15N TRNA. SYNTHESIS AND STRUCTURE

tRNA是普遍存在的小的稳定分子， 与蛋白质合成有关的作用，其中包括翻译 遗传密码，氨基酸转运，氨基酸调控 在病毒感染动物中抑制蛋白质生物合成 细胞，致癌状态下蛋白质合成的控制，引发 来自RNA肿瘤病毒的逆转录酶和氨酰转移 在细胞壁生物合成过程中。 tRNA的拓扑结构及其影响因素 这决定了拓扑结构对于所有这些功能来说显然是至关重要的。 的 这个项目的目标是将tRNA的结构和功能联系起来。 E.杆菌 用13 C和15 N区域特异性标记的tRNA将在 使用表达载体过量产生 营养缺陷型菌株中的分子。 修饰不足的tRNA将被 以类似的方式在营养缺陷型菌株中制备， 对假尿嘧啶酸合酶I（hisT）、巯基化因子A对s4 U的活性 二甲基烯丙基焦磷酸：tRNA转移酶（miaA），和 tRNA-（m5 U）甲基转移酶（trmA）。 天然和未修饰的tRNA 将在500 MHz下使用核磁共振光谱进行研究， 异序二维化学位移关联，核Overhauser 影响和弛豫测量，以确定结构和动力学 参数 将对以下基因进行tRNA的生化比较： 氨酰化和体外转录。 tRNA-蛋白质的NMR研究 复合物将用未修饰的E. coli tRNAPhe和 修饰酶假尿苷酸合酶I和 tRNA（m5 U）甲基转移酶。

tRNAs are ubiquitous small stable molecules that perform several important roles related to the synthesis of proteins, among which are translation of the genetic code, transport of amino acids, regulation of amino acid biosynthesis, inhibition of protein biosynthesis in virally infected animal cells, control of protein synthesis during carcinogenic states, priming for reverse transcriptases from RNA tumor viruses, and aminoacyl transfer during cell wall biosynthesis. The topologies of tRNAs and the factors which dictate topology are clearly critical to all of these functions. The goal of this project is to relate structure and function in tRNAs. E. coli tRNAs regiospecifically labeled with 13C and 15N will be produced in multimilligram quantities using expression vectors which overproduce the molecules in auxotrophic bacterial strains. Undermodified tRNAs will be prepared in a similar manner in auxotrophic strains which also lack activity for pseudouridilate synthase I (hisT), thiolation factor A for s4U (nuvA), dimethylallyl pyrophosphate:tRNA transferase (miaA), and tRNA-(m5U)methyltransferase (trmA). The native and undermodified tRNAs will be studied by nuclear magnetic resonance spectroscopy at 500 MHz using heteronuclear 2-dimensional chemical shift correlation, nuclear Overhauser effects, and relaxation measurements to determine structural and kinetic parameters. Biochemical comparisons of tRNAs will be made for aminoacylation and in vitro transcription. NMR studies of tRNA-protein complexes will be conducted with undermodified E. coli tRNAPhe and the modification enzymes pseudouridylate synthase I and tRNA(m5U)methyltransferase.