Nickel- and cobalt-dependent reagents identify structural features of RNA that are not detected by dimethyl sulfate or RNase T1.

Ribosomal 5S RNA presents a particular challenge to structural investigations since this polynucleotide is too large for complete NMR characterization but lacks significant tertiary structure to modulate, for example, diagnostic alkylation of guanine N7 by dimethyl sulfate. Nickel- and cobalt-dependent reagents that are sensitive to the N7 and aromatic face of guanine have now been applied to 5S rRNA (Xenopus lavis) and provide structural information that was not previously available from traditional chemical or enzymatic probes. Although G75 had repeatedly demonstrated an average reactivity with dimethyl sulfate and minimal reactivity with RNase T1, this residue was the major target of both metal-dependent reagents. Such reactivity provides crucial support for a structural model of loop E identified by prior physical, but not chemical, methods. Similarly, the tetraloop structure of loop D was more accurately reflected by the reactivity of G87 and G89 in the presence of the nickel reagent rather than in the presence of RNase T1. In addition, nickel-dependent modification of guanine residues surrounding the three-helix junction of loop A suggests an organization that is less compact than previously considered.