Functional silencing of TATA-binding protein (TBP) by a covalent linkage of the N-terminal domain of TBP-associated factor 1
Functional silencing of TATA-binding protein (TBP) by a covalent linkage of the N-terminal domain of TBP-associated factor 1
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DOI：
10.1074/jbc.m702988200
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发表时间：
2007-07-27
影响因子：
4.8
通讯作者：
Ikura, Mitsuhiko
中科院分区：
文献类型：
--
作者：
Mal, Tapas K.;Takahata, Shinya;Ikura, Mitsuhiko
转录因子TFIID由TATA结合蛋白（TBP）和TBP相关因子（TAFs）组成，共同参与转录起始的调控。酵母TAF 1的TAF N-末端结构域（TAND）含有两个亚结构域，TAND 1（残基10-37）和TAND 2（残基46-71），足以与TBP相互作用并抑制TBP的TATA结合活性。然而，酵母TBP和TAND 12（残基6-71）之间的复合物的详细结构分析因其在溶液中的溶解度和稳定性较差而受到阻碍。在这里，我们报告了一种分子工程方法，其中TBP的N末端通过含有（GGGS）（n）序列的各种长度的接头与TAND 12的C末端融合，（n = 1，2，3）。TAND 12-TBP融合物内接头的长度对溶解度和稳定性（SAS）具有显著影响。具有（GGGS）（3）接头的构建体产生最佳质量的单量子相干（HSQC）NMR光谱，具有显著改善的SAS。与这些观察结果平行，TAND 12-TBP融合体表现出TBP与TAF 1结合的功能以及体内酵母细胞生长中的温度敏感性的显著降低。值得注意的是，温度敏感性与融合体中接头的长度成比例：具有（GGGS）（3）接头的构建体在20 ℃下不生长，而具有（GGGS）（1）和（GGGS）（2）接头的构建体在20 ℃下生长。这些结果共同表明，TBP和TAND 12之间的天然相互作用在TAND 12-（GGGS）（3）-TBP融合物中得到很好的保持，并且这种融合方法提供了研究TBP-TAF 1相互作用的结构细节的极好的模型系统。
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 degrees 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.