Characterization of Physical Interactions of the Putative Transcriptional Adaptor, ADA2, with Acidic Activation Domains and TATA-binding Protein

Nickolai A Barlev,Reyes Candau,Lian Wang,Paula Darpino,Neal Silverman,Shelley L Berger

doi:10.1074/jbc.270.33.19337

Nickolai A Barlev, Reyes Candau + Show 4 more

Open Access

https://doi.org/10.1074/jbc.270.33.19337

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Abstract

RNA polymerase II transcription requires functional interactions between activator proteins bound to upstream DNA sites and general factors bound to the core promoter. Accessory transcription factors, such as adaptors and coactivators, have important, but still unclear, roles in the activation process. We tested physical interactions of the putative adaptor ADA2 with activation domains derived from acidic activator proteins and with certain general transcription factors. ADA2 associated with the herpesvirus VP16 and yeast GCN4 activation domains but not with the activation domain of yeast HAP4, which previously was shown to be independent of ADA2 function in vivo and in vitro. Furthermore, the amino terminus of ADA2 directly interacted with the VP16 activation domain, suggesting that ADA2 provides determinants for interaction between activation domains and the adaptor complex. Both TATA-binding protein (TBP) and TFIIB have previously been shown to interact directly with the VP16 activation domain in vitro (Stringer, K. F., Ingles, C. J., and Greenblatt, J. (1990) Nature 345, 783-786; Lin, Y. S., Ha, I., Maldonado, E., Reinberg, D., and Green, M. R. (1991) Nature 353, 569-571). Interestingly, when binding was tested between VP16 and these general factors in yeast nuclear extracts, both factors interacted with VP16, but only the TBP/VP16 association was dependent on ADA2. In addition, ADA2 physically associated with TBP, but not with TFIIB. These results suggest that the role of ADA2 in transcriptional activation is to promote physical interaction between activation domains and TBP.

Highlights

From The Wistar Institute, Philadelphia, Pennsylvania 19104 and the Wepartment of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
ADA2 Interacts with VP16 and GCN4 Activation Domains, but Not with That of HAP4-We have previously shown that certain acidic activation domains, such as herpesvirus VP16 and yeast GCN4, require ADA2 for activation both in vivo and in vitro, while other activation domains, such as yeast HAP4, are relatively independent of ADA2 (Berger et al, 1992; Pifia et al, 1993)
Proteins containing GST (Smith and Johnson, 1988) fused to activation domains derived from VP16, GCN4, and HAP4 were coupled to glutathione-Sepharose beads

Summary

Introduction

The activation domain of VP16 has been shown to contact directly three general transcription factors, TBpl (TATA-binding protein (Stringer et al, 1990)), TFIIB (Roberts et al, 1993) and TFIIH (Xiao et al, 1994). These interactions may not be sufficient for transcriptional activation, and several lines of evidence suggest that additional factors are required. In yeast are SRB proteins, which interact with the carboxyl-terminal tail of the largest subunit of RNA polymerase II and are required for activated transcription from many promoters (Thompson et al, 1993) These factors, along with several general factors, may constitute a holoenzyme RNA polymerase complex (Kim et al, 1994; Koleske and Young, 1994) which includes a transcriptional stimulatory activity (Kim et al, 1994)

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