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

Abstract

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 tRNA(fMet) in solution. The procedure involves conversion of cytosine residues to N(4)-[(14)C]methylcytosines by treatment with (14)CH(3)NH(2) and sodium bisulfite at pH7. Ribonuclease digestion of the modified tRNA produces (14)C-labeled oligonucleotides which comigrate with the corresponding unlabeled oligonucleotides, facilitating structural analysis. By this procedure, E. coli tRNA(fMet) has been found to contain only six reactive cytosines: C(1), C(16), C(17), C(35), C(75) and C(76). In addition, slow reaction at C(m) (33) was observed. These results are in excellent agreement with previously reported data on the sites of exposed cytosine residues in tRNA(fMet) 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.

Attachment of protein affinity-labeling reagents of variable length and amino acid specificity to E. coli tRNAfMet

“…A plot of the number of side chains attached to tRNAfMet vs. methionine acceptor activity shows that the tRNA containing derivatives of type IIb can be aminoacylated to a greater extent than those containing adducts Ia ( Figure 6). The sites of the modified cytidine residues in tRNAfMet are the same as those previously observed to be deaminated by treatment with sodium bisulfite alone at pH 6 (18) and to undergo transamination with methylamine in the presence of bisulfite at pH 7 (19). These include the unpaired cytidine at the 5' terminus, two cytidine residues in the dihydrouridine loop, the cytidine in the wobble position of the anticodon, and the two cytidine residues in the 3' terminal CCA sequence.…”

Study of the interaction of Escherichia coli methionyl-tRNA synthetase with tRNAfMet using chemical and enzymic probes

“…Figure 1 shows the results of cytosine-specific methylation protection experiments. E. coli tRNA™" contains six cytosines that react readily with single strand specific reagents: C,, C16, C17, C34, C74, and C75 (Goddard & Schulman, 1972;Chang, 1973;Schulman et al, 1974). We were unable to determine whether Cj was reactive with dimethyl sulfate using 3'-32Plabeled tRNA™"; however, the other cytosine residues were methylated as expected in the absence of methionyl-tRNA synthetase.…”

RNA structure

“…Under what circumstances is the tertiary structure of tRNA present in solution? From the chemical reactivity studies (see Robertus et al 1974;Schulman et al 1974;Rhodes, 1975) and *H NMR of hydrogenbonded protons, it is reasonable to identify the form of tRNA, in the presence of excess Mg 2+ , neutral pH and low temperature, with the X-ray model. In the absence of Mg 2+ it is found that the stability of this structure is diminished, although in the presence of c. 0-2 M-NaCl the tertiary structure is present (Cole, Yang & Crothers, 1972).…”