RNA Splicing in Archaea

转录后RNA加工可以调节基因表达，这对于控制细胞代谢、生长和分化至关重要。该AREA应用程序的广泛，长期目标是表征古细菌中的各种RNA加工事件。古生菌和细菌都是原核生物，但古生菌表现出几个类似于真核生物的分子特征。这项提议专门涉及嗜盐古菌Haloferax volcano ii中的前tRNA剪接。一个长期的目标是克隆和过表达H. volcanii剪接连接酶。本建议的具体目标是：H。volcanii tRNA剪接连接酶; H. volcanii剪接连接酶; H. volcanii剪接连接酶反应;确定在H.体内产生的tRNA的摆动位置是否存在2 ′-O-甲基胞苷（Cm）修饰。volcano ii通过无内含子tRNA基因;和，确定在tRNA摆动位置的Cm修饰对H.火山H. volcano II剪接连接酶在含低盐浓度的溶液中失活。因此，连接酶将通过连续保持高浓度钠/钾盐的方法纯化。将使用硫酸铵或有机溶质代替这些盐的某些程序进行纯化。将在连接反应中测试几种经修饰的体外生产和市售底物，以确定连接酶底物的结构要求。连接酶-底物结合将通过使用这些底物中的一些的凝胶位移测定来表征。将检查各种不同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，使用特异性修饰的报告蛋白系统和内含子缺失的tRNA基因。该无内含子基因的tRNA产物将通过RNase T1指纹分析来表征在tRNA的摆动位置处存在或不存在Cm修饰。

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.