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J. Biol. Chem., Vol. 282, Issue 30, 22228-22238, July 27, 2007

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Functional Silencing of TATA-binding Protein (TBP) by a Covalent Linkage of the N-terminal Domain of TBP-associated Factor 1^*

Tapas K. Mal, Shinya Takahata¹, Sewon Ki, Le Zheng, Tetsuro Kokubo, and Mitsuhiko Ikura²

From the Division of Signaling Biology, Ontario Cancer Institute, Department of Medical Biophysics, University of Toronto, Toronto Medical Discovery Towers, Toronto, Ontario M5G 1L7, Canada and the Division of Molecular and Cellular Biology, International Graduate School of Arts and Sciences, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan

General transcription factor TFIID is comprised of TATA-binding protein (TBP) and TBP-associated factors (TAFs), together playing critical roles in regulation of transcription initiation. The TAF N-terminal domain (TAND) of yeast TAF1 containing two subdomains, TAND1 (residues 10-37) and TAND2 (residues 46-71), is sufficient to interact with TBP and suppress the TATA binding activity of TBP. However, the detailed structural analysis of the complex between yeast TBP and TAND12 (residues 6-71) was hindered by its poor solubility and stability in solution. Here we report a molecular engineering approach where the N terminus of TBP is fused to the C terminus of TAND12 via linkers of various lengths containing (GGGS)_n sequence, (n = 1, 2, 3). The length of the linker within the TAND12-TBP fusion has a significant effect on solubility and stability (SAS). The construct with (GGGS)₃ linker produces the best quality single-quantum-coherence (HSQC) NMR spectrum with markedly improved SAS. In parallel to these observations, the TAND12-TBP fusion exhibits marked reduction of TBP function in binding to TAF1 as well as temperature sensitivity in in vivo yeast cell growth. Remarkably, the temperature sensitivity was proportional to the length of the linker in the fusions: the construct with (GGGS)₃ linker did not grow at 20 °C, while those with (GGGS)₁ and (GGGS)₂ linkers did. These results together indicate that the native interaction between TBP and TAND12 is well maintained in the TAND12-(GGGS)₃-TBP fusion and that this fusion approach provides an excellent model system to investigate the structural detail of the TBP-TAF1 interaction.

Received for publication, April 9, 2007 , and in revised form, June 6, 2007.

^* This project was supported in part by grants from Canadian Institutes of Health Research (to M. I.) and from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to T. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1 and S2.

¹ Present address: Dept. of Pathology, University of Utah, 15 N. Medical Dr. East, Salt Lake City, UT 84112.

¹ Holds the Canada Research Chair in Cancer Structural Biology. To whom correspondence should be addressed: Division of Signaling Biology, Ontario Cancer Institute, Dept. of Medical Biophysics, University of Toronto, Toronto Medical Discovery Towers, 101 College St., 4th floor, Rm. 804, Toronto, Ontario M5G 1L7, Canada. Tel.: 416-581-7550; Fax: 416-581-7597; E-mail: mikura{at}uhnres.utoronto.ca.

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