Virtually unidirectional binding of TBP to the AdMLP TATA box within the quaternary complex with TFIIA and TFIIB

Abstract

Background: The TATA box binding protein (TBP) is required by all three RNA polymerases for the promoter-specific initiation of transcription. All eukaryotic TBP–DNA complexes observed in crystal structures show the conserved C-terminal domain of TBP (TBP c) bound to the TATA box in a single orientation that is consistent with assembly of a preinitiation complex (PIC) possessing a unique polarity. The binding of TBP to the TATA box is believed to orient the PIC correctly on the promoter and can function as the rate-limiting step in PIC assembly. Previous work performed with TBP from Saccharomyces cerevisiae (yTBP) showed that, despite the oriented binding of eukaryotic TBP observed in crystal structures, yTBP in solution does not orient itself uniquely on the adenovirus major late promoter (AdMLP) TATA box. Instead, yTBP binds the AdMLP as a mixture of two orientational isomers that are related by a 180 degree rotation about the pseudo-dyad axis of the complex. In addition, these orientational isomers are not restricted to the 8 bp TATA box, but rather bind a distribution of sites that partially overlap the TATA box. Two members of the PIC, general transcription factor (TF) IIB and TFIIA individually enhance the orientational and axial specificity of yTBP binding to the TATA box, but fail to fix yTBP in a single orientation or a unique position on the promoter. Results: We used an affinity cleavage assay to explore the combined effects of TFIIA and TFIIB on the axial and orientational specificity of yTBP. Our results show that the combination of TFIIA and TFIIB affixes yTBP in virtually a single orientation as well as a unique location on the AdMLP TATA box. Ninety-five percent of the quaternary TBP–TFIIA–TFIIB–TATA complex contained yTBP bound in the orientation expected on the basis of crystallographic and genetic experiments, and more than 70% is restricted axially to the 8 bp sequence TATAAAAG. Conclusions: Although yTBP itself binds to the TATA box without a high level of orientational or axial specificity, our data show that a small subset of general TFs are capable of uniquely orienting the PIC on the AdMLP. Our results, in combination with recent data concerning the pathway of PIC formation in yeast, suggest that transcription could be regulated during both early and late stages of PIC assembly by general factors (and the proteins to which they bind) that influence the position and orientation of TBP on the promoter.