RNA SPLICING IN ARCHAEA

古细菌中的 RNA 剪接

• 批准号: 2628362
• 负责人: Ramesh Gupta
• 金额: $ 10.58万
• 依托单位: SOUTHERN ILLINOIS UNIVERSITY CARBONDALE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2628362
• 关键词: Archaea; RNA splicing; affinity chromatography; bacterial genetics; introns; phosphoester ligase; protein biosynthesis; protein purification; transfer RNA

DESCRIPTION: Studies of gene regulation help us in understanding cellular metabolism, growth, and differentiation. Post-transcriptional RNA processing is one of the several ways by which gene expression is regulated. The long-term objective of this AREA application is to characterize RNA processing in archaea, especially in the halophilic and then thermophilic archaea. Archaea are prokaryotes like bacteria; yet in several of their molecular features, they resemble eukaryotes more than the bacteria. Therefore, these studies besides providing information about the archaeal systems will also help in understanding the corresponding eukaryotic systems. The specific aims of this proposal are: Determination of the source of splice junction phosphate in Haloferax volcanii spliced tRNAs; Purification of H. volcanii tRNA splicing ligase; Determination of the mechanisms of ligation and ligase binding in tRNA splicing in H. volcanii; Determination of the presence or absence of 2'-O-r;ethylcytidine (Cm) modification at the wobble position of in vivo generated products of recombinant intron-deleted tRNA genes in H. volcanii; and Determination of the relationship among tRNA intron splicing, modifications at the wobble position of corresponding tRNAs, and accuracy of translation in H. volcanii. These aims are planned to determine the mechanism of pre-tRNA splicing and the reasons for the existence of tRNA introns in halophilic archaea. H. volcanii cell extracts containing splicing ligase activity will be used with unlabeled T7 RNA polymerase produced transcripts and [gamma 32P]GTP in one set of experiments and labeled transcripts (using [alpha -32P]GTP) and unlabeled ATP in another set. The products of the reaction will be analyzed by specific combinations of RNase Tl, T2, nuclease P1 and venom phosphodiesterase digestions to determine the source of splice junction phosphate. Ligase will be purified by the methods in which high concentrations of salts can be maintained continuously. Partially purified ligase will be used to ligate modified in vitro produced exons, in the studies determining mechanism of ligation. A potential role for the intron in modification at the wobble position of tRNA, and the effect of the modification on the accuracy of translation will be tested by genetic methods, using specifically modified reporter protein systems and intron-deleted tRNA genes. The tRNA products of these intronless genes will be characterized for the wobble modification by a specific combination of RNase T1 and T2 digestions.

描述：基因调节的研究有助于我们理解细胞 代谢，生长和分化。 转录后RNA 处理是调节基因表达的几种方式之一。 该区域应用的长期目标是表征RNA 在古细菌中加工，尤其是在卤素中，然后在嗜热中 古细菌。 古细菌是诸如细菌之类的原核生物。然而在他们的几个 分子特征，它们比细菌更类似于真核生物。 因此，这些研究除了提供有关古细菌的信息 系统还将有助于理解相应的真核生物 系统。 该提案的具体目的是：确定 Haloferax火山剪接的TRNA中的剪接连接磷酸盐的来源； 纯化火山trNA剪接连接酶的纯化；确定 H.火山中的tRNA剪接中的连接和连接酶结合的机理； 确定2'-O-R;乙基丁胺（CM）的存在或不存在 在体内生成的产品的摇摆位置进行修改 火山绿色绿oi中的重组内含子删除的tRNA基因；和决心 tRNA内含子剪接之间的关系，摇摆处的修改 相应的TRNA的位置，以及在H. dolcanii中翻译的准确性。 这些目标是计划确定pre-tRNA剪接的机制， 卤素古细菌中存在tRNA内含子的原因。 H. 含有剪接连接酶活性的火山细胞提取物将与 未标记的T7 RNA聚合酶在一个中产生的转录本和[Gamma 32p] GTP。 一组实验和标记的转录本（使用[alpha -32p] GTP）和 另一组未标记的ATP。 反应的产物将进行分析 通过RNase TL，T2，Nuclease P1和毒液的特定组合 磷酸二酯酶消化以确定剪接连接的来源 磷酸盐。 连接酶将通过高高的方法纯化 盐的浓度可以连续保持。 部分纯化 连接酶将用于在体外产生的外显子进行结合，在 研究确定结扎机制。 内含子的潜在作用 在tRNA的摇摆位置的修改中，效果 修改翻译准确性将通过遗传测试 方法，使用特定修改的报告基因蛋白系统和 内含子删除的tRNA基因。 这些无源基因的tRNA产物将 以特定组合的特定组合为特征 RNase T1和T2消化。