Hysteresis and conformational changes in ribosomal ribonucleic acid

Abstract

Both rat liver and Escherichia coli rRNA in 0.1m-sodium chloride were titrated with acid or alkali over the range pH3-7 at approx. 0 degrees C. rRNA did not bind acid reversibly and hysteresis was observed, i.e. the plot of acid bound to rRNA against pH had the form of a loop showing that the amount of acid bound at a particular pH depended on the direction of the titration. Although the boundary curves were reproducibly followed on titration from pH7 to 3 and from pH3 to 7, points within the loop were ;scanned', e.g. by titration from pH7 to a point in the range pH3-4 followed by titration with alkali to pH7. It is inferred that the ;lag' in the release of certain bound protons is at least 1 pH unit, that at least about 9-15% of the titratable groups (adenine and cytosine residues) that are involved in this process and that the free energy dissipated in completing a cycle is approx. 4.2kJ/mol (1kcal/mol) of nucleotide involved in hysteresis. The interpretation of the ;scanning' curves was illustrated by means of a cycle of possible changes in the conformation of a hypothetical nucleotide sequence that allows formation of poly(A).poly(AalphaH(+))-like regions in acidic solutions. It is also inferred that the extent of ;hysteresis' might depend on the primary nucleotide sequence of rRNA as well as on secondary structure.

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

“…Hysteresis loops for rRNA reflect the presence of long-lived metastable conformational states and non-equilibrium transitions in the system (Cox & Katchalsky, 1972). We shall show that the phenomenon is an intramolecular one so that analysis of the hysteresis can in principle yield information about the structure of individual RNA molecules.…”

“…An unusual feature of certain ribosomal RNAs is the existence of true hysteresis phenomena in their pH titrations in the acid range (Everett, 1967;Cox & Littauer, 1963;Cox, 1966;Cox & Katchalsky, 1972). That is, the optical density (or proton binding) versus pH data lie on one curve when the pH is lowered by addition of acid, and on a different curve when the pH is raised by addition of base.…”

“…The loop exists between detinite pH limits, while above and below these limits only a single reversible curve is observed. "Scanning" curves (Everett, 1967;Cox $ Katchalsky, 1972) and loops within the main loop can also be obtained.…”

“…It seems likely that at lower pH values there may be rearrangements to different types of base-paired regions, and we may infer which structures are likely by considering the properties of model polynucleotides. Cox & Katchalsky (1972) have discussed rRNA hysteresis qualitatively by using a hypothetical nucleotide sequence devised by Cox (1966). This hypothetical sequence has the same over-all base composition as rRNA, and the arrangement of the residues allows the formation of acid-stable regions at low pH values.…”

“…All features of the real hysteresis cycle, such as the smooth loop and the existence of scanning curves, were interpreted in terms of possible conformations of the hypothetical RNA. Cox & Katchalsky (1972) observed that the hysteresis may well be due to the prevention of the nucleation step in the formation of a (equilibrium) double-helical region; the barrier could arise because of metastability in the "low pH" structures which prevents nucleation by immobilizing the appropriate bases in non-equilibrium configurations.…”