Autonomous Function of the Amino-Terminal Inhibitory Domain of TAF1 in Transcriptional Regulation

Abstract

The general transcription factor TFIID is composed of TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). TFIID mediates the transcriptional activation of a subset of eukaryotic promoters. The N-terminal domain (TAND) of TAF1 protein (Taf1p) inhibits TBP by binding to its concave and convex surfaces. This study examines the role of the TAND in transcriptional regulation and tests whether the TAND is an autonomous regulator of TBP. The TAND binds to and regulates TBP function when it is fused to the amino or carboxy terminus of Taf1p, the amino or carboxy terminus of Taf5p, or the amino terminus of Taf11p. However, a carboxy-terminal fusion of the TAND and Taf11p is not compatible with several other TAF proteins, including Taf1p, in the TFIID complex. These results indicate that there is no or minimal geometric constraint on the ability of the TAND to function normally in transcriptional regulation as long as TFIID assembly is secured.Transcription of eukaryotic protein-coding genes is regulated by the concerted action of a number of distinct transcription factors (reviewed in references 34 and 38). These transcription factors are recruited to the promoter region in a precise gene-specific order (reviewed in reference 10). Transcription factor IID (TFIID) is a general transcription factor complex that is composed of the TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs) (39, 41). It recognizes and binds to several core promoter elements and is thought to be a platform for the subsequent assembly of the preinitiation complex (reviewed in reference 3). TFIID also plays a role in transcriptional activation (reviewed in references 3 and 34). Extensive studies using in vitro reconstitution systems have shown that the involvement of the TAFs that make up TFIID in transcriptional activation can vary depending on the experimental conditions, e.g., whether mediator components are included and/or nucleosomal DNA is used as a template (16,35,43). In good agreement with these observations, genetic studies of yeast revealed that the requirement for TAFs in in vivo transcription depends on the promoter structures of the target genes (reviewed in reference 13).Yeast promoters are classified as TAF dependent (TAF dep ) or TAF independent (TAF ind ) (23, 25). In vivo cross-linking and chromatin immunoprecipitation analyses indicate that TAFs interact infrequently with TAF ind promoters but frequently with TAF dep promoters at an approximately equimolar ratio to TBP. In the case of the TAF dep promoters of ribosomal protein (RP) genes, TAF recruitment is directed by upstream activating sequences (UAS) but not by the core promoter and it does not depend on the function of TBP and pol II (26, 32).In contrast, the TAF ind UAS in the PGK1 and PYK1 promoters do not efficiently recruit TAFs to TAF dep or TAF ind core promoters (32). These observations indicate that an activator(s) bound to a TAF dep UAS may specifically interact with TAF(s) and recruit TFIID onto the core promoter. In addition, there are some f...

A Random Screen Using a Novel Reporter Assay System Reveals a Set of Sequences That Are Preferred as the TATA or TATA-Like Elements in the CYC1 Promoter of Saccharomyces cerevisiae

“…All strains used in S1 Fig were derived from Y22.1, which carries a deletion of the chromosomal TAF1 coding region and the wild-type TAF1 gene in a URA3 -based low-copy-number vector (pYN1) [ 39 ]. YTK2741 [ 40 ] and YTK3778 were generated from Y22.1 by replacing pYN1 with pM1169 (HA-tagged wild-type TAF1 /pRS314) [ 41 ] and pM1746 (HA-tagged taf1-N568Δ /pRS314), respectively. The latter plasmid was constructed by site-directed mutagenesis of pM1169 using the primer TK176 [ 42 ].…”

Functional Silencing of TATA-binding Protein (TBP) by a Covalent Linkage of the N-terminal Domain of TBP-associated Factor 1

“…We previously reported that TAND of TAF1 could act independently from the rest of the molecule (46). The functional autonomy of TAND was tested by fusing it to the N or C termini of several other components of TFIID and it was found to have similar function as at the N terminus of TAF1.…”

“…1). Our recent studies suggest that TAND of TAF1 is an autonomous regulator of TBP function (46). TAND can interact with and regulate TBP function when it is fused to either termini of any TFIID subunit.…”

Saccharomyces cerevisiae Med9 comprises two functionally distinct domains that play different roles in transcriptional regulation

“…Immunoblotting and preparation of the polyclonal antibodies directed against Spt3, Taf11, and TBP were described previously (Tsukihashi et al. 2000; Takahata et al. 2004).…”

“…Two dimensional blue-native PAGE (BN-PAGE) (Wittig et al 2006) was conducted with precast Native PAGE 3%-12% gel in the first dimension and precast NuPAGE 4%-12% Bis-Tris Zoom gel in the second dimension, according to manufacturer's instructions (Invitrogen). Immunoblotting and preparation of the polyclonal antibodies directed against Spt3, Taf11, and TBP were described previously (Tsukihashi et al 2000;Takahata et al 2004). Polyclonal antibodies directed against Toa1 (aa 1 to 286), Sua7 (aa 1 to 345), Med4 (aa 1 to 284), Med6 (aa 1 to 295), Med7 (aa 1 to 222), Med9 (aa 1 to 149), Med11 (aa 1 to 132), Med15 (aa 1 to 300), Med17 (aa 1 to 300), Med19 (aa 1 to 220), Med20 (aa 1 to 210), Med22 (aa 1 to 121), Cdk8 (aa 1 to 555), Rpb3 (aa 1 to 318), Tfa1 (aa 1 to 482), and Tfg2 (aa 1 to 400) were raised in rabbits using recombinant, gel-purified His-tagged polypeptides expressed in E. coli as antigens.…”