Metastable secondary structures in ribosomal RNA: a new method for analyzing the titration behavior of rRNA.

The pH titration behavior of E . coli rRNA in the acid range has been analyzed by combining spectrophotometric and potentiometric titration data. The “simplest” model for the system, which considers as possible reactions the protonation of adenine (A), cytosine (C), and guanine (G) residues along with the opening of A-U and G . C base pairs, does not adequately account for the titration properties. I t is postulated that extra reactions may occur in addition to those in the “simplest” model, and a new analytical method was developed to deal with this situation. Our approach yields the ultraviolet spectral changes which accompany the extra reactions, from which the nature of these reactions can in principle be deduced. The calculations also give, a t each pH, the extents of the extra reactions as well as the extents of those reactions which comprise the “simplest” model. We infer that in acidic RNA solutions of 0.lM ionic strength there occur a t least two extra reactions, each of which involves G residues. We propose that in the pH range 6.0 2 pH 2 3.8 triple-stranded helical sequences, presumably protonated G .C .G, are formed. These regions are replaced at lower pH by acid-stable structures involving G .G and A.A base pairs. In solutions of lower ionic strength (Z = 0.OlM) no triple strands are formed, but G.G and A. A regions seem to develop even a t pH values as high as 6.0. At Z = O.lM, an acid-base titration cycle between pH 7 and 2.8 is not reversible; rRNA shows true hysteresis behavior. We conclude that in ribosomal RNA’s, which are generally G-rich, guanine residues may participate in hitherto unpredicted conformations, some of which may be metastable while others are equilibrium structures.