RNA Splicing in Archaea

古细菌中的 RNA 剪接

• 批准号: 6316235
• 负责人: Ramesh Gupta
• 金额: $ 14.1万
• 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 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6316235
• 关键词: Archaea; RNA splicing; bacterial genetics; cytidine; enzyme mechanism; enzyme substrate; gel mobility shift assay; introns; phosphoester ligase; posttranslational modifications; protein purification; transfer RNA

Post-transcriptional RNA processing can regulate gene expression, which is essential for the control of cellular metabolism, growth, and differentiation. Broad, long-term objectives of this AREA application are to characterize various RNA processing events in archaea. Both archaea and bacteria are prokaryotes; yet archaea exhibit several molecular features resembling eukaryotes. This proposal specifically deals with pre- tRNA splicing in Haloferax volcanii, a halophilic archaeon. A long-term goal is to also clone and overexpress the gene for H. volcanii splicing ligase. The specific aims of this proposal are: Purification of H. volcanii tRNA splicing ligase; Determination of the structural requirements for the substrates of H. volcanii splicing ligase; Characterization of H. volcanii splicing ligase reaction; Determination of the presence or absence of 2'- O-methylcytidine (Cm) modification at the wobble position of the tRNA produced in vivo in H. volcanii by an intronless tRNA gene; and, Determination of the effect of Cm modification at the wobble position of tRNA, on the accuracy of translation in H. volcanii. H. volcanii splicing ligase is inactivated under low salt concentrations containing solutions. Therefore, the ligase will be purified by the methods where high concentrations of sodium/potassium salts are maintained continuously. Certain procedures where ammonium sulfate or organic solutes replace these salts, will be used for the purification. Several modified in vitro produced and commercially available substrates will be tested in ligation reactions for determining the structural requirements for the substrates of ligase. Ligase-substrate binding will be characterized by gel-shift assays using some of these substrates. Various different substances and modified substrates will be examined for their effect on the ligase reaction. Role of the intron in modification at the wobble position of tRNA and its effect on the accuracy of translation will be tested by genetic methods, using a specifically modified reporter protein system and an intron-deleted tRNA gene. The tRNA product of this intronless gene will be characterized for the presence or absence of Cm modification at the wobble position of the tRNA, by RNase T1 fingerprinting.

转录后RNA处理可以调节基因表达，这对于控制细胞代谢，生长和分化至关重要。该区域应用的广泛的长期目标是表征古细菌中各种RNA处理事件。古细菌和细菌都是原核生物。然而，古细菌表现出类似于真核生物的几个分子特征。该提案专门讨论了在卤素古老的Haloferax Volcanii中剪接的剪接。一个长期的目标是克隆和过表达火山链球链接酶的基因。该提案的具体目的是：纯化火山trNA剪接连接酶；确定火山梭菌剪接连接酶的底物的结构要求；火山链球菌剪接连接酶反应的表征；通过无固有的tRNA基因在H. opert of Vivo产生的tRNA的摇摆位置确定2'- O-O-甲基胞苷（CM）修饰的存在或不存在。并且，确定tRNA摇摆位置CM修饰的影响，对H. to h. to trnai the trna的准确性。 H. h.火山剪接连接酶在含有溶液的低盐浓度下被灭活。因此，将连续保持高浓度的钠/钾盐含量的方法纯化。硫酸铵或有机溶质取代这些盐的某些程序将用于纯化。在连接反应中将测试几种经过体外生产和市售底物的经过修饰，以确定连接酶底物的结构要求。连接酶 - 基底结合将使用其中一些底物进行凝胶转移测定。将检查各种不同的物质和修饰的底物对连接酶反应的影响。内含子在tRNA的摇摆位置的修饰中的作用及其对翻译准确性的影响将通过遗传方法进行测试，使用特定修饰的报告基因蛋白系统和内含子删除的tRNA基因。该内含子基因的tRNA产物将以RNase T1指纹识别在tRNA的摇摆位置存在或不存在CM修饰的特征。