The Gcn4p Activation Domain Interacts Specifically In Vitro with RNA Polymerase II Holoenzyme, TFIID, and the Adap-Gcn5p Coactivator Complex

Abstract

The Gcn4p activation domain contains seven clusters of hydrophobic residues that make additive contributions to transcriptional activation in vivo. We observed efficient binding of a glutathione S-transferase (GST) Transcription initiation by RNA polymerase II (Pol II) requires assembly of a large complex consisting of Pol II and general transcription factors (GTFs) at the promoter. It has been proposed that assembly of this complex begins when TFIID, consisting of TATA box-binding protein (TBP) and its associated factors (TAF II proteins), binds to the core promoter, followed by sequential binding of other GTFs and Pol II itself (9). In another scenario, Pol II, certain GTFs, and coactivator proteins bind to the promoter as a preformed holoenzyme complex (46). Transcriptional activators bind to the promoter, generally upstream of the TATA element, and stimulate the assembly or function of the transcription initiation complex. Binding of TFIID to the core promoter appears to be rate limiting for initiation (12,43,88), and certain activators stimulate this step in initiation complex formation (3,11,21,39,40,50,91). Several activators bind TBP in vitro in a manner that depends on amino acids in the activation domain that are critical for transcriptional activation in vivo (7,11,26,35,38,51,(61)(62)(63), suggesting that direct interactions between the activator and TBP are involved in recruiting TFIID to the core promoter. Certain activation domains also bind TFIIB in vitro in a sequence-specific manner (4,7,14,41,56,91) and may stimulate recruitment of this GTF to the initiation complex (15,41,55,56).-Other studies suggest that activator function is mediated by one or more of the TAF II coactivator proteins associated with TBP in TFIID. Different activators may require specific TAF II proteins for activation (13,(74)(75)(76), and indeed, certain activation domains bind preferentially to specific TAF II proteins in vitro (24,37,57,83). The interactions between activators and TAF II proteins may serve primarily to recruit TFIID to the promoter (75). The human TAF II 250 subunit (and its Saccharomyces cerevisiae homolog yTAF II 130) has histone acetyltransferase (HAT) activity that may also promote initiation complex formation by destabilizing a repressive nucleosome structure at the promoter (64). A yeast Pol II-TAF II complex was shown to be required for transcriptional activation of a Gcn4p-regulated promoter in vitro (44); however, recent studies indicate that yTAF II proteins are not essential for transcriptional activation in vivo by Gcn4p and by several other yeast activator proteins (65,85).

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

“…This indicates that the vast majority of VP16-dependent cross-linking to the TAFs reflects interaction with SAGA not TFIID. This result agrees with GST pulldown experiments, showing that the Gcn4 activation domain interacts with TAFs present in both SAGA and TFIID but not with TFIID-specific TAFs (39).…”

“…In vitro, transcriptional activators can interact with TATAbinding protein (TBP) 1 (25)(26)(27), TBP-associated factors (TAFs) (28,29), TFIIA (30), TFIIB (31), TFIIH (32), components of the mediator subcomplex of RNA polymerase II holoenzyme (33)(34)(35)(36), Swi/Snf (34,37,38), SAGA (39,40), and NuA4 (40). However, it is generally not understood which of these interactions occur under physiological conditions and are relevant for transcriptional activation in vivo.…”

The Coactivator p/CIP/SRC-3 Facilitates Retinoic Acid Receptor Signaling via Recruitment of GCN5

“…Interestingly, each of these complexes also contain several subunits found in the TFIID complex including histone-like TAFs (47,65,69). It is interesting to note that the ADA2 binding region of GCN5 is required for interaction with p/CIP.…”

A Small Ubiquitin-related Modifier-interacting Motif Functions as the Transcriptional Activation Domain of Krüppel-like Factor 4

“…It is, therefore, of interest to note that although an activation domain of a transcription factor becomes ␣-helical upon binding to basal transcription factors such as TATA-binding protein (TBP) (1), SIM usually assumes a ␤-sheet structure (6,9,10,66) and can bind in either a parallel or antiparallel orientation to the ␤2-strand of SUMO (6,10). Because an activation domain often has multiple binding surfaces with several possible orientations (1,53,(67)(68)(69), it is conceivable that a transcriptional activation domain is induced to become ␣-helical upon binding to basal transcription factors but becomes a ␤-sheet upon binding to SUMO. Thus, a SIM may influence protein conformation (6).…”