TRANSACTIVATION ABILITY OF P53 TRANSCRIPTIONAL ACTIVATION DOMAIN IS DIRECTLY RELATED TO THE BINDING-AFFINITY TO TATA-BINDING PROTEIN
TRANSACTIVATION ABILITY OF P53 TRANSCRIPTIONAL ACTIVATION DOMAIN IS DIRECTLY RELATED TO THE BINDING-AFFINITY TO TATA-BINDING PROTEIN
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DOI：
10.1074/jbc.270.42.25014
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发表时间：
1995-10-20
影响因子：
4.8
通讯作者：
SUNG, YC
中科院分区：
文献类型：
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作者：
CHANG, J;KIM, DH;SUNG, YC
肿瘤抑制蛋白P53是一种有效的转录激活因子，负向调节细胞生长。为了研究p53的转录激活域(TAD)，在Ga14 DNA结合域的背景下构建了不同的点突变株，并检测了它们的反式激活能力。我们的结果表明，与单纯疱疹病毒VP16和其他反式激活剂共享的位置保守的疏水残基对于反式激活是必不可少的。此外，带负电荷的残基和脯氨酸残基是充分活性所必需的，但对P53 TAD的活性不是必需的。缺失分析表明，p53 TAD可分为1-40和43-73两个亚区。体外谷胱甘肽S转移酶下拉实验建立了体内P53 TAD介导的反式激活与P53 TAD与TATA结合蛋白结合活性之间的线性关系。突变降低了Ga14-P53 TAD的反式激活能力，也严重损害了TBP与TBP的结合活性，我们的结果表明，至少TBP是P53 TAD的直接靶点，TAD与TBP的结合强度(TFIID)是控制P53 TAD活性的重要参数。圆二色谱表明，P53 TAD多肽在溶液中没有任何规则的二级结构，野生型TAD和反式激活缺陷突变型TAD的光谱没有显著差异。
Tumor suppressor protein p53 is a potent transcriptional activator and regulates cell growth negatively. To characterize the transcriptional activation domain (TAD) of p53, various point mutants were constructed in the context of Ga14 DNA binding domain and tested for their transactivation ability. Our results demonstrated that the positionally conserved hydrophobic residues shared with herpes simplex virus VP16 and other transactivators are essential for transactivation. Also, the negatively charged residues and proline residues are necessary for full activity, but not essential for the activity of p53 TAD. Deletion analyses showed that p53 TAD can be divided into two subdomains, amino acids 1-40 and 43-73. An in vitro glutathione S-transferase pull-down assay establishes a linear correlation between p53 TAD mediated transactivation in vivo and the binding activity of p53 TAD to TATA-binding protein (TBP) in vitro. Mutations that diminish the transactivation ability of Ga14-p53 TAD also impair the binding activity to TBP severely, Our results suggest that at least TBP is a direct target for p53 TAD and that the binding strength of TAD to TBP (TFIID) is an important parameter controlling activity of p53 TAD. In addition, circular dichroism spectroscopy has shown that p53 TAD peptide lacks any regular secondary structure in solution and that there is no significant difference between the spectra of the wild type TAD and that of the transactivation-deficient mutant type.