Molecular genetic dissection of TAF25, an essential yeast gene encoding a subunit shared by TFIID and SAGA multiprotein transcription factors.

mRNA gene transcription is mediated by RNA polymerase II working in concert with multiple general transcription factors (GTFs). The basal mRNA gene transcription machinery, as originally defined in vitro, is comprised of the GTFs TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, which function with RNA polymerase II to promote preinitiation complex (PIC) formation in vitro and in vivo (see references 29 and 55 for reviews). GTFs contribute to PIC formation in multiple ways, either acting sequentially in a stepwise fashion (7) to form a PIC or acting as a single unit comprising the RNA polymerase II holoenzyme (35, 39). Although the composition of the basal transcription machinery and its possible modes of action have been fairly well characterized, the mechanisms of transcriptional activation are still poorly understood (44). Transactivator proteins have been shown to interact with a variety of targets, including the protein components of the RNA polymerase II transcription machinery as well as chromatin constituents and activities which modify chromatin. Among these putative transactivator targets are the GTF TFIID and the SAGA (Spt-Ada-Gcn5 acetylase) histone acetyltransferase complex. Both of these multisubunit complexes have been extensively studied in yeasts and metazoans (see references 1, 4, 18, and 66 for recent reviews). Yeast TFIID is composed of 14 TATA box DNA binding protein-associated factors (TAFs) exhibiting molecular masses ranging from 150 to 17 kDa (61). Although the identifies of TFIID subunits are known, the exact stoichiometry of these multiple subunits within the complex is not. With the exception of TAF30p (30), all TFIID subunits are encoded by single-copy essential genes, and all display a high degree of sequence conservation among eukaryotes. One of these subunits, yeast TAF130p (also known as TAF145p) (58), and its metazoan counterparts (human and Drosophila TAF250p) contain intrinsic enzymatic activities that contribute to transcription (15, 16, 47, 49, 53, 54, 56). Genetic and biochemical experiments have indicated that direct interactions between the activation domains (AD) of transcriptional activators and the subunits of TFIID play key roles in transactivation (11, 22, 23, 38, 40, 45, 62, 63, 70, 73, 74, 77). This coactivator function may be manifested at the molecular level by DNA-bound activators either stabilizing (recruiting) TFIID on the TATA box-core promoter (TATA-INR-DPE) (8, 42, 64, 65) of cis-linked genes or, perhaps, by (also) activating latent enzymatic activities residing within the subunits of TFIID itself. Regardless of the exact mechanisms through which transactivation occurs, only by a systematic molecular genetic dissection of the components comprising the GTFs, particularly TFIID, will this complex process be fully understood. The yeast SAGA complex contains at least 14 subunits with molecular masses ranging from 430 to 17 kDa. The Gcn5p subunit of SAGA carries the catalytic activity capable of acetylating nucleosomal histones (24). Interestingly, five of the subunits of TFIID, TAF90p, TAF61(68)p, TAF60p, TAF25p, and TAF17p, are shared with SAGA (25). Except for TRA1, which encodes the largest subunit, none of the other known, non-TAF SAGA subunits are encoded by essential genes. Presumably, this genetic nonessentiality reflects the redundant nature of the function(s) of SAGA with other chromatin-modifying complexes (see references 36, 66, and 75 for recent reviews). Like TFIID, SAGA has been shown to play a key role in gene induction and transcriptional regulation. Recent in vivo (13, 26, 41, 69) and in vitro (46, 52, 72) studies have demonstrated that SAGA specifically associates with target genes early in the transcriptional activation process. The association of these chromatin-modifying complexes is thought to be a direct effect of specific, high-affinity AD-SAGA interactions. Complex formation between AD and SAGA is mutationally sensitive and can be readily observed in vitro (33, 72). Clearly, a thorough and detailed understanding of the mechanisms of gene activation will also require extensive dissection of the components comprising SAGA. Certain TAFp-TAFp interactions are understood at the molecular level. The cocrystal structures of two TAFp-TAFp (core) complexes have been solved by X-ray crystallography (5, 76). From this and other work it is now quite clear that a highly conserved protein-protein interaction motif, the histone fold (HF), is found in many interacting proteins (68), and TAFps are no exception. Indeed, Burley and Roeder proposed a key role for histone-like TAFps in mediating TFIID functions some years ago (9). The HF motif mediates protein interactions both within TFIID (i.e., between TAF61p-TAF48p, TAF60p-TAF17p, TAF25p-TAF47p, and TAF25p-TAF65p) and within SAGA, where HF-mediated interactions between TAF25p and Spt7p as well as TAF68p and Ada1p have been identified and characterized (18, 19, 20). Dissecting the molecular rules defining these protein-protein interactions will prove crucial to understanding the roles that these multisubunit transcription factors play in regulated mRNA gene transcription. TAF25p, an HF-containing protein, provides a unique insight into the study of the mechanisms of transcriptional regulation because this TAF is an integral subunit of both TFIID and SAGA. We originally cloned TAF25 and characterized the encoded protein, TAF25p, due to its presence in our TFIID preparations (37, 57). It has been shown that TAF25p plays a key role in mediating transcription both in vitro (37) and in vivo (43, 60). However, since TAF25p is resident in both TFIID and SAGA, it was not possible to unambiguously determine which TAF25p-containing complex was responsible for the observed transcription effects in the aforementioned studies. In order to address this and other gaps in our understanding of TAF25p function, we initiated a systematic analysis of this protein, including a detailed analysis of the structure-function relationships of TAF25. In this report, we describe our efforts to dissect TAF25p into functional domains through genetic and biochemical experimentation.