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：基因调控的研究帮助我们理解细胞 新陈代谢、生长和分化。转录后RNA 加工是调控基因表达的几种方式之一。 该领域应用长期目标是表征RNA 在古生菌中的加工，尤指在嗜盐菌和嗜热菌中 古生菌。古生菌是像细菌一样的原核生物；但在它们的几个 分子特征，它们更像真核生物，而不是细菌。 因此，这些研究除了提供有关古生物的信息外 系统也将有助于理解相应的真核生物 系统。这项建议的具体目标是：确定 盐生植物火山剪接tRNAs中剪接接头磷酸盐的来源； 火山嗜血杆菌tRNA剪接连接酶的纯化 火山杆菌tRNA剪接中的连接和连接酶结合机制； 2‘-O-r；乙基胞苷(Cm)存在与否的测定 在体内产生的产物的摆动位置的修饰 重组内含子缺失tRNA基因在火山杆菌中的表达 TRNA内含子剪接、摆动修饰之间的关系 相应的tRNAs的位置，以及在H.Volcanii中翻译的准确性。 这些目的是为了确定前tRNA剪接的机制和 嗜盐古菌中存在tRNA内含子的原因。H. 含有剪接连接酶活性的Volcanii细胞提取物将用于 未标记的T7 RNA聚合酶产生转录本和[γ~(32)P]GTP 一系列实验和标记的成绩单(使用[α-32P]GTP)和 另一组中未标记的ATP。将对反应的产物进行分析 通过核糖核酸酶T1、T2、核酸酶P1和毒液的特定组合 用磷酸二酯酶消化确定剪接连接的来源 磷酸盐。连接酶将通过高纯度的方法进行纯化 盐的浓度可以连续保持。部分提纯 连接酶将用于连接在体外产生的修饰的外显子，在 研究结扎的决定机制。内含子的潜在作用 在tRNA摆动位置的修饰中，以及 对翻译准确性的修改将通过基因测试 方法，使用特定修改的报告蛋白系统和 内含子缺失的tRNA基因。这些无内含子基因的tRNA产物将 通过特定的组合来表征摆动修正 RNaseT1和T2酶。