A simplified method for study of RNA conformation - reaction of formylmethionine transfer RNA with [14c]methylamine-bisulfite

摘要

A new chemical method for radioactive labeling of single-stranded regions of RNA has been used to probe the three-dimensional structure of E. coli tRNAfMet in solution. The procedure involves conversion of cytosine residues to N4-[14C]methylcytosines by treatment with. 14CH₃NH₂ and sodium bisulfite at pH7. Ribonuclease digestion of the modified tRNA produces 14C-labeled oligo-nucleotides which comigrate with the corresponding unlabeled oligormcleotides, facilitating structural analysis. By this procedure, E. coli tRNAfmet has been found to contain only six reactive cytosines: C₁ C₁₆, C₁₇, C₃₅, C₇₅ and C₇₆. In addition, slow reaction at C₃₃m, was observed. These results are in excellent agreement with previously reported data on the sites of exposed cytosine residues in tRNAfmet obtained by two other chemical methods. The methylamine-bisulfite procedure is recommended for studying the ordered structure of more complex polyribonucleotides such as viral and ribosomal RNAs.