Molecular mechanismn of transcription termination factor Rho as a hexameric RAN/DNA helicase

转录终止因子 Rho 作为六聚体 RAN/DNA 解旋酶的分子机制

• 批准号: 13680762
• 负责人: SHIGESADA Katsuya
• 金额: $ 2.05万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680762/
• 关键词: Rho factor; Transcription termination; F1-ATPase homology; Helicase; Troidal hexamer; Chemical cross-linking; 3-D structure; X-ray crystallography; 転写終結因子; アミノ酸変異; RNA移送モデル; F_1-ATPase; ヒドロキシラミン分解

The E. coli transcription termination protein Rho is a hexameric helicase, and is believed to function by separating an RNA-DNA hybrid in an ATPdependent manner. To further elucidate the molecular mechanism of Rho, we have focused on its structural similarity to F1-ATP-ase and conducted the following two lines of studies1) Our previous cross-linking study suggested that the Rho hexamer has a pseudo-C3 symmetry, in which its subunits take dual conformanonal states in an alternating manner. We further tried herein to map the positions of cross-links on Rho by digestion with hydroxylamine, which singly cuts the rho polypeptide between Asn 151 and Gly 152. The result revealed that a lysine on the N-terminal fragment from one subunit is cross-linked to a lysine on the C-terminal fragment from an adjacent subunit at all subunit-subunit interfaces. On the other band, a three-dimensional reconstruction of Rho hexamer predicted that Lysl23 is positioned in close proximity to Lys224 and Lys249 on an adjacent subunit. Thus, Lysl23 would make a good candidate for crosslinks to Lys224 or Lys2492) Toward determining the exact three-dimensional structure of Rho, we also set out for its X-ray crystallographic analysis. Initially, we could only obtain Rho crystals of poor qualities that showed no clear diffractions. Through subsequent repeated trials, we have identified several conditions that could improve the quality of Rho crystal: a) addition of a high concentration of KC1 (0 5 M); b) a prolonged aging of crystal; c) methylation of lysine residues; and so on. Even with these conditions combined, however, best crystals so far made yielded diffractions at unsatisfactory resolutions of at most 10 Angstrom. Most recently, we have found that the addition of ligands such as ATP analogs and oligo (dC) help to maintain Rho protein in a mono-disperse state, a prerequisite for making good crystals. Thus trials are in progress to crystallize complexes containing such ligands

大肠杆菌转录终止蛋白Rho是一种六聚体解旋酶，被认为是通过以ATP依赖的方式分离RNA-DNA杂交体而发挥作用的。为了进一步阐明Rho的分子机制，我们重点研究了它与F1-ATP-ase的结构相似性，并进行了以下两方面的研究：1)我们先前的交联性研究表明，Rho六聚体具有伪C3对称性，其亚基交替地处于双构象状态。在这里，我们进一步尝试通过羟胺的消化来定位Rho上的交联链的位置，这将在Asn 151和Gly 152之间单独切割Rho多肽。结果表明，在所有亚基-亚基界面上，来自一个亚基的N-末端片段上的一个赖氨酸与来自相邻亚基的C-末端片段上的一个赖氨酸发生交联。在另一条带上，Rho六角体的三维重建预测Lysl23位于邻近亚基上的Lys224和Lys249附近。因此，Lys23将是与Lys224或Lys2492进行交联的一个很好的候选者)为了确定Rho的确切三维结构，我们还对其进行了X射线晶体分析。最初，我们只能得到质量较差的Rho晶体，没有明显的衍射峰。通过随后的反复试验，我们确定了几个可以提高Rho晶体质量的条件：a)添加高浓度的Kc1(0.5M)；b)延长晶体的老化时间；c)赖氨酸残基的甲基化；等等。然而，即使将这些条件结合在一起，迄今为止最好的晶体产生的衍射分辨率最高可达10埃，令人不满意。最近，我们发现ATP类似物和寡核苷酸(DC)等配体的加入有助于保持Rho蛋白的单分散状态，这是形成良好晶体的先决条件。因此，结晶含有这种配体的络合物的试验正在进行中。

项目成果

Joelle Michaud: "in vitro analyses of known and novel RUNX1/AML1 mutations in dominant familial platelet disorder with predisposition to FPD/AML"Blood. 99・4. 1364-1372 (2001)

Joelle Michaud：“对 FPD/AML 易感性的显性家族性血小板疾病中已知和新型 RUNX1/AML1 突变的体外分析”血液 99·4 (2001)。

Motomi Osato: "Point mutations of the RUNX1/AML1 gene in sporadic and familial myeloid leukemias"Int J Hematol. 74・3. 245-251 (2001)

Motomi Osato：“散发性和家族性骨髓性白血病中 RUNX1/AML1 基因的点突变”Int J Hematol 74·3（2001）。

Taketoshi Yoshida: "Functional analysis of RUNX2 mutations in Japanese patients with cleidocranial dysplasia demonstrates novel genotype-phenotype correlations"Am J Hum Genet. 71・4. 724-738 (2002)

Taketoshi Yoshida：“日本锁骨颅骨发育不良患者的 RUNX2 突变的功能分析表明了新的基因型-表型相关性”Am J Hum Genet 71・4 (2002)。

Taketoshi Yoshida et al.: "Functional analysis of RUNX2 mutations in Japanese patients with cleidocranial dysplasia demonstrates novel genotype-phenotype correlations"Am J Hum Genet. 71(4). 724-738 (2002)

Taketoshi Yoshida 等人：“日本锁骨颅骨发育不良患者 RUNX2 突变的功能分析证明了新的基因型-表型相关性”Am J Hum Genet。

Taketoshi Yoshida: "Functional analysis of RUNX2 mutations in cleidocranial dysplasia : novel insights into genotype-phenotype correlations"Blood Cells Mol Dis.. 30・2. 184-193 (2003)

Taketoshi Yoshida：“锁骨颅骨发育不良中 RUNX2 突变的功能分析：基因型-表型相关性的新见解”Blood Cells Mol Dis.. 184-193 (2003)。