MISSING CYSTEINYL-TRNA SYNTHETASE IN METHANOGENS

产甲烷菌中缺失半胱氨酰-TRNA 合成酶

• 批准号: 6385184
• 负责人: LIANG FENG
• 金额: $ 3.48万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6385184
• 关键词: Archaea; Escherichia coli; RNA binding protein; RNA biosynthesis; aminoacid tRNA ligase; aminoacyl tRNA; gene complementation; intermolecular interaction; molecular cloning; site directed mutagenesis

Sequence analysis of two newly sequenced Archaea genomes, Methanococcus jannaschii and Methanobacterium thermoautotrophicum, fails to identify a recognizable homolog of existing cysteinyl-tRNA synthetases (CysRS). This raises the possibility that cysteine is charged by a novel pathway or by a novel cysteine tRNA synthetase in the methanogens. The goal of this proposed research is to investigate the mechanism of cysteinyl tRNA biosynthesis in M. jannaschii and M. thermoautotrophicum. Genes responsible for cysteine charging activities will be cloned by reverse genetics and genetic complementation. An overexpression system in E. coli will be used to produce sufficient material for biochemical characterizations. If a novel pathway is responsible for cysteine charging, the focus will be to characterize the pertinent enzymes and the reactions they catalyze. If direct charging exists, studies will focus on the comparison of the novel CysRS with its bacteria and eukaryotes relatives in terms of catalysis and substrate recognition. The unique RNA:protein interactions will also be studied by site-directed mutagenesis and attempts will be made to crystallize the proteins for future structural studies. The results should provide us insight into the evolution of the genetic code and the limits on the flexibility of the translational apparatus in prokaryotes and eukaryotes.

对两个新测序的古细菌基因组甲烷甲虫和嗜热雄杆菌的序列分析未能鉴定出现有Cysteinyl-tRNA合酶（CYSRS）的可识别同源性。 这增加了半胱氨酸被新型途径或甲烷元中新型半胱氨酸tRNA合成酶收取的可能性。 这项拟议的研究的目的是研究Jannaschii和M. heletoautotrophicum中胱氨酸tRNA生物合成的机制。 负责半胱氨酸充电活性的基因将通过反向遗传学和遗传互补来克隆。 大肠杆菌中的过表达系统将用于生产足够的生化特征材料。 如果新型途径负责半胱氨酸的充电，则重点是表征相关酶及其催化的反应。 如果存在直接充电，则研究将重点放在新型CYSR与其细菌和真核生物亲戚在催化和底物识别方面的比较。 独特的RNA：蛋白质相互作用还将通过定点诱变进行研究，并尝试将蛋白质结晶以进行未来的结构研究。 结果应该使我们深入了解遗传密码的演变以及对原核生物和真核生物转化设备的灵活性的限制。