TRANSFER RNA PROCESSING IN THE ARCHAEA

古细菌中的转移 RNA 加工

• 批准号: 3308149
• 负责人: CHARLES J DANIELS
• 金额: $ 16.69万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3308149
• 关键词: Archaea; RNA binding protein; RNA splicing; aminoacyl tRNA; bacterial genetics; biochemical evolution; endonuclease; enzyme structure; enzyme substrate; isoleucine; molecular cloning; mutant; nucleic acid sequence; transfer RNA

Recent work from this laboratory has focused on the characterization of RNA processing enzymes from the Archaea (formerly called the Archaebacteria). As expected from their unique evolutionary position, these cells contain a mosaic of molecular characteristics. The enzymes that participate in tRNA maturation are a good example of this diversity. We have observed that the tRNA intron endonuclease from Haloferax volcanii has a substrate recognition mechanism that is distinct from its eukaroytic counterpart. This enzyme does not require mature tRNA structure, rather it recognizes a specific structure, and possibly sequences, a the exon-intron boundaries. We have also found that the RNaseP RNA of this organism is similar in structure and sequence to the bacterial RNaseP RNA. However, this RNA does not appear to have catalytic activity in the absence of protein as do the bacterial RNAs. The specific aims of the proposed research include a determination of the precise recognition properties of the Archaea tRNA intron endonuclease using in vitro and in vivo assays developed in this laboratory. Methods are described for the purification of the Methanosarcina barkeri tRNA intron endonuclease protein, and the cloning of its gene. The endonuclease purification studies are being done in collaboration with Dr. R. Garrett at the University of Copenhagen. Studies on the RNaseP RNA are also planned. We propose to reinvestigate the catalytic capability of the halobacterial RNaseF RNA using the small H. trapanicum RNaseP RNA, and to isolate the gene encoding the M. barkeri RNaseP RNA. These RNaseP RNAs will also be used to develop methods for the reconstitution of their holoenzyme complexes. These experiments will provide a valuable perspective on the evolution of RNA processing enzymes, and further our understanding of the factors that govern RNA protein interactions.

该实验室的最新工作重点是 来自古细菌的RNA加工酶（以前称为 考古细菌）。正如他们独特的进化立场所期望的那样， 这些细胞包含分子特征的镶嵌物。酶 参与tRNA成熟是这种多样性的一个很好的例子。 我们已经观察到haloferax火山的tRNA内含子核酸内切酶 具有底物识别机制，其与其Eukaroytic不同 对准。该酶不需要成熟的tRNA结构，而是 识别特定的结构，甚至可能是序列A外显子内龙 边界。我们还发现，该生物的rnasep rna是 在结构和序列上与细菌RNASEP RNA相似。然而， 在没有的情况下，该RNA似乎没有催化活性 蛋白质和细菌RNA一样。提议的具体目的 研究包括确定的确切识别特性 使用体外和体内测定的古细菌tRNA内含子核酸内切酶 在这个实验室中开发。描述了纯化的方法 甲状腺菌的barkeri tRNA内含子核酸内切酶蛋白的蛋白质和 克隆其基因。核酸内切酶纯化研究正在进行 与哥本哈根大学的R. Garrett博士合作。 还计划了RNASEP RNA的研究。我们建议对 使用小的 H. trapanicum rnasep rna，并分离编码barkeri的基因 rnasep rna。这些RNASEP RNA也将用于开发用于 重构其全酶复合物。 这些实验将为演变提供宝贵的观点 RNA加工酶，进一步我们对这些因素的理解 控制RNA蛋白相互作用。