Enzymatic approaches to probing of RNA secondary and tertiary structure.

To make strong statements about possible tertiary structure or the relative stability of regions of secondary structure, the structure-probing experiments must go further than single-hit reactions. Some elements of the environment of the RNA molecule must be altered systematically. Knowledge of the effects of ions or other interacting factors on the activity or physical parameters (e.g., NMR and melting cooperativity) of the RNA help in experimental design. For example, the copious work on tRNA(Phe) compared the crystal and solution structures and allowed the direct correlation of Mg2+ stabilization of the tertiary structure of that molecule. Figure 3 demonstrates that pre-tRNA(Leu-3) responds to Mg2+ depletion in the same manner as detected by the appearance of highly sensitive RNase cleavage sites in the D and T psi C loops. Similar experiments titrating polyamine concentrations suggested that secondary structure was more efficiently stabilized by polyamines than by Mg2+. The variation of Mg2+ concentrations has been used to gain additional information about other RNA structures. Others have used protein-RNA interactions to approach the question of the functional structure of a RNA (for examples, see Ref. 3). Thus, the ideal parameters to choose would be those known to affect the function of the RNA. The variation of Mg2+ and polyamine concentrations would minimally suggest regions of greater or lesser secondary or tertiary structure stability.