RNA Splicing in Archaea

描述（由申请人提供）：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应用的广泛的长期目标是表征在Escherichia中的各种RNA加工事件。古细菌和细菌都是原核生物，但古细菌有几个类似真核生物的分子特征。该建议具体涉及含内含子的前体tRNA在嗜盐古菌Haloferax volcanii中的加工。 本研究的具体目标是：体外sRNP系统在H. volcanii pre-tRNATrp;分别在H中tRNATrp的34和39位产生2 ′-O-甲基胞苷（Cm）和2 ′-O-甲基尿苷（Um）的体内表征。volcanii和研究，在体内，这些修饰反应和pre-tRNA剪接之间的关系;和确定的影响，在tRNA的摆动位置的Cm修饰的翻译在H的准确性。火山 H. volcanii pre-tRNATrp核苷酸的体外研究将通过使用重组盒C/D RNP核心蛋白和H. volcanii细胞提取物沿着与32 P标记的T7 RNA聚合酶一起产生前体tRNA及其内含子。核心蛋白与正常和各种修饰的底物的结合将通过凝胶位移测定来确定。2 '-O-甲基化核苷酸的产生将通过RNA的RNA酶T2或核酸酶P1底物的薄层色谱法（TLC）测定。tRNATrp基因的修饰版本将用于在体内研究盒C/D引导的修饰反应及其与剪接的关系。该修饰基因的tRNA产物与正常基因组基因的tRNA产物可以区分。该修饰基因的各种突变形式的产物将在体内研究中表征。含有该修饰基因的突变形式的细胞提取物也将用于体外甲基化研究。内含子在tRNA的摆动碱基修饰中的作用及其对翻译准确性的影响将通过使用特异性修饰的报告基因和内含子缺失的tRNA基因来测试。该无内含子基因的tRNA产物将通过变性凝胶或指纹法分离其RNA酶T1底物，并通过TLC确定适当寡核苷酸的组成，来表征是否存在特定的Cm和Um修饰。

DESCRIPTION (provided by applicant): 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 processing of intron-containing pre-tRNAs in Haloferax volcanii, a halophilic archaeon. The specific aims of this proposal are: Characterization of in vitro sRNP system for site-specific modifications in H. volcanii pre-tRNATrp; Characterization, in vivo, of the production of 2'-O-methylcytidine (Cm) and 2'-O-methyluridine (Um) at positions 34 and 39, respectively, of the tRNATrp in H. volcanii and study, in vivo, the relationship between these modification reactions and pre-tRNA splicing; and Determination of the effect of Cm modification at the wobble position of tRNA on the accuracy of translation in H. volcanii. Mechanism of 2'-O-methylation of H. volcanii pre-tRNATrp nucleotides, in vitro, will be studied by using recombinant box C/D RNP core proteins and H. volcanii cell extracts along with 32P-labeled T7 RNA polymerase generated pre-tRNAs and their introns. Binding of core proteins to normal and variously modified substrates will be determined by gel-shift assays. Production of the 2'-O-methylated nucleotides will be determined by thin layer chromatography (TLC) of the RNase T2 or nuclease P1 digests of the RNAs. A modified version of tRNATrp gene will be used to study, in vivo, box C/D guided modifications reactions and their relationship to splicing. The tRNA product of this modified gene and normal genomic gene can be distinguished. Products of variously mutated versions of this modified gene will be characterized in in vivo studies. Extracts of the cells containing mutated forms of this modified gene will also be used for the in vitro methylation studies. Role of the intron in wobble base modification of tRNA and its effect on the accuracy of translation will be tested by using specifically modified reporter gene and an intron-deleted tRNA gene. The tRNA products of this intronless gene will be characterized for the presence or absence of specific Cm and Um modifications by separating their RNase T1 digests by denaturing gels or fingerprinting, and determining the composition of the appropriate oligonucleotides by TLC.