Transfer RNA Structure During Protein Synthesis

这个项目的长期目标是确定转移RNA(TRNA)与其他核酸和蛋白质之间特定部位的结构和功能相互作用的性质，特别是关于修饰核苷周围的结构。将结合质子和异核核磁共振技术(用于探测其他方法不易获得的局部构象和结构)对修饰的核苷进行特定的异核浓缩。我们已经构建了一种独特的大肠杆菌表达载体，任何化学合成的tRNA基因都可以放入其中，用于体内tRNA的过量生产。无论是富含13C的野生型还是设计的核苷类突变tRNAs，都可以大量产生，用于生化功能分析和核磁共振研究。TRNA与蛋白质和核酸的相互作用取决于tRNA分子在溶液中的构象及其特定的柔韧性，这两者都不是完全了解的。TRNA构象和位点特异性迁移率的研究对于比较大肠杆菌和酵母的tRNA结构也很重要。在研究溶液中的大分子时，与其他物理技术相比，核磁共振的优势在于可以同时从分子许多不同部分的报告基团获得信号。

The long term goal of this project is to determine the nature of site specific structural and functional interactions of transfer RNA (tRNA) with other nucleic acids and proteins particularly with regard to the structure found around the modified nucleosides. Site specific heteronuclear enrichment of modified nucleosides combined with proton and heteronuclear NMR techniques (for the probing of local conformations and structure not readily accessible by other methods) will be carried out. A unique E. coli plasmid has been constructed into which any chemically synthesized tRNA gene can be placed for overproduction of the tRNA in vivo. Both 13C-enriched wild type and designed mutant tRNAs with site selected nucleoside changes can be produced in quantity for analysis of biochemical function and for NMR studies. The interaction of tRNA with proteins and nucleic acids depends upon the tRNA molecule's conformation in solution and its specific flexibilities, neither of which are fully understood. A study of tRNA conformation and site specific mobility is also important for a comparison of E. coli and yeast tRNA structures. The advantage of NMR over other physical techniques in studying macromolecules in solution is that signals are obtainable from reporter groups in many different parts of the molecules simultaneously.