Basic research on double-stranded DNA-binding proteins

双链DNA结合蛋白的基础研究

• 批准号: 17370058
• 负责人: NISHIMURA Yoshifumi
• 金额: $ 8.99万
• 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: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17370058/
• 关键词: Nucleic acid; Genome; Protein; Regulation of gene expression; Aging; NMR; General transcription factor; Structural biology; タンパク質; 転写調節

We determined the structures of both free hTFIIEalpha C-tearminal acidic domain (AC-D) and its form bound to the N-terminal pleckstrin homology domain (PH-D) of the p62 subunit of hTFIIH. Intriguingly, hTFIIEalpha AC-D was found to share its binding surface on p62 PH-D with the acidic transactivation peptide fragment of the human tumor supressor protein p53. However, hTFIIEalpha AC-D employs an entirely novel binding mode, which differs from the amphipathic helix method used by many transcriptional activators. The binding surface between p62 PH-D and hTFIIEalpha AC-D is much broader than the specific binding surface between p62 PH-D and the p53 peptide fragment. Given the recent demonstration of TFIIE and TFIIH functions in post initiation events (i.e. promoter clearance) these data are of special relevance to the regulatory mechanisms involved.Essential Sas-related acetyltransferase 1 (Esa1) contains a presumed chromodomain in addition to a histone acetyltransferase (HAT) domain. We initially determined the solution structure of the Esa1 presumed chromodomain, and showed it to consist of a well folded structure similar to the tudor domain. The domain showed no RNA/DNA binding ability. Since the N-terminus of the protein forms a helical turn, we prepared an N-terminally extended construct, which was surprisingly found to bind to RNA and to be critical for in vivo function. This extended protein contains an additional β-sheet which acts as a knot for the tudor domain. The knot does not cause a global change in the core structure but induces a well defined loop in the tudor domain itself, which is responsible for RNA binding. We made point mutants in an esa1 mutant gene in yeast and their functional abilities examined. The knotted tudor domain mutations which were lethal to the yeast lost RNA binding ability.

我们测定了hTFIIH p62亚基的游离hTFIIEαC-撕裂酸性结构域(AC-D)及其与p62亚基的N-末端Pleckstrin同源结构域(PH-D)的结合形式。有趣的是，hTFIIEpha AC-D被发现与人肿瘤抑制蛋白P53的酸性反式激活肽片段在p62 PH-D上有相同的结合面。然而，hTFIIEpha AC-D采用了一种全新的结合模式，这与许多转录激活剂使用的两亲性螺旋方法不同。P62 PH-D与hTFIIEpha AC-D的结合表面比p62 PH-D与p53多肽片段的特异性结合表面宽得多。鉴于最近对TFIIE和TFIIH在启动后事件(即启动子清除)中的功能的研究，这些数据与所涉及的调控机制特别相关。必需的SAS相关乙酰基转移酶1(ESA1)除了组蛋白乙酰转移酶(HAT)结构域外，还含有一个推测的染色质结构域。我们最初确定了Esa1推测的染色区域的溶液结构，并表明它由一个类似于Tudor结构域的良好折叠结构组成。该结构域不具有与RNA/DNA结合的能力。由于蛋白质的N-末端形成螺旋状转折，我们制备了一个N-末端延伸的结构，令人惊讶地发现它与RNA结合，对体内功能至关重要。这种延伸的蛋白质含有一个额外的β-折叠，作为都铎结构域的一个结。该结不会导致核心结构的全局变化，但会在负责RNA结合的Tudor结构域本身中诱导一个定义良好的环。我们在酵母中对esa1突变基因进行了点突变，并对其功能进行了检测。对酵母致死的打结的Tudor结构域突变失去了RNA结合能力。

项目成果

Comparison between TRF2 and TRF1 on their telomeric DNA-bound structures and DNA-binding activities.

TRF2和TRF1端粒DNA结合结构和DNA结合活性的比较。

• 发表时间: 2005
• 期刊: Protein Sci. 14
• 作者: Hanaoka;S.;Nagadoi;A.;Nishimura;Y.
• 通讯作者: Y.

蛋白質・核酸・酵素52蛋白質動的構造の新しいパラダイム:特集にあたって

蛋白质、核酸和酶 52 蛋白质动态结构新范式：针对特殊功能

• 发表时间: 2007
• 作者: 西村 善文;中村 春木
• 通讯作者: 中村 春木

蛋白質・核酸・酵素 52 蛋白質動的構造の新しいパラダイム:特集にあたって

蛋白质、核酸和酶 52 蛋白质动态结构新范式：关于特殊性

• 发表时间: 2007
• 作者: 西村善文;中村春木
• 通讯作者: 中村春木

Stmctural insight into the TFIIE/TFIIH interaction: TFIIEand p53 share the binding region on TFIIH.

TFIIE/TFIIH 相互作用的结构洞察：TFIIE 和 p53 共享 TFIIH 上的结合区域。

• 发表时间: 2008
• 期刊: EMBO J. 27
• 作者: Okuda;M.;Tanaka;A.;Satoh;M.;Mizuta;S.;Takazawa;M.;Ohkuma;Y.;and Nishimura;Y.
• 通讯作者: Y.

NMR dynamics distinguish between hard and soft hydrophobic cores in the DNA-Binding domain of PhoB and demonstrate different roles of the cores in binding to DNA

NMR 动力学可区分 PhoB DNA 结合域中的硬疏水核心和软疏水核心，并证明核心在与 DNA 结合中的不同作用

• 发表时间: 2007
• 期刊: J.Mol.Biol. 367
• 作者: Okamura;H.;Makino;K.;Nishimura;Y.
• 通讯作者: Y.