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

Abstract

SynopsisThe 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. It 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 at 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.

Properties of RNA in formamide

“…Very stable structures, such as that of poly(rG) and twostranded RNA, are destroyed only on heating, and at least in the case of the latter the melted state is indefinitely stable after heating. Hysteresis effects in RNA conformations have often been observed and their basis analyzed by Neumann and Katchalsky (1970) andRevzin et al (1973). The cycle which we have described in formamide represents a twofold hysteresis of a type previously observed by Geiduschek and Herskovits (1961) in DNA dissolved in methanolwater mixtures.…”

Molecular Complexity Favors the Evolution of Ribopolymers

“…Hysteresis effects in RNA conformation have been previously observed, and their basis was analyzed , . RNA meta-stability is based on acquired structures, from base stacking to complex foldings.…”

“…Samples were resuspended at a 0.025 M final concentration in water or in the appropriate formamide reaction medium (usually in 0.5-1.0 mL) and incubated at various temperatures (20,30,50,60,70,80, or 90 °C) at the indicated pH values for the appropriate periods of time. Aliquots (5 µL) were diluted to a final concentration of 50% formamide in a final volume of 10 µL and injected into a Supelcosil LC-18-T 3 µm high-performance liquid chromatography (HPLC) column (Supelco), 15 cm × 4.6 mm.…”