Purification of Active TFIID from Saccharomyces cerevisiae

Abstract

The basal transcription factor TFIID is composed of the TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). Although TBP alone binds to the TATA box of DNA and supports basal transcription, the TAFs have essential functions that remain poorly defined. In order to study its properties, TFIID was purified from Saccharomyces cerevisiae using a newly developed affinity tag. Analysis of the final elution by mass spectrometry confirms the presence of all the known TAFs and TBP, as well as Rsp5, Bul1, Ubp3, Bre5, Cka1, and Cka2. Both Taf1 and Taf5 are ubiquitinated, and the ubiquitination pattern of TFIID changes when BUL1 or BRE5 is deleted. Purified TFIID binds specifically to promoter DNA in a manner stabilized by TFIIA, and these complexes can be analyzed by native gel electrophoresis. Phenanthroline-copper footprinting and photoaffinity cross-linking indicate that TFIID makes extensive contacts upstream and downstream of the TATA box. TFIID supports basal transcription and activated transcription, both of which are enhanced by TFIIA.TFIID is a large, multisubunit complex that is essential for transcription by RNA polymerase II. It consists of the TATAbinding protein (TBP) 1 and at least 14 TBP-associated factors (TAFs) (1). Although the sizes of these TAFs differ by organism, conserved domains reflect that these proteins have been conserved over the eukaryotic evolution (2). TBP is necessary and sufficient for binding to the TATA box sequence, although this binding is stabilized by the addition of TFIIA and TFIIB (3). Yeast TFIIA is a complex of two proteins that binds TBP and makes contacts with the DNA immediately upstream of the TATA box (4 -6). Human TFIID also binds DNA in a TATAspecific fashion but makes additional downstream contacts that may allow binding to promoters lacking a TATA element (7). In human and Drosophila melanogaster promoters, a significant element is the initiator region about 25 bp downstream of the TATA box. The two largest TAFs, hTaf1 and hTaf2, may contact the initiator region directly (8). Within Drosophila TFIID, it has been proposed that some of the histone-like TAFs interact with a downstream promoter element situated at approximately ϩ30 relative to the transcription start site (9). In yeast, the TATA box is usually situated 50 -90 bp upstream of the transcription start sites, but no clear consensus sequences have emerged for a downstream promoter element or initiator region.Although the yeast system has been extremely useful for studying the genetics of TAFs, there have been comparatively few biochemical studies of yeast TFIID. Immunoaffinity purification of the complex has been successful, but yields are relatively low, and the procedure requires large amounts of antibodies. Nonetheless, analysis of immunopurified yeast TFIID by mass spectrometry led to the confirmation of TFIID composition and estimates of subunit stoichiometry (1). Another study using DNase I protection found that yTFIID binding was centered on the TATA box but made additional contacts up to 45 ...

Evidence for a Complex of Transcription Factor IIB (TFIIB) with Poly(A) Polymerase and Cleavage Factor 1 Subunits Required for Gene Looping

“…The presence of TFIID and TFIIH in the gradient fractions was detected by Western blot analysis using antibodies against the TBP and Kin28 subunits of TFIID and TFIIH, respectively. TFIID, which has a molecular mass of about 750 kDa (32), sedimented in fractions 7-15 with the peak in fraction number 11 (Fig. 3B, panel ii), whereas TFIIH with an approximate molecular mass of 500 kDa (33) sedimented in fractions 13-20 (Fig.…”

Atg19p Ubiquitination and the Cytoplasm to Vacuole Trafficking Pathway in Yeast

“…Ubp3p interacts physically with the chromatin silencing protein Sir4p, and ubp3-⌬ strains show enhanced silencing, but the molecular mechanism by which Ubp3p antagonizes silencing is unknown (68). Ubp3p and its cofactor Bre5p co-purify with transcription factor TFIID (69). Two TATA-binding protein-associated factors, Taf1p and Taf5p, are ubiquitinated, and the pattern of their ubiquitination is altered in a bre5-⌬ strain (and presumably in a ubp3-⌬ strain).…”

“…Two TATA-binding protein-associated factors, Taf1p and Taf5p, are ubiquitinated, and the pattern of their ubiquitination is altered in a bre5-⌬ strain (and presumably in a ubp3-⌬ strain). The function of these modifications, and thus of Ubp3p and Bre5p in the TFIID complex, is not known (69). Others have suggested that Ubp3p may be a general "proofreader" for the ubiquitin-proteasome system, removing ubiquitin from damaged or misfolded proteins before they can be destroyed (45, 70).…”

Identifying a TFIID interactome via integrated biochemical and high-throughput proteomic studies

“…A previous study on native yeast TFIID revealed co-purification of the ubiquitin E3 ligase Rsp5 with the observation of TAF1 and TAF5 being ubiquinated [34]. In addition, multiple human TFIID subunits (TAF1, 2, 7, 9 and TBP) are ubiquitinated by unknown E3 ligases [38,39].…”

“…A number of previous studies have used global mass spectrometry to identify factors interacting with the TBP subunit of TFIID [3,[32][33][34]. Alternative proteomic approaches utilizing the intact native human TFIID complex as bait to find direct interactors had not been assessed.…”