Study on the biosynthesis of the modified uridine derivatives at the first position of the anticodon of tRNA

tRNA反密码子第一位修饰尿苷衍生物的生物合成研究

• 批准号: 61571061
• 负责人: MURAO Katsutoshi
• 金额: $ 1.34万
• 依托单位: Jichi Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1986
• 资助国家: 日本
• 起止时间: 1986 至 1987
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-61571061/
• 关键词: tRNA; modified nucleoside; Anticodon; Biosynthesis; RNase H; RNaseH; アンチユードン

1. Bacillus subtilis was cultivated in IOL of a medium of Antibiotic Medium 3 120 times. Crude tRNA was extracted from B. subtilis cells with 88% phenol and purified by a combination of several column chromatography. Glycine(2 kinds),lysine, threonine, valine accepting tRNAs were purified.2. In order to change the anticodon nucleotides with unmodified oligonucleotide, each tRNA molecule was digested with RNase TI, RNase A or nuclease Sl. 3'- and 5' half molecules were purified by polyacrylamide gel electrophoresis.3. RNase H was very effective to obtain the specific half molecule of tRNA. The enzyme can split in RNA strand in the double strand region of RNA=DNA. The chimeric oligomers which is complimentary to the nucleotide sequence of stem and loop region of anticodon were synthesized. The phosphodiester bond between permanent U34 and C33 was specifically hydrolyzed.4. 5'-Half molecules from glycine, lysine, threonine tRNAs were ligated with the unmodified oligonucleotide, [32P]pUp, … More [32P]pUUU or [32P]pUA by RNA ligase. After 3'-terminal phosphate was eliminated by PMase, if any, they were annealed with corresponding 3'-half molecule. Two halves were ligated again by RNA ligase to give a whole tRNA molecule which contains unmodified anticodon nucleotide.5. Reconstructed tRNA molecules were incubated with B. subtilis S-100 or S-30 fraction. After incubation, tRNA was precipitated by alcohol and hydrolyzed with nuclease Pl. The modification of uridylic acid in the first position of the anticodon, which contains [32P] at the 5' phosphate was checked by thin layer chromatography. None of the reconstructed tRNA molecule was modified at the first nucleotide in the anticodon by crude enzyme fraction. It may be difficult to modify isotope-labeled tRNA as a substrate because of the low concentration of a substrate or cosubstrate.6. We have not obtained a much progress in this project in these two years. But, we hope to continue this research project to get an anticipated results. Less

1.枯草芽孢杆菌在抗生素培养基3的IOL中培养120倍。从B中提取粗tRNA。枯草杆菌细胞与88%苯酚的混合物，并通过几种柱层析的组合进行纯化。纯化了甘氨酸（2种）、赖氨酸、苏氨酸、缬氨酸接受的tRNA.为了用未修饰的寡核苷酸改变反密码子核苷酸，用RNase TI、RNase A或核酸酶S1消化每个tRNA分子。通过聚丙烯酰胺凝胶电泳纯化3 '-和5'半分子。RNase H是获得特异性半分子tRNA的有效方法。该酶可在RNA=DNA的双链区域中在RNA链中分裂。合成了与反密码子茎环区核苷酸序列互补的嵌合寡聚体。永久性U34和C33之间的磷酸二酯键被特异性水解。将来自甘氨酸、赖氨酸、苏氨酸tRNA的5 ′-半分子与未修饰的寡核苷酸[32 P]pUp连接， ...更多信息 [32 P]pUUU或[32 P]普阿。在用PMase消除3 '端磷酸后，如果有的话，它们与相应的3'半分子退火。再用RNA连接酶将两半连接起来，得到含有未修饰的反密码子核苷酸的完整tRNA分子。将重建的tRNA分子与B孵育。枯草杆菌S-100或S-30级分。孵育后，用乙醇沉淀tRNA并用核酸酶P1水解。通过薄层色谱法检查反密码子第一位尿苷酸的修饰，其在5'磷酸含有[32 P]。在反密码子的第一个核苷酸处，没有一个重建的tRNA分子被粗酶组分修饰。由于底物或辅助底物的浓度较低，因此可能难以修饰同位素标记的tRNA作为底物。在这两年里，我们在这个项目上没有取得多大进展。但是，我们希望继续这项研究项目，以获得预期的结果。少