TAF_II170 Interacts with the Concave Surface of TATA-Binding Protein To Inhibit Its DNA Binding Activity

Abstract

The human RNA polymerase II transcription factor B-TFIID consists of TATA-binding protein (TBP) and the TBP-associated factor (TAF) TAF_II170 and can rapidly redistribute over promoter DNA. Here we report the identification of human TBP-binding regions in human TAF_II170. We have defined the TBP interaction domain of TAF_II170 within three amino-terminal regions: residues 2 to 137, 290 to 381, and 380 to 460. Each region contains a pair of Huntington-elongation-A subunit-Tor repeats and exhibits species-specific interactions with TBP family members. Remarkably, the altered-specificity TBP mutant (TBP_AS) containing a triple mutation in the concave surface is defective for binding the TAF_II170 amino-terminal region of residues 1 to 504. Furthermore, within this region the TAF_II170 residues 290 to 381 can inhibit the interaction between DrosophilaTAF_II230 (residues 2 to 81) and TBP through competition for the concave surface of TBP. Biochemical analyses of TBP binding to the TATA box indicated that TAF_II170 region 290-381 inhibits TBP-DNA complex formation. Importantly, the TBP_AS mutant is less sensitive to TAF_II170 inhibition. Collectively, our results support a mechanism in which TAF_II170 induces high-mobility DNA binding by TBP through reversible interactions with its concave DNA binding surface.

ACKNOWLEDGMENTS

We thank A. Berk for the pSRaMSVtkneoTBP_AS convex mutants; H. Stunnenberg for pSG-ehTBP and pSG-ehTBPm3e; C. Kane for pET22-TFIIS, Y. Nakatani for pGST-dTAF_II230 (2-81), pGST-dTAF_II230 (82-156), and pGST-dTAF_II230 (2-156); M. Rabenstein for pGST-dTRF-2; R. Romier for pACYC; L. Tora for pGST-ceTLF; and N. Zak for pGST-dTBP. We are grateful to N. Zak for communication of unpublished results. We acknowledge the contributions of F. Holstege in providing TFIIH. We thank F. Kavelaars for technical assistance and members of our laboratory for discussions and critical reading of this manuscript.

This work was supported by a grant to H.T.M.T. from Human Sciences Frontier Program Organization (HSFPO) and The Netherlands Organization for Scientific Research-Medical Sciences (NWO-MW).