Production of specific site probes of tRNA structure by enrichment with carbon 13 at particular locations.
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-(13)C] adenine, and [2-(13)C] 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.
D000225:Adenine / Q000378:metabolism; D002247:Carbon Isotopes; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D015122:Mercaptopurine / Q000494:pharmacology; D008715:Methionine / Q000378:metabolism; D008968:Molecular Conformation; D009690:Nucleic Acid Conformation; D011687:Purines / Q000096:biosynthesis; D012343:RNA, Transfer / Q000378:metabolism; D014498:Uracil / Q000378:metabolism
D002247:Carbon Isotopes; D011687:Purines; D014498:Uracil; D012343:RNA, Transfer; D008715:Methionine; D015122:Mercaptopurine; D000225:Adenine