Abstract
Essentially all nuclear eukaryotic gene transcription depends upon the function of the transcription factor TATA-binding protein (TBP). Here we show that the abundant, multifunctional DNA binding transcription factor repressor activator protein Rap1p interacts directly with TBP. TBP-Rap1p binding occurs efficiently in vivo at physiological expression levels, and in vitro analyses confirm that this is a direct interaction. The DNA binding domains of the two proteins mediate interaction between TBP and Rap1p. TBP-Rap1p complex formation inhibits TBP binding to TATA promoter DNA. Alterations in either Rap1p or TBP levels modulate mRNA gene transcription in vivo. We propose that Rap1p represents a heretofore unrecognized regulator of TBP.
Highlights
MARCH 28, 2008 VOLUME 283 NUMBER 13 genes is bound via the TATA-binding protein (TBP) subunit of TFIID through a process facilitated by TBP-associated factors (TAFs) [7,8,9,10]
While conducting footprinting experiments to examine the binding of yeast TFIID to a battery of Rap1p-dependent and Rap1p-independent genes, control reactions showed that Rap1p addition blocked TBP-TATA DNA binding
Given the central role of TBP in nuclear gene transcription, the potency of inhibition of TBP-DNA binding by nanomolar concentrations of Rap1p, the nuclear abundance, and high concentration of both proteins (Ն100 M), we examined this phenomenon in more detail
Summary
Reactions were mixed 30 min at room temperature, beads were washed 3 times with binding buffer, resuspended in 20 l of SDS-PAGE sample buffer, heat denatured, and analyzed by SDS-PAGE on NuPAGE 4 –12% polyacrylamide gels followed by Sypro Ruby staining or immunoblotting. Primer Extension and Chromatin Immunoprecipitation (ChIP) Assays—Primer extension [67] and ChIP assays were performed essentially as described [55] except ChIP PCR products were fractionated by non-denaturing polyacrylamide gel electrophoresis in 1ϫ TBE buffer and DNA quantified by staining with Sypro Gold (Invitrogen)
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