Metastable secondary structures in ribosomal RNA molecular hysteresis in the acid-base titration of Escherichia coli ribosomal RNA

Abstract

The metastable conformational states which underlie the hysteresis displayed by Escherichia coli ribosomal RNA in its pH titration in the acid range have been analyzed in terms of acid-stable RNA secondary structures. Sedimentation measurements show that the phenomenon is ktramolecular, so that analysis of the hysteresis loops can, in principle, reveal details of molecular architecture. Hysteresis cycles obtained spectrophotometrically and potentiometrically were compared for RNA in solutions of different ionic strengths and ionic compositions. The effect is much smaller at lower ionic strength and disappears in the absence of magnesium ions. The curve followed upon addition of acid appears to reflect the equilibrium state of the system at each pH value. On the "base branch" of the loop, a slow absorbance change (complete in hours) was observed after the pH was raised by addition of a portion of base. This slow process is attributed to the annealing of "mismatched" multihelical regions of the ribosomal RNA. Certain regions, however, remain in metastable configurations for days and it is these long-lived non-equilibrium structures that underlie the hysteresis. Titration at 35°C gave hysteresis loops of the same size and shape as at 20°C ; indeed, we found that the metastabilities are not removed even at 80°C. Ultraviolet light absorbance difference spectra at 80°C between solutions at the same pH, but on different branches of the cycle, give insight into the nature of the metastable conformation(s).Our experimental observations lead us to propose that the hysteresis is due to the formation at acidic pH of double-helical structures involving protonated guanine and adenine base pairs. The G.G pairs seem especially important to account for the very high thermal stability, as well as for the fact that the structures formed at a given pH value as acid is added dissociate only at higher pH values when the solution is titrated with base. Titrations of transfer RNA, along with literature data on 16 S rRNA primary structure, imply that the metastable regions in rRNA may consist of perhaps 10 to 15 base pairs.

Configurational changes in ribosomal RNA as a function of ionic conditions

“…A possible interpretation of this phenomenon, especially in relation to the results discussed in the previous section, is that base-paired regions persist at pH 3 and high ionic strength, preventing complete condensation of the molecule. This is consistent with the proposal of Rezvin et al (1973) that metastable base-paired regions are formed under these conditions.…”

“…Indeed, Watson and Kidson (1969) have summarized the factors which appear to affect RNA tertiary structure: influence of cation concentration upon base stacking, intrachain electrostatic repulsion due to partially charged phosphate groups, and variable extents of base pairing. To these should probably be added the possible formation, at low pH, of metastable base-paired regions involving guanine residues (Rezvin et al, 1973). The results obtained here lead us to conclude that these influences result in several discrete configurations.…”

“…The third, a compact 50S sphere, is produced at low pH and low ionic strength. A fourth, consisting mainly of a small number of condensed regions, may also possess the metastable regions proposed by Rezvin et al (1973).…”

Localization of the structural change induced in tRNA fMet (Escherichia coli) by acidic pH

“…It will be of particular interest to observe the extent of variation in that region as further threedimensional structures are obtained for tRNA, especially if changes are observed under different crystallization conditions. Structural changes dependent on pH are known in protein crystallography (Vandlen and Tulinsky, 1973;Mavridis et al, 1974), and they can be inferred to occur in other RNAs from the hysteresis observed in acid-base titrations of RNA (Revzin et al, 1973).…”