A role of transcriptional activators as antirepressors for the autoinhibitory activity of TATA box binding of transcription factor IID

Abstract

. 2). An alternative model postulates that the holoenzyme enters the preinitiation complex as a preassembled unit (2, 3). Although the nature of the assembly pathway most relevant to the in vivo context remains unclear, access of TFIID to the core promoter is likely to be a critical step in any pathway, given that only TFIID binds to the promoter in a sequence-specific manner. In support of this view, in vitro recruitment experiments using immobilized promoters demonstrated that the holoenzyme is not recruited independently of TFIID and TFIIA (4).TFIID is a multimeric protein complex consisting of the TATA box-binding protein (TBP) and Ͼ10 distinct TBPassociated factors (TAFs), which are conserved from yeast to man (reviewed in ref. . Consistent with these observations in vivo, TAF-independent activation was demonstrated in transcriptional experiments in vitro, using TFIID-depleted HeLa nuclear extracts, which could possess activator targets other than TAFs (8), or even with a highly purified cell-free transcription system (9, 10). Furthermore, transcription activation is reconstituted by supplementing purified yeast holoenzyme with only TBP (11, 12). These observations suggest that activators contribute to activated transcription by interacting with multiple targets (e.g., basal factors, TAFs, SRB mediators, or other unknown cofactors).Although TAFs are well recognized as positive cofactors, in our view, at least certain TAFs are negative cofactors. Importantly, highly purified TFIID manifests lower transcriptional activity than TBP alone (13) and binds to the core promoter poorly (reviewed in ref. 14). This inhibitory activity for promoter binding in TFIID is suppressed by limited proteolysis of TFIID or by TFIIA (14,15). These results strongly suggest that this inhibitory activity could be intrinsic to TFIID and derived from TAF(s) and is sensitive to proteases. Consistent with this idea, Drosophila (d)TAF230, or the homologous yTAF145, inhibits TBP binding to the TATA box when these TAFs are mixed with TBP in vitro (16)(17)(18). Mutational analyses of dTAF230 indicate that the N-terminal 156 residues inhibit TATA box binding through direct interaction with TBP (19,20). This N-terminal domain (designated as TAND; TAF N-terminal domain), has been dissected into subdomain 1 (dTAND-1; residues 11-77) and subdomain 2 (dTAND-2; residues 82-156), which bind to the concave undersurface and the convex upper surface of TBP in This paper was submitted directly (Track II) to the PNAS office.

Fluorescence-based Analyses of the Effects of Full-length Recombinant TAF130p on the Interaction of TATA Box-binding Protein with TATA Box DNA

“…Indeed, Nakatani and colleagues (27) found that a fragment of dTAF250p competed with the VP16 activation domain for interaction with specific amino acid sequences on the concave surface of TBP. These data are consistent with their hypothesis that the TAF130p TAND domain and transactivators compete with DNA for interactions on TBP's DNA binding surface (79). This continuous negative action of the TAND may be antagonized, or derepressed, by the action of TFIIA (Refs.…”

The VP16 Activation Domain Interacts with Multiple Transcriptional Components as Determined by Protein-Protein Cross-linking in Vivo

“…This suggests that the VP16 activation domain may not interact with TBP and TFIIB in the context of a preinitiation complex, and in this regard our observed crosslinking occurs primarily (and perhaps exclusively) when the proteins are not bound to DNA. Our results are consistent with the idea that activation domains can function as antirepressors of the autoinhibitory activity of TBP (65). However, while our results establish that the VP16 activation domain directly interacts with TBP and TFIIB in vivo, the importance of these interactions for transcriptional activity in vivo remain to be determined.…”

“…First, unlike the case for SAGA, TBP and TFIIB are not recruited by the Gal4 activator bound to its genomic sites in the absence of a TATA element (23,24). Second, the VP16 activation domain interacts with surfaces of TBP (64,65) and TFIIB (31,66) that are critical for promoter binding, and mutations that abolish the interactions in vitro do not significantly affect the level of transcriptional activity in vivo (67,68). This suggests that the VP16 activation domain may not interact with TBP and TFIIB in the context of a preinitiation complex, and in this regard our observed crosslinking occurs primarily (and perhaps exclusively) when the proteins are not bound to DNA.…”

TATA-binding Protein and the Gal4 Transactivator Do Not Bind to Promoters Cooperatively

“…In vivo cross-linking experiments have shown clearly that TBP does not associate with the TATA box of the GAL1 promoter until Gal4p activity has been induced (12,13), so Gal4p either directly or indirectly plays an important role in this event. Since many inhibitors of TBP-TATA interactions are known (28, 36 -40), one model is that the activator could help to compete these negative regulators from TBP and then "hand-off" the protein to the TATA box (41). This type of model would be consistent with the inability of TBP to bind Gal4p and DNA simultaneously as well as the in vivo cross-linking result.…”