Role of RNA polymerase subunit RPB 5 in transcriptional complex formation and Dranscriptional control

RNA聚合酶亚基RPB 5在转录复合物形成和转录控制中的作用

• 批准号: 07458178
• 负责人: MURAKAMI Seishi
• 金额: $ 4.48万
• 依托单位: Kanazawa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07458178/
• 关键词: RNA polymerase; RPB5; Hepafitis B uirus X protein; Tramsactivation; Tramscirption initiation; General transcription fuctor TFIIB; Transcriptional cofactor; RPB5-mediating-factor; B型肝炎ウイルス; 肝細胞がん; X蛋白(HBx); 転写活性化

Our previous finding that HBx directly interacts with RPB5, a common subunit of RNA polymerases, implies that HBx modulates the function of RNA polymerase directly. This project addressed the molecular mechanism of transcriptional modulation of RPB5 by HBx. 1.We found that both HBx and RPB5 specifically bound to TFIIB in vitro and in vivo. We could detect the ternary complex consisting of RPB5, HBx, and TFIIB in vivo and in vitro. 2.Some HBx substitution mutants, which were severely impaired in transacting activity, exhibited reduced binding affinity with either TFIIB or RPB5 in a mutually exclusive manner, suggesting that HBx transactivation requires the interactions of both RPB5 and TFIIB.The results indicate that HBx is a novel virus modulator that facilitates transcriptional initiation by stabilizing the association between RNA polymerase and TFIIB through communication with RPB5 and TFIIB.3.We addressed whether HBx acts as a transcriptional activator when it is recruited to a distal cis-element, and whether HBx positively affects in vitro transcription. The results indicated that HBx can not act as a transcriptional transactivator but can act as a cofactor involving in transcription through protein-protein interaction. 4.To understand the function of RPB5, we tried to isolate RPB5-binding protein by direct cDNA cloning using far-Western blotting. We successed to isolate a cCNA redundantly which was found unreported and unique. The gene was designated as RPB5 mediating protein (RMP). Preliminarily experiments showed that RMP may interfer transactivation of HBx in mammalian cells. The results suggest that RMP may antagonize HBx. Further characterization of RMP is on going.

我们之前发现HBx直接与RPB5相互作用，RPB5是RNA聚合酶的一个常见亚基，这意味着HBx直接调节RNA聚合酶的功能。本项目研究了HBx对RPB5转录调控的分子机制。1.我们在体外和体内均发现HBx和RPB5特异性结合TFIIB。我们可以在体内和体外检测到RPB5、HBx和TFIIB组成的三元配合物。2.一些HBx替代突变体，其交易活性严重受损，表现出与TFIIB或RPB5的结合亲和力降低，以互排斥的方式，表明HBx的交易激活需要RPB5和TFIIB的相互作用。结果表明，HBx是一种新型的病毒调节剂，通过与RPB5和TFIIB的通信，稳定RNA聚合酶与TFIIB之间的关联，从而促进转录起始。我们研究了当HBx被募集到远端顺式元件时，它是否作为转录激活因子，以及HBx是否积极影响体外转录。结果表明，HBx不能作为转录反激活因子，但可以通过蛋白-蛋白相互作用作为辅助因子参与转录。4.为了了解RPB5的功能，我们尝试用远西印迹法直接克隆cDNA分离RPB5结合蛋白。我们成功地分离了一个cCNA冗余，这是发现未报告的和独特的。该基因被命名为RPB5介导蛋白（RMP）。初步实验表明，RMP可能干扰哺乳动物细胞HBx的转激活。结果表明，RMP具有拮抗HBx的作用。对RMP的进一步描述正在进行中。

项目成果

Nakamoto,Y.,Kaneko,S.,MURAKAMI,S.,et al.,: "B-cell epitopes in by pervariable region 1 of hepatitis C virus obtainece from patients with chsonic persistant hepatitis" J.Med.Virol. 50. 35-41 (1996)

Nakamoto,Y.、Kaneko,S.、MURAKAMI,S.等人，：“从慢性持续性肝炎患者中获得的丙型肝炎病毒常变区 1 中的 B 细胞表位”J.Med.Virol。

MURAKAMI,S.,Lin,Y.,et al.,: "Human hepatitis B virus X protein is a transciptional modilator that communicate with trancription factor,TFIIB and the RNA palipmerase subunit RPB5" J.Biol Chem.,. (印刷中). (1997)

MURAKAMI, S., Lin, Y. 等人：“人类乙型肝炎病毒 X 蛋白是一种与转录因子 TFIIB 和 RNA 棕榈酶亚基 RPB5 通讯的转录调节剂”J.Biol Chem.,。 1997）

Ohno,H.,Murakami,S.,et al.,: "Human hepatitis B virus enhance 1 is responsive to human interlaukin 6" J.Medical Virol. (印刷中). (1997)

Ohno, H.、Murakami, S. 等人：“人类乙型肝炎病毒增强 1 对人类白介素 6 有反应”J.Medical Virol（出版中）。

Yi, MK., Nakamoto, Y., Murakami, S., et al: "Delaieation of regions important for heteromeric association of Hepatitis C virus El and E2" Virology. (印刷中). (1997)

Yi, MK., Nakamoto, Y., Murakami, S., 等人：“对丙型肝炎病毒 E1 和 E2 异聚关联重要的区域的延迟”病毒学（1997 年出版）。

村上清史: "ヒトB型肝炎ウイルス(HBV)X蛋白のトランス活性化機構" 臨床科学. 31. 1671-1678 (1995)

Kiyoshi Murakami：“人类乙型肝炎病毒（HBV）X蛋白的反式激活机制”《临床科学》31。1671-1678（1995）。