Stimulation of RNA polymerase II transcription initiation by recruitment of TBP in vivo

Abstract

Eukaryotic transcriptional activators may stimulate RNA polymerase II activity by promoting assembly of preinitiation complexes on promoters through their interactions with one or more components of the basal machinery. On the basis of its central role in initiating transcription-complex formation upon binding to the TATA box, the general transcription factor TFIID, which includes the TATA-binding protein (TBP) and several TBP-associated factors, has been implicated as a target for activators. Consistent with this idea, an increasing number of activators have been reported to bind directly to TBP. To assess the functional importance of these in vitro interactions for transcriptional regulation in vivo, we made use of a novel strategy in yeast to show that a physical interaction with TBP is sufficient for a sequence-specific DNA-binding protein to increase initiation of transcription by RNA polymerase II. These results imply that binding of TFIID to promoter elements is a limiting step in transcription complex assembly in vivo.

Artificially Recruited TATA-Binding Protein Fails To Remodel Chromatin and Does Not Activate Three Promoters That Require Chromatin Remodeling

“…This may well account in part for the generally low transcription levels induced by artificially recruited TBP and also for part of its differential activity at different promoters (10,22,36,39,79). However, it does not readily explain the ability of RAP1 to synergize with GAL4-TBP at the modified HIS4 promoter of Fig.…”

“…For example, transcription initiated at a mutated TATA element by induced synthesis of TBP with altered specificity was enhanced in both rate and extent in the presence of an activator that could bind upstream of the relevant promoter, consistent with activator-mediated recruitment (40). Recruitment has also been inferred from the results of "activator bypass" experiments in which artificial recruitment of TBP to promoter sites near the TATA element resulted in transcriptional activation, implying that TBP recruitment is a rate-limiting step in transcriptional activation in vivo (10,39,79). Most convincingly, chromatin immunoprecipitation experiments revealed TBP to be physically associated with promoters of numerous genes under activated, but not nonactivated, conditions, implying that recruitment accompanies activation (43,46).…”

“…Thus, our findings suggest that in spite of the histone acetyltransferase activity of yeast TAF II 130 (50), TFIID is considerably poorer at remodeling chromatin than are true activators. Previous studies have also reported variable transcriptional activation by artificially recruited TBP at different promoters in yeast, but chromatin structure has not generally been examined (10,22,36,39,79). Similarly, experiments done in mammalian cells have been performed with transiently transfected DNA, which is not incorporated into organized chromatin in the same fashion as genomic DNA (2,48,57,80).…”

“…Similarly, experiments done in mammalian cells have been performed with transiently transfected DNA, which is not incorporated into organized chromatin in the same fashion as genomic DNA (2,48,57,80). Interestingly, however, sufficient activation by artificially recruited TBP is seen at the HIS3 locus to allow growth in the presence of 3-aminotriazole (which requires HIS3 activation) (10,36,39), and this promoter contains a poly(dA-dT) sequence that creates an open chromatin structure (34).…”

Conversion of the omega subunit of Escherichia coli RNA polymerase into a transcriptional activator or an activation target

“…Recent findings in both eukaryotes and prokaryotes indicate that arbitrary protein-protein contacts can trigger gene activation provided one of the protein partners is tethered to the DNA and the other is a component (or is tethered to a component) of RNA polymerase (RNAP) (Barberis et al 1995;Chatterjee and Struhl 1995;Klages and Strubin 1995;Xiao et al 1995;Apone et al 1996;Farrell et al 1996;Dove et al 1997;Gaudreau et al 1997;Gonzalez-Couto et al 1997;Lee and Struhl 1997; for review, see Ptashne and Gann 1997). Experiments in yeast have shown further that direct fusion of a DNA-binding domain to a component of the RNAP II holoenzyme can activate transcription from a promoter bearing a recognition site for the DNA-binding domain (Barberis et al 1995;Farrell et al 1996;Gaudreau et al 1997; for review, see Ptashne and Gann 1997), but analogous experiments have not been performed previously in bacteria.…”