Purification and Characterization of RNA polymerase from halophilic archaebacteria.

嗜盐古细菌 RNA 聚合酶的纯化和表征。

• 批准号: 04808043
• 负责人: NAKAYAMA Masashi
• 金额: $ 1.02万
• 依托单位: Osaka University of Education
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04808043/
• 关键词: Halobacterium halobium; RNA polymerase; halophilic archaebacteria

Halobacterium halobium, one of the halophilic archaebacteria, grows in extreme halophilic environments. The intracellular concentration of K^+ is also extremely high. The mechanisum of gene transcription in H.halobium and the nature of RNA polymerase involved in this process are still remained to study. We tried to purify the RNA polymerase which requires high salt concentrations for function.Zilling et al. (1978) identified a high-molecular-weight RNA polymerase from a related H.cutirubrum.In crude extract of H.halobium cells, we detected two RNA polymerase activities, one requiring high concentrations of salt for maximun activity and the other not requiring it. The high-requiring RNA polymerase was purified up to about 1,000-fold by PEI iteatment, Buty-Toyopearl and glycerol gradient centrifugation. The partially purified RNA polymerase required 3M NaCl for maximum activity DNA-dependent RNA synthesis continued at least until 90 min at 40ﾟC.The enzyme was resistant to both rifampicin and alpha-amanitin, but was sensitive to lignin, which is a potent inhibitor of influenza virus RNA polymerase. The molecular weight was estimated to be about 45 kDs by glycerol gradient centrifugation. Futher purification of the enzyme and detailed analysis of the enzyme structure are in progress.

盐生细菌是一种嗜盐古细菌，生长在极端的嗜盐环境中。细胞内K~(++)浓度也极高。盐藻基因转录的机制以及参与这一过程的RNA聚合酶的性质仍有待研究。我们试图纯化RNA聚合酶，它需要很高的盐浓度才能发挥作用。(1978)从相关的皮肤红假单胞菌中鉴定出一种高相对分子质量的RNA聚合酶。在盐杆菌细胞的粗提物中，我们检测到两种RNA聚合酶活性，其中一种需要高浓度的盐才能达到最大活性，另一种不需要。通过PEI处理、丁二醇酯和甘油梯度离心法，可将高要求的RNA聚合酶纯化约1000倍。部分纯化的核糖核酸聚合酶需要3M氯化钠在40ﾟC下持续90分钟才能获得最大活性。该酶对利福平和α-氨甲酸氨基都有抗性，但对木质素敏感，木质素是流感病毒核糖核酸聚合酶的有效抑制剂。甘油梯度离心法测得其相对分子质量约为45kds。该酶的进一步纯化和详细的结构分析正在进行中。