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加工可以调节基因的表达，这对于控制细胞的新陈代谢、生长和分化是必不可少的。这一领域应用的广泛的、长期的目标是表征古生菌中的各种RNA加工事件。古生菌和细菌都是原核生物，但古生菌表现出一些类似于真核生物的分子特征。这项提议专门涉及嗜盐考古菌Haloferax Volcanus II中的前tRNA剪接。一个长期目标是也克隆和过度表达火山杆菌剪接连接酶基因。这项建议的具体目的是：纯化火山杆菌tRNA剪接连接酶；测定火山杆菌剪接连接酶底物的结构要求；火山杆菌剪接连接酶反应的特征；确定无内含子tRNA基因在火山杆菌体内产生的tRNA的摆动位置是否存在2‘-O-甲基胞苷(Cm)修饰；以及确定tRNA摆动位置的Cm修饰对火山杆菌翻译准确性的影响。火山杆菌剪接连接酶在含有低盐浓度的溶液中失活。因此，连接酶将采用连续保持高浓度钠/钾盐的方法进行纯化。在某些步骤中，用硫酸铵或有机溶质代替这些盐，将用于提纯。为了确定连接酶底物的结构要求，将在连接反应中测试几种经过修饰的体外生产的和商业上可用的底物。连接酶与底物的结合将通过使用其中一些底物的凝胶移位分析来表征。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摆动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学方法进行测试，使用特定修改的报告蛋白系统和内含子缺失的tRNA基因。这个无内含子基因的tRNA产物将通过RNaseT1指纹图谱来表征tRNA摆动位置上是否存在Cm修饰。