I5N LABELED TRNA--SYNTHESIS AND STRUCTURE

将制备区域特异性标记15N的大肠杆菌RNA，并 分离出的数量足以进行核磁共振研究的 光谱学。多克量的[1-15N]-和[3-15N]尿嘧啶 合成并加入到所有生物合成的碱基中 从尿苷(尿苷、硫代尿苷、假尿苷、核胸苷)使用 S0187突变体。TRNAfMet，tRNAmMet，tRNAmMet， TRNAG1u2、tRNatyr和tRNAPhe)和5S rRNA将被提纯。高场 将使用质子核磁共振波谱来识别 并指定了与氢有关的尿苷亚氨基质子的共振 凝聚力。将确定与15N相连的质子的亚米诺共振 通过大的单键1H-15N耦合作用。1H和15N化学 位移将通过异核双共振实验进行关联。 这些研究，结合核奥豪泽实验，应该 允许我们在中指定与尿苷相关的二级和三级碱基对 上面列出的tRNA。将进行类似的实验过程 对于5S rRNA，对二级和三级结构知之甚少。

E. coli RNAs labeled regiospecifically with 15N will be prepared and isolated in quantities sufficient for study by nuclear magnetic resonance spectroscopy. Multigram quantities of [1-15N]- and [3-15N]uracil will be synthesized and incorporated into all of the bases derived biosynthetically from uridine (uridine, thiouridine, pseudouridine, ribothymidine using the S0 187 mutant. Multimilligram quantities of five tRNAs tRNAfMet, tRNAmMet, tRNAG1u2, tRNATyr, and tRNAPhe) and 5S rRNA will be purified. High field proton nuclear magnetic resonance spectroscopy will be employed to identify and assign resonances for the uridine imino protons involved in hydrogen bonding. Imino resonances for protons attached to 15N will be identified by the large one-bond 1H-15N coupling interaction. The 1H and 15N chemical shifts will be correlated by heteronuclear double resonance experiments. These studies, in conjunction with nuclear Overhauser experiments, should allow us to assign the secondary and tertiary uridine-related base pairs in the tRNAs listed above. A similar course of experiments will be undertaken for 5S rRNA where much less is known about secondary and tertiary structure.