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Nucleic Acids Research, 1979, Vol. 7, No. 3 765-779
© 1979

Articles

Production of specific site probes of tRNA structure by enrichment with carbon 13 at particular locations

Julia G. Tompson and Paul F. Agris*

Division of Biological Sciences, University of Missouri Columbia, MO 65211, USA

*Send all correspondence to: Paul F. Agris, Division of Biological Sciences, Tucker Hall, University of Missouri, Columbia, MO 65211, USA

Received July 5, 1979. Escherichia coli C6 rel met cys was cultured in a stringently defined minimal medium containing 13C-enriched metabolites in order to (1) achieve maximal 13C isotopic enrichment of tRNA; and (2) produce site specific but natural, non-perturbing NMR probes of tRNA structure and function. Growth conditions were manipulated to achieve optimal culture growth concomitant with maximal in vivo incorporation of various 13C-enriched nucleic acid precursors, including L-[methyl-13C] methionine, [2-13C] adenine, and [2-13C] uracil. Effective blockage of purine biosynthesis de novo was accomplished with the addition of the antimetabolite 6-mercaptopurine to the growth medium. Transfer RNAs specifically 13C-enriched in all methyl groups (57 atom %), C2 of adenine (60 atom %), and C2 of uracil (82 atom %) and C2 of cytosine (73 atom %) have been produced.


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