PROTON-EXCHANGE AND BASEPAIR KINETICS OF YEAST TRNA(PHE) AND TRNA(ASP1)

Abstract

Nuclear magnetic resonance measurements of proton exchange were performed on yeast tRNA(Phe) and yeast tRNA(Asp), at temperatures from 20 to 45 degrees C, in the presence of various levels of salt, phosphate, added magnesium, and pH. The dynamical changes of the tRNA molecule were interpreted, with the aid of firmly established assignments and the use of the saturation recovery technique. In tRNA(Phe), the exchange rates in zero magnesium indicated early melting of the acceptor stem, tertiary structure, and D stem. However, in the presence of even low levels of magnesium the D stem remained intact up to high temperature, stabilized by a Mg2+ ion. A similar unfolding sequence was observed in tRNA(Asp). The difference between these two tRNAs was the thermal behavior of the tertiary resonance U8-A14, In tRNA(Phe), this base pair showed sharp rate increases between 32 and 39 degrees C. However, in tRNA(Asp), it remained intact up to 36 degrees C and disappeared at 39 degrees C, even if there was not important kinetic broadening. By measuring the temperature dependence of the exchange rates, we obtained an activation energy of 40-60 kcal/mol for all the imino protons of yeast tRNAPhe in zero magnesium. The same activation energy was obtained for tRNA(Phe) with equimolar concentration of magnesium. By investigation of the dependence ofchange rates of these imino protons on solution conditions, we observed the transition from k(op) rate limiting in the absence of magnesium to k(ex) rate limiting in the presence of magnesium.