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.
