Die Temperaturabhängigkeit der Aminoacylierungsgeschwindigkeit von Phenylalanin- und Serin-spezifischer Transfer-Ribonucleinsäure aus Brauerhefe

Abstract

It is known that several physical properties in tRNA * are changed between 35°C and 45°C. These changes have been attributed to unfolding of tertiary structure. In this paper the correlation between these changes and the rate of aminoacylation is studied. With purified tRNASer yeast and tRNAPhe yeast and purified aminoacyl synthetases, aminoacylation reaches a plateau within 20 min. (Fig. 2). The rate of charging was determined in the linear region (5 min.) at varying temperatures. With tRNASer yeast there is a sharp drop in the rate of charging at 39° (Fig. 3 a) corresponding to the first melting of Ser yeast (Fig. 6 a). By changing the Mg2⊕ concentration, rate of aminoacylation and first melting are shifted in the same direction. With tRNAPhe yeast no stepwise decrease of aminoacylation with temperature is observed (Fig. 3 b). At the same time this molecule does not exhibit a two step melting profile (Fig. 6 b). The effects observed cannot be due to a change or irreversible inactivation of the enzyme since the thermal inactivation of the enzyme follows a normal pattern above 43°C and 38.5°C respectively (Figs. 4 a and 4 b). Likewise the amino acid activation reaction is not responsible for these effects. This reaction has been measured by the pyrophosphate formation to have a normal temperature profile up to 50°C. Only at this temperature the enzyme begins to become denatured (Fig. 5). The results indicate that the observed changes in aminoacylation are caused by a change in physical properties of the tRNA. This change yields a molecule having a conformation that can no longer be aminoacylated. Since at the higher temperature still the same plateau is reached, however, at a slower rate, an equilibrium between the two forms must exist. This equilibrium could be of the following manner: