcription activation, we have analyzed their contribution in TBP recruitment to several SAGA-dependent promoters by a formaldehyde-based in vivo cross-linking and chromatin immunoprecipitation (ChIP) assay. Our results reveal differences in the roles of various subunits of the SAGA complex.