Structural analyses of transcriptional regulation in stress response

应激反应转录调控的结构分析

• 批准号: 13480222
• 负责人: NISHIMURA Yoshifumi
• 金额: $ 7.17万
• 依托单位: Yokohama City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13480222/
• 关键词: transcription factor; telomeres; DNA-binding protein; TRF1; TRF2; NMR; TFIIE; Zn binding domain; 転写因子; Rap1; Myb; DNA認識; テロメア結合タンパク質; DNA結合ドメイン; 表面構造; hTRF1; hRap1

The zinc finger domain in the large subunit of TFIIE is phylogenetically conserved and is essential for transcription. We have determined its NMR structure. It consists of one alpha helix and six beta strands showing novel features distinct from previously determined zinc-binding structures. We created point mutants in this domain and examined their binding abilities to other general transcription factors as well as their transcription activities. Interesting functional asymmetry of Zn^<2+>-ligand mutants was observed: the N-terminal two mutants remained approximately 20% activity on a supercoiled template. CD and NMR studies showed that those two equilibrate mainly with the random coil structure, although all four mutants possessed partially folded characteristic structures coordinating Zn^<2+>-. And also, highly conserved D 164 mutants (D 164A and D 164K) were found to increase transcription.In addition, we have determined the solution structure of the DNA binding domain of hTRF2 bound to a telomeric double-stranded DNA with the sequence of GTTAGGGTTAGGG and compared it with the corresponding DNA complex structure of hTRF1. Telomeres are the ends of eukaryotic linear chromosomes consisting of repetitive G-rich sequence and telomeric repeat binding factors. In mammalian telomeres, TRF1 and TRF2 bind to double-stranded telomeric DNA. Both contain a central TRF-homology (TRFH) domain and a C-terminal DNA binding domain. Both DNA-bound structures are very close to each other, however, small but significant structural differences are observed. Based on the present structure of hTRF2 we could make several mutants of hTRF2, which have a stronger binding ability to telomeric DNA rather than the wild type.

TFIIE的大亚基中的锌指结构域在遗传学上是保守的，并且对于转录是必需的。我们测定了它的NMR结构。它由一个α螺旋和六个β链组成，显示出与先前确定的锌结合结构不同的新特征。我们在这个域中创建点突变体，并检查它们与其他一般转录因子的结合能力以及它们的转录活性。我们观察到了Zn^2+-配体突变体有趣的功能不对称性：N-末端的两个突变体在超螺旋模板上保持了大约20%的活性。CD和NMR研究表明，这两个平衡主要与无规卷曲结构，虽然所有四个突变体具有部分折叠的特征结构，配位Zn^<2+>-。此外，我们还测定了hTRF 2的DNA结合域与端粒双链DNA（序列为GTTAGGGTTAGGG）结合的溶液结构，并与hTRF 1的相应DNA复合物结构进行了比较。端粒是真核生物线性染色体的末端，由富含G的重复序列和端粒重复序列结合因子组成。在哺乳动物端粒中，TRF 1和TRF 2与双链端粒DNA结合。两者都含有一个中央TRF同源（TRFH）结构域和一个C-末端DNA结合结构域。两种DNA结合结构彼此非常接近，然而，观察到小但显著的结构差异。根据hTRF 2的结构，我们可以构建几种突变体，它们与端粒DNA的结合能力比野生型更强。

项目成果

Hanaoka, S.: "NMR structure of the hRap1 Myb motif reveals a canonical three helix bundle lacking the positive surface charge typical of Myb DNA binding domains"J. Mol. Biol.. 312. 167-175 (2001)

Hanaoka, S.：“hRap1 Myb 基序的 NMR 结构揭示了典型的三螺旋束，缺乏 Myb DNA 结合域典型的正表面电荷”J.

A Novel zinc finger structure in the large Subunit of human general transcription factor TFIIE.

人类通用转录因子 TFIIE 大亚基中的新型锌指结构。

• 发表时间: 2004
• 期刊: The Journal of Biological Chemistry 279
• 作者: Okuda;M.;Tanaka;A.;Arai;Y.;Satoh;M.;Okamura;H.;Nagadoi;A.;Hanaoka;F.;Ohkuma;Y.;Nishimura;Y.
• 通讯作者: Y.

奥田昌彦, 西村善文: "転写における構造生物学"蛋白質・核酸・酵素. Vol.47. 926-940 (2002)

Masahiko Okuda，Yoshifumi Nishimura：“转录的结构生物学”，蛋白质、核酸和酶，第 47 卷（2002 年）。

NMR分光法-原理から応用まで 測定法シリーズ41

NMR 波谱 - 从原理到应用 测量方法系列 41

• 发表时间: 2004
• 作者: 西村 善文

神経特異的遺伝子発現に必須なアミノ酸配列

神经元特异性基因表达必需的氨基酸序列

• 发表时间: 2004